Principles of Cancer Biotherapy

5th Edition
Principles of Cancer
Biotherapy
5th Edition

Edited by
Robert K. Oldham
Hematology-Oncology Associates of Southeast Missouri Hospital
Southeast Medical Plaza
Cape Girardeau, MO
USA

Robert O. Dillman
Hoag Cancer Center
Newport Beach, CA
USA
University of California
Irvine, CA
USA
Robert K. Oldham Robert O. Dillman
Hematology-Oncology Associates Hoag Cancer Center
of Southeast Missouri Hospital Newport Beach, CA
Southeast Medical Plaza USA
Cape Girardeau, MO University of California
USA Irvine, CA
USA

ISBN 978-90-481-2277-6 e-ISBN 978-90-481-2289-9

DOI 10.1007/978-90-481-229-9
Springer Dordrecht Heidelberg London New York

Library of Congress Control Number: 2009929387

First edition published 1987 by Raven Press

Second edition published 1993 by Williams & Wilkins
Third and Fourth edition published 1998 Kluwer Academic Publishers
© Springer Science+Business Media B.V. 2009
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 Preface
The idea for the first edition of Principles of Cancer
Biotherapy was formulated in the early 1980s. As the
founding director of the Biological Response Modifiers
Program for the National Cancer Institute from 1980-
1984, one of us (rko) envisioned a textbook that would
embody the principles of the then fledgling fourth modal-
ity of cancer treatment - biotherapy. Contributing authors
were solicited in 1985, and the first edition came off the
presses in 1987. Principles represented the first compre-
hensive textbook on the use of cancer biotherapy and
summarized the work done in this field through 1986.
The second edition of Principles was published in
1991 about the time biotherapy was more broadly recog-
nized as the fourth major cancer treatment modality.
Subsequent textbooks by DeVita, Hellman and Rosenberg
[1] in 1991, Mitchell [2] in 1993 and Rosenberg [3] in
2000 validated the importance of this modality in
cancer care.
This third edition was published in 1998 and con-
firmed the tremendous progress that had been made in
the previous five years using biologicals in cancer treat-
ment. It was generally agreed that biopharmaceuticals
were producing major opportunities for new cancer
therapies. Cancer biotherapy was emerging as a more
specific and targeted form of systemic cancer treatment.
Cancer growth control was also becoming an effective
method of treatment complementing cancer destruction
as mechanisms of cancer treatment and “cure”.
The fourth edition of Principles was published in
2003. It was apparent that biotherapy and the use of bio-
pharmaceuticals had not only become recognized as the
fourth modality of cancer treatment, it was clear that
biopharmaceuticals had become the dominant form of
new cancer therapeutics which in the future will replace
less selective, more toxic forms of therapy.
For years, the chemical manipulation of small mole-
cules has been pursued in drug development. We now
have all the tools for the biological manipulation of natu-
ral substances for therapeutic use. In fact, as we better
understand the interaction between biological molecules
and their receptors, it is clear that biological manipula-
tion and chemical manipulation are coming together to
bring molecular medicine to the bedside. Many biologi-
cal molecules are large and have functions other than
those mediated by their active sites. There is increasing
evidence that drug development will focus on the inter-
action between the smaller active regions of these large
biological molecules and their receptors. This opens up
a broad field of molecular design for extending and
improving the therapeutic activities of natural biological
molecules. This has recently been extended to small
molecules interacting with DNA/RNA (anti-sense), with
enzymes such as tyrosine kinase and with growth and
vascular factor receptors. The last decade has been
extraordinarily productive in the development of new
anticancer drugs through chemical and biological manip-
ulation of these natural molecules. Thus, the body itself
has become the “medicine cabinet” of the future.
In the 1990s and beyond into this millennium, medi-
cine will face extraordinary demands. While technol-
ogy brings us tremendous opportunity, it also highlights
problems in our medical care system. Most new tech-
nology is expensive and, as it comes from the laboratory
to the clinic, is by its very nature untried and unproven.
Our medical care system involves a private and govern-
ment insurance reimbursement system that favors pay-
ing for marginally effective medical care of the past
rather than innovative medical treatments of the future.
Such a system is inhibitory to the development of effec-
tive new anti-cancer medicines.
To more rapidly and efficiently exploit the opportuni-
ties in cancer biotherapy in the future, patients, employ-
ers, insurers, universities, and government must come
together and redefine the system of reimbursement to
maximize the patient’s opportunity for access to new
and potentially effective cancer therapies. To simply
reimburse for old ineffective or marginally effective
treatment is not the answer. Provisions must be made to
fund clinical research and afford these new approaches
broader use at the bedside. We must develop methods to
allow our patients access to the opportunities of the
future, while maintaining solid support for effective
therapies of the past. No longer is it acceptable to pay
only for medical care that utilizes old technology, such
as chemotherapy, that is approved but only marginally
effective. Across the broad spectrum of human malig-
nancies, most chemotherapeutic drugs are toxic and of
limited medical value. We must support clinical research
v
vi
in its efforts to bring newer methods of cancer treatment
to the clinic, methods that are less toxic and more
effective.
We believe cancer biotherapy will ultimately replace
much of what we utilize today in cancer treatment. In
light of this view, we want to thank all the authors for
their dedication to purpose in writing this fifth edition of
Principles. This book summarizes an evolving science
and a rapidly changing medical practice. As we progress
into the millennium, it now becomes possible to envision
a much more diversified system of cancer research and
treatment that will afford greater opportunities for our
patients. As indicated in some of the chapters in Principles,
there is increasing evidence that our historical “kill and
cure” outlook in cancer treatment is in need of modifica-
tion. Some forms of cancer biotherapy use the strategy
of tumor growth stabilization and control through con-
tinued biological therapy over a longer period of time,
akin to the use of insulin in the treatment of diabetes.
Preface
These chapters illustrate some of these new methods of
thinking and illustrate new strategies for the treatment
and control of cancer. It is always difficult to move from
past dogmas to future opportunities, but this fifth edi-
tion of Principles of Cancer Biotherapy illustrates why it
is so important for researchers, regulators and clinicians
to explore and apply these new opportunities in cancer
biotherapy to the benefit of our patients.
Robert K. Oldham, M.D. Robert O. Dillman MD
References
1. DeVita VT Jr, Hellman S, Rosenberg SA. Biologic Therapy of
Cancer. J.B. Lippincott, 1991.
2. Mitchell MS. Biological Approaches to Cancer Treatment.
McGraw-Hill, 1993.
3. Rosenberg SA. Principles and Practices of the Biologic Therapy of
Cancer. J.B. Lippincott, 2000.
 Contents

Preface v
Robert K. Oldham

Contributors ix

1. Cancer biotherapy: general principles 1

Robert K. Oldham

2. The pathogenesis of cancer metastasis: relevance to therapy 17

Sun-Jin Kim, Cheryl Hunt Baker, Yasuhiko Kitadai, Toru Nakamura,
Toshio Kuwai, Takamitsu Sasaki, Robert Langley, and Isaiah J. Fidler

3. Developmental therapeutics and the design of clinical trials 41

Robert K. Oldham

4. Recombinant proteins and genomics in cancer therapy 53

Kapil Mehta, Bulent Ozpolat, Kishorchandra Gohil, and Bharat B. Aggarwal

5. Current concepts in immunology 85

Robert K. Oldham

6. Therapeutic approaches to cancer-associated immune suppression 101

Robert K. Oldham

7. Cancer vaccines 147

Kenneth A. Foon and Malek M. Safa

8. Cytokines 155
Walter M. Lewko and Robert K. Oldham

9. Interferons: therapy for cancer 277

David Goldstein, Robert Jones, Richard V. Smalley, and Ernest C. Borden

10. Monoclonal antibody therapy 303

Robert O. Dillman

11. Immunotoxins 407

Arthur E. Frankel, Jung-Hee Woo, and David M. Neville

12. Drug Immunoconjugates 451

Malek Safa, Kenneth A. Foon, and Robert K. Oldham

13. Targeted radionuclide therapy of cancer 463

John M. Pagel, Otto C. Boerman, Hazel B. Breitz, and Ruby Meredith

vii
viii Contents

14. Stem cell/bone marrow transplantation as biotherapy 497

Robert K. Oldham

15. Cellular immunotherapy (CI), where have we been and where are we going? 505
John R. Yannelli

16. Growth and differentiation factors as cancer therapeutics 527

Kapil Mehta, Robert K. Oldham, and Bulent Ozpolat

17. Granulocyte colony-stimulating factor: biology and clinical potential 569

MaryAnn Foote and George Morstyn

18. Granulocyte-macrophage colony-stimulating factor 581

Maryann Foote and George Morstyn

19. Cancer gene therapy 589

Donald J. Buchsbaum, C. Ryan Miller, Lacey R. Mcnally, and Sergey A. Kaliberov

20. Regulatory process for approval of biologicals for cancer therapy 613
Antonio J. Grillo-López

21.1. Cancer biotherapy: 2009 disease-related activity 631

Robert K. Oldham and Robert O. Dillman

21.2. Biological therapy of melanoma 633

Robert K. Oldham

21.3. Biological therapy of genitourinary cancer 645

Robert K. Oldham

21.4. Biological therapy of colon cancer 659

Robert O. Dillman

21.5. Biological therapy of breast cancer 669

Robert O. Dillman

21.6. Biological therapy of lung cancer 679

Robert O. Dillman

21.7. Biological therapy of B and T cell lymphoproliferative disorders 693

Robert O. Dillman

21.8. Biological therapy of multiple myeloma 711

Robert K. Oldham

21.9. Biological therapy of squamous cell cancers of the head and neck 713
Robert O. Dillman

21.10. Biological therapy of glioblastoma 723

Robert O. Dillman

22. Speculations for 2009 and beyond 733

Robert K. Oldham

Index 739
 Contributors

Robert K. Oldham Toshio Kuwai

Hematology-Oncology Associates of Southeast Department of Cancer Biology
Missouri Hospital M. D. Anderson Cancer Center
Southeast Medical Plaza The University of Texas
Cape Girardeau, MO Houston, TX
USA USA

Robert O. Dillman Takamitsu Sasaki

Hoag Cancer Center Department of Cancer Biology
Newport Beach, CA M. D. Anderson Cancer Center
USA The University of Texas
University of California Houston, TX
Irvine, CA USA
USA
Robert Langley
Sun-Jin Kim Department of Cancer Biology
Department of Cancer Biology M. D. Anderson Cancer Center
M. D. Anderson Cancer Center The University of Texas
The University of Texas Houston, TX
Houston, TX USA
USA
Isaiah J. Fidler
Cheryl Hunt Baker Department of Cancer Biology
Department of Cancer Biology M. D. Anderson Cancer Center
M. D. Anderson Cancer Center The University of Texas
The University of Texas Houston, TX
Houston, TX USA
USA
Kapil Mehta
Yasuhiko Kitadai Department of Experimental Therapeutics
Department of Cancer Biology M. D. Anderson Cancer Center
M. D. Anderson Cancer Center The University of Texas
The University of Texas Houston, TX
Houston, TX USA
USA
Bulent Ozpolat
Toru Nakamura Department of Experimental Therapeutics
Department of Cancer Biology M. D. Anderson Cancer Center
M. D. Anderson Cancer Center The University of Texas
The University of Texas Houston, TX
Houston, TX USA
USA

ix
x Contributors

Kishorchandra Gohil Jung-Hee Woo

Department of Internal Medicine Cancer Research Institute of Scott &White
The University of California Temple, TX
Davis, CA USA
USA
David M. Neville
Bharat B. Aggarwal Angimmune LLC,
Department of Experimental Therapeutics Bethesda, MD
M. D. Anderson Cancer Center USA
The University of Texas
Houston, TX John M. Pagel
USA The Fred Hutchinson Cancer
Research Center
Kenneth A. Foon Seattle, WA
Department of Hematological Malignancies USA
Nevada Cancer Institute
Las Vegas, NV Otto C. Boerman
USA University Medical Center
Nijmegen
Malek M. Safa The Netherlands
Division of Hematology / Oncology
University of Cincinnati Hazel B. Breitz
Cincinnati, OH Poniard Pharmaceuticals, Inc.
USA Seattle, WA
USA
Walter M. Lewko
Cancer Cellular Therapeutics, Inc. Ruby F. Meredith
Cape Girardeau, MO Department of Radiation Oncology
USA University of Alabama Birmingham
Birmingham, Alabama
David Goldstein USA
Department of Medical Oncology
Prince of Wales Hospital John R. Yannelli
Randwick, Sydney Department of Microbiology,
Australia Immunology and Molecular Genetics
Markey Cancer Center
Robert Jones University of Kentucky,
Centre for Oncology and Applied Pharmacology School of Medicine
CRUK Beatson Laboratories Lexington, Kentucky
Glasgow, Scotland USA
UK
MaryAnn Foote
Richard V. Smalley (deceased) Thousand Oaks, CA
Synertron Inc. USA
Madison, WI
USA Dr George Morstyn
Department of Microbiology
Arthur E. Frankel Monash University
Cancer Research Institute of Scott &White Victoria
Temple, TX Australia
USA
Contributors xi

Donald J. Buchsbaum C. Ryan Miller

Department of Radiation Oncology Department of Pathology
University of Alabama at Birmingham University of North Carolina
Birmingham, AL Chapel Hill, NC
USA USA

Lacey R. McNally Antonio J. Grillo-López

Department of Radiology Rancho Santa Fé, CA
University of Alabama at Birmingham USA
Birmingham, AL
USA
Sergey A. Kaliberov
Department of Radiation Oncology
University of Alabama at Birmingham
Birmingham, AL
USA
1 Cancer biotherapy: general principles
ROBERT K. OLDHAM
The term biotherapy encompasses the therapeutic use of
any biological substance, but more specifically, it con-
notes the use of products of the mammalian genome.
With modern techniques of genetic engineering, the
mammalian genome represents the new “medicine cabi-
net.” Biological response modifiers (BRM) are agents
and approaches whose mechanisms of action involve the
individual’s own biological responses. Biologicals and
BRM work through diverse mechanisms in the biother-
apy of cancer. They may (a) augment the host’s defenses
through the administration of cells, natural biologicals, or
the synthetic derivatives thereof as effectors or mediators
(direct or indirect) of an antitumor response; (b) increase
the individual’s antitumor responses through augmenta-
tion or restoration of effector mechanisms, or decrease a
component of the host’s reaction that is deleterious; (c)
augment the individual’s responses using modified tumor
cells or vaccines to stimulate a greater response, or
increase tumor cell sensitivity to an existing biological
response; (d) decrease transformation and/or increase dif-
ferentiation or maturation of tumor cells; (e) interfere
with growth-promoting factors and angiogenesis induc-
ing factors produced by tumor cells; (f) decrease or arrest
the tendency of tumor cells to metastasize to other sites;
(g) increase the ability of the patient to tolerate damage
by cytotoxic modalities of cancer treatment; and/or (h)
use biological molecules to target and bind to cancer cells
and induce more effective cytostatic or cytocidial antitu-
mor activity.
While several of these approaches involve the aug-
mentation or use of biological responses, an understand-
ing of the biological properties of immune response
molecules, growth and maturation factors, and other
biological substances will assist in the development of
specific molecular entities that can act on biological
responses and/or act directly on tumor cells. Thus, one
can visualize the development of biological approaches
with response-modifying as well as direct cytolytic,
cytostatic, growth-inhibiting (antiproliferative), or mat-
urational effects on tumor cells.
Biotherapy is the fourth modality of cancer therapy
and can be effective alone or in association with surgery,
radiotherapy, and chemotherapy. To put biotherapy into
perspective, it is important to dispel a historical miscon-
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
ception associated with immunotherapy: biotherapy can
be effective against clinically apparent, even bulky, can-
cer, and treatment should not be restricted to situations
where the tumor cell mass is imperceptible [64, 71].
Thus, the clinical trial designs for biotherapy can be
similar to those used previously for other modalities of
cancer treatment, as long as one measures both pharma-
cokinetics and the biological responses affected by these
approaches [65]. Testing is continuing for biotherapy
using the interferons, lymphokines, chemokines and
cytokines, growth and maturation factors, angiostatic
factors, monoclonal antibodies and their immunoconju-
gates, vaccines, and cellular therapy [68].
Historical Perspectives
The use of chemical and biological compounds to mod-
ulate biological responses has been under active investi-
gation for over 30 years. Although various chemicals,
bacterial extracts, and viruses have been found to modu-
late immune responses in experimental animals, and, to
a more limited extent, in humans, these “nonspecific”
immunomodulators have not been highly effective in
clinical trials [71]. Molecular biologists have recently
developed techniques for the isolation of genes and their
subsequent translation into appropriate production sys-
tems. These methods make available virtually unlimited
quantities of highly purified biological compounds for
experimental and therapeutic use. As a result, several
classes of biologicals are being evaluated in preclinical
models and clinical trials (Table 1).
The continued investigation of nonspecific immuno-
modulators and adjuvants, as well as the recent advent
of genetically engineered biologicals, makes the need
for predictive preclinical assays of biological activity
and efficacy apparent [33]. In vitro assays of biological
activity (bioassays) are generally used to define and
quantitate the activity of a given biological substance.
Subsequently, flow cytometry, immunoperoxidase staining,
enzyme-linked immunosorbent assays (ELISA), tetramer
assays, radioimmunoassays (RIA), and variations of these
methods allow the precise determination of levels of these
molecules and activities in appropriate fluids and tissues.
1
2 Cancer biotherapy: general principles
Table 1. Biologicals and BRM
Immunomodulators (chemicals, bacterial extracts, viruses, etc.)
Lymphokines/cytokines α, β, γ-interferon; IL-1–32, tumor
necrosis factor (TNF); etc.
Growth/maturation factors (CSF, IL-2, EPO, etc.)
Effector cells (cytotoxic and helper T cells, NK, and LAK cells,
gene-engineered cells, etc.)
Tumor-associated antigens and gene-engineered cellular
vaccines
Monoclonal antibodies
Immunoconjugates
Finally, there is the need to assess the in vivo activity of
these materials in preclinical models to develop predic-
tive assays for clinical efficacy and provide information
useful in the rational selection of agents and the design of
clinical trials [33, 54].
Given the variability in the biological behavior of
cancer and its interface with the human outbred host, it
is not surprising that trials of nonspecific and specific
immunotherapy, as translated from artificially con-
structed animal models, have not been uniformly suc-
cessful in cancer treatment [4, 33]. Naturally occurring
cancers arise in a particular organ from one cell or a few
cells under some carcinogenic stimulus. In humans,
these initial foci of cancer cells may grow – and some-
times lie dormant – over very long periods of time (from
1% to 30% of the human life span) before there is clini-
cal evidence of cancer. Dissemination of cells from the
initial focus may occur at any time during the develop-
ment of the primary tumor. Subsequently, growth and
metastasis occur over periods of months to years from
primary and secondary foci, causing complex biological
interactions to occur.
In contrast, experimentally induced cancer is an arti-
ficial (even artifactual) situation. A short-duration, high-
dose carcinogen may be used to induce cancer quickly
so that experiments can proceed rapidly. In transplant-
able models, the tumor cells are injected into young,
normal, syngeneic animals, thereby circumventing the
influences of environmental or genetic factors that may
be operative in the natural host during tumor develop-
ment. Many of the experimental models are transplant-
able tumors that have been maintained for decades and
have only the most remote relevance to cancer in
humans. The injection represents a single, instantaneous
point source for a defined tumor load that has been
manipulated in vitro. Regardless of whether that tumor
load is 10 or 106 cells, it is being placed artificially in a
single site and allowed to grow and metastasize from
that selected and artificial, single site. Thus, these trans-
plantable cancers are simply not analogous to clinical
cancer and the conclusions drawn from them are unlikely
to be broadly applicable to human cancers [33].
The modern era of cancer treatment began in the
1950s with the recognition that most cancers were sys-
temic problems. It became obvious that lymphatic and
blood-borne metastases often occurred simultaneously
with local growth and regional spread. The early success
of chemotherapy in leukemia and lymphoma prompted a
massive and enthusiastic search for chemicals that might
have cytolytic or cytostatic effects on cancer cells.
Millions of compounds have been “screened” for antitu-
mor activity [11], and this effort has given clinicians less
than 75 “approved and active” anticancer chemotherapy
drugs [84]. In addition, there are about 15 approved hor-
monal agents, ten targeted small molecules, and 25 bio-
logical drugs. There is now widespread recognition that
only a few drugs in cancer treatment can effectively pal-
liate and sometimes cure [19]. The development of three
modalities (surgery, radiotherapy and chemotherapy)
and their subsequent integration into what is now multi-
modal cancer treatment has been summarized [18, 20].
Between 1975 and 1990 a plateau was reached in cancer
treatment. New surgical techniques (e.g., debulking,
intra-operative methods for radiotherapy, and catheter
isolation/infusion for chemotherapy) and new methods
of radiotherapy (e.g., neutrons, protons, interstitial ther-
apy, isotopes) continue to be developed, but these two
modalities are primarily useful in local and regional can-
cer treatment. There has been continued, but slow, prog-
ress in the treatment of highly replicative, drug-sensitive
malignancies over the past 15 years. It is now apparent
that further progress with chemotherapy will depend on
a greater understanding of the metabolic and enzymatic
processes of cancer cells and the differences between
these and normal cells. In addition, there are the prob-
lems of drug resistance, selectivity of action, and drug
delivery. Cancer cells are more like than unlike normal
cells with respect to chemotherapy sensitivity but they
are more likely to become chemotherapy resistant with
drug exposure. Many chemotherapeutic agents are
highly cytolytic, but the problems of normal tissue tox-
icity, drug delivery, and tumor-cell resistance remain
[19, 46]. Thus, cancer remains a systemic problem, and
further systemic but more selective approaches are
required for more effective treatment [20].
In addition to standard chemotherapy drugs that are
approved and in clinical use, there are approximately 15
hormonal drugs available. These are primarily used in
breast cancer and prostate cancer, and many are varia-
tions on the theme of blocking hormonal receptors. The
evolution in this area has been primarily testing new
Robert K. Oldham
hormonal agents for superiority in therapy and for lesser
degrees of toxicity when used in large-scale trials against
hormonally sensitive tumors.
Then newest area of drug development is in targeted
therapy. Here, small molecules are tested for the ability to
block a particular receptor, enzyme, or target which may
be involved in cell replication or cell metabolism. Eight
targeted agents are currently available in the clinic, and a
very large number of these small molecules are currently
in clinical trials, most of which are variations on the
theme of blocking a particular receptor or enzyme system
with many of these drugs being multifunctional in that
they can block more than one receptor or system.
The scientific basis for biotherapy as the fourth modal-
ity of cancer treatment is now firm [21–23, 68, 70, 81, 82,
94, 97]. Historically, there was an attempt to establish
immunotherapy in this role. Whereas immunotherapy
was reproducible under specific experimental protocols,
it was not strikingly effective in animals bearing palpable
tumors and did not translate well to patients. Given the
observation that immunotherapy was more effective with
small tumor burdens, investigators began to study both
“specific” and “nonspecific” immunotherapy as treatment
for minimal residual disease. Although it became widely
accepted that the treatment of animals with minimal
residual disease was analogous to the postsurgical treat-
ment of cancer in humans, this analogy was often
stretched beyond reason. Immunotherapy in young, nor-
mal, syngeneic animals was often begun on the day of the
tumor transplant (or within 1 or 2 days), using a trans-
plant of a very small number of tumor cells (1–1,000) to
a single site. In many of these studies, and in studies in
which the tumor was surgically resected and no evident
disease remained, the effects of immunotherapy were rea-
sonably reproducible; the therapy was most beneficial
when the tumor mass was less than 106 cells.
These experimental results produced a dogma that
immunological manipulation or immunotherapy could
work only when the tumor cell mass was imperceptible
[4, 71]. This posed real problems for clinical immuno-
therapy, since the tumor cell mass at clinical diagnosis
or after surgery is usually three orders of magnitude (or
more) greater than 106 cells. Despite the obvious diffi-
culties with the experimental models and the translation
to humans, clinicians began larger scale immunotherapy
trials in the 1970s. The results of initial, small, uncon-
trolled trials were often reported as positive. Larger, ran-
domized, controlled studies were done to confirm the
efficacy of a particular immunotherapeutic regimen in a
particular type of cancer. Although some of the con-
trolled studies were positive, most yielded marginal or
3
negative results. Thus, immunotherapy had a poor image
by the end of the 1970s [71, 109].
Immunotherapy failed to establish itself as a major
mode of cancer treatment for several reasons. One impor-
tant factor was the lack of definition and purity of immu-
notherapeutic agents. Many of the nonspecific approaches
involved the use of complex chemicals, bacteria, viruses,
and poorly defined extracts in an attempt to “stimulate”
the immune response. Thus, molecular definition of the
actual stimulating entity was not available. Given the lack
of analogy between model systems and humans, the
poorly characterized reagents, and the problems of vari-
ability in experimental procedures, the lack of demon-
strable clinical efficacy was predictable [71].
Immunotherapy is not an appropriate term for the
modern use of biologicals and BRM in medicine.
Biological control mechanisms should be envisioned on
a much broader basis than the immune system. While
immunotherapy remains a subcategory of biotherapy,
growth and differentiation factors, chemokines and
angiostatic factors, the use of synthetically derived
molecular analogs, and the pharmacological exploita-
tion of biological molecules is much broader than
immunotherapy (Table 1) [68].
Certain specific developments led to biotherapy
becoming the fourth modality of cancer therapy.
Advances in molecular biology have given scientists the
capability to clone individual genes and produce signifi-
cant quantities of highly purified genomic products as
medicines. Unlike extracted and purified biological
molecules, available in small quantities as semi-purified
mixtures, the products of cloned genes have a level of
purity on a par with drugs. They can be analyzed alone
or in combination as to their effects in cancer biology. In
addition, progress in nucleic acid sequencing and trans-
lation, protein sequencing and synthesis, the isolation
and purification of biological products, and mass cell
culture has given the scientific community the opportu-
nity to alter nucleic acids and proteins at the nucleotide
or amino acid level to manipulate then optimize their
biological activity [42]. The elucidation of the human
genome and the encoded products broadened the oppor-
tunities for advancements in biotherapy.
As a result of gene cloning, a major approach in can-
cer treatment has evolved. Interleukin-2 can be used to
stimulate the growth of a broad range of lymphocytes
(T, NK, and LAK cells). This has given clinicians the
ability to have large quantities of specific subclasses of
effector cells for cancer treatment. Emerging evidence
suggests that these effector cells can be helpful in the
regional and systemic treatment of advanced, bulky
4 Cancer biotherapy: general principles
cancer. It was the availability of Interleukin-2 that
allowed this technology to prosper [83]. In addition,
IL-2 is now being used as the T cell growth factor for
gene engineered lymphocytes containing new genes to
enhance their cancer treatment capacity [80, 103].
Another major technical advance was the discovery
of hybridomas. A major limitation on the use of anti-
bodies had been the inability to make reproducible high-
titer, specific antisera and to define these preparations
on a molecular basis. Immunoglobulin reagents can now
be produced with the same level of molecular purity as
cloned gene products and drugs. Processes to easily
“humanize” antibodies have produced therapeutic anti-
bodies with excellent specificity, low immunogenicity
and optimal pharmacokinetics. These antibodies are
also powerful tools in the isolation and purification of
tumor-associated antigens, lymphokines/cytokines, and
other biologicals, which can then be used in biotherapy.
The advances in molecular biology and hybridoma tech-
nology have eclipsed previous techniques for the isola-
tion and purification of biological molecules [63, 77].
Technical advances in instrumentation, computers,
and computer software have been critically important
in the isolation and purification of biological molecules.
The construction of nucleotide or amino acid sequences
to fit any biological message is now possible. While
this synthetic capability is currently limited to smaller
gene products, techniques by which analysis and con-
struction of nucleotide sequences will occur in an auto-
mated way, making enormously complex molecules
possible to synthesize and manufacture, are rapidly
becoming available.
Preclinical Models
Biological Activity in Preclinical Models
Central to the identification of biotherapy that might be
useful in cancer patients is the recognition that, in the
main, the challenge in humans is the eradication of
metastases. Metastases can result from the dissemina-
tion of different subpopulations of cells within the pri-
mary neoplasm [32]. This may explain the observation
that cells within a metastasis can be antigenically dis-
tinct from those that predominate in the parental tumor
[32]. Metastases may also emanate from other metasta-
ses. The implications of cellular heterogeneity as it
relates to the outcome of the specific immunotherapy
are obvious. In addition, normal animals are not com-
parable to animals or humans bearing autochthonous
neoplasms [33]. There may exist in animals and in
humans specific or nonspecific defects important in the
development of their autochthonous tumors. Corrections
of such defects may require a form of biotherapy totally
different from that required to assist the normal host in
controlling a transplanted cancer.
Model Screening Criteria
Theoretically, an ideal procedure for screening new bio-
logicals should employ a system of sequential and pro-
gressively more demanding protocols designed to select
a maximum number of effective agents.
The term screening denotes a series of sequential
assays through which promising agents are tested for
therapeutic potential. For some biotherapies, a general
screening procedure may be inappropriate. For exam-
ple, the activity of a monoclonal antibody with antitu-
mor specificity would not be detected by use of the
general screen of biological activity. Design consider-
ations for general screening in biotherapy have been
extensively reviewed [33, 54, 56, 61]. A step-by-step
approach to the screening of potential BRM was devel-
oped to define their effects on T-cell, B-cell, NK-cell,
and macrophage functions. A progressive in vitro and
then in vivo sequence allows the variables of dose,
schedule, route, duration and maintenance of activity,
adjuvanticity, and synergistic potential to be explored
in an orderly fashion [33]. Unfortunately, the screening
program was used only briefly by the NCI and no
replacement program is now in use.
Evaluation of Screening Programs
Screening programs for chemotherapeutic agents were
initiated in the mid-1950s, and attempts have been
made to randomly examine thousands of compounds
for antitumor activity [11]. Such large screening pro-
grams are empirically rather than rationally based, and
are no longer appropriate [4, 5, 20, 33, 71]. Whether
induced or transplantable animal tumor systems are
valid models for testing therapeutic approaches for
human cancer has been a controversial issue [33].
In patients, therapy successful against one type of can-
cer may not be successful against another type, or even
for another patient with the same histological type of
cancer. Unlike the model systems, in which treatment
can be given with precise timing relative to the meta-
static phase of a resected tumor or injected tumor cells,
cancer diagnosis in humans is generally late, and micro-
metastases (and often macro-metastases) have become
Robert K. Oldham 5

well established before treatment can be initiated. Thus,
screening programs can only provide tentative indica-
tions on agents and approaches of interest.
Biotherapy: Specific Agents
and Approaches
Nonspecific Immunomodulators
Since the early 1900s, immunotherapy with bacterial or
viral products has been utilized with the hope of “non-
specifically” stimulating the host’s immune response
[71]. These agents had been useful as adjuvants and as
nonspecific stimulants in animal tumor models, but
human trials have been disappointing.
Perhaps purified viruses or specific chemicals will lead
to the development of more effective adjuvants or stimu-
lants of the immune response. Bacillus Calmette-Guerin
(BCG) and other whole organisms were used early in
immunotherapy. The use of a purified derivative of bacte-
rial components, such as muramyl di- or tripeptide, “pack-
aged” in liposomes as a method to stimulate macrophages
to greater anticancer activity has also been tried. Such
adjuvants may prove useful with genetically engineered or
synthetic tumor-associated antigens, active specific
immunotherapy, or immunoprophylaxis.
Multiple agents that appear capable of augmenting
one or more immune functions already exist. Several of
these have been associated with prolongation of sur-
vival in prospectively randomized trials involving
patients with a wide variety of malignancies (Table 2).
Although some of these agents have produced modest
clinical benefits, and do represent a potential method of
immune augmentation, it is doubtful that they will have
a major role in future cancer therapy. Great problems

Table 2. Biologicals and biological response modifiers

Immunomodulating agents Lymphokines, cytokines, growth/maturation factors
Alkyl lysophospholipids (ALP) Antigrowth factors
Azimexon Chalones
BCG Colony-stimulating factors (CSF)
Bestatin Growth factors (transforming growth factor, TGF)
Brucella abortus Lymphocyte activation factor (LAF-interleukin 1 [IL-1])
Cornyebacterium parvum Lymphotoxins (TNF, α, β, LT)
Cimetidine Macrophage activation factors (MAF)
Sodium diethyldithiocarbamate (DTC) Macrophage chemotactic factor
Endotoxin Macrophage cytotoxic factor (MCF)
Glucan Macrophage growth factor (MGF)
‘Immune’ RNAs Migration inhibitory factor (MIF)
Krestin Maturation factors
Lentinan Interleukin 3–18, etc.
T-cell growth factors (‘TCGF’ – interleukin 2 [IL-2]) Thymocyte mitogenic factor (TMF)
Levamisole Transfer factor
Muramyldipeptide (MDP), tripeptide (MTP) Transforming growth factor (TFR, α, β)
Maleic anhydride-divinyl ether (MVE-2)
Mixed bacterial vaccines (MBV) Antigens
Nocardia rubra cell wall skeletons (CWS) Tumor-associated antigens
Picibanil (OK432) Molecular vaccines
Prostaglandin inhibitors (aspirin, indomethacin) Cell-engineered cellular vaccines
Thiobendazole Effector cells
Tuftsin Macrophages
Interferons and interferon inducers NK cells
Interferons (α, β, γ) Cytotoxic T cells
Poly IC-LC LAK cells
Poly A-U T helper cells
GE-132 Miscellaneous approaches
Brucella abortus Allogeneic immunization
Tilorone Liposome-encapsulated biologicals
Viruses Bone-marrow transplantation and reconstitution
Pyrimidinones Plasmapheresis and ex vivo treatments (activation
Thymosins columns, immunoabsorbents, and ultrafiltration)
Thymosin alpha-1 Virus infection of cells (oncolysates)
Thymosin fraction 5
Other thymic factors
6 Cancer biotherapy: general principles

exist for most of the agents, including lack of chemical Table 3. Studies of immunotherapy with random designs
definition, low purity, and poor reproducibility from Specific cancer
one lot to another. An additional problem has been the and type of Positive Equivocal Negative
inability to define clearly a mechanism of action for immunotherapy studies studies studies
these agents in humans. The preclinical screening Leukemia
established by the Biological Response Modifiers BCG/AML +
Program (BRMP) of the National Cancer Institute was BCG/Cells/AML + +
one mechanism to do this [31]. Data from this type of MER/AML +
screening with subsequent phase I and phase II clinical CP/Cells/AML +
data could have provided interesting insights and cor- All/Hodgkin’s
relations [98, 104, 105]. Unfortunately, this approach NHL/MM
has been abandoned. Lev/ALL +
The ability to specifically control and manipulate BDG/NHL +
immune responses with highly purified, defined mole- Lev/MM +
cules obtained by genetic engineering is the future. Lung cancer
Thus, it seems probable that nonspecific immunotherapy IT BCG + +
as a sole modality has become obsolete, although as IP BCG + +
adjuvants some may find a role in active specific immu- IP BCG & Lev +
notherapy (Chapter 6). IP CP +
Lev + +
Thy Fr V +
Active Specific Immunotherapy BCG/Cells +
TAA/Freund’s
There has been a substantial effort to produce active Adjuvant +
immunization of autochthonous or syngeneic hosts with
Breast cancer
irradiated or chemically modified tumor cells in an
Poly A/Poly U +
attempt to use active specific immunotherapy (AST) [46]. BCG + +
Inherent is the assumption that tumor cells express immu- Lev +
nogenic tumor-associated antigens (TAA). Treatment of
Colon cancer
tumor cells with a variety of unrelated agents, such as
BCG +
irradiation, mitomycin, lipophilic agents, neurominidase, MER +
viruses, or admixtures of cells with bacterial or chemical CP +
adjuvants, has produced non-tumoroigenic tumor cell Lev +
preparations that are immunogenic upon injection into
Melanoma
syngeneic hosts (Table 3). IL/BCG +
AST using BCG-tumor cell (“antigens”) vaccines has BCG/BCG + Cells + +
been reevaluated using a syngeneic guinea pig hepato- BCG + +
carcinoma. The definition of several variables of vac- CP +
cine preparation, such as a ratio of bacterial organisms Lev +
to viable, metabolically active tumor cells, the proce- Genitourinary cancer
dures of tumor cell dissociation and cryobiologic pres- IC BCG/bladder +
ervation, and the irradiation attenuation of cells, has BCG/prostate +
resulted in the development of an effective non-tumori- Gynecological cancer
genic vaccine. It has proven effective in both micro- and CP/cervix +
macro-metastatic disease [47]. CP/ovary +
The nature of the anatomic alteration in metastatic BCG/ovary +
nodules after AST was explored using a specific Other cancers
monoclonal antibody to assess vascular permeability Lev/H & N +
within these tumor nodules [50, 51]. Immunohistologic CP/H & N +
analysis demonstrated significantly more antibody in BCG/Cells/Sarcoma +
tumors from vaccinated animals than in comparable MER/Neuroblastoma +
tumors from unvaccinated guinea pigs. These data sup-
port the hypothesis that the anatomic characteristics of tecting tumors not only from immunotherapy but from
tumor foci restrict drug and antibody access, thus pro- other forms of treatment as well [45].
Robert K. Oldham
The regulation of the blood supply to neoplastic tissue
may be unique in comparison to normal tissue. Tumor
metastatic nodules may have a vascular “barrier,” which
contributes to a limitation in delivery for chemothera-
peutic agents, monoclonal antibodies, and immune
effector cells. Such vascular barriers may provide an
environment in which some tumor cells survive
blood-borne chemotherapeutic and biologic agents.
Thus, solid tumor nodules may serve as “pharmacologic
sanctuaries,” allowing even drug-sensitive tumor cells
to continue to grow [45–47].
Hanna and co-workers [47] demonstrated that strategi-
cally timed chemotherapy subsequent to immunotherapy
can effectively double the number of survivors attainable
with immunotherapy alone. This effect was not drug spe-
cific. These results suggest a new basis for AST in the treat-
ment of solid tumors. Inflammatory disruption of anatomic
barriers of metastatic nodules combined with strategically
administered chemotherapy or biotherapy may prove use-
ful in the design of future biotherapy trials in humans.
Another approach used more recently involves the deliv-
ery of lymphokines and cytokines, such as interleukins,
colony stimulating factors, tumor necrosis factor, lympho-
toxins, macrophage cytotoxic factors, and activated com-
plexes (such as those generated by plasma perfusion over
protein A columns) to the tumor and its vascular bed. These
substances are known to have powerful effects on tumor
vasculature and cellular infiltrations, something leading to
tumor necrosis. This approach may, in addition, increase
the access of antibody, immunoconjugates, drugs, and acti-
vated cells to the cancer nodule [45].
A more recent method to influence the blood supply
of tumor nodules has been the use of a monoclonal anti-
body, Bevacizumab, which targets the vascular endothe-
lial growth factor (VEGF) receptor. Bevacizumab is
primarily used with chemotherapy, and it may be there
are similar mechanisms at play with regard to increasing
chemotherapy drug access to tumor nodules by the use
of a monoclonal antibody targeting the VEGF receptor
[38, 49, 52].
A major limitation of AST has been the availability of
purified TAA. Whereas whole cell vaccine preparations
contain viable, non-tumorigenic cells prepared from
individual tumors, purified TAA is now prompting large-
scale immunization. TAA purification and characteriza-
tion followed by genetic engineering of the antigen for
vaccine production is underway and several purified
antigens are in clinical trials, especially in melanoma.
Alternatively, synthetic peptide sequences of the active
portion of TAA may prove useful in the near future.
Even the combining site of antibody to TAA has recently
been suggested as a potential vaccine. These technologies
7
are now undergoing extensive preclinical and clinical
evaluation (see Chapter 7).
Thymic Factors
It has been known for years that thymic extracts have
immunological activity [40, 41]. Thymosin fraction 5 and
thymosin alpha-1 have received the most attention in the
laboratory and the clinic. Thymosin fraction 5 is an extract
containing a variety of thymic polypeptides, and alpha-1 is
a synthetic polypeptide component present in many thy-
mic extracts. Thymic preparations have been shown to
enhance and suppress immune responses in both intact
and thymectomized animals. Many investigators have
reported that the thymosins can correct selected immuno-
deficiency states, both natural and experimentally induced.
There have also been reports that thymic factors can aug-
ment suppressed or depressed T-cell responses in patients
with cancer. Studies in preclinical screening have demon-
strated stimulation of T-cell activity [99], but clinical stud-
ies have not shown striking effects [25, 98, 99], and none
have been approved for clinical use.
Recombinant DNA Technology
Recombinant DNA technology, commonly referred to as
genetic engineering, has provided us with the tool for the
biosynthesis and subsequent mass production of a sig-
nificant number of biologicals [8, 29, 39]. This is highly
relevant to lymphokines/cytokines as well as growth and
maturation factors, and should revolutionize the treat-
ment of cancer over the next 10 years. The process
involves the incorporation (recombination) of a segment
of a DNA molecule containing a desired gene into a vec-
tor, usually a plasmid, which in turn is inserted into a
host organism, usually an Escherichia coli, although
other bacteria, yeasts, insects, and mammalian cells have
been utilized. The cells are cloned and the cells produc-
ing the desired protein or polypeptide are selected. This
clone is mass produced using fermentation techniques,
and the protein molecule is harvested and purified. The
resultant product is a highly purified (95–99%) protein
solution, and has a high specific activity (i.e., biological
activity per weight of protein).
The relevant DNA can be obtained by a variety of
methods. Once messenger RNA (mRNA) is isolated,
complementary DNA (cDNA) can be produced through
the use of reverse transcriptase. Alternatively, an artificial
DNA molecule can be constructed once the nucleotide or
amino acid sequence is known. This can be used to isolate
the complementary sequence, which is then isolated and
cloned. RNA molecules can also be used in cell-free systems
8 Cancer biotherapy: general principles
Table 4. Clinical lymphokines/cytokines
Colony-stimulating factors
Erythropoietin
Interferons: α, β, γ
Interleukins 1–32, etc.
Lymphotoxincs: α, β (TNF)
Macrophage-activating factors
Thymosins
Transfer factor(s)
to produce these biologicals. There are over 200 restric-
tion enzymes that can cut desired fragments of DNA and
lead to their isolation. These enzymes can uniquely cleave
a DNA molecule at specific, predictable sites relative to
the nucleotide sequence. These fragments are then incor-
porated into the plasmid, and combine with plasmid
DNA. Plasmids have a symbiotic relationship with
selected bacteria inducing resistance to a variety of anti-
biotics, which allows for selection of engineered clones.
A number of alpha interferons, as well as beta and gamma
interferon, have been genetically engineered [24, 39, 106,
111]; multiple interleukins, colony-stimulating factors,
and tumor necrosis factor have been cloned [107]. The
number of cloned biological products increases yearly
(see Chapter 4). These biological products and their
receptors (Table 4) are rapidly being translated into high-
quality pharmaceuticals for clinical testing and approval
for general use.
Lymphokines/Cytokines
Lymphokines and cytokines are molecules secreted by a
variety of cells (Table 4). They provide one means
through which the cells involved in the immune process
communicate with one another and direct the overall
process [40]. Lymphokines/cytokines with specific
effects on cell proliferation have been identified and
may prove useful as anticancer agents. Interleukins (IL)
1–32 are among the multiple lymphokines that appear to
be involved in a cascade phenomenon leading to the
induction of a variety of immune responses [86]. Other
examples include multiple subclasses of colony-stimu-
lating factors (CSF) chemokines and ligands. The list of
lymphokines/cytokines is long, and the potential for
therapeutic manipulation is great [73, 74]. The identifi-
cation of these biological activities is the start of a pro-
cess that should ultimately provide us with the knowledge
and the tools to identify more accurately and control a
number of immune responses. Physicians may then be
able to manipulate the immune system (Fig. 1) intelli-
gently, in favor of the host. Further, by selective activation
and subsequent cloning in vitro, T-cell lines with spe-
cific cytotoxic and helper capabilities can be obtained
and utilized in autologous and allogeneic adoptive
immunotherapy [16, 27, 93, 103, 112].
Many investigators have held the rather simplistic
view that the immune system (Fig. 1) might be manipu-
lated in vivo and corrected to better deal with the cancer
problem. Evidence to date suggests that the pharmaco-
logic use of biologicals, in rather high doses, is a more
effective method for cancer treatment, with immunoac-
tivation and immunomodulation, playing the dominant
roles [69, 70].
Another specific use of lymphokines may be in the
pharmacological regulation of tumors of the lymphoid sys-
tem. Although many of these tumors are considered to be
generally unresponsive to normal growth-controlling
mechanisms mediated by lymphokines, it is possible that
large quantities of pure lymphokines administered as
medicinals, or the use of certain molecular analogs of
these naturally occurring lymphokines, may be useful in
the treatment of lymphoid malignancies.
The use of certain lymphokines/cytokines has been
extended to other cancers, in that in vitro observations
suggest an antiproliferative activity in some solid
tumors. These antiproliferative effects might be maxi-
mized by testing the tumor cells of each patient to “cus-
tom tailor” the treatment rather than giving these
biologicals as general treatment, as has been done with
anticancer drugs [70].
IL-1, originally known as lymphocyte activating fac-
tor, is a macrophage-derived cytokine that has an
enhancing effect on murine thymocyte proliferation.
Both IL-1 and viable macrophages are necessary for the
initial step in activation of IL-2 (Fig. 2). Cloning of IL-1
and IL-2 has made large quantities of highly purified
materials available for clinical studies.
Preclinical studies with IL-2 have been oriented around
in vitro cell production protocols and induction or mainte-
nance of antitumor T-cell effects in vivo [15, 16, 67, 70].
IL-2 has been used to activate peripheral blood cells and
initially stimulated much interest [93, 104]. These acti-
vated cells are generally more cytotoxic against cancer
cells than normal cells; however, their lineage can be T
cell or NK cell (LAK cells) depending on the technique
employed. This approach, though expensive and techni-
cally demanding, illustrates that cellular therapy is feasible
and can be effective [67, 70, 85, 103].
A lymphotoxic product of antigen/mitogen-stimulated
leukocytes was called lymphotoxin [92]. Lymphotoxin
may be a principle effector of delayed hypersensitivity
and, although conflicting data have been reported, may
also be involved in the cytoxic reactions of T-cell-mediated
Robert K. Oldham
Figure 1. Immune response
lysis and NK- or K-cell lysis. Depending upon the type
of tumor cell involved, the in vitro effect of lymphoto-
xin may be either cytolytic or cytostatic. Mouse tumor
cells are frequently killed by homologous and heterolo-
gous lymphotoxins, whereas in other species reversible
inhibition of tumor cell proliferation is more common
[48].
Human lymphotoxin is of at least two species, alpha
and beta [30, 37, 60, 88, 108]. Alpha lymphotoxin is
tumor necrosis factor (TNF). Both have been cloned, and
TNF has undergone rather extensive clinical trials [31,
91, 101]. While some antitumor responses have been
seen, the lymphotoxins have not been highly efficacious
as single agents in the treatment of advanced human
tumors and the toxicity of systemic administration has
been unacceptable. Continued trials are underway com-
bining lymphotoxins such as TNF with other lym-
phokine/cytokines and with chemotherapy. Targeted
delivery of these molecules may prove more efficacious
since they have high toxicity administered intravenously
with what is probably minimal delivery to the tumor cell
9
site [60, 28]. Thus, intratumoral and regional perfusion
studies with TNF have yielded positive results in patients
with melanoma and sarcoma [17, 53, 59, 60, 90].
There is now evidence that the combined use of various
lymphokines may produce enhanced antiproliferative
effects. Selective assays for lymphokine antiproliferative
cocktails may prove useful in “tailoring” such preparations
for individual patients [6, 95, 97, 102, 100, 110].
More than 100 biological molecules have already been
described and named as lymphokines/cytokines (Table 5).
Biologicals such as the interferons, lymphotoxins, TNF,
CSF, IL-1–22 are now under evaluation (see Chapter 8).
The studies require quantities of material obtained
through genetic engineering, using sensitive and rapid
assay procedures to monitor production, purification, and
bioavailability.
Interferons
Interferons are small, biologically active proteins with
antiviral, antiproliferative, and immunomodulatory
10
Monocytic Macrophage
Ag Stimulated
T Cell
? CSF
Activated Macrophage
IL-1
Activated T cells Ag Stimulated T cell
IL -2
Effector T cell
Mature Effector T Cells
Precursor
Figure 2. Model for interleukin stimulation of T-cell immune
responses
activities (see Chapter 9). Each interferon has
distinctive capabilities in altering a variety of
immunological and biological responses. As a class,
the interferons appear to have some growth-regulating
capacity in that antiproliferative effects are measur-
able with in vitro assays and in animal model systems.
The relative efficacy of the mixtures of natural inter-
ferons that occur after virus stimulation as compared
to the cloned interferons remains to be precisely deter-
mined. There are more than 20 interferon molecules
(and theoretically hundreds of recombinant hybrids
there from), and efforts are underway altering individ-
ual interferon molecules in specific ways, so the range
of biological activities of the interferons as antiviral
agents, as immunomodulating agents, and as antipro-
liferative agents may be very broad [66, 69].
In addition to antiviral and antiproliferative activity,
the interferons have profound effects on the immune
system. Low doses enhance antibody formation and
lymphocyte blastogenesis, while higher doses inhibit
Cancer biotherapy: general principles
these functions. Low to moderate doses may inhibit
delayed hypersensitivity while enhancing macrophage
phagocytosis and cytotoxicity, natural killer activity,
and surface antigen expression. Interferons prolong and
inhibit cell division (both transformed and normal cells).
In addition, interferons stimulate the induction of sev-
eral intracellular enzyme systems with resultant pro-
found effect on macromolecular activities and protein
synthesis.
We can draw some preliminary conclusions about
interferon therapy for human cancer [9, 36, 43, 44, 57,
69, 96] (Tables 6 and 7). One is that the Cantell, lym-
phoblastoid, and recombinant alpha-interferons are
surprisingly similar, both quantitatively and qualita-
tively, in their toxicity, antitumor efficacy, and other
biologic effects. Second, objectively defined antitumor
responses in phase I alpha-interferon trials (mostly
involving lymphoma, myeloma, Kaposi’s sarcoma,
melanoma, and renal cancer) were observed in approx-
imately 10% of all patients treated. That level of activ-
ity may not seem impressive, but it does exceed the
average response rate of 1–2% in phase I trials of che-
motherapeutic agents. We should also note that very
few responses in patients with tumors of the breast,
colon, lung, or lower genitourinary system have been
seen with alpha-interferon as a single agent. Overall,
even though interferons have toxicities, they were
more tolerable and less permanent than those observed
in early-phase chemotherapy testing.
A third impression, suggested by increased response
rates with higher alpha-interferon doses, is that
interferons may produce their acute antitumor effect by
a direct cytostatic action, rather than an indirect immu-
nomodulatory mechanism [12, 23, 57, 66, 69, 72, 98].
Finally, clinical experience with beta- and gamma-
interferons indicated that both produce response rates
and response patterns similar to those obtained with
alpha-interferon, although beta-interferon is only
approved for multiple sclerosis and gamma only for
chronic granulomatous disease. What is clear from the
current preclinical and clinical studies is that the inter-
ferons have antitumor activity even in bulky, drug-resis-
tant cancers [102]. Clinical activity has been seen most
reproducibly in several lymphomas and leukemias
(Table 6), but responses in many other tumor types with
approval in melanoma and renal cancer [36].
Growth and Maturation Factors
Using technology similar to that employed for
lymphokine/cytokines, scientists have cloned and pro-
duced a variety of growth and maturation factors. The
Robert K. Oldham 11

Table 5. Antigen-nonspecific mediators,a unrestricted by MHC

Helper factors ESP (eosinophil stimulation factor)
LAF (lymphocyte-activating factor, IL-1) LCF (lymphocyte chemotactic factor)
NMF (normal macrophage factor) LTF (lymphocyte trapping factor)
BAF (B-cell-activating factor)
TRF (T-cell-replacing factor) Factors acting on vascular endothelium
MP (mitogenic protein) SRF (skin reactive factor)
TDF (thymus differentiation factor) TPF (thymic permeability factor)
Transferrin LNPF (lymph node permeability factor)
MF (mitogenic [blastogenic] factor) LNAF (lymph node activating factor)
NSF (nonspecific factor) AIPF (anaphylactoid inflammation promoting factor)
TDEF (T-cell-derived enhancing factor) IVPF (increased vascular permeability factor)
TEF (thymus extracted factor) Factors acting on other cells
Complement components Interferons
DSRF (deficient serum-restoring factor) TMIF (tumor cell migration inhibition factor)
Suppressor factors OAF (osteoclast activating factor)
Inhibitor(s) of DNA synthesis Fibroblast chemotactic factor
AIM (antibody-inhibitory material) Pyrogens
IDA (inhibitor of DNA synthesis) FAF (fibroblast activating factor)
LTF (lymphoblastogenesis inhibition factor) Growth-stimulating factors
FIF (feedback inhibition factor) BCGF (B-cell growth factor)
MIFIF (Mif inhibition factor) BCDF (B-cell differentiation factor)
SIRS (soluble immune response suppressor) MGF (macrophage growth factor)
IRF (immunoregulatory gamma-globulin) MF (mitogeni [blastogenic] factor)
Chalones LIAF (lymphocyte-induced angiogenesis factor)
IF (interferons) CSF (colony-stimulating factor)
AFP (alpha-fetoprotein) TDF (thymus differentiation factor)
LDL (low-density lipoproteins) Thymopoietin, thymosin
CRP (C-reactive protein) TCGF (T-cell growth factor, IL-2)
Fibrinogen degradation products IL-3 (interleukin 3)
NIP (normal immunosuppressive protein) EGF (epidermal growth factor)
LMWS (low-molecular-weight suppressor)
HSF (histamine-induced suppressor factor) Direct-acting factors
TCSF (T-cell suppressive factor) Lysosomal enzymes
CTF (cytotoxic factors)
Factors acting on inflammatory cells MTF or MCF (macrophage toxic [cytotoxic] factor)
MIF (migration inhibitory factor) SMC (specific macrophage cytotoxin)
MCF (macrophage chemotactic factor) MCF (macrophage cytolytic factor)
MSF (macrophage slowing factor) ACT (adherent cell toxin)
MEF (migration enhancement factor) Chromosomal breakage factors
MAF (macrophage activating factor) Microbicidal factors
MFF (macrophage fusion factor) LT (lymphotoxin)
PRS (pyrogen-releasing substance) PIF (proliferation inhibitory factor)
LIF (leukocyte inhibition factor) CIF (cloning inhibition factor)
NCF (neutrophil chemotactic factor) IDS (inhibitor of DNA synthesis)
PAR (products of antigenic recognition) Transforming factors
BCF (basophil chemotactic factor) TNF (tumor necrosis factor)
ECF (eosinophil chemotactic factor)
a
These names/factors are based on biological activity. Several may represent the activity of a single molecule.

clinical trials have focused mainly on erythropoietin
and the colony-stimulating factors. The former is a drug
now approved for use in refractory anemia, and GM-CSF
and GCSF are approved for the treatment of bone marrow
dysplasia and chemotherapy-induced marrow suppres-
sion. Oprelvekim to stimulate platelet production is now
approved. These factors are reviewed in later chapters,
but it should be noted that they represent only the early
beginnings of the very broad field of growth and matu-
ration factors (See Chapter 16). It is now clear that a
variety of biological substances up- and down-regulate
growth of both normal and neoplastic cells. These sub-
stances may stimulate or inhibit growth and may change
the maturation cycle of various normal and neoplastic
cells. Contained within this broad category of factors
are the tumor growth factors, colony-stimulating fac-
tors, and a variety of still to be defined factors important
in the regulation of cell growth and maturation. Future
12 Cancer biotherapy: general principles

Table 6. Apha-interferon activitya antigens on the cell surfaces, which will improve our
Active understanding of cell differentiation and of cancer biol-
Hairy-cell leukemia ogy. Major problems in understanding the biology of the
Chronic myelogenous leukemia cancer cell have been the difficulties of isolating, purify-
Myeloproliferative disorders ing, and characterizing tumor-associated antigens (TAA).
Non-Hodgkin’s lymphoma
The use of monoclonal antibody technology will improve
Cutaneous T-cell lymphoma
Kaposi’s sarcoma the definition of the neoplastic cell surface and identify
Multiple myeloma its differences from the normal counterpart. This will be
Melanoma of great value in cancer diagnosis and histopathologic
Renal cell carcinoma classification, and will be useful in the imaging of tumor
Bladder cancer (intravesical)
cell masses and in the therapy of cancer [1, 7, 13, 14, 32,
Inactive 33, 34, 35, 50, 51, 55, 63, 75, 84, 89]. Finally, antibodies
Breast cancer may be a useful reagent in treating certain immune defi-
Colon cancer
Lung cancer ciencies and in altering immune responses. The removal
Prostate cancer of T cells from bone marrow to improve bone marrow
Acute myelogenous leukemia transplantation techniques is an example of using anti-
a
body as a BRM [63]. A more recent example is the use
As a single agent.
of anti-CTLA-4 antibodies to regulate the immune
response. Two monoclonal antibodies are in develop-
Table 7. Malignancies: Summary of responses to alpha- ment that have influence on T-regulatory cells, which
interferon with date of FDA approval influence the immune response to cancer. It is already
Tumor type Response rate (%) Approved clear that this class of monoclonal antibodies can influ-
Multiple myeloma 18–27 ence T-regulatory cells in a yet undefined manner, such
Hairy cell leukemia 80–90 1986 that regression of bulky melanoma can occur. At the
Low-grade lymphoma 38–73 1997 same time, they give a range of interesting toxicities with
High-grade lymphoma 0–10 regard to uveitis, colitis, and depigmentation syndromes
Kaposi’s sarcoma 25–40 1988 relative to their use in melanoma [26].
Chronic myelogenous 80–90 1995 In spite of encouraging data from the use of anti-
leukemia
bodies and, especially, immunoconjugates to target
Melanoma 20–30 1995
Renal cancer 10–20
toxic substances to cancer, the heterogeneity of cancer
is an important consideration [2, 71–78, 87]. If a sin-
gle antibody or a fixed combination of a few antibod-
ies covering only a portion of the tumor cells is used,
therapeutic use of these factors may regulate growth and and if that preparation does not eliminate the true rep-
spread of cancer. Such a chronic growth restraining licating cell population (stem cell) from the patient’s
strategy may not “cure” cancer; rather treatment may be tumor population, eventual outgrowth of viable, per-
more analogous to using insulin in diabetes. This is a haps resistant cells is inevitable. Therefore, it seems
field that is undergoing explosive growth and should be logical to proceed with attempts to type human tumors
watched carefully over the coming decade for molecules and to deliver toxic substances to them utilizing “cock-
with therapeutic activity. tails” of antibodies sufficient to cover all the tumor
cells suspected of replication in each patient. This
type of approach may require a considerable amount
Monoclonal Antibodies of testing for each patient and a “typing” of one or
The advent of hybridoma technology in the late 1970s more tumors from each patient [3, 58, 62, 78, 79, 87].
made available an important tool for the production of Such approaches may be more individually designed
monoclonal antibodies for therapeutic trials [10] (see than is easily approachable through the product devel-
Chapter 10). These antibodies are now being produced in opment paradigm that has been used in the develop-
huge quantities and in highly purified form for cancer ment of new anticancer drugs. If however, the spectrum
treatment. The humanization of murine antibody combin- of human tumor heterogeneity is great, the goal of the
ing sites has yielded a whole new class of therapeutic anti- ideal antibody conjugated to the ideal toxic agent may
bodies [84]. They will undoubtedly define a new range of not be achievable.
Robert K. Oldham
Future Perspectives
How rapidly will biotherapy develop and what role will
it have in cancer treatment in the next decade? It is cer-
tain that we now have much more powerful tools for
improving cancer therapy in the future. We now have
the techniques to decipher the major problems in cancer
biology down to the genetic level. These techniques,
along with the recognition that biotherapy can provide
increased selectivity in cancer treatment, support the
belief that new and highly effective approaches are near.
Biotherapy provides an additional technique, which
may work effectively in combination with surgery or
radiotherapy (to decrease the local and regional tumor)
or with chemotherapy (to reduce the systemic tumor
burden). It may work very effectively via antibodies in
directing radioisotopes selectively to the tumor site, and
with chemotherapy, toxins, and other cytostatic or cyto-
toxic molecules in directing the agent to the tumor bed,
enhancing activity and selectivity.
The use of biotherapy is at an early stage. We have
already seen that highly purified biologicals can be effec-
tive in patients with clinically apparent, even advanced,
bulky cancer. Clinical studies with alpha-interferon have
demonstrated the responsiveness of radiation- and drug-
resistant lymphoma, melanoma, and renal carcinoma.
IL-2 with effector cells or alone (in high doses) produces
partial and complete remissions in melanoma and kidney
cancer. These results, along with the clinical results using
monoclonal antibody alone and conjugated to toxic sub-
stances in selected cancers (lymphoma, melanoma, gas-
trointestinal, leukemia and breast cancer), confirm the
concept that we need not think of biotherapy as a tool that
can be used only in patients with undetectable and mini-
mal residual tumor burdens [84]. While this modality
may work best with minimal tumor burdens (a situation
that is also true for chemotherapy), biotherapy can be
used as a single modality in clinically apparent disease. It
may be even more effective in multimodality treatment
regimens. Biotherapy offers the hope for selective treat-
ment to enhance the therapeutic/toxic ratio and lessen the
problem of nonspecific toxicity, a major impediment to
the development of more effective cancer treatment.
This decade will provide many opportunities to
pursue new approaches in cancer treatment. These
approaches will employ new techniques in the laboratory
and clinic, requiring special training and expertise. The
medical oncologist trained in the administration of che-
motherapy drugs, is not necessarily well prepared to
give biologicals for cancer treatment. Biotherapy uses
biological substances that are often active on, or work in
13
association with, the immune system. The tremendous
diversity of this system is best understood by clinical
immunologists and cell biologists who are well suited to
assist in the translation of biotherapeutic approaches to
the clinic.
Given these new techniques and new approaches, we
must redesign many of the mechanisms for develop-
mental therapeutics [59]. It may well be that the speci-
ficity of biologicals will require that biotherapy be
developed in an individualistic fashion and applied to
each patient in a specific, personalized way. This con-
cept was conceptualized in 1977 by the Nobel laureate
Sir Peter Medawar:
The cure of cancer is never going to be found. It is far more likely
that each tumor in each patient is going to present a unique research
problem for which laboratory workers and clinicians between them
will have to work out a unique solution.
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2 The pathogenesis of cancer metastasis:
relevance to therapy
SUN-JIN KIM, CHERYL HUNT BAKER, YASUHIKO KITADAI, TORU NAKAMURA,
TOSHIO KUWAI, TAKAMITSU SASAKI, ROBERT LANGLEY, AND ISAIAH J. FIDLER
Introduction
Metastasis, the spread of malignant tumor cells from a
primary neoplasm to distant parts of the body where
they multiply to form new growths, is a major cause of
death from cancer. The treatment of cancer poses a major
problem to clinical oncologists, because by the time
many cancers are diagnosed, metastasis may already have
occurred, and the presence of multiple metastases makes
complete eradication by surgery, radiation, drugs, or
biotherapy nearly impossible. Metastases can be located
in different organs and in different locations within
the same organ. These aspects significantly influence the
response of tumor cells to therapy and the efficiency of
anticancer drugs, which must be delivered to tumor lesions
to destroy cells without leading to undesirable side effects.
Similarly, immune effector cells of current biotherapeu-
tic regimens may not reach or localize in metastases with
different organs.
Exacerbating the problems of treating metastatic dis-
ease is the fact that tumor cells in different metastases
and in some instances even in different regions within a
single metastasis may respond differently to treatment.
Tumor cell resistance to current therapeutic modalities
is the single most important reason for the lack success
in treating many types of solid neoplasms. In part, the
emergence of treatment-resistant tumor cells is due to
the heterogeneous nature of malignant neoplasms. This
diversity, which permits selected variants to develop
from the parent tumor, implies not only that the primary
tumor and metastases can differ in their response to
treatment, but also that individual metastases can differ
markedly from one another.
Insight into the molecular mechanisms that regulate
the pathogenesis of cancer metastasis as well as a better
understanding of the interaction between metastatic
cells and the host microenvironment should provide a
foundation for the design of new therapeutic approaches.
Moreover, the development of adequate models that
allow for the isolation and characterization of cells
possessing metastatic potential will be invaluable in the
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
design of more effective therapeutic modalities. In this
chapter, we discuss the biology of cancer metastasis
with special emphasis on recent data demonstrating that
the microenvironment of different organs can influence
the biological behavior of tumor cells at different steps
of the metastatic process and the development of bio-
logic diversity in malignant neoplasms. These findings
have obvious implications for the therapy of neoplasms
in general and metastases in particular.
The Pathogenesis of Cancer
Metastasis
The process of cancer metastasis is dynamic, complex
and consists of multiple, sequential, and interrelated
steps shown schematically in Fig. 1. To produce a clini-
cally relevant lesion, metastatic cells must survive all
the steps of the process. If the disseminating tumor
cell fails to complete any one of these steps, it will fail
to produce a metastasis. Thus, the successful metastatic
cell has been likened to a decathlon champion who must
be proficient in all ten events to be successful, not just a
few [58]. The outcome of this process depends on both
the intrinsic properties of the tumor cells and their inter-
actions with host factors [48, 56, 57, 99].
The essential steps in the formation of a metastasis
are: (1) After the initial transforming event, either uni-
cellular or multicellular, growth of neoplastic cells must
be progressive, with nutrients for the expanding tumor
mass initially supplied by simple diffusion. (2) Extensive
vascularization must occur if a tumor mass is to exceed
1–2 mm in diameter. The synthesis and secretion of
proangiogenic angiogenesis factor probably plays a key
role in establishing a neocapillary network from the
surrounding host tissue. (3) Local invasion of the host
stroma by some tumor cells could occur by several
nonmutually exclusive mechanisms. (4) Thin-walled
venules, like lymphatic channels, offer very little resis-
tance to penetration by tumor cells and provide the most
17
18 The pathogenesis of cancer metastasis: relevance to therapy

THE PATHOGENESIS OF A METASTASIS

TRANSFORMATION ANGIOGENESIS MOTILITY & INVASION

Capillaries,
ARREST IN Venules, lymphatic vessels
CAPILLARY
BEDS EMBOLISM &
CIRCULATION

TRANSPORT

ADHERENCE
MULTICELL AGGREGATES
(lymphocytes,platelets)

EXTRAVASATION
INTO ORGAN RESPONSE TO
PARENCHYMA MICROENVIRONMENT

METASTASIS OF
METASTASES
TUMOR CELL
PROLIFERATION
& ANGIOGENESIS
METASTASES

Figure 1. The pathogenesis of cancer metastasis

common pathways for tumor cell entry into the circula-
tion. Although clinical observations have suggested that
carcinomas frequently metastasize and grow via the
lymphatic system, whereas malignant tumors of mesen-
chymal origin more often spread by the hematogenous
route, the presence of numerous venolymphatic anasto-
moses invalidates this concept. (5) Detachment and embo-
lization of small tumor cell aggregates occurs next, the
vast majority of circulating tumor cells being rapidly
destroyed. (6) Once the tumor cells have survived the
circulation, they must (7) arrest in the capillary beds of
organs, either by adhering to capillary endothelial cells
or by adhering to subendothelial basement membrane,
which may be exposed. (8) Extravasation occurs next,
probably by the same mechanisms that influence initial
invasion. (9) Proliferation within the organ parenchyma
completes the metastatic process (10). To continue
growing, the micrometastases must develop a vascular
network and continue to evade the host immune system.
Moreover, the cells can invade, penetrate blood vessels,
and enter the circulation to produce additional metasta-
ses (Fig. 1).
Neovascularization-
Angiogenesis
Oxygen can diffuse from capillaries for only 150–200 μm.
When distances of cells from a blood supply exceed
this, cell death follows [80]. Thus, the expansion of tumor
masses beyond 1 mm in diameter depends on neovascu-
larization, i.e., angiogenesis [78, 79]. The formation of
new vasculature consists of multiple, interdependent
steps. It begins with local degradation of the basement
Sun-Jin Kim et al.
membrane surrounding capillaries, followed by invasion
of the surrounding stroma and migration of endothelial
cells in the direction of the angiogenic stimulus. Proli-
feration of endothelial cells occurs at the leading edge
of the migrating column, and the endothelial cells begin
to organize into three-dimensional structures to form
new capillary tubes. Differences in cellular composi-
tion, vascular permeability, blood vessel stability, and
growth regulation distinguish vessels in neoplasms from
those in normal tissue [1, 3, 79, 156].
The onset of angiogenesis involves a change in the local
equilibrium between proangiogenic and antiangiogenic
molecules [55]. Some of the common proangiogenic
factors include bFGF, which induces the proliferation of
a variety of cells and has also been shown to stimulate
endothelial cells to migrate, to increase production of
proteases, and to undergo morphogenesis [80]. Likewise,
VEGF/VPF has been shown to induce the proliferation
of endothelial cells, to increase vascular permeability,
and to induce production of urokinase plasminogen acti-
vator by endothelial cells [46]. Additional proangiogenic
factors include IL-8 [268], platelet-derived endothelial
cell growth factor, which has been shown to stimulate
endothelial cell DNA synthesis and to induce production
of FGF, hepatocyte growth factor (HGF), or scatter fac-
tor, that increases endothelial cell migration, invasion,
and the production of proteases, and platelet-derived
growth factor (PDGF). The production of bFGF, VEGF,
and IL-8 by tumor or host cells or the release of proan-
giogenic molecules from the extracellular matrix is
known to induce the growth of endothelial cells and
formation of blood vessels. Further, the organ microen-
vironment can directly contribute to the induction and
maintenance of the proangiogenic factors bFGF [226,
227] and IL-8 [228].
The production of angiogenic molecules, e.g., VEGF,
bFGF, and IL-8 by melanoma cells is regulated by com-
plex interactions with keratinocytes in the skin [106].
Reports from our laboratory showed that IL-8 is an
important molecule in melanoma growth and progres-
sion. Constitutive expression of IL-8 directly correlated
with the metastatic potential of human melanoma cells.
Further, IL-8 induced proliferation, migration, and inva-
sion of endothelial cells and, hence, neovascularization
[97]. Several organ-derived cytokines (produced by infla-
mmatory cells) are known to induce expression of IL-8
in normal and transformed cells [106]. Since IL-8 expres-
sion in melanocytes and melanoma cells can be induced
by inflammatory signals, the question of whether specific
organ microenvironments could influence the expression
of IL-8 was analyzed. Melanoma cells were implanted
into the subcutis, the spleen (to produce liver metastasis),
19
and intravenously (to produce lung metastasis) of athy-
mic nude mice. Subcutaneous tumors, lung lesions, and
liver lesions expressed high, intermediate, and no IL-8,
respectively, at both the mRNA and protein levels.
Melanoma cells established from the tumors growing
in vivo exhibited similar levels of IL-8 mRNA transcripts
as continuously cultured cells, thus demonstrating that
the differential expression of IL-8 was not due to the
selection of a subpopulation of cells [97].
IL-8 expression can be upregulated by coculturing
melanoma cells with keratinocytes (skin) and inhibited
by coculturing melanoma cells with hepatocytes (liver).
We also investigated the effects of two cytokines pro-
duced by keratinocytes (IL-1, IFN-β) and two cytokines
produced by hepatocytes (TGF-α, TGF-β) on the regu-
lation of IL-8 in human melanoma cells. IL-1 upregu-
lated the expression of IL-8 in human melanoma cells at
both the mRNA and protein levels in a dose- and time-
dependent manner in the presence of de novo protein
synthesis. IFN-β did not affect constitutive IL-8 mRNA
and protein production in human melanoma cells, but it
did block the induction of IL-8 by IL-1. TGF-β inhib-
ited the expression of IL-8, while TGF-α had no effect
on IL-8 expression [226].
Patients with renal cell carcinoma exhibit high levels of
bFGF in the serum or the urine that inversely correlates
with survival [181, 184]. Human renal cancer cells
implanted into the kidney of nude mice were highly meta-
static to the lung, whereas renal cancer cells implanted
subcutaneously remain local [227]. The subcutaneous or
intramuscular tumors expressed a lower level of mRNA
transcripts for bFGF than did tumor cells growing in cul-
ture, whereas the renal tumors had 20 times higher levels
of bFGF mRNA and protein levels as compared with the
cultured cells. A histopathologic examination of the tumors
demonstrated that the subcutaneous tumors had few blood
vessels, whereas the renal tumors had many [227, 228].
Direct correlation between the level of bFGF and
advanced disease was also reported for patients diagnosed
with colon carcinoma. Patients with Duke’s C or D tumors
had markedly higher levels of bFGF in the blood than
patients with Duke’s B. In situ hybridization analysis
revealed that bFGF was overexpressed at the tumor
periphery associated with cell division. Northern blot
analysis detected no mRNA transcripts for bFGF [129]. In
a follow-up study of patients with colon cancer, bFGF
expression was found to be highest in the primary tumors
of patients who presented with metastatic disease and
therefore identify a cohort of patients who appeared to be
free of metastatic disease at the time of surgery (low bFGF
expression) as compared to another cohort of patients who
did develop metastatic disease (high bFGF) [130].
20
Tumor Cell Invasion
To reach blood vessels or lymphatics, tumor cells must
penetrate host stroma that includes basement membrane.
The interaction with the basement membrane consists of
attachment, matrix dissolution, motility and penetration
[154]. At least three nonmutually excluding mechanisms
can be involved in tumor cell invasion of tissues. First,
mechanical pressure produced by rapidly proliferating
neoplasms may force cords of tumor cells along tissue
planes of least resistance [154, 155]. Second, increased
cell motility can contribute to tumor cell invasion. Most
tumor cells possess the necessary cytoplasmic machinery
for active locomotion and increased tumor cell motility
is preceded by a loss of cell-to-cell cohesive forces.
In epithelial cells, the loss of cell-to-cell contact is associated
with downregulation of the expression of E-cadherin, a
cell surface glycoprotein involved in calcium-dependent
homotypic cell-to-cell cohesion. Reduced levels of
E-cadherin are associated with a decrease in cellular/
tissue differentiation and increased grade in carcinomas
[120]. Many differentiated carcinomas express higher
levels of E-cadherin mRNA, as do adjacent normal epi-
thelial cells, whereas poorly differentiated carcinomas
do not. Mutations in the E-cadherin gene and abnormal-
ities of α-catenin, which is an E-cadherin-associated
protein, have been associated with the transition of cells
from the noninvasive to the invasive phenotype [261].
Third, invasive tumor cells secrete enzymes capable
of degrading basement membranes, which constitute a
barrier between epithelial cells and the stroma. Epithelial
cells and stromal cells produce a complex mixture of col-
lagens, proteoglycans, and other molecules, which contains
ligands for adhesion receptors and is permeable to mole-
cules but not to cells [230].
To invade the basement membrane, a tumor cell must
first attach to extracellular matrix (ECM) components
by a receptor-ligand interaction. One group of such cell
surface receptors are the integrins which specifically
bind cells to laminin, collagen, or fibronectin [218].
Many integrins that bind to different components of the
ECM are expressed on the surface of human carcinoma
cells. Tumor progression has been associated with a
gradual decrease of integrin expression suggesting that
the loss of integrins, coupled with the loss of E-cadherin,
may facilitate detachment from a primary neoplasm.
Subsequent to binding, tumor cells can degrade
connective-tissue ECM and basement membrane compo-
nents [177]. The production of enzymes such as type
IV collagenase (gelatinase, matrix metalloproteinase) and
heparinase in metastatic tumor cells correlates with invasive
capacity of human carcinoma cells. Type IV collagenolytic
The pathogenesis of cancer metastasis: relevance to therapy
metalloproteinases with apparent molecular masses of 98,
92, 80, 68, and 64-kDa have been detected in highly meta-
static cells. Poorly metastatic cells, on the other hand,
appear to secrete very low amounts of only the 92-kDa
metalloproteinase [171, 172].
Lymphatic Metastasis
Early clinical observations led to the impression that
carcinomas spread mainly by the lymphatic route and
mesenchymal tumors spread mainly by means of the
bloodstream. We now know, however, that the lymphatic
and vascular systems have numerous connections and
that disseminating tumor cells may pass from one
system to the other [27]. For these reasons, the division
of metastasis into lymphatic spread and hematogenous
spread is arbitrary. During invasion, tumor cells can
easily penetrate small lymphatic vessels and be passively
transported in the lymph. Tumor emboli may be trapped
in the first lymph node encountered on their route, or
they may bypass regional draining lymph nodes to form
distant nodal metastases (“skip metastasis”). Although
this phenomenon was recognized in the late 1800s
[193], its implications for treatment were frequently
ignored in the development of surgical approaches to
treat cancers [263, 264].
Regional lymph nodes (RLN) in the area of a primary
neoplasm may become enlarged as a result of hyperplasia
or growth of tumor cells in the node. Although the use
of morphologic criteria for assessing prognoses based
on lymph node appearance is debatable, lymphocyte-
depleted lymph nodes are believed to indicate a less
favorable prognosis than those demonstrating reactive
morphologic characteristics [16]. Hyperplastic responses
could indicate reactivity to autochthonous tumors, and
this could benefit the host.
Whether the RLN can retain tumor cells and serve as
a temporary barrier for cell dissemination is controver-
sial. In most experimental animal systems used to inves-
tigate this question, normal lymph nodes were subjected
to a sudden challenge with a large number of tumor cells,
a situation that may not be analogous to RLN at the early
stages of cancer spread in humans, when small numbers
of cancer cells continuously enter the lymphatics. This
issue is important because of practical considerations
for surgical management of such neoplasms as cutaneous
melanoma. It raises the question, is elective prophylactic
lymph node dissection appropriate for the treatment of
micrometastases? The biologic justification for elective
lymph node dissection in patients with melanoma
presumes that metastasis of some cutaneous melanomas
Sun-Jin Kim et al.
occurs first in the RLN, and that only at a later time do
tumor cells gain access to the circulation to reach distant
organs. If this is the case, and RLN can act as a tempo-
rary barrier to the spread of cancer, removing the RLN
with micrometastases could clearly increase the cure rate
in subgroups of patients with melanoma. Some evidence
exists that patients with melanomas of intermediate
thickness (1–4 mm) do in fact have an improved survival
rate subsequent to elective lymph node dissection.
Similarly, some data suggest that an improved survival
rate can be achieved for selected patients with head and
neck cancers by elective lymph node dissection or local
treatment with x-irradiation [25].
Recent advances in mapping of the lymphatics draining
cutaneous melanoma (by the use of dyes or radioactive
tracers) have allowed surgeons to identify the lymph
node draining the tumor site (i.e., the sentinel lymph
node) [36]. The presence of melanoma micrometastases
in sentinel lymph nodes is correlated with poor progno-
sis and hence indicates wide field dissection. A series of
more than 500 melanoma cases with longer than 4 years’
median clinical follow-up concluded that absence of
disease in sentinel lymph node correlates with increased
disease-free status (in other nodes) and few or no skip
metastases [119, 175]. These data suggest that elective
lymph node dissection when metastatic cells are present
in sentinel lymph nodes produces beneficial results in
patients with melanoma.
Hematogenous Metastasis
During blood-borne metastasis, tumor cells must survive
transport in the circulation, adhere to small blood
vessels or capillaries, and invade the vessel wall. The
mere presence of tumor cells in the circulation does not
in itself constitute metastasis, since most cells released
into the bloodstream are eliminated rapidly [60, 263,
264]. Using radiolabeled tumor cells, we found that by
24 h after entry into the circulation, less than 1% of the
cells are still viable, and less than 0.1% of tumor cells
placed into the circulation eventually survive to produce
metastases [60].
Although most tumor cells are destroyed in the blood-
stream, it seems that the greater the number of cells
released by a primary tumor, the greater the probability
that some cells will survive to form metastases. The
number of tumor emboli in the circulation appears to
correlate well with the size and clinical duration of the
primary tumor, and the development of necrotic and hemo-
rrhagic areas in large tumors facilitates this process by
providing tumor cells easy access to the circulation [263].
21
To a large degree, the rapid death of most circulating
tumor cells is probably due to such simple mechanical
factors as blood turbulence. Tumor cell survival can be
increased by aggregation. Tumor cells can aggregate
with each other or with host cells, such as platelets and
lymphocytes [67].
Once metastatic cells reach the microcirculation, they
interact with cells of the vascular endothelium. These
interactions include nonspecific mechanical lodgment
of tumor cell emboli as well as formation of stable adhe-
sions between tumor cells and small-vessel endothelial
cells. The organ distribution of metastatic foci is believed
to depend, in part, on the ability of blood-borne malig-
nant cells to adhere to specific endothelium and produce
endothelial cell retraction [185, 186].
The formation of fibrin clots at sites of tumor cell
arrest in the microcirculation can result in blood vessel
damage [45, 46]. In some tumor systems, fibrin forma-
tion is not essential for tumor cell implantation or
metastasis formation. The increased coagulability often
observed in patients with cancer may be related to the
high levels of thromboplastin found in certain tumors or
to production of high levels of procoagulant-A activity,
which can directly activate factor X in the clotting pro-
cess. Since reduced blood flow could lead to increased
trapping of circulating tumor cells and perhaps to their
increased survival, the use of anticoagulants in the treat-
ment or control of metastasis has been tried, albeit to a
limited success.
The adhesion of tumor cells to the vascular endothe-
lium is regulated by mechanisms similar to those used by
leukocytes. The initial attachment of leukocytes to vas-
cular endothelial cells is regulated by the selectin family
of adhesion molecules which consists of three closely
related cell surface molecules. E-selectin, which is
expressed by endothelial cells, mediates initial attach-
ment of lymphocytes (and tumor cells) by interaction
with specific carbohydrate ligands that contain sialylated
fucosylated lactosamines. The expression of mucin-type
carbohydrates on the surface of human colon carcinoma
has been correlated with their metastatic potential [161],
perhaps through differential interaction with E-selectins
expressed on specific endothelial cells. The development of
firm adhesion requires the interaction of other adhesion
molecules, another selective process in metastasis. Several
classes of cell-to-cell adhesion molecules regulate this
adhesion. These include the hyaluronate receptor CD44
and its splice-variants [183], the integrins α5β1, α6β1,
and α6β4, and the galactoside-binding galectin-3 [218].
The arrest of tumor cells in capillary beds leads to the
retraction of endothelial cells and the exposure of the
tumor cells to the ECM. The adhesion of metastatic cells
22
to components of the ECM, such as fibronectin, laminin,
and thrombospondin, facilitates metastasis to specific
tissues, and peptides containing sequences of these com-
ponents of the ECM can reduce formation of hematogenous
metastases [246].
Extravasation of arrested tumor cells is believed to
operate by mechanisms similar to those responsible for
local invasion. Tumor cells can grow and destroy the
surrounding vessel, invade by penetrating the endothe-
lial basement membrane, or they can follow migrating
white blood cells [50]. The ability of malignant cells to
extravasate into surrounding tissues of particular organs
seems to arise, in part, from their selective adherence to
and invasion of certain tissues [187]. Malignant cells
frequently penetrate thin-walled capillaries but rarely
invade arteries or arteriole walls, which are rich in elas-
tin fibers. This resistance to invasion is not necessarily
mediated by mechanical strength alone. Connective
tissues have been shown to produce protease inhibitors,
and these may block enzyme-dependent processes of
invasion.
The invasion, survival, and growth of malignant cells
at particular secondary sites also involve their responses
to tissue or organ factors. Tumor cells can recognize
tissue-specific motility factors that direct their move-
ment and invasion [196]. After tumor cells invade organ
parenchyma, they must also respond to organ-specific
factors that influence their growth [187].
Metastasis of Metastases
The tumor cells proliferating within metastases can
invade host stroma, penetrate blood vessels, and enter
the circulation to produce secondary metastases, the
so-called “metastasis of metastases” [73, 235, 236].
Hart and Fidler used the preferential growth of B16
melanoma metastases in specific organs. Following the
intravenous injection of B16 melanoma cells into syn-
geneic mice, tumor growths developed in the lungs and
in fragments of lung or ovarian tissue implanted intra-
muscularly into the quadriceps femoris but not in renal
tissue implanted as a control [101]. Tumor growth in the
specific transplanted organ could have been caused by
the arrest and growth of tumor cells immediately follo-
wing intravenous injection, i.e., “initial metastases.”
Alternatively, tumor cells injected intravenously could
have been arrested in the lungs, where they developed;
once metastases were established, tumor cells could enter
the circulation to be arrested at other organs and produce
“secondary metastases” [235]. To distinguish between
these possibilities, Nicolson and Fidler performed
The pathogenesis of cancer metastasis: relevance to therapy
several experiments [73]: Two weeks after normal,
tumor-free mice were joined parabiotically to metastasis-
bearing animals, there was no evidence of any tumor
growth in the “guest” animals. However, when the para-
biont animals were allowed to survive for 4 weeks after
separation from the metastasis-bearing animals, 40%
developed lung metastases. Since the host mice did not
have primary tumors at the time of parabiosis, the
metastases in the guest mice could have only arisen as
metastasis from metastases [101].
The Biologic and Metastatic
Heterogeneity of Neoplasms
Only a few tumor cells that enter the circulation can
produce metastases. In fact, since less than 0.01% of
circulating cells are likely to produce a secondary
growth, the development of metastases could represent
the fortuitous survival of a few tumor cells or the selec-
tion from the parent tumor of a subpopulation of meta-
static cells endowed with properties that enhance their
survival [65, 66, 71]. Data generated by our research
group and many others prove that neoplasms are bio-
logically heterogeneous and that metastasis is indeed a
selective process.
The first experimental proof of metastatic heteroge-
neity of neoplasms was provided by Fidler and Kripke
in 1977 working with the murine B16 melanoma [71].
Using the modified fluctuation assay of Luria and Delbruck
[157], they showed that different tumor cell clones, each
derived from an individual cell isolated from the parent
tumor, varied dramatically in their ability to produce
pulmonary nodules after intravenous inoculation into
syngeneic recipient mice. Control subcloning proce-
dures demonstrated that the observed diversity was not
a consequence of the cloning procedure [71]. The find-
ing that preexisting tumor cell subpopulations prolifer-
ating in the same tumor exhibit heterogeneous metastatic
potential has since been confirmed in many laboratories
with a wide range of experimental animal tumors of dif-
ferent histories and histologic origins [reviewed in 58,
63, 64, 208, 209]. In addition, studies using young nude
mice as models for metastasis of human neoplasms have
shown that several human tumor lines and freshly iso-
lated tumors such as colon carcinoma, renal carcinoma,
and prostate cancer also contain subpopulations of cells
with widely differing metastatic properties [64].
We studied the biologic and metastatic heterogeneity
in a mouse melanoma induced in C3H mice by chronic
exposure to ultraviolet B radiation and painting with
Sun-Jin Kim et al.
croton oil [134, 135]. One mouse thus treated developed
a melanoma designated by Kripke as K-1735 [134]. The
original K-1735 melanoma was established in culture
and immediately cloned [69]. In an experiment similar
in design to the one described for the B16 melanoma,
the clones differed greatly from each other and from the
parent tumor in their ability to produce lung metastases.
In addition to differences in number of metastases, we
also found significant variability in the size and pigmen-
tation of the metastases. Metastases to the brain, heart,
liver and skin were found as well; those growing in the
brain were uniformly pigmented, whereas those grow-
ing in the lymph nodes, heart, liver, or skin generally
had no pigment.
To determine whether the absence of metastasis pro-
duction by some (but not all) clones of the K-1735 was
a consequence of their immunologic rejection by the
normal host [134–136], we examined their metastatic
behavior in young nude mice. In addition to the lack of
a functional T-cell system, unstressed 3-week-old nude
mice are also deficient in natural killer cell activity. In
such recipients, the immunologic barrier to metastatic
cells that also may be highly immunogenic is removed,
and they may thus successfully complete the process.
This was true for cells of two clones that did not pro-
duce metastases in normal syngeneic mice but produced
tumor foci in the young nude recipients. Most of the
nonmetastatic clones were nonmetastatic in both normal
syngeneic and in the nude recipients. Therefore, the
clones’ failure to metastasize in syngeneic mice proba-
bly was not caused by their immunologic rejection by
the host but by their inability to complete one or another
step in the complex metastatic process.
Enhanced Metastatic Potential
of Tumor Cells Harvested from
Metastases
Our studies and most data reported by others have led us
to conclude that metastasis is a highly selective process
regulated by a number of as yet imperfectly understood
mechanisms. This belief is contrary to the once widely
accepted notion that neoplastic dissemination is the ulti-
mate expression of cellular anarchy. In fact, suggesting
that cancer metastasis is a selective process is a more opti-
mistic view in terms of cancer therapy than one that
postulates that tumor dissemination is an entirely random
event. Belief that certain rules govern the spread of neo-
plastic disease implies that elucidation and understanding
of these rules will lead to better therapeutic interventions.
23
We addressed the question of whether the cells that
survive to form metastases possess a greater metastatic
capacity than most cells in the parent neoplasm [243].
Some support for this possibility comes from the initial
in vivo selection experiments of the highly metastatic
B16-F10 cell line derived from the parent B16 mela-
noma [66]. Comparable results have been obtained with
the K-1735 tumor. When cells derived from the parent
tumor were injected intramuscularly into the hind foot
pads of syngeneic mice, the resulting skin tumors
produced spontaneous pulmonary metastases. Four cell
lines were established from four individual lung nod-
ules harvested from four different mice. The finding that
all the lines derived from metastatic deposits produced
significantly more metastases than cells of the parent
line was good evidence for the hypothesis that metastasis
is a selective process, that is, cells populating metasta-
ses have an increased metastatic capacity [243].
Our studies demonstrate that the K-1735 melanoma is
heterogeneous and contains both nonmetastatic and met-
astatic cells. In contrast, individual metastases (sponta-
neous pulmonary metastases) are more uniform. This
suggests that metastatic foci could develop by a clonal
expansion of a few surviving metastatic cells. Moreover,
it explains the observations showing that tumor cells in
primary and metastatic lesions differ in their antigenic
properties, biochemical characteristics, and response to
cytotoxic drugs [reviewed in 61–63].
Clonal Origin of Cancer
Metastases
Multiple metastases proliferating in a host, even in the
same organ, often exhibit diverse biological characteris-
tics of, for example, hormone receptors, antigenicity or
immunogenicity, and response to various chemothera-
peutic agents. This diversity may result from the nature
of the pathogenesis of metastasis, the process of tumor
evolution and progression, or both.
Pathologists have long been aware that neoplasms
frequently exhibit different morphological appearances
in different areas. For this reason, the malignant or benign
nature of a tumor cannot be determined with confidence
unless multiple sections obtained from all parts of the
tumor are examined. The zonal differences in tumors
are not restricted to morphology alone but include bio-
logical characteristics such as growth rates, sensitivity
to cytotoxic drugs, antigenicity, and pigmentation [74].
Since primary tumors are not uniform, it is possible that
tumor cell aggregates entering the circulation from one
24
zone of the tumor may be different from those entering
from another zone. If an embolic aggregate originates
from a primary tumor’s homogeneous zone, regardless
of whether only one cell or several cells survived to pro-
liferate in distant organs, the resulting metastasis would
be like a primary tumor of unicellular origin. If a mixed
embolus derived from an area of zonal junctions enters
the circulation, the unicellular or multicellular origin
of the metastasis would depend on whether a single cell
or multiple cells survived to proliferate. To determine
whether individual metastases are clonal and whether
different metastases can be produced by different pro-
genitor cells, Talmadge et al. [244] performed a series
of experiments using the fact that x-irradiation of tumor
cells induces random chromosome breaks and rear-
rangements. Analyzing the karyotype composition of 21
individual melanoma lung metastases after cultivating
cells from individual lesions, this research group found
unique karyotypic patterns of abnormal, marker chromo-
somes in most of the lines established from metastases,
which suggested that each metastasis originated from a
single progenitor cell. Similar results have been obtained
in other rodent tumor systems. These studies revealed
that the majority of metastases are of clonal origin.
Moreover, variant clones with diverse phenotypes are
formed, rapidly resulting in the generation of significant
cellular diversity within individual metastases [244].
Cancer metastases of a clonal origin can be produced
by two different mechanisms, proliferation of a single
cell or of many cells. In the case of the second possibility,
the cell aggregate at the metastatic site must have a
homogeneous composition. To determine which of these
possibilities is responsible for the generation of clonal
K-1735 melanoma metastases, we injected C3H mice
intravenously with aggregates of K-1735 cells consisting
of two distinct subpopulations [75]. Cells of line K-1735-M2
are highly metastatic and exhibit a stable normal karyo-
type. Cells of the X-met-21 line are also highly meta-
static but exhibit a stable, submetacentric chromosomal
marker. After mixed aggregates (>20 cells) of these two
cell types were injected, individual lung metastases
were recovered and cultured, and each metastatic line
was subjected to chromosome analysis. We reasoned
that if an experimental metastasis originated from a
single proliferating cell, all tumor cells within the meta-
static focus should express either the K-1735-M2 or the
X-met-21 chromosomal profile. This indeed was the
case. Analysis of the distribution and fate of circulating
tumor emboli has demonstrated that multicellular aggre-
gates are more likely to give rise to a metastasis than a
single-tumor-cell embolus. This is probably so because
tumor cells not on the periphery of circulating emboli
The pathogenesis of cancer metastasis: relevance to therapy
can be protected from destruction in the circulation, and
a large aggregate of cells can more readily arrest in the
capillary bed of an organ. Since the aggregates we injected
were large, each containing more than 20 cells, the results
suggest that the melanoma lung metastases resulted from
the proliferation of a single viable cell within the embolus.
Thus, regardless of whether an embolus is initially homo-
geneous or heterogeneous, metastases can be unicellular
in origin.
Collectively, these observations indicate that different
metastases arise from different progenitor cells and
account for the well-documented differences in behavior
of different metastases. Among individual metastases of
proved clonal origins, however, heterogeneity can develop
rapidly to create significant intralesional heterogeneity.
Development of Biological
Diversity within and Among
Metastases
Clinical and histologic observations of neoplasms have
suggested that tumors undergo a series of changes during
the course of the disease. A tumor initially diagnosed as
benign, for example, can evolve over a period of many
months into a malignant tumor. This can best be demon-
strated in the case of human cutaneous melanoma where
the transformation of normal melanocytes and their
conversion into metastatic cells has been studied in detail
by Clark and coworkers [32, 33]. This progression is
gradual and consists of a series of discrete irreversible
steps [107]. To explain the process of tumor evolution
and progression as originally defined by Foulds in 1954
[81], Nowell [190] suggested that acquired genetic
variability within developing clones of tumors, coupled
with host selection pressures, can result in the emer-
gence of new tumor cell variants that exhibit increasing
growth autonomy or malignancy. Nowell’s hypothesis
[190] predicted that accelerating tumor progression
toward malignancy can be accompanied by increasing
genetic instability of the evolving cells. To test this
hypothesis, we have examined the metastatic stability
and rates of mutation of paired metastatic and nonmeta-
static cloned lines isolated from four different mouse
neoplasms. We found that highly metastatic cells were
phenotypically less stable than their nonmetastatic
counterparts. Moreover, in highly metastatic clones, the
rate of spontaneous mutation was found to be several-
fold higher than in low-metastatic clones. These results
are in accord with the hypothesis that tumor progres-
sion occurs as a result of acquired genetic alterations.
Sun-Jin Kim et al.
Similar data have been reported for other neoplasms
[reviewed in 62, 65]. Evidence that genetic mechanisms
can be responsible for tumor progression comes from
mutagenesis experiments using nitrosoguanidine.
The finding that metastatic cells exhibit higher muta-
tion rates than nonmetastatic cells [31], and that hetero-
geneity develops more rapidly in tumors containing few
subpopulations of cells [186, 208, 210] suggest that
accelerated tumor evolution and progression will result
in the rapid development of biologic diversity in metas-
tases, especially when such lesions are of clonal origin.
Intratumoral Heterogeneity for
Expression of Tyrosine Kinase
Growth Factor Receptors
It is well recognized that tumor progression, angiogene-
sis, and metastasis are regulated by the interaction of
tumor cells with the host organ microenvironment [70].
The expression of growth factors and their receptors
varies within different zones of any given organ and may
also differ among different cells within a tumor [62, 63].
Overexpression of transforming growth factor-alpha
(TGF-α), epidermal growth factor (EGF), and the EGF
receptor (EGFR) has been reported to be associated with
poor prognosis in different neoplasms [2, 111, 170, 253,
271]. Expression of vascular endothelial growth factor
(VEGF) is associated with increased vascular permeabil-
ity, cell proliferation, and survival of endothelial cells
[85, 86, 148, 272]. The expression of VEGF has also
been correlated with microvessel density of neoplasms
[52, 242] metastasis, and, hence, poor prognosis [53, 116,
140, 234, 262]. The expression of PDGF and its receptor
(PDGFR) by tumor cells, tumor-associated endothelial
cells [126, 251], and pericytes and myofibroblasts [127,
131, 151, 269, 270] is common to many neoplasms.
In clinical specimens of human colon carcinomas, while
PDGF is produced by tumor cells, the expression of
PDGFR is restricted to stromal cells, including tumor-
associated endothelial cells [131], i.e., tumor-associated
stromal cells generate a microenvironment favorable to
the survival and progressive growth of tumor cells [213].
Specifically, PDGFR signaling is related to recruitment
of pericytes and control of interstitial fluid pressure [14,
192, 202, 213], which are favorable to the survival and
progressive growth of neoplasms [155].
The expression of TGF-α, EGF, VEGF, and PDGF-β
and their respective receptors was examined in 12 human
colon cancer surgical specimens and in orthotopic tumors
in nude mice produced by two distinct human colon
25
carcinoma cell lines. Inter- and intratumoral heterogeneity
in expression of these growth factors and their receptors
was reported. Different proteins produced by tumor cells
can regulate the interaction between tumor cells and
the organ microenvironment [57]. Many protein tyrosine
kinase receptors on tumor cells and tumor-associated endo-
thelial cells can be induced or upregulated by ligands
produced by tumor cells via autocrine and paracrine path-
ways [55]. In many clinical trials, the presence of targets
in patients was not confirmed, and the response rate was
unpredictable [247]. For inhibition of phosphorylated
EGFR, the presence of mutated receptors was reported
to be a predictable variable [158], but significant thera-
peutic responses of cancer cells with wild-type receptors
has also been reported [23, 269, 270]. These confusing
criteria for selection of patients for treatment strongly
indicate the need for better methodologies.
The data demonstrating inter- and intratumoral het-
erogeneity for expression of EGFR, VEGFR2, and
PDGFR-β in different human colon cancer specimens
of different stages suggest that targeting a single tyrosine
kinase receptor is not likely to provide significant thera-
peutic results. In other words, targeted therapy is effec-
tive only against its target and eliminating tumor cells
that are dependent on one pathway, e.g., EGFR, is not
likely to prevent the proliferation of tumor cells that are
independent of this pathway. These results agree with a
recent report demonstrating biological heterogeneity of
tyrosine kinase receptors in other cancers [30, 173].
Indeed, simultaneous blocking of two protein tyrosine
kinase pathways produce more efficient therapeutic
effects in prostate [128] and pancreatic [269] carcinoma
than did blocking of a single such pathway. Therapeutic
efficacy was further increased when three protein
tyrosine kinase pathways were inhibited [270].
Cancers are biologically heterogeneous for multiple
properties, including antigenicity, sensitivity to chemo-
therapeutic agents, invasion, and metastasis [76, 77].
The progressive growth, metastasis, and survival of
tumor cells depend on their interaction with the organ
microenvironment. The expression of multiple tyrosine
kinase receptors by different tumor cells within a single
neoplasm indicates that targeting a single tyrosine
kinase may not produce eradication of the disease.
Zonal Heterogeneity for Gene
Expression
A better understanding of the cross-talk between cancer
cells and the organ microenvironment involving multiple
genes first requires the identification of gene expression
26
profiles within the tumor. Among current methods for
establishing these profiles, microarray analysis is the most
powerful, and several such studies have been under-
taken to predict disease outcome for individual patients
[9, 10, 29, 115, 207, 216, 258, 259] and to identify
cancer patients who should receive chemotherapy [17].
For example, pretherapeutic gene expression profiling
has been undertaken to identify breast cancer patients
who should receive specific chemotherapy [17], to
predict response of rectal cancer patients to preopera-
tive chemoradiotherapy [89], and to predict response of
breast cancer patients to docetaxel [28], and a specific
gene expression pattern was correlated with recurrence
in Dukes’ B colon carcinomas [265]. None of these
studies, however, accounted for the biologic heteroge-
neity of gene expression within a single tumor. Tumor
cells depend on multiple and redundant pathways for
growth, survival, and adaptation to the host microenvi-
ronment [70, 76, 77, 155]. In malignant neoplasms,
genetic instability of cancer cells frequently yields
extensive intratumoral heterogeneity in the pattern of
structural chromosome aberrations [90, 92, 93, 95].
Three dimensional tumor growth and uneven fractional
division of tumor cells within a single tumor mass can
give rise to biologically different central and peripheral
zones. The microenvironment of the central zone differs
significantly from that of the peripheral zone, and
zonal heterogeneity for several molecules in different
tumor systems has been reported [102, 105, 129, 130,
138, 139, 191, 225]. Among these, bFGF, matrix metal-
loproteinase-2 (MMP-2), and MMP-9 have been shown
to be highly expressed at the invasive edge of tumors
[105, 130, 131, 138, 139], whereas the cell-to-cell cohe-
sion molecule, E-cadherin, was downregulated at the
periphery of the tumors [105, 130, 131, 138, 139, 191].
In fact, the ratio of expression of MMP-2 and MMP-9 to
E-cadherin (MMP/E-cadherin ratio) at the periphery of
the tumors correlated with metastatic potential and recur-
rent disease [138, 139].
Affymetrix HG-U133-Plus 2.0 array and laser capture
microdissection techniques, demonstrated that different
zones of the same pancreatic tumor exhibit differential
expression of genes [179, 180]. The study of 1,222 genes
demonstrated statistically significant differences. Bioinfor-
matic functional analysis revealed that 346 upregulated
genes in the peripheral zone were related to cytoskeleton
organization and biogenesis, cell cycle, cell adhesion,
cell motility, DNA replication, localization, integrin-
mediated signaling pathway, development, morpho-
genesis, and IκB kinase/NF-κB cascade. In the central
zone, 876 upregulated genes were related to the regula-
tion of cell proliferation, transcription, transmembrane
The pathogenesis of cancer metastasis: relevance to therapy
receptor protein tyrosine kinase signaling pathways,
response to stress, small GTPase-mediated signal trans-
duction, hexose metabolism, cell death, response to
external stimulus, and carbohydrate metabolism. These
data clearly demonstrate zonal heterogeneity for gene
expression profiles within single tumors and suggest
that characterization of zonal gene expression profiles is
essential if microarray analyses of genetic profiles are to
produce reproducible data, predict disease prognosis,
and allow design of specific therapeutics [179, 180].
Tumor-Organ Interaction: The
“Seed and Soil” Hypothesis
Clinical observations of cancer patients and studies with
experimental rodent tumors have led cancer biologists
to conclude that the metastatic pattern of certain tumors
is organ-specific and independent of vascular anatomy,
rate of blood flow, and number of tumor cells delivered
to each organ [reviewed in 62, 65, 263, 264]. Indeed, the
distribution and fate of hematogenously disseminated,
radiolabeled melanoma cells in experimental animals
conclusively demonstrated that tumor cells can reach
the microvasculature of many organs, but growth in the
organ parenchyma occurs in only specific organs [60].
These findings, however, were not new.
More than 100 years earlier, Paget reached a similar
conclusion. In 1889, he asked, “What is it that decides
what organs shall suffer in a case of disseminated
cancer?” Paget’s study was motivated by the discrepancy
between considerations of blood flow and the frequency
of metastases in different organs. He examined the
autopsy records of 735 women who died of breast cancer
and many other patients with different neoplasms and
noticed the high frequency of breast cancer metastasis to
the ovaries and the variations in incidence of skeletal
metastases produced by different primary tumors. These
findings were not compatible with the view that meta-
static spread was due to “a matter of chance” or that tissues
“played a passive role” in the process. Paget [193] con-
cluded that metastasis occurred only when certain favored
tumor cells (the “seed”) had a special affinity for the growth
milieu provided by certain specific organs (the “soil”). The
formation of metastasis required the interaction of the right
cells with the compatible organ environment.
In 1928, Ewing [50] challenged Paget’s seed and soil
theory and hypothesized that metastatic dissemination
occurs purely by mechanical factors that are a result of
the anatomical structure of the vascular system. Both
of these explanations have been evoked separately or
Sun-Jin Kim et al.
together in order to explain the metastatic site preference
of certain types of neoplasms. In a review of clinical
studies on site preferences of metastases produced by
different human neoplasms, Sugarbaker [235] concluded
that common regional metastatic involvements could be
attributed to anatomical or mechanical considerations,
such as efferent venous circulation or lymphatic drain-
age to regional lymph nodes, but that metastasis in distant
organs from numerous types of cancers were indeed site-
specific.
Experimental data supporting Paget’s 1889 seed and
soil hypothesis were provided a century later by Hart and
Fidler [101], who studied the preferential growth of B16
melanoma metastases in specific organs. Following the
intravenous (i.v.) injection of B16 melanoma cells into
syngeneic C57BL/6 mice, tumor growths developed in
the natural lungs and in grafts of pulmonary or ovarian
tissue implanted either subcutaneously (s.c.) or intramus-
cularly (i.m.). In contrast, neoplastic lesions failed to
develop in control grafts of similarly implanted renal
tissue or at the site of surgical trauma. Parabiosis experi-
ments suggested that the growth of the B16 melanoma in
ectopic lung or ovarian tissue was due to the immediate
arrest of circulating neoplastic cells and not to shedding of
malignant cells from foci growing in the natural lungs.
Quantitative analysis of tumor cell arrest and distribution
using cells labeled with [125I]-5-iodo-2´-deoxyuridine
indicated that the growth of tumors in the implanted organs
was not due to an enhanced initial arrest of B16 cells. No
significant differences in immediate tumor cell arrest were
detected between implanted fragments of lungs (tumor-
positive) and kidney (tumor-negative) or between organ-
bearing and contralateral control limbs. These data
demonstrated that the outcome of metastasis is dependent
on both tumor cell properties and host factors and sup-
ported the seed and soil hypothesis as an explanation of
the nonrandom pattern of cancer metastasis.
The introduction of peritoneovenous shunts for palli-
ation of malignant ascites provided an opportunity to
study some of the factors affecting metastatic spread in
humans. Tarin et al. [245] described the outcome in
patients with malignant ascites draining into the venous
circulation, with the resulting entry of viable tumor cells
into the jugular veins. Good palliation with minimal
complications was reported for 29 patients with various
neoplasms. The autopsy findings in 15 patients substan-
tiated the clinical observations that the shunts did not
significantly increase the risk of metastasis. In fact,
despite continuous entry of millions of tumor cells into
the circulation, metastases in the lung (the first capillary
bed encountered) were rare. These results provide com-
pelling verification of the seed and soil hypothesis.
27
A clear demonstration of organ-site specific metastasis
comes from studies of experimental brain metastasis.
Two murine melanomas were injected into the carotid
artery to simulate the hematogenous spread of tumor
emboli to the brain. The K-1735 melanoma generated
lesions only in the brain parenchyma, whereas the B16
melanoma produced only meningeal growths [219].
Similarly, different human melanomas [220] injected
into the internal carotid artery of nude mice also pro-
duced unique patterns of brain metastasis. Distribution
analysis of radiolabeled melanoma cells injected into
the internal carotid artery ruled out the possibility that
the patterns of initial cell arrest in the microvasculature
of the brain predicted the eventual sites of growth.
Rather, the different sites of tumor growth in the brain
involved interactions between the metastatic cells and
brain endothelial cells, and the response of tumor cells
to local growth factors. In other words, site-specific
metastases were produced by tumor cells that are recep-
tive to their new environment.
Regulation of Tumor Cell Gene
Expression by the Organ
Microenvironment
Tumor cells with different metastatic capabilities have
been shown to differ in expression of proteins, such as
collagenases, E-cadherin, IL-8, bFGF, and many others
[65]. In most cases, however, these differential expres-
sions were most evident in tumor cells growing at
anatomically correct sites. Many biologic investigations
of solid tumor cells often use cell lines growing in vitro
as monolayer cultures. While easy to accomplish, mono-
layer cultures are not subjected to any cross talk, e.g.,
paracrine signaling pathways associated with growth
in vivo [37].
Clinical observations of cancer patients and studies in
rodent models of cancers have concluded that certain
tumors tend to metastasize to certain organs [264]. The
concept that metastasis results only when certain tumor
cells interact with a specific organ microenvironment
was originally proposed in Paget’s venerable “seed and
soil” hypothesis [193]. Indeed, spontaneous metastasis
is produced by tumors growing at orthotopic sites,
whereas the same tumor cells implanted into ectopic
sites fail to produce metastasis [124].
To determine the influence of the microenvironment
on changes in gene expression, microarray analyses
were done on three variant lines of a human pancreatic
cancer with different metastatic potentials [141, 179].
28
The variant lines were grown in tissue culture in the
subcutis (ectopic) or pancreas (orthotopic) of nude mice.
Compared with tissue culture, the number of genes
whose expression was affected by the microenviron-
ment was upregulated in tumors growing in the subcutis
and pancreas. In addition, highly metastatic pancreatic
carcinoma cells growing in the pancreas expressed
significantly higher levels of 226 genes than did the low
metastatic variant cells. Growth of the tumor cells in the
subcutis did not yield similar results, indicating that
the orthotopic microenvironment significantly influences
gene expression in pancreatic cancer cells [141, 179].
Regulation of the Invasive
Phenotype by the Microenvironment
The metastatic capacity of human colon cancer cells grow-
ing in orthotopic tissues of nude mice directly correlates
with the level of collagenase type IV activity [233].
Histological examination of the human colon carcinomas
growing in the subcutis, wall of the colon, or kidney of
nude mice revealed a thick pseudocapsule around the
subcutaneous but not cecal or kidney tumors [61, 70, 76].
These differences suggested that the organ environment
could influence the ability of metastatic cells to invade
host stroma. Significant differences were found in the
levels of secreted type IV collagenases between human
colon cancer cells growing subcutaneously or in the
cecum of nude mice. In the medium conditioned by
human colon cancer cells derived from subcutaneous
implants, we detected only a latent form of the 92-kDa
type IV collagenase. In contrast, both latent and active
forms of the 92-kDa type IV collagenase were found in
culture medium conditioned by tumor cells harvested
from cecal tumors. Moreover, cancer cells grown in the
cecum secreted more than twice as much enzymes as
the subcutaneous tumors [178].
The invasive ability of human colon cancer cells is
directly influenced by organ-specific fibroblasts. Primary
cultures of nude mouse fibroblasts from skin, lung, and
colon were established, and invasive and metastatic
human colon cancer cells were cultured alone or with
the fibroblasts. The cancer cells grew on monolayers of
all three fibroblast cultures but did not invade through
skin fibroblasts [51]. Cancer cells growing on plastic
and on colon or lung fibroblasts produced significant
levels of latent and active forms of type IV collagenase,
whereas colon cancer cells cocultivated with nude mouse
skin fibroblasts did not. One possible explanation is that
fibroblasts from the skin produce IFN-β, whereas those
The pathogenesis of cancer metastasis: relevance to therapy
from the kidney or colon do not. The incubation of
human colon cancer cells [51] and human renal cancer
cells [91] with IFN-β significantly reduced the expres-
sion and activity of collagenase type independently of
antiproliferative activity.
Regulation of Angiogenesis by
the Organ Microenvironment
The intensity of the angiogenic response varies consid-
erably among different types of tumors and within
different zones of a single tumor [48]. The rate of tumor
cell division is still several orders of magnitude greater
than the rate of neovascularization. As tumors expand,
their microenvironment is often hypoxic [260]. Hypoxia
is often associated with the activation of the transcrip-
tion factor hypoxia-inducible factor-1 alpha (HIF-1α) to
initiate the transcription of genes, e.g., VEGF/VPF [54].
VEGF/VPF increases the permeability of blood vessels
by stimulating the functional activity of vesicular-vacuolar
organelles, clusters of cytoplasmic vesicles and vacuoles
located in microvascular endothelial cells [45–47].
VEGF also induces migration, protease production, and
endothelial cell proliferation [54]. In addition, VEGF/
VPF regulates endothelial cell survival by activating the
phosphatidylinositol-3 kinase/Akt signal transduction
pathway and stimulating expression of the antiapoptotic
proteins Bcl-2 and A1 [110].
HIF-1α signaling also encodes the polypeptide chains
of PDGF [100]. The functional activity of PDGF is, to a
large extent, determined by the anatomical location of a
specific tumor. For example, in pancreatic tumors,
PDGF has been shown to stabilize developing vascular
networks by recruiting pericytes to support the imma-
ture blood vessel walls [13, 14]. In tumors of the central
nervous system, PDGF stimulates the release of VEGF
from the tumor-associated endothelium [96]. In contrast,
tumors in the skin rely on PDGF signaling to regulate
the level of interstitial fluid pressure in the tumor [199].
In prostate cancer bone metastasis (see next section),
PDGF functions as a survival factor for tumor endothe-
lial cells by activating the intracellular effectors MAPK
and Akt [142, 143].
The vascular endothelium is regarded as structurally
and functionally heterogeneous [88]. To examine this
diversity, we generated a broad panel of microvascular
endothelial cells from various organs of H-2Kb-tsA58
transgenic mice [142]. cDNA expression profiles generated
on the endothelial cells predicted significant organ-
specific differences in expression levels of tyrosine kinase
Sun-Jin Kim et al.
receptors, chemokine receptors, and proteins that regu-
late the efflux of toxic substrates; these were confirmed
at the protein level. Endothelial cells derived from the
mouse brain expressed measurable levels of PDGF-Rβ,
the chemokine receptor CXCR-2, and P-glycoprotein,
whereas endothelial cells from the pulmonary circula-
tion did not express detectable levels of these proteins.
The organ-derived endothelial cells also exhibited vast
differences in response to stimulation with endothelial
cell mitogens. Endothelial cells originating from the
brain and liver showed the greatest increase in cell divi-
sion in response to basic fibroblast growth factor, while
EGF was the most potent mitogen for endothelial cells
derived from the lung and uterus.
Influence of Lymphoid Cells
on Angiogenesis
The regulation of angiogenesis by T lymphocytes,
macrophages, and mast cells, is well established [55, 59,
68, 83, 160, 164, 169, 204, 223, 240]. For example,
invasive cutaneous melanoma is often associated with
a local inflammatory reaction involving T lympho-
cytes and macrophages, a condition often associated
with an increased risk of metastasis [19, 217]. The
relatively slow growth of tumors in aging mice has been
closely linked with decreased vascularization [133]
associated with a diminished immunological response
[38, 92]. The role of tumor vascularization and its effect
on tumor growth in immunosuppressed mice was inves-
tigated and it was concluded that the growth of the
immunogenic B16 melanoma was delayed in myelosup-
pressed mice as compared to control mice [98]. Similarly,
tumor growth in mice pretreated with doxorubicin
(DXR) and then injected with normal splenocytes 1 day
before tumor challenge was comparable to that in the
control mice, implicating myelosuppression as a cause of
retardation of tumor growth and vascularization. Similar
results were obtained with athymic nude mice [98].
Many other studies have recognized the importance of
macrophages in tumor angiogenesis [5, 204–206, 239].
Macrophages can produce more than 20 molecules that
influence endothelial cell proliferation, migration, and
differentiation [204, 205]. Macrophages may modify the
extracellular matrix, thereby modulating angiogenesis
either through direct production of extracellular matrix
components or proteases that alter the structure and
composition of the extracellular matrix [240]. Macro-
phages have also been shown to produce antiangiogenic
molecules, such as thrombospondin-1 [40, 152, 153, 205].
29
Macrophage-derived metalloelastase has been shown to
be responsible for the generation of angiostatin in Lewis
lung carcinoma [42], and the addition of plasminogen to
3LL Lewis lung carcinoma cells cultured in vitro did not
result in the generation of angiostatin. However, its addi-
tion to co-cultured macrophages and carcinoma cells did
[42], suggesting that elastase activity in macrophages
was significantly enhanced by the cytokine GM-CSF
which was secreted by the tumor cells [137], leading to
the generation of plasminogen.
Regulation of Response to
Chemotherapy by the Organ
Microenvironment
Clinical observations suggest that the organ environment
can influence the response of tumors to chemotherapy.
For example, in women with breast cancer, lymph node
and skin metastases are more sensitive to chemothera-
peutic agents than are metastases residing in either the
lung or bone [41]. Several intrinsic properties of tumor
cells can render them resistant to chemotherapeutic
drugs, including increased expression of the mdr genes,
leading to overproduction of the transmembrane trans-
port protein P-glycoprotein (P-gp) [18, 249]. Expression
of P-gp often parallels increases in dose or duration of
chemotherapy. Indeed, increased levels of P-gp can be
induced by selecting tumor cells for resistance to natural
product amphiphilic anticancer drugs. Nevertheless,
elevated expression of P-gp accompanied by develop-
ment of the multidrug resistance (MDR) phenotype has
also been found in many solid tumors of the colon,
kidney, and liver that had not been previously exposed
to chemotherapy [249].
One of the most striking examples of site-specific
variations in therapeutic response was observed following
implantation of colon carcinoma cells into different
anatomic locations of nude mice (using the highly meta-
static KM12L4 human colon carcinoma cell line) or
syngeneic BALB/c mice (using the CT-26 murine colon
carcinoma). Mice received injections of the KM12L4a
cells into either the subcutis (ectopic site), spleen (lead-
ing to experimental liver metastasis), or cecum (growth
at the orthotopic site). Tumor-bearing mice were given
doxorubicin and subsequently evaluated for response to
treatment. Up to 80% growth of subcutaneous tumors
was inhibited by two i.v. injections of doxorubicin as
compared to about 40% inhibition of the intracecal
tumors and less than 10% inhibition of lesions in the
liver [267]. Similar results were obtained with murine
30
colon cancer cells. Subcutaneous tumors were sensitive
to treatment with doxorubicin; lung metastases were
insensitive [42]. However, the tumor cells at both of
these sites were equally sensitive to 5-FU, a drug whose
activity is not influenced by expression of the MDR
phenotype. Northern blot analysis showed that the rela-
tive expression of the mdr1 and mdr3 genes was great-
est in the cecum, liver, and lungs of mice, and this
expression correlated with the relative resistance of
tumor cells. Indeed, this expression of mdr was transient
and the subsequent culture of cells from a liver metasta-
sis for 7–10 days resulted in a decrease of mdr expres-
sion to the level in tumor cells maintained in culture
[43]. These events resulted from organ-specific modula-
tion of tumor cell properties. These findings are not
restricted to experimental systems. In patients with
colon carcinoma, elevated P-gp expression is found on
the invasive edge of the primary tumor (growing in the
colon) and in metastases located in lymph node, lung,
and liver [129]. Whether this finding is due to selection
or adaptation is unclear.
Targeting the Vasculature
Because systemic antitumor therapy fails primarily because
of the genetic instability and biologic heterogeneity
of neoplasms, therapeutic agents that target a tumor’s
vasculature, a genetically stable and essential compo-
nent of tumors, have been explored as an alternative to
conventional therapy. The structure and architecture of
tumor vasculature can differ dramatically from those of
normal organs [48, 182, 189]. Modern techniques, such
as phage display targeting, have defined “vascular
addresses” that may be distinct for different organs and
for tumors in those organs [194]. In normal human
tissues, for example, endothelial cells are long lived
and recycle every 3–5 years, whereas tumor-associated
endothelial cells can recycle every 30–40 days. The rate
of endothelial cell turnover has been reported to be
<0.1% in normal human tissues, whereas in malignant
human tumors, it is 2–9.9% [48, 49].
Endothelial cells that line blood vessels in different
organs are also phenotypically distinct. The expression
of various cytokines and growth factors and their rele-
vant receptors on both tumor cells and organ-specific
endothelial cells differs in different organ microenvi-
ronments, and the interaction of these growth factor
ligands (i.e., EGF, TGF-α, VEGF, and PDGF) with their
relevant receptors is essential for endothelial cell
survival and growth [6, 7, 128, 142, 143]. For example,
tumor cells that produce growth factor ligands are likely
The pathogenesis of cancer metastasis: relevance to therapy
to up-regulate the growth factor receptors and the acti-
vation of these receptors on tumor cells (autocrine effect)
and on tumor-associated endothelial cells (paracrine
effect). These effects can lead to downstream signa-
ling that activate mitogen-activated protein kinases
and phosphatidylinositol-3 kinase pathways and lead to
downregulation of the apoptotic proteins caspases 3 and
8 [8, 142, 143, 229]. Activation of growth factor recep-
tors on tumor cells and on the tumor-associated endothe-
lial cells is likely to increase the expression of
antiapoptotic proteins, causing endothelial cell resis-
tance to chemotherapy, even though these cells divide
every 30–40 days. Inhibition of this growth factor recep-
tor activation by receptor antagonists in tumor-associ-
ated endothelial cells (and tumor cells) can lead to BAX
induction, activation of caspase-8, and down-regulation
of BCL-2 and NF-κB [1, 24], thereby increasing the
susceptibility of tumors to chemotherapy. If the tumor-
associated endothelial cells continue to cycle, the use of
receptor antagonists [6, 7, 125–128] combined with
anticycling drugs can cause the destruction of the vascu-
lature within neoplasms, leading to the apoptosis of
adjacent tumor cells [20, 21, 193, 232].
Targeting the Expression of
Platelet-Derived Growth Factor
Receptor by Reactive Stroma
Colon carcinoma cells produce various growth factors
and cytokines that contribute to progressive growth and
metastasis [117]. One example is the family of PDGFs,
members of a family of dimeric disulfide-bonded growth
factors exerting their biological effects through activa-
tion of two structurally related tyrosine kinase recep-
tors, the PDGF-α- and -β receptors [104]. PDGF consists
of dimeric forms, including PDGF-AA, PDGF-BB,
PDGF-AB, PDGF-CC, and PDGF-DD [15, 144, 149].
The α-receptor binds all possible forms of PDGF except
PDGF-DD, whereas the PDGF-β receptor preferentially
binds PDGF-BB. PDGF-Rβ is expressed on many tumor
types, and PDGF-BB is an important autocrine growth
factor for many cell types, including gliomas, sarcomas,
pancreatic carcinoma, and prostate cancer [103]. PDGF-R
signaling has also been reported to stimulate angiogenesis
[213], recruit pericytes [14, 192], and control the intersti-
tial fluid pressure in stroma, thereby affecting transvas-
cular transport of chemotherapeutic agents [200–203].
A recent study of human colon cancer clinical specimens
revealed that PDGF-Rβ and phosphorylated PDGF-Rβ are
not expressed on tumor cells but, rather, are predominantly
Sun-Jin Kim et al.
expressed in stromal cells and in pericytes surrounding
the tumor microvessels [131, 132, 238, 241].
Imatinib, a derivative of 2-phenylaminopyrimidine,
was originally developed as a competitor for an ATP-
binding site of the Abl protein tyrosine kinase [44];
however, it is also a potent tyrosine kinase inhibitor of
c-Kit and PDGF-R [22]. We have reported that imatinib
can slow both the progressive growth of human pancre-
atic carcinoma in nude mice [114, 270] and the growth
of experimental bone metastasis of human prostate
cancer [127, 251].
To examine the role of PDGFR on colon cancer stromal
cells, we examined the therapeutic effect of imatinib
administered as a single agent or in combination with the
chemotherapeutic irinotecan against human colon carci-
noma cells growing in orthotopic (cecum and liver) and
ectopic (subcutis) organs of nude mice. Blockade of
PDGF-Rβ signaling by oral administration of imatinib or
imatinib combined with irinotecan significantly inhibited
the growth of orthotopic tumors and the incidence of
lymph node metastasis in nude mice. Histopathological
analysis of human colon carcinoma growing in the cecum
and liver of nude mice treated with imatinib alone or
with imatinib combined with irinotecan demonstrated
decreased stromal reaction and inhibition of phosphory-
lated PDGF-Rβ in the tumor-associated stromal cells.
These effects were associated with the inhibition of tumor
cell proliferation, an increase of apoptosis in stromal
cells, and a decrease in the number of pericytes surrounding
the tumor-associated microvessels. In contrast, treatment
of mice with imatinib alone or imatinib combined with
irinotecan did not affect the growth of colon carcinoma
cells in the subcutaneous space, demonstrating once again
that the biology of tumors differs with the organ microen-
vironment [58, 76].
In general, tumor cells in a neoplasm are biologically
heterogeneous and their phenotype can be modified by
the organ microenvironment [63, 74]. Histologically,
human carcinoma tissues are composed of both paren-
chyma and stroma. Tumor stroma consists of fibroblasts,
smooth muscle cells, inflammatory cells, microvessels,
and abundant extracellular matrix (ECM) [99]. Tumor-
associated stroma at both primary sites and metastatic
sites generate a favorable microenvironment for the
survival of cancer cells [155]. Fibroblasts are activated
by various growth factors and cytokines that are released
by cancer cells, for example TGF-β, PDGF, and bFGF.
Activated fibroblasts express αSMA, leading to the term
‘myofibroblasts’ [45]. Stromal reaction (desmoplasia),
such as myodifferentiation of fibroblasts [109, 145, 215]
clearly alters the stromal phenotype. In the liver, hepatic
stellate cells are the only mesenchymal cells present in
31
the parenchyma. These cells are activated by various
stimuli from tumor cells and undergo transformation
into myofibroblasts, which are characterized by expres-
sion of αSMA [211]. In general, tumor-associated stroma
is an abundant source of tumor-promoting growth
factors and cytokines, and stromal cells activated by
cancer cells can in turn regulate the growth and progres-
sion of carcinoma cells [112, 165, 222, 250]. The pro-
gressive growth of colorectal tumors in experimental
animals has been correlated with proliferation of myofi-
broblasts, whereas regression of colon tumors has been
linked to a fibrous capsule, suggesting that the presence
of reactive myofibroblasts may inhibit growth of tumor
cells [150]. Wounding has been associated with tumor-
promoting effects [224] and, functionally, tumor-associ-
ated stromal cells are similar to stromal cells in healing
wounds insofar as expression of myodifferentiation
markers, production of ECM, expression of growth fac-
tors and cytokines, and neovascularization [121–123].
The combination of imatinib and irinotecan com-
pletely inhibited tumor cell growth at the abdominal
wound healing site induced at the time of tumor cell
injection into the cecum [131]. Imatinib may inhibit
stromal reaction at the orthotopic primary site and the
surgical wound. PDGF-R signaling pathway also plays
an important role in increasing tumor interstitial hyper-
tension, which may block the accumulation of antitu-
mor drugs [201–203].
The microvasculature in both tumor tissue and normal
colon mucosa consists of endothelial cells, pericytes (mural
cells or smooth muscle cells), and basement membranes.
All of these components are thought to be abnormal in
tumor vessels. Pericytes are key cells in vascular develop-
ment, stabilization, maturation, and remodeling [12, 87].
Functional-blocking antibodies that target PDGF-Rβ
block pericyte recruitment during vascular development
[252]. In our study, desmin-positive pericytes were found
on microvessels in both normal organs and colon cancers,
albeit with different morphological characteristics. Speci-
fically, pericytes in tumor-associated vessels, but not those
in vessels of normal colon mucosa, were enlarged and
overexpressed PDGF-Rβ and p-PDGF-Rβ [131]. In agree-
ment with earlier reports [14, 269, 270], we found that
treatment with imatinib decreased pericyte coverage on
tumor-associated endothelial cells. The inhibition of
PDGF-R signaling by a protein tyrosine kinase inhibitor
decreased pericyte recruitment and attachment to endothe-
lial cells and destabilized tumor vasculature by killing
pericytes. In the absence of decreased vessel density in
tumors treated with imatinib combined with irinotecan,
functional rather than quantitative changes of the tumor
vasculature by imatinib may diminish tumor growth [84].
32
Recently, targeting the VEGF pathway to inhibit tumor
angiogenesis is attracting attention as a novel cancer
therapy [113]; however, it has been suggested that VEGF-
targeting therapies are mostly active against immature
vessels [13], i.e., normalization of the tumor vasculature
[118]. Because imatinib inhibits pericyte coverage on
tumor vasculature, it will be very interesting to deter-
mine if the combined inhibition of PDGF-R and VEGF-R
signaling may produce synergistic antivascular effects
[45, 118, 200]. Multi-target tyrosine kinase inhibitors are
under current investigation in clinical trials. Sorafenib
and sunitinib target not only multiple VEGF-Rs but also
PDGF-Rβ, and phase II studies show promising activity
of these molecules in renal cell carcinoma [180], likely
due to inhibition of angiogenesis [266].
Anti-Vascular Therapy of Prostate
Cancer Bone Metastasis
In clinical specimens of human prostate cancer bone
metastasis and in experimental models of orthotopic
human prostate cancers in the long bones of nude mice,
the expression of PDGF and PDGF-R correlates with
the growth of metastatic tumor cells in the bone paren-
chyma. Specifically, prostate cancer cells growing adja-
cent to bone tissue express high levels of the PDGF
protein and the PDGF-R, which is phosphorylated, and
tumor-associated endothelial cells express high levels
of phosphorylated PDGF-R [127, 128]. Oral adminis-
tration of the PDGF-R tyrosine kinase inhibitor imatinib
(STI571) to nude mice blocks the PDGF signaling path-
way by inhibiting phosphorylation of its receptors [251].
Oral administration of STI571 or STI571 plus injectable
taxol to male nude mice reduced the incidence and size
of primary tumors and bone lesions and prevented bone
lysis as measured by digital radiography. Immuno-
histochemical analysis of control, untreated bone
lesions, demonstrated that human prostate cancer cells
growing adjacent to the bone expressed high levels of
PDGF and activated (phosphorylated) PDGF-R, whereas
tumor cells growing in the adjacent musculature follo-
wing lysis of the bone did not. Treatment with STI571
and more so with STI571 plus taxol significantly inhib-
ited phosphorylation of PDGF-R on tumor cells and
endothelial cells, decreased tumor cell proliferation,
and induced significant apoptosis in tumor cells and
tumor-associated endothelial cells. These data indicate
that targeting PDGF-R phosphorylation can produce
significant therapeutic effects against prostate cancer
bone metastasis [125–128].
The pathogenesis of cancer metastasis: relevance to therapy
To determine whether the PDGF-R expressed on
tumor-associated endothelial cells could be the primary
target of imatinib, we selected a multidrug-resistant
variant of the human PC-3MM2 prostate cancer. These
cells were implanted into the tibia of nude mice, and
when the lesions were growing progressively, the mice
were randomized to different treatment regimens. The
bone tumors were resistant to taxol, whereas treatment
with imatinib and taxol produced a significant decrease
in tumor incidence, bone lysis, and incidence of lymph
node metastasis. Initially, this treatment produced apop-
tosis of tumor-associated endothelial cells (but not
tumor cells), followed 1 week later by apoptosis of the
tumor cells. Collectively, these studies demonstrated
that the primary targets for imatinib treatment are the
tumor-associated endothelial cells, i.e., an anti-vascular
therapy [126].
Targeting Platelet-Derived
Growth Factor Receptor on
Endothelial Cells of Multidrug
Resistant Prostate Cancer
The major cause of death from prostate cancer is due to
metastases that are resistant to conventional therapies.
Genetic instability of tumor cells leading to biological
heterogeneity is largely responsible for the development
of hormone-refractory prostate cancer cells [10, 11, 56,
197, 198, 254–257], and selection pressures by chemo-
therapeutic agents results in the emergence of cells that
are resistant to chemotherapy [122, 147, 159, 198, 221,
231]. These resistant cells often express the MDR1 gene
and its product, P-glycoprotein (P-gp) [94, 248, 257].
Methods to overcome multidrug resistant activity by
blocking transmembrane drug efflux pump action [82,
166–168, 176], liposomal encapsulation of cytotoxic
drugs [18], immunotherapeutic monoclonal antibody
targeting P-glycoprotein [214] or protein toxins selec-
tively killing cells expressing P-glycoprotein on their
surfaces [168] are under clinical investigation.
Multidrug resistance-associated protein (MRP) was
found in multidrug resistant prostate cancer cell lines
that do not express P-glycoprotein [34]. MRP1 [237,
256] and MRP2 [255] have been reported to be expressed
in prostate cancer cell lines, but there are very few
compounds with proven MRP1-associated multidrug
resistant-modulating capacity at present. Glutathione and
glutathione-S-transferase (GST) detoxification systems
are another drug-resistance mechanism reported to
Sun-Jin Kim et al.
protect cancer cells against the lethal effects of chemo-
therapy [174]. GST-π inactivation is related to early steps
of prostate carcinogenesis [146, 237], whereas the hor-
mone-independent disseminated prostate cancers clearly
expressed GST-π [256]. However, reversal of multidrug
resistance in prostate cancer by challenging the glutathi-
one pathway has been observed in vitro [212]. Many
changes in the regulatory processes of apoptosis appar-
ently contributed to the malignant phenotype of prostate
cancer cells [35, 39, 188, 256], and methods to stimulate
apoptosis are currently under development. However,
despite progress in our understanding of the biology of
MDR, therapeutic approaches that directly target tumor
cells have not been successful.
To overcome multidrug resistance of prostate cancer,
one may wish to affect the host factors in the organ
microenvironment, such as the vasculature [26, 63, 72,
78–80, 122]. Because all cells in the body depend on an
adequate supply of oxygen and nutrients and the ability
to remove toxic molecules, therapy directed against
tumor-associated endothelial cells can destroy tumor
cells, regardless of their biological heterogeneity [77].
Recent reports have demonstrated statistically signifi-
cant therapeutic results in experimental human prostate
cancer bone metastasis in nude mice treated with a com-
bination of paclitaxel and various tyrosine kinase inhibi-
tors directed toward PDGFR or EGFR [125–128, 251].
These experimental data have been successfully translated
to clinical trials [45, 46]. Since both tumor cells and tumor-
associated endothelial cells can express these activated
growth factor receptors, it has not been clear whether
the combination treatment targeted the tumor cells or the
endothelial cells or both. We reported that multidrug resis-
tant PC-3MM2-MDR human prostate cancer cells growing
in the prostate of nude mice are resistant to systemic
administration of paclitaxel. Targeting the phosphorylated
PDGF-R on tumor-associated endothelial cells leads to
regression of the multidrug resistant prostate cancer and
inhibition of lymph node metastasis.
As stated previously, regardless of sensitivity or resis-
tance to hormones or chemotherapeutic drugs, all tumor
cells are dependent on a viable vasculature for growth
and survival [56, 62, 123]. Tumor cells interact with
host factors in the microenvironment to induce growth
and expansion of vasculature [70, 72, 121]. We have
reported [126–128] that PDGF produced by prostate
cancer cells growing adjacent to bone can increases and
activate the expression of PDGF-R on tumor-associated
endothelial cells by a paracrine mechanism. The systemic
administration of imatinib inhibited the phosphoryla-
tion of PDGF-R (but not EGF-R) [128], and the combi-
nation of imatinib and paclitaxel induced apoptosis of
33
tumor-associated endothelial cells as well as PDGF-R-
expressing tumor cells. The expression of phosphorylated
PDGF-R is known to activate anti-apoptotic pathways
involving Akt, PI3K, MAPK, and Bcl-2 [142, 143], and
treatment with imatinib can inhibit this effect. These data
suggest that a major target for imatinib and paclitaxel
therapy could well be the tumor-associated endothelial
cell. We tested this possibility by using MDR human
prostate cancer cells.
The acquisition of multidrug resistance by leukemia
cells [162], hepatocellular carcinoma [273], and human
lung carcinoma [163] has been shown to decrease
tumorigenicity and prolong tumor cell doubling time.
In agreement with these reports, the in vivo growth of the
human prostate cancer PC-3MM2 selected for multidrug
resistance (PC-3MM2-MDR) was slower than the paren-
tal tumor cells. The ‘seed and soil’ theory [63, 193] held
true regardless of the MDR phenotype. Similar to parental
cells, MDR tumor cells growing in the bone (but not in
the muscle) expressed IL-8, bFGF, EGF, EGF-R, PDGF,
and PDGF-R. Endothelial cells of tumor-associated vessels
in bone lesions also expressed PDGF-R on their surface,
and treatment with imatinib and paclitaxel inhibited the
phosphorylation of the PDGF-R on both tumor cells and
endothelial cells. The PC-3MM2-MDR bone lesions
responded to systemic administration of imatinib and
paclitaxel (but not to paclitaxel administered alone),
raising the possibility that imatinib could have sensi-
tized the tumor cells to paclitaxel. Immunohistochemical
analyses revealed that early apoptosis (after 2–3 weeks
of chemotherapy) was mostly limited to tumor-associ-
ated endothelial cells. The multidrug resistant tumor
cells were still treatment-resistant. In contrast, parental
cells underwent extensive apoptosis in mice treated with
imatinib and paclitaxel.
The PDGF-R has been shown to regulate the intersti-
tial hypertension and transcapillary transport of mole-
cules [108], and the administration of imatinib to mice
bearing tumors can increase the concentration of a
systemically administered chemotherapeutic drug within
a tumor by twofold to threefold [201–203]. Since the
PC-3MM2-MDR cells were >50-fold more resistant to
paclitaxel even in the presence of imatinib, the in vivo
therapeutic response of the PC-3MM2-MDR bone
tumors to imatinib and more so to imatinib plus pacli-
taxel could not have been due to changes in interstitial
fluid pressure. Endothelial cells in normal tissues rarely
divide, whereas 2–3% of endothelial cells in prostate
cancer divide daily [4, 48]. These dividing endothelial
cells should be sensitive to anticycling drugs such as
paclitaxel. As stated above, the first wave of apoptosis
in bone tumors from mice treated with imatinib and
34
paclitaxel for only 2 weeks occurred in tumor-associated
endothelial cells, followed by apoptosis of tumor cells
and ultimately necrosis. By the fourth week of treatment
with imatinib and paclitaxel or imatinib alone, concurrent
apoptosis of tumor cells and tumor-associated endo-
thelial cells was observed. Without paclitaxel, imatinib
may produce therapeutic effects by the blockade of
PDGF-R, which serves as a survival factor [142, 143].
Therefore, targeting the PDGF-R on tumor-associated
endothelial cells by imatinib and paclitaxel can produce
therapeutic results in multidrug resistant human prostate
cancer experimental bone metastasis.
Conclusions
A current definition of the seed and soil hypothesis
consists of three principles. First, neoplasms are biologi-
cally heterogeneous and contain subpopulations of cells
with different properties. Second, the process of metas-
tasis is selective for cells that must succeed in all the
steps of this complex process. Although some of the
steps in this process contain stochastic elements, as a
whole, metastasis favors the survival and growth of a
few subpopulations of cells that preexist within the
parent neoplasm. Thus, metastases can have a clonal
origin, and different metastases can originate from the
proliferation of different single cells. Third, the out-
come of metastasis depends on multiple interactions
(“cross-talk”) of metastatic cells with homeostatic
mechanisms, which the tumor cells can exploit. A con-
vincing example of this principle is the establishment of
tumor-associated vasculature.
Tumor growth and metastasis depend on an adequate
blood supply. The extent of angiogenesis within and
around tumors is regulated by the balance between
proangiogenic and antiangiogenic molecules. The cross-
talk between tumor cells, normal cells, leukocytes, and
stromal cells occurs through growth factors, growth
factor receptors, and cytokine signaling, all of which are
released and act on cells within the tumor microenviron-
ment. These interactions enhance the establishment of
angiogenesis that in turn regulates the proliferation and
survival of metastatic cells. Interruption of one or more
of these interactions can lead to the inhibition or regres-
sion of metastasis. Because the growth of primary
tumors is often controlled with surgery and/or irradia-
tion, antiangiogenic agents may be most beneficial to
prevent widespread and metastatic disease. To succeed,
several principles must be considered. First, it is essen-
tial to remember that all neoplasms are biologically
heterogeneous. One cannot treat a heterogeneous disease
The pathogenesis of cancer metastasis: relevance to therapy
using homogeneous therapy. In other words, it is unlikely
that therapy of cancer in general and metastasis in
particular can be accomplished using a single modality.
Second, cancer is a chronic disease. A chronic disease
must be treated chronically, i.e., by management. Third,
cancer is the disease of the “seed” and the “soil”. Since
the outcome of metastasis depends on multiple interac-
tions of metastatic cells with homeostatic mechanisms
in the organ microenvironment, therapy for metastasis
should be targeted not only against tumor cells, but also
against the homeostatic factors that helps metastatic
cells grow and survive.
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3 Developmental therapeutics
and the design of clinical trials
ROBERT K. OLDHAM
New techniques in biotechnology and the use of biolo-
gicals in cancer treatment have made it apparent that
developmental therapeutics for biotherapy are different
from standard drug development. Millions of chemicals
have been screened as anticancer agents, but less than 75
have reached the clinic as commercial pharmaceuticals.
Perhaps 20 of these drugs can be classed as moderately
effective; the rest are only marginally useful and all can
be highly toxic. With the discovery of monoclonal anti-
bodies and conjugates thereof, the exploitation of bioen-
gineering to produce purified, characterized lymphokines/
cytokines and other biologicals, and further information
on the mechanism of action of these natural molecules,
the rate of development for biological therapeutics has
risen dramatically. Because of their selectivity and with
the implicit biological diversity of cancer, new approaches
are needed to efficiently bring biotherapy to the clinic.
Previously, there have always been fewer promising agents
(drugs) to test than patients who needed new approaches.
In fact, the drug development paradigm has been a slow
and laborious, with each drug taking some 8–12 years to
gain commercial approval, at a cost of more than US$500
million per drug. Taxol, a drug approved for general use,
entered clinical trials in the late 1980s and only became
generally available in 1998; taking more than a decade to
pass through our current system of drug development.
On average, fewer than four new chemotherapy drugs
per year have been approved for general use by oncolo-
gists. This expensive and slow paradigm reflects both the
toxicity and marginal effectiveness of chemotherapeutic
drugs as well as a bureaucratic regulatory system more
fearful of criticism over toxicity than a willingness to pursue
opportunities for seriously ill patients [33, 70, 71, 83].
More recently, a large number of biological substances
and targeted therapies have been making their way to
the clinic, increasing the difficulty of decisions as to the
order and amount of preclinical and clinical testing.
Hundreds of biologic agents are in clinical testing with
more agents to test than there are easily available patients
for testing. The current system for drug development is
not sufficiently flexible, and needs major changes in
direction and technique to optimize clinical testing and
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
speed the translation of new approaches to patients [6, 8,
17, 21, 22, 24, 30, 32, 33, 41, 55, 57, 61, 76–82, 90, 91,
94, 98, 99, 103].
Although the concept of biotherapy is not new, the
use of recombinant genetics to produce highly purified
biologicals as medicinals dates from about 1980 [31].
A member of the alpha-interferon family was the first
biological produced by recombinant methods to be used
as an anticancer medicine in humans [103, 105]. In the
25 years since the first alpha-interferon molecule was
prepared by recombinant methods, a large number of
recombinant molecules (lymphokines, cytokines, mono-
clonal antibodies, growth and differentiation factors,
angiogenesis factors and cell receptors) have become
available or are being prepared for testing in the clinic
(see Chapter 8).
Historical aspects in the development of immunother-
apy have been reviewed [12, 75]. Before the 1980s, the
term immunotherapy was considered to be synonymous
with biological therapy by most investigators. However,
it is now clear that there are many biological approaches
that may affect cancer growth and metastases, yet are
not within the immune system. Thus, biotherapy now
refers to agents derived from biological sources and/or
the use of agents that affect biological responses. The term
“biologicals” describes agents extracted from or pro-
duced from biological materials. With biotechno-logy,
this involves the use of recombinant genetics to isolate
the gene, transfect it into an appropriate producer organ-
ism, and then the isolation and purification of the protein
product. Genomics and proteonomics are processes to
yield the “code” to eventually prepare biological therapy
by chemical synthesis. The types of materials that alter
biological responses for the benefit of the patient have
been called biological response modifiers (BRM). The
use of biologicals and BRM in the treatment of cancer
and other diseases is biotherapy (a term I initially used
in 1984 to describe this fourth modality of cancer treat-
ment) [84, 85]. As is always the case, nomenclature
can be confusing and terms may be variously defined
by different individuals. In the broadest sense, biothe-
rapy includes blood products, transplanted organs, bone
41
42
marrow/stem cell transplants, antibiotics (often derived
by extraction from biological organisms and later syn-
thesized), and a variety of other agents and approaches.
However, in this chapter, the focus will primarily be on
BRM, recombinant biologicals and gene engineered or
activated cells being developed as medicinals.
Immunotherapy has had a checkered past. Kari Cantell
said it well when describing some of the early interferon
research: “Much second class research was carried out
with third class preparations slightly contaminated
with interferon” [5]. In addition, there have been ques-
tionable approaches (“alternative medicine”) used by
certain practitioners, and even frank quackery by others
who purported to deliver “immunotherapy” for cancer
patients. Even for the very dedicated scientists who have
explored immunotherapy over the past several decades,
there have been many pitfalls relating to the purity
of their preparations, the source of the materials, and
the assays and techniques by which their measurements
were made.
Huge expenditures by the National Institute of Health
(NIH) in the area of molecular, biological and viral onco-
logy over the last 40 years led, in large part, to the current
technology of “genetic engineering.” In contrast to previous
attempts to develop biological therapy, we now have
techniques available that can isolate a single gene and use
it in a bio-manufacturing process to produce absolutely
pure proteins identical to those found in the body. It is
with these techniques that thousands of biological com-
pounds and their synthetic analogs and components are
being developed. Many of these biologicals will be
candidates for use as medicinals. A major value of the
research done thus far with interferon is that it may be
viewed as a model for the development of other biologi-
cal substances as medicinals [61, 76]. Interferon research
in particular and biotherapy in general must be seen as a
new challenge in developmental therapeutics, with agents
to be tested appearing at a still accelerating rate. No
longer can the historical drug development paradigm be
used [55]. The methods used by the Food and Drug
Administration [11], National Institutes of Health, and
the pharmaceutical industry in the development of drugs
must be drastically changed to a new paradigm that will
accommodate the realities relevant to biologicals and
their use in medicine [33, 55, 71, 82, 86, 87, 90].
Drug Development
The current process of drug development involves a
very long and costly set of procedures [33, 55, 80]. This
includes the initial concept, extraction or synthesis,
Development therapeutics and the design of clinical trials
formulation, documentation of biological activity and
purity, early studies in the laboratory and in experimental
animals to determine the mechanism of action and
toxicity, and compilation of all the preclinical data into an
investigational new drug application (INDA). It costs mil-
lions of dollars for a pharmaceutical company to file a
single INDA. During preclinical development, companies
make projections about the potential market size and
profitability as justification for the investment. These
projections are difficult, and are most accurate when
related to an existing drug in a known market. It follows
that predicting the market size for a totally new agent or
new approach is much more difficult.
Subsequent to the INDA, further preclinical work on
the mechanism of action and preclinical toxicology of a
new drug is done. In addition, early-phase clinical studies
are begun to determine biological activity in humans.
Although the process is not uniform for all classes of
pharmaceuticals, studies are termed “phase I” when the
dose of the drug is escalated to determine its biological
activity and its toxicity. Based on the preclinical toxicology
information in small animals and sometimes in primates,
projections are made for the starting dose in humans. A low
starting dose is selected to avoid severe toxicity in the
initial patients in a phase I clinical trial. The rate of dose
escalation in these trials and the acceptability of toxic
side effects vary with the population at risk and are
related to the seriousness of the disorder and the treat-
ment alternatives for the patient. Because the starting
doses are very low and because of phase I trial design,
using different patients at each dose level (to avoid
cumulative toxicity), the chance of a therapeutic effect
for the initial patients receiving a new agent is nearly
zero. It is axiomatic that the phase I trials are designed
to accumulate the maximum amount of clinical infor-
mation with the least toxicity. Generally, the end point
for phase I trials is the achievement of a maximum toler-
ated dose (MTD), the dose at which side effects are
unacceptable to the physician and the patient within the
design of that clinical trial. These trials may require
100–500 patients to reach agreement on the MTD in
two to four schedules and routes of administration. In
addition, pharmacokinetic and metabolism studies are
done as part of Phase I. Once the MTD is established,
subsequent investigators can be reasonably assured that
the upper limit for the dose to be administered is well
defined and that therapeutic activity will not be missed
in later phase trials because of sub-therapeutic doses.
Phase II studies are then conducted to determine the
therapeutic activity of the new drug in various types of
cancer. Patients with specific cancers are selected in
order that these trials can be conducted in reasonably
Robert K. Oldham
uniform patient groups. Based on the preclinical infor-
mation and the clinical toxicology studies, as well as
pharmacokinetic and metabolic considerations, thera-
peutic doses are selected that represent the investigator’s
“best guess” as to the therapeutically active dose range.
In addition, schedule and route of administration must be
considered. In phase II trials, it is often necessary to
administer an agent at different doses by different routes
(e.g., intramuscular, intravenous, subcutaneous, oral)
and on different schedules (e.g., once a day, three times a
day, 24-h infusion, etc.). Phase II trials are considered
complete when a substantial body of data exists concer-
ning the therapeutic activity of a new drug with refer-
ence to best dose, route of administration, and schedule
for therapeutic efficacy. If one accepts classical criteria
for cancer subtypes, over 100 histologic types of cancer
exist. If one assumes a standard statistical criterion of at
least 14 patients treated to look for one response so as
not to miss a 20% response rate, then a new drug would
need to be tested in a minimum of 1,400 patients by each
schedule and route to assure clinicians of its inactivity in
each tumor type; and if one assumes three routes and five
schedules must be tested, over 21,000 patients would
need to participate just to prove a specific drug ineffective.
Such studies demonstrating inactivity sacrifice patients
“for the good of the system.” Such FDA-mandated test-
ing stretches ethical standards to the limit.
Following the completion of adequate phase I and
phase II trials, phase III trials to compare a new agent
with standard treatment are conducted. The extent of
phase III trials depends on the treatment alternatives
available. In the case of diseases for which other thera-
pies are effective, such trials may need to be extensive,
controlled, and employ random designs, sometimes with
blinding of the study both to the physician and to the
patient. Such phase III trials have produced an enormous
literature on the subject of ethics, trial design, end points,
and the assessment of efficacy [9, 23, 28, 38, 71, 82, 91,
92, 95, 106, 107, 110]. If, in such phase III trials, a new
agent proves therapeutically superior without unaccept-
able side effects, in comparison with or in addition to
“standard” therapy, the new agent is very likely to receive
FDA approval. Since “standard therapy” is often only
marginally effective, large randomized trials proving a
new treatment is significantly (usually marginally) better
means most patients derive little or no real therapeutic
benefit in the trials. Where efficacious therapy with an
approved drug is not available, fewer phase III data may
be needed to gain approval, and patients with advanced
cancer are often randomized against best supportive care
or a placebo. This latter practice also stretches ethical
practice to the limit [47, 71, 95].
43
Most drugs fail at the phase I/II level, being either too
toxic or inactive. Drugs that complete phase II trials
with acceptable toxicity and activity in at least one
cancer have a greater than 50% chance of successfully
passing phase III with eventual approval by the FDA.
For this reason, it has been suggested that drugs should
be approved for general use at the end of phase II to
speed up the process of bringing new drugs to the clinic
[47, 83].
The final step in the commercial development of a
new drug requires the filing of a new drug application
(NDA). All the information available under the INDA
and all of the data from the phase I, II, and III testing are
made available to the FDA for review and consideration.
Only the FDA is authorized to approve a new drug for
sale in the United States; this usually involves defining
the drug dose, route of administration, schedule, toxic-
ity, and therapeutic activity in specific diseases (indica-
tions). After FDA approval, the company may begin
to advertise the agent for the approved use only. Uses
outside the specific FDA approval indication(s) are termed
“off label” uses. Perhaps 50% of all cancer drug therapy
is “off label” use by physicians but the pharmaceutical
industry is specifically prohibited by the FDA from
providing clinical trial information to clinicians on “off
label” drug activity.
The long time and enormous expenditure in the
current drug development system have been justified as
being in the public interest inasmuch as the FDA requires
extensive testing so active agents with reasonable toxi-
cities (safe drugs) can be made available as medicinals.
The definitions of reasonable toxicity and of therapeutic
efficacy have been the subject of considerable debate,
both in the general sense and relative to specific drugs.
Often, the long-term effects and/or toxicities of approved
drugs have prompted secondary revisions in the regula-
tory process, further lengthening and expanding the steps
necessary for new drugs to be approved. This process
would seem to be totally in the public interest. However,
when viewed in another context, the process clearly has
some exclusionary aspects [47, 66, 71, 83]. The FDA
defines a single standard for drug development in the
United States. Marked differences exist between coun-
tries as to a regulatory body’s role in drug development,
and there are marked differences in the number of drugs
available to patients in different countries. The regula-
tory agency of each country has its own view of what is
ethical and in the public interest (different standards,
different ethics). Thus, there can be reasonable debate
on which rules are the best for the development of new
drugs. Finally, it is obvious that the long development
times and the huge expenditures required effectively
44
restrict competition in the area of drug development.
Such restriction of competition gives major pharmaceu-
tical firms a virtual monopoly on drug development.
Although most pharmaceutical firms would undoubtedly
say that they would prefer a lower cost for drug deve-
lopment, one may view the process as being in their best
interests (barrier to entry), since it restricts competition
from smaller firms that cannot marshal the resources to
carry out these extensive and expensive studies [47, 55,
71, 79, 80, 82, 83].
Thus, our drug development paradigm is highly restric-
tive. It has worked reasonably well over the past 50 years
only because of public acceptance of a conservative
regulatory structure and because of the small number of
relatively toxic drugs that actually were available for
clinical testing. The advent of biologicals has placed great
pressure on this paradigm. Indeed, the effective develop-
ment of biologicals will require changes in the regulations
and policies for the development of new pharmaceuticals
[47, 55, 71, 78, 82, 90, 91].
Biologicals and BRM
Development
A process of developmental therapeutics similar to that
described for chemicals (drugs) is now being applied to
biopharmaceuticals. Historically, for new biologicals,
the information on composition, formulation, and purity
was often imprecise. In contrast to drugs, which are
generally small, synthesized molecules with a chemical
definition that is quite straightforward, biologicals have
had a more variable developmental process. Preparing a
biological for testing has involved extraction from a
microorganism, from a fraction of a cell culture, or from
a tissue (complex chemical mixture). Such extractions
yielded complex mixtures with defined biological activity,
where the precise chemical composition was incomplete
or unknown [75]. Biotechnology is now making avail-
able a range of biologicals (lymphokines/cytokines,
monoclonal antibodies, antigens, growth and maturation
factors) that are pure and as well defined as small mole-
cule drugs [55, 57].
Market size, the potential for profit, the long develop-
ment period of preclinical and clinical testing, and the
large investment necessary to develop new therapeutic
agents have defined the scope of drug development. The
development and testing of new biologicals will require
extensive procedural and regulatory changes if we are to
be successful in bringing these agents to the clinic
quickly and efficiently [47, 55, 83, 92].
Development therapeutics and the design of clinical trials
Perhaps the most cogent example relates to the devel-
opment of monoclonal antibodies. For years there have
been sufficient data to indicate that monoclonal antibodies
are eventually going to be very useful both diagnosti-
cally and therapeutically [10, 26, 36, 40, 51, 52, 54, 62,
73, 94, 102, 103]. Now there are therapeutic antibodies
approved for colon and breast cancer, lymphoma and
leukemia with hundreds more in testing [8, 19, 21, 41,
94, 103]. The problems of developing new monoclonal
antibodies for therapy is quite different from those
previously encountered with drugs. The most striking of
these problems relate to market size. Generally speaking,
one looks at the population afflicted with a particular
disease and makes the presumption that a new drug will
be active in a certain percentage of patients with that
disease. The percentage is often reasonably high and
allows the market forecast to be applicable to a substan-
tial number of patients. For example, the number of
patients at risk per year with lung cancer is reasonably
well defined, and once some evidence of clinical activity
of a new drug in lung cancer is available, the market size
for that drug can easily be calculated. For monoclonal
antibodies, the situation is very different. At the extreme
is anti-idiotypic monoclonal antibody therapy. It has
been demonstrated in preclinical models and, to a more
limited extent, in humans that anti-idiotypic antibody
can be made to the specific tumor antigen (idiotype)
of the neoplastic cell [11, 29, 39, 51, 65]. Such anti-
idiotypic antibodies or idiotype vaccines derived therefrom
have proven useful in controlling the growth of the B-cell
lymphomas. However, a critical problem for the devel-
opment of these reagents is market size; here, it is the
individual patient with that particular neoplasm. Thus,
the market size might be as small as one. Even with some
cross-reactivity, many of these antibodies or vaccines
are expected to apply only to very small populations,
much smaller than even the “orphan drugs” envisioned
by FDA policy. Obviously, most pharmaceutical com-
panies will never develop these kinds of “individualized
or personalized medicines” under existing FDA guide-
lines [37, 71, 93, 94].
A less extreme example relates to the development of
monoclonal antibodies for tumor-associated antigens
that may be restricted to subpopulations of cancer cells
[19, 20]. Considerable heterogeneity exists within any
one histologic type of cancer (between patients) and
even within a single patient’s cancer [54, 64]. It may be
that for any one cancer only a small portion of the patient
population will have a particular antigen or array of
antigens on the cancer cell surface. Therefore, a mono-
clonal antibody might be applicable only to 1%, 5%, or
10% of the patients with a particular type of neoplasm.
Robert K. Oldham
Because of clonal heterogeneity, both between patients
and in different clones within individual patients, there
may be the need to use multiple antibodies (“cocktails”)
in treatment [1, 46, 69, 90]. Given the over 100 histologic
types of cancer and the heterogeneity within each cancer
type, market calculations may define very narrow appli-
cations [55, 63]. These considerations restrict market size
to a level unapproachable given drug development costs
under the current drug development paradigm.
Perhaps less obvious, but equally problematic, will be
the development of other biologicals for treatment. For
lymphokines and cytokines, there is already evidence of
both antigen-specific and non-antigen-specific signals
that may have growth regulatory effects (See Chapter 8).
One can visualize, with an antigen-specific lymphokine,
how the signal may relate to the specific antigen and be
applicable in a single patient, or a very few patients (e.g.,
transfer factor, IgE suppressor factors). Less restricted,
but still highly restricted compared to standard drug
development, is the development of non-antigen-specific
lymphokines (e.g., interferons, lymphotoxins, interleu-
kins), which are active against certain classes of cells,
thus making them clinically applicable only in selected
populations [55, 57].
Interferons: the Early Model
Interferons represented early models for new biological
approaches in cancer treatment [57, 61, 76]. “Natural”
extracted interferons from stimulated white blood cells
were used in initial clinical trials. The low purity, lot-
to-lot variation, and expense of stimulating leukocytes
to produce interferon, along with the difficulties in the
extraction and purification methods, limited the clinical
use of these materials. These preparations were typical of
early forms of nonspecific immunotherapy [5, 75]. With
the advent of increased interferon availability through
recombinant genetics, and with the very high purity
(greater than 99%) of these preparations, extensive trials
were completed for alpha-interferon preparations which
led to the approval of alpha-interferon as the first geneti-
cally engineered anticancer biopharmaceutical [42, 76].
The design of phase I biotherapy trials should differ
markedly from those for drugs. The dose-response curve
for these agents may be very broad (and sometimes mul-
tiphasic), with peak effects at different doses for each
system responding to the biopharmaceutical. For exam-
ple, the immunomodulatory activity of alpha-interferon
can be seen at very low doses, whereas the antiprolifera-
tive activity appears to be more reproducible at higher
doses. In biotherapy, there is a need to measure biological
45
responses in the context of the clinical trials [37, 48, 50,
62, 105]. Since one may be administering the biological
to stimulate a particular biological response, for which
the dose-response curve may not be known a priori, one
must perform studies with pharmacokinetics to assay
serum availability, and also measure the desired biologi-
cal effects to determine the optimal dose at which it
might alter a particular biological response (optimal bio-
logical response modification, OBRM). Finally, sched-
ule and route of administration have already proven
important [42, 76]. The pharmacokinetics after intrave-
nous and intramuscular administration differ for the
alpha interferons [43], and there has been a variable lack
of absorption of intramuscularly administered beta- and
gamma-interferons [34, 74]. Thus, the proper design of
phase I biotherapy trials must take into account appro-
priate measurements of bioavailability, pharmacokinet-
ics, biological response modification, and toxicity, all in
the context of escalating doses, to determine the dose–
response curves for each of these properties. Responses
to biologicals vary substantially between patients, and
escalating doses in individual patients can yield valu-
able data without subjecting patients to “tests” with no
therapeutic opportunity.
Like most biologicals, the interferons may act through
a diverse set of biological mechanisms. Cell surface recep-
tors for their activity exist, and responses to the adminis-
tration of biologicals are somewhat predetermined by the
condition and biological receptor repertoire of the patient.
This situation is in direct contradistinction to that of drugs,
in which a totally new chemical is often administered to a
patient in whom no standard biological response mecha-
nism existed a priori. Thus, biologicals may be viewed
in their physiologic role of correcting immunodeficiency
states, as well as in the pharmacologic role of augment-
ing host responses and perhaps having direct antitumor
effects.
Clearly, it was rational to test chemical drugs to MTD
and to treat just below this dose in a “kill or cure” approach
to cancer therapy. Current evidence suggests the OBRM
dose and the MTD dose should be determined in cancer
biotherapy to properly design effective therapeutic trials.
Once determined, the design of therapeutic trials for bio-
therapy may differ greatly from classical chemotherapy
studies.
Biotherapy Trial Strategies
Many strategies exist or can be envisioned for conduc-
ting clinical trials with new biologicals. Some of these
strategies have already been utilized in the early-phase
46
testing of the interferons [4, 27, 59, 60, 103]. Two under-
lying principles are apparent wherein biologicals and
drugs differ: when biologicals are administered, patients
already have physiologic mechanisms and receptors
that respond to them; and biologicals are derivatives of
natural products of the mammalian or human genome,
and may be expected to have less acute and chronic toxi-
city than drugs at similar biologically effective doses.
However, when high doses are used to exploit a certain
action of a biological, acute and chronic toxicities may
appear. These two considerations will not necessarily
apply to those BRM that are well-defined chemical enti-
ties and behave more in the manner of drugs with regard
to toxicity.
Empirical Clinical Testing
In many early interferon trials a set dose of a “natural”
interferon was given to a variety of patients with neo-
plastic or viral disorders [57, 62, 76]. The chosen dose
was the one expected, based on preclinical information
or other clinical data, to be relatively nontoxic and yet to
have sufficient biological effects to be therapeutically
active. In this context, most early clinical trials with
leukocyte interferon preparations were conducted at doses
under five million units per day, although it became
apparent later that doses up to 60 million units per day
could be tolerated by the patients for a certain number of
days. These trials were conducted as preliminary feasibi-
lity trials or pilot phase II trials to gain some information
on the biological effects of the interferon preparations.
Much of the information derived from them was anec-
dotal, but they yielded preliminary information about
the biological effects and toxicities of alpha-interferons.
Now that the development of biotherapy is proceeding
more rapidly, other strategies are to be preferred.
Escalating dose Trials Using Different
Groups of Patients
Phase I trials for biologicals have been modeled after
the standard phase I trial design used for drugs. Groups
of patients (usually three to five) are treated with a par-
ticular dose of a new biological with a single route of
administration [103]. These trials are begun with very
low (sub-therapeutic) doses based on preclinical infor-
mation, and the schedule is designed to increase the
dose gradually to levels where toxicity occurs [55, 74].
Generally, new patients are entered at each higher dose
level after all the patients have been entered on the
previous dose and have received at least several treat-
ments. In this way, each patient group provides toxicity
Development therapeutics and the design of clinical trials
information before the higher dose is initiated in new
patients. By utilizing different groups of patients at each
dose level, cumulative toxicity for any one patient is
avoided. Patients receive a predetermined number of
doses at each dose level and then are followed. During this
type of study, biological response modification and clini-
cal toxicity are assessed [4, 48–50, 53]. Pharmacokinetics
are also done in selected patients, so that the bioavailability
can be determined [103].
The dose-escalation scheme can be a modified
Fibonacci series, or some variation of this classical dose-
escalation method. The considerations involved in dose-
escalation methods dictated by drug toxicities do not
necessarily apply to biologicals. Thus, some inves-tigators
have used much faster dose escalations for biologicals.
Often, the dose escalation schedule is rather empirical,
the one selected being based on the best guess of the
investigators involved. This phase I drug development
strategy is based on historical data using new drugs and
has several potential disadvantages. Since each patient
receives only a particular dose for a defined period of
time, it is highly likely that a large percentage of the
patients will receive sub-therapeutic doses if antitumor
effects are observed only at higher dose levels. Early
clinical trials with biologicals have not produced severe
cumulative toxicity. This clinical trial strategy, which
was designed to avoid the cumulative toxic effects seen
with drugs, is inappropriate for biotherapy. Since toler-
ance (tachyphylaxis) may develop for some effects
(fever), entering different patients on progressively
higher doses may expose patients to avoidable acute
toxicities. Finally, the patients who are exposed to only
one dose level are not individually assessed for biologi-
cal response or for antitumor response over a wide
dosage range. This could give them a greater opportunity
for optimization of biological and therapeutic response
[59]. This may be particularly true in the very-early-phase
trials in which biological effects and antitumor effects
are totally unknown.
Escalating-dose Trials within Individual
Patients
An alternative clinical trial strategy is the use of an esca-
lating-dose trial within individual patients [58, 59, 74, 94].
There are several variations of this theme, but each
involves starting patients at a low dose and escalating
the dose in each patient to determine the biological
response modifying effects and toxicities over a broad
dose range. This clinical trial strategy is very conserva-
tive of patients, in that studies in small numbers of
patients can give a large amount of information over a
Robert K. Oldham
broad dose range in each biotherapy trial. In the context
of this dose escalation, pharmacokinetics and biological
response modification can be measured in each patient.
It is important in such a clinical trial strategy that
bioavailability studies be done and the information be
available concomitantly with the study. Appropriate
“wash-out” periods between doses can be utilized so
that the administered biological does not circulate in
increasing quantities as the trial continues. For example,
with the interferon trials, one can determine the serum
level after each dose and administer the next dose when
interferon is no longer detectable. This allows the patient
to avoid the possibility of severe acute toxic effects based
on cumulative serum levels. Obviously, biological and
therapeutic effects can still be cumulative and must be
monitored by appropriate clinical and biological mea-
surements throughout this type of trial.
This strategy offers the possibility of low-, medium-,
and high-dose therapy for the individual patient in the
context of a single clinical trial. It maximizes the oppor-
tunity for the investigator to learn the optimal biological
response modifying dose and the toxic dose, and per-
haps to gain therapeutic information, all in the context of
a single trial. Theoretically, rational maintenance regi-
mens could be designed based on the observations made
during the escalating dose trial for each patient.
For monoclonal antibody, this strategy may be particu-
larly important, in that the delivery of the monoclonal anti-
body to the tumor site may be the most important
consideration in developmental therapeutics [25, 36, 54,
63, 94]. Giving a low dose and then progressively higher
doses to the same patient, with subsequent determination
of antibody localization, is a useful way of determining
the correct dose for delivery of the antibody and/or its con-
jugates to the appropriate target organ in the context of a
toxicity study. This trial design rationally ties targe-ting
(delivery), toxicity, and therapeutic effects together in
combined phase I/II studies in a manner perfectly appro-
priate for antibody and conjugates, in direct contradistinc-
tion to drug development [2, 3, 17, 18, 59, 96, 98].
In studies that employ escalating doses, a useful vari-
ation is to enter individual patients for a limited number
of doses. With this trial design, the initial three or four
patients may enter at the lowest dose level and progress
through dose level 4 or 5, at which time the second
group of patients may be entered at dose 3 and escalated
upward in a manner that allows the second group to fol-
low the first group in dosage escalation toward the
MTD. A third group may be entered at dose level 6 and
so on. This strategy allows one to avoid administering
the full dose range to all patients, avoids most cumu-
lative toxicity, and helps avoid sub-therapeutic doses.
47
It increases the patient’s therapeutic opportunity without
undue risk of toxicity. This strategy is ethically prefe-
rable, most acceptable to patients and quite easy to
describe in the informed consent [71].
Schedule
In the initial clinical trials, the schedule of administration
is generally empirical or is based on preclinical observa-
tions. Once there is information on bioavailability, diffe-
rent schedules of administration can be designed rationally.
Both the bioavailability of the molecule and its biological
effects are relevant. There are some biologicals that have
a very short serum half-life (measured in a few seconds to
minutes) but may produce much longer lasting biological
effects. It may be useful to compare the biological effects
and toxicities of biologicals when administered under
conditions of rather constant exposure as against inter-
mittent exposure in order to gain a preliminary sense of
which schedule is most relevant [35, 74, 108]. Once these
data are available, schedules of administration for phase
II studies can be more rationally designed.
Route
The route of administration and delivery of biotherapy
to selected target organs is critical. There are data indi-
cating that certain biologicals are inactivated, or poorly
absorbed, when given intramuscularly or subcutane-
ously. For such biologicals intravenous administration
is quite important. When data on bioavailability are not
known from preclinical models, it is probably important
to conduct early clinical trials with the intravenous
route, since such trials can provide early data on serum
pharmacokinetics and provide the best opportunity for
broad biodistribution of the administered biological. It is
important in phase I biotherapy trials to determine if
serum levels are measurable and if biological effects are
seen in the presence or absence of serum levels for
agents given by any route of administration. There may
be instances in which second mediators are involved,
with useful biological effects occurring without apparent
serum bioavailability.
Effusions may contain both tumor cells and reactive
immunological elements, in which case the administra-
tion of a biological into a restricted space such as the
thoracic or peritoneal cavity might be appropriate. There
are indications that certain immunomodulators may be
effective in this setting [56]. Patients with tumors that
remain confined to a single compartment for a prolonged
period of time may offer unique opportunities for bio-
therapy [72, 88]. Ovarian cancer, with its propensity for
48
remaining localized in the abdominal peritoneum, may
be ideal for evaluating the antitumor activity of cells
[72], biologicals, and BRM in a relatively closed space.
Interleukin-2 with activated cells is active when infused
into selected anatomical sites and visceral cavities where
tumor is present [44, 88]. It seems clear that targeting of
monoclonal antibody and its conjugates may be improved
by selective organ or region perfusion or infusion.
Considerations for the design of early-phase trials in
these spaces differ markedly from those using systemic
administration.
Patient Selection
Historically, patient selection for phase I drug trials has
been broad, and patients bearing many tumor types have
been entered into the trials. While this may be appropriate
for drugs, and for selected lymphokines that act broadly
on immunological responses or may act in a general anti-
proliferative manner across a broad spectrum of tumors,
it is not appropriate for those biologicals that act more
specifically. Thus, a monoclonal antibody that has been
specifically designed to recognize a particular type of
cancer can be appropriately tested only in patients with
that type of cancer [45, 59].
In vitro determinations of specificity and activity may
play a greater role in the selection of the patient popula-
tions for phase I biotherapy trials. The escalating-dose
phase I trials, with the dose being escalated within indi-
vidual patients, preselected on the basis of in vitro speci-
ficity and/or activity, appear to be most relevant and
efficient for determining the distribution, biological
effects, and toxicity of monoclonal antibodies. The same
considerations may apply to certain lymphokines/cytok-
ines, in which case selection as to activity may be on
an individual patient basis. These trials may be more
appropriately termed phase I/II trials. In fact, given the
heterogeneity of cancer, both with respect to the specificity
of recognition by antibody and with respect to activity by
immunoconjugates and lymphokines/cytokines, in vitro
determinations may play a major role in clinical trial
design. Biological systems are diverse, and single patients
may require specifically designed treatments when spec-
ificity and activity restrict the applicability of biotherapy.
The design of early-phase [biotherapy] clinical trials needs
to be radically changed [55, 58, 71].
There has been much speculation concerning the
types of patients most appropriate for trials with bio-
logicals. Many believe that these agents should be tested
primarily in the adjuvant setting, where the tumor
burden is quite small and the biological responses to be
modified are still healthy [75]. Although this sort of
strategy may be optimal, it is also prohibitively expensive
Development therapeutics and the design of clinical trials
and very difficult to design. It would be virtually impos-
sible to investigate a significant number of biologicals in
phase II or III trials utilizing this type of clinical trial
design. The number of patients required for trials in
the adjuvant setting is enormous, in that the recurrence
rate cannot be precisely predicted without a concurrent
random control group. The lack of certainty for recurrence
prompts ethical questions in designing phase II or III trials
for these patients, since dose, route, schedule, OBRM and
therapeutic efficacy have not been determined.
It has now been well demonstrated that certain bio-
logicals have activity in patients with bulky and resistant
disease [4, 7, 13–16, 27, 57, 58, 67–68, 76, 89, 94, 108].
Even though biotherapy may be more effective, as
chemotherapy and radiotherapy are, when the tumor
burden is small, that does not mean that it is totally inef-
fective in patients with bulky disease. Given the large
number of biologicals available to the clinic, there is a
need to develop methods of rapid clinical testing in
early-phase trials, and this will necessitate testing in
patients with apparent disease [55, 57, 75, 108]. Indeed,
the initial clinical trials with interferons, interleukins,
monoclonal antibodies, and cellular therapies generally
selected such patients with a good performance status
and a reasonably functional immune system. These
patients have shown evidence of biological response
modification, and they have shown antitumor responses.
Thus, such clinical trials can be used as indicators for
biological activity of new agents [57, 67, 94].
Future Prospects
Developmental therapeutics for biotherapies is well
underway. From the inception of this field in 1980 to the
present, a great number of new approaches have become
available in biotherapy [42, 57, 71, 94, 108]. Unlike the
field of drugs, where very large numbers of compounds
are screened in a preclinical testing program and very
few reach the clinic, a much higher percentage of the
biologicals selected rationally and tested actually go on
to clinical trials. This is due to selection based on the
known physiologic activities of biologicals as opposed
to random testing of chemicals in drug development.
The strategies for these clinical trials need to be cost effi-
cient, advantageous to the patient, ethical [71, 97],
and scientifically interpretable to the clinician/scien-
tist [64, 75]. Phase I strategies are most important in
that they give the initial leads on which the design of
early phase II and III trials are based. With proper selec-
tion clinical trial strategies, new biologicals can be more
effectively screened and evaluated as potential anti-
cancer agents [59, 71].
Robert K. Oldham 49

Biotherapy is the fourth modality of cancer treatment
[57, 58, 84, 85, 86, 94]. These agents and this technol-
ogy have far broader applications in medicine than can-
cer therapeutics. Given the large number of cloned
biologicals and the virtually unlimited number of recom-
binant molecules that can be produced therefrom, along
with unlimited variety of monoclonal antibodies that
can now be produced, it is apparent that the coming
years will produce new challenges for those involved
in developmental therapeutics [33]. This tremendous
expansion of agents and approaches available for cancer
therapeutics will increase our opportunities and amplify
the complexity in the preclinical evaluation of these
agents and their translation to the clinic. The numbers of
biologicals to be evaluated and their inherent selectivity
call for new methods in the selection of the “most likely
to succeed.” Additionally, the methods used historically
by pharmaceutical manufacturers and the regulations
established by governmental agencies for approving
anticancer drugs are inhibitory to the rapid development
of biotherapy [47, 55, 83]. Novel approaches will be
necessary if these biologicals are to be effectively and
rapidly translated to clinical trials [47, 55, 64, 95].
Alpha-interferon, IL-2 and monoclonal antibodies
and their conjugates have anti-tumor effects, even in
patients with bulky disease. This finding is likely to
emerge for other forms of biotherapy [42, 57, 59, 67, 71,
104, 108, 109]. This should give pause to those who
believe the immunological dogma that biotherapy can
only be active in minimal residual disease. Like chemo-
therapy and radiotherapy, biotherapy may be more
active with lesser tumor burdens, but current data indi-
cate that activity and hence selection of compounds for
further study can be assessed in patients with advanced
disease.
The potential for the use of biotherapy is great. We
are in a new era in cancer therapeutics. To develop bio-
therapy, we must begin to approach this new era in phar-
maceutical research and to contemplate novel methods
for the efficient and timely development of biotherapy
rather than simply continuing in old paradigms (Fig. 1)
[33, 55, 71, 82, 83]. Some would still say no new treat-
ment can be judged without randomized, clinical trials.
Such trials are valid in searching for small differences
between a new treatment and an ethically acceptable
control (standard treatment) [101]. Whether placebo
controls are ever acceptable in cancer treatment is debat-
able; however, pilot studies without controls can be very
useful even to the point of defining efficacy if the treat-
ment effect is large and obvious. A recent and innova-
tive “n of 1” trial also bears consideration in trials of
biotherapy [35].

Preclinical Testing (in vitro/animal model)

Phase I Clinical Trial

Phase II Clinical Trial

Phase III Clinical Trials

Unresponsive or poorly responsive neoplasm;

Less than partial responses

Partial responses to newer active single agents

Increased partial responses and rare complete responses

Figure 1. Although radically different,
chemotherapy and biotherapy share a
systemic approach to cancer treatment. For
those cancers that chemotherapy has been
able to cure, reaching that goal required a
sequence of steps in which new and increas-
ingly more effective agents and combinations
of agents had to be identified. Biotherapy can
be expected to travel a similar path
To some single and combined agents
Increased complete responses to more effective
Combination therapy
Increased survival in complete responders with or
Without continued treatment during remission
A portion of long-term survivors are cured
50
We must not just continue to simply do what has been
done historically [71]. Development therapeutics for
biologicals certainly represents the kind of science
Lewis Thomas described:
It is hard to predict how science is going to turn out, and if it is
really good science it is impossible to predict. This is in the nature
of the enterprise. If the things to be found are actually new, they are
by definition unknown in advance, and there is no way of telling in
advance where a really new line of inquiry will lead. You cannot
make choices in this matter, selecting things you think you’re going
to like and shutting off the lines that make for discomfort. You
either have science or you don’t, and if you have it you are obliged
to accept the surprising and disturbing pieces of information, even
the overwhelming and up heaving ones, along with the neat and
promptly useful bits. It is like that.
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4 Recombinant proteins and genomics
in cancer therapy
KAPIL MEHTA, BULENT OZPOLAT, KISHORCHANDRA GOHIL,
AND BHARAT B. AGGARWAL
Abbreviations ADA, adenosine deaminase; AML, acute
myeloid leukemia; bFGF, basic fibroblast growth factor;
CML, chronic mylogenous leukemia; CSF, colony-stimu-
lating factor; EGF, epidermal growth factor; EPO, eryth-
ropoietin; FDA, Food and Drug Administration; HIV,
human immunodeficiency virus; IFN, interferon; IL,
interleukin; LIF, leukemia inhibitory factor; MAb,
monclonal antibody; PDGF, platelet-derived growth
factor; PE, Pseudomonas exotoxin; PEG, polyethylene
glycol; TGF, transforming growth factor; TNF, tumor
necrosis factor
Introduction
Recently published sequence of the complete human
genome represents a major milestone in the era of the
modern molecular biology [318, 132]. The sequencing
of approximately 3.2 billion nucleotides of the human
genome that is estimated to contain about 20,000–25,000
protein-encoding genes signifies the first step down the
long road. Gene identification does not necessarily trans-
late into an understanding of gene function. Although
mapping and cloning of several genes have linked them
to heritable genetic disorders, the normal function of a
majority of these genes remains unknown. Recombinant
DNA technology has made it possible to generate large
amounts of many biologically active proteins and to deli-
neate their functions. The novelty of recombinant techno-
logy is the precision and efficiency with which scientists
can manipulate single gene. The ability to isolate human
genes and insert them into microorganisms, which then
produce human proteins, thereby serving as biological
factories, has revolutionized the field of biology.
Interferons have special significance to recombinant
DNA technology as paradigm modifiers of immune
response. The interest in the therapeutic potential of inter-
feron against cancer and viral diseases has served as
catalyst to the emerging recombinant DNA industry.
Despite its great promise as an antiviral agent, the clinical
application of interferon had been rather slow, mainly
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
because of the lack of methods for producing adequate
amounts of the pure protein. Interest in interferon beyond
the field of virology began in the early 1960s, when
workers began to recognize its growth-inhibitory and
immune-activation properties. During the 1960s and
early 1970s, reports of interferon’s antiviral and antitumor
activity in laboratory animals and humans stirred up this
interest and several groups decided to purify human inter-
feron for clinical use.
The first practical method of producing sufficient
quantities of interferon was developed by Cantell et al.
[31]. They were able to isolate 100–200 mg of interferon
from 1,000 l of starting material that contained 2–5 kg of
other contaminating proteins. The purified material had
a specific activity of greater than 108 U/mg protein [69]
and was sufficient to treat only a few patients [282], but
the initial clinical results stimulated wider interest in
expanding production of interferon for more extensive
clinical trials. Enthusiasm intensified when interferon-
alpha was successfully cloned and the purified recombi-
nant protein became available [225, 226].
The first trial to test dose levels and side effects of the
purified bacterial product in human beings began in 1981
[227, 112]. The availability of pure recombinant pro-
tein led researchers to crystallize interferon, the first step
toward analysis of the protein’s three-dimensional struc-
ture by X-ray crystallography. This permitted the produc-
tion of individual molecular species of interferon free
from other species and other proteins that were simulta-
neously induced in human cell cultures. By using this
technique, our knowledge of the varied biological proper-
ties of interferons, previously determined with relatively
crude preparations, has been confirmed and extended.
Isolation, Cloning and Expression
of Genes
A gene is a defined region of a chromosome comprising
a specific sequence or part of a long polynucleotide.
It codes for some specific function or characteristic
53
54 Recombinant proteins and genomics in cancer therapy

GENE

-A-G-A a b c d
C-T-A-G- -
-T-C-T G-A-T-C- - Eukaryotic DNA Genome
Intron exon
Transcription

a b c d
Primary RNA transcript

a b c d
Splicing

a b c d
mRNA

Translation

Protein
NH2 ------ COOH

Protein processing, folding

Mature functional protein

Figure 1. The organization of a eukaryotic gene and processing of the RNA transcript

(phenotype) of a cell. The eukaryotic genome contains
up to 109 nucleotides in 50–100,000 genes [40, 97]. To
study the events in such a complex system it is neces-
sary to be able to isolate and study a single gene in a
purified form. Molecular cloning provides a method for
isolating a single discrete segment of DNA from a popu-
lation of genes and amplifying the DNA segment to pro-
duce enough pure material for chemical, genetic, and
biological analysis. Cloning and expression of foreign
genes has permitted access to such complex biological
mechanisms as RNA splicing, oncogene dynamics and
antibody diversity. It has also been the foundation for
the new biotechnology industry.
The organization of genes in higher organisms is
more complex than in bacteria and viruses. The linear
array of information in a complex gene contains one or
more stretches of non-coding sequences, called introns.
The remaining sequences, which encode information
for proteins, are called exons. When these genes pro-
duce a protein, the DNA is transcribed into a large RNA
molecule from which the introns are removed; this
mRNA molecule is then translated to produce the cor-
responding protein (Fig. 1).
The organization of bacterial genes is simpler. They
do not produce the enzymes necessary for RNA splicing,
so a eukaryotic gene containing introns, if introduced
into a bacterial cell as in recombinant techniques, will
not be properly expressed. This problem can be circum-
vented by making a DNA copy (cDNA) from the appro-
priate mRNA by using the enzyme reverse transcriptase
(Fig. 2). As an alternative, the DNA can be fragmented
at specific target sequences with the help of restriction
endonucleases [249]; the DNA fragment that contains
the gene of interest is inserted into the purified DNA
genome of a self-replicating genetic element, generally
a virus or a plasmid. A DNA fragment containing a human
gene, for example, when inserted into such a virus or
plasmid, can be joined in a test tube to the chromosome
of a bacterial cell. Starting with only one such recombi-
nant DNA molecule which can infect only a single
cell, the normal replication mechanism of the virus
can produce more than trillion identical molecules in
Kapil Mehta et al. 55

Genomic DNA
GENE Restriction nuclease digestion
------ ------

Transcription
mRNA

Reverse Transcriptase

DNA Fragments
Alkali degradation
Single-stranded DNA

DNA Polymerase

Complementary DNA (cDNA)

cDNA molecules are joined to

pBR322
vector DNA for propagation in
bacteria

Selection of clone(s) carrying desired cDNA

sequence, using a nucleic acid or antibody probe

Expression of cloned gene in host organisms such as

bacteria, yeast or mammalian cells

Figure 2. Gene cloning from mRNA or genomic DNA

less than a day, thereby amplifying the amount of the
inserted human DNA fragment by the same factor, making
it possible to infect millions of cells. The virus or plas-
mid used in this way is known as a cloning vector, and
the DNA propagated by insertion into it is said to have
been cloned.
Cloning the gene or cDNA encoding a particular
protein is only the first of many steps needed to produce
a recombinant protein for medical and industrial use.
Expression of foreign genes in a host organism requires
vectors which contain specific control sequences gov-
erning transcription and efficient splicing of RNA. The
most popular expression systems used for this purpose
are the bacteria Escherichia coli and Bacillus subtilis,
yeast, cultured insect cells and mammalian cells. The
choice of expression system depends on the properties
of the protein to be produced. Bacterial cells are most
common and convenient host organisms. They are simple
to grow, have short generation times, provide large yields,
and are most cost-effective. Particularly in the case of
B. subtilis, the cells can be induced to secrete the product
into culture medium, which facilitates the purification
of the cloned protein.
However, there are some disadvantages of using bacte-
rial cells for gene expression (Table 1). Though most pro-
teins are expressed in large amounts in bacteria, some
of them fail to fold properly, leading to formation of inso-
luble ‘inclusion bodies.’ Protein extracted from these
inclusion bodies is sometimes biologically inactive. Small
proteins can be refolded into their native form, but larger
ones containing several cysteine residues, in general, stay
inactive, most likely because of the improper formation
of disulfide bridges. Moreover, the expressed foreign pro-
tein is sometimes toxic to the bacteria, so that the culture
56 Recombinant proteins and genomics in cancer therapy

Table 1. Posttranslational processing of proteins in various using mammalian expression systems [61], but this pro-
expression systems cess is often lengthy and costly.
Insect Mammalian Expression of foreign proteins in cultured insect cells
Event Bacteria Yeast cells cells by baculovirus vectors is an alternative to this problem.
Proteolytic +/− + + This relatively new system is becoming the system of
Cleavage choice for expressing mammalian and viral proteins.
Glycosylation − + + + Baculovirus promoters are among the strongest known
Secretion +/− + + + and can drive the expression of target genes at high levels
Folding +/− +/− + + (1–500 mg/l). The baculovirus expression system offers
Phospho- − + + + several other advantages over prokaryotic, yeast, and
rylation
mammalian expression systems [178]. Some of the advan-
Acylation − + + +
Amidation − − + +
tages include: high-level expression, correct folding, the
% yield 1–5% 1% 30% <1% ability to catalyze post-translational modifications like
mammalian cells (Table 1), quick and easy growth in
monolayers or suspension cultures without CO2, and safest
producing the protein cannot be grown to high cell density. – they do not infect humans, animals or plants. More than
This problem can be overcome by using an inducible 400 different proteins have been expressed by using bacu-
promoter that is turned on to begin the transcription of the lovirus vectors, and in most cases the protein produced is
foreign gene only after the culture has been grown. Unlike similar in structure, biological activity and immunological
eukaryotic cells, bacterial cells lack enzymes that cata- reactivity to the authentic protein [177]. Although the cost
lyze posttranslational modifications of proteins such as of culturing insect cells is currently more than that of
phophorylation, acylation and glycosylation. These post- culturing bacteria or yeast, it is still significantly less than
translational modifications are sometimes essential for that of culturing mammalian cells.
the normal functioning of a protein. Despite the significant advantages of producing
Yeast cells may be preferable to bacteria for the human proteins in heterologous host cells, in some cases
production of some proteins biological by recombinant the best method for producing a mammalian protein is
DNA procedures. Yeast is a simple eukaryote that resem- a mammalian cell system. Transient expression in
bles mammalian cells in many ways but, like bacterial cells, mammalian cells is often used to check the function of
can be grown conveniently and economically. Yeast cells a newly cloned gene and as a quick method to assess
are capable of catalyzing many post-translational modi- the function of engineered proteins. The extracellular
fications that are found on mammalian proteins [126]. domains of cell-surface receptors have been engineered
Also, they process the signal peptides needed for the for secretion from cells by introduction of a stop codon
secretion of protein and thus can be induced to secrete into the gene before the sequence of the transmembrane
certain proteins into the growth medium for harvesting. domain. These soluble receptors, as we will discuss later
Moreover, unlike bacteria, yeast cells do not have endo- in this chapter, are valuable reagents for the study of
toxins. However, yeasts do produce active proteases ligand binding and for screening receptor agonists or
that can degrade the foreign proteins, thereby reducing antagonists that may eventually be used as therapeutics
the yield of the final product. To overcome this problem, themselves. Although transient transfections yield enough
the construction of yeast strains with deleted protease protein for laboratory experiments, stably integrated
genes is being attempted. amplified genes in mammalian cells are necessary for
Until recently, the expression of a biologically active large-scale production of proteins [2]. With this goal in
protein from a complex eukaryotic gene in large amounts mind, great improvements have been made in recent
was a problem. The problems with prokaryotic expres- years in identification of appropriate promoters, vectors,
sion systems have already been described. Even in yeast transformation protocols and host cell systems.
(eukaryotic) expression systems, the low biological
activity of complex eukaryotic proteins remains a com-
mon problem. Mammalian expression systems provide Recombinant Proteins as Cancer
all the post-translational modifications that may be
necessary for full activity of eukaryotic proteins, but the
Therapeutics
yields from these systems are generally much lower Biotherapy, as an alternative to the rigorous cytotoxic
than can be obtained from E. coli or yeast. Recently, regimens used in the treatment of various malignant dis-
protein yields as high as 10 mg/l have been obtained by orders, offers distinct and attractive advantages over
Kapil Mehta et al.
Antiproliferative
IFN
TNF
TGF
Amphiregulin
Oncostatin M
Lymphotoxin
FAS/APO ligand Approaches to the
Biological Therapy of
Cancer
Differentiation Immunomodulatory
CSFs
IFN-gamma IFNs
TNF TNF
LIF
EPO
Figure 3. Model for recombinant DNA-derived protein therapeutics for cancer
conventional chemotherapy. Biological therapy is based
on the principle of stimulating the body’s own immune
response and/or using biological substances against a dis-
ease. A perception of the malignant cell as one whose dif-
ferentiation has been blocked due to the lack, deficiency or
mutation of some key element led to the emergence of a
biological therapy. The strategy of biological therapy con-
trasts with the immediate cell death induced by cytotoxic
drugs, where there is no attempt to restore homeo-stasis.
Biotherapy may offer the opportunity to use relatively
nontoxic agents, the body’s own elements, to correct the
underlying problem. It has long been recognized that the
immune system plays a pivotal role in the patient’s response
to disease. Therefore, recent cancer therapies have been
directed at modulation of components of the immune
system (Fig. 3). In particular, “immune messengers” or
cytokines may play a vital role in regulating host antitumor
defense mechanisms. Knowledge of cytokines and their
functions is expanding rapidly [3, 6, 15, 122, 123, 126, 135,
179]. In the following section we will discuss the potential
role of cytokines in the treatment of malignant disease.
Cytokines
In general, cytokines are low molecular weight (10–
50 kDa) proteins secreted by cells of the immune system
that bind with great specificity and affinity to receptors
57
Anti-angiogenic
Anti-VEGF antibody
Endostatin
Angiostatin
Cytotoxic
Immunotoxins
antibodies
Fas/APO
on target cells and regulate the proliferation, differentia-
tion and metabolism of either the same cell (autocrine)
or another cell (paracrine). Cytokines are different from
endocrine hormones in that they are produced by any
number of different cells rather than by specialized
glands. Different cytokines exhibit considerable overlap
in their biological activities. Because of the advent of
recombinant DNA technology, cytokines have become
available in highly pure form and in sufficient quanti-
ties. This has accelerated the elucidation of in vitro and
in vivo biological activities of these proteins and led to
their rapid testing in the patients as therapeutic agents
for the treatment of cancer. The cytokines that have been
shown to have therapeutic potential in cancer include
the interferons (IFNs), interleukins (ILs), colony- stim-
ulating factors (CSFs), and tumor necrosis factors
(TNF). Table 2 lists some recombinant cytokines that
are currently being used for the treatment of malignant
diseases.
Interferons
Interferons are a family of regulatory glycoproteins pro-
duced by many cell types in response to viral infections
and a variety of mitogenic and antigenic stimuli. Three
major classes of IFNs have been described: IFN-α and
IFN-β, which share components of the same receptor
58 Recombinant proteins and genomics in cancer therapy

Table 2. Cytokines Approved or in Development for Human EPO Anemia associated with chronic USA
usea renal failure
Countries Malignancy, chemotherapy
Cytokine Target disease approved AIDS, AZT treatment
rheumatoid arthritis
r.Protein biotherapeutics approved by FDA for human use anemia of prematurity
IFN-α Chronic myelogenous leukemia USA, Europe, Autologous blood donation prior to
Japan surgery
Hairy cell leukemia USA, Europe, Compensation of surgical blood
Japan loss
AIDS-related Kaposi’s sarcoma USA Hepatitis B hepatomas surface
Chronic non-A/non-B/C USA antigen
hepatitis
Condylomata acuminata USA r.Protein biotherapeutics currently under clinical trials
Lymphoma IL-1αβ Malignant disorders
Essential thrombocythemia Bone marrow transplantation
Melanoma Severe aplastic anemia
Certain solid tumors Allotransplant patients
Multiple myeloma IL-3 Advanced neoplasms
Acute hepatitis B Secondary hematopoietic failure
IFN-β Renal cell carcinoma Europe Bone marrow recovery
Advanced solid tumors Breast cancer
Soft tissue carcinoma IL-4 Metastatic renal cell carcinoma
Adult T cell leukemia Metastatic breast carcinoma
IFN-γ Chronic granulomatous USA Metastatic melanoma
disease (CGD) Disseminated cancer
Advanced solid tumors Advanced malignancies
Renal cell carcinoma IL-7 Refractory solid tumors
Adult T cell leukemia Metastatic melanoma
Chronic myelogenous leukemia Metastatic kidney cancer
Lepromatous leprosy IL-11 Relapsed non-Hodgkin lymphoma
IL-2 Renal cell carcinoma Europe Leukemia
Metastatic melanoma Japan, Europe Melanoma
Advanced malignancies IL-12 Lymphoma
G-CSF Non-myeloid malignancies USA Advanced solid tumors (mela-
associated with chemo- noma, neuroblastoma, breast
therapy-induced cancer)
myelosuppression IL-18 Ovarian carcinoma
Myelodysplastic syndromes IL-21 Melanoma
Severe chronic neutropenia Ovarian carcinoma
(cyclic, idiopathic, congenital) IL-29 Hepatitis C (associated with
Acute myelogenous leukemia hepatocellular carcinoma)
Bone marrow transplantation CSF-1 Endometrial carcinoma
GM-CSF Autologous bone marrow USA, Japan IFN-α Metastatic renal carcinoma,
transplantation for non- Metastatic melanoma
Hodgkin’s lymphomas, Multiple myeloma
Hodgkin’ s disease, acute Lymphoma
lymphocytic leukemia TNF-α Advanced neoplasms
Allogeneic bone marrow trans- Reduction of ovarian ascites
plantation for leukemias M-CSF Solid tumors
Myelodysplastic syndromes/ Breast cancer
aplastic anemia
Fungal infections
Cancer chemotherapy associated
Acute myelogenous leukemia
myelosuppression
IL-1 AML, CML, sepsis, septic shock
Acute myelogenous leukemia
Receptor
AIDS, anti-AIDS drug treatment
antagonist
Associated myelosuppression
a
Disease in bold indicates approved use of cytokine.
Kapil Mehta et al. 59

and are referred to as type I; and IFN-γ, which uses a shown to induce remissions in some patients with well-
separate receptor system and is referred to as type II differentiated B-cell tumors [282].
(Table 3). Additional IFNs have been discovered, but The most remarkable effects of recombinant IFN-α
they are not well characterized [259]. Crude IFN was were observed in patients with hairy cell leukemia
probably the first cytokine used in patients and was [237]. Treatment with partially purified or recombinant
shown to delay recurrent growth of tumors in patients IFN-α suppressed peripheral blood cell production and
who had undergone surgery for osteogenic sarcoma rapidly increased platelet counts in these patients [112].
[282, 283]. Pharmacological doses of partially purified Their immune status improved and the number of leuke-
IFN-α were reported to induce regression of tumors in mia cells in the bone marrow and blood declined. These
significant numbers of patients with metastatic breast patients stopped having opportunistic infections and
cancer, low-grade lymphoma, or multiple myeloma required no further platelet and erythrocyte transfusions.
[110]. In 1981, IFN-α was successfully cloned by two These studies led to the approval of IFN-a for treatment
groups, and the purified protein was immediately tested of hairy cell leukemia in June 1986 by the United States
in the clinic. This was the first study of a recombinant Food and Drug Administration (FDA), an action adopted
cytokine in patients with cancer and, for the most part, by regulatory agencies from 31 other countries. The mecha-
the biological activity seen with the partially pure natu- nisms of IFN-α action in the compromised survival of
ral form was reproduced with the recombinant DNA- the patients with hairy cell leukemia are not fully under-
derived form [111]. Treatment with IFN-α was also stood but may include differentiation, cell-cycle arrest
and/or apoptosis.
Table 3. Recombinant DNA-derived proteins relevant to Other B-cell neoplasms also show variable degrees of
treatment of cancer sensitivity to IFN-α [110, 111, 282]. In patients with
Cytokine Receptor(s) used Reference multiple myeloma or low-grade lymphoma, the cytokine
often has demonstrated a positive clinical impact on
Interferon-α (IFNα) IFNα/β-R [2, 225]
Interferon-ß (IFN-β) IFNα/β-R [293]
survival when combined with chemotherapy [182, 272,
Interferon-γ (IFN-γ) IFNγ-R [103, 241, 261] 275]. Clinical results with IFN-α in patients with chronic
Transforming growth TGF-β -R1 and -RII [2, 56] myelogenous leukemia (CML) have been rather inter-
factor-β (TGF-β) esting. During the chronic phase of CML, IFN-α treatment
Thymosin α1 [329] causes hematologic remission [112]. Approximately 75%
Epidermal growth EGF-R [2, 101, 261] of the patients in the benign phase of the disease achieve
factor (EGF) complete normalization of blood counts. Moreover,
Platelet derived growth PDGF-RI and RII [116]
IFN-α had the astonishing capacity to suppress selec-
factor (PDGF)
Fibroblast growth factor FGF-R [98] tively the cells bearing the Philadelphia chromosome,
(Basic) (bFGF) resulting in partial or complete restoration of the normal
Fibroblast growth factor FGF-R [98] clone [290].
(Acidic) (aFGF) While showing great promise for leukemia, IFN-α
Transforming growth EGF-R [57] therapy of solid tumors has been rather discouraging.
factor-a (TGF-α) Only 10–15% of patients with renal cell carcinoma or
Insulin like growth IGF-RI and IGF-RII [54]
factor(IGF-I & II)
malignant melanoma undergo regression in response to
Hepatocyte growth HGF-R [199] IFN-α. However, the responses of carcinoid tumors to
factor (HGF) the cytokine were encouraging. A majority of patients
Macrophage colony- M-CSF-R [2, 145] showed improvement in symptoms, and a smaller frac-
stimulating factor tion experienced tumor regression [210]. Both squamous
Granulocyte colony- G-CSF-R [2, 120, 276] and basal cell carcinomas of the skin show sensitivity to
stimulating factor
IFN-α, as a single agent or in combination with retin-
GM-colony stimulating GM-CSF-R [2, 335]
factor oids [170, 229]. Mycosis fungoides, a malignancy of the
Leukemia inhibitory LIF-R [2, 99] T helper cells, is also sensitive to IFN-α alone or with
factor (LIF) other modalities including retinoids [140, 247]. Kaposi’s
Stem cell factor (SCF) SCF-R [2, 331] sarcoma, an angioproliferative disease that commonly
Erythropoietin (EPO) EPO-R [2, 78] develops in individuals infected with human immuno-
B cell growth factor BCGF-R [260] deficiency virus (HIV), has responded well to IFN-α
Hepatitis B surface ? [65, 314] therapy: 40% of patients experienced significant regres-
antigen
sion of lesions [282]. This work led to the approval of
60 Recombinant proteins and genomics in cancer therapy

IFN-α in the U.S. and 21 other countries for the treat- Table 5. Interleukins with relevance to cancer treatment
ment of AIDS-related Kaposi’s sarcoma. Interleukin Receptor Reference
After isolation of its gene and 10 years’ work identif-
Interleukin-1-α (IL-1α) IL-1R type I; IL-1R [59]
ying several of its biological activities, IFN-α was tested type II
in human subjects for a wide variety of diseases includ- Interleukin-1-β (IL-1ß) IL-1R type I; IL-1R [59]
ing cancer. It was found to have impressive effects against type II
chronic granulomatous disease, leading to its approval Interleukin-1 receptor IL-1R type I; IL-1R [17]
by the FDA for human use [298]. More recently, IFN-β antagonist type II
was approved for treatment of ambulatory patients with Interleukin-2 (IL-2) IL-2R. α, IL-2R. β, [162]
relapsing/remitting multiple sclerosis [4]. Clinical toxic
IL-2R.γ
Interleukin-3 (IL-3) IL-3R. α, IL-3γ [131]
effects associated with administration of IFNs and other Interleukin-4 (IL-4) IL-4R, IL-2R . γ [205]
cytokines are summarized in Table 4. The IFNs, like Interleukin-5 (IL-5) IL-5R. α, IL-5R. γ [288]
most other cytokines, are produced by the body to act Interleukin-6 (IL-6) IL-6R, gp130 [8]
locally. When used as a systemic pharmaceutical, they Interleukin-7 (IL-7) IL-7R (CDw127), [46]
can have substantial toxic effects [282]. IL-2R. γ
Interleukin-8 (IL-8) IL-8R, Type I & [128, 193]
Type II
Interleukins Interleukin-9 (IL-9) IL-9R, IL-2R . γ;
R [239, 240]
Interleukin-10 (IL-10) IL-10R1; IL-10R2 [188]
The interleukins are a family of cytokines that are essen-
Interleukin-11 (IL-11) IL-11R, gp130 [64, 222]
tial to both cellular and humoral immune responses. Interleukin-12 (IL-12) IL-12R [304]
To date, at least 34 interleukins have been identified Interleukin-13 (IL-13) IL-13R . α, IL-4R, [187, 347]
and cloned (Table 5). Many of these molecules exhibit IL-2R. γ
Interleukin-15 (IL-15) IL-15Rα, IL-2R . β, [90]
IL-2R. γ
Interleukin-16 (IL-16) CD4 [49]
Table 4. Common toxic effects associated with administra- Interleukin-17 (IL-17) IL-17R [166]
tion of cytokines Interleukin-18 (IL-18)/ IL-18R [200, 201]
Fever IGIF
Chills Interleukin 19 (IL-19)a IL-19R [86]
Nausea Interleukin-20 (IL-20) IL-20Ra. & IL-20Rβ [24]
Vomiting Interleukin-21 (IL-21) IL-21R [217, 353]
Headache Interleukin-22 (IL-22) IL-22R [149,
Anorexia 336–349]
Fatigue Interleukin-23 (IL-23) IL-12Rα1, IL-12Rβ2 [213]
Myalgias Interleukin-24 (IL-24) IL-20R1,IL-20R2, [347, 354,
Arthralgias IL-22R, IL-22R2 355]
Bone pain Interleukin-27 (IL-27) IL27RA [349, 350]
Flush
Interleukin-32 (IL-32) [351, 352]
Local erythema
Inflammation at the site of injection a
IL-19 is a novel homologue of human IL-10.
Capillary leak syndrome
Granulocytopenia
Thrombocytopenia
Anemia
Hypotension antineoplastic activity. Currently, IL-2, IL-12, IL-15
Liquid accumulation in the lung, spleen, kidneys and IL-18 are the most potent inducers of anti-tumor
Reversible increase in body weight
activity in preclinical and clinical studies. Recently, new
Reversible increase in the serum creatinine
Oliguria interleukins have been characterized, including IL-21,
Malaise IL-23, IL-24 and IL-27, which exert potent immuno-
Asthenia modulatory and anti-tumor effects in vitro and in vivo
Rigors settings, indicating that they may have considerable
Diarrhea
Hepatocytotoxicity
potential as future cancer therapeutics [348].
Lethargy, depression Interleukin 1 (IL-1): IL-1 was originally described as
Mental confusion an “endogenous pyrogen” in 1940 because of its ability
EEG-abnormalities to cause fever when injected into animals. Two forms of
Kapil Mehta et al.
IL-1 are now recognized: IL-1a is cell-associated and is
involved in antigen presentation, whereas IL-1β, the
predominant form, is readily secreted by macrophages.
Though the two forms have limited amino acid homol-
ogy, IL-1α and IL-1β bind to the same receptor and
share several biological properties. Other cell types that
produce IL-1 are endothelial cells, keratinocytes, neu-
trophils and B lymphocytes. Constitutive expression of
IL-1 occurs in cells lining the external environment, i.e.,
skin and mucosal surfaces [58]. The cDNAs coding for
both human IL-1s were reported in 1985 [39, 83].
Recombinant IL-1s induced fever, hepatic protein syn-
thesis, production of prostaglandin E2, cartilage break-
down, bone resorption, and elevated ACTH and
augmented the T lymphocyte response to antigens and
mitogens [58]. Recombinant IL-1 exhibits cytostatic
activity toward human melanoma tumor cells in vitro
and direct cytotoxic effects against human melanoma
cell line A375 [157]. Recently, recombinant IL-1 was
shown to enhance the recovery of platelets in ovarian
cancer patients following carboplatin therapy, suggesting
a potential role for IL-1 in attenuating thrombocytopenia
associated with chemotherapy [309].
Interleukin-2 (IL-2): IL-2 is a 15.5-kDa glycoprotein
produced by peripheral blood lymphocytes and is a
potent growth factor for activated T lymphocytes. It acts
as a cofactor in development of cytotoxic T lymphocyte
activity against tumors and has been shown to participate
in tumoricidal activity by inducing the growth of natural
killer (NK) cells and lymphokine-activated killer (LAK)
cells [242]. Several cancers show sensitivity toward
recombinant IL-2, both in animal models and in the
patients. LAK cells are peripheral blood lymphocytes
that can be generated in vitro by incubation with high
doses of IL-2. They have the ability to kill tumor cells
specifically while leaving normal cells unharmed. IL-2
has been used in combination with LAK cells to achieve
more potent antitumor response [242–245]. In 1985,
Rosenberg and associates published the results of their
first study documenting tumor regression in patients with
melanoma following administration of IL-2 and LAK
cells [243]. An update of the results in 180 patients was
published in 1991 [242]. Antitumor responses were seen
in patients with advanced melanoma, renal cell cancer,
colon cancer or non-Hodgkin’s lymphoma.
Like LAK, tumor-infiltrating lymphocytes (TILs), the
lymphoid cells that infiltrate solid tumors, can be grown
in vitro in the presence of IL-2. These cells have unique
lytic activity against autologous tumors. Treatment with
TILs in combination with IL-2 was shown to mediate
substantial tumor regression in some patients with advanced
malignant melanoma [174]. Objective responses were
61
observed that lasted for 3–14 months in 29% patients
with renal cell cancer and 23% of those with melanoma.
Further potential of IL-2 in cancer therapy has been
demonstrated by using recombinant IL-2 in combination
with other cytokines [244, 306]. For example, recombi-
nant IL-2 in combination with IFN-α elicited a potent
antitumor response in several animal tumor models [175].
The most significant antitumor activity seen with IL-2
therapy has been in malignant melanoma and renal cell
carcinoma. Other tumors treated with IL-2 include
glioma, bladder carcinoma, ovarian carcinoma, neuro-
blastoma, lung carcinoma, head and neck carcinoma,
breast carcinoma, lymphoma, colon carcinoma and
mesothelioma [123]. As shown in Table 4, IL-2 adminis-
tration in patients is associated with a wide range of toxic
effects, the most common being fluid retention, anemia,
thrombocytopenia and hypotension [175]. IL-2 is the
first cytokine that has been employed so widely in clinical
trials. However, because of its toxic effects, its clinical use
has been limited.
Interleukin-3 (IL-3): Recombinant IL-3 is a 15–17-
kDa polypeptide that is known to stimulate mast cells,
neutrophils, macrophages and megakaryocytes. No direct
antitumor activity has been observed for IL-3, but this
cytokine has a role in increasing platelet and neutrophil
counts in patients with advanced malignancy [87]. Phase
I and II clinical trials with recombinant IL-3 have been
carried out in patients with advanced malignancy. A dose-
dependent increase in platelet counts and substantial
increases in the numbers of circulating neutrophils,
eosinophils, monocytes and lymphocytes were observed
in these patients [87]. Hematopoietic failure caused by
prolonged chemotherapy, radiotherapy or infiltration of
bone marrow by tumor cells could be restored by recom-
binant IL-3 treatment. The side effects of rIL-3 therapy
in patients include fever, bone pain and headache [87].
Thus, recombinant IL-3 is a multilineage hematopoietic
cytokine with promising effects on platelet and neutro-
phil counts.
Interleukin-4 (IL-4): IL-4 is a T cell-derived glyco-
protein of 20 kDa. The gene for human IL-4 has been
cloned [16]. The antitumor functions of IL-4 include
increased T cell, NK cell and monocyte proliferation.
IL-4 has also been shown to enhance the generation of
cytotoxic T lymphocytes and to participate in induction
of LAK cell activity, and to synergize with I-2 in this
activity [194]. Furthermore, IL-4 can stimulate the gen-
eration of TILs in human melanoma, increase the anti-
gen-presenting ability of mouse and human monocytes
and augment the expression of tumoricidal activity
in murine macrophages. It appears to inhibit the release
of TNF, IL-1 and IL-6 by human monocytes [296].
62
Interleukin-4 has been shown to exert potent antitumor
activity against several transplantable tumors in a
murine model. Using IL-4-transfected tumor cells, the
potential of transfecting lymphokine genes into tumor
cells as a method of cancer therapy has been demon-
strated [93]. Because of its antitumor effects in vitro and
in murine models, IL-4 may be useful in inhibiting the
growth of solid tumors and cell lymphomas.
Other Interleukins
The family of interleukins has continued to grow and
new members have been included (Table 5). IL-18 is the
most recently cloned member of this family [200],
which is synthesized as a 24 kDa proform that is pro-
cessed into an 18 kDa mature form. The mature form of
IL-18 induces IFN-gamma secretion and plays an
important role in antitumor immunity [219]. IL-18-
transgenic mice show increased CD8+ CD44 high T
cells and macrophages, while B cells are decreased in
these mice [129]. Similarly, IgE, IgG1, IL-4 and IFN-γ
levels are substantially increased in the sera of IL-18-
transgenic mice. In contrast, the IL-18 knock out mice
exhibit impaired clearance of neurovirulent influenza A
virus-infected neurons from the brain [190]. Another
relatively newer member of the interleukin family is
IL-15 that acts as a strong cofactor for Th1 T cell devel-
opment and as a growth factor for T lymphocytes and
TILs [112]. Like IL-2, it binds to the beta and gamma
chains of the IL-2 receptor to exert its action [100].
However, specificity for IL-15 versus IL-2 resides in the
unique private α-chain receptors that complete the
IL-15Rαβ.g and IL-2αβγ heterotrimeric high-affinity
receptor complexes and thereby allow differential
responsiveness depending on the ligand and high affin-
ity receptor expressed [323]. A recent review discusses
the role of IL-15 in human diseases and its potential
clinical implications as a therapeutic target [70]. Other
important members of the interleukin family include
IL-5, IL-6, IL-7, IL-12, IL-21 and IL-24. IL-5 is an
18-kDa product of T lymphocytes that has been cloned
and shown to be a lineage-specific eosinophil growth
and differentiation factor [30, 250]. Murine IL-5 induces
antibody-dependent killing of tumor cells by blood
eosinophils and enhances phagocytosis by eosinophils.
Interleukin-6, initially described as β2-interferon, is a
19–28-kDa protein produced by a variety of cells,
including mononuclear phagocytes, fibroblasts, kerati-
nocytes and endothelial cells. The experimental data
support a potential clinical role for IL-6. Its hematopoi-
etic activity and thrombopietic activity, in particular,
may make this cytokine a useful agent for inducing the
Recombinant proteins and genomics in cancer therapy
recovery of bone marrow in patients with myelosup-
pression that usually follows aggressive chemotherapy
regimens. The results of phase I clinical studies of IL-6
have recently been reported [325]. IL-7 is a 22 to 25-kDa
glycoprotein that was originally characterized on the
basis of its ability to promote the growth of precursor B
lymphocytes [96]. Recombinant IL-7 induces the prolif-
eration of both thymocytes and mature T cells and is
known to activate macrophages for tumor cell killing. In
human monocytes, IL-7 induces the expression of IL-8,
IL-6, IL-1a, IL-1.b and TNF-α. IL-12, a product of B
cells and mononuclear phagocytes, has multiple effects
on both T cells and NK cells. It induces IFN-γ produc-
tion in T and NK cells and sustains the cell-mediated
immune response [304].
Interleukin-21 (IL-21) is a cytokine with structural
and sequence homology to IL-2 and IL-15 that has anti-
tumor activity alone in experimental tumor models and
a tolerable safety profile in phase I trials in patients with
metastatic melanoma and renal cell carcinoma [353].
Several monoclonal antibodies targeted at tumor-asso-
ciated antigens also have improved antitumor activities
in mice when used in combination with IL-21, suggest-
ing that the use of IL-21 in adjuvant settings induces a
strong T cell-mediated immune responses [353].
Interleukin-24 (IL-24) is a cytokine belonging to the
IL-10 family of cytokines that signals through two het-
erodimeric receptors (IL-20R1/IL-20R2 and IL-22R1/
IL-20R2). IL-24, a recently identified cancer gene thera-
peutic, is melanoma differentiation associated gene-7/
IL-24 (mda-7/IL-24) that has the unique ability of induc-
ing apoptosis in a variety of cancer cells with no toxicity
to normal cells or tissues. It exerts immunomodulatory,
anti-angiogenic, and radiosensitizing effects when applied
as adenovirus expressing mda-7/IL-24 (Ad.mda-7) in
human xenograft animal models and has been success-
fully used in a Phase I clinical trial in patients with
advanced cancers [347, 354].
Colony-Stimulating Factors
The colony-stimulating factors (CSF) are a family of
glycoproteins that have the ability to induce prolifera-
tion and differentiation of progenitor hematopoietic
cells and have effects on the functional status of their
mature progeny. Multi-colony stimulating factor (IL-3),
macrophage colony-stimulating factor (M-CSF), gran-
ulocyte colony-stimulating factor (G-CSF), granulo-
cyte-macrophage colony-stimulating factor (GM-CSF),
erythropoietin (EPO), stem cell factor (SCF), and leuke-
mia inhibitory factor (LIF) are some of the clinically
important members of this family (Table 3). All the
Kapil Mehta et al.
known CSFs have been produced by recombinant DNA
methods and tested in human subjects (Table 2). The
potential for using hematopoietic growth factors in the
treatment of disease is enormous. Their ability to con-
trol the production of blood cells has been realized, and
the results of clinical trials to date suggest that the side
effects of these growth factors are relatively minor
[329]. Three recombinant hematopoietic growth factors,
G-CSF (filgrastim), GM-CSF (Sargramostim), and EPO
(epoetin alfa), are now commercially available for
clinical use in the United States.
Extensive clinical and preclinical data on recombi-
nant human G-CSF and GM-CSF indicate that both
these cytokines are effective in accelerating neutrophil
recovery and shortening the duration of neutropenia fol-
lowing chemotherapy with or without bone marrow
transplantation [48, 77, 185, 193, 206]. Administration
of recombinant human G-CSF as an adjunct to cyclo-
phosphamide, doxorubicin and etoposide chemotherapy
for small cell lung carcinoma significantly reduced
duration and severity of neutropenia and associated
clinical sequelae [303]. Similarly, patients with transi-
tional cell carcinoma of the bladder treated with metho-
trexate, vinblastin, doxorubicin and cisplatin experienced
up to fourfold increases in neutrophil count on adminis-
tration of G-CSF with few or no toxic effects [84].
GM-CSF may also exert antitumor effect by inducing
tumoricidal activation of macrophages. Administration
of GM-CSF has been shown to decrease the tumor bur-
den in a murine Lewis lung carcinoma model [121].
Clinical trials of M-CSF have been performed in an
attempt to ameliorate leukopenia. A phase I trial of
M-CSF in patients with metastatic melanoma showed
an increase in the number and function of circulating
monocytes [18]. In a non-randomized, controlled study
(32 patients with urinary tract malignancies) and a ran-
domized controlled study (98 patients with gynecological
malignancies), M-CSF administration reduced the period
of post-chemotherapy leukopenia [192, 207].
Among the cytokines whose role can be predicted
from in vitro studies, EPO is perhaps the best example.
EPO is produced mainly by the kidneys and is respon-
sible for regulating the production of erythrocytes. EPO
acts on erythroid precursors in the bone marrow, spleen
and fetal liver and stimulates the colony formation of
the burst-forming unit-erythroid. When infused in mice,
EPO markedly increases both peripheral blood erythro-
cytes levels and the number of erythroid progenitor
cells present in bone marrow. These results led to the
clinical trials with a human recombinant EPO, the find-
ings suggested that EPO can reverse anemia in patients
with end-stage renal cell disease. EPO produced dose-
63
dependent increases in hematocrit and hemoglobin
levels, and in most cases eliminated the need for regular
blood transfusions [67]. The major side effect reported
is increased blood pressure. EPO also increases the abil-
ity of patients undergoing elective surgery to donate
autologous blood [94]. Double-blind placebo-controlled
studies with recombinant EPO suggested that it is an
effective treatment for predialysis patients [168].
Stem cell factor (SCF) has recently been used in the
clinic as a single agent following chemotherapy. SCF by
itself appears to have limited efficacy and significant
toxicity-mainly due to mast cell stimulation at higher
doses. However, Tong et al. [300] showed that patients
receiving CSF have an increase in primitive progenitor
cells suggesting that SCF might be highly effective in
combination with later acting hemopoietins. From these
data it is clear that recombinant CSFs are effective in
correcting hematopoietic disorders of various etiolo-
gies. Whether these mediators improve morbidity and
mortality in patients will be decided by further clinical
results. However, combinations of cytokines, for exam-
ple, those with relatively restricted biological activity
(EPO, G-CSF, M-CSF etc.) and those that have a broad
range of action (SCF, IL-3, GM-CSF, IL-6 etc.), are
likely to show more promising effects on hematopoiesis
than any single cytokine alone [114].
Tumor Necrosis Factors
Tumor necrosis factor (TNF) is a proinflammatory
cytokine that is produced primarily by mononuclear
phagocytes in response to endotoxin. In recent years,
several new members belonging to the TNF superfamily
of molecules has been described and the list has continued
to grow (Table 6). There are two most studied forms of
TNF; TNF-α is a cytotoxic factor with a molecular weight
of 17-kDa, and TNF-β, also known as ‘lymphotoxin,’ has
a molecular weight of 25-kDa and is released from stimu-
lated lymphocytes [102, 203, 204, 223]. Both forms have
been produced by recombinant DNA technology and
appear to have antiproliferative, cytostatic and cytolytic
effects against human tumor cells in vitro as well as in
vivo when injected into nude mice [212]. TNF-α exerts
synergistic effects with different cytokines, such as IFNs,
IL-1, IL-2 etc., and can induce secretion of series of
mediators, including IL-1, IL-6, prostaglandins etc. It has
been implicated in both the generation and the cytotoxic-
ity of LAK cells and cytotoxic T lymphocytes [37].
Based on these observations, a large number of phase
I and II studies were initiated to investigate the antitu-
mor properties of TNF-α. Unfortunately, in clinical
settings the efficacy of TNF-α has been very limited,
64 Recombinant proteins and genomics in cancer therapy

Table 6. Members of the TNF family and their receptors for its use. In a recent study, Lienard and co-workers
Cytokine Other names Receptor [167] used an intra-arterial route to administer of high
doses of TNF-α in conjunction with melphalan, hyper-
TNF-α TNFR1 (CD120a);
TNFR2(CD120b) thermia and IFN-γ. Of the 23 patients treated (19 with
LT-α3 (TNF-β) TNFR1,TNFR2 melanoma and four with sarcoma), all responded, 21
LT-β1α2 LTβR with complete remission and two with partial remission.
LIGHT (HVEML) HVEM (TR2, ATAR, Eleven of these patients were previously unresponsive
LIGHTR), LtbR, DcR3
to melphalan alone and one had failed to respond to cis-
OX40L CD134L OX40 (CD134)
4-1BBL CD137L 4-1BB, TNFRSF9, CD137, platin therapy. The toxic effects observed (neutropenia,
ILA hypotension, thrombocytopenia and kidney failure)
CD27L CD70) CD27 were reversible. The overall rate of survival was 70%
CD30L CD153 CD30 and of disease-free survival over 12 months, 76%.
CD40L CD154, TRAP, gp39) CD40
FasL Apo-1L, CD95L Fas (Apo-1, CD95); DcR3
These results suggested that further understanding of
TRAIL Apo-2L TRAIL-R1 (DR4, Apo-2) the mechanisms of TNF-α’s antitumor action could help
TRAIL-R2 (DR5, KILLER) improve its clinical efficacy. For example, decreasing the
TRAIL-R3 (DcR1, TRID) agent’s systemic toxicity without reducing its anticancer
TRAIL-R4 (DcR2, effects could lead to substantial therapeutic advantages.
TRUNDD)
OPG (OCIF) TNF-α mediates its effects by interacting with two dif-
RANK TRANCE, OPGL RANK (TRANCE-R) ferent surface receptors, p55 and p75 [296]. Studies have
OPG (OCIF) suggested that TNF-α’s interaction with p75 may be
TWEAK Apo-3L Fn14 (TWEAK-R), DR3 responsible for its systemic toxicity [118, 296]. Thus,
APRIL TALL-2 BCMA mutant TNF molecules that interact with p55 but not p75
TACI
BAFF (BlyS, THANK, could induce antitumor effects with reduced systemic
TALL-1, zTNF4) TACI toxicity, permitting higher doses of TNF-α. Indeed, such
MCMA mutant human TNF molecules that specifically bind to
GITRL AITRL GITR p55 have already been described and shown to exert
EDA-A1 EDAR
cytotoxic effects against transformed cells in vitro [214].
EDA-A2 XEDAR
? TAJ (TROY); TRAIN In addition, concomitant use of drugs that are able to
VEGI ? decrease TNF-α systemic toxicity could permit use of
? DR6 higher, more effective doses of this cytokine in cancer
75NTR NGFR (p75, NTR) therapy. Combination regimens of TNF-α with other
? RELT
cytokines, concomitant use of TNF toxicity inhibitors
APRIL, A proliferation-inducing ligand; BCMA, B cell maturation
and use of mutant TNF molecules may provide better
antigen; BAFF, B cell activating factor; BlyS, B lymphocyte stimu- clinical outcomes. Moreover, regional therapy with
lator; THANK, TNF homologue that activates apoptosis, NF-kB TNF-α requires further exploration in view of the fact
and JNK; TACI, Transmembrane activator and CAML-interactor; that such regimens have already produced some very
TWEAK, a weak homologue of TNF; HVEM, herpes virus enetery promising results [167].
mediator; LIGHT, homologous to lymphotoxin, shows inducible
expression and competes with HSV glycoprotein D for HVEM;
TRANCE, TNF-related activation-induced cytokine; RELT, receptor Soluble Cytokine Receptors
expressed in lymphoid tissues.
For details, see references [5, 52, 109, 262, 269, 324, 339, 340]. Certain membrane receptors are enzymatically cleaved
from the cell-surface and released into the extracellular
and its use is associated with serious toxic effects [125]. medium in the form of soluble fragments. Soluble recep-
Of 127 eligible patients enrolled in nine different phase tors corresponding to the ligand-binding domains of
II protocols between 1988 and 1990 for the treatment of many polypeptide hormones and cytokine receptors
diverse malignancies, including breast, colon, gastric, have been described (Table 7). The function of soluble
pancreatic, endometrial, and bladder cancers, multiple receptors is not yet known [73, 115]. However, it is
myeloma and sarcomas, only one patient responded likely that this process represents an important mecha-
(response rate, 0.8%), whereas 13% experienced grade nism for regulation of surface expression of such recep-
four or fatal toxic effects. Despite the initial disappointing tors and may determine the effects of cytokines and
results with TNF-α as an antitumor agent, investigators growth hormones on the target cells. For example, the cell
have continued working on new and improved approaches growth, differentiation and immunomodulatory effects
Kapil Mehta et al. 65

Table 7. List of soluble receptors identified of activation of specific proteolytic enzyme or enzymes.
for various cytokines The identity of enzymes involved in proteolytic cleavage
Receptor Reference of the receptors is not known; however, it is a highly
Proteolytic cleavage: regulated process and appears to be controlled by phos-
IL-1R [115, 267, 268, 279] phatases and kinases [4]. The treatment of cells with
IL-2R [274] ligand can also lead to down modulation of the receptors
IL-4R (murine) [74] and their subsequent shedding.
IL-6R [195, 198] The significance of soluble cytokine receptors as a
IL-6R (gp 130) [195, 198, 202] therapeutic modality for treatment of cancer will be
TNFR [12, 13, 88, 139] determined by further research and evaluation. Since
IFN-α R [208] soluble receptors can provide highly specific biological
IFN-γ R [209] inhibitors for cytokines and growth factors, and because
NGFR [60]
the majority of transformed cells require cytokines for
M-CSFR [62]
Hergulin R [161]
their growth and survival, soluble receptors may have
EGFR [108] therapeutic potential as antagonists to cytokine action.
EPOR [21, 196] For example, hematopoietic growth factors are known
PDGFR [299] to maintain the viability of hematopoietic cells through
Alternative splicing: the prevention of apoptosis [332]. Several investigators
IL-4R [191] have reported that autocrine production of hematopoi-
IL-5R (murine) [178] etic growth factors such as IL-1β [82, 104] or GM-CSF
IL-7R [95] [344] supports the growth and survival of acute myeloid
LIFR [159] leukemia (AML) cells in vitro. In contrast, their soluble
GM-CSFR [251]
receptors and receptor antagonist could inhibit the
G-CSFR [79]
growth of leukemic cells including AML [343], chronic
EGFR [228]
c-erbB3 [143] myelocytic leukemia (CML) [68], and juvenile CML
[257]. Receptor proteins for most of the cytokines have
been cloned and expressed [2]. However, the informa-
of cytokines are exerted in response to their binding to tion available on their therapeutic potential in cancer is
specific cell-surface receptors. The presence of soluble very limited.
receptors in the biological milieu may thus promote Like the soluble and membrane-bound forms of cytokine
direct binding of the ligand to the soluble receptor, neu- receptors, the cytokine ligands also exist in these two
tralizing and preventing its action. forms. For example, the cytokines IL-1, TNF, FGF, TGF-
The in vivo relevance of soluble cytokine receptors is α, TGF-β and SCF have been reported to exist in both the
well illustrated by several viruses. Vaccinia and cowpox soluble and membrane-bound forms. This process, com-
viruses encode a protein that displays homology with monly initiated by cell stimulation, may regulate the sur-
soluble IL-1 receptor and is able to bind IL-1β [279]. face expression of such cytokines and play an important
Furthermore, proteins that bind to TNF have been identi- role in determination of cytokine activity.
fied in the open reading frame of pox viral strains [274].
Herpes and myxoma viruses encode proteins that can Genomics and Proteomics in Cancer
effectively bind IL-8 and IFN-γ ligands, respectively [7,
307]. Such soluble receptors assist virus infection by sup-
Therapeutic
pressing host defense mechanisms. From the studies on Advances in the analytical tools and recombinant
induction of antiviral soluble LDL receptors by IFNs, it DNA technology have improved our understanding of
seems that host organisms make use of a similar mecha- the signaling pathways encoded in cytokine, growth
nism for the opposite role of controlling viral infection. factors and hormones [158, 85]. In most cases, the
During the release of cell-surface receptors, it is usu- focus of such investigation is often a single molecular
ally the extracellular domain of the receptor that is shed; target. Compilation and integration of this voluminous
thus, soluble receptors act as inhibitors of cytokines. The literature demonstrates a high level of complexity in
soluble receptors may originate via two separate mecha- cell’s repertoire for the detection and processing of
nisms, one involving alternate splicing in which a receptor molecular signals. Despite all this information, the
gene lacks a transmembrane domain. As an alternative, relative contribution of each and more importantly,
the receptors can be shed from the cell-surface as a result the cross-talk among different pathways that influence
66 Recombinant proteins and genomics in cancer therapy

the process of differentiation, proliferation or apoptosis
remains unclear.
Recent advances in the techniques for quantitative
and comprehensive analysis of all the mRNAs (tran-
scriptome, ∼40,000 unique molecules [53, 317]) or all
the proteins (proteome, >40,000 unique proteins) of the
cell, offer a promise for defining the cellular functions
in terms of causative molecular changes. In the follow-
ing sections we define some of the basic concepts of
these recent techniques and their early contribution in
our understanding of cancer cells. These early data suggest
considerable molecular heterogeneity in an apparently
homogeneous group of tumors classified by conventional
tools, such as immuno- and histochemistry [92, 9].
Interpretation of such results offers possible explanation
for the observed differences in the outcome of a specific
anticancer therapy. In addition, the spectrum of molecu-
lar targets for a therapeutic agent can also be defined by
the application of global gene expression analysis [181].
Such treatment specific mRNA profiles offer the oppor-
tunity to predict diverse effects of anticancer drugs and
contribute towards selection and design of tumor spe-
cific therapies.
The Fig. 4 represents some essential molecular players
that are involved in gene expression pathways and are
recruited during changes in cell proliferation, differen-
tiation and apoptosis initiated by extracellular signals.
mRNAs and proteins are obligatory intermediates in the
Cytokines
Growth factors Micronutrients
Inactive activated
transcription factors transcription factors
Translocation to
?
histone acetylases nucleus
histonedeacetylases
heterochromatin Euchromatin
(inactive) (active)
RNA
Splicing protein
mRNAs
Proteins
signal transduction pathways [277]. It is now abundantly
clear that single transcription factors such as NF-κB
which is translocated to the nucleus from cytoplasm in
response to binding of a cytokine to its receptor, acti-
vates a large number of genes [19, 255]. Similarly intra-
nuclear p53 [234, 345] and MYC [41] proteins also
affect the expression of large numbers of genes that are
important determinants of cell proliferation and cell
death. Such large-scale gene profiling studies begin to
address the relative importance of individual genes in
the determination of cell’s destiny.
Oligonucleotide Arrays for Quantitative
Analysis of >30,000 Unique mRNAs
GeneChips commercialized by Affymetrix offer a reli-
able tool for the evaluation of distinct mRNAs present in
extracts of total RNA or mRNA by selection with oligo
dT. The GeneChips are unique with respect to at least
two notable features: the first that they have oligonucle-
otide probes prepared by a combination of photolitho-
graphic technologies and combinatorial chemistry [169].
The second unique feature of GeneChips is the distribu-
tion of the probes on the glass slide. There are 16–20
pairs of antisense oligonucleotides arrayed on a glass
slide for each of ∼30,000 unique mRNAs. Each pair of
oligonucleotides contains a set of perfectly matching
antisense sequence of 23 nucleotides and a set of identi-
cal sequence of nucleotides with one “mismatch” base at
the 13th nucleotide. Hence each mRNA is probed 16–20
times for specific binding and an equal number of times
for non-specific binding. A comprehensive statistical
analysis of 16–20 pairs of data for each target then evalu-
ates the specific mRNA to be either present or absent and
gives a numerical value for absolute hybridization. A major
disadvantage of the technique is its inability to detect
low abundance mRNA. For example, in authors’ experi-
ence, the GeneChips were unable to detect mRNAs for
nitric oxide synthase genes that could be detected by
RT-PCR. The application of oligonucleotide arrays to
human cancers have initiated a more detailed compilation
of molecular changes that are cancer specific [285–328].
Such a data-base will prove useful in diagnosis and treat-
ment of cancers.

cDNA arrays
Oligonucleotide arrays
Proteomic analysis cDNA Arrays are Dotted Arrays of PCR-
(2D separation-Mass Spec)
SAGE
Differential display
Amplified Products of Cloned Genes
Subtraction hybridization
cDNA arrays of large numbers of genes were invented
Figure 4. Molecular players involved in gene expression and pioneered in laboratories of Patrick Brown and
pathways David Botstein [28, 256]. Unlike oligonucleotide arrays,
Kapil Mehta et al.
cDNA arrays are constructed by robotic dotting of PCR
amplified fragments of cDNA on to glass slides or nylon
membranes. The cDNA arrays of human genes have
been used to define disease specific gene profiles. Such
analyses have contributed towards a better understand-
ing of cancer and normal cells [9, 10]. The techniques
for constructing cDNA arrays are more readily available
to laboratories and tissue or cell-specific arrays can be
constructed in a specialized and dedicated core facility
[254]. The application of cDNA arrays to various can-
cers cells ex vivo [254] and in vivo [92] have resulted in
a better classification of cancers and will lead to
improved paradigms of anti-cancer drug development
in the near future.
Proteomic Analysis and Cancer
One major aim of scientists is to understand mutations
that can cause cancer. To accomplish this aim, a thorough
understanding of the workhorse of all biological systems,
the proteins, is required. Currently, a major effort is being
made to understand protein–protein interactions. There is
an equal emphasis on understanding the structural fea-
tures of proteins and the structural basis of protein–pro-
tein, protein–ligand, and protein–drug interactions.
However, initially lack of techniques that enable compre-
hensive and quantitative labeling and separation technol-
ogy for proteins, have been limiting factors these efforts
[1]. The separation of cellular proteins by two dimen-
sional, denaturing gel electophoresis combined with
determination of peptide sequence to identify proteins
has been used to define changes in proteins in response to
oxidative stress in yeast [91]. More recent developments
of analytical techniques that use novel methods of label-
ing and separating large numbers of proteins [1] are
beginning to define the pathways of flow of molecular
information from the genome to the proteome [130].
Various stable isotope labeling techniques have recently
emerged to improve the efficiency and accuracy of pro-
tein quantitation by mass spectrometry. We have devel-
oped a mass-tagging strategy to incorporate stable isotope
tagged amino acids into cellular proteins in a residue-
specific manner during cell growth [356].
The major emphasis for new drug development in the
modern era is based on thorough understanding of the
relationship between the drug and its target protein,
whether it is an enzyme, receptor, structural, or transport
protein. Detailed information regarding the physical,
chemical, biochemical, genetic, and physiological charac-
teristics of that relationship must be well understood.
Hence, a major efforts is being devoted to enhance the
pace and resolution of classical crystallographic and NMR
67
methods for structural determination, and to improve
methods for predicting protein structure. While efforts to
improve the crystallographic and NMR techniques have
continued to receive a major share of intellectual effort,
structural prediction studies have received new impetus
from the development of new models and methods [273].
The molecular modeling serves several functions: (1) it
provides the tool to visualize protein structure in three
dimensions; (2) it permits protein-to-protein comparisons;
(3) predicts the structure of proteins; and (4) facilitates the
computer-aided drug design. Such integrated information
that can define the transcriptome and the proteome will
help identify novel molecular targets for therapeutic inter-
ventions of malignant growth.
Antibodies and Conjugates
Antibodies are highly selective proteins that can bind to
a single target among millions of irrelevant sites. Because
of this specificity, the antibodies have been used exten-
sively to target drugs, prodrugs, toxins and other agents
to particular sites in the body. It is this use of antibodies
as targeting devices that led to the concept of “magic
bullets,” a treatment that could effectively seek and
selectively destroy tumor cells wherever they resided.
The major problem in the therapeutic use of antibodies
was their production in large quantities, but the develop-
ment of ‘monoclonal antibody’ technology changed the
situation dramatically [113, 186, 333]. Monoclonal anti-
bodies (MAb) are already widely used for the diagnosis
and treatment of cancer and for imaging of tumors for
radiotherapy.
Despite the rapid progress being made in application
of MAbs as therapeutic agents, their use has been limited
because of their immunogenicity problem. MAbs are
usually mouse proteins; when injected into patients they
are eventually recognized as foreign proteins and cleared
from the circulation. To overcome this problem, research-
ers set out to engineer fully “humanized MAbs” that will
be indistinguishable from natural proteins [32, 334].
Humanizing MAbs is a technology that uses recombi-
nant DNA techniques to improve or change the function
of these antibodies [233]. The first fully humanized MAb
recognizes an antigen on the surface of human lympho-
cytes and is being evaluated as an immunosuppresant
and for treatment of lymphoid tumors. Another poten-
tially useful humanized MAb recognizes a growth factor
receptor in large numbers on the surface of several breast
tumor cells. This MAb successfully inhibited tumor cell
growth in culture and is currently being evaluated in
patients [322]. In the following section, we will briefly
discuss the potential use of Mab-based immunotherapies
68
that have been used for the treatment of malignant dis-
eases. Detailed aspects of this approach will be discussed
somewhere else in this book.
Monoclonal Antibodies as Agonists
Antibodies directed against cell-surface molecules on
many types of tumor cells can act as ligands, resulting in
powerful antitumor effects mediated by signal transduc-
tion [320]. For example, MAb 4D5 against erbB-2,
when added to breast or ovarian carcinoma cells that
overexpress erbB-2, induces a strong antiproliferative
effect [164]. The erbB-2 protein product is a member of
the EGF receptor family and is shown to act as a signal-
ing receptor for a recently identified ligand, heregulin,
in regulation of growth and differentiation of breast cancer
and other cell types [47]. Herceptin is a humanized
monoclonal antibody directed against Her-2/neu protein
that is abnormally abundant in 25–30% breast cancer
patients [141]. In view of the encouraging results
observed in Her-2/neu positive breast cancer, Herceptin
(trastuzumab) was recently approved by the FDA for
treatment of metastatic breast cancer [165]. Data from
pivotal trials suggested that trastuzumab is active when
added to chemotherapy in patients with advanced meta-
static breast cancer [271]. In particular, the combination
significantly prolonged the median time to disease pro-
gression, increased overall response rate, increased the
duration of response, and improved median survival
time by approximately 25% compared with chemother-
apy alone [271, 22]. As a single agent, trastuzumab
induces durable objective response in women with
HER-2 positive metastatic breast cancer and is rapidly
becoming a standard of care for these patients [23].
Similarly, the ligation of CD95 (APO-1/Fas) protein
with an anti-Fas MAb resulted in apoptosis (pro-
grammed cell death) of malignant cells. Using MAbs,
the human Fas and APO-1 proteins were identified as
cell-surface proteins of 200- and 48-kDa molecular
mass, respectively, in two different laboratories. Both
induced apoptosis in a variety of cell types upon binding.
Subsequent isolation of cDNAs encoding the two proteins
revealed that they were identical despite a difference in
apparent molecular weight [133, 211]. Apoptosis triggered
by the anti-CD95/Fas/APO-1 Mab has been successfully
used for the treatment of mice bearing human hematopoi-
etic tumors [302, 151]. The clinical use of anti-Fas/
APO-1 therapy for cancer treatment will be based on
further studies.
Besides negative signaling, Mabs have other poten-
tial uses in tumor therapy. Some MAbs can block inter-
actions between tumor cells and neighboring cells,
Recombinant proteins and genomics in cancer therapy
stroma, or matrix that are necessary for tumor growth or
metastases. For example, injection of anti-CD44 MAb
or its F(ab’)2 fragment 1 week after inoculation of
human melanoma cells in mice with severe combined
immunodeficiency (SCID) prevented metastases but not
the development of primary tumor [78]. The antibody
had no effect on growth of tumor cells in vitro. MAbs
against growth factors or their receptors can also exert
significant antitumor effects. For example, antibodies
against IL-6 and IL-6 receptor were effective in the
treatment of human myeloma in SCID mice [287] and
produced transient responses in patients bearing IL-6-
dependent tumors [146]. MAbs against the IL-2 receptor
have been used to treat adult T-cell leukemia with some
partial or complete remissions [322]. Thus MAbs
selected against tumor surface antigens to exert either
potent growth-inhibitory effects or host-tumor interactions
should lead to new strategies for selecting the antitumor
activity of Mabs.
Monoclonal Antibodies-Conjugated Drugs
The clinical progress with conjugates of Mabs and cyto-
toxic drugs has been rather slow. An important factor
that has limited the use of this approach for treatment of
cancer is the relatively low potency of standard chemo-
therapeutic agents. The potency of these compounds is
further reduced by their conjugation with MAbs [148].
However, a recent report that such a conjugate, BR96-
doxorubicin (BR96-Dox), is highly effective in curing
xenografted human carcinoma-bearing mice [301], has
rejuvenated great interest in Mab-drug conjugates. BR96
is a chimeric monoclonal antibody that contains a frame-
work region of human immunoglobulin and the binding
region of a murine antibody. The antibody binds to an
antigen that is expressed on the surface of many human
carcinomas. Treatment of tumor-bearing athymic mice
with BR96-Dox induced complete regressions and cures
of xenografted human lung, breast, and colon carcino-
mas and cured 70% of mice with extensive metastases
from a human lung carcinoma [301]. Clinical trials with
BR96-Dox were recently initiated to determine its safety
in patients. Similar results with Mab-vinblastine conju-
gates were reported years ago, but evaluation of this
Mab-drug conjugate was discontinued because of unac-
ceptable gastrointestinal toxic effects [14, 230].
Recently, several groups have concentrated their
research on more potent immunotoxins conjugated with
agents such as calicheamicins, maytansines and trichoth-
ecenes. Calicheamicin is a family of antibiotics that pro-
duce double-stranded DNA breaks; when conjugated
with Mab CT-M-01, which recognizes PEM antigen and
Kapil Mehta et al.
is located on the surface of human cancerous epithelial
cells, and injected into breast carcinoma-xenografted
mice, it significantly inhibited tumor growth and pro-
duced long-term tumor free survivors [124]. In a more
recent study, 40 patients with relapsed or refractory
CD33+ AML were treated with humanized anti-CD33
antibody linked to the calicheamicin [266]. In eight of
the 40 patients, leukemia was eliminated from the blood
and marrow whereas, in 3 additional patients the blood
counts returned to normal. Similarly, patmaytansinoids,
which are 100 to 1,000-fold more potent than doxorubi-
cin and vinblastine, when conjugated to Mab A7, which
recognizes an antigen expressed on human colon cancer
cell lines, showed high antigen-specific in vitro cytotox-
icity against cancer cells and low systemic toxicity in
mice [34]. Similar specificity and potency have been
observed in Mab-trichothecenes conjugates (protein syn-
thesis inhibitors) has been observed in terms of their
tumor cell-killing ability [189].
More recently, Mabs have been used to deliver enzyme
inhibitors to tumor cells. Thus, conjugation of Geninstein
(an inhibitor of Src protooncogene family protein tyrosine
kinases) to MAb B43, which recognizes the B cell-
specific receptor CD19, selectively bound to B-cell pre-
cursor leukemia cells, inhibited CD19-associated tyrosine
kinases and triggered rapid apoptotic cell death [307].
Treatment of B-cell precursor leukemia-xenografted
SCID mice with less than one tenth the maximum toler-
ated dose of B43-Geninstein resulted in more than
99.999% killing of human leukemia cells, which led to
100% long-term even-free survival from an otherwise
Immunotoxin (IT)
Receptor Ligand (Ricin, Pseudomonas
exotoxin, gelonin etc.)
Cell surface MAb or
antigen or cytokine A chain
Cytokine hormone
Receptor
Killing
Domain
TUMOR CELL
Figure 5. Schematic representation of immunotoxin
69
invariably fatal leukemia of these mice [307]. It remains
to be seen whether antibody-drug conjugates will be
effective anti-cancer agents in clinical settings as well.
Immunotoxins
Immunotoxins are chimeric molecules in which antibod-
ies or the ligand that interacts with the cell-surface mol-
ecules are coupled to toxins or their subunits (Fig. 5).
The antibody or growth factor binds with high selectivity
to the target cells. The toxins are derived from plants or
bacteria. DNA sequences encoding the bacterial toxins;
Pseudomonas exotoxin (PE) and diphtheria toxin and
the plant toxins; ricin and gelonin have been cloned and
expressed in E. coli, [2, 221]. The topic of immunotoxins
is discussed in detail elsewhere in this book, therefore,
we will concentrate only on the therapeutic potential of
recombinant immunotoxins in this section.
Initially, the toxins produced in bacteria were chemi-
cally linked to antibodies to make immunotoxins. In recent
years, significant progress has been made in engineering
recombinant immunotoxins by fusing the cell-binding
ligand genes to modified toxin genes [221]. For example,
a truncated form of Pseudominas exotoxin (PE40) has
been produced by deleting the first 252 amino acids; this
toxin has extremely low toxicity because of its inability to
bind to cellular receptors [136]. However, when chemically
conjugated to an antibody [76, 156] or the recombinant
chimeric toxin generated by fusing the PE40 gene to DNA
fragments encoding growth factors, antibody-binding
sites, or other target-recognition elements, Pseudomonas
Toxin
B chain
Translocation Native
enhancing domain binding
domain
NORMAL CELL
70 Recombinant proteins and genomics in cancer therapy

exotoxin becomes highly specific and potent in killing that express IL-6 receptors at high numbers and also
target cells [220, 221]. The gene encoding this chime- several other carcinomas [150, 263, 264]. The first clinical
ric toxin is expressed in E. coli. Table 8 lists some of trial of a genetically engineered immunotoxin (B3LysPE38)
the recombinant immunotoxins that have been pro- was initiated in April 1993 in breast and colon carci-
duced by fusing the PE40 gene to cDNAs encoding noma patients. Some responses were evident, but toxic
different targeting molecules. Anti-Tac(Fv)-PE40 is effects appeared to be greater in human patients than
one such recombinant immunotoxin that was gener- seen in mice and primates [270].
ated by fusing the truncated form of the Pseudomonas In contrast to Pseudomonas exotoxin-immunotoxins,
exotoxin (PE40) gene with the variable region of an the immunotoxins composed of ricin or its A subunit and
antibody against the IL-2 receptor [36, 155]. This MAbs have generally been constructed using chemical
immunotoxin is highly toxic to cells from patients with cross-linking reagents [89, 173]. Recombinant ricin-
adult T-cell leukemia and induced regression of IL-2 based chimera molecules have been difficult to produce,
receptor-bearing carcinoma tumors in athymic mice because the A chain of the plant toxins must be attached
[66, 153, 154]. TGFα-PE40 recombinant chimera toxin to the cell recognition domain by a disulfide bond, and
targets PE40 to cells with EGF [66, 258]. Although disulfide-linked subunits are difficult to produce in bac-
many normal cells contain EGF receptors, tumor cells teria [238]. Many reviews have already described the
often have extremely large number of receptors because activities and properties of immunotoxins made with
of amplification and overexpression of the EGF recep- ricin and other plant toxins [89, 142, 249, 308, 315, 320].
tor gene [238]. When administered systemically, Ricin-containing immunotoxins have been used to elimi-
TG&Fbdot;α-PE40 caused regression of subcutaneous nate selected populations of lymphocytes. Vitetta, Uhr
epidermoid carcinoma and prostate carcinoma tumors and associates have produced ricin conjugates of anti-
in mice [221]. bodies to B cell-specific antigens and shown such conju-
Interleukin-2-PE40 is a recombinant chimeric protein gates to cause complete regression of B-cell lymphomas
designed to deliver the toxin to cells with IL-2 receptors in mice [80]. Significant antitumor activity of ricin A
[172]. Normal resting lymphocytes do not express IL-2 chain immunoconjugates has been observed against solid
receptors, but when they are activated with an antigen or or ascites tumors in animal models [75, 105].
IL-2, the receptors are induced. IL-2-PE40 has been Because of encouraging results in preclinical studies,
shown to be highly toxic to activated mouse and rat T several ricin-containing immunoconjugates have been
cells and had some therapeutic effect against mouse developed and approved for human trials, and two kinds
lymphoma [150]. Similarly, IL-6-PE40 chimeric toxin of human trials have been conducted. The first involves
killed many human myeloma and hepatoma cell lines the ex vivo treatment of harvested bone marrow to elimi-
nate contaminating tumor cells prior to re-infusion in
patients undergoing autologous bone marrow transplan-
tation. The second kind of trial involves the parenteral
Table 8. Recombinant toxins derived from Pseudomonas administration of immunotoxins [231, 308, 315, 320].
exotoxin Some patients with B-cell lymphoma responded to immu-
Immunotoxin Target Reference notoxin therapy [89]. Currently, an anti-CD22 dgA immu-
notoxin is being evaluated in phase II clinical studies in
PE40
lymphoma patients.
Anti-Tac (Fv)-PE40 Human IL-2 receptor [36,
(leukemia) 153–155] The side effects observed with administration of immu-
TGF-α-PE40 EGF receptor, [66, 113, notoxins are different from those of conventional chemo-
epidermoid carcinomas 258, 252] therapy; immunotoxins do not exert cytotoxic effects
adenocarcinomas, against normal rapidly dividing cells. Immunotoxins such
glioblastomas smooth as the bacterial toxins, Pseudomonas exotoxin and diph-
muscle cells theria toxin, induce hepatotoxicity, whereas the ricin-
IL-2-PE-40 IL-2 receptor (leukemia) [150, 172]
based immunotoxins cause reversible vascular leak and
IL-6-PE40 IL-6 receptor myelomas, [264, 265]
hepatomas, prostate myalgias [320]. Several groups of researchers are cur-
B3(dsFv)-PE38KDEL Many carcinomas [25, 26] rently working on second- and third-generation immuno-
e23(Fv)PE40 erbB2 toxins to eliminate the immunogenicity and side effects
lung, breast, ovary and [20] of the first generation immunotoxins. Continued refine-
stomach ment in design of these pharmaceuticals may eventually
adenocarcinomas prove useful in the treatment of cancer.
Kapil Mehta et al.
Monoclonal Antibodies and
Radioimmunotherapy
The use of MAbs to deliver radioisotopes for treatment of
certain cancers has yielded some encouraging results [106,
137, 138]. Some of the most promising of these results
radioimmunoconjugates in bone marrow transplantation.
In the treatment of leukemia, radioiodine-MAb conjugates
ablate marrow safely, delivering up to fourfold more radi-
ation to the marrow than to other normal tissues. Responses
have been less frequent in solid tumors treated with radio-
immunoconjugates in clinical trials [183, 184]. Objective
tumor regressions, however, were seen in four patients
with non-bulky disease who were treated with rhenium-
186-labeled Mab NR-LU-10 [134]. Because of their size
and high molecular weight, diffusion of MAbs within
bulky tumors can be a problem, as illustrated by the results
of a clinical trial that used radiolabeled anti-CD20 and
anti-CD21 MAbs to treat patients with B-cell lymphomas
[236, 253]. Among the most promising approaches to
overcoming this problem is the use of genetically engi-
neered, low molecular weight F(ab’)2, Fab and single-
chain Fv fragments that may diffuse better at tumor sites.
Early results of clinical trials employing humanized MAbs
appear encouraging. In addition to reduced immunogenic-
ity, F(ab’)2, Fab and Fvs, with their improved ability to
penetrate tumors, may prove useful carriers for radiother-
apy. Newer regimens combining radioantibody-based
therapy with other treatment modalities may prove even
more effective.
Chimeric Proteins
One interesting aspect of recombinant DNA technology
is the potential for producing of new proteins with novel
properties. For example, the hybrid proteins formed by
fusion of two or more genes (chimera) offer several
advantages in terms of their stability, affinity, efficacy and
pharmacology over the individual component proteins.
A chimera protein formed by fusion of the IFN-γ and LT
genes was shown to have better antiproliferative activity
than IFN-γ or LT alone [71]. Similarly, PIXY321, a
genetically engineered hybrid of the GM-CSF and IL-3
proteins exhibits greater colony-stimulating effects
in vitro than the combination of GM-CSF and IL-3 [50].
In preclinical studies, PIXY321 has been shown to accel-
erate both neutrophil and platelet recovery in rhesus mon-
keys subjected to sublethal irradiation [330]. Because of
the preclinical observations, PIXY321 was tested in
patients for its ability to ameliorate disease- or treatment-
related bone marrow suppression. The clinical results
were encouraging and suggested that the hybrid protein
71
elicits the biological effects of both its component cytok-
ines [29, 311, 312]. Thus, PIXY321 became the first
recombinant fusion of two hematopoietins to enter the
clinic. Early clinical experience has shown great potential
in the prevention and treatment of hematopoietic suppres-
sion. The other recombinant fusion proteins have been
made, including chimeric toxins constructed by fusion of
genes encoding human cytokines and Pseudomonas exo-
toxin [35, 55, 284]. For example, IL-4-PE, a chimera of
human IL-4 and Pseudomonas exotoxin proteins, is
highly potent against many cancer cells [55], suggesting
that it might be useful in the therapy of many cancers.
Generation of homologues and analogues of natural
biotherapeutic proteins is another potentially important
application of recombinant DNA technology. The tech-
nology involves alteration or deletion of key nucleotide
sequences in the gene that will result in the modification
of only a few amino acid sequences in the resultant pro-
tein, compared with the natural protein. Synthesis of
novel human TNFα, IFNα and IF&Nbdot;γ homologues
has already been reported [11, 214]. All three of these
homologues are distinct from their parent protein.
Prolonged activity of insulin has been achieved by various
substitutions that increase the isoelectric point. The pro-
longed activity seems to occur because the novel homo-
logues precipitate when they encounter the neutral pH of
the body [180]. These examples indicate an interesting
way by which protein modifications can be exploited for
therapeutic potential.
Furthermore, the ability afforded by newer techniques
in recombinant DNA technology such as “phage display”
to correlate protein structure and function in a systematic
way makes it possible to design novel drugs [38, 51,
176]. Such a technique could be used to design or even
select a small peptide that binds to the receptor with
the same affinity as the larger protein. And then, using
computer modeling to display the molecular contacts
between ligand and receptor, small nonprotein mole-
cules that make the same contacts could be designed
and synthesized. The end product would be a small
organic molecule that could be produced more cheaply
than the recombinant protein, yet would retain the full
biological activity of the protein hormone. What’s more,
such molecules could be administered orally, eliminat-
ing the major disadvantage of most recombinant protein
therapeutics, which must be delivered directly into the
blood stream by injection.
Cancer Vaccines
The issue of genetically engineered vaccines for cancer
treatment will be discussed in detail in another part of
72 Recombinant proteins and genomics in cancer therapy

this book. We will, therefore, focus only on the novel specific cell-surface antigens, and monoclonal antibody
aspects of recombinant vaccines that may have some technology permitted identification of tumor-associated
potential as cancer therapeutics. The function of a vaccine antigens, their characterization and tissue distribution.
is to give the immune system a boost, thus helping it The polymerase chain reaction (PCR) brought the tech-
recognize and destroy the ‘non-self’ antigens on the sur- nology of cloning and expressing gene products. These
face of cancer cells. Though the idea of inciting the technologies jointly led to the development of the sub-
immune system to fight cancer has been around for a unit vaccines of the 1990s.
long time, recent developments in biotechnology and The human melanoma antigen MAGE-1 was the first
better understanding of the immune-system network tumor-specific antigen identified [315]. Poxvirus con-
have caused an explosion of research and development taining the MAGE-1 gene is currently being evaluated
in the field of cancer vaccines. Table 9 lists some cancer as a candidate vaccine for treatment of melanoma and
vaccines that are currently undergoing clinical trial. breast cancers in humans [163]. Similarly, the recombi-
Prior to the advent of recombinant DNA technology, nant vaccinia virus containing carcinoembryonic anti-
two types of vaccines were used, inactivated vaccines gen (CEA) is being evaluated for treatment of certain
(chemically killed derivatives of actual infectious agents) cancers. CEA is expressed on the surface of virtually all
and attenuated vaccines (actual infectious organisms colorectal cancers, 70% of lung cancers, and 50% of
altered so that they do not multiply). However, these breast cancers. Clinical results if these phase I trials of
types of vaccines are potentially dangerous, as they can this vaccine in late-stage cancer patients were promising
carry over the infectious contaminations. For example, a [152]. A recombinant fusion of a tumor-derived idio-
small number of children each year contract polio from type and GM-CSF, yielded a strongly immunogenic
their live polio vaccinations. Thus, one of the most prom- protein that was capable of inducing idiotype-specific
ising applications of recombinant DNA technology is the antibodies and protected the recipient animals from
production of subunit vaccines, consisting solely of the challenge with an otherwise lethal dose of B-cell lym-
surface protein to which the immune system responds phoma [295]. These results can be applied not only to
and thus eliminating the risk of infection [27]. The arrival B-cell lymphoma but perhaps can be generalized to
of biotechnology in the late 1970s enabled targeting of other classes of tumor antigens as well.
The discrete peptide fragments from certain tumor-
Table 9. Cancer vaccines under clinical trials specific oncoproteins (such as mutant p53 and the pro-
tein products of the ras and HER-2/neu genes) are rapidly
Immunogen Target Cancer Reference
progressing as potential vaccine candidates for cancer
Recombinant vaccinia Colorectal, lung, breast [152] treatment [72, 342]. This strategy is based on the princi-
encoding for CEA ple that intracellular proteins are degraded and presented
Gene therapy using Renal cell, carcinoma, [152, 287]
back on the cell surface as small peptides in the groove
patient’s own cells melanoma prostate,
colorectal of class I major histocompatibility complex (MHC) anti-
Recombinant poxvirus Melanoma [315] gens. A nine amino acid peptide from the HER-2/neu
encoding for MAGE oncoprotein was shown to be recognized by both breast
antigen and ovarian cytotoxic T lymphocytes, and this small
Heat shock protein Melanoma [163] peptide was able to induce a tumor-specific immune
Naked DNA Lymphoma, Cervical [163, 232, response [224]. It is now possible to isolate and custom
prostate, 346]
Prostate, Melanoma,
synthesize tumor-specific immunogenic oncopeptides
Renal, Colorectal by using the patients’ own mutation to prepare an autolo-
Synthetic peptides Melanoma, Cervical [72, 224, gous peptide vaccine that is selectively targeted to tumor
342] cells containing the mutant gene product [342]. The abil-
Synthetic antigens Ovarian, Breast, [44, 163] ity of human oncopeptide vaccines to generate a peptide-
Melanoma, specific CD8+ cytotoxic T-lymphocyte response in
Colorectal
animal models has formed the rationale for clinical trials
Anti-idiotypic Melanonoma, Colorectal, [278, 295]
antibodies gastric, ovarian of autologous peptide immunizations in patients with
Inactivated tumor cells Colon [163] diverse epithelial malignancies (breast, lung, gastroin-
with the cytokine IL-2 testinal) that are commonly accompanied by p53 and ras
Recombinant antigens Colorectal, lung [63, 119, mutations [142]. It is likely that similar considerations
prostate 278] will apply to other recently identified oncoproteins such
Gene transfer Melanoma [163] as the product of the BRCA-1 gene in breast cancer.
Kapil Mehta et al.
Recently, there have been several attempts to gener-
ate tumor cell vaccines engineered to secrete various
cytokines [42, 43, 127, 235]. The strategy seeks to alter
the local immunologic environment of the tumor cell so
as to enhance either the antigen presentation of tumor-
specific antigens to the immune system or the activation
of tumor-specific lymphocytes. Many cytokine genes
have been introduced into tumor cells with varying
effects on both tumorigenicity and immunogenicity.
Some of these cytokines, when produced by tumor,
induce a local inflammatory response that results in
elimination of the injected tumor. The local inflamma-
tory response is, in general, dependent on leukocytes
other than classical T cells. Many cytokine genes have
been introduced into tumor cells, including IL-1α, IL-2,
IL-4, IL-5, IL-6, IL-7, IL-12, GM-CSF, IFNα, IFNγ and
TNFα [42, 215]. Preclinical data from various animal
models suggest that tumor cells engineered to produce
cytokines indeed provide a novel approach for tumor
therapy [42, 107, 216]. Clinical trials are currently in
progress to assess the therapeutic efficacy of cytokine-
transduced tumors as vaccines for the treatment of
established solid tumors.
A remarkably straightforward and potentially useful
approach in the field of cancer vaccines was recently
developed. It involves the direct in vivo delivery of
MHC-associated tumor antigens to provoke a tumor-
directed immune response. This approach for treatment
of diverse malignant diseases is currently under clinical
investigation (Table 9). In essence, this approach is
based on the ability of some viral and human ‘naked
DNA’ genes to transfect certain cells without the need
for elaborate genetic engineering maneuvers, sometimes
using modified DNA/liposome complexes to deliver
genes by direct injection at the tumor site or by systemic
administration [232, 346]. For example, injection of
naked DNA or mRNA transcripts encoding human CEA
in mice has been shown to elicit strong cytotoxic
T-lymphocytes and antibody responses against this anti-
gen [44, 45]. The development of this simple and direct
approach for in vivo gene therapy-based immunotherapy
is an extremely important avenue for continued clinical
and preclinical investigations.
Most tumor vaccines must be employed subsequent
to the development of cancer. However, in some instances,
for example, in those geographic areas in which human
papilloma virus infection is highly endemic and thus
rates of cervical carcinoma are high, it may be useful to
vaccinate children prophylactically. In China, where
endemic hepatitis virus is causally related to hepatic
cancer, broad immunization to prevent hepatitis and
subsequent cirrhosis and hepatic cancer is underway.
73
Ultimately, as we discussed earlier in this chapter, devel-
opment of multiple strategies that could be applied in
synergy are most likely to yield beneficial results in cancer
treatment and control. It remains to be seen what place
the immunotherapy will have in this armamentarium.
However, it is obvious that availability of recombinant
DNA technology has made it possible to design vaccines
on a molecular basis.
Problems Unique to
Recombinant Biotherapeutics
The development of protein therapeutics by using recom-
binant DNA technology has presented many new and
interesting challenges to pharmacologists and drug deliv-
ery scientists. Many of these biotherapeutics have multiple
biological effects [6]; thus, an important priority in their
development is the evaluation of their potency, pharmaco-
logical profile and toxic effects. One strategy to under-
stand the potency and toxicity of anticancer agents is the
use of appropriate animal models. However, many animal
models that have been developed for testing conventional
low molecular-weight drugs may not be useful for testing
rDNA-derived therapeutic proteins. Species specificity of
protein therapeutics further narrows down the choice for
appropriate animal models. Certain in vitro biological prop-
erties of the interferons for example, did not translate to their
efficacy in intact animals or in patients [280].
The lack of information on preclinical pharmacological
behavior also limits a general analysis of the toxic effects
of therapeutic proteins. For example, agents such as IFÑγ,
IFN-α and IL-2 are produced in E. coli and are nongly-
cosylated. EPO, in contrast, is produced in Chinese ham-
ster ovary cells and is glycosylated. Rats, dogs, hamsters
and monkeys were used to study the toxicity of these
agents. Comparison of results on gross morphology,
histology, blood chemistry and hematology obtained
from these studies demonstrated no general responses
that might be attributed to the use of biotherapeutic
proteins. The effects observed were primarily related to
the known or anticipated biological effects of the agent
and, as expected, occurred only in the species in which
the agents were known to be biologically active.
Considering these results, it is apparent that toxicologic
findings in animals essentially reflect the pharmacologi-
cal effects of biotherapeutic agents. Therefore, the use
of biotherapeutic agents in species in which they are
active should be most informative. However, observa-
tions of the lack of toxic effects in other species may be
important in addressing some concerns about nonspecific
74
toxic effects. Cross-species activity may be seen for
some if not all biological effects. For example, human
IFN-α has pyrogenic activity in rabbits. Clearly, the
pharmacological effects observed with materials that
lack pronounced species specificity are likely to be more
dependable, especially if different species have mani-
fested the same toxicity profile. Similarly, human TNF
was found to be less toxic in mice than in human sub-
jects because one of the receptor subunits with which
TNF interacts is species-specific and the other is not. In
contrast, the pharmacological effects of agents like EPO
are highly cell type and species specific.
There have been relatively few preclinical studies of
the immunogenicity of recombinant therapeutic pro-
teins. The useful information has come from the clinical
studies. The generation of antibodies to proteins admin-
istered over long periods may result in formation of
soluble immune complexes. These immune complexes
can induce vascular and tissue injury, particularly glom-
erulonephritis [281]. As an alternative, immune com-
plexes can elicit the release of inflammatory mediators
from cells. However, to date there have been no exam-
ples of antibody responses to any recombinant therapeu-
tic protein that have been shown to clearly cause clinical
pathologic effects.
Another potential problem is incorporation of the
wrong amino acids when a high level of expression of
recombinant proteins is enforced. Such errors are gener-
ally difficult to pinpoint, since current analytical meth-
ods for amino acid composition and sequence are not
really amenable to detecting variations below 10% of
the major constituents. It is conceivable that such altered
sequences may resemble a toxic peptide or a protein
with different biological functions. Also, the altered
sequences in the protein may render them immunogenic
and may provoke enhanced immunogenic responses.
Delivery of Therapeutic Proteins
The potential use of therapeutic proteins in medicine is
severely limited because of their poor activity when
administered orally. Proteins are rapidly degraded by
proteolytic enzymes in the gastrointestinal tract and have
been primarily administered by injection. Moreover,
proteins are generally characterized by short biological
half-lives in the circulatory system, so that the repeated
injections are generally needed. Even after intramuscular
or subcutaneous administration, their bioavailability is
often low because of their small size and the widespread
distribution of proteolytic enzymes. In addition, most
proteins pass through biological barriers rather poorly
because of low diffusion and a low partition coefficient.
Recombinant proteins and genomics in cancer therapy
These considerations have led to the development of dif-
ferent strategies to prolong the bioavailability of thera-
peutic proteins.
Conjugation of certain proteins to synthetic polymers
can circumvent the problems of rapid clearance from the
circulation, immunogenicity and instability. The general
requirements of any polymer used for this purpose are
that it be water-soluble, biocompatible, nonimmuno-
genic and devoid of biological activity. Zoladex® and
Nafarelin®, the decapeptide agonists of luteinizing hor-
mone releasing hormone (LHRH), have been formulated
in slow-releasing polymer base and used effectively in
clinical studies [81, 117]. Attempts have also been made
to stabilize the proteins against degradation at the site of
injection as well as in the circulation, but these studies
are still preliminary in nature [160].
Covalent conjugation of certain proteins with water-
soluble polyethylene glycol (PEG) enhances their solu-
bility and permits the design of stable formulations
suitable for clinical use. This is particularly important for
recombinant proteins produced in E. coli that are usually
recovered as insoluble refractile bodies, and unlike many
of their native counterparts, are not glycosylated. For
example, conjugation of both IL-2 and IFN-β with PEG
increased their solubility, and aqueous solutions were
stable for long periods of time [144]. Moreover, a recently
published study revealed that PEGlated IFN-α is better
tolerated and may be more effective in treating CML
patients [291]. Similarly, PEGylation of TNF-α alters its
pharmacokinetics and reduces its in vivo toxicity [144].
G-CSF conjugated to PEG has a four times slower clear-
ance rate in rats than unmodified G-CSF. In addition,
PEGylated G-CSF administration exerted a sustained
biological effect on peripheral blood neutrophils [294].
PEGylated adenosine deaminase (ADA), an enzyme
unrelated to cancer, is now approved for use as replace-
ment therapy for severe combined immunodeficiency
diseases that are due to inherited ADA deficiency.
Patients who received PEG-ADA did not develop neu-
tralizing antibodies to ADA activity [33].
The key factor with any drug delivery system is to
achieve adequate concentrations of the drug at the desired
sites while avoiding significant concentrations at sites
that mediate toxic effects. Novel delivery systems such
as liposomes may prove to be useful in achieving this.
The ability of IFN-γ to stimulate the tumoricidal activity
of monocytes was increased 1,000-fold by its encapsula-
tion in liposomes [147]. Encapsulation of TNF-α in lipo-
somes ameliorated the systemic toxicity of this cytokine
in dogs [171]. Anti-HER2 immunoliposomes, consisting
of long circulating liposomes linked to anti-HER2 MAb
fragments proved highly efficient for intracellular delivery
Kapil Mehta et al.
of the drugs and showed superior anti-tumor activity
in animal models [218]. Delivery can also be modified
by a combination of the biotherapeutic protein with an
antibody [61]. For example, the in vivo clearance of
human IFN-α in rats is threefold slower when it is
combined with a specific MAb [246]. However, it
remains to be seen whether such delivery systems will
confer any advantages to biotherapeutic proteins in vivo
in terms of local delivery, reduced toxicity or altered
pharmacokinetics.
Conclusions
It is clear that recombinant DNA technology has pro-
duced a revolution in the field of cancer therapeutics.
The dream of biological therapy, thought to have a great
potential for cancer, can now be realized. The ability to
manufacture cancer drugs by using genetically engi-
neered organisms has given rise to a novel biotechnol-
ogy industry within the last decade that has earned as
much as $15 billion. The revolution has been not only in
the industrial sector; but also in the academic sector. It
has enabled scientists to discover new molecules, rede-
sign molecules for lower toxicity and more efficacy, and
investigate the pathology of disease at the molecular
level. This technology, however, has given rise to new
set problems in the area of drug manufacturing and
delivery. One of the major problems in the manufactur-
ing area is ensuring that the recombinant biological
product is identical or very similar to the natural coun-
terpart. Another problem is the rapid degradation of pro-
teins in the circulation and their short circulating
half-lives, which restrict oral delivery of biotherapeutic
proteins and may require continuous injection.
It is now possible to design from the crystal structures
of the ligand and the receptor small molecules that can
mimic these large proteins and thus may help to over-
come delivery problems. Some of these problems might
be circumvented by directly injecting the gene for a
given protein: although this approach seems attractive,
it also suffers from the delivery and organ/cell-specific-
ity problems. Treatment with antisense DNA and RNA
to inhibit the expression of oncogenes in tumor cells and
the new technologies based on viral vectors for the
delivery of vaccines and genes may find widespread
application in the near future. Many of these approaches
work effectively in the test tube, and the main challenge
now is to translate these laboratory techniques into com-
mercially viable processes to produce active, effective
biotherapies with acceptable toxicity.
75
Acknowledgements The research in authors’ laboratories
was supported in part by a grants from Food and Drug
Administration, Clayton Foundation, and Department of
Defense.
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5 Current concepts in immunology
ROBERT K. OLDHAM
The main function of the immune system is to protect
the host from certain death due to numerous potential
pathogens present in the environment. The development
and maintenance of immunity is dependent on a com-
plex and highly sophisticated defense organization
functionally divided into the innate and adaptive immune
systems.
Innate immunity provides the first line of defense
against most pathogens. Phagocytes, including neutro-
phils and monocytes/macrophages, and natural killer
cells are the most important cell types participating in
this form of immunity. In addition, several soluble fac-
tors, including the complement cascade, lysozyme, and
acute-phase reactants, contribute to and reinforce the
physical barriers that prevent most infectious organisms
from penetrating the body. These factors also promote
the activation of the inflammatory reaction that contains
the injury caused by an invasive infectious agent. These
cells and factors are suitable to accomplish this function
because their activity does not depend on a prior encoun-
ter with the antigens in the microbial agent or tumor
cells, and they lack the fine specificity of the cells and
humoral factors of the adaptive immune system.
Specificity and memory are two cardinal features of
the adaptive immune system. The main cellular compo-
nents are the T and B lymphocytes. Each clone of these
cell populations has an extraordinary and unique antigen
specificity via the expression of genetically programmed,
antigen-specific receptors on the cell surface. The adap-
tive immune system has the ability to differentiate
between foreign and self-molecules and in this way,
most destructive reactions against the host’s own tissues
are prevented. When the immune system loses its capac-
ity to differentiate self from nonself, autoimmunity
develops.
The innate and adaptive systems communicate with
each other directly by cell–cell interactions and through
soluble mediators termed cytokines. For example, mac-
rophages are important not only in innate immunity but
also in specific immune responses, since they can pres-
ent antigen and activate T and B cells. In turn, T cells
regulate macrophages, B cells, and natural killer cell
activity through the synthesis and release of cytokines.
Some of the cytokines also behave as messengers that
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
mediate the communication between cells of the immune
system and other organs of the body, such as the central
nervous and endocrine systems.
This chapter presents a summary of the structure and
function of the immune system with emphasis in those
areas relevant to the defense against tumors and to can-
cer biotherapy. The following sections will briefly
describe the characteristics and functions of the major
cells and mechanisms that control immune responses
and the clinical methodologies for assessing the general
state of immunocompetence.
Current Concepts of Immunity
All the major cells of the immune system, that is, mono-
cytes/macrophages, B and T lymphocytes, natural killer
cells, polymorphonuclear cells, etc., originate in the
bone marrow from pluripotent stem cells [58]. Although
the exact mechanisms and mediators involved are not
completely understood, it is now accepted that stem
cells, under the influence of growth and differentiation
factors such as erythropoietin, colony-stimulating fac-
tors, and interleukins, differentiate into two main pro-
genitors: stem cells that have the potential to originate
erythrocytes, eosinophils, platelets, granulocytes and
monocytes/macrophages (GEMM-CFC); and stem cells
that are the precursors for mature lymphoid cells
(L-CFU) [37, 78, 109] (Fig. 1).
We now know many of the factors that induce prolif-
eration of the committed stem cells. Most of these
cytokines have been purified, sequenced, and are currently
produced by recombinant techniques [37, 78, 109, 134].
The availability of these recombinant cytokines has
contributed significantly to defining and characterizing
their biological activities (Fig. 1).
Four separate colony-stimulating factors (CSFs) have
been recognized. They are interleukin 3 (IL-3; multi-
CSF), granulocyte-macrophage colony-stimulating fac-
tor (GM-CSF), monocyte-colon-stimulating factor
(M-CSF) and granulocyte-stimulating factor (G-CSF)
[37, 109]. These factors, together with interleukin 1 (IL-1),
interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 6
(IL-6), are essential for the proliferation and differentiation
85
86 Current concepts in immunology

Bone Marrow
GEMM–CFC Pluripotent Stem Cell L-CFU

GM–CSF
IL–3
CFU–GM
GM–CSF Bursa of Fabricius
IL–3 Thymus
or equivalent
IL–5
IL–1 IL–2
IL–2 IFN–γ
IL–3 IL–3 IL–4
IL–4 Eosinophil GM–CSF
IL–3 IL–6
M–CSF B
GM–CSF
G–CSF
GM–CSF IL–1
IL–3 Basophil IL–2
IL–4 LGL IL–4
EP Mφ IL–6
BFU–E

RBC
GM–CSF Pc
IL–3
IL–4 Th/i
PMN

Platelets Ts Tc Ab
Megakaryocyte

Figure 1. Origin of the cells of the immune system. All the cells of the immune system are derived from the multipotential stem
cell in the bone marrow. Proliferation, maturation, and differentiation of the different cell lineages are driven by a multitude of
cytokines. GEMM-CFC, granulocyte/erythroid/megakaryocyte/monocyte colony-forming cell: L-CFU, lymphocyte colony-form-
ing unit; CFU-GM, granulocyte/monocyte colony-forming unit; B, B lymphocyte; Th/i, T helper inducer; Ts, T suppressor; Tc, T
cytotoxic; Mo, macrophage; LGL, large granular lymphocyte; Pc, plasma cell; Ab, antibody; PMN, polymorphonuclear cell; EP,
erythropoietin; BFU-E, burst-forming unit – erythroid; RBC, red blood cell; GM-CSF, granulocyte/monocyte colony-stimulating
factor; G-CSF, granulocyte colony-stimulating factor; M-CSF, monocyte colony-stimulating factor; IL-1, interleukin 1; IL-2,
interleukin 2; IL-3, interleukin 3; IL-4, interleukin 4; IL-6, interleukin 6; IFN-γ, interferon-γ

of committed stem cells, such as GEMM-CFC, GM-CFC,
and L-CFU. For example, IL-3 and GM-CSF are required
to induce GEMM-CFC to differentiate into granulocyte/
monocyte-colony forming cells (GM-CFC), while
M-CSF, GM-CSF, and IL-3 are necessary for GM-CFC
to differentiate into precursors of the monocytic lineage
(Fig. 1) [37, 78, 109]. Some of these cytokines act on
targeted cell lineages; for example, IL-5 regulates
eosinophilic maturation and B-cell activity while eryth-
ropoietin (EP) has its predominant effects on committed
erythroid cells (BFU-E) (Fig. 1). Macrophages, activated
T lymphocytes, fibroblasts, and endothelial cells are the
major sources of GM-CSF, G-CSF, M-CSF, IL-1, IL-3,
IL-5, and IL-6 [16, 37, 44, 51, 78, 90, 109, 134].
Some of these cytokines (i.e., C-CSF, GM-CSF) are
already approved for the treatment of patients with
chronic severe neutropenia associated with cancer che-
motherapy, AIDS, bone marrow transplantation, myelo-
dysplasia, and aplastic anemia [27, 37]. Platelet growth
activation factors have been approved and others, along
with receptor agonists, are in clinical testing.
Lymphocyte-precursors originating from the L-CFU
migrate to the thymus where they differentiate into
mature T lymphocytes. This cell population is com-
prised of several subpopulation, including T helper/
inducer (Th/I) lymphocytes, which modulate the activity
of virtually all other cell types of the immune system
(Fig. 1), T-cytotoxic (Tc) lymphocytes, which are the
Robert K. Oldham 87

effectors of the cytotoxic responses against tumors, for-
eign tissues, virus-infected cells, etc. and T-suppressor
or regulatory (Ts or Treg) lymphocytes, which are
involved in the down-regulation of the immune response.
In addition to T lymphocytes, the bone marrow
lymphocyte-precursor cells also give rise to natural
killer cells (NK), which are involved in the defense
against tumors, and B lymphocytes (B), which after
maturing in the bursa of Fabricius (in birds) or bursa
equivalent (bone marrow in humans), migrate to periph-
eral tissues, where in response to antigens, they differ-
entiate into antibody-producing plasma cells (Fig. 1).
Mature lymphoid cells circulate in blood, from where
they populate peripheral lymphoid tissues, such as the
paracortical areas of the lymph nodes and the periarte-
riolar sheaths of the spleen, the skin, and the mucosal
linings of the digestive, respiratory, and genitourinary
tracts. The migration of circulating B and T lympho-
cytes into specific lymphoid tissues appears to be gov-
erned by complementary adhesion molecules present on
lymphocytes and on endothelial cells of high endothe-
lial venules (HEV) and postcapillary venules (homing
receptors, specialized adhesion molecules) [115, 129].
The expression of some specialized adhesion molecules
by endothelial cells is induced by cytokines produced at
the inflammatory sites. For example, the intracellular
adhesion molecule 1 (ICAM-1), which serves as a
receptor for the leukocyte-function-associated antigen-1
(LFA-1) molecule present on leukocytes [5, 23, 110], is
induced by IL-1 in endothelial cells. The rapid increase
in these surface proteins facilitates the adhesion of leu-
kocytes to the endothelium, thus accelerating the initial
stages of the inflammatory reaction (Fig. 2).
In addition to traffic to and from secondary lymphoid
organs, a small number of lymphocytes travel through
most non-lymphoid tissues of the body. This traffic

Macrophage
IL-6
TGF-β

Plt
Monocyte
IL-1
PDGF PMN TNF-a

LOCAL IMMUNE SYSTEMIC

EFFECTS RESPONSE EFFECTS

Vascular Permeability Fever

IL – 1 IL – 6 TNF – a
Leukocyte Adherence Sleep
Anticoagulant T Lymphocyte Activation Acute Phase Reactants
IL-1/ Chemotaxis Cytokine Production Insulin
PGI Synthesis Antibody Sythesis Lipoprotein Lipase
Prolif. Endothelial Cells Steroid Synthesis
Shock
Neutrophilia
Proliferation GM – CSF
TGF – β
Proteolytic Enzymes Bone Marrow Stimulation
Collagen Synthesis Appetite
T Lymphocyte Proliferation
PGE Production Hypotension

Figure 2. Cytokine participation in inflammatory and immune responses. Early monouclear cell recruitment is induced by
platelet-derived transforming growth factor β (TGF-β). Activated macrophages produce additional TGF-β and other cytokines,
including interleukin 1 (IL-1), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6), that have local and systemic effects as
well as effects on the immune response. PDGF, platelet-derived growth factor; PMN, polymorphonuclear cell; Plt, platelets
88 Current concepts in immunology

pattern is designed to optimize the interaction of foreign
antigens with the appropriate receptor specificities in T
and B cells and to assure the fast development of a spe-
cific immune response. This process is also facilitated
by migration of antigen presenting cells (APC) from the
peripheral tissues to local lymph nodes [115]. Following
cognitive interaction (i.e., involving antigen and class II
histocompatibility complex molecules) between APC
and T cells and T-B cell cooperation, lymphocyte prolif-
eration and maturation take place, with the generation
and subsequent recirculation of antigen specific effector
and memory cells (Fig. 3). During these events, lym-
phoid cells secrete a number of cytokines, such as trans-
forming growth factor β (TGF-β), IL-1, IL-6, tumor
necrosis factor α (TNF-α), and platelet-derived growth
factor (PDGF), that affect the inflammatory and healing
processes by modulating the activity of endothelial cells
and fibroblasts, thus linking the inflammatory and the
immune response (Fig. 2) [124].
T lymphocytes
T cells, which comprise 70–80% of all circulating lym-
phocytes, are central to the development of normal
immune responses. They play a critical role in cellular
immune reactions, including delayed-type hypersensi-
tivity (DTH), resistance against certain bacteria, viruses
and tumor, and as effector cells mediating the rejection
of organ transplants [2, 54]. T cells also have important
regulatory function, due to their ability to produce
cytokines that act on other T cells, B cells and

Tr GM–CSF
M–CSF Mφ B
TGF–β IL–1
IL–6

Ag II
IL–1 IL–4; IFN–γ
TGF–β
IL–6 GM–CSF

Ag
Tact
IL–2; IL–4
IFN–γ
Tind IL–2; IL–3; IL–4
IL–5; IL–6; IFN–γ Bact

IL–2; IL–4
IFN–γ

Proliferation Proliferation Proliferation

Differentiatioin Differentiatioin Differentiation
LGL

IL–1; IL–2; CSF Pc Pc

IFN–γ ; TNF–β
Tumor cell killing

Tm Th
Ts Ts Tc Tc
Ab

Figure 3. Simplified scheme of the lymphokine cascade and its role in the modulation of immune response. Various cytokines
with unique and overlapping biological activities are produced during the immune response. These cytokines act in concert to
regulate the activation, proliferation and maturation of cells participating in the specific and nonspecific arms of the immune
system. Mo, macrophage; Tr, T activated lymphocyte; Tind, T inducer; Tm, T memory; Th, T helper; B, B cell; Ts, T suppressor; Tc,
T cytotoxic; Pc, plasma cell; LGL, large granular lymphocyte; IL-1, interleukin 1; IL-2, interleukin 2; IL-3, interleukin 3; IL-4,
interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; CSF, colony-stimulating factor; TNF-β, tumor necrosis factor β/lymphoto-
xin; IFN-γ, interferon γ TGF-β, transforming growth factor β; GM-CSF, granulocyte/monocyte colony-stimulating factor; M-CSF,
monocyte colony-stimulating factor; Ag, antigen; Ab, antibody; II, class II histocompatibility complex molecules
Robert K. Oldham
macrophages, and by directly interacting with other
lymphocytes [18, 67, 74, 112, 113, 116]. In general
terms, the functions of T cells can be divided into regu-
latory and effector categories. The positive and negative
regulatory functions are mediated by different lympho-
cyte subsets. The positive signals are associated with the
helper/inducer subpopulation of T cells (Th/I) and the
negative with the suppressor T-cell subpopulations (Ts)
[2, 19, 95, 105, 125]. The effector category includes the
cytotoxic (killer) T cells (Tc) reactive against virally
infected cells and foreign histocompatibility antigens,
and the sensitized Th(TDTH) cells, mediating delayed
hypersensitivity reactions [24, 91].
T cell subpopulations can be identified by immuno-
fluorescence or flow cytometry with the use of specific
monoclonal antibodies (mAb), which react with spe-
cific surface markers. An International Nomenclature
Subcommittee has examined a large number of mAb
directed to leukocyte antigens and clustered them into
groups of antibodies with the same reactivities (clus-
ters of differentiation or CD), and a number was
assigned to each group [46]. Helper/inducer T lympho-
cytes bear the CD4 marker, while cytotoxic/suppressor
T cells are recognized by the presence of the CD8
marker [41, 46, 95, 96]. Other mAb, which recognize
the T3 portion of the T-cell antigen receptor complex
(CD3), and others that recognize the CD2, react with
virtually all T cells and are widely used for the deter-
mination of total T-cell numbers [41, 46, 95, 96]. In
human peripheral blood, there are twice as many CD4
as CD8, a proportion that is altered in many diseases
including immunodeficiencies and cancer. More
recently, the phenotypes of the two main subsets of
CD-4 lymphocytes have been defined as helper-inducer
(CDw29+) and suppressor-inducer (Treg) (CD25 +
FOXp3 +, CD127neg) Th/I subpopulations, respectively
[60, 107].
The T-cell antigen receptor (TCR) has been isolated
and characterized [1, 12]. It is a disulfide-linked het-
erodimer composed of an α and β polypeptide chain,
each containing a constant and a variable (antigen binding)
region somewhat similar to the structure of immuno-
globulins (Fig. 4). In a small number of T cells present
in peripheral blood (0.5–10% T cells), thymus, epidermis,
and gut epithelium, a different type of T-cell receptor
consisting of two distinct polypeptide chains (γ and δ)
has been recognized [92]. The majority of these cells do
not express CD4 or CD8 on the cell surface and their
functions are not completely understood, but they are
believed to represent a mature functional lineage of
lymphocytes that can be activated by triggering through
the γ/δ receptor.
89
APC
MHC
Class
I or II Processed
Antigen
T cell receptor
CD4
or
CD8 CD3
Th/I or Tc/s
Figure 4. A current model for T-cell antigen recognition.
Antigen-presenting cells (APC) incorporate, process, and
express the modified antigen in conjunction with MHC mol-
ecules for presentation to T cells. The interaction of T helper
(Th/i) cells with APC is class II restricted; that is, it occurs
when the antigen is presented to the Th/i cell together with
class II molecules that interact with the T-cell receptor and
CD4 molecules, respectively. Class I restriction occurs
between APC expressing class I and the T-cell receptor in
CD8+ (Tc/s) cells. In both cases the delivery of the transduc-
tion signal for cell activation appears mediated by the CD3
molecular complex
Both α/β and γ/δ receptors are non-covalently associ-
ated with the CD3 molecular complex, which is com-
posed of at least four polypeptide chains, γ,δ,ε,and ζ one of
which (the γ chain) has a long intracytoplasmic portion
with several phosphorylation sites [12]. It is believed that
the CD3 complex is involved in mediating signal trans-
duction during the interaction of antigen with the specific
binding site in the α/β receptor (Fig. 4) [12].
Antigen recognition by T cells requires that the antigen
be presented to the T cells in association with the appro-
priate major histocompatibility complex (MHC) mole-
cules [32] (Fig. 4) and is amplified by co-stimulatory
molecules T-helper cells recognize antigen in the context
of class II MHC molecules expressed by antigen-present-
ing cells and B cells (class II restriction), while
T-suppressor and most cytotoxic T cells recognize anti-
gen in association with class I molecules (class I restric-
tion). Recent work has demonstrated that CD4 molecules
present on T cells bind with low affinity to certain invari-
able regions in the class II molecules. These findings sup-
port the theory that a complex involving the TCR α/β-CD3
and CD4 or CD8 molecules is formed on the surface of
the T cells during antigen recognition (Fig. 4). In this situ-
ation, the specificity is determined by the variable region
of the α/β receptor, the reaction is stabilized by binding
90
of CD4 and CD8 to class II or Class I molecules, respec-
tively, and the activation signal is transduced by the CD3
complex [64, 106].
T-helper Cells
T-helper cells were first described as the T lymphocyte
subpopulation that “helps” B lymphocytes mount an
optimal antibody response and “induces” the generation
of cytotoxic T cells. These T cells recognize processed
antigen presented by macrophages or other APC in the
context of Class II (DR) products (Fig. 4). The induc-
tion of lymphoid cell proliferation upon stimulation by
mitogens or antigens is characterized by two distinct
phases: competence and progression. The activating
signals (competence signals) cause the resting T cells to
move into the early G1 phase of the cell cycle. During
this “competence stage,” binding of antigens to the
TCR/CD3 (TCR complex) induces the generation of
intracellular signals, such as increases in intracellular
free calcium, membrane depolarization, generation of
diacylglycerol (DG) and phosphatidyl inositol (PI)
turnover, activation of protein kinase C (PKC), and
changes in the levels of cAMP, cGMP, protein phos-
phorylation, and express of c-fos, c-myc, and other
proto-oncogenes [13, 93, 94, 108, 111, 126]. These
events (some of which are triggered by IL-1) lead to the
expression of specific genes [including interleukin 2
and IL-2 receptor (IL-2R)] critical for the progression
phase [6, 126]. During the progression stage, binding
of IL-2 to its high-affinity receptor is a critical event
leading to passage of competent T cells from early G1
through the other phases of the cell cycle, culminating
in cell proliferation [6, 7, 29]. Other changes noted dur-
ing activation are increased numbers of interleukin 1
receptors (IL-IR), de novo expression of Class II mol-
ecules, and acquisition of transferrin receptors (TR) to
ensure incorporation of iron, since this element is
essential for cell division [75].
The rate of T-cell proliferation is tightly regulated
by the transient expression of IL-2R and the limited
production of IL-2. The high-affinity IL-2R
(HA-IL-2R), a heterodimer consisting of an α chain
(p55) associated with a β chain (p70), is rapidly inter-
nalized in the presence of the ligand and is widely rec-
ognized as the IL-2R species that mediates the
biological responsiveness to IL-2 [20, 53, 97, 98, 118,
122]. Small numbers of α-chain (MW 55 kD) mole-
cules are normally expressed by resting CD4 + T cells.
Normally, IL-2R expression lasts for about 1 week,
and IL-2 production for 2 or 3 days.
Current concepts in immunology
Antigen-activated CD4 T-cells release a number of
cytokines including IL-2, gamma-interferon (IFN-γ),
colony-stimulating factors (CSFs), B-cell growth factors
(IL-4, IL-5, IL-6), etc. [38, 40, 49, 73, 79, 87, 88, 117,
132] (Fig. 3). These cytokines, in turn, induce activa-
tion, proliferation and differentiation of other antigen-
specific T and B cells resulting in a specific immune
response and the production of memory T cells (Fig. 3).
There is some evidence that lymphokine release from
TH cells can lead to tumor regression (see Chapter 8).
Although human counterparts are not fully character-
ized, there are two well-defined subpopulations of
murine Th/I lymphocytes, termed TH1 and TH2 [69].
These subpopulations are characterized by the secretion
of different sets of cytokines, leading to different func-
tional properties [69]. Although some cytokines such as
GM-CSF, TNF-α, and IL-3 are produced by both cell
types, TH1 but not TH2 clones produce IL-2, γ-interferon,
and lymphotoxin (TNF-β) [69]. In contrast, TH2 but not
TH1 cells synthesize IL-4, IL-5 and IL-6. TH2 cells
induce growth and immunoglobulin (Ig) secretion of B
cells in response to specific antigens and polyclonal
activators. These functions require not only IL-4 and
IL-5 synthesis but also TH2-B cell-cell interactions. In
addition, IL-4 is essential for IgE production, and IL-5
appears to play a role in B-cell hyperactivation observed
in mice prone to develop a severe lupus-like auto-
immune syndrome.
TH1 cells appear to be involved in proliferation (but
no Ig synthesis) of B cells, induction of T-cell activity,
and generation of cells participating in the late phase of
delayed hypersensitivity reactions. Immune responses
characterized by predominant activation of TH1 cells
may result in strong induction of macrophage-mediated
cytotoxic reactions induced by IFN-α and TNF-β and
increased expression of Fc receptors for IgG2a in mac-
rophages. Thus, these responses would result in effec-
tive killing of target cells with intracellular viral or
parasite infections and strong DTH reactions. In con-
trast, activation of TH2 cells lelads to immune responses
characterized by high levels of antibody production.
Normal immune responses probably involve the partici-
pation of both cell types [69].
T-suppressor Cells
T-suppressor (regulatory) cells suppress (down-regulate)
T cell activity, antibody production by B cells, DTH,
contact sensitivity, cytolytic T-cell function, prolifera-
tion of T cells, and immune responses against tumors
[2, 19, 105]. The Ts cells are activated during normal
Robert K. Oldham
responses to a variety of antigens, thereby providing a
safety mechanism that continuously controls the magni-
tude of the immune response. Although T-suppressor cells
are antigen-specific, they are also able to bind antigen in
the absence of accessory cells or specific products of the
MHC. T-suppressor cells appear to be selectively acti-
vated when the antigen is presented in certain routes, that
is, intravenously or orally, or when administered in very
low doses (low zone tolerance). T-cell induced suppres-
sion might be mediated directly by Ts cell or by antigen-
specific and non-antigen-specific soluble factors released
by them (TSF). Ts binding consists of an antigen-binding
portion and an I-J molecule (28 kD) that binds to the
acceptor cell through the antigen molecule and an I-J
binding site [68]. Regulatory T cells inhibit tumor immu-
nity in some experimental systems [105], and recent
clinical trials [20] utilizing antibodies to Treg cells have
demonstrated that these biological drugs have clear ther-
apeutic activity (see Chapter 6).
T-cytotoxic Cells
T-cytotoxic cells were originally described as the effector
cells of specific cell-mediated cytotoxicity against
allografts, virus-infected cells, bacteria, tumor cells, etc.
The Tc lymphocytes also interact with the antigen (most
commonly a foreign cell, tumor cells, or virus-infected
cells) through the T-cell receptor [54, 71]. The interaction
of specific cytotoxic T cells with the target structure
results in lysis of the latter. “Killing” of a target cell,
including tumor cells, consists of a number of mecha-
nisms that can be divided in three phases [35, 36, 131].
Initially, binding between target and effector cells must
take place. The majority of cytotoxic T cells are CD8+
and therefore recognize the antigen in the context of
MHC 1 molecules (class I restriction [32]). In addition,
they can also recognize foreign class I antigens alone,
indicating that they can be effective in the destruction of
allogeneic transplanted tissues. A small percentage (10%)
of cytotoxic T cells bearing the CD4+ phenotype recog-
nize antigens in association with class II molecules and
might play a role in lysis of virus-infected cells [36].
The second phase (“programming for lysis”) involves
the reorganization of cytoplasmic organelles, including
polarization of the microtubule organizing center and
tubulin and actin. This leads to an increased area of con-
tact between the target and effector cells, leading to an
increase in the efficiency of the cytolytic process. This
is followed by reorientation of the cytoplasmic granules
toward the binding region, fusion to the membrane, and
release of lytic molecules (perforins) contained in those
91
granules. The presence of calcium is the limiting factor
in this phase, since polymerization of the perforin mol-
ecules on the membrane of the target cell with channel
formation does not occur in the presence of calcium
chelators [42, 66, 71]. The granules also contain other
factors known to mediate cytotoxic and cytostatic effects
on tumor cells, such as esterases and proteoglycans [89].
The third phase includes the delivery of the lethal hit
and death of the target cell. Following interaction with
the cytotoxic T cell, disturbances in the ion concentra-
tions and DNA fragmentation and fluid extrusion occur,
culminating in target-cell disintegration. Interestingly,
cytolytic cells are resistant to the cytolytic mechanisms
that they generate, and in this way each effector Tc may
kill more than one target cell.
Studies of Tc cells infiltrating human tumors has
helped define therapeutically active T cells leading to
tumor regression. These cells have proven exceedingly
useful in defining relevant human tumor associated antigens
for vaccine formulation.
B lymphocytes
B cells are responsible for humoral immunity, and, like
T cells, are present in peripheral blood where they rep-
resent about 10–20% of the circulating lymphocyte
pool. After activation, B cells mature into specific anti-
body-secreting cells or plasma cells [48, 128, 133].
Mature B cells express surface immunoglobulins (Ig)
with identical specificity to the antibodies (Ab) they
secrete [128]. The majority of peripheral blood B cells
express both IgM and IgD, while most B lymphocytes
present in body tissues express IgG, IgA, and IgE. Most
B cells also express Class II molecules in the cell mem-
brane. These molecules participate during the physical
cell–cell interaction that takes place during T-B cell
cooperation. In addition, receptors for the Fc portion of
IgG, the third component of complement (C3b), IL-2
and a large number of other markers (CD19, CD20,
CD21, CD22, etc.) have been found on the surface of
mature B lymphocytes [48, 128, 133].
The process of activation and maturation of B lym-
phocytes into plasma cells involves the interaction of
antigens with the specific immunoglobulins on the sur-
face of B lymphocytes, which triggers cell activation
and proliferation. The cooperation between B cells and
Th/I, macrophages, and other accessory cells, a process
requiring identity of Class II products of the MHC
among these cells, is necessary to induce optimal B
lymphocyte responses. However, binding of antigen to
B cells is necessary but not sufficient to initiate antibody
92
production. Nonspecific maturation and differentiation
factors (i.e., cytokines) mainly produced by Th/I cells are
also involved in the activation and progression of mature
B cells into antibody-forming cells [49, 128]. Among
these factors are IL-1 (produced by macrophages and
other accessory cells), IL-2, IL-4, IL-5, IL-6, IFN-γ and
other factors secreted by activated T lymphocytes [17,
22, 40, 44, 49, 52, 78, 79, 88, 90, 128]. Several of these
cytokines, including IL-1 and IL-4 are also produced by
B cells [22, 49]. Most of the circulating B lymphocytes
are in the resting state (G0 phase of the cell cycle), but
they become activated after interaction with the specific
antigen or antigen-presenting cells in the presence of
IL-4 and IL-1, progressing to the G1 phase of the cell
cycle. Activated B cells undergo cell division in the
presence of T cells and secreted cytokines. Differentiation
of proliferating B cells into plasma cells is mediated by
IFN-γ, IL-4, IL-5 and IL-6 [40, 78, 79]. Alternatively,
proliferating cells may return to the resting state and
remain as memory B lymphocytes (Fig. 3).
During the maturation process, they not only increase
their rate of Ig synthesis and actively secrete Ig, but they
also switch the class of heavy chains that carry the variable
region of Ig involved in antigen recognition [49, 128]. It
has been demonstrated that a single clone of proliferat-
ing B cells may switch at any division. Not all the pro-
liferating clones mature into secreting plasma cells.
Some return to the resting state and remain as long-lived
memory cells [128].
Monocytes and Macrophages
Monocytes are large cells (15–30 μm in diameter) that
comprise about 10–25% of peripheral blood mononu-
clear cells (PBMC). They originate in the bone marrow
from monoblasts, circulate in peripheral blood as mono-
cytes, and then enter various tissues where they are
termed resident tissue macrophages (Fig. 1). The large
degree of heterogeneity that exists within the macrophage
population is believed to represent different stages of the
maturational process and reflect environmental condi-
tions at the tissue level, rather than distinct macrophage
subpopulations [57]. Monocytes/macrophages can be
identified by a number of methods, including morphol-
ogy, ingestion of particles (such as latex), histochemical
staining of cytoplasmic enzymes (such as nonspecific
esterase), and by flow cytometry with a number of mAb
that recognize markers present on their membrane (i.e.,
CD40, CD54, CD80 and 86, etc.) [46].
Monocytes/macrophages play a critical role in the
defense against bacterial and other infections by ingest-
Current concepts in immunology
ing and killing the attacking microorganisms [57, 123].
They have also been shown to be very effective in
destroying neoplastic cells and removing dead or injured
cells (scavenger function). In addition, monocyte/mac-
rophages are critical for the development of normal
immune responses since they process and present anti-
gen (dendritic cells) to T lymphocytes and secrete
cytokines that play a major role in the initiation of spe-
cific immune responses, such as IL-1 and IL-6 [10, 11,
17, 22, 44, 51, 57, 90, 104, 119] (Fig. 3).
Monocytes/macrophages have been shown to secrete
more than a hundred different molecules that mediate
their functions. These products include (a) enzymes with
bactericidal capacities, such as lysozyme and lysosomal
hydrolases, neutral proteases (e.g., collagenase and plas-
minogen activator); (b) arachidonic acid metabolites
(e.g., prostacyclin, prostaglandin E2, and leukotrienes),
which have profound effects in the regulation of the
immune response; (c) reactive oxygen metabolites (e.g.,
superoxide, O2 radical, and H2O2), which are important
mediators in macrophage-mediated cytotoxicity; (d)
complement components; (e) coagulation factors; and (f)
cytokines, which in turn exert a multiplicity of regula-
tory actions including playing a critical role in antigen
presentation to T cells [10, 11, 17, 22, 44, 51, 57, 90, 104,
119, 123] (Fig. 3).
Antigen-presenting cells, which include dendritic
cells, Langerhans cells, veiled cells, interdigitating cells,
and others in addition to macrophages, are characterized
by their ability to process and present antigen to T cells
in conjunction with MHC class molecules, as well as
cytokine production, resulting in T-cell activation.
Macrophages incorporate antigens nonspecifically by
phagocytosis or by binding of immune complexes to the
Fc receptors. In contrast, activated B lymphocytes bind
antigen via the specific antigen receptor, and dendritic
cells are likely to process antigen directly in the cell
membranes. These short peptides (8–24 amino acids)
are then linked to the antigen cleft in the class II mole-
cules and returned to the cell membrane for interaction
with the Th/s cells [32] (Fig. 4).
Natural Killer Cells
Natural killer cells were first discovered by Oldham and
co-workers [80, 81]. Later studies defined their ability to
bind and lyse sensitive tumor and virus-infected normal
cells without the need of previous sensitization [31, 59, 84,
121]. These cells, which constitute approximately 15% of
peripheral blood lymphocytes, are a relatively homoge-
neous cell type identified as large granular lymphocytes
Robert K. Oldham
(LGL). LGL are nonphagocytic, express receptors for the
Fc portion of IgG (FcR-positive), and have a high cyto-
plasm to nucleus ratio, indented nucleus, and a few dis-
crete azurophilic granules in the cytoplasm [84, 121].
Several monoclonal antibodies have helped define the
surface markers and the phenotype of natural killer
cells. Some human NK cells express the following T-cell
markers: CD2, CD8; and after activation they express
T10 (a marker present in thymocytes and activated T
cells) [46, 59, 84, 121].
NK cell function is regulated by stimulatory and
inhibitory mechanisms. The generation and function of
NK cells are regulated mainly by IFN-α, IFN-γ, IL-2 and
IL-4 [59]. IL-2 and IFNs increase the lytic activity of
mature NK cells, promote the recruitment and activation
of nonlytic cells, and induce proliferation of precursors
and mature cells either in vivo or in vitro [121]. In addi-
tion to their nonspecific cytotoxic activity against tumor
cells, NK cells may exert a regulatory role in specific
immune responses mediated by T and B cells because of
their ability to produce a variety of cytokines including
IFN-α and γ, IL-2, IL-1, CSF, and TNF-α [59, 84, 121].
Natural killer cells play an important role in the resis-
tance to growth and metastasis of malignant tumors [81].
Results obtained using several in vivo experimental
models suggest that NK cells are of paramount impor-
tance during the early stages of tumor development. For
example, NK-sensitive clones of tumor cell lines take
longer to develop into palpable tumors when injected in
the footpads of syngeneic hosts than NK-insensitive
clones with similar doubling times [4, 39, 47].
Furthermore, selective depletion of natural killer cells in
experimental animals has been correlated with increased
frequency of spontaneous tumor metastasis.
Lymphokine-activated
Killer Cells
The lymphokine-activated killer phenomenon (LAK)
was originally defined as the ability of PBMC incubated
for several days in vitro in the presence of IL-2 to lyse
freshly isolated tumor cells and NK-resistant targets,
such as the HL-60 cell line [33, 34, 76, 100, 102, 130].
The LAK phenomenon, which is not MHC restricted
[85], can be mediated by cells expressing markers pres-
ent in both T and NK cell populations. For example, the
presence of cell populations with LAK activity express-
ing CD16 (Leull, an NK marker) and/or CD56 (Leu19,
a marker present in both Tc and NK cells) has been
reported in short-term cultures. Most of the LAK activity
93
in long-term cultures stimulated with IL-2 and anti-CD3
was observed in the CD3+ CD16−; CD3− CD16+;
CD3+ CD4− CD8−; CD16− and CD3− CD56+ cell
populations. On the contrary, the CD3+ CD4+ or CD3+
CD8+ populations present in these cultures exhibited a
significantly lower level of LAK activity [77].
Interleukin-2 mediated induction of LAK cells can be
enhanced by the addition of IL-1α or IL-1β, probably
by rendering LAK precursors more susceptible to the
activity of IL-2 [14, 21]. Interestingly, recent evidence
indicates that the generation of LAK cells is dependent
upon the expression of the p70/75 (intermediate affin-
ity) IL-2 binding protein in the cell membrane. The
expression of p70 occurs in the absence of the p55 (low
affinity, Tac) IL-2 binding protein, which is critical for
the expression of high-affinity IL-2R in activated T lym-
phocytes [86]. In addition to their remarkable tumori-
cidal activity, LAK cells are able to secrete IL-1α,
IL-1β, IFN-γ, TNF-α, and TNF-β (lymphotoxin) [56].
Experiments in murine models have clearly established
the in vivo antitumor activity of the cells mediating LAK
activity (i.e., LAK cells) when administered in conjunc-
tion with high doses of IL-2 [55, 56, 70, 103]. Based on
these observations, a number of clinical trials in advanced
cancer patients were done and these have demonstrated
clear, but limited, antitumor activity [16, 101, 127].
Clinical Assessment
of Immune Competence
In Vivo
Delayed Type Hypersensitivity (DTH)
DTH is a test of cell-mediated immunity based on the
response against a test antigen after it is injected intrader-
mally, or applied topically to the skin (Table 1). Reddening
and induration of the test site occur in 8–12 h, reaches a
peak at 48–72 h, and thereafter slowly subsides. The DTH
lesion is characterized by the accumulation of mononu-
clear cells in the subcutaneous and deep and superficial
dermis [8]. It should be stressed that DTH reactions are
complex immunological phenomena requiring the par-
ticipation of effector T lymphocytes as well as mono-
cytes/macrophages as accessory cells. Thus, a deficit of T
cells or monocyte/macrophages, or the presence of cer-
tain serum inhibitory factors that impair T cell or mono-
cyte/macrophage activity, or active suppressor cells, leads
to impaired DTH reactivity.
Antigens used in DTH testing can be divided into two
classes: recall antigens and neoantigens (an antigen to
94 Current concepts in immunology

Table 1. Selected immunologic tests patients, has been used as an indication of the existence
Parameter Test of alterations of immunoregulatory processes or of
defects in the production of Ig. Quantitation of the
T Cells
Total and subpopulations Flow cytometry levels of Ig is usually measured by the single radial
Responses to antigens Cell proliferation elicited by immunodiffusion method, ELISA, or by nephelometry
PHA, Con A, MLR [62, 63]. Among the antigens used to elicit primary and
Delayed-type hypersensitivity secondary antibody responses are brucella endotoxin,
Cytokine production Bioassays/RIA/ELISA
salmonella extract, hemocyanin, etc. Antigens have
T-receptor gene Southern blot analysis
rearrangement been divided into T-cell dependent or T-cell-independent
Cytotoxicity Release of radiolabeled depending upon whether or not Th/I cells are required for
compounds from target cells maximal antibody production by B cells.
B Cells
Total number Flow cytometry
Surface Ig Flow cytometry
Ig gene rearrangements
Serum immunoglobulins
Southern blot analysis
Serum electrophoresis
Reticuloendothelial System
Serum Ig classes Nephelometry, RID The in vivo phagocytic cell function can be assessed by
Reticuloendothelial system: measuring the rate of clearance from the bloodstream
Monocytes, macrophages after intravenous injection of a variety of materials
Cytokine production Bioassays, RIA, ELISA
Phagocytosis including colloidal gold, bacterial proteases, lipid emulsions,
Chemotaxis or aggregated human albumin labeled with radioactive
Tumor cell killing Cytotoxicity in vitro iodine [72, 114]. Another technique that has been used
Activation Production of oxygen radicals to study reticuloendothelial function is the Rebuck skin
window. In this procedure, a microabrasion of the skin
surface is made, followed by application of a cover slip
which the subject has never been previously exposed). to the raw area, and assessment of the accumulation of
One of the most commonly used neoantigens is 2,4-dini- macrophages onto the coverslip [45].
trochlorobenzene (DNCB), which has been used exten-
sively in cancer patients. Among the recall antigens,
tuberculin (PPD), mumps, tricophytin, and candida have In Vitro
been the most commonly used [8]. In general, several
recall antigens are studied simultaneously to ensure that Immune Cell Quantitation
a patient will have been exposed to at least one or more The accuracy in the quantitation of the levels of the dif-
of them. The interpretation of skin tests can vary widely ferent cells and cell subsets involved in the immune
between studies, since antigen concentration, reader response has improved dramatically with the advent of
variability, patient’s prior exposure to the antigen, boosting the monoclonal antibody technology. It is possible to
effects of repeated antigen administration, time course accurately estimate the numbers of monocytes/mac-
of the reaction, and definition of a positive reaction are rophages, Th/I, Tc, and Ts (regulatory) lymphocytes, LGL,
all variables that can influence the final assessment of a B lymphocytes, granulocytes, etc. with the use of flow
positive or negative response. cytometry using mAb that recognize specific markers
Of the available agents, only DNCB has been a con- on the surfaces of individual cell types [46, 99].
sistently useful skin test agent. However, DNCB is an
awkward reagent to employ, as repeated testing will
clearly yield anamnestic responses. Thus, only the original Lymphoproliferative Responses
test on an individual can provide clear information on This has been one of the most widely applied tests for the
the responsiveness to a neoantigen. determination of lymphocyte function. It is based on the
property of lymphocytes to undergo blast transformation
and proliferate in response to mitogenic or antigenic stim-
Humoral Immunity ulation [61]. The most commonly used mitogens include
phytohemagglutinin (PHA) and concanavalin A (Con A),
The testing of the levels of total and different classes of which primarily stimulate T cells, and pokeweed mitogen
Ig, as well as the primary or secondary antibody (PWM), which stimulates both T and B cells. Whereas
responses to a variety of antigens in the sera of cancer mitogens nonspecifically activate broad subpopulations of
Robert K. Oldham
cells, antigens activate specifically sensitized antigen-
reactive clones. The assay can be performed with whole
blood but most laboratories usually first isolate PBMC
(from whole blood, lymph nodes, or tumor specimens) by
density-gradient fractionation on Ficoll-Hypaque fol-
lowed by incubation with the appropriate mitogen at vari-
ous concentrations. Seventy-two hours later cultures are
pulsed with radiolabeled [3H]thymidine, which is incorpo-
rated into the cellular DNA. Cells are then harvested and
the radioactivity quantitated in a liquid scintillation coun-
ter and expressed as counts per minute (cpm). A similar
technique has been extensively employed to measure pro-
liferative responses to antigenic stimulation. In these studies,
mitogen is replaced by the specific antigen under study
and longer incubation periods are usually required [61].
Antigen responses are usually lower in magnitude that
those observed with mitogens, since they represent the
activation of specific T-cell clones, rather than most lym-
phocytes as is the case with mitogens.
Another proliferative response assay is the mixed leu-
kocyte reaction (MLR). This test is based in the ability of
T cells to proliferate in response to alloantigenic stimula-
tion (that is cells expressing different MHC antigens).
This is the in vitro correlate of in vivo graft rejection, and
is usually performed by incubating PBMC obtained from
the patient (responder cells) with irradiated PBMC
obtained form one or more histoincompatible individuals
(stimulator cells) for 6 days. Response is measured by
[3H]thymidine incorporation as described above [61].
Cytotoxicity
Cytotoxic reactions mediated by antigen-specific T
cells, NK cells, antibody-dependent killer cells, lym-
phokine-activated killer cells (LAK), and activated
macrophages are measured most commonly in microcy-
totoxicity assays [9, 30]. In general, target cells are
labeled with a radioactive element able to bind to some
intracytoplasmic structure, which is released to the
media when the cell dies. Fixed numbers of labeled tar-
get cells are incubated in microtiter plates with different
numbers of effector cells. The assays last for from 4 to
48 h, after which the supernatants are collected and the
radioactivity counted. The extent of radioactivity pres-
ent in the supernatants is in direct relationship with the
amount of killing. The results are expressed as percent
cytotoxicity [9, 31].
Lymphokine Production
As the roles of some of the soluble mediators in the
immune response have become clearer, assays have
95
been developed for their quantitation in the clinical set-
ting [28]. For example, the measurement of IL-2 is
based on (a) the fact that T lymphocytes produce IL-2 in
response to mitogenic and antigenic stimulation and (b)
the ability of IL-2 to induce lymphocyte proliferation
and maintain in vitro T cell lines (called IL-2 dependent
cell lines). The first step is the generation of cell culture
supernatants containing IL-2 by incubating PBMC with
mitogens (usually PHA or Con A) for 24–48 h. The IL-2
content in these supernatants is then determined by their
ability to support the in vitro growth of an IL-2 dependent
T-cell line, as assessed by [3H]thymidine incorporation
after 24–48 h of culture [30]. Currently, the concentrations
of a variety of cytokines, including IL-1, IL-2, IL-4,
IL-6, GM-CSF, TNF-α, IFN-γ, etc., are measured using
commercially available immunoassays [radioimmuno-
assay (RIA) and/or ELISA], which employ specific
monoclonal antibodies (see Chapter 8).
Immunoglobulin Production
Immunoglobulin production can be assessed in vitro by
exposing a suitable effector cell population (e.g., PBMC)
to either a polyclonal B-cell mitogen (e.g., PWM) or spe-
cific antigen and following a period of incubation, detect-
ing the immunoglobulin produced. Secreted Ig can be
detected either in the culture supernatants by using
ELISA or radial immunodiffusion (RID) techniques.
Phagocytic Cell Function
Both mononuclear (e.g., monocytes/macrophages)
and polymorphonuclear (PMN; e.g., granulocytes) leu-
kocytes exhibit phagocytic activity. Well-established tech-
niques exist for the assessment of monocyte phagocytosis,
hemotaxis (motility), and response to lymphokines (e.g.,
migration inhibition factors, MIF) [25, 26, 65, 70, 108].
Established techniques also exist for the assessment of
monocyte/macrophage cytotoxicity [50]. Various tests are
also available to measure PMN functions, including tests of
phagocytic cell activity, bactericidal capacity, and the ability
to take up and reduce nitroblue tetrazolium dye (NBT) [3].
Immunoregulatory Cell Functions
These assays are technically difficult to perform and are
not usually part of the routine assessment of immune
competence. Three broad classes of immunoregulatory
cell assays have been utilized:
1. Coculture assays. In these assays effector cells (e.g.,
B cells, T cells) are exposed to a polyclonal activator
96
(e.g., PWM, MLR) in the absence or presence of test
cells. The test-cell population might induce either
enhancement (e.g., increases in antibody production
or enhanced MLR responses) or reduction (suppres-
sion) of the response of the effector cells [125].
2. Mitogen-induced suppressor T-cell activity. In this
system suppressor cells are generated from resting T
cells by exposing them to a polyclonal T-cell lectin
such as Con A. Con A induced suppressor cells are
then added in coculture to an effector-cell assay as
described above.
3. Adherent suppressor cells. The presence of suppres-
sor monocytes can be assessed by either a positive or
negative effect. In the former situation, removal of
adherent cells (e.g., monocytes) leads to augmenta-
tion of effector cell function (e.g., lymphoproliferative
responses to mitogens), while in the latter case, read-
dition of adherent cells leads to suppression of the
response [120].
Quality Control of in vitro Assays
In practice, it has been difficult to standardize lymphop-
roliferative or cytolytic assays to insure the universal
validity and reproducibility of results. A number of con-
ditions affect the comparability of in vitro assays as per-
formed in different laboratories such as assay incubation
time, presence or absence of physiological buffers and/
or serum supplementation, choice of methodology to
prepare the responder cell population (i.e., purified
lymphocytes or unfractionated mononuclear cells), and
the purity and/or concentrations of mitogen/antigen, or
radioactive label.
In addition, various in vivo confounding variables,
such as the influence of concurrently administered drugs,
or seasonal and/or diurnal effects, provide further sources
of assay variability. A number of procedures have been
employed in an attempt to minimize interassay variabil-
ity such as the use of cryopreserved reference lympho-
cyte preparations [43, 82] or indices based on results
from a panel of reference lymphocytes [15]. Nevertheless,
because of the inherent variability of in vitro assays, only
limited successes have been achieved in clinical trials to
date. Indeed, neither appropriate guidelines for interpret-
ing the results of clinical trials employing immunorestor-
ative agents nor identified statistically significant end
points upon which to select appropriate sample sizes for
such clinical trials currently exist [83].
Acknowledgment Dr. Susana A. Serrate-Sztein (National
Institutes of Health, Bethesda, Maryland) and Dr. Marcelo
B. Sztein (University of Maryland School of Medicine,
Current concepts in immunology
Baltimore, Maryland) contributed much of this chapter in
the second edition, which was revised by the current
author in the third, fourth, and fifth editions.
I would especially like to acknowledge the assistance
of Dr. Richard S. Schulof, who co-authored this chapter
in the second edition, but died in an accident during the
preparation of the third edition.
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6 Therapeutic approaches to cancer-associated
immune suppression
ROBERT K. OLDHAM
It is now well established that many cancer patients
exhibit in vivo and in vitro evidence of immune suppres-
sion, which often correlates with tumor-cell burden, stage
of disease, and prognosis. Except in transplantation
associated cancers, cancer-associated immune suppres-
sion appears to be a direct result of the presence of
disease, or follows treatment for it, rather than being an
antecedent or predisposing condition. However, the pre-
cise role that nonspecific and/or specific antitumor
immunity plays in the control of human cancer remains
controversial. Indeed, there is some evidence that sug-
gests that the development of certain immune responses
may lead to augmented tumor cell growth rather than
tumor regression [178, 287, 406, 407, 582].
The clinical relevance of the relative state of general
immunocompetence in determining whether or not a
patient can be cured of cancer also remains controver-
sial. A case in point is the dissociation between immu-
nodeficiency and curability of patients with Hodgkin’s
disease. Despite the well-recognized immune suppression
associated with this malignancy, and the immunosup-
pressive therapies used to treat patients (e.g., lymphoid
irradiation and steroid-containing combination chemo-
therapy), it is one of the most curable of all cancers. Such
a discrepancy suggests that unique biological properties
of malignant cells, rather than the general immune com-
petence of the patient, are the more critical factors in
determining the curability of cancer.
Even though the precise relationship between the
general state of immune competence and cancer curabil-
ity has not been established, investigators have adminis-
tered a variety of agents to cancer patients including
biologicals, vitamins, hormones, and drugs, hoping that
the reversal or prevention of general immune suppres-
sion might translate into prolonged disease-free remis-
sions and improved patient survival. This chapter will
review the multifactorial basis of cancer-associated
immune suppression and the therapeutic strategies that
have been utilized. The sections on immune suppression
will focus exclusively on the general assessment of
immunity, and not specific antitumor immunity. The
sections on therapy of immune suppression will be lim-
ited to those biological response modifiers (BRMs),
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
both chemical and biological, that were administered
either to restore depressed immunity to normal, or to
prevent the deterioration of immune competence due to
surgery, radiation therapy, or chemotherapy. BRMs that
have been employed as adjuvants along with tumor cell
or tumor antigen vaccines to boost specific anti-tumor
immune responses, or whose primary mechanisms of
action are by activating effector cells directly, such as
interferons and/or interferon inducers, muramyl dipep-
tide and cogeners, or interleukin-2 and other interleu-
kins (e.g., tumor necrosis factor), will not be covered,
nor will more “traditional” whole organisms from the
older literature [e.d., Bacillus Calmette-Guerin (BCG),
Corynebacterium parvum, or mixed bacterial vaccine].
Immunosuppression and Cancer
It is clear that preexisting immunodeficiency plays a
permissive role in the development of certain cancers,
such as malignant lymphoma or Kaposi’s sarcoma [393,
448]. Patients with primary (e.g., Wiscott-Aldrich syn-
drome, at ataxia-telangiectasia) or secondary (e.g.,
acquired immune deficiency syndrome, AIDS) immu-
nodeficiency syndromes in which defects in cell-medi-
ated immunity predominate and patients receiving
immunosuppressive drugs following organ transplanta-
tion all exhibit an increased incidence of Burkitt’s and
non-Burkitt’s lymphoma, Kaposi’s sarcoma, and a vari-
ety of other tumors not otherwise commonly seen. In
several instances, following the cessation of immuno-
suppressive therapies, cancer regressions have been
noted, suggesting that when immunocompetence is
restored, control of tumor growth may occur. Patients
with primary (e.g., Bruton’s-type agammaglobuline-
mia) and secondary (e.g., chronic lymphocytic leuke-
mia) immunodeficiency syndromes in which defects in
humoral immunity predominate also exhibit an
increased incidence of malignancies including skin can-
cer, primary brain neoplasms, sarcomas, carcinomas,
and leukemias [167, 240, 394, 436].
Since the malignancies associated with underlying
immunodeficiency states are not those common in the
101
102 Therapeutic approaches to cancer-associated immune suppression

general population, it would appear that most adult
malignancies do not reflect underlying immunodeficiency.
It is more likely that a combination of genetic factors,
chronic immune system stimulation (possibly as a result
of recurrent infections), the presence of infectious
carcinogenic agents (e.g., viruses), chronic chemical
carcinogenesis, and other undefined factors leads to the
high incidence of certain cancers in patients with pri-
mary or secondary immunodeficiency states. There is
evidence to indicate that certain carcinogens – for example,
asbestos – can suppress immune functions such as NK
activity [430]. However, for the majority of common
cancers, the overwhelming evidence suggests that
immunodeficiency arises secondarily as a consequence
of cancer and the therapies used to treat it; that is,
cancer itself is an immunosuppressive disease. Cancer-
associated immunodeficiency is further influenced
by other factors, including age and genetic background,
as well as environmental factors, such as nutritional
status, stress, and infections. For example, nutritional
status is frequently impaired in patients with head and
neck cancer [87], which accounts for many of the immu-
nodeficiencies reported in these patient populations such
as decreased T cell numbers. Thus, it is the balance of
many different endogenous and exogenous factors that
ultimately contributes to the overall immune deficiency
state of cancer patients.
Multifactorial Basis of Immunodeficiency
in Cancer Patients
Because of subtle variations in methodologies employed
by different investigators, it is often difficult (and some-
times impossible) to directly compare the results of in
vitro and in vivo immunologic assays from different
studies. Nevertheless, a number of general conclusions
have been reached concerning cancer-associated immune
suppression. No single explanation, or generally agreed-
upon concept, has emerged to explain the immunodefi-
ciency. Rather, there is a complex set of interactions
involving a number of different mechanisms. This multi-
factorial basis of immunodeficiency is outlined concep-
tually in simplified form in Fig. 1. The scheme is equally
applicable to T−, B−, NK−, or phagocytic effector-cell
immune mechanisms.
Five major factors each play a role in determining
the immune responsiveness of cancer patients: (a) the
proportions and absolute numbers of circulating, tis-
sue-derived, or intratumoral effector cells; (b) the
intrinsic functional capabilities of the effector cells on
a per-cell basis; (c) the influence of immunoregulatory
helper and suppressor cells; (d) the influence of local
systemic-circulating and immunomodulatory soluble
factors; and (e) the influence of systemic treatment. In
Fig. 1, it is shown schematically how the relative con-
tributions of these five factors modulate immune
responsiveness of cancer patients as assessed with in
vitro and in vivo assays.
Effector Cell Numbers and Function
Any basic immune response reflects both the number
(or relative proportion) of effector cells present and the
intrinsic functional capability of the effector cells. Thus,
impaired immunity can result purely from a deficiency
in absolute numbers (or proportions) of effector cells

Local
Immunomodulatory Immune Boosters
Helper Cells Factors (adjuvants)

Tumor-derived
Ontogenesis Lymphokines
Factors In Vivo
Assays

EFFECTOR CELLS
Precurser (Stem) Cells EFFECTOR CELLS IMMUNITY
FUNCTION

In Vivo
Assays

Suppressor Cells Serum Inhibitory Factors Cancer

Therapy
(i.e. RT, Chemo)

Figure 1. Multifunctional basis of immunodeficiency in cancer patients

Robert K. Oldham
that otherwise exhibit normal function on a per-cell
basis, from effector cells that exhibit intrinsic functional
defects despite normal cell numbers, or from a combi-
nation of both decreased cell numbers and impaired
function of the individual cells.
The primary immunodeficiency syndromes, such as
the DiGeorge syndrome, ataxia-telangiectasia, and
severe combined immunodeficiency syndrome (SCID),
are examples of immune deficiency resulting from
defective ontogenesis of the immune system. However,
since the development of the immune system occurs
during fetal and neonatal life, defects in ontogeny are
not a consideration in the immune deficiency of cancer
patients. Cancer patients do, however, often exhibit
lymphocytopenia and decreased T-cell numbers [75,
101, 320, 352, 376, 384, 416, 481, 547], which contrib-
ute to their overall state of immunodeficiency. In con-
trast to primary immunodeficiency states, the effector
cell abnormalities detected in cancer patients arise sec-
ondarily, probably as a result of the suppressive effects
of cancer-derived factors on effector cell production
and/or survival.
When PBMC is used as the source of effector cells in
a lymphocyte or monocyte function assays, it cannot be
established whether a depressed functional immune
response results from a decreased proportion of effector
cells within the PBMC mixture, from intrinsic functional
defects of the effector cells themselves, from the pres-
ence of excessive suppressor cell activity, or from a com-
bination of abnormalities in cell proportions, functions,
and immunoregulation. There is considerable evidence
indicating that cancer patients exhibit depressions in
mitogen and antigen-induced proliferative assays [75,
94, 97, 101, 102, 141, 171, 174, 197, 239, 284, 310, 320,
333, 352, 376, 408, 420, 429, 481, 547, 574] and in
cytolytic activity [29, 287, 409, 496, 542] using PBMC
as effector cells. In some cancers, for example, head and
neck cancer [399] and Hodgkin’s disease [466], it has
been possible using purified T cells to document intrinsic
functional T-cell defects. Studies in cancer patients have
shown an impaired ability of T cells to produce the
lymphokine IL-2 [190, 321, 353] following activation by
mitogens; but that spontaneous IL-2 production (reflecting
possible stimulation due to circulating tumor antigens) is
increased [235]. However, in Hodgkin’s disease, although
IL-2 production and/or IL-2R expression of PBMC has
been reported to be low [48, 359, 490, 580], the abnor-
mality in T-cell lymphoproliferation does not appear to
be related to defects in the IL-2 system [48].
Peripheral blood monocytes and polymorphonuclear
leukocytes isolated from patients with a variety of cancers
also exhibit depressed functional activity [31, 73, 103,
103
136, 172, 203, 487]. NK-cell activity is also frequently
depressed in cancer patients [158, 409, 542]. At the local
level, NK activity may be more impaired than other
lymphocyte functions such as LPRs [17].
Thus, many studies now suggest that intrinsic func-
tional defects of effector cells contribute, at least in part,
to the immunodeficiency of cancer.
Immunoregulatory Cells
The intrinsic functional capabilities of T, B, NK, and
phagocytic cells are modulated by interactions with a
variety of immunoregulatory cells. The T- and B-cell
functions can be influenced either positively or nega-
tively, depending upon the relative balance between Tind
and Tc/s cells, as well as by number and function of
T-regulatory cells (Treg)[178, 540, 582]. Once again,
both absolute numbers (or proportions) and immuno-
regulatory function per cell are considerations in deter-
mining the overall influence of immunoregulatory cells
on effector cell function.
Another important immunoregulatory cell is the mono-
cyte/macrophage. Most normal immune responses require
the presence of monocytes/macrophages as accessory
cells for optimal processing of antigen and appropriate
activation of effector lymphocytes. In many disease
states, including cancer, monocytes/macrophages exhibit
suppressor-cell activity rather than helper-cell activity
[540]. It is likely that such “suppressor” cells may develop
in cancer patients to release factors capable of suppress-
ing tumor growth as their primary action and thus that the
suppression of immune reactivity is an “innocent
bystander” effect. The influence of monocytes/mac-
rophages is dependent upon cell numbers (or proportions)
and their state of activation [540].
There is considerable evidence for immune suppres-
sion mediated by activated monocytes/macrophages in
cancer patients [5, 28, 30, 50, 80, 193, 194, 195, 250, 257,
330, 461, 523, 532, 540]. For example, monocyte-mediated
suppression of lymphocyte-proliferative functions have
been demonstrated in Hodgkin’s disease [154, 175, 189,
336, 454, 535] as well as in patients with lung cancer [5,
93, 250], breast cancer [194, 253], malignant melanoma
[363], colorectal cancer [28, 194, 524], head and neck
cancer [30, 50, 550], bladder cancer [195], and a variety
of other malignancies [86, 540]. Monocyte-mediated
suppression of cytolytic effector-cell activity has also
been demonstrated in cancer patients [14, 18, 123, 151,
228, 229, 540], including NK-cell activity [246], and
autologous T-cell-mediated anti-tumor cytotoxic
responses [151, 461]. In comparative studies, it has been
demonstrated that such suppressor-cell activity is of
104 Therapeutic approaches to cancer-associated immune suppression
greater magnitude at the local level (i.e., intratumoral,
effecting TIL cells and in draining lymph nodes) than in
the systemic circulation [14, 18, 123, 228, 229]. Of
interest is the finding that monocyte/macrophages acti-
vated to kill cancer cells can suppress T-cell cytotoxicity
toward the very same cells [532].
The contribution of suppressor lymphocytes to the
generation of cancer-associated immunodeficiency is
less certain and still somewhat controversial. Several
studies that identified helper and suppressor T-cells on
the basis of the presence of surface Fc receptors for IgM
(Tμ cells) or IgG (Tγ cells), respectively, have suggested
that the Tind/Tc/s ratio is depressed in cancer patients [204,
267, 531]. However, more recent studies using monoclo-
nal antibodies to detect surface antigens indicate that the
relative proportions of Tind and Tc/s cells are preserved in
most untreated cancer patients, unless they have
extremely widespread disease [130, 200, 266, 310, 462].
Few reports have focused on functional aspects of sup-
pressor lymphocyte activity in cancer patients. Definitive
conclusions are lacking, although such activity has been
reported in patients with Hodgkin’s disease [217, 480]
and various solid tumors [151, 236, 237, 283, 537].
Inducible suppressor T-cell activity has generally [104,
183, 236, 464] but not always [521] been found to be
depressed in cancer patients.
Over the last 10 years, studies of immunoregulation
have focused increasingly on a subset of T-cells called
T-regulatory cells (Treg). Tregs have been well defined as a
distinct population of CD4 T-lymphocytes, which con-
stitutively express CD-25 and are further characterized
by the molecules CTLA-4, FOXP-3, and a TNF receptor.
Treg cells also produce immunosuppressive cytokines,
such as IL-10 and TGF beta [307, 444, 467]. Treg cells
with the phenotype CD-25+, FOXP-3+, CD-127− have
been found in tumor specimens and are suspected as
cells which suppress the activation of CD-4− and CD-8+
tumor-infiltrating lymphocytes active in tumor destruction.
The presence of these cells correlate with poor clinical
outcome in cancer patients [218, 450, 467]. Clinical
studies are now underway, using antibodies to CTLA-4,
to attempt to lessen the immunosuppressive effects of
Treg and, thereby, enhance the effectiveness of the CD-4
and CD-8 T-cells and immune effectors [573].
Immunomodulatory Factors
It is now well established that, in normal immune reac-
tions, the influence of immunoregulatory cells is medi-
ated at least in part by the local release of cytokines such
as IL-1 by monocytes, and IL-2, 6 and 10, as well as
γ-interferon by helper T cells (see Chapter 8). In a vari-
ety of disease states including cancer, there may be a
deficiency in the production of cytokines by cells that
normally produce them [159, 190, 206, 279, 353, 360,
548, 572, 579].
The release in cancer patients of a variety of immuno-
modulatory factors produced by monocytes/mac-
rophages and/or the tumor cells themselves has been
described. Immunosuppressive factors, for example, the
E series of prostaglandins, are released in excessive
quantity from both monocytes/macrophages [5, 28, 30,
50, 80, 154, 189, 195, 363, 386, 453, 523] and tumor
cells [72, 139, 140, 221, 261, 358, 523], which could
suppress cytolytic activities [358] and proliferative
functions [123] of TIL cells as well as of circulating
lymphocytes. However, prostaglandins alone do not
mediate the complete suppressor cell activity of mono-
cytes [28, 30, 154, 453, 523]. Monocyte-derived toxic
oxygen metabolites, for example, hydrogen peroxide
[336], and other as yet undefined mediators also appear
to play a role. Increased monocyte production of prosta-
glandins has been shown to directly impair lymphocyte
proliferative and cytolytic functions [139, 140] as well
as the phagocytic function of monocytes themselves
[150]. A variety of other factors have been identified
that are shed by tumor cells and modulate both local as
well as systemic immune responses. These include
tumor-associated glycoproteins [536] and lipids [196].
For example, various melanoma-associated ganglio-
sides have been shown to both up-regulate and down-
regulate lymphocyte responses to IL-2 [230]. Thus, it
has been suggested that the unique tumor-antigen-asso-
ciated phenotype of each individual tumor (based on the
proportion of various immune-suppressing and aug-
menting factors released by it) determines whether the
individual tumor will exhibit immune stimulation or
suppression. In this regard, it has been demonstrated
that whereas primary melanomas stimulate autologous
lymphocyte responses, metastatic melanomas suppress
immune responsiveness [510].
It is also apparent that a number of different substances
with immunosuppressive properties can be detected in
the blood of patients with cancer [469]. Many investiga-
tors have shown that serum from cancer patients is
immunosuppressive in that it suppresses mitogen and
antigen responsiveness of lymphocytes from normal
donors [11, 55, 98, 164, 182, 188, 226, 416, 421, 475,
482, 502]. Cancer serum inhibitory factors include
acute-phase reactants, such as α1-acid glycoprotein [39,
509], α-globulins [234], C-reactive protein [383], and
immune complexes [35, 259]. A serum factor in young
cancer patients has been reported to inhibit serum thy-
mic-hormone bioactivity [121]. Circulating immune
Robert K. Oldham
complexes have been shown to produce immunosup-
pressive effects by a variety of mechanisms including
(a) blocking of B-cell differentiation and antibody pro-
duction [435, 516]; (b) stimulating the production of
anti-idiotype antibody which then interferes with the
immune response to the original antigen [435, 516]; (c)
inducing suppressor T-cells [105]; (d) reducing IL-2
levels [418]; and (e) blocking Fc receptors on effector
cells [168]. Theoretically, the quantitative removal of
tumor antigens, antitumor antibodies, and/or immune
complexes could lead to a specific or nonspecific stimu-
lation of the immune system, leading to an increase in
general immune competence as well as in specific anti-
tumor immune responsiveness. This has formed the
basis for therapeutic trials with extracorporeal treatment
of cancer with immobilized staphylococcal protein A
[335, 362] and for attempts at plasma ultrafiltration. The
mechanism of antitumor activity of such approaches has
still not been defined. However, plasmapheresis has
been associated with increased LPRs and antitumor
immune responses [468, 470], probably due to removal
of immunosuppressive serum factors.
In conclusion, it is clear that the state of immunocom-
petence of an individual cancer patient is dependent
upon a number of complex interactions among effector
cells, immunoregulatory cells, and local and systemic
immunomodulatory factors. A simplified explanation
for the immunosuppression of cancer is that products
released from the malignant cells themselves lead to (a)
activation of suppressor cells; (b) impaired effector cell
production and survival; and (c) direct inhibition of
effector cell function. In assessing the state of immune
competence with in vitro and in vivo assays, the under-
lying basis of immunodeficiency may or may not be
identified.
Immunosuppression and Tumor Cell
Burden
In newly diagnosed, untreated cancer patients, the degree
of immunodeficiency generally parallels the extent of dis-
ease [564]. The most reasonable explanation for such an
association is that the release of tumor-derived immuno-
suppressive factors relates directly to the tumor cell bur-
den. Immune parameters of which impairment correlates
with extent of disease include DTH to recall antigens,
blood effector cell levels (e.g., T-lymphocyte levels),
lymphocyte functions, including proliferative responses
to mitogens and antigens, cytotoxic activity, phagocytic
cell activity, serum immunoglobulin levels, and primary
antibody responses to a variety of immunizing antigens.
Whereas quite early cancer is associated with only subtle
105
abnormalities of immune competence, more advanced
disease, particularly after treatment, leads to abnormali-
ties in all measurable immune parameters [131].
Solid Tumors
Delayed-Type Hypersensitivity (DTH)
Many reports have confirmed that as the extent of dis-
ease increases, the incidence of positive DTHS reactions
decreases [457]. For example, in one large study of 234
patients with various types of cancer, patients without
metastases exhibited positive reactions to DNCB (83%)
more often than those with regional metastases (67%) or
with distant metastases (41%) [401]. Similar findings
using both DNCB as well as recall antigens have been
noted in a wide variety of solid tumor-bearing patients,
including those with breast cancer [2, 34, 36, 75, 76, 146,
296, 496], gastrointestinal cancer [76], head and neck
cancer [495], lung cancer [8, 227, 389, 350, 561], renal
cancer [86, 355], gynecologic cancer [561], urologic
cancer [39, 85, 456], malignant melanoma [145, 408],
sarcomas [145], and primary brain cancers [84, 317].
The incidence of positive DTH responses varies
according to type of cancer. For example, patients with
localized head and neck cancer had a much lower reac-
tivity (42%) than patients with sarcomas (73%) and
lung cancer (80%) [76]. This result has been explained,
at least in part, by the alcohol intake and general state of
malnutrition associated with head and neck cancer
patients [54, 311]. In another study, it was found that the
correlation between DNCB reactivity and clinical status
was more pronounced in patients with squamous-cell
carcinomas of the head and neck than in those with
sarcomas or melanomas [562]. These findings suggest
that although DNCB reactivity generally correlates with
extent of disease, the relationships are modified depend-
ing upon the particular type of cancer.
It has been easy to demonstrate the marked degree of
impairment in DTH reactions found in patients with
metastatic cancer; however, it has been much more dif-
ficult to discern relative differences in reactivity of
patients with similar stages of disease whose tumors dif-
fer in size, regional lymph-node involvement, or local
invasiveness. Therefore, DTH responses are insensitive
immunological tests [101].
Lymphoproliferative Responses (LPR)
An exhaustive literature has accumulated in which vari-
ous in vitro lymphoproliferative assays were employed
to assess the state of immune competence of cancer
106 Therapeutic approaches to cancer-associated immune suppression
patients. In general, PBMC were the responder cells,
and T-cell mitogens (e.g., PHA, Con A) were used to
induce blastogenic transformation. Many reports have
documented an inverse correlation between PHA
response and stage of disease for patients with a variety
of solid tumors. For example, among 179 patients with
gastric carcinoma, 87% with stage I-II, 49% with stage
II, and only 24% with stage IV disease could exhibit
morphological evidence for blastogenic transformation
of PBMC following exposure to PHA [376]. A study of
154 patients with carcinoma of the lung revealed that
lymphoproliferative responses were significantly
decreased in patients with stage III disease, but not in
those with stages I and II [547]; other studies have
reported similar results [102, 141, 197, 420]. Depressed
LPR to alloantigens in mixed leukocyte culture (MLR)
were observed in 46% of patients with small (TINOMO)
stage I lung cancers [97]. Depressed mitogenic respon-
siveness has been correlated with advancing stage of
disease in breast cancer [49, 239, 320, 333, 464] and, in
one study, lymphocyte responses to PHA were impaired
in earlier stages of disease than was DTH reactivity
[496]. Similar inverse correlations between stage of dis-
ease and LPRs have been noted in colorectal cancer
[171], malignant melanoma [174, 284, 481], and head
and neck cancer [101].
Although many reports have documented that LPR
generally decrease with advancing stage in solid tumor
patients, this finding has not been universal. For exam-
ple, lack of correlation between blastogenic responses
and disease stage has been reported in patients with
breast cancer [71, 310, 429], malignant melanoma [94,
174, 408], and colorectal cancer [352]. These reported
inconsistencies have probably resulted from differences
in techniques and quality control procedures for assays
with a great deal of inherent variability. Nevertheless,
the same general conclusion holds for in vitro LPR and
DTH skin testing, namely, that there tends to be an
inverse correlation between immune reactivity and
tumor burden and/or stage of disease.
Immune Cell Quantitation
There are many monoclonal antibodies available to
quantitate blood, organ, and intratumoral levels of vari-
ous immune effector cells; however, most of the early
observations concerning lymphocyte quantitation and
cancer were made using more primitive assay methods.
The quantitation of lymphocytes in the peripheral blood
of cancer patients has been studied as a measure of
immunocompetence since the early 1920s, when lym-
phocytopenia was found to be common in patients with
advanced malignancies [578]. Other studies found an
inverse correlation between total lymphocyte count and
stage of disease in breast [416] and lung cancer [547].
The observation that human T lymphocytes could be
easily identified by a binding reaction to sheep red blood
cells to form E rosettes (E-RFC), coupled with the
emerging recognition of the importance of T cells in
immune responsiveness, led to numerous studies on
cancer patients. Using E-rosette techniques, the percent-
age of circulating T cells has been determined for
patients with a wide variety of solid tumors and corre-
lated with clinical stage of disease [320, 481, 547, 567].
Other studies have not found such a correlation [75,
352, 376]. In general, however, as with the in vitro
assays of T-cell functions, peripheral blood T-cell num-
bers tend to decrease in association with more advanced
stages of cancer.
Although assessments of B cells, monocytes, and Tind
and Tc/s ratios have been performed less frequently in
cancer patients, in general, B-cell numbers have paral-
leled T-cell numbers [266, 310, 352, 440], whereas
absolute monocyte counts tend to increase with advanc-
ing disease [310]. T-cell subset abnormalities have been
found in some patients with malignant melanoma [266],
head and neck cancer [130, 204], and lung cancer [13,
527, 565]. T-cell subset abnormalities were more pro-
nounced in lung lavage cells than in PBMC from patients
with lung cancer [160], suggesting that such perturba-
tions occur first at a local level before systemic abnor-
malities become detectable. In general, abnormalities of
the Tind and T c/s ratios are found in patients with advanced
or progressive disease. The ratio between Treg and CO8+
cytotoxic T cells correlates with immunosuppression
and clinical outcome [218, 450].
Cytolytic Functions
The measurement of nonspecific lymphocyte-mediated
cytotoxicity has been employed with increasing fre-
quency as part of the general assessment of immune
competence in patients with solid cancers. In general,
cytotoxic activity (most often NK activity) was
depressed in cancer patients, particularly those with
metastatic disease, but clear correlations have not been
identified between impaired function and clinical stage
of disease [262, 307, 505, 542]. Studies in patients with
small-cell lung cancer and stage I and II malignant mel-
anoma have found an inverse correlation between NK
activity and amount of clinically detected tumor [158].
Similarly, NK-cell activity was more likely to be
depressed in stage II breast cancer than in stage I dis-
ease. A large study of 247 cancer patients showed that
Robert K. Oldham
circulating NK cell numbers, assessed by monoclonal
antibody methods, were significantly reduced in patients
with colon, lung, and breast cancer, but not in those with
melanoma or sarcomas [29]. Thus, a depression of NK
cell numbers could explain the depressed NK function
reported in some, but not all, cancer patients [3].
The relationship between NK activity and other mea-
sures of immunocompetence has been explored. A lack
of association was found between DTH reactivity to
PPD and NK activity [409]. A comparison between TIL
activity and PBMC revealed that TIL expressed dimin-
ished NK activity compared with PBMC [225]. In one
study, although PBMC exhibited depressed NK activ-
ity, proliferative responses to PHA and in MLR were
maintained [409]. This suggested that cytolytic and
proliferative effector-cell mechanisms represent dis-
tinct functional entities.
Antibody Formation
A variety of humoral immune abnormalities have been
found to occur in association with solid tumors. A large
study of 984 patients with nonhematopoietic cancers
found no general trends, but did find increases in serum
IgG and IgA in males with skin and lung tumors,
increases in IgM in males with sarcomas and females
with melanoma, decreases in IgM in patients with ovar-
ian cancer, and increases in IgA in patients with oral,
gastrointestinal, and uterine cancers [238]. Increased
IgA has also been demonstrated in head and neck cancer
[319, 552] and prostate cancer [6], but to date, no cor-
relations have been observed between serum immuno-
globulin level and clinical stage of disease [125].
The serologic response to several different B-cell
immunogens has also been studied in solid-tumor
patients. Patients with nonlymphomatous malignancies
were found to exhibit decreased specific antibody
responses to Salmonella extract [297]. Patients with
stage III squamous-cell lung cancer exhibited deficits in
IgG and IgA production following immunization with
Helix pomatia hemocyanin, a T-cell-dependent antigen
[248]. A significant impairment in the ability of certain
patients with solid tumors to produce both IgM and IgG
antibody in response to primary challenge with mono-
meric S. Adelaide flagellin has been reported [297]. Both
complete and incomplete primary antibody responses to
heat-killed Brucella were reduced in patients with breast
and lung cancer [544]. However, precise kinetic data for
humoral antibody production in most patients with solid
tumors are lacking, as are correlations between humoral
immunity and extent of disease [143].
107
Phagocytic Cell Function
Other impairments of immune responsiveness in solid-
tumor patients have been documented. For example, the
inflammatory response of patients with advanced cancer
is associated with a reduced capacity to mobilize mono-
cytes [31, 137, 172, 546]. Reticuloendothelial function
in patients with breast and colorectal cancer is depressed
[138]. Depressed monocyte chemotaxis has been corre-
lated with disease stage for a variety of tumor types [73,
203, 487]. Detailed evaluations of monocyte function in
solid-tumor patients reveal abnormalities in patients
with malignant melanoma, breast cancer, colorectal
cancer, and head and neck cancer, but no consistent cor-
relations have been identified between tumor type,
monocyte defect, and clinical stage of disease.
Correlations Among Immune Cell
Numbers and Function
A number of studies have attempted to find correlations
between in vitro and in vivo abnormalities of immune
cell numbers and function. For example, both in vitro
PHA responses and in vivo DNCB reactivity were normal
in patients with early bladder cancer, but significantly
impaired in patients with advanced disease. Similar cor-
relations have been seen in patients with lung cancer
[424] and breast cancer. In contrast, in a study of 48
patients with lung cancer, LPRs were more often sup-
pressed than were skin test responses [8], and no corre-
lations between DNCB reactivity and in vitro immune
functions were found in patients with head and neck
cancer. It has also not been possible to identify any con-
sistent association between alterations in effector-cell
numbers and function [103, 251].
Hematopoietic Malignancies
Reed’s report in 1902 that patients with Hodgkin’s dis-
ease (HD) failed to react to tuberculin skin tests even if
they were known to have had active tuberculosis [410]
led to a vast number of immunologic studies for this
malignancy. Many of the immune abnormalities first
reported – for example, alterations of absolute lympho-
cyte counts, impairments of T-cell LPR, and the presence
of suppressor monocytes/macrophages – were subse-
quently also found in patients with a variety of other can-
cers. Even though the primary immune defects described
in untreated patients with HD involve T-cell immunity,
other defects have also been documented [58, 264, 433]
including impaired in vitro B-cell production of antibod-
ies, phagocytic cell function, and NK-cell activity [161].
108 Therapeutic approaches to cancer-associated immune suppression
The clinical relevance of the T-cell immunodeficiency
in patients with HD has been recognized for many years
[419]. Patients with HD have an increased susceptibility
to infections associated with defective T-cell immunity,
including Pneumocystis carinii pneumonia and viral
infections such as herpes simplex, herpes zoster, and
cytomegalovirus [22, 433]. In most of these studies,
however, it has been difficult to assess the role of the
treatment for HD (radiation therapy, chemotherapy) in
exaggerating the T-cell immune deficiencies, since many
of the infectious complications occurred only during or
after the completion of therapy.
A number of studies have now attempted to correlate
defects in T-cell functions with depression in T-cell
numbers in untreated patients with HD [71, 224, 460].
In general, no such relationships could be identified,
although lymphocytopenia patients were more likely to
exhibit impaired LPR. There is an easily identifiable
correlation between defects in immune responsiveness
and clinical stage of disease, in that most immune abnor-
malities are more readily apparent in patients with
advanced stages of disease than in those with localized
disease [460].
Immune deficiencies have also been shown to corre-
late with lymphoma subtype and extent of disease in the
non-Hodgkin’s lymphomas. These include impaired in
vitro LPR to mitogens [12, 312] and impaired in vivo
DTH responses [12]. In general, patients with high-
grade lymphomas exhibit more profound abnormalities
than those with more favorable histologies [258].
It has been very difficult to relate the extent of immu-
nodeficiency to tumor burden in patients with cancers
involving the bone marrow (e.g., leukemia, multiple
myeloma) since, with the exception of multiple myeloma,
staging systems based on tumor cell burdens do not exist.
However, a wide variety of in vitro as well as in vivo
abnormalities have been documented in patients with
lymphoid and nonlymphoid leukemias [199, 209].
Furthermore, a spectrum of intrinsic functional abnor-
malities has been identified in B cells and also purified
T-cell populations of patients with chronic lymphocytic
leukemia and multiple myeloma [236, 247]. Both of these
diseases are often associated with profound depressions
of normal serum immunoglobulin levels and impaired
ability to mount primary humoral immune responses.
Prognostic Implications of
Immunosuppression
Since immunodeficiency generally correlates with the
stage and extent of disease, and since stage and extent of
disease are the most important prognostic indicators for
any particular cancer, it is logical that the general state of
immunocompetence and prognosis should be closely
related. The potential importance for correlating immu-
nocompetence and prognosis at the time of diagnosis or
at the onset of therapy is obvious [155]. Assessment of
immune status could, in theory, help to select patients
with a poor prognosis who might require more aggressive
therapy than usual, or adjuvant therapy even if all disease
appeared to be surgically excised. In a now classic study,
it was observed that patients who could be sensitized to
DNCB and freed of disease by surgery had a good prog-
nosis, whereas those who were apparently freed of dis-
ease by surgery but could not react to DNCB had a poor
prognosis and relapsed within 6 months to 1 year [562].
The DNCB responses of patients in the poor prognosis
group were identical to those of patients who were found
to be inoperable at surgery. The two groups of patients
were otherwise comparable with regard to type of tumor,
extent of disease, and all other usual clinical prognostic
factors. This report formed the basis for a number of sub-
sequent studies attempting to correlate immunodeficiency
with prognosis for previously untreated patients with
solid tumors and hematopoietic malignancies.
Solid Tumors
Since 1970, there have been numerous attempts to cor-
relate impaired DTH skin reactions with prognosis.
Associations between impaired DNCB reactivity and
disease recurrence have been observed in lung cancer
[299], head and neck cancer [318, 441], gastrointestinal
cancer [77], and breast cancer [146]. A number of reports
have suggested that patients with or without metastases
but without reactivity to DNCB have a poor prognosis
[74, 85, 147, 265, 298, 318, 402]. Lack of reactivity to
common skin test recall antigens has also been corre-
lated with poor prognosis in breast [160, 349], lung [15,
244, 256, 299], gastrointestinal [382], urologic [334],
and head and neck cancer [494], although this has not
been a universal finding [54, 296, 350, 361, 397, 406,
452]. Several studies have subcharacterized patients
with identical clinical stages of disease on the basis of
impaired skin test reactivity to define further the rela-
tionship between anergy and prognosis. Such correla-
tions have been found, for example, in patients with
stage I [19] or stage III and IV [295] malignant mela-
noma, and in limited-disease small-cell lung cancer
[256]. However, other studies of accurately staged
patients with breast cancer, although DNCB-negative
patients had a worse overall survival, when survival dis-
tributions of DNCB-positive and –negative patients
Robert K. Oldham
with either primary operable or advanced breast cancer
were compared separately, significant differences were
not seen. Thus, studies to date have yielded conflicting
conclusions concerning the correlation between impaired
in vivo immunity and prognosis.
Associations between poor prognosis and impaired
in vitro LPR have also been reported [97, 392]. Other studies
have not produced such correlations [94, 174, 429, 446].
Studies correlating absolute lymphocyte counts or
absolute T-cell levels with prognosis have provided
conflicting results [376, 479]. Furthermore, the use of
multiparameter immunological assessments has not
generally improved the ability to assess prognosis for
previously untreated solid tumor patients [128, 174,
305, 320, 352]. However, some investigators have
developed predictive indices based on multiple immu-
nological parameters. For example, in one study, an
integrated score of immunocompetence based on vari-
ous in vivo and in vitro assays showed that the recur-
rence of breast cancer was significantly higher in
suboptimal (61%) as opposed to optimal (28%) respond-
ers [3]. Similarly, an immunological staging system
based on absolute lymphocyte counts and serum immu-
noglobulin level was found capable of predicting the
outcome off stage III head and neck cancer patients in
86% of cases [268]. However, in a report using logistic
regression methodology, it was demonstrated that only
the level of complement binding activity, which may
reflect levels of circulating immune complexes, corre-
lated with the likelihood of responding to induction che-
motherapy [452]. No associations could be determined
between response to chemotherapy and abnormalities of
a variety of other immune parameters, including lym-
phoproliferative responses, NK activity, lymphocyte
subset numbers and percentages, and serum immuno-
globulin levels.
Hematopoietic Malignancies
Among the hematopoietic malignancies, most attention
has focused on correlating defects in T-cell immunity
with prognosis in HD [488, 577]. More recent reports
suggest that there is a correlation between extent of
immune dysfunction and prognosis [57, 153, 533].
Relationships between immunocompetence and prog-
nosis have also been reported for a variety of other
hematopoietic tumors. For example, in acute leukemia,
patients with showed DTH skin reactivity and, to a lesser
degree, in vitro blastogenic responses to mitogens exhib-
ited the best overall survival, outranking such prognostic
variables as age, type of leukemia, and absolute blast cell
count [177, 303, 304]. In chronic lymphocytic leukemia,
109
both cell-mediated and humoral immune functions have
been found to correlate with prognosis [59, 120]. While
DTH was only moderately decreased, diminution in anti-
body responses and PHA responses correlated with the
duration of disease, status of therapy, degree of lympho-
cytosis, and immunoglobulin levels [120]. A correlation
has been reported between depressed absolute circulating
NK-cell levels and poor prognosis in patients with large-
cell lymphoma [38]. Patients with high-grade lymphomas
who have high levels of serum IL-2 receptor have been
shown to have more advanced disease [410] and a worse
prognosis [543]. However, in this instance, the IL-2
receptor is synthesized by the tumor cells so that serum
levels parallel tumor cell bulk. Nevertheless, the predic-
tive value of soluble IL-2 receptor was superior to that of
other markers that reflected tumor cell bulk such as lactic
dehydrogenase level (LDH) or clinical stage [410].
Perioperative Immunosuppression
Although it is perhaps not universally recognized, the
operative procedure and its associated general anesthesia
result in a variety of transient immunological defects that
can persist for several weeks after surgery [559]. During
operative procedures under general anesthesia for a vari-
ety of benign and malignant conditions, patients exhibit
inhibition of skin reactivity to DNCB [511] and DTH
recall antigens [527], suppression of circulating T-cell
levels [273, 528, 529], diminished LPR to PHA and
other mitogens [260, 437, 483], and depressed NK activ-
ity [456, 513]. Patients with cancer appear more likely to
experience these periods of postsurgical immunosup-
pression than do patients who undergo surgery for benign
conditions. Among the conditions associated with the
greatest degree of postoperative immunosuppression are
intraabdominal and intrathoracic procedures, blood trans-
fusions, and longer operating times [439, 471].
Perioperative Blood Transfusion
Several studies have suggested that perioperative blood
transfusion, possibly by inducing a greater degree of
immunosuppression, results in an adverse effect on
prognosis for postoperative patients with colorectal cancer
[222], breast cancer [514], non-small-cell lung cancer
[512], prostate cancer [205] and soft-tissue sarcoma [434].
However, three recent studies in breast cancer [541],
colorectal cancer [550], and lung cancer [271] have not
confirmed initial reports. Thus, it has not been conclu-
sively proven that perioperative blood transfusions worsen
the prognosis of patients with cancer. It is possible that
the requirement for transfusion is a marker for other risk
110 Therapeutic approaches to cancer-associated immune suppression
factors such as advanced stage of disease, need for more
extensive operation, or greater blood loss during surgery,
thus accounting for the worse prognosis following surgery.
The precise mechanisms for postoperative immune
suppression have not been fully defined. Among the
considerations are the immunosuppressive effects of
surgical stress [124], or of the anesthetic agents them-
selves [511], a relative decrease in circulating T-cell lev-
els compared with other cell types [180, 181], and/or the
generation of suppressor cells [360]. In nonsurgical
patients, chronic blood transfusion is associated with
depressed Tind/Tc/s ratios and impaired NK activity.
Theoretically, the associations between perioperative
blood transfusion and earlier cancer recurrence, and
between prolongation of renal allograft survival and
transfusion, may be attributed to a transfusion-induced
immune suppression resulting from a graft-versus-host
reaction mediated by transfused T-lymphocytes. Although
the precise mechanisms to explain perioperative immune
suppression have not yet been defined, this well-docu-
mented abnormality has nevertheless formed the basis
for a number of therapeutic trials in which various puta-
tive immunorestorative agents have been administered
as surgical adjuvants.
Radiation Therapy-induced
Immunosuppression
The immunological effects of external-beam radiation
therapy have been delineated in detail. Radiation ther-
apy to a variety of portals, including mediastinal [146,
273, 306, 371, 498], pelvic [65, 306, 375, 413, 498],
head and neck [306, 371, 483, 494, 551], lymphoid
[165, 185, 432], and breast [33, 191, 306, 413, 522, 556]
portals, results in similar acute and chronic changes
characterized by a generalized lymphocytopenia involv-
ing primarily a depletion of circulating T cells as well as
marked depressions in various T-cell functions such as
in vitro LPRs to mitogens and antigens and in vivo
DTHS reactions [265, 484].
The depression of T-cell numbers and function occurs
progressively with radiotherapy. The magnitude of
immune depression reflects both the dose of radiation
received and the volume of tissue, blood, lymph, or bone
marrow included within the radiation portals. This acute
immune suppression is short-lived, and substantial recov-
ery is apparent within 3 weeks of cessation of therapy.
However, most patients show a modest chronic depression
in both numbers and functions of T cells, which may last
for years following the completion of radiotherapy [252].
A number of other observations have been made con-
cerning the mechanism of functional immune impairment
following radiation therapy. Mediastinal irradiation for
treatment of localized lung cancer has been associated
with a decrease in proliferative responses of purified
peripheral blood T cells, suggesting that radiation
directly impairs their functional capabilities [462].
Several studies have assessed the effects of radiotherapy
on T-cell subset proportions. Although there was a drop
in the absolute numbers of both helper and cytotoxic/
suppressor T-cells following mediastinal irradiation, the
drop in cytotoxic/suppressor T-cells was greater, so that
treatment resulted in a significant increase in the Tind/Tc/s
ratio [462]. These observations are consistent with
in vitro functional data indicating that suppressor T-cells
are more radiation-sensitive than helper T cells [184,
398]. In contrast, radiotherapy for patients with breast
cancer [452, 397], head and neck cancer [249], and
Hodgkin’s disease [404, 432] has been associated with a
relative increase in the proportion of T cells bearing sur-
face antigens and/or Fc receptors (IgG) of cytotoxic/
suppressor cells, leading to a decrease in the Tind/Tc/s
ratio. Indeed, in postradiotherapy patients with breast
cancer [397] and Hodgkin’s disease [185], helper T-cell
levels remained low for years after irradiation.
Several studies have assessed the influence of thera-
peutic irradiation on suppressor cell function. They
indicate that radiation therapy can activate suppressor
monocytes [63, 68, 313] as well as increase the sensitivity
of lymphocytes to the suppressive effects of prostaglan-
dins [313].
External-beam radiotherapy, such as pelvic [375, 413,
498, 499] or chest wall [64, 513, 498, 499, 522, 556]
radiotherapy, has also been associated with a decrease in
B-cell and NK-cell numbers and functions in many, but
not all, reports. Mediastinal irradiation more severely
depressed NK activity than treatment to nonmediastinal
sites [332], possibly reflecting the relatively large volume
of blood and/or lymph node volume included within the
mediastinal radiation portal, leading to damage of NK
cells, or their more radiation-sensitive precursor cells.
However, within 3–4 months following the completion
of radiotherapy, NK-cell activity returned to pretreat-
ment levels, which then persisted for at least 2 years
[67]. There was a partial recovery in B-cell function,
which remained below pretreatment levels for 12–18
months after completion of therapy [438, 500]. Reports
concerning the effects of radiotherapy on absolute blood
monocyte levels have been conflicting [522, 549]. Other
immunological parameters have not been significantly
affected. For example, radiotherapy does not produce
changes in serum immunoglobulin levels or neutrophil
function. However, the ability of monocytes to differen-
tiate into macrophages was impaired in breast cancer
Robert K. Oldham
patients treated by radiotherapy [515], and absolute
monocyte levels increased after treatment [522], whereas
pelvic radiotherapy produced a decrease in both mono-
cyte numbers and functions in patients with colorectal
cancer [549].
In the earlier literature it was not possible to correlate
the extent of radiotherapy-induced immunosuppression
with relapse or survival [133, 462, 555]. In general, how-
ever, patients who exhibited skin reactivity to DNCB
prior to radiotherapy had a better prognosis than those
who did not [79]. In two more recent studies of patients
who received primary radiation therapy for breast cancer
[499, 555] and/or cervical cancer [499], those who
exhibited the greatest postradiotherapy depressions of
LPRs to mitogens or antigens had the shortest survival
[555]. Several studies on postradiotherapy patients with
lung cancer have serially assessed the immune status of
those who responded transiently to treatment but relapsed
at a later date [133]. Lung cancer patients who responded
clinically to radiotherapy showed some improvement of
absolute T-cell levels in the months following cessation
of treatment, whereas those who progressed did not show
such partial recovery [133]. In addition, for postradio-
therapy patients with locally advanced non-small-cell
lung cancer, there was a gradual and progressive decrease
in T-cell and helper T-cell percentages, and in the Tind/Tc/s
ratio that preceeded relapse [462].
Chemotherapy-induced
Immunosuppression
It has been much more difficult to assess the effects of
cancer chemotherapy than of radiation therapy on
immune responsiveness. In part, this is due to the fact
that a wide variety of different cancer chemotherapy
drugs are available for clinical use, many of which can
be administered by a variety of routes and doses, either
alone or in combination with other drugs. The majority
of anticancer drugs produce a dose-dependent pancy-
topenia, which itself is immunosuppressive because of
the associated granulocytopenia. In addition, most, if
not all, cancer chemotherapy drugs exhibit immunosup-
pressive properties as assessed with conventional in vivo
and in vitro assays. This includes the alkylating agents,
antimetabolites, and antitumor antibiotics.
Cancer chemotherapy may lead to profound depres-
sions of cellular as well as humoral immunity [202, 207,
213]. Newly acquired DTH and primary humoral immune
responses appear to be more sensitive to drug-induced
immunosuppression than are secondary responses. Less
often, the administration of selective drugs, for example,
cyclophosphamide (CTX), at low doses can lead to a
111
temporary enhancement of immune responsiveness due
to preferential inhibitory effects on suppressor cells.
Traditionally, it has been felt that intermittent chemo-
therapy schedules of administration using single or mul-
tiple drugs tend to be associated with relatively little
effect on DTH responses and with transient depressions
of lymphocyte numbers, in vitro LPR, antibody responses,
and inflammatory responses, with at least some recovery
several weeks after discontinuation of treatment [210,
417]. In contrast, continuous therapy, if given in adequate
doses, has been shown to lead to a progressive decline in
all phases of immune reactivity [166, 207, 210, 331, 417],
which is reversible after the cessation of administration
of drug(s). In several more recent studies, however, it has
been demonstrated that intermittent combination chemo-
therapy regimens lead to cumulative depressions of lym-
phocyte numbers and functions over months to years
[315, 501], which may or may not be reversible. These
changes include depressions of absolute T- and B-cell
levels, depression in the Tind/Tc/s ratio, and depressions of
LPR to mitogens and antigens.
Another phenomenon that has been described is the
recovery or transient, rebound overshoot of immune
responsiveness after a single cycle of chemotherapy
[81, 108, 201]. Rebound is found in patients who are
responding clinically to treatment. Patients whose
immune functions remain suppressed either have not
responded clinically to treatment or are at high risk for
relapse compared with those who exhibit recovery to
levels above those pretherapy. It has been suggested that
the rebound overshoot in immune reactivity following
chemotherapy may be due to a reduction in monocyte
suppressor cell function [81]. The effect can result from
a transient, chemotherapy-induced decrease in relative
numbers of monocytes/macrophages, from a reduction
in suppressor cell function on a per-cell basis, or from a
combination of both. Recovery of suppressor monocyte/
macrophage function is responsible for the eventual
decline in immune function following drug-induced
rebound overshoot.
Antimetabolites
All of the antimetabolites commonly used for treating
cancer patients, including thiopurines such as 6-mercap-
topurine (6-MP) and 6-thioguanine (6-TG), antifolates
such as MTX, fluorinated pyrimidine analogs such as
5-fluorouracil (5-FU), cytosine arabinoside (Ara-C), and
DTIC (5-[3-3-dimethyl-1-triazeno]-imidazole-4-carbox-
amide) are immunosuppressive in humans. 6-MP has
been one of the most extensively studied agents [207,
213, 300]. Azathioprine, a nitroimidazole derivative of
112 Therapeutic approaches to cancer-associated immune suppression
6-MP, is a potent immunosuppressive drug, and has found
more use in clinical transplantation than as a cancer che-
motherapy drug [343]. 6-MP, 6-TG, MTX, and 5-FU
have all been shown to preferentially depress primary
antibody responses and the development of new DTH
reactivity [213, 343, 345, 346]. However, 5-FU treatment
restored DTH to recall antigens such as PPD, mumps,
and trichophytin [62, 345], but caused a transient decrease
of in vitro LPRs to PHA and PPD [370]. Intravenous
5-FU produced a rapid (within 1–2 days) decrease in
absolute T- and B-lymphocyte levels, which returned to
baseline over the ensuing 1–3 weeks [152]. Ara-C has
been shown to partially suppress primary and secondary
antibody responses [117]. Although both MTX and
Ara-C abolished established DTHS to recall antigens,
Ara-C was much more potent in inhibiting DNCB reac-
tivity [346]. In vitro MTX did not affect the phagocytic or
cytolytic activities of human neutrophils [255]. DTIC has
been considered only weakly immunosuppressive in
humans, based on studies in patients with malignant mel-
anoma in which only a minority of treated patients exhib-
ited depressions in antibody production to typhoid vaccine
or in DTHS reactions to primary sensitization [89]. More
recent studies with DTIC have indicated that treatment is
associated with a cumulative decrease of T-cell numbers,
but no change in B-cell numbers [52]. A normalization of
Tind cell numbers occurred after cessation of treatment.
Alkylating Agents
The alkylating agents comprise a variety of drugs includ-
ing cyclophosphamide (CTX), nitrogen mustard (HN2),
chlorambucil (CLB), thiotepa (TT), and busulfan (BS).
These compounds have found widespread clinical use, par-
ticularly for the treatment of leukemias and lymphomas.
Their antiproliferative effects also extend to normal host
hematopoietic precursor cells of all lineages, including
lymphoid precursors. In animals, these agents are potent
immunosuppressants of antibody formation [447].
Recent clinical interest has focused on the novelty of
CTX as an immunomodulatory agent [37, 144, 202,
207, 213, 216, 325, 380]. Administration of oral daily
CTX maintenance therapy to patients with lymphomas
did not result in interference with the development of
normal humoral and cell-mediated immune response to
keyhole limpet hemocyanin. Other studies have revealed
that prolonged oral therapy with CTX can produce lym-
phopenia and suppress in vitro LPRs to PHA. CTX
administered intravenously at conventional doses for 7
days inhibited the production of primary antibody
responses, but did not significantly interfere with DTHS.
Low intravenous doses of CTX (100–600 mg/m2) pref-
erentially decreased circulating B-cell numbers. Doses
from 200 to 600 mg/m2 selectively depleted cytotoxic/
suppressor T cells, leading to a transient increase in the
Tind/Tc/s ratio, whereas higher doses affected all T-cell
subsets equally. Intravenous administration of conven-
tional doses of CTX for 5 days has been shown to
depress established DTH by 7–10 days after the cessa-
tion of therapy, at a time when the peripheral white
blood cell count was at its nadir.
Perhaps the most intriguing aspect of CTX-induced
immune changes has been the elucidation of the selective
immunopotentiating effects of CTX [144, 325]. For
example, in a study of 22 patients with metastatic cancer,
CTX pretreatment significantly augmented the develop-
ment of DNCB reactivity as well as DTHS to new anti-
gens [44], even though absolute lymphocyte counts fell
within 1–2 days and did not recover for 21 days [46]. The
T-cell, B-cell, and T-cell subset numbers were all affected
equally. Lymphoproliferative responses to mitogens and
alloantigens also fell significantly within a day, but recov-
ered to pretreatment levels by day 3; some cases exhibited
rebound overshoot by day 7. Inducible T-cell suppressor
cell activity was also diminished within 1 day after CTX
administration; however, in contrast to LPRs, suppressor-
cell activity remained significantly impaired on day 3 and
only partially recovered by day 7. Thus, between 3 and 7
days after intravenous administration of CTX, there
appears to be a preferential impairment of nonspecific
T-cell-mediated suppressor cell activity, which could
account for the augmented DTHS noted in CTX-treated
patients. Most recently, a single intravenous low dose
(300 mg/m2) of CTX was shown to inhibit the generation
of inducible suppressor cell activity in cancer patients for
up to 19 days without affecting lymphoproliferative
responses to T-cell mitogens or the Tind/Tc/s ratio [45].
This CTX dose (300 mg/m2) 3 days prior to immunother-
apy has been employed clinically in a variety of circum-
stances in an attempt to abrogate suppressor cell activity,
for example, as an adjunct to active specific immunother-
apy with a melanoma tumor cell vaccine [43], and in
combination with low-dose intravenous IL-2 [344].
Although the mechanism by which CTX augments the
immune response has not yet been elucidated, the leading
hypothesis, at present, is an abrogation of suppressor-cell
function, with blockage of the subset of helper T-cells
that is the inducer of suppressor cells [144].
Antitumor Antibiotics
Surprisingly, clinical information concerning the immu-
nosuppressive effects of this class of anticancer drugs
that includes such widely employed drugs as adriamy-
Robert K. Oldham
cin (ADR), daunomycin (DNR), mitomycin (MTC),
and bleomycin (BLEO) is limited. However, in animal
models, these agents affect a variety of immune cells, in
particular, macrophages and immunoregulatory cells
[144, 340], suggesting that they should exert a spectrum
of immunomodulatory effects in humans. On the other
hand, ADR has been shown to augment both monocyte-
and lymphocyte-mediated cytotoxicity of human PBMC
[20, 21, 282]. Following a single intravenous dose
(25 mg/m2), PBL from cancer patients showed an
increase in lymphocyte cytotoxicity as well as an
increase in Tc/s levels [20]. In cancer patients, intrave-
nously administered ADR has been shown to produce a
rapid but reversible lymphocytopenia of both T and B
cells [152]. In vitro, ADR has been shown to impair
phagocytic function of human polymorphonuclear leu-
kocytes [536], whereas similar effects were not seen
with a variety of other drugs, including MT, VCR, 5-FU,
and cisplatin. In addition, ADR has also been shown to
inhibit granulocyte degranulation and release [95].
Some evidence has been provided that low doses of
ADR can also augment both T- and B-cell mediated
immune responses [144]. Thus, it now appears as if
ADR, like CTX, can exhibit a variety of immunomodu-
latory activities.
Vinca Alkaloids and Podophyllotoxins
The vinca alkaloids, vincristine (VCR) and vinblastine
(VLB), possess a unique mechanism of antitumor activ-
ity involving drug-induced microtubular dysfunction
leading to mitotic arrest. VCR has been shown to depress
granulocyte aggregation, lysozyme release, and chemot-
axis [95, 492]. However, there remains a substantial
lack of studies concerning the immunomodulatory
effects of these agents in humans. They are often admin-
istered in conjunction with steroids as treatment for
hematopoietic cancers, making it impossible to define
their selective immunosuppressive effects. Not unex-
pectedly, they inhibit human LPR in vitro, and they
should exert similar effects in vivo. Both VCR and VLB
have not been particularly immunosuppressive in
humans [208]. Vindesine, a new semisynthetic vinca
alkaloid, was devoid of immunosuppressive activity in
preliminary clinical trials [425]. Podophyllotoxins such
as VP-16 have mild immunosuppressive effect, similar
to the vincas.
Other Drugs
Other frequently employed anticancer drugs, such as
cisplatin, nitrosoureas (BCNU, CCNU) and taxol also
113
exhibit varying degrees of immunomodulatory activity
when incubated in vitro with human PBMC [492], but
clinical data are sparse. Single intravenous doses of cis-
platin have been associated with a rapid depression of
LPR, with recovery in 1–2 days, whereas a single dose of
CCNU leads to depressed PHA responsiveness for up to
6 weeks, in the absence of changes in T- and B-cell pro-
portions. Long-term therapy with CCNU has been asso-
ciated with cumulative depressions of absolute levels of
both and T cells [52]. The reduction of T-lymphocyte
levels was due mainly to a depletion of Tind cells. Taxanes
have moderate immunosuppressive effects.
Hormones
Hormonal agents such as tamoxifen (TAM) and
medroxyprogesterone acetate (MPA) are used widely in
the treatment of breast cancer. Both estrogen and pro-
gesterone receptors have been identified in human lym-
phocytes [117, 126]. There is considerable experimental
and clinical evidence suggesting that hormones can
modulate immune mechanisms [114, 371, 381]. Long-
term treatment of patients with TAM has not produced
significant immunosuppression [254, 451], whereas
MPA depressed LRP as well as the Tind Tc/s ratio of treated
patients (451).
Combination Chemotherapy
When multiple cancer chemotherapy drugs are adminis-
tered together, it is impossible to predict what effects
their interactions will have on their individual immuno-
suppressive properties. Nevertheless, at the present
time, most cancer chemotherapy regimens include a
combination of drugs. A number of recent studies have
begun to define the immunological consequences of
combination chemotherapy as currently used for the
treatment of patients with advanced disease, as well as
for patients treated in the adjuvant setting.
A comparison of intravenous doses of single drugs,
namely ADR and 5-FU, with combinations such as
COBAM (CTX, VCR, BLEO, ADR, MTX) or DOMF
(DTIC, VCR, methyl-CCNU, 5-FU × 5) revealed that
both the single agent and multiagent regimens produced a
rapid drop (within several days) in the percentage and
absolute numbers of circulating B and T cells, which was
more pronounced for the combined drug regimens [152].
Such decreases were transient, and either a partial or
complete recovery (with or without rebound) to baseline
levels was then observed over the 1 or 2 weeks after drug
administration [152]. Similar acute effects of multiagent
chemotherapy regimens have been noted with respect to
114 Therapeutic approaches to cancer-associated immune suppression
functional immune parameters such as in vitro LPRs [87],
B-cell activation [232], reticuloendothelial cell function
[151], and cytotoxic cell (NK) activity [83, 442]. In gen-
eral, there has not been a good correlation between the
depressions in immune cell numbers and functions.
Cyclic combination chemotherapy can result in cumu-
lative immunosuppressive sequelae [315, 396, 501].
Three similar studies have all involved patients receiving
cyclic adjuvant chemotherapy for breast cancer who did
not exhibit evidence of immune suppression before treat-
ment. Serial immune assessments revealed that cyclic
chemotherapy was associated with a marked initial
(within 1 month) decrease in T- and B-lymphocyte num-
bers, and then gradual progressive further decreases
[501], the same being observed in Tind Tc/s ratios [396].
There was also a rapid initial decrease in functional
immune parameters, such as in vitro LPRs to antigens,
with stabilization or some improvement over the ensuing
year of treatment. In general, the functional immunologi-
cal parameters tended to normalize during or, more often,
within several months of stopping chemotherapy, whereas
the abnormalities of immune cell numbers were still
apparent 1 year after cessation of treatment [315]. Thus,
it appears that prolonged administration of cyclic combi-
nation chemotherapy results in cumulative impairments
of immune cell numbers and functions, some of which
are long-lasting [439, 501]. In none of these studies were
investigators able to predicate relapses in individual
patients based on immune parameters.
Recent attempts to manipulate the immune system
have involved the combination of total body irradiation
with aggressive, but non-ablative, chemotherapy. Such
therapeutic studies are being done to deplete regulatory
cells that negatively influence T-cell function followed
by the reinfusion of T-lymphocytes derived from the
patient’s own tumor, so-called tumor-infiltrating lym-
phocytes (TIL). These studies, which will be described
in more detail elsewhere in this book, are producing
clinical responses in 50% or more of patients with
advanced melanoma [573]. Such aggressive approaches
indicate that the immune system can be manipulated
with chemotherapy and radiation therapy, but the future
will probably in the area of more selective immune
depletion with monocloncal antibody of the targets
agents imparting less general toxicity.
Immune Status of Patients in Clinical
Remission
A number of studies have evaluated the immune status
of patients in remission following the successful treat-
ment of their cancer. In general, there tends to be
improvement and/or normalization of immune cell
numbers and function for patients who achieve clinical
remission following chemotherapy or radiation therapy.
Nevertheless, some immunologic impairment persist
for years after completion of successful treatment. Thus,
in many cases, it may be extremely difficult to dissoci-
ate the chronic immunologic consequences of therapy
from the presence of persistent immune defects while in
clinical remission.
Solid Tumors
Patients surgically cured of their cancers generally have
a restoration of immune cell numbers and function after
the perioperative periods [260, 437, 483]. Although
patients in clinical remission following potentially cura-
tive radiation therapy may have some normalization of
immunity, it does not usually become apparent until
years after the completion of treatment [375]. Finally, as
discussed previously, it has also become apparent that
adjuvant chemotherapy for breast cancer results in
cumulative depressions of both lymphocyte numbers
and functions [315, 396, 501].
Hematopoietic Malignancies
Much important information concerning the long-term
effects of chemotherapy and radiation therapy in cured
patients has resulted from studies of children who
received maintenance chemotherapy for acute lympho-
blastic leukemia and of adult patients with HD. In acute
lymphoblastic leukemia patients who remained in clini-
cal remission for 1–3 years after completion of chemo-
therapy, immune competence was found to recover.
Those patients destined to relapse showed a subsequent
deterioration in immune status, which was detectable
some months before clinical relapse [178, 210, 211,
212, 214, 472]. The immune status of patients with HD
in long-term clinical remission after treatment with che-
motherapy or radiation therapy has also been evaluated
in detail. At the completion of radiotherapy, DNCB
reactivity was lost in almost all patients who were ini-
tially sensitive. However, many patients regained their
DTHS responses during the first year after discontinua-
tion of chemotherapy [7, 427] or radiotherapy [165,
428]. NK-cell activity also improved following success-
ful therapy for HD. [161]. Several studies have shown
that, following the completion of successful MOPP che-
motherapy, patients in remission from HD exhibited
gradual improvements of in vitro T-cell functions [7,
427]. In contrast, other studies have revealed a persistent
depression of LPRs at 1–10 years after the completion
Robert K. Oldham 115

of treatment, with no evidence for recovery [56, 57, 100, Table 1. Biological response modifiers with immunorestor-
154, 189, 428], along with a preferential chronic deple- ative properties
tion of Tind cells [29, 185, 404, 427, 432]. In some of Chemicals Biologicals
these studies, patients had received radiation therapy, Azimexone Bestatin
which was felt possibly to contribute to the prolonged Cimetidine CSFs
Copovithane FK-565
immune deficiency [165, 185, 404, 432]. Several authors Coumarin IMreg-1
have argued, however, that patients with HD in pro- DTC ImuVert
longed clinical remission continue to manifest immune Ibuprofen Interleukin 1-–32
abnormalities as a reflection of their underlying disease Indomethacin Interferon gamma
Interferon inducers Lentinan
[56, 57, 100, 154, 291]. For example, persistent defects
Isoprinosine OK-432
in T-cell functions were more severe in irradiated Levamisole Retinoids
patients with HD than in similarly treated patients with NPT 15392 T-Cell reconsti-
testicular cancer [47, 432]. Although some improve- Oxyphenbutazone tuting factor
ments were noted on serial immune assessments follow- Piroxicam (SR 270258)
Ranitidine Thymic factors
ing discontinuation of treatment, there have been no Transfer factor
correlations between the status of immunity during clin- Tuftsin
ical remission and likelihood of relapse [427, 428]. It
has been suggested that an increased sensitivity of
T-cells to the inhibitory effects of suppressor cells may
account for the persistent depressions of T-cell func- the protective effect of a Brucella vaccine [423] led to
tions in cured HD patients [535]. widespread clinical investigation of the immunomodu-
latory activity of both tetramisole and levamisole. In
various animal models, and also following in vitro incu-
Treatment of Cancer-associated bation with effector cells from human donors, levami-
sole has been found to increase both T-cell numbers and
Immunodeficiency functions (e.d., LPR), if initially depressed [424, 574],
The term immunotherapy was introduced to clinical as well as phagocytic and chemotactic activities of poly-
oncology 2 decades ago following the independent morphonuclear leukocytes and monocytes [111]. Its
observations that Bacillus Calmette-Guerin (BCG) immunorestorative mechanism of action is currently
administration could prolong the survival of patients unknown [220]. It has been shown to induce thymic
with acute lymphoblastic leukemia [326] and induce factor-like activity, which has been attributed to the
regressions of injected as well as noninjected malignant presence of a sulfur atom in its structure [186].
melanoma lesions [357]. Since these reports, a wide Levamisole, which contains an imidazole ring, may
variety of chemicals and biologicals have been adminis- function like imidazole in affecting enzymes that con-
tered to immunosuppressed cancer patients in the hope trol cyclic nucleotide levels in lymphoid precursors of
of reconstituting or boosting host immune mechanisms lymphocytes. Both imidazole and levamisole, which
(Table 1). A broader term, biotherapy, is now being used themselves are not mitogenic, elevate cyclic GMP levels
since there are many biological substances that stimu- in lymphocytes in vitro and enhance their proliferative
late cells outside of those from the immune system. responses to mitogens or foreign antigens [111].
These stimulations with colony stimulating factors, growth Only a limited number of dose- and schedule-seeking
and maturation factors may have secondary effects on trials for cancer patients were performed with levami-
cancer and immune function. sole. In general, it has been administered intermittently,
using two or three daily doses every 1–2 weeks [9]. The
drug is well absorbed, and a single oral dose of 150 mg
Immunorestorative Agents produces a peak plasma level in 2 h (0.49 + 0.05 μg/ml),
which is the concentration required for in vitro activity.
Chemicals
The plasma half-life of levamisole is 4 h. It is widely
Levamisole, an orally active synthetic phenylimidazole, distributed and can be detected in all tissues and fluids,
2,3,5,6-tetrahydro-6-phenyl-imidazol[2, 1-6]thiazole, is with the highest levels in liver and kidneys. It is excreted
the levo isomer of tetramisole, a potent, broad-spectrum primarily in the urine, most of it by 24 h, although much
antihelminthic agent introduced in 1966 [517]. The of the excreted product has already undergone extensive
demonstration in mice that tetramisole could augment metabolic changes.
116 Therapeutic approaches to cancer-associated immune suppression
A number of side effects of levamisole have been
reported [411, 414, 415, 431, 503, 568]. In studies on
3,900 patients with a variety of diseases (including
rheumatic and inflammatory diseases and cancer), reac-
tions included idiosyncratic or allergic ones, such as a
rash or febrile influenza-like illness, sensorineural reac-
tions such as alterations of taste and smell, and gastroin-
testinal symptoms. Rashes and fever resulted in the
cessation of levamisole treatment in 7% and 1.5% of
cases, respectively, but were quickly reversible. The
major serious side effects have been agranulocytosis
and/or neutropenia and, less commonly, thrombocy-
topenia, which have been observed in between 0.2% and
2% of all treated patients. Agranulocytosis could not be
related to dose or schedule of administration, and was
always spontaneously reversible following discontinua-
tion of treatment [9]. The incidence of side effects in
various clinical trials has varied from insignificant to
major, requiring interruption of therapy in up to 21% of
cases [385].
Levamisole was approved for the adjuvant treatment
of stage III colon cancer as an immunomodulator to be
used with adjuvant chemotherapy. It enjoyed some use
for several years in this setting, but is no longer used
because of improved regimens for the adjuvant treat-
ment of stage III colon cancer.
Isoprinosine (IPS), a synthetic antiviral agent, is a
complex of inosine and the p-acetamidobenzoate
(PacBA) salt of N,N-dimethylamino-1-propanol (DIP)
is a 1:3 molar ratio [111, 186]. In early clinical trials
with rhinovirus-infected humans, IPS increased the
titers of circulating antiviral antibody, suggesting it had
B-cell immunomodulatory activity [489]. Subsequent in
vivo studies in animal models and in vitro studies with
human PBMC have indicated that IPS enhances T-cell
functions such as LPR to mitogens and alloantigens
[111, 186, 356]. Helper T cells appear to be the main
target for the drug in humans. IPS has also been shown
to induce the appearance of T-cell surface markers in
mouse prothymocytes, similar to that of thymic factors,
as well as increase the proportions of various T-cell sub-
populations following incubation with human PBMC.
In vitro, IPS at a concentration of 100 μg/ml restored
LPRs, NK activity, and monocyte chemotaxis of PBMC
isolated from cancer patients [526]. Because the immu-
nomodulatory activity of IPS is similar to that of a variety
of thymic factors, it has been classified as a thymomi-
metic drug.
Although IPS has been investigated clinically in several
different viral diseases (including human immunodefi-
ciency virus [HIV] infections), only a limited number of
studies have been performed with cancer patients [111,
517]. Extensive tolerance and safety studies have been
conducted in which IPS was administered orally for peri-
ods of 1 week to 2 years at doses of 1–8 g/day. Minimal
side effects have been noted, including transient rises in
serum and urine uric acid levels and, occasionally, tran-
sient nausea associated with higher daily dosages.
Following oral or intravenous administration to rhesus
monkeys, the inosine moiety of IPS is rapidly metabo-
lized, with a half-life of less than 4 h.
Azimexone (BM 12.531) is an orally active aziridine
dye, 2-[2-cyanaziridinyl-(1)-2-[2-carbamoylaziridinyl-
(1)]-propane, which has been found to increase the
number of cytotoxic autoreactive cells. It exhibits anti-
tumor activity in animal models [53, 111, 186] and
exerts a variety of immunomodulatory effects on T cells,
monocytes, phagocytic cells, and NK cells. Azimexone
has exhibited immunorestorative activity in various ani-
mal models of infectious diseases. In vitro studies using
PBL from advanced cancer patients revealed that
azimexone at various concentrations (0.2–10 μg/ml)
enhanced the LPR to PHA with a maximal effect at
0.2 μg/ml. Other studies have indicated that azimexone
also enhances the percentage of activated T lympho-
cytes in vitro.
In a limited number of clinical studies, the only sig-
nificant side effect observed with intravenous adminis-
tration has been a dose-dependent self-limiting hemolysis
[358]. Oral absorption of azimexone is almost complete,
and the serum half-life is 6 h.
Histamine Receptor Antagonists
Cimetidine is a histamine type II receptor antagonist
widely used for the treatment of gastrointestinal ulcers.
A growing body of evidence has suggested that suppres-
sor T cells that possess histamine receptors (H2 type)
may play an important immunoregulatory role in nor-
mal immunologic responses [88, 149, 377]. The ratio-
nale for administering H2 blockers to cancer patients is
based on the observation that cimetidine could abrogate
in vitro histamine-induced suppressor T-cell activity
using human PBL. Results of in vitro preclinical testing
with cimetidine have been summarized [329]. It has
been shown to augment LPRs and IL-2 production of
PBMC from cancer patients to mitogens and alloanti-
gens [148, 289, 513]. In vitro cimetidine also enhances
NK-cell activity of PBMC from cancer patients, proba-
bly through its inhibitory effects on suppressor cells
[156]. No effects have been seen on transformation of
peripheral blood monocytes to macrophages [169].
Cimetidine was employed in a phase I/II study [323]
and in combination with coumarin [322, 518, 519].
Varying degrees of antitumor activity have been noted
Robert K. Oldham
in patients with malignant melanoma [518, 519] and
renal cancer [322] treated with the combination of
cimetidine plus coumarin. The mechanism of antitumor
activity has not been established but the doses used are
approximately 2x that for treatment of ulcers.
Histamine has been shown to suppress human LPR to
mitogens, and cimetidine might block the inhibition.
More recently, histamine was used in clinical trials as a
drug to assist IL-2 induced T cell function with some
evidence of useful clinical effects in melanoma metastases
to the liver. The clinical effects were marginal.
Nonsteroidal Anti-inflammatory and Antipyretic
Agents (NSAID)
Indomethacin and ibuprofen are prototype inhibitors of
prostaglandin synthesis. A large number of in vitro
studies using PBMC from patients with solid tumors
and HD have suggested that (a) the depressed LPRs of
cancer patients result, at least in part, from the immu-
nosuppressive effects of prostaglandins released by
activated monocytes/macrophages, and (b) indomethacin
is capable of abrogating the suppressor cell influence.
In addition, it has been shown in vitro that indometha-
cin also acts directly on T cells of patients with malig-
nant melanoma to augment mitogen responsiveness
[520]. It has been postulated that this direct immuno-
modulatory action results from specific pharmacologic
effects such as alteration of intracellular cyclic AMP
levels. Finally, a correlation has been observed between
tumor spread and content of prostaglandin E2 of the
tumor [42]. These observations have formed the basis
for administering prostaglandin inhibitors to cancer
patients [96, 405, 563].
NPT 15392
Studies with IPS suggested that the inosine moiety
might be responsible for its immunomodulatory activity
on LPRs. Subsequently, a number of purines with inos-
ine-like structures were synthesized, one of which is
NPT 15392 (9-erythro-2-hydroxy-3-nonylhypoxan-
thine) [157, 186, 578]. In animals, NPT 15392 has
exhibited effects on T-cell and monocyte/macrophage
functions and T-cell differentiation similar to those of
the parent compound [157]. Toxicological studies have
shown that it is nontoxic at oral doses up to 35 mg/day.
This drug has been tested in clinical trials in cancer
patients [575].
DTC, sodium diethyldithiocarbamate (Immunothiol),
was developed as a result of preliminary studies with
levamisole in an attempt to synthesize a chemically
defined sulfur-containing compound that would be la
more potent immunorestorative agent than the parent
117
compound [186, 422]. DTC is a chelating agent in use
for the treatment of heavy-metal poisoning. In animal
models, it exhibits a variety of immune-augmenting
effects on T-cell dependent immune responses as well as
on the induction of T-cell differentiation. No significant
toxicity has been noted following long-term administra-
tion. Clinical trials in cancer patients and in patients
with HIV disease have not been promising.
Coumarin (1,2 benzopyrone) has been reported to
exhibit immunomodulatory activity [49, 581]. Unlike
warfarin, coumarin is devoid of anticoagulant activity. In
cancer patients, coumarin administration was reported to
enhance LPRs to PHA but did not effect T-cell numbers
[49]. In vitro coumarin has augmented HLA DR expres-
sion and NK activity [581]. It has been administered in
combination with cimetidine to patients with malignant
melanoma [518, 519] and renal cancer [322] with varying
degrees of antitumor activity reported. The mechanism of
antitumor activity of the combination has not been
established.
Copovithane (BAYi7433) is a copolymer of 1,3-bis
(methyl amino carboxy)-2 methylene propane carbamate,
has exhibited antitumor activity in a variety of pre-clinical
models [458]. A phase I trial in advanced cancer patients
using weekly intravenous dosing revealed minimal
fatigue, and occasional nausea and proteinuria, as the
only side effects, some antitumor effects, and some
improvements in Tind/Tc/s and in vitro toxicity responses
and LPRs [233].
Biologicals
Thymic Factors
A functioning thymus gland is an essential requirement for
the normal development and maintenance of cell-mediated
immunity [73]. The thymus is responsible for the nor-
mal maturation of all the various subclasses of
T-lymphocytes, including various effector cells, as well
as immunoregulatory cells. The thymus exerts its influ-
ence during the ontogenesis of the immune system of
releasing, in situ from its epithelial stroma, a variety of
differentiating factors that induce the maturation of resi-
dent pre-T stem cells (thymocytes) into mature T cells,
which ultimately circulate in the peripheral blood and
lymph. It is now well established that the thymus gland
is an endocrine organ and that at least several of its
locally released differentiating factors are also secreted
into the bloodstream. Thymic hormone-like bioactivity
has been demonstrated in the blood of animals and
humans. This activity decreases following thymectomy,
as well as with age, in parallel to the physiological age-
dependent involution of the thymus. The presence of the
118 Therapeutic approaches to cancer-associated immune suppression
thymus is important well into adulthood in order to
maintain immune T-cell mechanisms.
A number of factors with thymic hormone-like activity
have been prepared from thymus tissue and blood, and
these are in varying stages of characterization [173, 372,
463, 478, 485]. The best studied are thymosin fraction 5
(TF5), thymosin α1 (Tα1), prothymosin α (Pro Tα), thy-
mostimulin (TP-1), thymulin (FTS-Zn), thymopoietin
(TP), thymic humoral factor (THF), and thymic factor x
(TFX). These agents all exhibit a broad spectrum of
immunorestorative effects on T-cell numbers and func-
tions in animal models and humans. In some bioassays,
many of the well-characterized thymic preparations have
identical, or even opposite, effects. An increasing num-
ber of thymic factors have been employed therapeuti-
cally in treating a variety of diseases – predominantly
cancer and HIV disease [134].
The well-characterized thymic preparations are listed
in Table 2 [173, 192, 463]. Among the thymic factors
that have been administered to cancer patients are TF5,
Tα1, TP-1, THF, and TFX. Both TF5 and TP-1 are par-
tially purified extracts of calf thymus glands. They
include a mixture of different biologically active as well
as inactive polypeptides. The purification procedures
for TF5 and TP-1 are similar but not identical. TF5 con-
sists of ten major – and at least 30 minor – polypeptides
on analytical isoelectric gel focusing with molecular
weights ranging from 1,000 to 15,000. The first biologi-
cally active polypeptide isolated from TF5 is Tα1.
Biologically active Tα1 has a molecular mass of 3,108
daltons. It has been synthesized by classical chemical,
Table 2. Chemical properties of thymic hormones
Name Chemical properties
Thymosin fraction 5 (TF5) Heat-stable, acidic Peptides MW
1,000–15,000
Thymosin α1 (Tα1) Peptide of 28 residues, MW 3108
Prothymosin α (Proα) 113 Amino acids, MW 13,500
Thymosin α9 Acidid peptide, MW 2000, pl 3.5
Thymosin α11 (T α11) Peptide of 35 residues, 28 residues
identical
To Tα1
Thymosin β3 (T β3) Peptide of 49 residues, MW 5700,
43 residues
Identical to Tβ4
Thymosin β4 (Tβ4) Peptide of 43 residues, MW 4963
Thymic factor X (TFX) Mixture of peptides; active
compound is a peptide, MW 4200
Thymostimulin (TS/TP-1) Mixture of peptides
Thymulin (FTS-Zn) Nonapeptide, MW 857,
Thymic humoral factor Peptide MW 3200
(THF)
Thymopoietin (TP) Peptide of 49 residues, MW 5562
solid-phase, and recombinant DNA techniques, but only
the chemically synthesized material has been employed
in clinical trials. Thymopoietin, THF, and TFX are purified
thymic peptides with molecular weights ranging from
3,220 to 5,562. TP-5 is a biologically active synthetic
pentapeptide that represents amino acids 32–36 of thy-
mopoietin. In contrast to all other thymic preparations,
thymulin (FTS-Zn) has usually been isolated from pig
blood rather than thymus tissue. Although thymulin,
Tα1, and thymopoietin are all detectable in the blood,
only thymulin levels drop significantly following
thymectomy, and are restored by thymic grafts.
None of the well-characterized thymic peptides exhibit
any significant homology with the other characterized pep-
tides. However, a 50% homology has recently been identi-
fied between a 35 amino acid region of Tα1 and that of the
p17 core protein of the AIDS retrovirus (HIV) [449].
Thymic factors, which have been administered to
more than 1,000 cancer patients, have shown minimal
toxicity, but some have been approved for general clinical
use. The purified preparations that have been adminis-
tered by intramuscular or subcutaneous injection have
not produced any significant side effects [173, 463].
Partially purified bovine preparations, such as TF5, have
produced rare (less than 1%) allergic reactions and ery-
thema and/or pain at the sites of injection in about one
third of treated patients. However, potentially immuniz-
ing treatment schedules, such as daily injections for 1
week followed by 3 weeks of rest, and reinstitution of
treatment, have been associated with a high (10%) inci-
dence of anaphylactoid-like reactions. As a single agent,
TF5 has exhibited minimal antitumor activity in patients
with renal cancer in one study [465] and none in another
[135]. More recently, thymic factors have been used less
due to the availability of specific lymphokines with
more powerful activities.
T-cell Reconstituting Factor
This is a highly purified protein fraction isolated from
human serum that has been shown to exhibit immuno-
modulatory effects on T-cell numbers and functions
[364]. In preliminary phase I/II trials, no unexpected tox-
icities have been observed following SQ administration.
TsIF is a thymic isolate, distinct from other thymic
factors and cytokines, that induces immature bone mar-
row cells to differentiate into competent suppressor T
cells. It has a molecular mass of 75,000 daltons as deter-
mined by gel filtration and high-performance liquid
chromatography. Recent studies in mice indicated that
TsIF administration suppresses the development of
autoimmune disease in lupus-rheumatoid arthritis-prone
animals [341].
Robert K. Oldham
Transfer Factor
In 1955 it was demonstrated in humans that the transfer
of DTHS to streptoccal M substance and tuberculin could
be accomplished by administration of a suspension of
leukocytes disrupted by either distilled water or repeated
freeze-thaw cycles [294]. The substance (or substances)
responsible for this transfer was resistant to RNAase and
DNAase and was termed transfer factor. This initial work
was extended using dialyzable human leukocyte extracts
from patients sensitized to various antigens [25], includ-
ing skin allograft antigens [493]. The crude dialyzable
preparation has subsequently been shown to enhance
LPRs in vitro [24, 142]. The further chemical character-
ization of transfer factor has been hampered by the lack
of a unique biological assay to monitor final purification.
Preliminary fractionation studies of human leukocyte
extracts capable of transferring DTHS responses have
indicated that transfer factor is probably a low-molecular-
weight material (approximately 1,000) with the electro-
phoretic mobility of slow γ-globulin but with no reactivity
to anti-immunoglobulin antisera. Its possible composi-
tion, that is, a short polypeptide chain joined with a three-
or four-base segment of RNA, has been difficult to verify.
Unlike other nonspecific immunorestorative BRMs,
transfer factor appears to exert antigen-specific immune-
restorative effects.
Despite the lack of readily reproducible in vitro or
in vivo assays, the effects of transfer factor have been
studied following subcutaneous administration to patients
with a variety of immunodeficiency diseases, including
cancer [354].
Interleukins
Many of the BRMs currently being synthesized by recom-
binant DNA techniques are products of lymphocytes
(lymphokines), monocytes (monokines), or other cells
(cytokines). Various interferons and interleukins have
been purified to homogeneity produced by genetic engi-
neering to make large quantities available for clinical tri-
als. Various other cytokines are undergoing active clinical
investigation [187]. The characteristics and results of clini-
cal trials with these materials are discussed in Chapter 8.
Retinoids
Considerable interest has recently been focused on the
influence of vitamin A (retinol) and its natural and syn-
thetic derivatives (Aretinoids) on the growth and differ-
entiation of neoplastic cells. Although retinoids have
been administered to cancer patients primarily as
chemopreventive agents [74, 308], accumulated evi-
dence also indicates that they have beneficial effects on
the host immune system. For example, in animals they
119
have been shown to restore PHA responsiveness, to
induce augmentation of cytotoxic and helper T-cell
numbers and functions, and to inhibit prostaglandin
synthesis by host tumor-activated macrophages.
However, the role that altered immune mechanisms play
in the chemopreventative action of retinoids, such as
13-cis-retinoic acid, remains unclear (see Chapter 16).
FK-565 [heptanoyl-8-D-Glu-(L)meso-α1-A2pm(L)
AlaOH] is a heptanoyl tripeptide analogue of FK-156, a
biologically active acylpeptide isolated from fermenta-
tion products of Streptomyces. Preclinical studies in mice
have shown that FK-565 augments NK-cell activity and
macrophage functions as well as T-cell functions both in
vitro and in vivo [507]. However, in animals, mitogen-
induced IL-2 production is decreased [4]. Clinical trials
in cancer patients have not been promising.
Bestatin [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
L-leucine] is a low-molecular-weight immunomodifier
found in supernatants of Streptomyces olivoreticuli
[327]. It is a competitive inhibitor for the enzymes
aminopeptidase B and leucine aminopeptidase. These
enzymes are associated with the outer membranes of
most mammalian cells, including lymphocytes. In ani-
mal models, bestatin augmented both humoral as well
as cell-mediated immune responses [60, 69], particu-
larly in immunosuppressed mice. Macrophage activa-
tion, but not NK activity, was also observed both in vitro
and in vivo [506]. It has also been shown to enhance
T-cell numbers and cytotoxic functions following in
vitro incubation with human lymphocytes, but it has not
significantly influenced LPR [69]. It has been shown to
augment phagocytic cell function in vitro.
Bestatin has now been chemically synthesized and
is in clinical trials. The drug has been well tolerated in
cancer patients at daily doses of 30 mg for up to 2
years [60, 69, 369, 576], but has shown little clinical
activity.
Tuftsin is a naturally occurring tetrapeptide (Thr-Lys-
Pro-Arg), found normally in human and animal plasma,
and represents residues 289–292 of the heavy chain of
γ-globulin [121, 157, 365, 395]. It is released enzymati-
cally from a protein carrier (Leukokinin) and is capable
of activating various functions (particularly phagocyto-
sis) of polymorphonuclear leukocytes and monocytes/
macrophages, T-cell cytolytic activity, and IL-2 produc-
tion at physiologic concentrations [125, 327]. It has also
been shown to enhance antibody production to thymic-
dependent as well as thymic-independent antigens in
animals and Ia-suppression. It has also been able to
reduce tumor necrosis factor in animals [571].
Lentinan is a purified polysaccharide obtained from
the extracts of the edible mushroom Lentinus edodes
120 Therapeutic approaches to cancer-associated immune suppression
[66, 110, 243, 373]. Chemically, lentinan is a β-(1,3)-
glucan with some β-(1,6)-glucoside side chains. It has a
molecular weight of about 500,000. Evidence has been
presented that lentinan is a T-cell adjuvant, but a variety
of immunomodulatory activities have been observed
[109, 327]. In animal models, it has been shown to aug-
ment antibody production only in the presence of T
cells. It has also been shown to augment a variety of cyto-
toxic effector-cell mechanisms following administration
to animals and to trigger production of various kinds of
serum factors including IL-1, CSF, and IL-3 [109]. In
vitro, it activates monocytes/macrophages and NK-cell
activity of PBMC from cancer patients but does not
enhance LPR or LAK-like cytotoxicity [263].
OK-432 is a heat-killed substrain of Streptococcus
pyogenes that has been studied extensively in Japan.
OK-432 predominantly acts by augmenting NK-cell
activity [378, 539, 545], as well as augmenting mac-
rophage [269] and T-cell [223] cytotoxicity. It has also
been shown to induce various cytokines such as inter-
feron, IL-1, and IL-2 [242, 368, 443]. A variety of
Japanese phase I, II, and III studies have been performed
in cancer patients in which it has been administered intra-
dermally [106, 115, 339, 545], intramuscularly or intral-
esionally [539]. The major toxicities have been fevers
and local inflammatory reactions at injection sites.
ImuVert is prepared from the bacterium Serratia
marcesseus. Its primary components are vescicles
derived from the bacterial membrane and ribosomes
[554]. It augments natural killer-cell activity and has
antitumor cell activity in animals. It has demonstrated
poor activity in clinical trials in patients with malignant
gliomas and other cancers.
Imreg-1 is a natural, leukocyte-derived immunomod-
ulator containing two low-molecular-weight peptides: a
dipeptide (tyrosine-glycine) and a tripeptide (tyrosine-
glycine-glycine). In vitro Imreg-1 enhances the production
of various cytokines, including IL-2 [176]. Clinical trials
to date have been limited to patients with HIV disease
and have not shown much promise.
Treatment of Cancer-associated
Immunosuppression: Phase I and II Studies
Most of the agents described in the preceding section
have already been employed therapeutically in patients
with cancer. Of the chemical BRMs, levamisole has
been by far the most exhaustively studied, whereas
among the biologicals, the interleukins have received
the most attention. No organized approach has been
employed for the clinical evaluation of the immu-
norestorative BRMs. In general, a small phase I or phase
II study to assess the tolerability and immunomodula-
tory effects of the agent in advanced cancer patients has
been followed by phase III trials with random experi-
mental designs in which survival was the end point and
immunological monitoring was minimal, or even omitted.
Thus, it has been difficult to make any firm conclusions
concerning the immunorestorative properties of these
agents in cancer patients.
A number of preliminary small-scale phase I and II
studies have indicated that levamisole [111, 302, 349,
366, 558], isoprinosine [111, 356, 403], azimexone [53,
388], bestatin [69, 288, 328, 369, 560, 576], OK-432
[530, 545], retinoids [337], NPT 15392 [575], DTC
[422], coumarin [49], various thymic factors [173, 463],
lentinan [245], cimetidine [289, 329, 426, 488], and
transfer factor [493] could improve T-cell, NK-cell, or
B-cell numbers and/or functions in advanced cancer
patients with pretreatment abnormalities. In general,
however, the effects have not been striking. When
administered to patients without immunodeficiencies,
however, therapy was often followed by a deterioration
of immune competence. In several reports, no immuno-
modulatory effects of levamisole were observed on
T-cell numbers or functions [200, 558]. A study of
patients with colorectal cancer suggests that the major
in vivo effect of levamisole is augmentation of mono-
cyte chemotaxis [179]. Of the thymic factors, THF and
TFX have been employed predominantly in patients
with infectious complications, and reports have been
mostly anecdotal. Cimetidine has increased an in vivo
local graft-versus-host reaction of advanced cancer
patients [508]. However, in other studies, it had no
effects on immune cell numbers or functions, or on DTH
[316]. Similarly indomethacin has been of only limited
utility in restoring immunity in advanced cancer patients
[219]. The administration of cimetidine or ranitidine to
advanced cancer patients has been associated with
improvements in performance status [92]. In one study,
treatment with the combination of coumarin plus cime-
tidine increased the percentage of monocytes and of
DR+ monocytes of treated patients [323].
Chemicals
Hodgkin’s Disease
HD was chosen because of the well-documented abnor-
malities of T-cell immunity that are present prior to treat-
ment and that persist in patients during clinical remission.
Levamisole has been shown in vitro to enhance T-cell
proportions [50, 451] among PBMC of HD patients.
Treatment of patients with HD in remission for less than
2 years after the completion of radiotherapy resulted in
Robert K. Oldham
improvement in T-cell percentages and functions [301].
DTH reactions and PHA responses also increased [50].
Continuation of treatment beyond 3 months led to a
slight but consistent decline of these parameters.
Solid Tumors
A pilot study was performed with levamisole in surgi-
cally resected patients with malignant melanoma and
squamous-cell carcinoma of the head and neck [57].
Patients treated with levamisole (150 mg orally twice a
week) for 6 months exhibited improvements in absolute
circulating T-cell levels, and only one of eight mela-
noma patients treated with levamisole relapsed. Based
on these results, large-scale phase III were designed, the
results of which have been reported as negative in one
and marginally positive in the others [412, 491].
Based on phase III data in resected colon cancer,
levamisole was approved for use with 5-fluoruracil as
adjuvant treatment of resected stage III colon cancer.
However, more active regimens have recently been
available, making levamisole little used in the current
treatment of colon cancer.
Biologicals
Hodgkin’s Disease
In one study, 19 consecutive untreated patients with
HD received TP-1 at a dose of 1 mg/kg daily for 7 days,
and immunological monitoring was performed prior to
treatment and again on day 8 [324]. A majority of
untreated patients exhibited depressed T-cell percent-
ages as well as depressed DTHS and LPRs to PHA. The
mean T-cell percentage increased significantly, from
47% to 55.7% (normal is 58.9%) following treatment
with TP-1, whereas the mean PHA response improved
but did not totally normalize. Similarly, DTH responses
to recall antigens were positive in 53% of patients
before therapy, and in 95% after therapy. The patients
most likely to respond to TP-1 were the most lym-
phopenic, for whom an in vitro enhancing effect of
TP-1 was observed on T-cell percentages and LPRs of
PBMC prior to therapy. In a similar study, 15 patients
with HD (in remission and off therapy for at least 1
year) were treated with 50 mg TP-1 by daily intramus-
cular injections for 60 consecutive days [303]. For
patients with initially depressed T-cell numbers, includ-
ing depressed helper (Tind) cell numbers, reconstitution
to normal occurred within 30 days after the onset of
therapy, but returned to pretreatment levels after the
discontinuation of therapy. There was also a significant
increase in NK activity, but in vivo DNCB reactivity
did not become positive in any patient who had been
121
negative prior to treatment. In a more recent report from
the same group, 19 patients in remission for at least 6
months were randomized to receive TP-1 at 50 mg IM
daily, every other day for 35 days, or no treatment
[304]. Patients who received TP-1 were then main-
tained on TP-1 twice weekly. Before treatment, patients
exhibited depressed percentages and absolute numbers
of circulating T cells and Tind cells. Following 5 weeks
of daily TP-1, the proportions and numbers of all T-cell
subpopulations increased significantly, while alternate-
day treatment was not as effective. Maintenance TP-1
therapy did not produce any further improvements in
T-cell or B-cell numbers or proportions. PBMC from
some patients also exhibited improvements in mitogen-
induced lymphokine production (e.g., IL-2, IFN-γ), but
the improvements were not statistically significant
overall. No significant changes were observed in a vari-
ety of serum markers including neopterin and
β2-microglublin. It was concluded that TP-1 has the
potential to expand the T-cell pool, and, in particular,
Tind cells, in patients with HD in remission, but that
intensive (daily) induction therapy is required. In this
small sample, no conclusions could be established and
large-scale clinical trials have not been done.
Malignant Melanoma
TP-1 has been studied in a novel random-design trial
involving 32 patients with localized stage I malignant
melanoma who were rendered disease-free following
surgery [51]. Subjects were selected from a cohort of
211 postsurgical patients who were monitored at
3-month intervals in the first 2 years after surgery. In
prior work, it was demonstrated that patients who devel-
oped low circulating T-cell levels were at high risk for
relapse. Patients who either exhibited presurgical
depressions of total T-cell numbers or on serial monitor-
ing developed a depression of absolute circulating
T cells to less than 1,000/mm3 were considered eligible
for random assignment to one of three treatment arms:
TP-1 alone (25 mg IM once a week, eight patients); che-
motherapy alone (DTIC, 200 mg/m2 intravenously for
5 days, repeated monthly, eight patients); or no further
therapy (16 patients). In the eight patients who received
TP-1, T-cell numbers began increasing within 3 days
after the first injection, and weekly immunological
monitoring revealed that levels returned to normal and
were maintained at that level. Only inconsistent effects
on T-cell functions were seen in the group that received
TP-1, and no changes were observed in any parameter
in patients who were untreated or who received chemo-
therapy. Overall survival was better for the TP-1 group,
but the improvement was not statistically significant.
122 Therapeutic approaches to cancer-associated immune suppression
Chronic Lymphocytic Leukemia (CLL)
Patients with Cll have also been treated with TP-1 [290].
TP-1 significantly increased Tind-cell proportions, lead-
ing to an improvement in the Tind/Tc/s ratio accompanied
by an increase in LPRs to PHA and helper-cell function.
In this study, the in vitro incubation of purified T-cells
with TP-1 did not produce modifications of subset pro-
portions or of immune functions. In contrast, in vitro
incubation of T-cells with Tα1 from patients with stable-
phase CLL led to an increase in T-cell proportions and
an improvement in T-cell functions [41].
Phase II/III Surgical Adjuvant Studies
Chemicals
Lung Cancer
The vast majority of large-scale phase III surgical adju-
vant studies have been performed with levamisole. Two
randomized, double-blind trials in patients with non-
small-cell lung cancer have been reported [10, 16]. In
the first trial, a fixed dose of levamisole was adminis-
tered preoperatively for 3 days, and then for 3 days
every 2 weeks [10]. Treatment was continued for 2
years or until relapse. Among 211 patients, although
there was no difference in overall survival, levamisole
treatment was associated with a reduction in distant
metastases and in cancer deaths. When analyzed on the
basis of adequacy of drug dosage, it was found that the
greatest benefit of levamisole was seen with patients
who received a daily dose of 2.1 mg/kg or more. The
second study was designed as a confirmatory trial, and
a weight-related dose of levamisole was employed,
with a schedule identical to that in the original study
[16]. Nevertheless, among the 217 evaluable patients,
overall survival was decreased in those who received
levamisole, because of non-cancer-related deaths in the
perioperative period.
Gastrointestinal Cancer
In the early 1980s, results of several small-scale trials
were reported that suggested that levamisole could pre-
vent perioperative immune suppression. A British report
in 1979 first indicated that the administration of levami-
sole on 3 postoperative days could accelerate the recov-
ery of antitumor immunity (in an LMI assay) but not
LPRs to PHA [570]. In a Japanese trial of 50 patients with
colon cancer, 15 were treated with levamisole at a dose of
150 mg daily for 2 consecutive days, every other week,
beginning 3 days before surgery. Treatment with levami-
sole appeared to prevent the postoperative depression of
LPRs to PHA, as compared with a control group of
patients, but did not affect DNCB reactivity [477]. A
Belgian colorectal trial used levamisole (150 mg for 3
days every 2 weeks) in a matched group of patients oper-
ated on by the same surgeon. In both the overall group
and the 40 patients with Dukes B2 and C cancers, survival
was prolonged with levamisole [538]. Improved survival
of resected patients was reported in a broad trial of 177
patients with various gastrointestinal cancers treated with
levamisole [347]. There was also improvement in DTHS
reactions and in vivo LPRs. Similar results from the
Japanese literature have been reported for patients with
advanced gastric cancer treated with levamisole [348].
An interesting recent report of a randomized trial
involving 181 resected patients with gastric cancer
showed an increase in median survival for patients who
received cimetidine [524].
Phase III Levamisole Trials in Colon Cancer
Within the past decade, results of a series of phase III
trial results have been reported, in which levamisole
was administered alone, or in combination with adju-
vant chemotherapy in patients with resected colorectal
cancer [23, 32, 113, 244, 292, 293, 351, 474, 569].
A large-scale trial that was designed to compare treat-
ment with levamisole to surgery only reported by the
European Organization for Research and Treatment of
Cancer (EORTC) Gastrointestinal Tract Cancer
Cooperative Group [23]. This double-blind placebo-
controlled trial involved 297 patients with Dukes C
colon cancer. Levamisole dosage was based on body
weight and ranged from 100 to 250 mg/day for 2 con-
secutive days each week for a period of 1 year. Treatment
was started as soon as possible after surgery, usually
within 2 weeks. With a median follow-up time of 3
years, no benefit was seen in disease-free survival for
patients who received levamisole. Although the proportion
of patients alive at 5 years was 51% in the levamisole
group versus 39% in the placebo group, this difference
was not statistically significant.
Other large-scale trials convincingly demonstrated
that postoperative adjuvant therapy with levamisole plus
5-FU impacts significantly on survival of patients with
colon cancer [244, 292, 293, 351]. Thus, levamisole plus
5-FU was considered standard adjuvant treatment for
resected Dukes C colon cancer until folinic acid plus
5-FU proved as effective, less expensive and easier to
administer. Levamisole is currently not used in the treat-
ment of colon cancer or any other human malignancy.
Gynecologic and Urologic Cancer
Positive surgical adjuvant studies have been reported
with levamisole in postoperative patients with cervical
Robert K. Oldham
cancer [447] and bladder cancer [486] but is no longer
being used.
Malignant Melanoma
Only one study has focused on assessing in detail the
immunorestorative effects of levamisole in patients with
locally advanced malignant melanoma following surgi-
cal resection [253]. Levamisole was administered at
150 mg/twice weekly. No improvements were noted in
T-cell numbers, or in LPRs to mitogens or antigens.
A large-scale phase III trial with levamisole involved
203 postsurgical patients with malignant melanoma. No
improvement in either relapse rates or overall survival
was noted in patients who received levamisole [491]. In
stage I patients, there was a trend in favor of levamisole
regarding time-to-recurrence and survival. More
recently, a large study involving 548 patients with com-
pletely resected malignant melanoma having a poor
prognosis (Clark levels 3, 4, or 5, greater than 0.75 mm,
satellite lesions, in-transit metastases, or regional lymph
node metastases) revealed that patients treated postop-
eratively with levamisole (2.5 mg/kg orally on 2 con-
secutive days weekly for 3 years) survived significantly
longer than those who received either no postoperative
therapy or weekly BCG, or a combination of BCG alter-
nating with levamisole [412]. Median follow-up time
for this trial was 5.1 years. In a randomized trial involv-
ing 156 stage I Clark level 3, 4, and 5 patients, there was
a trend for a delay in appearance of distant metastases in
patients receiving levamisole (30 months versus 9
months for patients receiving placebo), but overall sur-
vival was not significantly improved [309].
Biologicals
Lung Cancer
Several studies have focused on the effects of transfer
factor administered as an adjuvant to surgery. A tumor
antigen-specific preparation, prepared from household
contact family members with positive reactivity to lung
cancer antigen, was administered to 28 resected patients
with non-small-cell lung cancer twice by subcutaneous
injection. Treatment was associated with a significant
improvement in a variety of specific as well as nonspe-
cific immune parameters, including DTH reactions and
LPR to PHA [163]. In a randomized study of 63 postop-
erative patients with non-small-cell lung cancer (some
of whom received mediastinal radiotherapy), those who
received transfer factor from normal donors beginning 1
month after surgery and continuing every 3 months
showed a significant improvement in survival at 2 years
compared with nontreated patients [276].
123
Head and Neck Cancer
The impact of preoperative perilesional therapy with
OK-432 was studied in 13 patients [539]. Treatment
was associated with less pronounced decreases in NK
activity of PBMC, and higher NK activity in draining
lymph nodes, than in patients treated by surgery alone.
Malignant Melanoma
TP-1, transfer factor [90], isoprinosine [403], and TF5
[463] have been administered to postoperative patients
with malignant melanoma. Each of these trials involved
random treatment assignments and varying degrees of
immune monitoring. No adjuvant effects could be dem-
onstrated in these small studies. The trial with TF5 was
complicated by the fact that only 45 patients were allo-
cated to treatment with either a low dose (4 mg/m2) or
high dose (40 mg/m2) administered subcutaneously,
concurrently with BCG, with or without DTIC chemo-
therapy. A large-scale, randomized, double-blind phase
III trial that involved 168 resected patients with high-
risk stage I and stage II malignant melanoma has been
reported [342]. Therapy (normal donor transfer factor or
placebo) was initiated within 90 days of resection and
continued for 2 years. With a median follow-up period
of 25 months, patients who received placebo exhibited a
trend for improved disease-free survival and overall sur-
vival. Thus, this large-scale trial has indicated that normal
donor transfer factor is not an effective postsurgical
adjuvant therapy for malignant melanoma.
Treatment of Radiotherapy-induced
Immunosuppression
Although a number of clinical trials using immunorestor-
ative BRMs in irradiated patients have been reported,
little information is available concerning whether or not
treatment can prevent and/or reverse radiotherapy-induced
immunosuppression.
Chemicals
Levamisole
Of the putative chemical immunorestorative agents,
levamisole has been most extensively evaluated as an
adjunct to radiation therapy. Since the larger trials have
focused almost exclusively on survival as an end point,
very little information has been obtained concerning its
immunorestorative properties.
Several of the smaller trials have included serial
immune monitoring. In a study involving 57 patients
with colorectal cancer treated with surgery and radia-
tion therapy, levamisole at a dose of 150 mg/day for
124 Therapeutic approaches to cancer-associated immune suppression
2 consecutive days every other week, beginning concur-
rently with postoperative radiation therapy, enhanced
LPRs to PHA as compared with an untreated control
group [293]; no effects were observed on DNCB reac-
tivities. In a randomized double-blind study involving 71
postoperative patients with stage II breast cancer, 38% of
those who received levamisole (2.5 mg/kg/day on 2 con-
secutive days, twice weekly, beginning with the initia-
tion of radiation therapy) exhibited improvement in
DTHS to at least three recall antigens, as compared with
only 19% of those given a placebo [277]. Similar results
were noted in an earlier trial using DNCB reactivity
[473]. Thus, such studies suggested that levamisole may
improve some aspects of T-cell functions in irradiated
patients.
A number of large-scale cooperative group trials have
now demonstrated that levamisole administration does
not improve the survival of patients treated with radio-
therapy. Four separate negative trials have been reported
involving patients with non-small-cell lung cancer
(NSCLC). The Southeastern Cancer Study Group
reported that levamisole was without significant clinical
benefit in a large randomized, placebo-controlled trial
of 251 patients undergoing radiotherapy for inoperable
non-small-cell lung cancer [285]. In this study, levami-
sole was administered at a dose of 2.4 mg/kg twice
weekly beginning at the initiation of radiotherapy. The
median survival of patients treated with levamisole was
shorter than that of those who received placebo. Negative
results have also been reported in a SWOG similar trial
[566]. The Radiation Therapy Oncology Group (RTOG)
reported results of two separate randomized, placebo-
controlled trials, one involving 74 patients with resected
NSCLC and positive lymph nodes [215], and a second
involving 285 patients with unresectable tumors [395].
Levamisole (2.5 mg/kg on days 1 and 2, weekly) or pla-
cebo was initiated at the beginning of radiation therapy
and continued for up to 2 years. Accrual to the first trial
was terminated prematurely when survival data became
available from the second study indicating a worse survival
(9 months versus 12 months). Thus, it appears to be con-
clusively established that levamisole combined with
radiation therapy has no benefit in the treatment of
NSCLC [400].
In the randomized trial involving 71 stage II breast
cancer patients treated with radiotherapy postoperatively,
those who received levamisole exhibited a slight prolon-
gation of disease-free survival [277]. Among postmeno-
pausal patients, levamisole significantly increased both
disease-free and overall survival, and the levamisole
group showed fewer distant metastases as the first sign
of recurrence. In another trial involving 150 patients ran-
domized to postoperative radiotherapy, chemotherapy,
or both with or without levamisole, patients receiving
radiotherapy plus levamisole exhibited improved disease-
free and overall survival [278, 280]. These results are in
contrast to two other similar studies. In a randomized but
not double-blind study, levamisole treatment was associ-
ated with an increased recurrence rate [127]. In a trial
involving 198 patients with resectable axillary node-
positive disease, levamisole, when begun following
completion of postoperative radiotherapy, had no effect
on either disease-free or overall survival [525]. Nearly a
third of patients treated with levamisole had to terminate
therapy prematurely because of toxicity (primarily leu-
kopenia, skin rash, nausea, fever, and mucosal infection).
It has been argued that differences in patient characteristics
led to these discordant results.
In head and neck cancer, three reports from the same
Italian group have indicated that radiotherapy led to a
decrease in both T-cell numbers and functions (LPRs),
and that treatment with levamisole accelerated the resto-
ration of T-cell counts, compared to patients who
received placebo [26, 27, 382]. With a median follow-up
of 30 months, there was some improvement in disease-
free interval for patients who received levamisole.
Another similar trial failed to demonstrate an improve-
ment in survival for patients with head and neck cancer
treated with levamisole [374].
Isoprinosine
In a double-blind, placebo-controlled trial designed to
evaluate the immunorestorative effects of isoprinosine
following radiotherapy, one half of 106 irradiated
patients with breast, head and neck, or uterine cancers
were randomly assigned to receive isoprinosine or pla-
cebo in discontinuous courses for 5 months. After 3
months of treatment, 64% of the isoprinosine-treated
patients exhibited evidence of improvement of DTHS
and in vitro functional tests, whereas only 23% of pla-
cebo-treated patients did [356]. No correlations have yet
emerged between immune reconstitution and clinical
status.
Prostaglandin Antagonists
The nonsteroidal anti-inflammatory drug oxyphenbuta-
zone was found to improve survival of patients with
stage III cervical cancer when administered during radi-
ation therapy [563]. This trial involved 160 patients.
Thirty of 73 patients with stage III disease were treated
with 100 mg oxyphenbutazone three times daily.
Treatment was associated with an improved survival,
but no studies were performed to assess the drug’s effect
on immune responsiveness.
Robert K. Oldham
Biologicals
Thymic Factors
Several clinical trials with TF5 and Tα1 have indicated
that thymic factors may accelerate the reconstitution of
T-cell functions following radiation therapy to head and
neck or mediastinal portals. The first clinical trial
involved 75 patients with localized but unresectable head
and neck cancer [553]. Patients were randomly assigned
to receive TF5 (60 mg/m2) subcutaneously in a loading
dose (daily for 10 days, then twice weekly) schedule or
no further therapy. Treatment with TF5 began concur-
rently with the initiation of radiotherapy and continued
for 1 year or until relapse. TF5 administration did not
prevent, nor could it restore, the marked T-cell lympho-
cytopenia that followed radiotherapy. Patients treated
with TF5 exhibited a prolongation of disease-free sur-
vival, but of only borderline statistical significance.
Similar findings have been reported with Tα1 in
postradiotherapy patients with non-small-cell lung
cancer [459]. This study was a double-blind, random-
design trial involving 42 patients with localized, unre-
sectable disease who had just completed a course of
radiotherapy to the primary tumor and mediastinum.
Patients who received mediastinal radiation were cho-
sen specifically as the study population, based on an in
vitro study, indicating that TF5 could increase T-cell
proportions of PBMC from patients who had received
mediastinal radiation to other portals [272]. Patients
whose disease regressed or remained stable at the com-
pletion of radiotherapy were randomized to receive
Tα1 (900 μg/m2 subcutaneously) either by a loading
dose (daily for 14 days, then twice a week) or on a
twice-weekly schedule; treatment began within a week
after completion of radiotherapy. All patients exhib-
ited a marked depletion in absolute circulating T-cell
levels and in T-cell LPRs at the completion of radiation
therapy. Serial immunological monitoring revealed
that the patients who received Tα1 had a complete nor-
malization of T-cell function in MLR, which became
apparent following 7 weeks of treatment. However,
only patients treated on the twice-weekly schedule
maintained normal Tind/Tc/s ratios throughout the study
period. The Tα1 administration, however, did not influ-
ence absolute circulating T-cell numbers or T-cell sub-
set numbers, and all treated patients remained
lymphocytopenia over the 15-week follow-up period.
These results were interpreted to indicate that more
intensive schedules of Tα1 administration are optimal
for inducing restoration of T-cell functions, whereas
less intensive schedules are optimal for maintaining
immune balance of T-cell subsets.
125
Transfer Factor
In a randomized, double-blind trial involving 100
patients with nasopharyngeal carcinoma, transfer factor
(derived from young adults with a proven history of
infectious mononucleosis and from normal blood donors
with high anti-Epstein-Barr virus capsid antigen anti-
body levels) was administered for 18 months in con-
junction with radiotherapy [170]. This trial was based
on the association of Epstein-Barr virus with nasopha-
ryngeal cancers. No significant effects of transfer factor
were noted on disease-free and overall survival. Immune
competence studies were not performed.
A study was reported involving 111 patients with
NSCLC who had surgery followed by radiotherapy [93].
Twenty-six patients received transfer factor (normal donor)
bimonthly beginning after RT was completed. Increased
DTHS and a lower relapse rate were observed in patients
who received transfer factor, but the small sample size of
this trial precludes any definitive interpretations.
In a small randomized, placebo-controlled trial of 47
patients receiving radiotherapy with or without chemo-
therapy for Hodgkin’s disease, DTHS skin responses
were markedly enhanced in 22 patients who received
transfer factor (prepared from normal donor buffy coat
cells), but no improvements were noted in a variety of
other immunologic parameters, nor in the prevention of
infection, including varicella/zoster [198].
T-cell Reconstituting Factor
A pilot clinical trial has been performed in which this
factor was administered by SQ injection (2 mg/m2 TIW)
for 1 month to 11 patients who had just completed radio-
therapy for a variety of locally advanced solid tumors. A
variety of immune parameters were followed serially
and compared to those of five patients randomized not
to receive treatment. Treatment produced a generalized
lymphocytosis involving both Tind and Tc/s cell numbers,
but no effects were noted on DTHS or LPRs at the dose
and schedule employed [112].
Retinoids
Therapeutic and immune restorative effects of vitamin
A have been evaluated in a randomized trial of 42
patients undergoing radiation therapy for inoperable
cervical carcinoma [338]. Vitamin A palmitate was
administered orally at a daily dose of 1.5 × 106 IU on
days 1–5, 8–12, 16–20, and 23–27, and radiotherapy
began on day 22 for 8 weeks. Vitamin A was well toler-
ated, although most of the 21 treated patients developed
scaling of the skin. Serial immunological assessments
were performed on some of the patients. No effects were
found on DTHS reactivity, in that only about 50% of
126 Therapeutic approaches to cancer-associated immune suppression
patients reacted in both the treatment and control groups.
Vitamin A treatment increased in vitro LPRs (albeit to a
low degree) from pretreatment values, whereas the con-
trol patients showed no change or decrease in LPRs dur-
ing radiation treatment. Thus, it was concluded that
vitamin A administered concurrently with radiation
could prevent the radiation-induced depression of at
least one T-cell function.
Bestatin
Several studies have explored the in vivo effects of
bestatin in irradiated cancer patients [61, 66, 70, 373].
In a prospective randomized trial, the clinical efficacy
of bestatin was evaluated in 151 evaluable patients who
had completed a course of local radiotherapy for blad-
der cancer. Patients were randomly assigned to receive
10 mg bestatin orally, three times daily, for at least 1
year, or no further treatment. The recovery of LPRs to
PHA and PPD proceeded at an accelerated rate for 9
months in the bestatin-treated patients [70]. Patients
treated with bestatin exhibited a greater percentage of
circulating T cells after 1 month of treatment, but levels
declined to control values within 3 months. Similar
transient changes were noted in NK-like activity. It was
concluded that the schedule or dose of bestatin was not
optimal, and that either higher doses or intermittent
schedules require evaluation. An update of the trial has
shown that the bestatin-treated patients exhibited an
improvement in disease-free survival compared to the
radiotherapy-only group, but no improvement in overall
survival [65]. The disease-free survival benefit was
more apparent in patients with earlier stages of disease.
OK-432
A large multicenter trial in Japan randomized 382
patients with cervical cancer, stratified by presence or
absence of surgery and clinical stage, to radiotherapy
with or without intradermal OK-432 starting concur-
rently at 2-day intervals with the initiation of radiation
treatment [106], and continuing biweekly for 2 years.
Patients who received OK-432 exhibited a decrease in
3-year recurrence rate, a more rapid restoration in DTHS
to PHA, a-polysaccharide antigen, and peripheral blood
lymphocyte counts than the control patients.
Combined-modality Studies with
Chemotherapy
Three major problems in combined-modality studies
(Table 3) have made it difficult to draw conclusions
concerning the influence of putative immunorestorative
agents on chemotherapy-induced immunosuppression:
(a) the changing and varied combination chemotherapy
regimens available for clinical use; (b) the lack of well-
defined doses and schedules of administration for the
immunorestorative agents; and (c) the almost universal
omission of detailed serial immunologic assessments
of patients receiving treatment. For the most part, these
studies have emphasized conventional chemotherapy
parameters as their end point, that is, tumor regression
rates and overall patient survival, on the assumption
that if improvements were noted in patients who
received an immunorestorative agent, they would result
from undefined immunomodulatory effects on the
immune response.
Advanced Disease
Three broad approaches are feasible for the treatment of
advanced cancer patients with combined chemotherapy-
immunorestorative therapy. The putative immunorestor-
ative agent could be administered concurrently with
chemotherapy, following the completion of chemotherapy
when clinical remission has been achieved, or during
maintenance chemotherapy if it is continued. All three
approaches have been applied, and most studies have
employed levamisole and/or thymic factors.
Chemicals
Levamisole
This compound has been evaluated in several large-
scale clinical trials as an adjunct to conventional che-
motherapy, with mixed results. In general, levamisole
has been administered as either 150–200 mg or 2.5 mg/
kg on 2 consecutive days each week between chemo-
therapy courses. Serial immunological studies have
usually not been performed. In 82 patients with meta-
static colorectal cancer, survival of those receiving
5-FU and levamisole was significantly greater than
those receiving 5-FU alone [78]. Improvements in
response rates and/or survival have also been seen in
patients receiving levamisole in addition to combina-
tion chemotherapy for advanced breast cancer [278,
281, 497]. In one study, an improvement in DNCB
reactivity was noted with levamisole treatment; how-
ever, negative reports have also appeared concerning
breast cancer [99, 387], colorectal cancer [91], non-
small-cell lung cancer [107, 129], and malignant mela-
noma [122]. A report in 669 patients with non-small-cell
lung cancer indicated that patients who received com-
bination chemotherapy along with levamisole plus
warfarin plus tranexamic acid survived longer than
those receiving chemotherapy alone [473].
Robert K. Oldham 127

Table 3. Summary of combined modality studies designed to treat chemotherapy-induced immunosuppression

Cancer types Outcome References
Advanced disease
Chemicals
Levamisole Colon + [78]
− [91]
Breast + [278, 281, 497]
− [99, 387]
Non-small-cell lung + [475]
− [107, 129]
Melanoma − [122]
Multiple myeloma + [445]
Acute lymphoblastic leukemia + [390, 391]
Acute myeloblastic leukemia − [441]
Isoprinosine Colon − [119]
Azimexone Breast + [111, 286]
Piroxicam Non-small-cell lung + [82]
Cimetidine Ovarian + [295]
Biologicals
Thymostimulin Gastrointestinal + [478]
Melanoma − [51]
Small-cell lung + [314]
Non-small-cell lung − [132, 312]
Thymosin Fr. 5 Small-cell lung + [118]
− [455, 476]
Non-small-cell lung − [40]
OK-432 Non-small-cell lung + [557]
Lentinan Gastric + [231, 504]
Bestatin Acute nonlymphoblastic leukemia + [379]
Adjuvant treatment
Chemicals
Levamisole Colon + [113, 292, 293]
− [244, 351]
Breast + [279, 280]
− [270]
Gastric + [367]
Ovarian − [274]
Biologicals
Thymostimulin Breast + [241]
Transfer factor Non-small-cell lung + [162]
OK-432 Non-small-cell lung + [557]

Levamisole has also been evaluated as an adjunct to
maintenance chemotherapy for a variety of hematologic
malignancies. Improvements in survival from the start of
maintenance chemotherapy have been noted in patients
receiving levamisole in cases of multiple myeloma [445]
and acute lymphoblastic leukemia [390, 391]. Thus,
levamisole did appear to have potential as an adjunct to
maintenance chemotherapy for patients with hematopoi-
etic cancers; however, no effects were observed when
levamisole was administered concurrently with intensive
induction chemotherapy for ANLL [534].
Isoprinosine has been administered concurrently with
intravenous 5-FU at various doses without any observ-
able antitumor effects [119].
Azimexone has been administered to ten patients
with breast cancer after remission was induced by
chemotherapy (CTX, MTX, 5-FU, VCR, predni-
sone) to assess whether it could ameliorate the
immunosuppressive effects of treatment. Detailed
weekly serial immunological assessments, per-
formed while patients were receiving 100-mg weekly
intravenous injections, revealed that chemotherapy-
induced immunosuppression was markedly reversed
during azimexone administration. Significant
increases in peripheral blood lymphocyte counts and
in vitro LPR were noted without change in T- or
B-cell percentages [111, 286].
128 Therapeutic approaches to cancer-associated immune suppression
Prostaglandin Inhibitors
It has been demonstrated that the concurrent administra-
tion of nonsteroidal anti-inflammatory drugs can prevent
some aspects of chemotherapy-induced immune sup-
pression [82].
Cimetidine
A study of the immunomodulatory effects of cimetidine
was performed in patients with advanced ovarian cancer
who received concurrent chemotherapy (cisplatin, ADR,
CTX) [275]. Treatment with chemotherapy produced a
decrease in CD4 cell counts and in IL-2 production of
PBL, which was significantly improved in patients who
received concurrent cimetidine.
Biologicals
Thymic Factors
Two studies performed in the late 1970s with TP-1 and
TF5 suggested both a rationale and role for the use of
thymic factors in conjunction with combination chemo-
therapy [478]. Although overall response rates were not
altered by TF5, its administration at a high dose (60 mg/
m2) led to a significant improvement in overall median
survival [116]. Improved survival was limited to patients
who had exhibited pretreatment depressions of total
T-cell levels below 775/mm3 and serum HS glycopro-
tein levels below 60.5 mg/dl. Although serial immuno-
logical assessments were not performed, it was
concluded that, whereas TF5 had no detectable direct
antitumor effects, its administration to immunosup-
pressed patients may have improved survival by ame-
liorating the immune defects due to the presence of
tumor, and exacerbated by the chemotherapy.
These two reports culminated in the performance of a
number of confirmatory trials, as well as a variety of
other studies in which TF5 and TP-1 were administered
concurrently with combination chemotherapy for
patients with solid tumors, generally small-cell or NSCLC
[40, 51, 132, 312, 314, 455, 476]. In only one of these
studies did the administration of thymic factors improve
the overall response rate to the chemotherapy and sur-
vival [314]. Although this trial involved very small patient
numbers, the favorable effects of TP-1 on both myelotox-
icity and survival indicate that further studies should be
performed adding TP-1 to conventional chemotherapy.
In a randomized trial involving 91 of patients with
small-cell lung cancer [455, 476], TF5 (60 mg/m2 sub-
cutaneously, twice weekly) had no effect on survival
when combined with chemotherapy, compared to che-
motherapy alone (CTX, ADR, VCR alternating with
VP-16, cisplatin). An analysis based on pretreatment
immune function, total white blood cell count, and abso-
lute lymphocyte count revealed no difference in survival
distributions. These results could not confirm the prior
reported study [118]. However, this latter trial differed
from the original study in that it used different chemo-
therapy regimens and included prophylactic chest and
whole-brain radiotherapy for patients who responded to
chemotherapy. Thus, no firm conclusions can be reached
regarding the role of TF5 as an adjunct to chemotherapy
for small-cell lung cancer.
Several different trials in patients with advanced
non-small-cell lung cancer treated with combination che-
motherapy with or without TF5 [41] or TP-1 [132, 312]
and in metastatic melanoma patients treated with TP-1
[51] failed to find improvements in survival for patients
treated with thymic factors. In one of the lung cancer tri-
als, only immunosuppressed patients were considered
eligible for study; however, no significant immunorestor-
ative effects were noted in the TP-1-treated patients except
transient mild improvement in absolute T-cell levels.
In vitro LPR to mitogens remained suppressed following
treatment, and no improvements in DTHS were observed.
Thus, follow-up combined modality studies with TF5
and TP-1 have not provided evidence that thymic factors
could either prevent chemotherapy-induced immuno-
suppression or improve survival.
Lentinan
In small studies, administration of lentinan in combina-
tion with tegafur [504] or 5-FU and mitomycin [231] to
patients with advanced gastric cancer has been reported
to improve patient survival.
Bestatin
In a randomized controlled trial of 101 patients with
ANLL, patients over 50 years of age who received
bestatin along with induction chemotherapy exhibited a
better remission duration and survival than the chemo-
therapy-only group [379].
Adjuvant Chemotherapy
Adjuvant chemotherapy has become an accepted treat-
ment modality for patients with breast and colon cancer.
Evidence is also accumulating that postsurgical adju-
vant chemotherapy for other solid tumors may also
improve patient survival. The acute and chronic immu-
nosuppressive effects of contemporary adjuvant chemo-
therapy regimens have recently been evaluated [315,
396, 501].
Robert K. Oldham
Chemicals
Levamisole
In a randomized trial of 135 postoperative breast can-
cer patients with positive axillary nodes, patients
received L-phenylalanine mustard with levamisole
(150 mg for 3 days every 2 weeks) or placebo for up to
2 years [270]. No significant effects of levamisole were
noted, although trends for improved survival were seen
in postmenopausal patients and in those with four or
more positive lymph nodes. In other studies involving
patients with stage III breast cancer treated by either
radiotherapy or adjuvant chemotherapy (ADR, CTX,
VCR), there was a high incidence of toxicity (primar-
ily transient agranulocytosis), requiring discontinua-
tion of levamisole. Follow-up is continuing on this
trial. In a study comparing postoperative radiation
therapy and adjuvant chemotherapy (vincristine, adri-
amycin, and cyclophosphamide), levamisole adminis-
tered concurrently appeared to improve both
disease-free, as well as overall, survival for patients
who received chemotherapy alone or combined radia-
tion therapy plus chemotherapy [279]. Improvements
were also noted in various immune parameters in the
levamisole treatment group [305]. Although this trial
involved 150 patients, the multiple randomizations
made it difficult to analyze.
In other recent studies, levamisole was shown to
improve the survival of resected patients with gastric
cancer treated with MIT and tegafur [367], but there
was a deleterious effect of levamisole in a trial of 140
patients with ovarian cancer who received adjuvant che-
motherapy following maximal surgical reduction of
tumor [274].
Biologicals
Transfer Factor
The administration of transfer factor, prepared from leu-
kocytes from household contacts, has been reported to
improve survival for stage I and II patients with resected
non-small-cell lung cancer who were treated further
with a variety of adjuvant combination chemotherapy
regimens [162].
OK-432
In a trial of 311 patients, OK-432 was shown to improve
survival of patients with resected stage I, II and III non-
small-cell lung cancer when combined with three-drug
combination chemotherapy, compared to chemotherapy
[557].
129
Thymic Factors
In a randomized trial 51 patients received adjuvant CMF
chemotherapy with or without TP-1 (50 mg/m2 IM daily
× 2 weeks, then twice weekly for a minimum of 3
months) following radical mastectomy for breast cancer
[241]. Although details of the patients’ clinical charac-
teristics were not reported, patients who received TP-1
exhibited a significant decrease in the incidence of
infections (mostly cystitis, conjunctivitis, and mucosi-
tis), and an increase in Tind/Tc/s compared to the control
(no treatment) group. There was also a lower incidence
of myelotoxicity in the TP-1 treated patients. These
results have not been confirmed with a large-scale trial.
Current Status of Therapeutic
Alterations for Cancer-associated
Immune Suppression
The results with levamisole in combination with 5-FU
as adjuvant therapy for Dukes C colon cancer have pro-
vided strong evidence that drugs with immunorestor-
ative properties can play a role in the treatment of human
cancer. What has been learned about the reversibility or
prevention of cancer-associated immunosuppression?
All of the agents discussed in this chapter, at least in
small phase I and II studies, appear to have the capabil-
ity of improving immune cell numbers and/or functions
in immunosuppressed patients. The large-scale phase III
surgical adjuvant trials for the most part have provided
only limited information concerning whether or not
perioperative immunosuppression could be prevented.
Part of this relates to the fact that it is extremely difficult
– and probably impossible – to perform adequate, qual-
ity control assays of immunity in multiple different
institutions participating in the same large-scale trial.
The mechanism by which levamisole improved survival
was not addressed, and it is not clear whether the drug
exerted its effects as a result of immunomodulation or
by other, as yet undefined, mechanisms. Several smaller
studies in patients with GI cancers suggested that certain
aspects of T-cell immunity (i.e., LPR) could be main-
tained during the perioperative period by treatment with
levamisole. Since these studies, FA/5-FU and later
FOLFOX regimens have become the regimen of choice
in adjuvant therapy of colon cancer.
Studies with levamisole, TF5, Tα1, bestatin, and vita-
min A have suggested that various immunorestorative
agents could ameliorate radiotherapy-induced depres-
sion of T-cell numbers or functions, or accelerate the
130 Therapeutic approaches to cancer-associated immune suppression
reconstitution of immunity following radiotherapy. In
no case, however, did treatment totally normalize both
T-cell numbers and functions, and so efficacy can be
considered partial at best. In several clinical trials,
levamisole has not improved survival in irradiated
patients with non-small-cell lung cancer. Analysis of the
trial in postradiotherapy patients with non-small-cell
lung cancer has suggested that Tα1 can improve overall
patient survival when used as an adjunct to radiotherapy.
However, the mechanism by which Tα1 improved sur-
vival is not known. In addition to accelerating the recon-
stitution of T-cell dependent immunity, it is possible that
thymic factors can protect bone marrow stem cells from
the myelotoxic effects of radiotherapy or chemotherapy
[241]. Such a mechanism has been proposed to explain
the lower incidence of myelotoxicity in breast cancer
patients who received TP-1 along with adjuvant CMF
chemotherapy [241].
Because radiation therapy results in a uniform and
marked depression of immune cell numbers and func-
tions, patients who have received this treatment are ideal
candidates for studies of the immunorestorative effects
of BRMs. However, it must be recognized that if puta-
tive immunorestorative agents are administered concur-
rently with radiation, their effects on immunity might be
negated to some degree. An agent could exert a benefi-
cial effect during radiation treatment only if it provided
a direct protective action on immune cells from the del-
eterious effects of radiation.
Although a number of large-scale clinical trials have
been performed with patients receiving concurrent che-
motherapy, there are only very limited data concerning
the prevention of chemotherapy-induced immunosup-
pression. Several studies have suggested that levamisole
may have a role as a therapeutic adjunct for patients
with hematopoietic malignancies who are receiving
maintenance chemotherapy, yet the mechanism by
which levamisole exerts its effects remains unknown.
Although none of the immunorestorative agents have
proven effective when administered concurrently with
intensive combination chemotherapy for patients with
advanced metastatic cancers, their potential role in
patients treated with adjuvant chemotherapy remains to
be explored.
The success or failure of future studies with BRMs
will be determined primarily, if not exclusively, by the
selection of suitable phase II and III clinical models for
study and by the avoidance of confounding variables or
inadequate sample sizes, which could lead to uninter-
pretable results. It is likely that a two-phased approach
will be necessary to evaluate BRMs with immunorestor-
ative properties: focusing initially on phase I/II,
single-institution pilot studies to establish the pharma-
cokinetics and immunomodulatory potential of the
agent, and then in properly stratified, large-scale phase
III randomized trials performed by a cooperative cancer
group to establish clinical efficacy using the optimal
immunomodulatory dose and schedule and an appropri-
ate patient population.
Acknowledgment Dr. Richard Schulof co-authored this
chapter in the second edition, but died in an automobile
accident during the preparation of the third edition.
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7 Cancer vaccines
KENNETH A. FOON AND MALEK M. SAFA
Introduction
Immune approaches to the therapy of cancer have sub-
stantially evolved over recent years, from treating
patients with nonspecific immune stimulants to a focus
on the use of tumor-associated antigens (TAAs) either
by passive immune therapy with antibodies targeted
directly to tumor cells or by active immune therapy via
vaccinations with tumor cells, tumor cell lysates, pep-
tides, carbohydrates, gene constructs encoding proteins,
or anti-idiotype antibodies that mimic TAAs.
Approaches to Cancer Vaccines
Specific active-immunotherapy differs from nonspecific
immune-based therapies such a BCG in that the goal is
not general but rather specific activation of the immune
system to eliminate tumor cells and not affect surround-
ing normal tissue. Theoretically, specific immunotherapy
through vaccines activates a unique lymphocyte (B-and/
or T-cell) response, which has an immediate antitumor
effect as well as memory response against future tumor
challenge. The first and most obvious type of vaccines is
autologous or allogeneic tumor cell preparations (Table 1).
Alternatively, membrane preparations from tumor cells
have been used. In either instance these vaccines have
been combined with a variety of cytokines. Gene-
modified tumor cells expressing antigens designed to
increase immunogenicity or gene modified to secrete
cytokines have been a valuable tool for vaccination. In
addition, increase in our knowledge of TAA biology has
led to the use of purified TAAs, DNA-encoding protein
antigens, and/or protein-derived peptides. All of these
approaches are being tested in the clinic.
Mechanistically, the ultimate aim of a vaccine is to
activate a component of the immune system such as
antibodies or lymphocytes against TAAs presented by
the tumor (Table 2). Antibodies must recognize antigens
in the native protein state at the cell surface. Once
bound, these molecules can mediate ADCC or comple-
ment-mediated cytotoxicity. T lymphocytes, on the
other hand, recognize proteins as fragments or peptides
of varying size, presented in the context of MHC anti-
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
gens on the surface of the cells being recognized (Fig. 1)
[1–3]. The proteins from which the peptides are derived
maybe cell surface or cytoplasmic proteins [4, 5]. MHC
antigens are highly polymorphic, and different alleles
have distinct peptide-binding capabilities. The sequenc-
ing of peptides derived from MHC molecules has led to
the discovery of allele-specific motifs that correspond to
anchor residues that fit into specific pockets on MHC
class I or II molecules [6, 7].
There are two T lymphocyte, helper and cytotoxic,
which recognize antigens through a specific TCR com-
posed of both α and β subunits arranged in close conjunc-
tion to the CD3 molecule, which is responsible for
signaling. CD4 helper T cells secrete cytokines and lym-
phokines that enhance immunoglobulin production as
well as activate CD8 CTLs. CD4 helper T cells are acti-
vated by binding via their TCR to class II molecules,
which contain 14–25 amino acid (mer) peptides in their
antigen-binding cleft [8–10]. Extracellular proteins are
endocytosed and degraded (exogenous processing into
14–25 mer peptides in endocytic compartments (acidified
endosomes) ) and bind to newly synthesized MHC class II
molecules. The MHC peptide complex is transported to
the cell membrane, where it can be recognized by specific
CD4 helper T cells. In most cased the MHC class II anti-
gen-containing peptide is presented to the CD4 helper T
cells by a specialized cell called an antigen presenting cell
(APC). More specifically, a variety of cells are capable of
processing and presenting exogenous antigen including B
cells, monocytes, macrophages, and the bone marrow
derived derndritic cells (DC). DCs are the most efficient
APCs and express high levels of MHC class I and II mol-
ecules, costimulatory molecules such as CD80 and CD86,
and specific markers such as CD83. After antigen uptake,
DCs migrate peripherally to lymph nodes, where antigen
presentation to CD4 helper T cells takes place [11, 12].
There are two types of CD4 helper T cell capable of
generating either an antibody-or a cell-mediated immune
response. This is based on the type of signaling they
receive. Th1 CD4 helper T cells stimulate cell-mediated
immunity by activating CTLs thought the release of lym-
phocytokines such as IL-2. Th2 CD4 helper T cells medi-
ate an antibody response through the release of
lymphocytokines such IL-4 and IL-10. In some instances
147
148 Cancer vaccines

Table 1. Tumor vaccines the generation of one type of response may serve to inhibit
Autologous tumor vaccines the generation of the other (ref. 13, i.e., IL-10 secretion by
Allogeneic whole cell vaccines Th2 helper T cells inhibits the generation of CTLs).
Dendritic cell vaccines CD8-positive CTLs are activated in most cases by pep-
Viral oncolysates tides derived from intracellular proteins that are cleaved
Polyvalent shed antigen vaccines
Peptide vaccines to 9–10 mer peptides in the cytosol of tumor cells or
Anti-idiotype vaccines APCs by proteosomes. The peptides are then transported
Genetically modified vaccines via specialized transporter molecules called TAP proteins
Recombinant viral vaccines to the endoplasmic reticulum, where they become associ-
DNA vaccines
ated with newly synthesized MHC class I molecules [14].
The complex is then transported via the Golgi apparatus
to the cell surface membrane, where the complex is rec-
Table 2. Characteristics of different vaccines
ognized by CD8 cytotoxic T cells via a specific TCR. Any
Response characteristics endogenously processed protein can be presented to the
Multiple Single Antibody T cell immune system in this way. Several reports suggest a
Vaccine antigens antigen response response subset of APCs can present exogenously processed pro-
Autologous cells + + + + teins on MHC class I molecules to CTLs [15–19].
Allogeneic cells + − + +
Shed antigens + − + +
Carbohydrate − + + −
Peptide − + + + Pitfalls in Developing Cancer
Anti-idiotype − + + +
antibody Vaccines
Dendritic cell NA NA + +
DNA − + + +
Tumor cells have developed a variety of mechanisms to
escape immune surveillance. DC’s are actively inhibited
+, present; −, absent; NA, not applicable in the tumor milieu. Both immature and defective DCs
Vaccine (cells, lysates, protein, peptide, cDNA)

14 25mer
peptide CD4 T -cell
TCRS
Antigen Presenting Cell CD
(APC)
MHC IL - 2
Class II
TC
CD R
Exogenous protein S IL - 4
from lysed tumor 9 10 mer
MHC
peptide CD8 T -cell IL - 5
Class
IL - 10
R
TC S
CD
Tumor cell
undergoing lysis
Tumor cell

B -cell

Figure 1. T cell activation: T cells recognize antigens as fragments of proteins (peptides) presented with major histocompatibility
complex (MHC) molecules on the surface of cells. The antigen-presenting cell processes exogenous protein from the vaccine or
from the lysed tumor cell into a peptide, and presents the 14/25-mer peptide to CD4 helper T cells on a class II molecule. There
are also data to suggest that exogenous proteins can be processed into 9/10-mer peptides that may be presented on MHC class I
molecules to CD8 cytotoxic T cells. Activated Th1 CD4 helper T cells secrete Th1 cytokines such as IL-2 that up-regulate CD8
cytotoxic T cells. Activated Th2 CD4 hepler T cells secrete Th2 cytokines such as IL-4, IL-5, and IL-10 that activate B cells
Kenneth A. Foon and Malek M. Safa
are described in a variety of tumors. In addition, DC’s
that present tumor antigens, may fail to reach the T cells
in lymph nodes that generate active immune responses
against tumors. The immune response may be skewed
toward a Th2 response or T cells may be anergic. Immune
regulatory cells may contribute to immune tolerance.
CD4 positive T cells (T-reg) with a high affinity receptor
for CD25 that co-express the intracellular marker Foxp3
also play an important role in immune tolerance [20,
21]. Mutation or down regulation of immunodominant
tumor antigens, MHC molecules, or molecules involved
in the antigen processing machinery may also in part
explain the escape of tumor cells from immune recogni-
tion [22–24]. Down regulation or mutation of pro-apop-
totic molecules and expression of anti-apoptotic
molecules may also render tumor cells resistant to apop-
tosis. Tumor cells may acquire mechanisms that may
actively contribute to immune tolerance. For instance,
Fas ligand (FasL) – expressing tumors can deliver an
apoptotic signal to activated T cells and natural killer
(NK) cells expressing Fas receptor. The tumor micro-
environment may also contain soluble factors that
inhibit T cell function. Factors such as TGF-beta, pros-
taglandins, IL-10, and catabolizing enzymes are pro-
duced by tumor cells themselves or by stromal cells that
may lead to T cell hyporesponsiveness. Countering these
various tumor escape mechanisms is a key component to
successful vaccine therapy.
Mechanisms to Improve
the Immune Response
Potent adjuvants improve the effectiveness of vaccines
by accelerating the generation of immune responses and
sustaining responses for extended periods of time.
Commonly used adjuvants such as alum or Freund’s are
effective in elevating antibody titers but do not elicit
significant Th1 responses or activate cytolytic T lym-
phocytes (CTL’s). The current focus is on the generation
of adjuvants that are designed to specifically elicit cel-
lular immune responses. Toll-like receptors (TLRs) are
known to be involved in the initiation of immune
responses. CpG stimulates TLR9 and has been used
with vaccines to augment immune responses [25]. TLR8
may be involved in the activity of T-reg cells. Strategies
aimed at TLR8 are proposed to neutralize T- reg cells
[26]. One approach to decrease the role of T-reg cells is
to use immunotoxin directed against the high affinity
IL-2 receptor [27, 28]. Monoclonal antibodies specific
for the negative regulatory signals mediating the CTL
149
antigen 4 (CTLA4) on T-reg cells has also been tested to
enhance the anti-tumor immunity to vaccines [29].
Cyclophosphamide has been used for many decades to
boost immune response [30–32], as have other chemo-
therapy agents [33, 34].
Cancer Prevention Vaccines
A major success story in cancer vaccinology is cancer
prevention targeting the human papillomavirus (HPV)
to prevent cervical cancer. Cervical cancer is the second
most common cancer in women responsible for over
250,000 deaths annually worldwide. Seventy percent of
cervical cancers are caused by the two most common
oncogenic HPV types, HPV-16 and HPV-18, while
another 10% are caused by HPV-45 and HPV-31 [35].
The Food and Drug Administration has approved the
cancer prevention vaccine HPV-16/18/6/11 (Gardisil,
Merck & Co. Inc, Whitehouse Station, NJ). This vaccine
uses recombinant DNA technology to develop subunit
vaccines which include only the epitopes from the
pathogen recognized by the immune system. Copies of
the L1 viral capsid protein, the same protein which anti-
bodies are generated against in the natural immune
response to HPV, spontaneously self-assemble into non-
infectious virus-like particles which are used as the antigen
in the prophylactic vaccine. The vaccine is formulated
with ASO4 (aluminum hydroxide and monophosphoryl
lipid A) adjuvant [36, 37]. Only half the women with
HPV infection develop protective immunity because the
natural infection by HPV evades detection of the
immune system. Clinical studies demonstrated seropos-
itivity in women who receive the HPV 16/18/6/11 vaccine
was 100% for HPV-16 and HPV-18 1 month after vac-
cination and remained at 100% 4.5 years after vaccination
[38–40]. Efficacy against cervical intraepithelial neo-
plasia associated with HPV-16 was 100%. Of course,
many women are already infected with HPV and will
develop cervical intraepithelial neoplasia and remain at
risk for developing cervical carcinoma. A number of HPV
based cervical vaccines are in development that are designed
to eliminate HPV induced disease after infection.
Therapeutic Cancer Vaccines
There has not yet been a therapeutic cancer vaccine
approved by the US FDA, however, there are a number
of success stories and near success stories that should lead
to approval in the near future. Melacine is composed of
lyophilized melanoma lysates from two melanoma cells
150 Cancer vaccines

lines and the adjuvant Detox. In a phase 3 trial of 604 Table 3. Selected therapeutic vaccines in phase 3 development
patients with resected stage 3 melanoma, patients were Vaccine Company Indication
administered Melacine and low dose interferon alpha-
Antigen-loaded
2b versus high dose interferon alpha-2b [41]. Patient’s dendritic cells
were stratified by sex and number of nodes and ran- Sipuleucal-T Dendreon Corp Prostate
domly assigned to receive either 2 years of treatment DCVax-prostate Northwest Prostate cancer
with Melacine and low dose interferon alpha-2b or high Biotheraputics
Inc
dose interferon alpha-2b alone for 1 year. The median
Monified tumor cells
overall survival exceeded 84 months on the melancine OncoVax-CL Intracel Corp Colon cancer
low dose interferon alpha-2b (arm 1) versus 83 months GVAX Cell Genesys Inc Prostate cancer
in the high dose interferon alpha-2b (arm 2) (p = 0.56). Idiotype vaccines
Five year overall survival was 61% in arm 1versus 57% Favid Favrille Inc Follicular
lyphoma
in arm 2 and estimated 5 year relapse-free survival was BiovaxID Biovest Follicular
50% in arm 1 versus 48% in arm 2 with median relapse- International Inc lyphoma
free survival times of 58 months in arm 1 and 50 months MyVax Genitope Corp Follicular
in arm 2. Overall survival and relapse-free survival were lymphoma
clearly indistinguishable in the two arms. The incidence Viral vector
INGN-201 Introgen Head and neck
of neuropsychiatric severe adverse experiences were Therapeutics Inc cancer
similar, although they were more severe in the high dose TroVax Oxford BioMedica Renal cell cancer
interferon alpha-2b arm. The primary aim of this study Peptides
was that Melacine plus low dose interferon alpha-2b TV-1001 GemVax AS Pancreatic
would prolong overall survival compared with high dose (telomerase) cancer
BLP-25 (MUC1) Merck KGA Non-small cell
interferon alpha-2b. Unfortunately, this primary aim lung cancer
was not met, and their results failed to demonstrate Carbohydrate
rejection of the null hypothesis, with nearly identical GMK Progenics Melanoma
survival curves. Melacine was approved in Canada Pharmaceuticals
Inc
based on quality of life improvements.
DC vaccines are an attractive approach to vaccine
therapy although they are labor intensive requiring
prostate cancer. In the first study, patients were treated
unique autologous DC preparations from individual
at two dose levels with median survival at the low-dose
patients (Table 3). Sipuleucel-T and DCVax-Prostate
of 24 months and at the high dose of 35 months. A second
are both vaccines based on DC’s engineered to present
study used five vaccine doses. The median survival at
T-cell antigens associated with prostate cancer [42, 43].
the low and middle doses was 23 months and 20 months
Sipuleucel-T consists of autologous DC and a fusion
respectively and was not yet reached at the high dose
prostatic acid of prostates and phosphatase and granulo-
(>29 months). Phase 3 trials are ongoing.
cyte macrophage colony stimulating factor (GM-CSF).
There are a number of non-cell based patient specific
A randomized phase 3 placebo controlled trial in patient
vaccines that are currently in phase 3 trials. Favid,
with metastatic asymptomatic androgen-independent
BiovaxID and MyVax are idiotype vaccines that use KLH
prostate cancer was completed. Eighty-two patients
as a carrier. All three are patient specific for patient’s with
were randomized in a 2:1 ratio [44]. The primary end-
B-cell lymphoma. Anti-idiotype immune responses have
point of this study which was progression-free survival
been shown to correlate with better clinical outcomes in
was not reached (p = 0.052). However, overall survival
follicular lymphoma patients who received idiotype vac-
was significantly different for vaccine (26 months) ver-
cines [47, 48]. MyVax is also being studied in phase 2
sus placebo (21 months) with a hazard ratio of 1.7 and p
trials for mantle cell lymphoma, diffuse large B-cell lym-
= 0.01. A second study showed a trend toward increased
phoma and chronic lymphocytic leukemia.
survival (19 months versus 16 months) but did not reach
INGN-201 is a recombinant adenovirus-p53-based
significance. A phase 3 clinical trial using survival as an
vaccine for head and neck cancer. The adenovirus theo-
end point is ongoing.
retically functions to deliver the p53 protein in large
GVAX consists of two irradiated allogeneic prostate
quantities to the tumor cells. While this is classified as a
cancer cell lines engineered to secrete GM-CSF [45,
gene therapy, there is evidence that suggests an immune
46]. Two phase 2 trials have been completed in patients
response is elicited by p53 which is overexpressed in
with asymptomatic metastatic androgen-independent
Kenneth A. Foon and Malek M. Safa 151

endocytosis of anti-Id by APC

processed to processed to
9/10 mer peptides 15/25 mer peptides

MHC class II
MHC class I+
peptide MHC class I MHC class II+
CD8 peptide
APC TCR
TcR
IL-2r
CD4

CD8 T cell Th1

CD4
cytokines
T cell
clonal proliferation (IL-2, IFN-γ)
TCR
Mφ Th2 CD4 MHC class II+
NK cytokines peptide
T (IL-4, IL-10)
direct ADCC
Activated lysis B cell recognition
CD8 T cell TcR MHC of anti-Ιd and
processed to
class II endocytosis of anti-Ιd
15/25 mer
CD8
peptides
MHC class I+ Anti-anti-
peptide Id (Ab11) B Cell
Granzymes
Preforin
INF-γ
TNF-β Tumor cell mediates ADCC and
direct anti-metastatic effect

Figure 2. Putative immune pathways for anti-id vaccines

the tumor. Therefore, p53 may be considered a tumor-
associated antigen and may aid in the elimination of
tumor cells [49]. Interestingly, an adenovirus-p53 gene
therapy vector (Gendicine) has been approved in China
for head and neck cancer. TroVax is a vaccine in which
the tumor associated antigen 5T4 is expressed in a mod-
ified vaccinia Ankara (MVA) vector, which induces
strong immune response similar to the live virus. It is
expected that the recombinant 5T4 antigen will be
included in the antiviral immune response. This vaccine
is in phase 3 trials for renal cell cancer. TV-1001 is a
telomerase peptide based vaccine that is in phase 3 trials
for pancreatic cancer. Telomerase is over expressed in
many cancers and is theoretically a good target antigen.
GMK is a ganglioside conjugate vaccine in which gan-
glioside GM2 is coupled to KLH and formulated with
QS-21 adjuvant [50]. The goal of this vaccine is to
induce an antibody response rather than a T-cell response
[51]. This vaccine is in phase 3 trials for melanoma.
BLP-25 is a liposome-encapsulated synthetic peptide
that corresponds to the variable number 10 tandem
repeat region of the mucin (MUC)-1 molecule. MUC-1
is overexpressed and underglycosylated on tumor cells.
The MUC-1 target is highly represented on most epithe-
lial tumors. There is currently a phase 3 trial in non-small
cell lung cancer using BLB-25.
Conclusion
The platform for therapeutic vaccines is broad including
a variety of antigens, both non specific antigens repre-
sented by whole cell based vaccine approaches and
recombinant antigens as represented by the protein and
virus based approaches. DC’s are an extremely appeal-
ing vaccine approach, however, they are limited by the
152
difficulties associated with patients specific cell therapies.
No specific approach to vaccine therapy has stood out as
clearly superior. Strategies to enhance the immune response
will be the next most important step in therapeutic can-
cer vaccines. Monoclonal antibodies inhibiting T-regs,
the use of a variety of cytokines and TLR stimulation
are among the strategies that will be employed.
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8 Cytokines
WALTER M. LEWKO AND ROBERT K. OLDHAM

Cytokines are regulatory proteins, produced and secreted book. Table 1 summarizes cell sources and effects of
by various cells, which control immune response, cytokines discussed.
hematopoiesis, inflammation, wound repair and tissue
morphogesis. Cytokines may be secreted or membrane
bound. Secreted cytokines may act locally as autocrine
or paracrine factors or over some distance as would a
Cytokine Receptors: Many
hormone. Membrane bound cytokines act by cell–cell Belong to Receptor Families
contact, communicating information from one cell to
Cytokines usually induce their effects by binding to
another, often bidirectionally. There are cell surface
target cell receptors, which generate intracellular signals.
receptors for each cytokine that bind the cytokine spe-
Often the result is gene activation. The receptors for the
cifically. Receptor subunits may be shared between dif-
various cytokines tend to be grouped into families
ferent cytokines. Binding the cytokine brings about
reflecting common genetic origin, structure, signaling
signaling and a series of cell activating events. For many
and cell response. Some of the major receptor groups
cytokine receptors (not all), this involves increased
include the IL-1 family, cytokine/hematopoietin recep-
phosphorylation of certain tryrosine residues on key cel-
tor family, the IL-6 family, the IL-17 family and the
lular proteins. Kinases are the enzymes that carry out
tumor necrosis factor family. For example, the receptors
phosphorylation. Receptors may themselves be kinases,
for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15,
which activate upon binding. More typically, the acti-
G-CSF, GM-CSF and Prolactin are all members of the
vated receptor may recruit cytosolic kinases, for exam-
hematopoietin receptor family. Receptors for these
ple, mitogen activated protein kinase (p38 MAPK)1 and
cytokines have been cloned and analyzed. Amino acid
Janus kinases (Jak1, Trk and Jak 3) [97, 533, 556, 1009,
sequence reveals segments that are similar between
1699]. As the kinases bind the receptor complex, their
members of the same family [1472]. The IL-6 family
enzymatic activities increase and an array of cellular
members share the gp130 signaling subunit [1943].
proteins is phoshorylated, in certain cases including the
Receptors are generally composed of two or more sub-
receptor itself. These modifications bring about changes,
units. In a basic model, one subunit binds the ligand; the
increases or decreases, in each protein’s activity. Among
other subunit generates the signal; together in a complex,
these proteins are the STAT proteins that control gene
the subunits bind the cytokine with higher affinity than
expression. When phosphorylated, STAT proteins trans-
they do separated. Different receptors may share subunits
locate from the cytoplasm to the nucleus and bind spe-
that carry out common functions. For example, several
cific enhancer segments allowing the expression of the
receptors (IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-21)
genes needed to bring about a cytokine’s response.
share a common gamma subunit (γc) that is involved in
In this chapter, several of the major cytokines will be
signaling. The importance of this gamma chain’s function
discussed. Regulation of cellular immunity will be empha-
is shown in various forms of combined immunodefi-
sized for its role in the elimination of tumor and virus
ciency disease that appear due to mutations [259, 1097,
infected cells. Inflammation is discussed for its important
1699]. Since this receptor subunit is shared by so many
role in not only in immune response but also cancer pro-
cytokines, these genetic defects have far reaching and
gression. Unfortunately, it is not possible to cover all of
debilitating results in the immune response system.
the interesting cytokine research in detail. We have tried to
Cytokine activity is of necessity transient. Uncontrolled
summarize past results and highlight certain interesting
activity may result in inflammatory diseases, allergy or
trends. The interferons, colony stimulating factors and
autoimmunity. There are several levels at which regula-
certain growth factors are covered in other chapters in this
tion may occur, from cytokine secretion to receptor bind-
ing to intracellular signaling to target cell responsiveness.
For example, there are cellular proteins that regulate
1
Abbreviations are listed at the end of the chapter. signaling [492, 1413, 1882, 2256]. Among them are the

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, 155
© Springer Science + Business Media B.V. 2009
156 Cytokines

Table 1. Summary of cytokine sources and effects related to the growth and treatment of cancera
Cytokine Cell source Cells influenced Effectb
IL-1 Mono/Macro Macrophages Activation, TNF, IL-6, PGE2, NO (w/IFNγ)
B cells B cells Growth/Ig (w/IL-4, IL-6), chemotaxis
T cells T cells Growth (w/IL-2), migration, IL-2, IL-2R, IFNγ
NK Th2 Growth, IL-4, IL-5, IL-6
DC Mast cells IL-3, IL-4, IL-5, IL-6, IL-9 (Th2-like cytokines)
Some tumor cells LAK Induction (w/IL-2)
Fibroblasts NK IFNγ (w/1L-12), cytotoxicity (w/IFNγ)
Keratinocytes Tumor cells Growth/inhibition/metastasis/no effect
Endothelial Fibroblasts Growth, PGE2, collagen
Glial cells Chondrocytes ↓ Growth, ↓ collagen, ↑ protease
Eosinophils Keratinocytes Growth
Neurons Endothelial PGE2, VCAM, ICAM, angiogenesis
Epithelial Vascular smooth muscle Growth, IFNβ
Synovial Eosinophils Degranulation
Basophils Degranulation, histamine release
Liver Metabolism, protein production
Synovial PGE2, collagenase, phospholipase A2
Glial cells Growth
Neurons Survival
Pancreatic Islet Insulin
Muscle Negative protein balance (w/insulin, IL-1, TNF)
DC Maturation (w/GM-CSF)
Stem cells Viability/survival (w/IL-3, G-, M-, GM-CSF)
Hypothalamus Corticotrophin releasing factor
Some tumors ↓ Growth
Melanoma B16 ↑ Metastasis (↑ endothelial VCAM)
IL-2 T cells (active) NK Activation/cytotoxicity
LAK Activation/cytotoxicity
T cells Growth/cytotoxicity/tolerance/AICD
PBL Growth/CTL generation
B cells Growth/differentiation (w/IL-6, IL-12)
Macrophages Activation, TNFα, β, NO
DC Proliferation
Keratinocyte Proliferation
Eosinophils Eosinophilia
Tumor cells Growth/inhibition/no effect ↓ ICAM/↓ MHCI
IL-3 T cells (active) Stem cells Growth
Thymic epithelium Megakaryocyte Platelet production
Mast cells RBC precursors RBC production
Keratinocytes Mast cells Survival/growth (w/IL-4)
Neurons Natural cytotoxic Growth/TNF
Eosinophils Granulocytes Growth/survival/differ
Macrophages Growth/differ/activation
NK Activation (w/IL-2)
T cells Activation/growth (w/IL-2)
Osteoclasts ↑ or ↓
Hematopoietic tumor Growth stimulation/inhibition/no effect
IL-4 Th2 B cells Growth, Ig secretion (IgG, IgE), MHCII
Mast cells T cells Growth stimulation/inhibition/no effect
Basophils Th0 Differentiation to Th2, inhibition formation Th1
NK Mono/Macro Growth/activation/AP/HLA DR
↓ Inflammatory cytokines
Eosinophils Endothelial Growth/VCAM
DC Fibroblasts Growth
γδ T cells PBL Growth (IL2-primed)
NK T cells NK Growth/IL5 secretion (w/IL-2, IL-12)
Mast cells Growth/ICAM (w/IL-3)
LAK Activation (IL-2 primed)
DC Growth/activation (w/IL-2, IL-7, GM-CSF)
(continued)
Walter M. Lewko and Robert K. Oldham 157

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
Hematopoietic cells Growth (+/−)
Certain tumors ↓ Growth, ↓ COX-2, ↓ PGE2
IL-5 Th2 Eosinophils Growth/differ/survival/activity/chemotaxis
Eosinophils B cells Growth/survival/Ig (IGA, IgM)
Mast cells Mast cells Growth (w/other factors)
NK T cells Growth/differentiation (w/IL-2), IL-2R
LAK Activation (w/IL-2)
NK Activation (w/IL-2), IL-2R
IL-6 Fibroblasts B cells Differentiation/Ig (w/IL-2)/survival
Macrophages T cells Growth/activation (w/IL-2)
Epithelial Megakaryocytes Growth/platelets
Endothelial NK Activation/cytotoxicity
Eosinophils Hepatocytes Acute phase proteins, fibrinogen
Neutrophils Intestine cells Acute phase proteins
Mast cells Fibroblasts Growth/collagen
B cells Osteoclasts Growth/activation
Keratinocytes Endothelial Growth
Th2/CD8 T Neurons Regeneration
Osteoblasts Melanoma ↓ Growth
Synoviocytes Leukemia/lymphoma↓ ↓ Growth
growth
Megakaryocytes Breast cancer ↓ Growth
Langerhans cells Cervical cancer ↓ Growth
Astrocytes DC Antigen presentation, especially self antigen
DC
Some tumors
IL-7 Stroma, marrow Pre-B cells Growth/differ (w/SCF/FLT3L)
Stroma, thymus Pre-T cells Growth/differ/survival (w/SCF/FLT3L)
Keratinocyte Mono/macro Growth/activation/antitumor activity
B cells NK Activation
Intestinal epithelium LAK Activation
DC T cells (w/IL-2) Growth/different/survival/memory/cytotoxicity
Endothelial DC Growth/antigen presentation
Some carcinomas TIL Activation/proliferation
Leukemia/lymphoma Pre-eosinophils Growth
Melanoma ICAM
Some leukemia/lymp Growth
IL-8 Macrophages Neutrophils Chemotaxis, superoxide, degranulation,
hydrolase
Endothelial T cells Chemotaxis
Neutrophils Macrophages Chemotaxis
Eosinophils Endothelial Chemotaxis/angiogenesis
Mast cells Eosinophils Chemotaxis
Epithelial cells NK Chemotaxis
Fibroblasts Cancer Autocrine growth/metastasis/angiogenesis
Keratinocyte
Some melanomas
Some carcinomas
IL-9 Th2 T cells (active) Growth (w/IL2), IL-22
Mast cells Fetal thymocytes Growth (w/IL2)
Mast cells Survival, growth (w/IL-3, IL-4), IL-22
Pre erythroid Growth (w/erythropoietin)
B cells IgE
Lymphoma Growth/survival
IL-10 Th2, mice T cells ↓ Growth/IFNγ/cytotoxicity/chemotaxis.
Th1 Th2, humans NK ↓ Cytotoxicity/IFNγ
Tr1 Mono/macro ↓ Activivity/IL12/AP/collagenase; ↑ TIMP
nTreg Mast cells Growth (w/IL-3, IL-4)
B cells B cells Growth/differ/survival/MHCII (w/IL-2)
(continued)
158 Cytokines

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
Monocytes Activated B cells Apoptosis
Eosinophils Fibroblasts ↓ Collagen/fibrosis
Mast cells DC ↓ Antigen presentation
Keratinocyte
Some tumors
IL-11 Stroma, marrow Progenitor cells Growth/colony formation
Fibroblasts Megakaryocytes Thrombopoiesis (w/IL-3)
Epithelial B cells Growth
Chondrocytes Macrophages ↓ IL-12
Synovial Th2 ↓ IL-4, IL-5, IL-13
Smooth muscle Fibroblasts ↓ Differ to adipocytes
Liver Acute phase proteins
Synovial cells TIMP
Osteoclasts Growth/differentiation
IL-12 B cells NK Growth/cytotoxicity/IFN (w/IL-1)
Macrophages CD8 T cells Growth/cytotoxicity/IFN (w/IL-2)
DC Th1 IFNγ, IL-2, growth
Neutrophils Th2 ↓ IL-4, IL-10, growth
Microglial PBL LAK induction/CTL growth cytotoxicity
Keratinocytes TIL Growth/cytotoxicity
Macrophages IFNγ
B cell Growth/differentiation (w/IL-2)
DC Activation/antigen presentation, IFNγ
IL-13 Th2 cells Mono/macro ↓ Cytokines/NO/PGE2/CD14/ADCC
Mast cells ↑MHCII/CD11b c/IL-1RA/CD23
Basophils B cells Growth/differ/Ig/IgE switching/CD23
Eosinophils Neutrophils ↑ IL1Ra, ↓ CD14
DC NK IFNγ, cytotoxicity
Some B lines Endothelial VCAM/MCP-1/angiogenesis
Tumor cells DC Growth/maturation,↓ IL-23, IL-1, IL-6.
Th17 Suppression
Epithelial ↓ NO
Synovial ↓ IL-1β, TNF
Macrophages ↓ HIV production
Renal ca ↓ Growth
IL-14 CD8 T cells B cells Growth/differentiation/memory
Th1, Th2 B cell lymphoma Autocrine growth
NKT cells
Follicular DC
B lymphoma
IL-15 Epithelial T cells Growth/cytotoxicity/memory
Stroma, marrow γδ T cells Growth/activation
PBMC B cells Growth/IgG, IgA, IgM
Fibroblasts NK Growth/cytotoxicity/chemotaxis
Keratinocyte LAK Induction/cytotoxicity
TIL Growth
Mast cells Growth/response
Keratinocyte Growth; psoriasis
IL-16 CD8 T CD4 T cells Migration/growth w/IL-2, IL-15
Macrophages Mono/macro Migration/activation/antigen presentation
CD4 T cells DC Migration/antigen presentation/tolerance (w/TPO)
B cells Eosinophils Migration
Fibroblasts HIV ↓ Replication/↓entry
Eosinophils NK Cytokines/migration
Mast cells
DC (immature)
Epithelial
Brain
IL-17 Th17 Macrophages Inflammatory cytokines, IL-10, IL-12.
Neutrophils Fibroblasts Inflammatory cytokines, GM-CSF
(continued)
Walter M. Lewko and Robert K. Oldham 159

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
CD8+ T cells Epithelial Inflammatory cytokines
Endothelial Inflammatory cytokines
Keratinocyte Inflammatory cytokines, ICAM
DC Maturation/cytokine production
Chondrocytes Inflammatory cytokines/MMP/NO
Granulocytes Production (w/GM-CSF)
Synoviocytes IL-6, LIF
CH ovary cells Invasiveness
Monocytes Osteoclast differentiation
Osteoclasts Activation
IL-17B Pancreas Monocytes IL-1β, TNFα
Intestine Neutrophils Migration (indirect effect)
Stomach
IL-17C Prostate Monocytes IL-1β, TNFα.
Fetal kidney
IL-17F CD4 T cells Endothelial ↓ Angiogenesis. ↑ IL-2, TGFβ, MCP-1
Monocytes Bronchial epithelium ICAM, IL-6, IL-8 (neutrophil recruitment)
Basophils
Mast cells
IL-18 Macrophages Th1 (w/IL-2, IL-12) IFNγ, growth/differ/IL-2,
FAS/apoptosis/escape, IL-13
DC B cells IFNγ (w/IL-12), IgG
Kupffer cells NK (w/IL-2, IL-12) IFNγ, growth, anti tumor activity
FAS/apoptosis/escape, IL-13
Keratinocyte Macrophages IFNγ (w/IL-2)
Airway epithelium DC IFNγ (w/IL-2)
Adrenal cortex NK T cells IL-4
Neutrophils Degranulation, cytokines, CD11b (complement R)
Endothelial Migration/regulation of angiogenesis
IL-19 Monocytes Th2 Activation, IL-4
Keratinocytes Monocytes Activation, IL-6, TNF, ROS
PBMC PBMC IL-10, IL-19
IL-20 Keratinocytes Keratinocyte Proliferation, psoriatic character
Monocytes Hemato progenitors Proliferation
Glial cells Endothelium bFGF, VEGF, IL-8, angiogenesis
Synovial fibroblasts Inflammatory cytokines IL-6, IL-8
IL-21 Th2 NK Proliferation, activity
Th17 T cells, Th17 Proliferation, activity
Follicular T cells Treg Inhibition
B cells Class switching, ↑IgG, ↓ IgE
DC Regulation, ↓maturation, ↓activation
Hematopoietic tumor Growth stimulation, inhibition, or no effect
IL-22 Th17 Liver Acute phase proteins
Mast cells Th2 ↓ IL-4
Th1 Epithelium Inflammatory cytokines
Th2 Keratinocytes Inflammatory cytokines, migration, β-Defensin
NK Hepatocytes Survival, acute phase proteins
IL-23 DC CD4+ T memory Proliferation
Macrophages Th IFNγ, Il-17, IL-6, IL-1, TNF, ICOS
Keratinocytes DC IL-12, IFNγ
Macrophages IFNγ
Th17 Activation (IL-17)
IL-24 Th2 PBL Proinflammatory, IL-6, TNF
Monocytes (activ) Melanocytes Differentiation, ↓Proliferation
Melanocytes Keratinocytes Proliferation
PBMC (activ) Vasc sm muscle ↓Growth, ↓migration, ↓angiogenesis
Some tumor cells Monocytes TNF, IL-6
Tumors ↓Growth, ↑apoptosis, ↓angiogenesis,
↑radiosens
Endothelium ↓ Angiogenesis
(continued)
160 Cytokines

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
IL-25 Th2 Eosinophils Growth, infiltration, ICAM, L-selectin,
Eosinophils IL-6, IL-8, MCP-1, MIP-1α
Mast cells Th2 Activation (IL-4, IL-5, IL-13)
Microglial cells Th17 Regulation
Basophils Cartilage Catabolism
Fibroblasts (Lung) Inflammatory cytokines
IL-26 T cells Epithelial cells ICAM-1, IL-8, IL-10
NK
IL-27 DC T cells Proliferation, IL-12R, IFNγ
Monocytes (LPS) NK IFNγ
Th1 Differentiation, proliferation, activity
Macrophages Th17 ↓ Differentiation response to TFGβ/IL-6
Granulocytes ↓ ROS response to endotoxin
Macrophages ↓ ROS response to endotoxin
Endothelium ↓ Angiogenesis
IL-28A, B, IL-29 Monocytes Most cells Antiviral, oligoadenylate synthetase, MHCI/II
Macrophages DC Maturation, Treg induction
DC Tumor cells ↓Growth, ↑apoptosis, ↑ Immune response
IL-31 Th2 Th2 ↓ Activation (↓ IL-4, IL-5, IL-13)
Myeloid progenitors Survival
Monocytes Inflammatory cytokines
Epithelial cells Inflammatory cytokines
Skin epithelium Dermatitis
Lung epithelium EGF, VEGF, inflammatory cytokines
Sensory neurons Itching sensation, development
IL-32 NK cells Monocytes/DC TNFα, IL-1β, IL-6, IL-8, PGE2
T cells T cells Apoptosis, TNF
Epithelium
Monocytes
Synoviocyte (RA)
IL-33 Endothelium Th2 Activation (IL-4. IL-5, IL-13)
Fibroblasts Mast cells IL-8, IL-4, IL-5, IL-13, survival
Endothelium Nuclear protein/transcription repressor
Fibroblasts, heart ↓ Collagen
Cardiomyocytes ↓ Hypertrophy
IL-35 nTreg nTreg Proliferation, IL-10
CD4+CD25− Suppression
Th17 cells Suppression, ↓ IL-17
4-1BBL T Cells CD8 T/CD4 Th1 Proliferation/cytokines/↓ AICD/↓ apoptosis
DC CD4 Th2 Suppression/anergy
Monocytes/macro Monocytes/macro Activation/survival
B cells B cells (active) Proliferation/survival
Stromal cells NK/NKT IFNγ/IL-2
Some tumor cells DC IL-12/1l-6
T cells ↓ Activation (by reverse signal)
CD27L B cells B cells Differentiation (+/−), memory/apoptosis/AICD
T cells B cells Activation/ab production (by reverse signal)
NK T cells Stimulate or inhibit, memory/apoptosis/AICD
DC γδT cells Activation/cytotoxicity (reverse signal)
B cell malignancy NK Activation/cytotoxicity (w/IL-2)
Renal cancer cells
Brain tumor
CD30L Macrophages B cells Memory/↓ class switching, Ig prod (by rev signal)
B cells Neutrophils Oxidative burst/IL-8 (by reverse signal)
T cells T cells Proliferation/apoptosis/AICD, IL-6 (by rev signal)
Megakaryocytes Thymocytes Apoptosis/negative selection
Neutrophils NK Apoptosis/AICD
Eosinophils Mast cells Chemokines (rev signal)
(continued)
Walter M. Lewko and Robert K. Oldham 161

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
pre-erythrocytes Eosinophils Apoptosis
Some leukemias Lymphomas Apoptosis/no effect/growth
Some lymphomas
CD40L CD4 B cells Proliferation/class switch/Ig/survival
CD8 DC Activation/B7.1/B7.2/ICAM/IL-12, survival
Mast cells Macrophages Activation/proinflammatory cytokines
Basophils T cells Costimulation/activation/growth/longevity.
Fibroblasts NK Activation/cytotoxicity
Platelets Fibroblasts Activation/proinflammatory cytokines/ICAM/
VCAM
Endothelium ICAM/VCAM/Angiogenesis factors
CD4 IL-4 (by reverse signal)
CD8 Activation/cytotoxicity (by reverse signal)
Platelets RANTES/Inflammation
Some tumors Apoptosis/AP
FASL T cells T cells Apoptosis/AICD/Immune privilege
B cells B cells Apoptosis/AICD/Immune privilege
NK cells Some tumors Apoptosis; chemotherapy response
Some tumors Endothelium Damage (VLS)
DC (Immature) Some tumors Apoptosis
FLT3L Hematopoietic Hematopoietic SC Growth/survival
Non- Progenitor cells Growth/survival
hematopoietic DC Growth/maturation (w/SCF, GM-CSF, IL-4, TNFα)
B cells Growth (w/IL-7, SCF)
NK/LAK Growth of progenitors; response to IL-2
Osteoclasts Differentiation
Neurons Survival (w/NGF)
Neuron SC Growth regulation
Some leukemias Growth
Some tumors Growth (especially neural crest origin)
Interferon γ Th1 Cells in general MHCI/II, FcR, P1 Kinase,
CD8 T cells 2 -5 oligoadenylate synthetase
NK Macrophages Activation, H2O2
NK Cytotoxicity
B cells Altered Ig isotype secreted
Inhibition of IL-4-induced functions
CD8 T cells Differentiation/cytotoxicity (w/IL-2)
Th1 Growth/cytokine production
Th2 ↓ Growth/cytokine production
Th17 ↓ Differentiation
Endothelium ↓ Growth/angiogenesis
Tumor cells ↓ Growth; increased differentiation
↑ MHC expression; ↓ NK sensitivity
LIF Fibroblasts T cells Differentiation
Endothelium Colon Colitis; progression to colon cancer
Macrophages Mammary Development
Epithelium, thymus Osteoclasts Growth/activation
Synoviocytes Endometrium Implantation/trophoblast differentiation
Neurons Survival/↓ maturation
Corticotropes ↓ Growth↑ differentiation, ACTH secretion
Myeloid leukemia Differentiation/↓ growth
Glioma Differentiation/↓ growth
Some carcinomas Growth (breast, colon, renal, prostate)
Embryonic SC ↓ Differentiation
LIGHT T cells T cells Proliferation, IL-2/IFNγ/IL-4/TNFα
Granulocytes DC Costimulation for AP to T cells
Monocytes NK T cell help
DC (immature) Hepatocytes Proliferation
HVEM+ LTβR+ Apoptosis
(continued)
162 Cytokines

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
Some tumors Apoptosis
Lymphotoxin α Th1 B/T/DC etc. Lymphoid organogenesis
(TNFβ) CD8 T cells Endothelial VCAM/ICAM
B cells (early) NK T cells Growth/development
NK Osteoblasts Growth/activation
Astrocytes Osteoclasts Growth/activation
Lymphotoxin β T cells Lymphoid cells Lymphoid organogenesis
B cells T cells Survival
NK cells B cells Survival
DC (Immature)
Novel LN cells B cells Growth/IgM/IgE/IgG
Neurotrophin Spleen cells Macrophages Growth
Neurons Survival/development
Oncostatin M T cells LN cells T cell development/migration
Mono Fibroblasts Growth/wound repair/collagen/fibrosis
Neutrophils Osteoclasts Growth/activation
Certain tumors Vascular sm muscle Growth/wound repair
Synoviocytes Inflammation regulation (+/−)
Some gliomas Differentiation/↓ growth
Sarcoma (Kaposi) Growth
Osteopontin T cells Th Activation (favors Th1)
Mono/Macro DC Activation (favors Th1)
Osteoblasts Osteoclasts Binding/recruit/remodel/↓Hydroxyapatite deposit
Endothelium Osteoblasts Binding/remodeling/↓Hydroxyapatite deposit
Some epithelial Mono/macro Chemotaxis/IL-1, TNF, IL8/↓ NO
Brain Endothelial Survival/angiogenesis/↓ NO
Certain tumors Epithelium, kidney ↓ NO
Melanocytes Survival
OX40L DC CD4 T cells Costimulation/IL-2/IL-4/IL-5/longevity/memory
B cell DC Maturation/TNF/IL-12/IL-1β/IL-6 (by back signal)
Endothelium B cell Growth/differ (by back signal)
T cells Endothelial Inflammation (by back signal)
T cells Leukemia (w/HTLV)
RANKL T cells DC Activation/AP/survival
B cells T, B, DC etc. Lymphoid organogenesis
DC Preosteoclasts Maturation; bone development
Stroma, BM Mono Osteoclast differ (w/GM-CSF, TGFβ)
B cells B development; osteoclast development
SCF Stroma, BM Stem cells Growth
Fibroblasts Progenitor cells Growth
Liver Mast cells Growth/differentiation/survival/fibrosis
Spleen γδ T cells Growth
Certain tumors Tumors Growth (breast, lung, testicular, utererine,
cervical, ovarian, melanoma)
TNFα Mono/macro T cells Growth/development (wIL-1)/IL-2/IFNγ
T cells (active) B cells Activation/Ig production
NK Endothelial NO/cytotoxicity
Endothelial Macrophages NO/cytotox/IL-12/IL-10/apoptosis
Mast cells DC Growth/maturation (w/GM-CSF)
Neutrophils Neutrophils Degranulation/complement R/
Keratinocytes adhesion/ADCC/apoptasis
Adipocytes TIL Cytotoxicity
Pancreatic β LAK Cytotoxicity
Osteoblasts γδT cells Growth
Astrocytes Endothelial VCAM, damage, vascular leak syndrome
Neurons Osteoblasts Mitosis
Adrenal cells Osteoclasts Activation
(continued)
Walter M. Lewko and Robert K. Oldham 163

Table 1. (continued)
Cytokine Cell source Cells influenced Effectb
DC (Immature) Synovial Enzyme secretion
Epithelial cells Fibroblasts Growth
TRAIL CD4 T cells Tumor cells Apoptosis; apoptotic bodies for AP
NK cells DC AP/T cell activation
Monocytes Autoimmune T cells Apoptosis
DC (Immature) Mast cells Apoptosis
Tweak Many cell types Fibroblasts IL-8, IL-6, RANTES, IP-10
Synoviocytes Inflammatory cytokines
Bronch epithelium Inflammatory cytokines
Macrophages IL-6, MCP-1, IL-8, MMP-9
Mesangial cells MCP-1, RANTES, IP-10, VCAM
Astrocytes IL-8, IL-6, ICAM
Keratinocytes RANTES
Liver Proliferation
Endothelium Proliferation, ICAM, E-selectin, IL-8, angiogenesis
Some tumors Proliferation, survival
a
This is a partial listing that emphasizes cancer related activities.
b
Activity increased unless otherwise indicated.

suppressors of cytokine signaling (SOCS) which are also
referred to as cytokine-inducible suppressor proteins
[332, 1882]. They act by binding Jak/STAT components,
interfering with their binding to the receptor and their
function [333, 492, 1882]. They are tightly regulated and
required for normal lymphoid development [1882].
Various cytokines induce SOCS to regulate their own
activities (a form of feedback inhibition) [1857] and the
activities of other cytokines [428]. For example, SOCS
may be increased by immune suppressor cytokines such
as IL-10 [256].
Cytokines may act synergistically. For example,
IL-18 and IL-12 are synergistic in the way they induce
IFN-γ secretion. Synergism may be explained in a num-
ber of ways, but frequently, it is due to the upregulation
of one cytokine’s receptor or its signaling pathway by
the other cytokine [521, 965, 1724, 2214, 2253].
Toll-Like Receptors
and the Response to LPS
There is a family of receptors, molecularly related to
cytokine receptors, called the pattern recognition recep-
tors [520, 1089]. They are better known as the Toll-like
receptors (TLR) for their genetic similarity to an impor-
tant class of Drosophila morphogenesis genes. In addition
to their role in fruit fly embryogenesis, Toll also has an
antifungal immune function in the adult flies [1089].
These receptors have the important role of sensing the
presence of microbial invaders and initiating immune
response. They do this by specifically binding molecules
(danger signals) shed from microbes, collectively called
pathogen-associated molecular patterns (PAMPs) [1282,
1654]. Bacterial lipopolysaccharide (LPS) (endotoxin)
is a well-studied PAMP. LPS is a major cell surface
component of Gram-negative bacteria. It binds TLR on
macrophages, endothelial cells and dendritic cells where
it is a potent activator and stimulator of cytokine pro-
duction [1983, 2081]. Activation may be very vigorous.
LPS induces toxic shock syndrome [1629]. Knockout
gene studies in mice have shown that TLR4 recognizes
LPS whereas TLR2 is essential for response to several
gram positive PAMPs [1586, 1956]. There are at least
six human Toll-like receptors. They are integral mem-
brane proteins. The intracellular region is homologous
with the signaling domain of the IL-1 receptor family
[1303]. TLR signaling activates several cells that are
important in immune response [516, 2081]. Immature
dendritic cells contain these receptors. Binding LPS
induces DC activation, maturation and secretion of
cytokines such as IL-12, allowing antigen presentation
to occur. Toll receptors are an important bridge between
the innate and specific immune response systems.
The Helper T Cell System
Mosmann and coworkers proposed a paradigm for the
differentiation of CD4+ helper T (Th) cells, cytokines
secreted and their roles in regulating of the development
164
of cellular versus humoral immunity [1375, 1376, 1659,
1709, 1937]. This paradigm has been expanded to
include additional cells and newly discovered cytokines.
The following is a very brief summary of an involved
system.
A model for the differentiation of Th1 and Th2 helper
T cells is shown in Fig. 1. Thp cells are naive precursors
to helper cells; IL-2 is the major cytokine they produce.
Antigen presentation stimulates their conversion to Th0,
the intermediate precursor cells. Th0 cells secrete IL-2,
IL-4 and IFN-γ [1314, 1376]. Depending on environmen-
tal conditions, Th0 cells differentiate into Th1, Th2, Th17
and regulatory T cells. This process is commonly referred
to as polarization; the cells become polarized to secrete
specific cytokines and perform certain functions.
Th1 cells favor the development of cellular immunity.
Intracellular microbes (such as viruses) are eliminated
by this path. Cancer cells are also killed by cellular
immune response. Th1 responses are important in
delayed type hypersensitivity and in certain autoim-
mune diseases. Th1 cells secrete several cytokines,
among them INF-γ, IL-2 and TNF-β, which are collec-
tively referred to as Th1 cytokines. In particular, IFN-γ
is considered a key marker for Th1 response. IL-12 and
IL-2 favor the Th1 path. IL-4, a product of Th2 cells,
generally inhibits the Th1 path.
Th2 cells favor the development of humoral immu-
nity. Th2 response also stimulates eosinophil recruit-
ment and macrophage function. This path eliminates
extracellular microbes and large parasites. Th2 responses
are important in infectious diseases and in allergy. Th2
cells secrete IL-4, IL-5, IL-6, IL-9 and IL-13. IL-4 is the
hallmark Th2 cytokine. Mouse Th2 cells secrete IL-10.
(In humans, both Th1 and Th2 cells produce IL-10).
IL-4 stimulates the Th2 path whereas IFN-γ, a major
Th1 cytokine, inhibits it [2, 1746]. In addition to cytokines,
there are further influences that fine tune Th1 versus
Figure 1. Differentiation of Th1 and Th2 helper T cells.
Antigen presentation (AP) stimulates the conversion of
naive precursor Thp cells to immediate precursor Th0 cells.
Th1 cells favor the development of cellular immunity and
response to intracellular microbes. Th2 cells foster humoral
immunity and response to extracellular microbes and para-
sites. The major positive and negative stimulators of each
path are indicated. Cytokines within the boxes are produced
by the indicated cells. IL-10 is a Th2 cytokine for mouse
cells; in humans, both Th1 and Th2 cells produce IL-10
Cytokines
Th2 path selection. (a) Antigen dose; generally, low
antigen dose favors the Th1 path, while higher antigen
dose favors Th2 cells [798]. (b) Route of antigen admin-
istration [1993]. (c) Type of antigen presenting cell
[1775]. (d) Type of costimulation: during antigen
presentation, B7.2 favors Th2 [565].
Th17 cells induce inflammation. This path responds
to extracellular microbes. Th17 cells have important
roles in autoimmunity. Th17 cells secrete IL-17A,
IL-17F, IL-21, IL-22, IL-6 and TNF-α. TGF-β and IL-6
induce differentiation of Th17 cells; IL-23 maintains
and stimulates cytokine production. IL-2 and IL-13
inhibit Th17 cells [55, 869].
Regulatory T cells (Treg) are defined functionally by
their suppressive effects on immune response, inflam-
mation and autoimmunity. Tregs are the subject of
intense research; they have important roles in the cause
and prevention of diseases including cancer. Many types
of cells show regulatory character. Here we will
mention three major types. Th3 cells secrete the immu-
nosuppressive cytokine TGF-β. Tr1 cells produce
immunosuppressive IL-10. Natural Treg cells have the
phenotype CD4+ CD25+ Foxp3+. IL-2 and TGF-β
induce nTreg cells. The nTreg mechanism of suppres-
sion involves cell contact (cell surface receptors) and
possibly the secretion of immunosuppressive IL-10 and
TGF-β [1714, 2002].
Polarization is not limited to Th cells. Based on the
cytokines secreted, NK cells [285], cytotoxic CD8
T cells [251, 1708], macrophages [1313] and dendritic
cells [1206, 1596, 1647] may develop type 1, type 2
and regulatory cell profiles. Imbalance in type cells
(exaggerated polarization/cytokine secretion) appears
to drive development of diseases including autoimmu-
nity (exaggerated Th1 or Th17) [6, 290], allergy
(exaggerated Th2) [923, 1101], and cancer (Treg)
[1590, 2056].
IL-12 + IFNγ IL-2 TNF
Th1
IL-4 - IL-13 IL-17
AP
Thp Th0
IFNγγ - IL-4 IL-5 IL-6
Th2
IL-2 IL-2 IL-4 + IL-9 IL-10 IL-13
IL-4 OX40 +
IFNγ B7.2 +
IL-13
Walter M. Lewko and Robert K. Oldham
Antigen Presentation;
Dendritic Cells
T-cells and B-cells do not typically respond to antigens
directly, rather a family of cells referred to as profes-
sional antigen presenting (AP) cells make the presence
of an antigen known to these effectors in a complex and
highly regulated process referred to as antigen presenta-
tion [1159, 1888]. Dendritic cells (DC), macrophages, B
cells, Langerhans’ cells, and eosinophils are antigen
presenting cells. These cells have the necessary ability
to migrate (between tissues and lymphnodes), they con-
tain the appropriate antigen processing machinery (anti-
gen is broken down to small peptides in the process) and
surface binding proteins (including MHC to display
antigen peptides) to help insure that an effective and
safe immune response occurs. AP cells also have the
necessary costimulatory molecules such as CD80 (B7.1)
and CD86 (B7.2). Among the AP cells, dendritic cells
are particularly good at inducing specific T cells with
anticancer and antiviral activity. Different subpopula-
tions of AP cells may favor different types of immune
response (e.g. Th1 vs. Th2) [1595, 1596]. Cytokines
control the proliferation and development of AP cells.
AP cells are also a source of cytokines which influence
innate as well acquired immunity. Abnormalities in anti-
gen presentation may result in autoimmune disease or,
in the opposite extreme, a failure to mount any immune
response at all.
Tumor Necrosis Factor Family
of Cytokines
Tumor necrosis factor (TNF) was originally discovered as
a component of blood, produced by host cells in response
to infection and bacterial products such as LPS [150, 151,
152, 250, 1471]. A number of related factors turned up [6,
150, 657, 685]. Now it is known that TNF (now called
TNF-α) is the founding member of a family of at least 19
cytokines and related virus proteins. The members of the
TNF family are involved in cell growth, differentiation,
inflammation, wound repair, costimulation of immune
response and the regulation of cell death.
There is homology in amino acid sequence among
most TNF family members, though it is not particularly
high [685]. Nerve growth factor (NGF) is not homolo-
gous but its receptor is related to the TNF receptor.
TNF-β and NGF are soluble cytokines. TNF-α and
TWEAK are mostly soluble, partly membrane bound.
The remaining TNF family members are membrane-
165
bound proteins [685]. The active, receptor binding form
of most TNF family members is a trimer. These trimers
induce receptor clustering and signal transduction [75].
Membrane-bound TNF family members act by direct
contact between the cytokine bearing cells and receptor
bearing target cells. Certain membrane-bound cytok-
ines, upon engaging receptor, may signal back (reverse
signaling) inducing effects in the parental cell; where
reverse signaling occurs, both cells are, at the same
time, targets and effectors.
TNF family members bind receptors that are related
molecularly. They are transmembrane glycoproteins.
Some TNF family receptors have soluble/shed forms
that are the result of proteolytic cleavage or alternative
mRNA splicing. The amino acid identity between the
human receptors is in the range of 25–35%. The extra-
cellular regions characteristically have three to six copies
of cysteine-rich pseudo repeats, each containing six
cysteines in a segment of about 40 amino acids. The
cytoplasmic region of certain receptors contains a con-
served “death domain,” involved in apoptosis (e.g.
TNFRI, Fas, DR3, DR4, DR5). There are intracellular
receptor associated proteins such as TRAF (TNFR-
associated factors), TRADD (TNFR1-associated death
domain protein), and RIP (receptor interacting protein)
which bind and communicate between the TNF receptor
family members to regulate apoptosis and other cytokine
responses [2137]. Members of the TNFR family share
these intracellular signaling proteins to activate similar
transduction pathways. Certain members of the TNF
receptor family are found in many types of cells (TNFRI,
TNFRII, Fas, Fn14) whereas others are restricted to
hematopoietic cells (CD27, CD30, CD40, HVEM, OX40,
4-1BB) or specific tissues (nerve growth factor receptor)
[75, 2137].
Interleukin-1
Inflammation, Immune Regulation,
Hematopoiesis, and Wound Repair
IL-1 was originally called lymphocyte activating factor
for its effects on mitogen-treated thymus cells [617].
It has also been called leukocyte pyrogen and endoge-
nous pyrogen, for its fever inducing effects [436, 1401].
Monocytes, macrophages, keratinocytes, T cells, B cells,
NK cells, eosinophils, dendritic cells, fibroblasts, epithe-
lial cells, endothelial cells, neurons, glial cells and
astrocytes produce IL-1 [551, 1170, 1735, 1972, 2140].
IL-1 is involved in inflammation, hematopoiesis, immune
regulation, wound healing and metabolic regulation.
166
It is responsible for, or at least involved in, a number of
inflammatory diseases. IL-1 also appears to influence
cancer growth and metastasis.
The IL-1 gene family has three main members: IL-1α,
IL-1β and IL-1 receptor antagonist (IL-1Ra). IL-1α and
IL-1β are synthesized by separate, distantly related
genes [1225, 1500]. The proteins have 26% homology.
Both cytokines are synthesized as 31,000 kDa pro-IL-1
molecules. They lack the usual signal peptides charac-
teristic of most secreted proteins.
Pro-IL-1α and the processed 17,000 mw product are
both active cytokines. Newly synthesized IL-1α accu-
mulates within the cytosol. Not much IL-1α is secreted.
Intracellular IL-1 may serve an autocrine function in
endothelial cells, keratinocytes and fibroblasts. Pro-IL-1α
is found on the cell surface, associated with lectin;
bound IL-1α has cytokine activity [201].
Pro-IL-1β is inactive. It is processed to a 17,000 mw
form, which is functional and the major secreted form of
IL-1. Processing is carried out by the protease, IL-1β
converting enzyme (ICE, caspase 1) [68, 160, 272, 299,
1992]. ICE is involved in the processing of other cytok-
ines (IL-16 and IL-18), but not IL-1α, which is cleaved
by another protease.
mRNA synthesis and posttranslational processing
control the production of IL-1β. Bacterial products
(LPS), TNF-α, IL-1 itself, and the immunostimulatory
drug OK-432 stimulate IL-1β secretion by blood mono-
cytes and other cells [782, 971, 435, 437]. Extracellular
ATP triggers activation of ICE and a burst of IL-1β
secretion [529, 528, 1557]. ATP is a signal that is
released by activated lymphocytes and from damaged
cells [782, 1925].
IL-1Ra is an inhibitor of IL-1. It competes with IL-1
for its receptor [454, 482, 658, 723]. Secretion of Il-1Ra
generally follows IL-1 production. In patients and in
cultures of human PBMC, IL-1Ra secretion is stimu-
lated by IFN-α, IL-4 and to a lesser extent by IFN-γ
[1997]. Patients treated with IL-1 had increased serum
IL-1Ra and with time the antagonist remained measur-
able after IL-1 was no longer detected [1007].
There are two receptors for IL-1, IL-1RI that is
responsible for activity and IL-1RII that is a regulatory
decoy receptor. They are distinct membrane bound pro-
teins, produced by separate genes [310, 1829].
The active signaling complex contains several com-
ponents. IL-1 binds the IL-1RI. IL-1 receptor accessory
protein (IL-1RAcP) binds and enhances affinity for
IL-1; it is required for signal transduction [663, 1008,
2144]. MyD88 is an adapter protein that joins the com-
plex [2145]. IL-1R associated kinase (IL-1RAK) then
binds; the kinase is phosphorylated and activated [354].
Cytokines
Then TNFR-associated factor-6 (TRAF-6) binds. Now
this multicomponent receptor complex is functional and
it induces the activation of transcription factors, in par-
ticular NF-κB, which translocates to the nucleus, binds
DNA response elements and induces the production of
mRNA’s responsible for IL-1 effects [243, 354, 1008].
The second receptor, IL-1RII binds IL-1 but cannot
signal due to the lack of a binding site for MyD88. There
is a soluble form of RII, which is found in tissue fluids;
it is increased during inflammation [628, 1593, 1938].
Soluble receptor II may be generated by proteolytic
cleavage [1506] or by alternative mRNA splicing
[1136]. The cellular form of RII binds IL-1α, IL-1β and
IL-1Ra; soluble RII binds IL-1α and IL-1β but not
IL-1Ra [65]. RII appears to function as a competitive
decoy for IL-1 [334, 1446, 1621] and IL-1RAcP [1056]
available to bind IL-1RI.
The biological properties of IL-1 are far reaching and
complex; it is often referred to as a two edge sword. On
one hand IL-1 functions in hematopoiesis, the regula-
tion of metabolism, wound healing and the control of
infection. On the other hand, IL-1 appears to be involved
in the debilitating effects of several inflammatory dis-
eases and septicemia [846, 1482].
IL-1 has a remarkable effect on hematopoiesis. In
early progenitor cells, IL-1 is survival factor; it protects
stem cells from environmental insults, cytotoxic chemi-
cals, radiation and the like. IL-1 also primes progenitor
cells for outgrowth, by placing them in the G0-G1 stage.
IL-1 by itself does not induce outgrowth [191, 530,
1442, 1501, 1762]. IL-1 increases IL-3, G-CSF, M-CSF
and GM-CSF; together with these cytokines, IL-1
synergistically stimulates the expansion of specific cell
lineages [100, 1329, 1349, 2306, 2309]. Patient treatment
with moderate doses of IL-1 caused elevated neutrophil
and platelet levels [1842]. Interestingly, prolonged treat-
ment and higher doses caused an apparent decrease in
neutrophils, platelets and erythrocytes. There are two
possible explanations for this decrease: First, IL-1
induces TNF and TNF has a depressive effect on
hematopoiesis [594, 845, 898]. Second, IL-1 induces
adhesion molecules on capillary endothelial cells.
Studies in culture showed that endothelial cells with
elevated adhesion molecules bind a number of different
types of cells; these cells may be sequestered in the cap-
illary bed resulting in an apparent decrease in cell num-
bers [406, 1380, 1681].
IL-1 stimulates dendritic cells. It acts together with
GM-CSF to promote maturation [744]. Dendritic cells
have an important function as antigen presenting cells in
the development of lymphocytes. T cells and B cells
synthesize IL-1 and have the IL-1 receptor [1163, 1504].
Walter M. Lewko and Robert K. Oldham
IL-1 stimulated T cells, induced IL-2 production, IL-2
receptors and cytokine production; IL-2R induction
appeared to be a key step [940, 1039, 1827]. Immature
T cells appeared to require IL-1 in order to synthesize
IL-2; IL-6 acts synergistically with IL-1 in T-cell matu-
ration and the production of IL-2 [1686]. In more mature
T cells, IL-1 favored Th2 cells. It stimulates the prolif-
eration of Th2 cells and the production of IL-4, IL-5 and
IL-6. IL-1 had no apparent effect on Th1 cells. IL-1 also
acts as a chemoattractant for T cells and it stimulates
production of IL-8 [822, 1039, 1335]. In B cells, IL-1
acts together with B cell growth and differentiation
factors IL-4, IL-6 and IL-2 to stimulate growth and the
production of immunoglobulin. IL-1 increases the levels
of glucocorticoids. Glucocorticoids stimulate B cell
IL-1 receptors. Glucocorticoids and IL-1 together stim-
ulate antibody production [14]. In natural killer cells,
IL-1 and IL-12 together stimulated the secretion of
IFN-γ in response to infection [824].
The activities of IL-1, TNF and IL-6 are interrelated.
IL-1 and TNF have similar biological properties and
their combined effects are usually synergistic [1216,
2093]. IL-1 induces TNF production [1077, 2173] and
TNF induces IL-1 production [197, 435]. The receptors
for IL-1 and TNF share a common accessory protein
and signaling path. As part of a regulatory loop, IL-1
tends to depress TNF receptors [788, 2093]. In mice
IL-1 induced IL-6 production. Blocking IL-1 by IL-1Ra
depressed IL-6 levels and inflammation [616, 1090].
TNF stimulates both IL-1β and IL-6 [550]. IL-6 regu-
lates IL-1 activity by increasing IL-1Ra levels [1749]
and by decreasing IL-1 formation [1742].
IL-1 stimulated mast cell production of IL-3, IL-4,
IL-5, IL-6, IL-9 and TNF. Thus, IL-1 may enhance Th2-
related events including humoral response and allergic
inflammation. IL-1 may also influence the natural
response of mast cells to parasites, viral infection [820]
and cancer.
The nervous system has receptors for IL-1 and brain
cells are capable of IL-1 production [196, 1072]. IL-1β
is secreted by microglial cells in response to brain
trauma. IL-1 induces production of nerve growth factor
[1873] and ciliary neurotrophic factor [742], which are
involved in nerve cell survival and wound repair. In this
way IL-1 released during brain inflammation may have
a beneficial effect on CNS regeneration [742]. IL-1
treated patients often suffer from sleepiness, fever and
lack of interest in eating. IL-1 activity in the nervous
system appears to be involved. IL-1 injections in ani-
mals induced slow wave sleep and depressed rapid eye
movement [1815]. IL-1 induces fever. There are several
other cytokines capable of doing so including TNF, IL-6
167
and IFN-α. IL-1 increases IL-6 levels; blocking IL-1
activity lowered IL-6 and depressed fever [327, 616,
2277]. IL-1 also induces anorexia in animals. The mech-
anism appears to involve increased brain cyclooxyge-
nase and prostaglandin production [733].
IL-1 has several effects on metabolism. It interacts
closely with a number of hormones, in particular the
glucocorticoids and insulin. IL-1 influences the brain-
pituitary-adrenal axis. IL-1, injected i.v., increased the
release of corticotropin releasing hormone and ACTH
as well as endorphins, vasopressin and somatostatin.
Glucocorticoid levels increased in response to injected
IL-1 and during stress and trauma [1639, 1812].
Glucocorticoids influence metabolism throughout the
body; hydrocortisone feeds back on the production of
IL-1 [144, 148, 410, 1080]. In this way glucocorticoids
appear to regulate inflammatory response. IL-1 pro-
motes bone resorption and cartilage degradation [424].
It induces the release of collagenase, phospholipase A
and prostaglandin E2 from synovial cells [154, 1326,
2304]. IL-1 altered the production of several liver pro-
teins; fibrinogen, clotting factors, metallothionein and
complement were increased. Albumin, transferrin and
lipoprotein lipase were decreased. Negative nitrogen
balance in muscle protein is associated with inflamma-
tory diseases. IL-1, IL-6, TNF and insulin are involved.
Pancreatic β islet cells have IL-1 receptors [706]. Both
IL-1 and IL-6 stimulate production of insulin [2182].
IL-1 and IL-6 together with TNF have insulin-like
effects on metabolism. In rats treated with endotoxin,
for example, IL-1 blockade using IL-1Ra spared muscle
protein [2272].
IL-1 stimulates the growth of several different types
of cells including keratinocytes, smooth muscle cells,
glial cells, mesangial cells and fibroblasts [996, 1126].
In this way, IL-1 has a role in wound repair and angio-
genesis. In fibroblasts, IL-1 increased proliferation
[1744] and the uptake of glucose [156]. IL-1 also stimu-
lates collagen production by fibroblasts and epithelial
cells. Tissue fibrosis is associated with inflammatory
disease; it appears to be related to paracrine and auto-
crine IL-1 secretion. In cultured fibrotic kidney cells,
IL-1 blockade using IL-1Ra depressed fibroblast prolif-
eration [1150].
There are interesting relationships between IL-1 and
endotoxin. IL-1 is one of several cytokines (IL-2, IL-15,
IFN-γ and TNF-α), which stimulate Toll-like receptor
(TLR) levels in macrophages [1247]. TLRs bind micro-
bial products and stimulate innate immunity and inflam-
matory responses to pathogens. Endotoxin and IL-1
have similar biological properties including the induction
of IL-1 and TNF levels. Interestingly, TLR and the
168
IL-1R have homologous intracellular signaling domains.
Activation of these receptors brings about similar proin-
flammatory responses [164].
IL-1 is involved in several inflammatory diseases.
In the intestine, IL-1 and TNF are produced by epithe-
lial cells and appear to prevent microbial invasion. In
inflammatory bowel disease, these cytokines were
increased. Somatostatin inhibited basal IL-1β produc-
tion and prevented induction by TNF and bacteria.
These results suggested that somatostatin regulates
IL-1 and may have a role in prevention of bowel inflam-
mation [314]. In patients with rheumatoid arthritis,
synovial tissues produce IL-1 [538] and the levels of
IL-1 are elevated in plasma. IL-1 is a chemotactic fac-
tor for neutrophils [1732]; IL-1 induces nitric oxide
synthetase and the production of nitric oxide, which
mediates many inflammatory processes [504]. IL-1 also
induces cyclooxygenase II and the production of pros-
taglandins, which have inflammatory effects [1217,
1360]. IL-1 directly stimulated levels of the proteolytic
enzymes involved in joint destruction, collagenase, tis-
sue plasminogen activator, and stromelysin [424, 1403].
In chondrocytes, chronic IL-1 inhibited proliferation
and the production of collagen and proteoglycan [1156,
1885, 1968]. IL-1 and tumor necrosis factor act mutu-
ally to stimulate joint inflammation [197, 2173].
Clinical and animal studies have shown that therapies
blocking TNF-α activity were beneficial. In mice with
collagen-induced arthritis, anti-IL-1 antibodies and
antibodies to the IL-1 receptor [607, 901, 2055, 2173]
reduced the severity of the disease. Blocking both IL-1
and TNF-α had an additive effect [2173]. IL-1Ra and
IL-1 RII are being studied clinically to see if they are of
benefit in treating inflammatory diseases. In patients
with rheumatoid arthritis, rhIL-1Ra reduced the pro-
gression of joint erosion [199]. Unfortunately, rhIL-
1Ra did not significantly increase survival in patients
with severe sepsis [1499].
In patients with arthritis, exercise has a beneficial
effect on joints. A biochemical rationale was provided in
an interestingly cell culture model for physical therapy.
Cyclic tensile strain on cultured chondrocytes decreased
the effects of IL-1β by interfering with IL-1 receptor
signaling. Markers for inflammation were decreased
and the extracellular matrix was restored [2217].
IFN is used to treat viral hepatitis. However, 60% of
patients do not respond well. IL-1 may be a reason for
this lack of response. IL-1β appears to depress the anti-
viral activity of type I interferon [1994]. When mice
were injected with IL-1β, liver cell antiviral response
was depressed. IL-1 appeared to interfere with IFN
signaling at the level of STAT 1 phosphorylation. It is
Cytokines
possible that IL-1 blockade may be a way to intensify
IFN response in the treatment of viral disease [1994]
and possibly cancer. IL-1 is of interest in the treatment
of cancer [1838]. In culture, IL-1 has a direct inhibitory
effect on the growth of several types of human cancer
cell lines [378, 959, 1498]. In animal studies, intratu-
moral treatment with IL-1 induced regression of injected
tumors but not distant metastases [1418]. Fibrosarcoma
cells expressing cell surface IL-1α, implanted in mice
showed decreased growth and induced protective immu-
nity [1858]. While preclinical studies showed promise,
little or no benefit has been observed using IL-1 in
patients with melanoma [1879] or renal cancer [1625].
The combination of IL-1 with IL-2 was explored in
clinical trials. Increases in NK and LAK activity were
observed and there were some responses in patients
with colon cancer, melanoma and renal cancer [2026].
IL-1 has also been tested in combination with chemo-
therapy to enhance drug effectiveness and decrease
blood cell suppression. IL-1 decreases the activity of
drug metabolizing cytochrome P-450 [619]. In cultured
cells, IL-1 had synergistic effects with certain forms of
chemotherapy [1417, 2046]. Similar synergistic effects
of IL-1 and chemotherapy were seen in animal tumor
models [897, 1051]. Thus far the results in clinical trials
have been rather disappointing. In ovarian cancer
patients treated with carboplatin and in osteosarcoma
patients treated with etoposide, the addition of IL-1
resulted in very modest increases in antitumor response
[2072, 2199]. In preclinical studies, IL-1 offered some
protection to progenitor hematopoietic cells undergoing
irradiation or treatment with cytotoxic drugs [191, 1441,
1495, 1762]. When cancer patients were given IL-1 in
phase 1 clinical trials, granulocytes and platelets were
stimulated showing that IL-1 could be beneficial for
blood cell recovery [357, 1842]. In children on chemo-
therapy (Ifosfamide-Carboplatin-Etoposide), unfortu-
nately, treatment with IL-1α produced no significant
hematoprotective benefit [576].
IL-1 produces significant though manageable side
effects. Low dose IL-1 causes headache, myalgia, arth-
ralgia, sleepiness and anorexia. Higher doses cause a
rapid loss in blood pressure. These symptoms mimic septic
shock. Highest doses produced grade IV hypotension and
neurological signs [1842]. In summary, IL-1 stimulates T
cells, B cells and hematopoiesis. Undoubtedly, it has a
role in the development of immunity. But at present its
toxicity and modest antitumor effects preclude approval
for use in cancer patients.
There is evidence that IL-1 stimulates the growth of
certain types of cancer. In patients with chronic myelog-
enous leukemia, Il-1β levels were elevated and blocking
Walter M. Lewko and Robert K. Oldham
IL-1 depressed cancer cell growth; IL-1 was determined
to be a negative prognostic factor [2148]. IL-1 is an
autocrine growth stimulator for certain human gastric
cancers [864]. In capillary endothelial cells, IL-1
increased cell surface adhesion molecules and the bind-
ing of tumor cells [406]. Pretreatment of mice with IL-1
increased the metastasis of B16 [2074] and human mel-
anoma cells [620]. When the mice were treated with
IL-1Ra, there were fewer metastases, smaller metasta-
ses and the animals lived longer [620]. Perhaps thera-
pies aimed at blocking IL-1 may be beneficial in
controlling the growth and spread of certain cancers.
Interleukin-2
Growth And Activation of T, B and NK
Cells; Activation-Induced Cell Death;
Elimination of Self-Reactive T Cells
IL-2 was originally described as a factor in the condi-
tioned medium of mixed lymphocyte cultures that stim-
ulated T cell growth [643, 936]. The following is a brief
review of this very well studied cytokine. The early
work is covered in greater depth in prior editions of this
book [1110].
IL-2 is a member of the helical cytokine family,
which includes IL-2, IL-4, IL-7 and IL-15 [1963]. IL-2
is produced mainly by T cells. Activated cytotoxic T
cells, Th0 and Th1 cells secrete IL-2 but Th2 cells do
not [1040, 1798].
Initial studies used natural IL-2 prepared from lym-
phoid cells [561, 1305, 1649, 2141]. It is a 133 amino
acid glycoprotein with variable molecular weight due to
the carbohydrate. The gene for IL-2 has since been
cloned [1666, 1963]. The recombinant protein made in
bacteria lacks carbohydrate. Certain forms of recombi-
nant IL-2 have an amino acid change to facilitate pro-
duction [210]. Recombinant IL-2 and the natural product
have similar biological activities and stabilities [1663].
Both forms of IL-2 have relatively short half-lives of
1–2 h following intravenous injection [696, 1158, 1160]
and 4 h following bolus sc injection [696]. IL-2 loss is
mainly by renal clearance rather than hepatic metabo-
lism or target cell binding [448]. Intraperitoneal injec-
tion has been used to treat abdominal malignancies.
Injection i.p. produces sustained, locally high levels of
IL-2; systemic toxicity is lower. Sometimes, repeat i.p.
injections induced fibrosis, likely due to the release of
secondary cytokines such as PDGF or TNF-α [145,
1091, 2044]. IL-2 has also been delivered by inhalation
[818] or and by mini-osmotic pumps [1458].
169
The IL-2 receptor is composed of three subunits:
IL2Rα, IL2Rβ and IL2Rγ (also called γC, common γ
subunit). The α subunit has high affinity for IL-2. The
β and γ chains are involved in signaling [968, 1002,
1097, 1823]. The three subunit complex exhibits high-
est affinity for IL-2 (Kd 10−10 M). In NK cells, the
IL-2R is in the form of a βγ dimer; this two-subunit
receptor has moderate affinity (Kd 10−9 M) and requires
higher IL-2 concentrations to produce effects [734,
945, 1024, 1508, 1823]. Janus kinase activity is associ-
ated with the γ chain. The receptor complex is activated
by phosphorylation. A major signal path involves the
STAT transcription factors (signal transducer and
activator of transcription), which bind to the activated
receptor complex and are in turn activated by phospho-
rylation. The activated STATs translocate to the nucleus,
bind an array of gene promoters and initiate transcrip-
tion associated with IL-2 response. Immune suppressive
drugs FK406 and Cyclosporin A act at the level of
transcription factors, which are activated by IL-2 and
other cytokines [491, 544, 656, 1262, 1263].
A peptide has been produced which comprises
amino acids 1–30 of human IL-2. It forms a tetra-
meric structure that binds IL-2R β dimers. The pep-
tide is an IL-2 agonist: it induces activation of CD8+
T cells and lymphokine-activated killer (LAK).
Interestingly, it acts synergistically with IL-4, IL-9,
IL-15 and with IL-2 itself. This peptide may have
therapeutic potential [476, 477].
IL-2 was originally discovered by its capacity to
stimulate proliferation in T cells. T cells produce IL-2
and it stimulates T cell growth. A defect in IL-2 produc-
tion appears responsible for a type of severe combined
immunodeficiency disease [2135]. IL-2−/− knockout
mice have been developed which lack IL-2. In initial
studies, proliferation response was weak in Con A or
anti-CD3 Mab activated mononuclear leukocytes; other-
wise the mice appeared rather normal [1754]. Further
studies showed IL-2 −/− mice had increased autoim-
mune disease, ulcerative colitis, inflammatory bowel
disease, anemia, progressive loss of B cells and altered
bone marrow cell profiles [1185, 1710, 1711]. The animals
suffered from uncontrolled T cell proliferation, appar-
ently due to a defect in Fas-induced apoptosis [990,
1092]. Related observations have been made in mice
lacking the IL-2 receptor α chain [2168] and β chain
[1933]. In germfree IL-2 −/− mice, autoimmune disease
developed but not colitis, which appeared to require
enteric microbial antigen [341]. Interestingly, allograft
rejection still occurred, not only in IL-2 −/− knockout
mice [1886] but also in IL-2 −/− IL-4 −/− double knock-
out mice [1119]. These results suggested IL-2 is not an
170
absolute requirement for rejection. Other cytokines such
as IL-7, IL-12, IL-15 or IL-21 may provide redundancy
in certain IL-2 functions [1886]. Nonetheless, IL-2
appears necessary for the normal regulation of T-cell
growth and involution. In particular, IL-2 has a role in
tolerance by inducing T cell suicide [1706, 1933, 2168],
in activation-induced T cell death [1092, 1626, 2062]
and it is involved in the inhibition of T cell memory
maintenance [1026]. Further, IL-2 induces regulatory T
cells (Treg: Foxp3+ CD4+ CD25+), which are antigen
specific cells that regulate immune response. In particu-
lar, they suppress effector T cells, which are self-antigen
specific and autoimmunogenic. In many cancer patients,
these regulatory T cells are enriched in the tumors and
blood. Since many tumor antigens are essentially over-
expressed self antigens, Treg cells may have a role in
tumor immune escape and poor response to cancer bio-
therapy [165, 1309, 1806].
IL-2 stimulates cytotoxicity in NK cells [734, 1386,
1387], thymocytes [1800] and in cytotoxic T lympho-
cytes (CTL) [1799]. Peripheral blood lymphocytes
treated with IL-2 generate LAK cells [176, 444].
Cytotoxic NK cells make up the major active compo-
nent of LAK [1507, 1567, 1995, 2133]. NK cells lyse
tumor cells by membrane channel forming perforin,
granzymes and receptor mediated cell death by apopto-
sis. IL-2 also enhances antibody dependent cellular
cytotoxicity (ADCC) mediated by lymphocytes against
tumor cells [1810].
IL-2 stimulates B cell growth, differentiation and the
production of Ig. IL-2 acts alone and together with
other B cell stimulating cytokines such as IL-6 [886,
1419, 1872]. IL-2 also activates production of several
cytokines including the interferons, tumor necrosis fac-
tor α (cachectin) and tumor necrosis factor β (lympho-
toxin) [478, 611, 1016, 1599]. In this way IL-2 may
induces a cascade of effects influencing immune
response.
Many tumor cell lines express the IL-2 receptor.
Some of these cells respond directly to added IL-2 with
decreased growth. Interestingly, IL-2 sometimes down
regulates surface molecules such as ICAM and MHC,
which are involved in antitumor immune response
[2234]. Denileukin diftitox (ONTAK) is a recombinant
cytotoxic chimeric protein composes of active domains
of diphtheria toxin fused with the receptor-binding
domain of IL-2. It binds to cells expressing the IL-2R, is
internalized and kills susceptible cells. ONTAK has
been approved for use in cutaneous T cell lymphoma
and is being tested in other hematologic malignancies.
It is also being studied as a means of controlling regula-
tory T cells in cancer patients [86, 380].
Cytokines
IL-2 is used to treat renal cell carcinoma and mela-
noma patients [190, 1674, 1675, 2146]. Its effects gen-
erally appear due to T cells and NK cells. Response
rates were related to IL-2 dose. Most patients tolerate
IL-2, but in addition to the usual flu-like discomforts
associated with cytokine therapy, high dose IL-2 induced
hypotension and vascular leak syndrome (VLS) which
may be life threatening [106, 1676].
VLS is due to capillary endothelial cell damage.
Possible sources of damage include cytotoxic lympho-
cytes, neutrophils [717], complement [1982], TNF [458,
1607] and inflammatory factors such as histamine, sero-
tonin and bradykinin [1211]. Toxicity may be due to
IL-2-induced IL-1. In a phase I trial, cancer patients
were treated with soluble IL-1 receptor, an antagonist,
in an attempt to lessen IL-2 toxicity [1266].
Unfortunately, soluble IL-1R provided no benefit. The
evidence for cytotoxic lymphocyte involvement is rather
strong. Immune suppressive agents including IL-10
(discussed later) [1676, 1115] and inhibitors of immune
cell outgrowth [1606] depress VLS. IL-2-induced LAK
cells adhere to endothelial cells and kill them [375].
Blocking the binding of leukocytes to capillary endothe-
lium [1481] and depletion of NK cells [1547] ameliorate
VLS. Finally, IL-2-induced endothelial cell damage is
significantly lower in mice, which are defective for
perforin or Fas ligand (discussed later), both involved in
lymphocyte-mediated cytotoxicity [1612].
IL-2 has been used ex vivo to stimulate patient periph-
eral blood lymphocytes to form LAK cells [1668, 1669].
Lymphocytes are collected by cytopheresis and cultured
with IL-2 for several days. IL-2-treated lymphocytes are
then reinfused back into the patient. Oldham and co-
workers later developed a protocol, which only required
a very short bedside incubation of lymphocytes with IL-2
prior to reinfusion [794]. LAK cells are preferentially
cytotoxic to neoplastic cells, though lysis of normal capil-
lary cells has been observed [76, 375, 1512, 1855]. LAK
cells act in a non-MHC-restricted manner [673, 1677].
In fact, the presence of strong MHC on the tumor cell
surface inhibits LAK. Initial reports indicated that LAK
appeared effective in metastatic renal cancer and mela-
noma with response rates of 15–25% [474, 541, 1667].
But subsequent reports for patients with renal cancer
questioned whether the cells provided any added benefit
to the IL-2 given systemically at the time of LAK therapy
[1669, 1674]. It appears that cancer patients treated with
IL-2 develop LAK-like cells in vivo [1670].
IL-2 has also been used ex vivo to stimulate out-
growth of tumor derived T cells. These expanded T cells
are called tumor infiltrating lymphocytes (TIL) [1018,
1671, 2011]. They are also referred to as tumor derived
Walter M. Lewko and Robert K. Oldham
activated T cells (TDAC) [1108, 1109, 1207, 1208,
1493, 1494]. In the original work in mice, Rosenberg
showed these cells were 100x more potent than LAK in
anticancer activity and TIL were effective against
certain LAK-resistant tumor cells [1672]. TIL are part
of the adaptive immune response system; they are cyto-
toxic in an MHC restricted manner; antigen presenting
cells were involved in their development. TIL are grown
to greater than 1010 cells and then reinfused back into
the patient. Response rates in melanoma and renal
cancer are on the order of 10–25% with some durable
remissions. Combinations with other cytokines are still
being tested for increased treatment efficacy.
Biochemotherapy, the combination of traditional che-
motherapy with biological response modifiers, produces
an unexpectedly high response in melanoma patients.
The reason for this is not understood. Response rate was
correlated with increased serum IL-6 and IL-10, both
Th2 cytokines, in patients receiving cisplatin, vinblastine
and dacarbazine followed by IL-2 and IFN-α-2b [674].
However, in spite of response rates exceeding 50%, it
is not clear that biochemotherapy adds any long term
survival benefit over IL-2 alone.
IL-2 is an effective adjuvant, stimulating immune
responses to tumor cells in mice. IL-2 is being tested as
a drug and as a product of genetically engineered cells,
in tumor cell and dendritic cell cancer vaccines [30, 587,
588, 1106].
In addition to cancer therapy, IL-2 has been used to
treat patients infected with human immunodeficiency
virus. CD4+ cell levels were increased with no increase
in plasma virus [1017]. IL-2 has also been used ex vivo
to generate antiviral T cells, which were reinfused into
patients [200, 2095].
Interleukin-3
Hematopoietic Cytokine
IL-3 is a 28,000 mw protein. It is produced mainly by
activated T cells [323, 324] but also by mast cells, thy-
mic epithelium, keratinocytes, neurons, monocytes,
neutrophils and eosinophils [835, 857]. In the past, IL-3
has been referred to as CFU stimulating activity, mast
cell growth factor, Thy1 inducing factor, multicolony
stimulating factor, P cell stimulating factor, and
hematopoietic growth factor [1327]. The gene for
human IL-3 is on chromosome 5 in close linkage with
IL-4, IL-5, IL-9 and IL-13 [2227, 2248]. IL-3 is released
during immune response and serves as a bridge between
the immune and hematopoietic systems.
171
The receptor for IL-3 has two subunits. The IL-3Rα
binds IL-3 [977]. It has homology with the GM-CSFRα
[1010]. The β subunit is shared with receptors for IL-5
and GM-CSF [726]. The β subunit does not bind
cytokine but as part of each receptor complex it enhances
affinity for the cytokine and it is responsible for signaling
(Jak2-STAT5) [1315]. IL-3, IL-5 and GM-CSF generate
similar signals [697, 1323]. There is also evidence that
the IL-3Rα chain signals [1314] adding complexity and
specificity to the response.
IL-3 stimulates the growth of most early multipo-
tential progenitor cells and early committed precursors.
IL-3 also stimulates macrophages, granulocytes and
mast cells through their most mature forms. IL-3 is a
major developmental cytokine for basophils [1061,
1230, 2033, 1296]. Only the later stages of the eryth-
roid [857] and megakaryocytic [2171] lines are no
longer sensitive. IL-3 thus increases the production of
macrophages, granulocytes, erythrocytes, and mega-
karyocytes. Effects on early erythroid and megakaryo-
cyte growth are rather distinctive to IL-3. IL-3 serves
as a primer and costimulator while other cytokines act
later to induce differentiation. It stimulates both cell
division and cell survival [407, 835, 2229, 924, 1919].
It acts synergistically with several cytokines including
IL-6 [843], IL-11 [1405], G-CSF [842], thrombopoi-
etin [1027], stem cell factor [2031] and Flt3 ligand
[1791]. In murine cell cultures, IL-3 tended to depress
the B lymphoid potential of lymphohemopoietic pro-
genitors [767]. In human cells, IL-3 increased the
production of B cell progenitors from uncommitted
CD34+ CD38− cells [353].
Osteoclast development in hematopoietic tissue is
influenced by colony stimulating factors. IL-3 has been
reported to be stimulatory and regulatory. In RANK
ligand mobilized osteoclast precursors, IL-3 inhibited
osteoclast differentiation, diverting the precursors to
macrophages [956].
Natural cytotoxic (NC) cells [1524, 1912] are mast
cell-like morphologically and they participate in immune
reactions such as tumor rejection and graft versus host
disease. They differ from standard NK cells in origin,
growth and kinetics of target cell lysis. Natural cyto-
toxic cells are IL-3 dependent. Unlike NK cells, they do
not respond to IL-2 [875–876]. Natural cytotoxic cells
appear to induce lysis by the release of TNF [876].
IL-3 has been studied extensively for possible clinical
use [479]. It has been tested in combination with other
cytokines such as GM-CSF, M-CSF, and EPO to stimu-
late hematopoietic recovery of chemotherapy patients
and bone marrow transplant recipients. IL-3 increased
platelets, leukocytes and reticulocytes. Side effects due
172
to IL-3 (fever, headache, myalgia) were generally mild
to moderate and manageable [589]. There were some
benefits but not sufficient to warrant becoming a part of
standard therapy. PIXY321 is a GM-CSF:IL-3 fusion
product, which has been tested in cancer patients with
similar results and toxicities [899]. Unfortunately, most
patients developed neutralizing antibodies suppressing
the effects of subsequent PIXY321 treatments [1311].
At present, the main clinical use for IL-3 is in the pro-
duction of cultured stem cells for patient reinfusion and
gene transfer protocols [214, 467, 475, 997].
It has been reported that IL-3 may stimulate [90] or
inhibit [1789] certain hematopoietic malignancies.
DT(388)IL3 is a diphtheria toxin-IL-3 fusion protein,
which is being tested in patients with acute myeloge-
nous leukemia. It kills malignant progenitors while
sparing normal progenitors [781].
There is interest in the use of particulate tumor anti-
gens for anticancer vaccination. Particulate antigens
tend to induce cytotoxic CD8+ cells while soluble
antigens induce CD4+ helper T cells. IL-3 appeared to
be beneficial in this type of vaccination. IL-3 increased
the number of antigen presenting cells, the percent of
these cells actually presenting particulate antigen and the
number of CTLs produced [1594, 2239].
Interleukin-4
B Cell and T Helper Response
Interleukin 4 (IL-4) is a 20 kDa glycoprotein. It was
originally called B cell stimulatory factor-1 (BCSF-1)
for its effects on B cell growth and Ig secretion [514,
801, 1544, 1608, 2249]. IL-4 is produced by activated
CD4+ Th2 cells [132, 1036, 1040, 1127, 1375], mast
cells [189, 204], basophils [1190], eosinophils [1463],
NK cells [461, 1154], γ/δT cells [2307], CD4+ CD8+ T
cells [1086] and NKT cells [1087, 1832, 2252]. The
gene for IL-4 is located on human chromosome 5 in a
region with other cytokines including IL3, IL-5, IL-9
and IL-13.
The IL-4 receptor is composed of two chains, IL-4Rα
and γc, the common γ chain found in several receptors
including the IL-2R [1372]. The α chain has the IL-4
binding site and conveys specificity as an IL-4 receptor.
The γ chain is involved in signaling. There is another
configuration of the IL-4 receptor in which IL-13Rα
replaces the γ chain, in which case the same receptor
complex binds and responds to both IL-4 and IL-13
[736, 1439]. Signaling involves phosphorylation of cer-
tain proteins including the receptor itself and phosphati-
Cytokines
dylinositol-3′ kinase [944]. In mononuclear and B cells,
IL-4 receptor levels are increased by IL-4; IFNγ inhibits
this increase [1853].
In addition to the membrane bound receptor, there is
a soluble form found in body fluids and in the medium
of cultured cells [526, 527]. The soluble receptor is
smaller in size but it retains the capacity to bind IL-4
[1374]. The soluble receptor is not due to proteolysis of
the membrane receptor, but rather differential mRNA
splicing [122, 1374]. IL-4 stimulates soluble receptor
production, apparently as a way of regulating IL-4
activity [307, 1638]. Soluble receptor competes with
the membrane receptor for IL-4 [527, 1212]. The func-
tion of the soluble receptor appears to be mainly inhibi-
tory [1212, 1730] but stimulatory [1730, 1871] effects
have also been observed. Further, the soluble receptor
may also serve as a carrier for IL-4, which protects IL-4
from metabolism and excretion, increasing its func-
tional half-life [526, 457]. Dissociation of IL-4 from
the soluble receptor would allow it to bind and activate
the membrane receptor.
IL-4 has a major role in immune response. It has
direct effects on target cells, which are often synergis-
tistic with other cytokines. IL-4 also has a major regu-
latory function in immunity by its influence on the Th1/
Th2 system [2, 254, 1821]. IL-4 induces precursor Th0
differention to Th2 cells [2]. In part, regulation in the
Th1/Th2 system involves apoptosis, programmed cell
death. Glucocorticoids favor apoptosis in thymocytes
and in mature T cells. Several cytokines including IL-4,
IL-2 and IL-1 inhibit glucocorticoid-induced apopto-
sis. IL-4 specifically rescues Th2 cells from death while
IL-2 rescues Th1 cells [1306, 2308].
As B cells depend on T cells for help, IL-4 is one of
the major cytokines for this purpose. IL-4 acts directly
on B cells and it is the main inducer of other Th2 cytok-
ines, most of which also stimulate B cells. IL-4 increases
pre-B cell growth, differentiation and Ig secretion
[1015, 1597, 1850, 1851, 1871, 2082]. IL-4 stimulates
class switching and the production IgG1 and IgE [321,
329, 409, 1552, 1851, 1871]. Among its other effects,
IL-4 increases MHC II on B cells for antigen presenta-
tion [1459].
In bone marrow IL-4 has a synergistic effect with
IL-11 on the induction and growth of hematopoietic
progenitor cells [1407, 1537, 1637]. IL-4 increases the
growth of monocytes [1974], macrophages [1447] and
increases macrophage antitumor activity and antigen
presentation [239, 351, 2302].
IL-4 enhances the growth of activated PBL but had
little effect on unprimed resting cells. Timing of the
cytokine appears to be a factor. While IL-4 stimulated
Walter M. Lewko and Robert K. Oldham
PBLs previously activated with IL-2, IL-4 added to
cultures simultaneously inhibited proliferation induced
by IL-2 [707, 2021].
IL-4 tends to stimulate the growth of thymoctyes
[2301] and T cells [2152]. But IL-4 effects are complex
and in certain circumstances may be inhibitory [2153].
In cultures of TIL, addition of IL-4 in combination with
IL-2, grew T cells from human tumors with specific
activity against autologous tumor [933, 934, 1108, 2034].
IL-2 was required; TIL could not be generated using
IL-4 only [934]. IL-4 induced the IL-2 receptor in mouse
T cells [254]. IL-4 together with IL-2 produced better
TIL growth and specific antitumor activity in many but
not all tumors [534, 933]. In certain TIL cultures, early
growth effects of IL-4 were lost or even reversed with
time [534, 933]. In another study, IL-4 in combination
with IL-2 tended to produce more T cells with increased
antitumor cytotoxicity compared to TIL induced with
IL-2 only (8,724). We developed an IL-4 dependent T
cell line from a node specimen of a lymphoma patient.
This tumor cell line required IL-4 in addition to IL-2
[1108]. Other reports did not show remarkable benefits
of IL-4. In one study using melanoma, the addition of
IL-4 to IL-2 promoted TIL growth in a minority of cases
(5/24), but decreased growth in most cases (17/24).
Likewise, specific lysis was enhanced by IL-4 in a
minority of cases but depressed in the majority (13/19)
[1132]. It may be that IL-4 stimulated those T cells,
which had been optimally activated by tumor antigen,
but inhibited those cells, which had not been properly
activated.
Treating PBLs with IL-2 typically induces LAK cells.
In mice IL-4 could induce LAK in the absence of added
IL-2 [1388]. When mouse PBLs were induced with
IL-2, IL-4 tended to enhance LAK activity [1388]. In
human PBLs, LAK may be induced by IL-4 in cancer
patients pretreated with IL-2 but not in unprimed cells
[752, 874, 932, 933]. In previously untreated cells, IL-4
added at the same time as IL-2 inhibited induction of
LAK by IL-2 [203, 707, 752, 932, 1870, 2152]. Priming
with IL-2 in vivo or in culture was necessary for IL-4
activity. Loss of the IL-2 receptor may be the mecha-
nism by which IL-4 inhibited LAK [1133].
Knockout mice have been developed which are defi-
cient in IL-4. IL-4 has many important and interrelated
immune functions, but it is interesting that these mice had
a relatively normal immune profile. Serum IgG1 was low.
In response to nematode infection, the normal rise in IgE
levels failed to occur. Otherwise, T and B cell develop-
ment appeared relatively normal [1032]. More recent
studies showed that IL-4 deficient knockout mice had
impaired antitumor cellular immunity (Schuler T. 1999).
173
This may be related to the observations that long term
IL-4 benefits IL-12 secretion that in turn stimulates
cellular immunity [911].
IL-4 appears to have a role in several inflammatory
diseases. IL-4 together with IL-3 stimulated prolifera-
tion of mast cells [1374, 2032, 2033], polymorphonu-
clear leukocytes [518] and fibroblasts [1342]. IL-4 also
stimulates ICAM-1. In allergic airway inflammation,
eosinophils accumulate and release IL-4 [1463]. IL-4
increases production of IgE [321, 329, 1552, 1852,
1872]. IL-4 also induces eotaxin, to further increase
eosinophil infiltration [2298]. In TNF-activated kerati-
nocytes, IL-4 enhances secretion of chemokines, which
in turn attract and activate effector cells [19]. While
IL-4 may have a role promoting inflammation, it also
modulates inflammation directly, or by way of the Th
system [1541]. In monocytes IL-4 inhibits production of
IL-1, TNF-α and IL-6 while it stimulates IL-1Ra [525,
1973]. Conversely, IL-1 inhibits the production of IL-4
[1720]. Mouse autoimmune allergic encephalitis is a
Th1-related disease. In knockout mice lacking IL-4, the
disease was exacerbated; treatment with IL-4 had a
protective effect [507]. There is, therefore, clinical
interest in IL-4 for the management of allergy, inflam-
mation, autoimmune disease and also cancer.
The receptor for IL-4 is expressed in several types of
cancer. In culture, IL-4 has inhibitory effects on certain
hematological malignancies [16, 399, 452, 850, 1214,
1630]. IL-4 also inhibited the growth of certain solid
tumor cells including breast [2004], stomach [1358,
1359], colon [2004, 2013] melanoma [790], lung [2012,
364], sarcoma [1602] and renal [791, 1473]. Antitumor
effects in culture were generally IL-4 receptor-dependent
[17, 1359, 1473, 1602].
IL-4 may be considered angiogenic in that normal
fibroblasts and endothelial cells are stimulated by IL-4
[574, 1342, 2005]. On the other hand, it has also been
reported IL-4 is antiangiogenic [2083].
In animal studies, IL-4 inhibited certain human head
and neck, glioma and colon cell lines growing as xeno-
grafts in nude mice. No remarkable immune cell infil-
tration accompanied depressed growth [2012, 2013]. An
acute lymphoblastic leukemia cell line from a child
grew in nude mice. IL-7 stimulated growth while IL-4,
TNF and IFN were inhibitory [664]. There are other
reports of inhibited acute lymphoblastic leukemia,
non-Hodgkin’s lymphoma and multiple myeloma cell
growth [16]. These animal studies showed IL-4 may be
able to inhibit tumor growth directly in addition to its
immune effects. But the growth inhibition is in most
cases just that; decreased growth rate; growth proceeds
but at a slower pace.
174
Phase II trials have been carried out using IL-4 in
cancer patients [85, 1157, 1171, 1226, 1971, 2021,
2150]. Unfortunately, response rates were very low and
toxicities were significant, particularly in the gastroin-
testinal tract [1157]. IL-4 may cause cardiotoxicity
[2020]. In vitro inhibitory effects of IL-4 on B cell
chronic lymphocytic leukemia could not be confirmed
in treated patients [1167]
Vaccines have been tested using mouse tumor cells
genetically engineered to produce IL-4. In mice vacci-
nated with IL-4 secreting tumor cells [637, 1979, 2264]
or IL-4 secreting fibroblast cells mixed with tumor cells
[1572], animals rejected the modified tumor cells and
subsequent challenges of parental tumor cells [2264].
Cures of established tumors have also been reported
[30, 636]. These approaches are now being tried in
patients [67, 1485]. IL-4 stimulates cell growth and
antigen presentation in dendritic cells, B cells and mac-
rophages [239, 351, 1974, 2301, 2302]. Cancer patients,
treated with daily sc IL-4 and GM-CSF had increased
numbers of circulating antigen presenting cells [1683].
In current dendritic cell protocols for tumor vaccination,
IL-4 is often used in culture together with GM-CSF to
prepare and grow the dendritic cells for patient treat-
ment [2039].
Interleukin-5
Eosinophil Growth/Differentiation;
Inflammation; Augmentation of T, B, NK
and Mast Cells
IL-5 is a 12–18 kDa protein, which was originally
known as B cell growth factor 2 (BCGF-2), T cell
replacing factor, eosinophil colony stimulating factor,
eosinophil differentiation factor and IgA enhancing
factor. The human gene is located on chromosome 5
where it is linked with several other cytokines includ-
ing IL-3, IL-4, IL-9 and IL-13 [969]. The molecule is a
homodimer held together by disulfide bonds [96, 969].
IL-5 is produced by activated Th2 cells [1935, 1936],
mast cells, eosinophils [202, 418, 459] and NK cells
[2121]. In NK cells, IL-5 production is enhanced by
IL-4 while IL-10 and IL-12 are inhibitory [2120].
The receptor for IL-5 is composed of two subunits, an
α chain (IL-5Rα) and a β chain, which is identical to the
β chain of IL-3R and GM-CSFR. The α chain binds
IL-5. Its structure is quite distinct from that of the α
chains of the IL-3R and GM-CSFR [1010, 1398, 1929,
1970, 2040]. The β subunit enhances the affinity of the
α chain for IL-5 and it is responsible for signaling.
Cytokines
Signaling is through the Jak-STAT pathway. IL-3, IL-5
and GM-CSF show similar patterns of signaling [1320,
1323]. There is evidence that the IL-5Rα chain also sig-
nals adding to the uniqueness and complexity of the
IL-5R signaling system [1316].
There are membrane bound or soluble forms of the
receptor, the result of alternative mRNA splicing [1970].
The soluble form appears to have a regulatory role in
eosinophilia in that it binds IL-5 and neutralizes its
activity [205, 421].
IL-5, IL-3 and GM-CSF downregulate the levels of
IL-5Rα. This happens rather rapidly, within hours. This
regulation occurs at the level of mRNA transcription as
the gene is turned off [2105].
IL-5 stimulates growth and differentiation of eosino-
phils [1723, 2222]. It activates eosinophil function
[1151] and potentiates chemotactic responses in eosino-
phils by IL-8 and RANTES [1769]. IL-5 also stimulates
B cell growth and the production of IgA and IgM [1722,
1935, 2246, 2247, 1953]; as such it is one of the main
factors from T cells, which provides help to B cells [96,
969]. IL-5 enhances IL-2 receptors and responsiveness
in T cells and NK cells [935, 1953]. IL-5 augments
IL-2-induced LAK activity in peripheral blood cells
[58] and promotes, together with other factors, T cell
differentiation and mast cells growth [2246].
Eosinophils have important proinflammatory effects,
particularly in asthma [635, 783, 938]. For this reason
there is interest in IL-5 and its antagonists in asthma and
other inflammatory diseases [1723, 1278].
Though IL-5 influences several cells in immune
response, significant antitumor activity in preclinical
models has not been demonstrated. No clinical trials
with IL-5 in cancer have been done.
Interleukin-6
Hematopoiesis, Thrombopoiesis,
Inflammation
IL-6 is the founding member of the IL-6 family of
cytokines which includes leukemia inhibitory factor,
oncostatin M, ciliary neurotrophic factor, cardiotro-
phin-1, novel neurotrophin/B cell stimulating factor-3
and IL-11. These cytokines share structure and their
receptors share a common gp130 subunit for signaling
[183, 601, 906, 2273].
IL-6 is a 22–27 kDa glycoprotein. It is also referred to
as B cell stimulating factor-2, B cell differentiation
factor, cytotoxic T-cell differentiation factor, hepatocyte
stimulating factor, IL-1 inducible 26 kDa protein, and
Walter M. Lewko and Robert K. Oldham
interferon β2 [2052]. The gene for IL-6 is located on
chromosome 7p21. Its primary structure has some
homology with G-CSF [766]. IL-6 is produced by
several types of activated cells including fibroblasts
[1257], macrophages, B cells [1868], CD4+ Th2 cells
[1043], CD8+ T cells [1708], epithelial cells [1023],
endothelial cells [2213], eosinophils [1287], astrocytes
[1079], neurons [1234, 1646], synovial cells [1324,
1940], megakaryocytes [1324], osteoblasts [859], mast
cells [580], keratinocytes [365], Langerhans’ cells
[365], neutrophils [496, 1287], colon epithelial cells
[907] and certain tumors [743, 1164, 1749].
As with many other cytokines, the regulation of IL-6
is complex and disregulation may be involved in disease.
IL-6 levels increase with bacterial and viral infection,
inflammation and trauma [2053, 2102]. Wound fluids
contain IL-6 and wound fibroblasts secrete IL-6 in
culture [1238]. IL-6 levels may increase remarkably
after surgery, up to several hundred-fold. Thrombin,
which induces clotting and wound healing, stimulates
the production of IL-6 by fibroblasts, epithelium and
other types of cells [1865]. Endotoxin and TNF-induce
the production of IL-6 and other inflammatory cytok-
ines [550]. IL-1 increases expression of IL-6 and IL-8 in
skin fibroblasts and arthritic synovial cells [612]; TGF-β
induced the production of IL-6 in several cells including
monocytes, keratinocytes and bone marrow stroma. In
plamacytoma cells TNF and IL-1 were both stimulators
of IL-6 production [2052]. IL-4 and IFN-γ acted syner-
gistically to enhance IL-6 production in endothelial
cells [803]. In lung fibroblasts, TGF-β induced IL-6
synthesis by a transient burst of H2O2 followed by an
increase in cellular calcium, which activated the signal
pathway for IL-6 expression [909]. And CD40 engagement
stimulated IL-6 in B cells and several non-hematopoietic
cells; activation of NF-κB was involved in the process
[487, 743].
Heparin and heparin sulfate selectively bind IL-6
and several other cytokines. Bound cytokine, upon dis-
sociation, is available to its receptor. Heparin protects
the cytokine from proteolysis and may serve to keep
the cytokine in high local concentration, close to where
it was secreted, available for paracrine effect [1392].
The receptor for IL-6 is composed of two subunits,
the IL-6Rα chain and gp130. IL-6 binds to the IL-6Rα
subunit [2225], which in turn binds, dimerizes and acti-
vates gp130 subunits [1566, 1943]. This leads to the
activation of tyrosine kinases, in particular Jak1. These
kinases phosphorylate gp130; gp130 then binds and
activates STAT transcription factors, in particular
STAT3. Phosphorylated transcription factors translocate
into the nucleus where target genes are activated [1566].
175
STAT 3 has different and even opposite functions
depending on the cell type and physiological status.
The gp130 subunit is shared with the receptor for
oncostatin M and other members of the IL-6R cytokine
family [602, 974, 1138]; the shared subunit appears
responsible for the common effects brought about by
these cytokines. Essentially all the cells of the body
produce gp130, whereas IL-6Rα is expressed by IL-6
target cells including B cells [766] hepatocytes [610],
monocytes, CD4 and CD8 T cells [2183], CD34+ stem
cells [1946, 2183], neurons [1752], neutrophils [1331]
and osteoblasts [2041]. There is a soluble form of
IL-6Rα. It is interesting in that unlike most soluble
cytokine receptors, it is active. The soluble IL-6Rα is
generated in two ways, by proteolytic cleavage of cell
surface receptor and by alternate splicing of mRNA
[1391, 1665]. TACE, the protease involved in TNF-α
production, appears to be responsible for proteolytic
shedding of IL-6R [716]. The soluble form binds IL-6
and mediates IL-6 responses in target cells that express
the gp130 but lack membrane bound IL-6Rα [1197,
1562]. Certain hematopoietic cells [1563], neurons [1234]
and smooth muscle cells [987] were responsive to IL-6
only in the presence of the soluble receptor subunit.
IL-6 has a major role in the regulation of hematopoi-
esis, inflammation and immunity. IL-6 −/− knockout
mice were defective in immune and acute phase protein
responses [991]. IL-6 stimulates proliferation of multi-
lineage hematopoietic stem cells. Mice that over
expressed both IL-6 and the soluble IL-6 receptor had
grossly enlarged livers and spleens, and overproduced
blood cells [1562].
IL-6 arrests the growth of cultured M1 cells while
inducing differentiation into macrophages. IL-6 stimu-
lates B cell differentiation and immunoglobulin pro-
duction [766, 1394, 1872, 2064]. IL-6 is a costimulator
of growth and cytokine production in thymocytes and T
cells [1155, 2016, 2064]. IL-6 stimulates T cell activation,
differentiation and antitumor activity [1385]. Fibroblasts
and epithelial cells secrete IL-6 in response to blood
clotting [1865]; along with other factors, IL-6 enhances
megakaryocyte colony formation and the production of
platelets [308, 755]. Natural killer cells are activated by
IL-6 in peripheral blood lymphocytes [584, 838, 1847].
IL-6 has direct effects on several types of non-
immune cells. IL-6 is a hepatocyte stimulating factor
[999]; it induced the production of acute phase proteins
including fibrinogen [258] and the receptor for anaphy-
latoxin C5a [1740]. IL-6 also induced acute phase
proteins in intestinal cells [1340]. IL-6 and the other
inflammatory cytokines TNF-α and IL-1 induce ACTH
and cortical release [1237]. IL-6 has a part in angiogenesis;
176
it stimulates endothelial cell growth and blood vessel
formation [1378]. IL-6 also increases fibroblast out-
growth and collagen production for wound repair [468].
Physiological levels of IL-6 appear to protect neurons
and support nervous system repair [682, 1037]. Mice
that over expressed both IL-6 and its receptor showed
accelerated nerve regeneration after damage [769]. In
bone, IL-6 stimulated osteoclast production and bone
resorption activity [1220]. IL-6 may be involved in
epithelium-stroma signaling during normal development,
for example, in breast tissue [831].
IL-6 appears to have a role in inflammatory condi-
tions. The blood of patients with gram-negative septic
shock contained elevated IL-6, TNF, IL-1 and LIF and
the levels of each were correlated with disease severity
[2077, 2118, 2119]. IL-6 and LIF were induced by
TNF-α [880]. IL-6 and its soluble receptor were ele-
vated in inflamed intestinal tissue [676, 1317]. IL-6 was
one of the proinflammatory cytokines required for the
induction of mouse colitis as a model for human Crohn’s
disease [2223], for experimental antigen-induced rheu-
matoid arthritis [1510] and autoimmune encephalomy-
elitis [1718]. IL-6 is among the several proinflammatory
cytokines expressed by synovial cells of arthritic patients
[1324, 1963]. IL-6 is elevated blood and cerebral spinal
fluid of patients with multiple sclerosis, Parkinson’s and
Alzheimer’s disease [168, 682, 1434]. Overexpression
of IL-6 in mouse brain induced acute phase proteins and
neurodegeneration [235]. In scleroderma patients, IL-6
was one of the cytokines required for the continued pro-
duction of autoimmune antibodies [1043]. IL-6 may
play a part in cardiac disease. Fibrinogen is considered
a cardiovascular risk factor [497]. Fibrinogen levels are
elevated during inflammation; IL-6 is one of the main
inducers of fibrinogen expression [1620].
IL-6 is generally referred to as a proinflammatory
cytokine, but it appears to be involved in the resolution
of inflammation as well. IL-6 attenuates the synthesis of
proinflammatory cytokines while it has little effect on
production of cytokines such as IL-10 and TGF-β. In
IL-6 deficient mice, inflammation, eosinopilia, the
secretion of chemokines and Th2 cytokines were
increased. In mice that produced excess IL-6, inflamma-
tion and Th2 cytokine secretion were decreased [2102].
IL-6 has also been shown to induce the production of
IL-1 receptor antagonist and the soluble TNF receptor,
both of which inhibit inflammation [1998]. In astro-
cytes, IL-6 inhibited expression of TNF-α and down
regulated levels of cell adhesion molecules [137, 1480,
1816]. One mechanism for the anti-inflammatory effects
of IL-6 (and the related cytokine IL-11) may involve
inhibition of transcription factor NF-κB [2023].
Cytokines
Human herpes virus 8, which is associated with
Kaposi’s sarcoma, produces a viral IL-6 molecule
that has 25% homology with human IL-6 [1437]. It
has certain effects that are similar to those of cellular
IL-6 [226]. The viral IL-6 appears to be responsible
for some of the virus’ pathology, including increased
angiogenesis and hematopoiesis [60]. Interestingly,
the viral IL-6 binds directly to gp130 and exerts its
effects on cells independently of the IL-6 receptor,
for which viral IL-6 appears to have little or no
affinity [1390].
In preclinical studies, IL-6 showed promise as an
anticancer agent. In vitro, IL-6 inhibited the growth of
certain breast cancer cells, leukemia, lymphoma and
other cell lines [296, 1357]. IL-6 increased ICAM-1
(CD54) in certain breast cancer and melanoma cell lines
[831, 973]. IL-6 inhibited growth in several mouse
tumor models; these mice developed tumor-specific
CTLs, but not LAK cells. In mice treated with IL-6,
growth and metastasis of Lewis lung [927] and mela-
noma [926, 1164, 1165] were inhibited. Resistance may
develop in IL-6 sensitive cells. Loss of sensitivity was
associated with loss of IL-6 receptors in some but not all
cases [1825]. IL-6 is involved in the inflammatory
response that produces tumor infiltrating lymphocytes
[1385]. In cultures of TIL from human renal cell carci-
noma, addition of IL-6 to the standard IL-2-containing
medium stimulated TIL growth though not tumor cell
kill; IL-6 alone did not induce TIL proliferation [1082].
In vaccination studies, IL-6 engineered mouse mam-
mary tumor cells offered some protection to subsequent
challenge by unmodified tumor cells.
IL-6 and other IL-6 family members (IL-11, LIF,
CNTF, NNT) induce appetite loss and wasting. In mice,
colon adenocarcinoma cells produced elevated serum
IL-6 and cachexia. The drug Suramin interfered with the
binding of IL-6 to its receptor and reversed cachexia in
tumor bearing mice [1900, 1901].
IL-6 is an autocrine growth factor in certain human
cancer cells including multiple myeloma [115, 724,
1478], B-cell leukemia [754], cervical carcinoma [503],
renal cell carcinoma and prostate cancer [1005, 1307,
1955]. In myeloma, growth stimulation appears to be
linked with several of the IL-6 family cytokines (OSM,
LIF, CNTF, IL-6) [689, 690, 2147]. In human melanoma,
there is evidence that with tumor progression, there is a
transition from IL-6 paracrine growth inhibition to auto-
crine growth stimulation. Serum IL-6 in melanoma
patients was found to be a prognostic factor for poor
survival and lack of response to IL-2 therapy [1966].
IL-6 was tested in cancer patients, including renal
cancer, in phase I and II trials [2057, 2126, 2136].
Walter M. Lewko and Robert K. Oldham
In addition to its potential anticancer activity, there was
interest in IL-6 for the stimulation of platelet and blood
cell levels. Patients treated with IL-6 demonstrated the
usual cytokine flu-like side effects plus reversible anemia,
leukosis, thrombocytosis, increased acute phase proteins,
heart problems, elevated bilirubin and confusion indica-
tive of neurotoxicity. There was a suggestion of benefit
in certain studies but toxicity was significant. One report,
a phase II study in advanced renal cell cancer, showed
lack of efficacy [1758]. Anti-IL-6 monoclonal antibodies
have been used to treat patients with myeloma [114, 2065]
and AIDS associated Kaposi’s sarcoma [1610] without
any apparent benefit. In a phase I trial involving pediat-
ric patients given IL-6 plus G-CSF after myelosuppres-
sive chemotherapy, there were increased hematological
responses but with a high incidence of toxicity [188].
IL-6, IL-10 and TGF-β are cytokines reported to be
secreted by tumors, which interfere with dendritic cell
differentiation and antigen presentation, resulting in
ineffective antitumor immune response [129].
Interleukin-7
T Cell, B Cell, Macrophage and Dendritic
Cell Development
IL-7 was first described as a factor produced by mouse
bone marrow, which stimulated B-lymphocyte progeni-
tor cells to multiply and develop [1422, 1423]. IL-7 is a
25 kDa glycoprotein. It is produced by bone marrow
stroma [641, 1422, 1423], thymic stroma [1716], kerati-
nocytes [69, 745, 1245], human intestinal epithelium
[2115], B cells [134], follicular dendritic cells and
vascular cells [1024]. IL-7 is also produced by tumors
including certain carcinomas [910] leukemias [569,
1578] and lymphomas [973, 1001]. IL-7 is not detected
in normal T cells [2263]. Upon secretion, IL-7 binds to
integrin in the extracellular matrix; this may be the form
utilized by thymocytes [980].
The IL-7 receptor is composed of two subunits,
IL-7Rα and γc. The IL-7Rα chain contains the IL-7
binding site [640]. The γc is shared between IL-2R,
IL-4R, IL-7R, IL-9R and IL-15 R [1002]. γc enhances
receptor affinity of IL-7 and it participates in signaling
[1002, 1461]. The activation response involves Jak1,
pI-3 kinase, STAT3 and STAT5 [556]. Receptors that
share γc have similar but not identical nuclear effects.
Lack of active γ chain in patients with severe combined
immunodeficiency disease results in loss of function in
these five receptors. Loss of IL-7R function is a major
reason for poor immune response in these individuals.
177
There is a soluble form of the receptor that is produced
by a specific mRNA [641].
Three different IL-7 binding affinities have been
observed [74, 558, 1523, 2017]. The highest affinity
receptor is generally found in activated cells and appears
to be the one responsible for cell division [73, 1523]. In
human T cells, response to IL-7 depends on the activa-
tion state. In resting cells, IL-7 induces certain cellular
proteins (CD25, IL2 R) but no proliferation. In activated
cells, IL-7 is a potent inducer of cell division [648, 1365].
Immunosuppressive drugs cyclosporin and FK506
inhibit IL-7 stimulated growth, apparently by interfering
with the function of the high affinity receptor [557].
IL-7 has important influences on several different
immune cell types. Genetically deficient mice, lacking
IL-7, were extremely lymphopenic [2084]; early lym-
phocyte development was severely impaired [1548]. In
mice there is an absolute requirement for IL-7 in the
development of B and T cells. Interestingly, it is unusual
for the loss of any one cytokine to cause such impair-
ment. This is probably due to redundancy of cytokine
functions, which is not the case for IL-7 in mice [2084].
In humans, IL-7 is not so strictly required for the devel-
opment of B cells [1592].
IL-7 induces the growth of early B lineage cells. In B
lymphocytes, IL-7R was found on pre-B cells but not on
mature B cells, consistent with its role in the early stages
of B cell maturation [371, 372, 1918]. Stem cell factor
and Flt3 ligand are costimulators [1229, 1424]. IFN-γ
blocks the stimulatory effect of IL-7 on pre-B cells
[592]. Mice deficient in IL-7 fail to support normal B
cell development [2084].
For prolonged B cell outgrowth, additional IL-7
related signals appear to be required, which occur by
way of physical contact with stromal cells. IL-7 supports
outgrowth of pre-B and pro-B cells but the pre-pro-B
cell population requires, pre-pro-B cell growth-stimulat-
ing factor. PPBSF is composed of at least two covalently
associated subunits, one of which is an IL-7 chain. The
other subunit is a protein that is attached to the stromal
cell surface. Pre-pro-B cells bind PPBSF during stromal
cell contact; PPBSF stimulates division and primes the
cells for IL-7 [1273]. Thymic stromal lymphopoietin is
another IL-7-related factor in early B cell development.
It was discovered in the conditioned medium of a thy-
mus cell line [568]. TSLP and IL-7 have overlapping
actions [568]. TSLP binds the IL-7Rα with another
receptor subunit; it does not use γc. TSLP induces the
transcription factor STAT5 as does IL-7, but TSLP did
not activate any known Janus kinases.
Normal T cell development depends on IL-7. Mice
lacking IL-7Rα [1205, 1223, 1548] and mice treated
178
with neutralizing antibodies to IL-7 [650] do not pro-
duce normal T cells. γδ T cells are missing entirely in
IL-7Rα (−/−) mice [728, 1548]. αβ T cells are present,
but they are not responsive [1548]. IL-7 is a growth/
differentiation factor for fetal pre-T cells, thymocytes
[143, 735, 1492, 2080, 2153] and mature CD4+ and
CD8+ T lymphocytes [73, 143, 293, 347, 564, 648,
1148, 1365, 2243]. An important effect of IL-7 in both
T and B cells appears to be its stimulation of the bcl-2
antiapoptotic pathway, for the maintenance of lympho-
cyte viability [15, 1222, 2085]. IL-7 was the only
cytokine among 16 factors tested capable of inducing
rearrangement (diversity) in the T cell receptor V(D)J
region. IL-7 activated expression of the RAG1 and
RAG2 genes involved in gene rearrangement [306,
1383]. In activated human peripheral blood T cells, IL-7
induced the secretion of several cytokines including
IL-2, IL-3, IL-4, IL-6, IFN-γ and GM-CSF [442, 443].
IL-7 also increased antitumor cytotoxicity in T cells [22,
143, 749, 776, 1180] and this cytotoxicity may persist
long term in culture without frequent antigenic restimu-
lation [1182].
IL-7 acts together with several cytokines in the devel-
opment of T cells [2139]. Flt3 ligand and stem cell fac-
tor [1350, 1364] are co-stimulators with IL-7 for early T
cells. IL-1 and GM-CSF induced outgrowth of mouse
thymocytes; IL-7 was required [735]. IL-2 acts together
with IL-7 to stimulate antigen-induced effector cells
from memory CD8+ T cells. IL-7 may act alone or with
IL-2 [125, 1283]. Together the effect is synergistic
[1283]. IL-7 stimulates IL-2 receptor (CD25) levels in
resting T cells [73]; this may explain the synergy.
Among its many other functions, IL-7 induced prolif-
eration and antitumor activity in human blood mono-
cytes and macrophages [24, 871]. In treated mice, IL-7
mobilized myeloid progenitor cells from bone marrow
to peripheral sites [686]. It supported eosinophil pro-
genitors in human bone marrow cell culture [2071].
IL-7 induced lymphokine activated killer cells [1181,
1736, 1896]. While IL-2-LAK exhibited better mela-
noma cell kill, IL-7 LAK killed with a different pattern
of cytokine release [1431, 1432], without TNF secretion
and without toxicity towards normal cells [1731].
IL-7 appears to have a part in antigen presentation. IL-7
was a growth factor for mouse dendritic epidermal T cells
[1245, 1246]. Dendritic cells produce IL-7 [1022].
Keratinocytes that surround the dendritic cells in skin pro-
duce IL-7 and TNF-α [1245, 1246]. Together these cytok-
ines stimulated dendritic cell growth. IL-7 also increased
levels of the co-stimulatory protein B7 on B cells [414].
Lymphopenia can occur in patients with HIV infec-
tion and chemotherapy. It has been shown that IL-7 may
Cytokines
be administered to patients to expand CD4+ and CD8+
lymphocytes. This occurs with a decrease in the per-
centage of regulatory CD4+ T cells [1673].
Several preclinical studies suggested IL-7 may be
useful in the treatment of cancer. Lung metastases of
Renca renal cancer cells were reduced in mice treated
with rhuIL-7; the mice showed increases in T cells,
CD8/CD4 ratio, B cells, macrophages and NK cells
[1001]. Nude mice with human colon cancer xenografts
lived longer when treated with rhuIL-7 plus human T
cells compared to mice treated with either alone. The
antitumor activity appeared due to interferon induced
in CD8+ cells, not cytolysis. Interestingly, injected
interferon was not that effective suggesting local con-
tinuous release was better than systemic interferon
therapy [1402].
IL-7 appears to be a good adjuvant during tumor vac-
cination. Tumorigenesis was decreased in mice inocu-
lated with IL-7 gene engineered plasmacytoma cells
[775], glioma cells [59] and fibrosarcoma cells [1261].
Animals that rejected engineered tumor cells developed
immunity to subsequent injections of the parental cells
[61, 1261]. The tumors were infiltrated with T cells.
Complement receptor rich macrophages, eosinophils
and basophils were also increased [776, 1261]. IL-7 was
one of several cytokines which upregulated intercellular
adhesion molecule-1 (ICAM-1) on human melanoma
cells. This protein is involved in immune recognition
and anticancer action [973].
Tumor infiltrating lymphocytes have been used to
treat cancer patients. Interleukin-2 is used to activate
and grow TIL in culture. IL-2 generally stimulates good
initial growth and tumor cell kill, but antitumor activity
is often difficult to maintain over the long culture time
needed to grow sufficient cells for therapy, especially
when tumor cells are not available as a source of antigen
to restimulate the T cells. There are reports of difficulty
maintaining CD4+ helper cells in IL-2-induced cultures.
IL-7 has been examined to determine whether it pro-
vides any benefit in culture. IL-7 alone stimulated the
growth of TIL cultures from certain renal cancer and
enhanced IL-2-induced growth in others [1182]. Growth
and antitumor activity persisted during long term culture.
Antigen restimulation was not required to maintain
antitumor activity. Others have reported IL-7 was not
beneficial at the initiation of TIL culture but rather IL-7
stimulated growth and cytokine secretion in cultures
that were already responding to IL-2 [1820].
There are reports that IL-7 stimulates certain types
of cancer. IL-7 increased the growth of several leu-
kemia cell lines [664, 1242, 1351, 1491, 1531, 2017].
Sezary lymphoma cell lines responded to IL-7 and
Walter M. Lewko and Robert K. Oldham
IL-2 with increased and in some cases synergistic
growth [374, 554]. The effect of IL-7 may be auto-
crine or paracrine [554, 664]. Abnormal cytokine
secretion by tumor cells may be responsible for
immune system abnormalities common in CLL
patients [569]. Transgenic mice have been developed
with increased IL-7 expression in lymphoid tissues.
These animals had remarkable skin T cell infiltrates,
the result of the growth stimulating effects of IL-7.
These transgenic mice also produced T and B cell
lymphomas. This model system shows that the IL-7
locus could behave as an oncogene.
There have been preliminary studies using IL-7-
transfected autologous tumor cells as vaccines. Phase I
trials in patients with melanoma showed that IL-7 gene-
transfected, irradiated cells administered subcutane-
ously were safe and produced tumor-specific CTL
responses. One minor clinical response was reported
[1338, 1339]. Another study, a phase I/II trial in ten
patients with metastatic carcinoma, used autologous
tumor cells transfected with IL-7, GM-CSF and double
stem-loop immunomodulating oligodeoxyribonucle-
otides (d-SLIM). One CR, one PR and one MR were
observed [2179].
Interleukin-8
Chemotaxis, Angiogenesis
IL-8 is a potent chemotactic and proinflammatory
cytokine [1250, 1384]. It is a member of the CXC
(ELR+) chemokine family (see below). IL-8 is a rela-
tively small, 6,000–8,000 mw glycoprotein [1755, 2096,
2257]. It is secreted by a variety of cell types including
macrophages [1755], endothelial cells [623, 1455, 1603,
1756, 1905], neutrophils [255, 1949, 2130], epithelial
cells [123, 913, 961, 1878], fibroblasts [1063], keratino-
cytes [1063, 2281], mast cells [1337] and eosinophils
[2261]. Certain tumor cells also produce IL-8 including
melanoma [1737], squamous cell carcinoma [2281]
colon adenocarcinoma [1757]. IL-8 secretion is con-
trolled by various factors such as IL-1 [1063, 1757],
IL-3 [2121], IL-5 [1769], GM-CSF [1264, 2121], TNF
[1063, 1904], vitamin D [2281], lipopolysaccharide
[1757] and fibrin [1603]. Mast cell secretion of IL-8 was
specifically stimulated by stromal cell factor-1 [1130].
There are two receptors for IL-8, CXCR1 and CXCR2
[5, 787, 1074, 1371, 1400]. These receptors bind IL-8
with high affinity and they also bind other members of
the chemokine family [1074]. There are receptors for
IL-8 on neutrophils, T cells, mast cells, macrophages,
179
endothelial cells and keratinocytes [950]. Like other
chemokine receptors, the IL-8 receptors are members of
the rhodopsin superfamily; these proteins characteristi-
cally contain seven transmembrane domains. Chemokine
receptors are linked to phospholipase C through G
proteins. Receptor activation results in increased diacyl-
glycerol, inositol triphosphate and increased intracellular
calcium released from cellular stores.
IL-8 was first described as a macrophage-derived
chemoattractant for neutrophils [1384, 1755, 2096]. IL-8
is also a chemoattractant for T cells [98]. It has little effect
on B cells. In dogs injected with human IL-8, the site
became infiltrated, mainly with neutrophils [1987]. IL-8
stimulates neutrophil production of superoxide anion,
degranulation with the release of hydrolytic enzymes
such as elastase, and migration through endothelium of
capillaries [101, 252, 815, 1565]. Mast cell-dependent
recruitment of neutrophils appears to be mediated by IL-8
[1130]. IL-8 also induces migration in IL-2 activated
peripheral blood NK cells [1774]. In rabbits, IL-8 block-
ing antibodies had an inhibitory effect on inflammation,
especially in the lung [1780]. IL-8 also stimulates angio-
genesis [1346]. IL-8 is an attractant to antigen presenting
cells such as macrophages and eosinophils [408, 1769,
1777]. As such, IL-8 may have an important role in the
initiation of immune response as well as the recruitment
of effector cells that carry out the response.
Fibrin formation is associated with trauma, inflam-
mation, wound healing and cancer. The addition of
fibrin to cultured endothelial cells induced the release of
IL-8 [1603]. The physical act of blood clotting may
stimulate cytokine release and cell migration. Besides
IL-8 there are several additional proteins and peptides
that are chemotactic to immune cells; macrophage
inflammatory protein α (MIP), IL-1α, and RANTES
(see below) to mention a few.
IL-8 appears to have a role in inflammatory diseases.
Arthritic synovial fibroblasts constitutively oversecrete
IL-8; IL-1β, itself elevated in synovial fluid, stimulates
secretion further. Specific inhibitor studies showed that
NF-κB, an important signal for inflammation genes,
was involved in both spontaneous and IL-1-induced
expression of IL-8 [612].
IL-8 or other chemokines released within tumors may
have anticancer effects by recruitment of macrophages,
granulocytes and lymphocytes. But there is also evi-
dence that IL-8 contributes to tumor development and
spread. In melanoma, IL-8 has been shown to be an
autocrine growth factor [1737, 1833]. IL-8 stimulated
tumor cell movement. Metastasis of melanoma cells in
nude mice was correlated with the tumor’s capacity to
produce IL-8 [1833]. Developing tumors require
180
adequate blood supply and may benefit from IL-8-
induced angiogenesis [994, 1905]. IL-8 secretion cor-
related with vascularity in human gastric cancer [976].
In a nude mouse model for human tumor progression,
IL-8 was one of several angiogenesis factors that corre-
lated with progression in ovarian cancer [2250]. Reduced
IL-8 expression or blocking antibodies administered to
nude mice decreased growth, invasiveness and angio-
genesis in melanoma, breast cancer and transitional cell
carcinoma cells [812, 966, 1299]. Therefore, IL-8 may
act directly to stimulate tumor cells. Alternatively, IL-8
may activate neighboring normal cells to produce factors
that tumors utilize for growth and metastasis.
Interleukin-9
Mast Cell, T Cell, Lymphoma Growth
Factor; Inflammatory; Hematopoietic
IL-9 is a 32–39 kDa, 144 amino acid protein. Its gene is
located on chromosome 5 in a region near genes for
GM-CSF, IL-3, IL-4 and IL-5. IL-9 is structurally
related to IL-2, IL-4, IL-7 and IL-15 and some of their
activities are similar. IL-9 was discovered as a factor in
the medium of HTLV-transformed T cells that stimu-
lated growth in a human leukemia cell line [2229]. IL-9
has also been referred to as P40 [2063, 2170], mast cell
growth enhancing activity [1332] and T cell growth fac-
tor III [1748]. It is produced by activated Th2 cells
[1634], by naive CD4+ cells [1747], and by mast cells
[820]. In CD4+ cells, IL-9 secretion is stimulated by
IL-1, IL-2, IL-4 and TGF-β and inhibited by IFN-γ
[1745, 1746]. In mast cells, IL-1, IL-10 and kit ligand
increase IL-9 secretion [820, 1899].
The receptor for IL-9 contains IL-9Rα and γc [968].
The signaling pathway involves Jak3, Jak1, the adaptor
protein IRS-1 [2245] and the transcription factors
STAT1, STAT2, and STAT5 [412, 116].
IL-9 usually acts together with other cytokines. With
IL-3 or GM-CSF, IL-9 induced the growth of hematopoi-
etic progenitor cells [784]. With erythropoietin, IL-9
supported erythroid colony (BFU-E) formation [447,
2170]. In mice, IL-9 with IL-2 stimulated fetal thymo-
cyte proliferation [1917]. IL-9 increased growth in T
cell lines and activated helper T cells [800]. Interestingly,
quality of growth induced in certain Th cell lines by
IL-9 was different from that of IL-2 in that IL-9 induced
growth was hardly affected by glucocorticoids while
IL-2-induced growth could be inhibited [1162]. IL-9
also increased granzyme B (protease involved in apop-
tosis and cytotoxicity) and high affinity receptors for
Cytokines
IgE in several Th cell clones. These are mast cell-like
characteristics that IL-9 induced in Th cells [1161]. In
cultures of mast cells, IL-9 enhanced survival; IL-9
together with IL-3 and IL-4 stimulated mast cell growth
and the secretion of IL-6 [819]. Proliferation of mast
cells was one of the remarkable features of IL-9 trans-
genic mice [631]. In B cells, IL-9 potentiated IL-4-
induced immunoglobulin secretion [461]. IL-9 increased
resistance to nematode infections [515].
Mice that oversecreted IL-9 showed increased allergen-
induced inflammation and airway hyperresponsiveness
[1279, 1977]. There is genetic evidence that IL-9 is
involved in human asthma [451, 1450, 1460].
Related to cancer, IL-9 has been shown to stimulate
growth and depress apoptosis in mouse lymphoma cells
[1633, 2079]. Transgenic mice overexpressing IL-9
tended to develop lymphomas [1632]. HTLV-transformed
T cells produce IL-9 [947]. IL-9 was an autocrine growth
factor for cultured Hodgkin’s lymphoma and Reed-
Sternberg cells [684]. It is possible that antagonists of
IL-9 may be useful therapeutically.
Interleukin-10
Regulator of Immune Response
IL-10 was first discovered in cultures of Th2 cells as a
factor that inhibited IFN-γ production by activated Th1
cells [535]. It is a 17,000 mw protein with relatively lit-
tle glycosylation [1177]. IL-10 is produced by several
types of cells including regulatory T cells (Tr1 and
nTreg) [2002, 2204], CD4+ Th2 cells [297, 535, 2262],
thymocytes [1198], B cells [629, 1470], monocytes
[423, 548], mast cells [1990], eosinophils [1416], and
keratinocytes [494]. In CD4+ T cells, activation
induces several cytokines including IL-2 as an early
response and IL-10 as a relatively late regulatory
response [332, 2262]. Blocking endogenous IL-2 pre-
vented the increase in IL-10 [332]. IL-4, IL-7, IL-15 and
IL-12 were costimulators of IL-10 production. IL-10
feeds back on the production of IL-2 and its own synthe-
sis by deactivating T cells [332]. In macrophages, activa-
tion induced several cytokines including an early
response for TNF-α (3.5 h) and a much later peak in
IL-10 (48 h) [388, 423, 2116]. Cortisol (immunosup-
pressive) stimulated plasma IL-10 levels in treated nor-
mal volunteers [376]. IL-10 feeds back on its own
production by deactivating macrophages and TNF-α
secretion [388, 423, 2167].
The IL-10R structurally resembles the IFN receptor
[773, 1014, 1869]. Signaling in monocytes and T cells
Walter M. Lewko and Robert K. Oldham
involves tyk2, Jak 1, STAT1α, STAT3 and a different
STAT3-like protein in monocytes, not observed in T
cells [533].
IL-10 is a key regulator of immune response. A major
effect of IL-10 is the suppression of Th1-dependent cel-
lular immunity and the promotion of Th2-dependent
humoral immunity [1347]. IL-10 deactivates a number
of macrophage activities. It inhibits macrophage pro-
duction of IL-12 and other cytokines that stimulate T
cells [173, 369, 388, 536]. Further, IL-10 depresses
macrophage production of peroxide [173] and nitrogen
oxide [600]. In activated monocytes, IL-10 also inhibits
the production of M-CSF.
IL-10 regulates neutrophil activity. It inhibits endotoxin-
induced release of proinflammatory cytokines [920]. IL-10
depresses neutrophil production of MIP-1α, MIP-1β and
IL-8 [920] and enhances the release of IL-1Ra [257].
IL-10 inhibits antigen presentation and T cell response
[537]. In target cells, IL-10 down-regulates MHC class I
[1243]. In monocytes, IL-10 depresses costimulatory
and adhesion molecules involved in antigen presentation
[2167]. IL-10 also inhibits dendritic cell-induced pro-
duction of IFNγ by Th1 and CD8+ T cells [1188]. It
decreases antigen presentation by macrophages to T
cells, in particular, to Th1 cells [388]. IL-10 also
depresses T cell growth directly [1942]. In mitogen stim-
ulated T cells, IL-10 inhibits cell division and production
of IL-2 and IFN-γ [1942]. In many ways IL-10 resembles
IL-4, but interestingly, IFN-γ levels are stimulated by
IL-4. This is the key difference in activities between IL-4
and IL-10 which otherwise act similarly as they tend to
suppress cell mediated immune response.
In NK cells, IL-10 depresses INF-γ secretion and
cytotoxicity induced by IL-12 and TNF-α [2027]. In
fibroblasts, IL-10 depresses the production of collagen,
enhances the production of collagenase and stromelysin
[1628]. In macrophages, it inhibits production of colla-
genase and stimulates the production of tissue inhibitor
of metalloproteinases (TIMP) [1052]. As already noted,
glucocorticoids, which are immunosuppressive, stimu-
late IL-10 levels in blood [376]. The overall effect of
IL-10 is antiinflammatory and it tends to limit tissue
damage associated with inflammation.
IL-10 has been described as a growth and differentia-
tion factor for CD8 T cells [297, 1198] and activated B
cells [1687]. In T cells, IL-10 induced the IL-2R and
thereby enhanced IL-2-dependent proliferation [1198,
331]. IL-10 is a chemotactic factor for CD8+ T cells (the
first described) but it is an inhibitor of IL-8-induced
migration for CD4+ cells [893, 895]. In a model for dia-
betes, IL-10 expression by islet cells caused the local
accumulation of T cells [2181]. In B cells, IL-10 and
181
IL-2 acted together synergistically, as IL-2R levels were
increased [547, 867]. IL-10 enhanced class II MHC
expression [629] and stimulated the production of IgG,
IgA and IgM [400, 867]. In B cells, IL-10 had opposite
effects on apoptosis that appeared to depend on the acti-
vation state of the cells. IL-10 depressed apoptosis in
germinal center B cells [1107] but it enhanced apoptosis
in chronic lymphocytic leukemia B cells [546]. In
Staphylococcus aureus-activated B cells, IL-10 added 3
days later inhibited apoptosis; when IL-10 was added at
the time of S. aureus, apoptosis was stimulated [866]. In
mast cells IL-10 acts together with IL-3 and IL-4, to
stimulate growth [1990].
IL-10-deficient knockout mice exhibited a number of
problems, in particular the mice developed enterocolitis
due to unregulated immune response to microbes [1031].
IL-10 protects mice against the effects of endotoxin
shock [613, 802].
Epstein Barr Virus genes code for vIL-10 (BCRF1).
The protein is highly homologous with hIL-10 [806,
1348, 2076]. It appears that at some point in evolution-
ary time, EBV picked up this gene from a mammalian
cell source. viral IL-10 is expressed soon after infection,
within 2–3 h, whereas host IL-10 appears 20–30 h after
infection. viral IL-10 expression interferes with antigen
presentation and the antiviral immune response [423].
IL-10 functions during pregnancy. The uterus pro-
vides a site of immune privilege for the development
of a fetus. Successful pregnancy is said to be a Th2
phenomenon [2127]. In an abortion-prone mouse
model system, a defect in IL-10 production appeared
to be responsible for fetal loss; treating the mice with
IL-10 prevented fetal resorption [313]. The pregnancy
hormone progesterone induces the production of
IL-10 [709]. In human trophoblasts, IL-10 is an inhib-
itor of matrix metallopoteinase-9 production, a pro-
tease believed to have a role in parturition [1682].
IL-10 production in the placenta was shown to
decrease at term. Lower IL-10 levels would allow the
expression of MMP-9 during parturition and a shift
from Th2 to Th1 immunity [709].
IL-10 may have a role in human diseases involving
inflammation and inappropriate immune response.
In volunteers treated with a single i.v. dose of IL-10, T
cell levels were decreased. In endotoxin treated volun-
teers, pretreatment with IL-10 lowered fever and sev-
eral proinflammatory cytokines [300, 1525]. IL-10
depresses lung granulocyte number and capacity for
degranulation [1525]. IL-10 inhibits collagen produc-
tion in fibroblasts. In patients with chronic hepatitis C,
IL-10 normalized serum ALT (marker for hepatic
inflammation), improved liver histology and reduced
182
fibrosis [1440]. Most patients with psoriasis benefited
from treatment with IL-10 [77, 1627]. In Crohn’s dis-
ease, responses to IL-10 were observed in 23.5%
patients. In another study, patients treated post surgi-
cally to prevent recurrence of Crohn’s disease did not
show any benefit [336]. IL-10 treatments were well
tolerated; observed toxicities, were moderate and
reversible. In another approach, blocking antibodies to
IL-10 were used to treat a small group of SLE patients.
The patients were treated for 21 days; five/six patients
benefitted from the antibody therapy [1142]. The
immunosuppressive and antiinflammatory properties
of IL-10 may be of value in tissue transplantation
[125]. But it should be noted, certain mouse studies
did not show any IL-10 benefit [1022]; in some cases
IL-10 exacerbated graft vs. host disease [163].
Although IL-10 is generally antiinflammatory, it has
certain proinflammatory effects, which may compli-
cate its use in therapy. For example, one study showed
that IL-10 potentiated IFN-γ release in LPS-treated
volunteers [1066].
IL-10 may be elevated in cancer patients, naturally
due to immune response or due to IL-10 production by
tumors. Increased IL-10 could be involved in tumor
growth and immune escape [882, 1173, 1574]. It has
been shown that Epstein-Barr viral IL-10 is an autocrine
growth factor for certain B cell lymphomas [121, 957].
IL-10 is also a growth factor for human myeloma cells,
apparently by induction of a gp130 R dependent (e.g.
LIF) process [689]. IL-10 may interfere with antigen
presentation. Using antigen-pulsed DCs, repetitive vac-
cination induced CD4+ cells, which produced IL-4 and
IL-10 [282]. IL-10 appeared to induce T cell anergy
[282, 1173, 1934]. In tumor cells, IL-10 decreased
expression of HLA class I, which would decrease recog-
nition by effector cells [1243]. In Hodgkin’s disease,
elevated IL-10 was determined to be an independent
prognostic factor for treatment failure [174, 1727]. It
thus appears IL-10 may have a role in tumor progression.
It should be mentioned that during immunotherapy,
IL-10 levels might rise [1996]. This could be a reason
for lack of response.
One might not expect IL-10 to be useful in a tumor
vaccine. However, interesting results were obtained in
mice vaccinated with mammary adenocarcinoma cells
engineered to produce IL-10 [30, 624]. Some mice
developed long term immunity. In another study, trans-
genic mice overexpressing IL-10 in antigen presenting
cells rejected an immunogenic melanocytoma [678]. In
another study, mice received tumor cells expressing both
IL-10 and IL-12. 50–70% showed remission of metasta-
ses to lung, colon and breast tumors. Vaccination with
Cytokines
cells expressing either IL-10 of IL-12 alone yielded little
or no response. CD8 and CD4 cells were both involved
and tumor specific antibodies were induced [677].
Interleukin-11
Hematopoietic Progenitor/
Thrombocytosis Stimulator;
Regulator of Inflammation
IL-11 was discovered as a factor produced by a bone
marrow stromal cell line, which supported the growth
of an IL-6-dependent plasmacytoma cell line [1543]. It
is a 22 kDa glycoprotein [455, 456]. IL-11 is a member
of the IL-6 cytokine family. It is produced by stromal
cells in hematopoietic tissues [1543], fibroblasts [486,
1653, 2290], epithelial cells [485], chondrocytes and
synovial cells [1200] and by airway smooth muscle
cells [484]. IL-11 is induced by IL-1, TGF-β, hista-
mine, eosinophil major basic protein and by certain
viruses [484, 485, 481]. The receptor for IL-11 shares
the gp130 subunit with receptors for the other IL-6
family members [1138, 1944].
IL-11 stimulates the growth and differentiation of
hematopoietic progenitor cells and megakaryocytes
[1404, 1405]. It influences very early stem cells and more
committed precursor cells. In mouse cells, IL-11 acted
synergistically with IL-3 and IL-4 to stimulate growth of
primitive blast colony forming cells [1504, 1405]. IL-11
stimulates B cells and the production of immunoglobulin
[1543, 2244]. IL-11 has a remarkable effect on platelet
production and this is its main clinical use [456].
IL-11 inhibited the differentiation of fibroblast lines
into adipocytes; IL-6 and TNF-α had similar effects
[937]. IL-11 inhibited the production of IL-12 by mac-
rophages [1104]. IL-11 is involved in bone metabolism;
it is said to be critical for osteoclast development [625].
Also IL-11 is among the several cytokines capable of
inducing acute phase protein release from hepatic and
nonhepatic cells [1340].
IL-11 regulates Th2 responses and the production of
inflammatory cytokines [2022, 2296]. IL-11 is secreted
during respiratory tract infections, airway inflammation,
and asthma [481, 1229, 2103] and certain other inflam-
matory responses such as Lyme disease [53]. IL-11 also
ameliorates inflammatory bowel disease and oral
mucositis [958]. IL-11 protects and restores damaged,
inflamed tissue by the secretion of protease inhibitors
[1202]. In an ovalbumin allergy model, lung overex-
pression of transgenic IL-11 decreased antigen-induced
eosinophilia, inflammation and endothelial VCAM-1 in
Walter M. Lewko and Robert K. Oldham
lung tissue. Th2 cytokines IL-4, IL-5 and IL-13 were
diminished [2102].
IL-11 also has protective effects during radiation-
induced thoracic injury [1623] and intestinal damage
[455]. IL-11 protects against immune complex lung
injury [1095] and oxygen-induced lung damage [2123].
In the care of cancer patients, IL-11 augments bone
marrow recovery and platelet production. It is approved
for use in patients with non-myeloid malignancies to
prevent thrombocytopenia and to reduce the need for
platelet transfusion following chemotherapy. It has been
shown in breast cancer patients, for example, that treat-
ment with rhIL-11 increased bone marrow megakaryo-
cytes [1503]. Blood platelet counts were increased and
patients experienced less thrombocytopenia [645]. In
patients with thrombocytopenia requiring platelet trans-
fusions, treatment with rh-IL-11 significantly decreased
the need for transfusions with subsequent chemotherapy
[1978]. RhIL-11 is well tolerated by adults and children.
Adverse events are generally mild or moderate and
reversible [1843].
Il-11 may have a role in progression of breast cancer.
IL-11 and its receptors were higher in tumors than normal
tissue and higher in node positive tumors than node
negative tumors. Higher expression of IL-11 in tumors
was linked to poorer survival of patients [708]. IL-11
also induces the formation of osteoclasts, which appear
to be involved in the metastasis of breast cancer to bone
[918, 1353, 1830].
Interleukin-12
Immune Stimulation, Inflammation,
Hematopoiesis, Regulator of Innate and
Adaptive Immunity
IL-12 is a key cytokine with far reaching influences. It
serves as a link between the innate and adaptive immune
response systems [993, 2025, 2186]. IL-12 is a well
studied cytokine. It was reviewed in some detail in a
previous edition [1110]. The following is a brief over-
view with a discussion of certain new reports related to
cancer.
IL-12 was discovered in B cell lines as a factor, which
acted with IL-2 to stimulate proliferation, IFN-γ secre-
tion and cytotoxicity in NK cells and T cells [596, 737,
1149, 1560, 2054]. It has been referred to as natural
killer cell stimulating factor [993], T cell stimulating
factor-1 [649], and cytotoxic lymphocyte maturation
factor [1891]. IL-12 is a 70,000 mw glycoprotein; it is
composed of two chains, α (also called p35) and β (p40)
183
[680]. These chains are produced from two distinct
genes, on separate chromosomes [680, 1822]. The α
subunit has homology with IL-6 and G-CSF [1293]. The
β subunit is unusual in that its structure is more like that
of a receptor than a cytokine [603]. Expression of these
two genes does not appear to be coordinated. Curiously,
many cells in the body produce the α subunit without
making β [368]. But both chains must be transcribed
within the same cell for the production of active IL-12
[2185]. Regulation of IL-12 secretion generally occurs
at the level of β chain synthesis [368] though this wasn’t
the case in DCs prepared from newborns where low
IL-12 production appeared to be due to lack of the α
chain. Low DC IL-12 may be one reason why newborns
have relatively poor cellular immune response [646].
Mouse cells secrete an IL-12 β-β homodimer that
acts as a physiological antagonist of IL-12 [1252, 595,
731]. It is not clear whether the β-β homodimer has a
function in humans.
IL-12 is produced by macrophages, dendritic cells,
neutrophils, microglial cells, keratinocytes and trans-
formed B cells [368, 1189, 804, 1608]. IL-12 is not typi-
cally secreted by tumors except certain cancers of B
cells origin [2185]. IL-12 production in macrophages is
stimulated by bacteria, viruses, parasites [368, 804,
2185] and by contact with T cells through CD40–CD40L
interaction [1817]. IL-12 production is enhanced by
IFN-γ (Th1 cytokine) and inhibited by IL-10, IL-4 (Th2
cytokines), TGF-β and IFN-α/β [2108].
IL-12 receptor contains two chains, IL-12Rβ1 and
IL-12β2. They have homology with the β chain (gp130)
of the IL-6R family [318]. Both subunits are required
for IL-12 function. β1 is responsible for binding. β2 is
the subunit that signals [2200, 2201]. IL-12R is
expressed mainly on NK cells and T cells [416]. It is not
expressed on Th2 cells [1939]. In PBMC, IL-12R was
upregulated by activation with PHA or IL-2 [416].
IL-12R signaling involves Tyk2, Jak2, STAT3 and
STAT4 and p38 MAPK [97, 326, 873, 2282, 2303].
IL-12 promotes the development of cellular immune
response. It does this by stimulating the production of
IFN-γ and Th1-related cytokines in PBL, T cells and
NK cells [284, 317, 824, 993, 1028, 1283, 1284, 1554,
2202]. IL-12 also stimulates hematopoiesis. It acts on
progenitor cells together with growth factors such as
GM-CSF [130, 1431, 1432, 1433]. IL-12 increases DC
production of cytokines (GM-CSF, IL-1β, IL-6, IL-12,
TNFα and IFN-γ) and antigen presentation [675, 1028,
1412]. IL-12, together with IL-2, stimulates the growth
and differentiation of B cells [885]. IL-12 induces LAK
cells when added to cultures of PBLs for 3–5 days. LAK
cells are also induced by IL-2. At least part of the IL-2
184
effect on LAK appeared due to IL-12 for antibodies to
IL-12 decreased the response [597, 1431].
In anti CD3 activated TIL, IL-12 stimulated growth
and tumor cell lysis [50]. The effect of IL-12 was not
prevented by antibodies to IL-2. When IL-12 was
added to cultures with a low, suboptimal dose of IL-2,
there was an additive effect on growth and cytotoxic-
ity [50]. Growth induced by IL-12 alone was tran-
sient. Maintenance of growth after 72 h required IL-2.
IL-2 appears to be more potent than IL-12. It remains
to be seen whether IL-12-treated TIL cells have added
clinical benefit.
IL-12 appeared to be involved in inflammatory dis-
eases such as multiple sclerosis, diabetes, and arthritis
[2025]. However, more recent studies suggest IL-23,
which is proinflammatory and structurally related to
IL-12, may be responsible for certain effects attributed
to IL-12, specifically in experimental allergic encephalitis.
Further, IL-12 may be more regulatory, suppressing
TNF and other proinflammatory cytokines [2279].
IL-12 secretion by antigen presenting cells may be
responsible for differences in immune response between
females and males [2161]. Females tend to have more
vigorous humoral and cellular immunity. Females also
have a higher incidence of autoimmune disease. An
interesting study showed that activated AP cells from
SJL female mice secreted IL-12 but not IL-10 and
favored Th1 response. These female mice suffered a
high incidence of experimental allergic encephalomy-
elitis. In males, AP cells produced IL-10 but not IL-12
and had a much lower susceptibility to the disease.
Castration or estrogen treatment increased IL-12,
decreased IL-10 and increased the incidence of this
autoimmune disease [2161].
Anticancer activity of IL-12 may be related to any of
a number of direct or extended effects that this cytokine
has on Th1 differentiation, CTLs, dendritic cells, mac-
rophages, NK cells, NK T cells, and vascular endothe-
lial cells [1848]. IL-12 induces several cytokines; IFN-γ
is a major down stream mediator of IL-12 antitumor
activity [1898, 2297]. As a single agent, IL-12 inhibited
a number of mouse model tumors [216, 1425]. Mice
bearing B16 F10 melanoma, M5076 sarcoma and Renca
renal carcinoma benefitted from treatment with IL-12.
The antitumor effect did not require NK cells but did
depend on T cells, specifically CD8+ T cells [216]. Mice
with sarcomas, treated with IL-12, showed decreased
tumor growth, increased longevity and in some cases
complete regressions. Interferon γ was required for the
IL-12 effect and it appeared to be mediated by CD4+
and CD8+ T cells [1425]. Downstream the chemokine
IP-10 (interferon inducible protein) was responsible for
Cytokines
IL-12-induced, CD8+ mediated immunity to mouse
neuroblastoma [1559]. In mice with brain tumors, IL-12
induced a T cell response, decreased tumor size, and
prolonged survival time [1690]. These studies suggest
that systemic IL-12 may be of benefit in the treatment of
CNS cancers.
Several studies have shown that IL-12 inhibits angio-
genesis in tumors [249, 349, 460, 606, 1898, 1964]. The
process involves a complex interaction between several
types of cells. IL-12 does not appear to act directly on
endothelial cells [460, 1898]. Rather IL-12 induces
IFN-γ, which in turn induces IP-10 and Mig; these two
chemokines act directly on endothelial cells, to induce
vascular damage, clotting and tumor necrosis [52, 64,
350, 1874, 1898, 1964]. Vasostatin is another antiangio-
genic factor. In nude mouse studies, treatment with
vasostatin or IL-12 inhibited tumor growth. Together
they effectively blocked Burkitt’s lymphoma, colon
carcinoma, and ovarian carcinoma [2230].
IL-12 engineered cells show promise as anticancer
vaccines. IL-12 transfected tumor cells [264, 1152] or
fibroblasts [2299] inhibited the growth of some estab-
lished tumors and induced the rejection of subsequent
tumor implants. In a model using an MHC I negative
small cell lung cancer growing in nude mice (no capac-
ity for CTL response), cells engineered to produce both
IL-15 and IL-12 did not grow; coinjected wild type
tumor was also rejected. This study showed that IL-12
could stimulate innate antitumor immunity and that
macrophages appeared to be involved [427].
Dendritic cells transfected by bombardment with
DNA-coated gold particles containing genes for tumor
antigens express antigen and presented it. Co-transfection
of these dendritic cells with IL-12 or IFN-α consistently
enhanced the induction of specific CTLs in vaccinated
mice [2039].
IL-2 and IL-12 interact; receptors are mutually stim-
ulated [416] and they have additive or synergistic
effects on NK cells [394], T cells [596, 1284] and mac-
rophages [2157]. Administration of IL-12 with pulse
IL-2 induced complete regression of established mam-
mary carcinoma in treated mice [2158]. IL-12 has also
been reported to potentiate the effects of tumor cell
vaccines engineered to secrete IL-2 for colon cancer
[2048] and glioma [981].
Time-release microcarriers have been used to deliver
a single dose of adjuvant IL-12 by intratumoral injection.
IL-12 alone and especially with GM-CSF, activated T
effector/memory cells, killed regulatory T cells and
increased CD8+ effector cells. Elimination of suppression
and development of cytotoxic T lymphocytes eradicated
metastatic tumors [960].
Walter M. Lewko and Robert K. Oldham
The preclinical studies with IL-12 were quite
encouraging. This cytokine had many effects similar to
those of IL-2 and appeared to be less toxic. For these
reasons and others, IL-2 and IL-12 may prove to be
effective in combination or sequential biotherapy.
Similarly, IL-12 gene insertion may prove an effective
strategy for increasing tumor immunogenicity. Finally,
ex vivo expansion of T cells with IL-12 in addition to
IL-2 might be rendered more specific and more cytolyt-
ically effective. Studies from our laboratory and others
indicate that IL-12 is effective in stimulating cytolytic
populations of tumor derived T cells and may be of
benefit in the development of T-cell based therapy.
There have been several reports on phase I/II studies
using IL-12. Patients with cutaneous T cell lymphoma,
injected with rhIL-12, had a response rate (CR + PR) of
50% [1661]. IL-12 has also been tested in patients with
renal cancer [84, 635, 1379, 1484, 1588, 1650], mela-
noma [84, 635, 1075, 1367, 1650, 1928] and ovarian can-
cer [829, 1096]. IL-12 has been used in combination with
Herceptin in breast cancer patients [1534] and with IFN-
α2b in with renal cancer and melanoma patients [832].
Vaccination studies have been carried out using IL-12 as
an adjuvant with peptide-pulsed antigen presenting cells
[581] and using transfected, IL-12-secreting autologous
tumor cells [1928]. Patients have also been injected intra-
tumorally with transfected, IL-12-secreting fibroblasts
[917]. Collectively, these studies showed that IL-12
induced immune response; NK cell and specific CTL
activities were increased and serum cytokine levels were
elevated, in particular, IFN-γ. Toxicities were significant
but manageable. Lymphopenia was the most common
problem. Clinical activity was observed but the response
rates were disappointingly low (generally less than 10%)
and not durable. In order to improve the response rate,
IL-12 may have to be administered in combination with
other cytokines, IL-2 for instance [635].
Interleukin-13
Regulation of Inflammation; Allergy
IL-13 is a Th2 cytokine that has a role in parasite infec-
tions, allergy and the regulation of inflammation.
Recombinant IL-13 has a molecular weight of about
14,000. It is related to IL-4 [1274, 2310]. Activated Th2
CD4+ T cells, CD8+ T cells [908, 1275, 1315, 1597],
mast cells [225], eosinophils [2180], basophils, dendritic
cells and certain B cell lines produce IL-13. The gene of
IL-13 is located on human chromosome 5 together with
genes for IL-3, IL-4, IL-5 and IL-9 [1271].
185
The receptor for IL-13 has two subunits, IL-4Rα and
IL-13Rα1 [741, 758]. This complex also binds and
responds to IL-4. Signaling involves Jak1 and Tyk2 [943,
944]. In addition, there is IL-13Rα2, which may be solu-
ble or membrane bound [244, 445, 1940]. The soluble
form appears to be a non-signaling decoy receptor [2197];
the membrane bound form may have certain functions
[302, 531].
IL-13 appears to have remarkable effects on T cells;
Th2 cells are stimulated and Th17 cells are inhibited.
But the receptor for IL-13 is not found on T cells; these
effects of IL-13 must be indirect. In DC for example,
IL-13 inhibits expression of IL-6, a key factor necessary
for Th17 differentiation [985, 1276, 2310].
In monocytes, IL-13 is anti-inflammatory and pro-al-
lergy. IL-13 decreased production of several pro inflam-
matory cytokines including IL-1α, IL-1β, IL-6, IL-8,
IL-10, IL-12, GM-CSF, G-CSF, MIP-1α, TNF-α, GROα
and prostaglandin E2 [422, 1315, 1598, 2198]. IL-13
also inhibited antibody-dependent cell-mediated cyto-
toxicity [422]. IL-13 caused remarkable changes in
monocyte phenotype and adherence. It stimulated levels
of complement receptors CD11b and CD11c. IL-13 also
stimulated MHC class II levels. IL-13 increased CD23
(an IgE receptor). IL-13 also increased CD49e and
CD29, which together form VLA5 (integrin α5β1, a
fibronectin receptor) [162]. IL-13 depressed levels of
CD64, CD32 and CD16 (IgG receptors) [422]. IL-13
decreased CD14 (LPS receptor) [162, 339]. IL-13 pro-
tected animals from the lethal effects of LPS-endotoxemia
[1382]. IL-13 increased production of IL-1 receptor
antagonist [422, 1408]. IL-13 is also a potent suppressor
of nitric oxide production in activated macrophages, epi-
thelial cells and mesangial cells [139, 172, 286, 1733].
IL-13 does this by regulating levels of nitric oxide syn-
thase and substrate arginine available for production
[286, 1733]. Further, IL-13 inhibited the production of
HIV-1 virus in cultured human macrophages [1343].
In B cells, IL-13 increased proliferation, the synthesis of
immunoglobulin, and IgE class switching [328, 398, 1275,
1597]. IL-13 also enhanced the expression of MHCII and
CD23 (an IgE receptor) [1277]. Essentially all of these
effects of IL-13 in B cells are similar to those of IL-4.
IL-13 has various additional activities. In neutrophils,
IL-13 induced the secretion of interleukin-1 receptor
antagonist [337]. IL-13 also stimulated the production of
IL-6 in human keratinocytes [415] and in human micro-
glial cells [1773]. In NK cells, IL-13 induced the produc-
tion of IFN-γ and cytotoxicity [1315]. IL-13 is a chemotactic
factor for human osteoblasts [1131]. In endothelial cells,
IL-13 stimulates production of VCAM enhancing T cell
adhesion [620]. IL-13 is a chemoattractant for monocytes
186
[1200] and stimulates production of monocyte chemoat-
tractant protein-1 in vascular endothelial cells [633].
IL-13 is involved in inflammatory diseases. IL-13 reg-
ulates rheumatoid arthritis. Cultured explants of synovial
tissue from patients contained elevated levels of IL-1β,
TNF-α and PGE2. Transfection with IL-13 depressed the
production of these inflammatory factors [2198]. While
IL-13 is generally antiinflammatory, it does have certain
proinflammatory effects. IL-13 has a central role in the
development of allergic asthma [681, 2175]. IL-13 is a
Th2 cytokine. It fosters humoral immune response and
the production of IgE. Bronchoalveolar lavage cells from
asthmatic patients challenged with ragweed allergen pro-
duced high levels of IL-13 [813]. Antigen stimulated
CD4+ cells of patients with allergic rhinitis produced
more IL-13 compared to normal controls.
There are some reports on IL-13, relating to cancer.
Renal cell carcinomas produce IL-13 and IL-13R
[1474]. Addition of IL-13 to the medium of cultured
cells inhibited growth up to 50% [1475]. Human glioma
cells contained unusually high levels of the IL-13R.
Tumor growth was inhibited by an IL-13-exotoxin con-
jugate [393, 830].
IL-13 is angiogenic. It stimulated the formation of
vessel-like structures from endothelial cells in collagen
culture and in vivo it stimulated outgrowth of vessels in
the rat cornea assay [574, 703]. The mechanism appeared
to involve the production of VCAM, which in its soluble
form is angiogenic.
IL-4 is used together with GM-CSF for in vitro pro-
duction of dendritic cells in human vaccination studies.
IL-13 substitutes for IL-4 in the production of dendritic
cells [33]. It remains to be seen whether IL-13 may be of
advantage in the development of anticancer vaccines.
Interleukin 14
B Cell Development and Memory
IL-14 was discovered in the conditioned media of
Burkitt’s lymphoma cell lines. It was originally referred
to as high molecular weight B cell growth factor (HMW-
BCGF) [39–41]. It is a rather large glycosylated protein
with a variable mw of 53–65 kDa, depending on carbo-
hydrate content [42]. IL-14 is produced by PHA stimu-
lated CD8+ T cells, CD4+ T cells (Th1 and Th2), NKT
cells, follicular dendritic cells [342, 1704] and B cell
lymphomas [552, 553]. Its mRNA has been detected in
unstimulated vascular endothelial cells [1455].
IL-14 stimulates B cell growth and differentiation
[39, 41]. It also appears to induce and maintain B cell
Cytokines
memory [40, 1479]. Mice that were transgenic for IL-14
developed autoimmunity and B cell lymphomas, which
resembled systemic lupus in patients [1803]. IL-14 is
secreted by B cell malignancies and may have a role in
the development of these diseases [552].
Interleukin 15
NK Cell Activity, Maintenance
of T Cell Memory
IL-15 was discovered as a factor in culture media of
monkey kidney epithelium that supported IL-2 depen-
dent growth in a T cell line. The same factor was identi-
fied in a human bone marrow stroma cell line [647].
IL-15 was also discovered independently, as the factor
IL-T in a T cell leukemia cell line [108, 228].
IL-15 is a 14–15 kDa, 114 amino acid protein. It a
member of the four α helix bundle cytokine family to
which IL-2 belongs [647]. Many of its activities are
similar to those of IL-2. While IL-2 is produced mainly
by activated T cells, IL-15 is produced by a rather wide
variety of cells types including peripheral blood mono-
nuclear cells, epithelial cells, fibroblasts, placenta, skel-
etal muscle, heart, lung, liver, kidney [647], and
keratinocytes [1334]. IL-15 was not detected in acti-
vated T cells [647]. While IL-2 synthesis is regulated at
the level of transcription and mRNA stability, IL-15 is
regulated at the translational level [107] which is less
common but has been shown to occur for certain cytok-
ines (e.g. IL-1β, TNF-α, TGF-β3, TGF-β1, GM-CSF).
A pool of mRNA, readily available for translational
activation, may allow rapid production of IL-15 when
required, as in response to an intracellular infectious
agent [107].
The IL-15R is composed of three subunits: IL-15Rα,
IL-2Rβ and γC. The β and γc subunits are shared with
IL-2 receptors; IL-2 and IL-15 have similar signaling
patterns and cytokine activities [626]. And although the
IL-15Rα chain binds IL-15 specifically, it is structurally
related to IL-2Rα [46, 627]. IL-15R is expressed in a
greater variety of cell types than the IL-2R [46, 627].
Interestingly, mast cells respond to IL-15; they have a
different IL-15 receptor and signal transduction path-
way [1945].
There is a soluble form of the IL-15Rα chain. It is
released from cells by proteolysis. It binds IL-15 with
high affinity and it acts as an IL-15 antagonist [1369]
There is evidence that IL-15 can exist in a mem-
brane-bound form, which is capable of reverse/bidi-
rectional signaling when engaged by its receptor.
Walter M. Lewko and Robert K. Oldham
This membrane bound IL-15 was upregulated in
monocytes by IFN-γ [219].
IL-15 stimulates proliferation and differentiation of
NK cells, B cells, and T cells. Knockout mice lacking
IL-15Rα were deficient in NK cells, NK-T cells, CD8+
T cells and γδ T cells [1145, 1457, 2172]. In NK cells,
IL-15 stimulated differentiation and activation [249,
1145, 1381, 1477, 2172]. IL-15 upregulated expression
of NKGD2 receptors, which are involved in antitumor
activity [1930]. IL-15 is also chemotactic for NK cells
[29]. In B cells, IL-15 stimulated proliferation and secre-
tion of IgM, IgG, and IgA [71]. In activated cytotoxic T
lymphocytes, it increased growth and cytotoxicity [617].
IL-15 stimulated the growth of an IL-2 dependent CTL
cell line [228]. IL-15 has been shown to rescue tolerant
CD8+ T cells for use in adoptive anticancer therapy
[1975]. IL-15 also increased growth and activity of γδ T
cells [591, 1457]. IL-15, like IL-2, induced formation of
LAK cells in cultures of peripheral blood lymphocytes
[228, 617]. In primary cultures of tumor infiltrating lym-
phocytes, IL-15 replaced IL-2, inducing outgrowth and
cytotoxicity of tumor derived activated T cells (TDAC)
[1111]. In TDAC cultures that were initially induced
with IL-2 and dependent on IL-2 for growth, IL-15 could
replace IL-2 for the maintenance of growth [1111]. With
increased awareness of the importance of IL-2 in Treg
immunosuppressive function, there has been consider-
ation for replacing IL-2, in the development of CTL for
immunotherapy, with IL-15 alone or in combination with
IL-21 [56, 2275].
IL-15 is synergistic with IL-12 in the induction of
mouse Th1 clones [92]; IL-15 appears to have a strong
pro-Th1 tendency. In transgenic mice, overexpression
of IL-15 augmented Th1 response to infection with
intracellular bacterium [1457]. Increased IL-15 is asso-
ciated with inflammatory diseases including rheumatoid
arthritis [1268, 1692], pulmonary sarcoidosis [12], mul-
tiple sclerosis [982] and inflammatory bowel disease
[972, 1715]. On the other hand, IL-15 has been shown
to inhibit allergy. IL-15 overproduction in transgenic
mice had a negative effect on the development of aller-
gic asthma, a Th2 disease; IL-15 depressed pulmonary
eosinophilia and the production of Th2 cytokines [860].
But it should be noted IL-15 may have some pro Th2
effects; in mice primed with dust mite allergen, IL-15
stimulated IL-5 production by allergen-specific human
Th2 clones and resulting eosinophil activation [1354].
While similar in function, there are differences
between IL-2 and IL-15. IL-2 has a role in tolerance by
inducing T cell suicide [1710, 1933, 2168], in activa-
tion-induced T cell death [1092, 1626, 2062] and it is
involved in the inhibition of T cell memory maintenance
187
[1026]. On the other hand, IL-15 has anti-apoptotic
effects [223] and increases the survival of CD8+ mem-
ory cells [1026, 2284]. In mast cells, IL-2 has little or no
effect while IL-15 stimulates growth and response to
IL-3 and stem cell factor [1945, 2091].
Human T cell lymphotropic virus codes for tax protein;
when T cells were infected, tax expression increased the
production of IL-15 [94, 95] and IL-15Rα [1227]. IL-15
appears to be responsible for the abnormal proliferation of
T cells associated with infection by this virus [95].
The anti-apoptotic effects of IL-15 may have a role in
psoriasis, a chronic proliferative inflammatory skin
disease. Keratinocytes produce IL-15 and the IL-15 R.
Compared with normal epidermis, biopsies of psoriatic
skin lesions were high in IL-15 and IL-15 binding
capacity [1693].
Cellular immunity is responsible for allograft rejec-
tion. A study showed that blocking the effects of IL-15
using soluble IL-15Rα chain prolonged the survival of
heart grafts. This indicated that IL-15 has a role in graft
rejection and that IL-15 antagonists may be therapeuti-
cally useful in the treatment of graft recipients [1845].
In a nude mouse xenograft tumor model, MHC I nega-
tive small cell lung cancer cells, engineered to secrete
IL-15, grew more slowly and had a slightly reduced take
rate; tumors were infiltrated with NK cells and the deple-
tion of NK prevented the antitumor effect. Interestingly,
when engineered to secrete both IL-12 and IL-15, the
tumors were completely rejected, as were coinjected
wild type tumors. Tumors were infiltrated with mac-
rophages, NK cells and granulocytes. Activated mac-
rophages appeared to be the major effector cells [426]. It
has also been shown that in NK cells, IL-15 potentiates
IL-12-induced secretion of IFN-γ, MIP-1, and IL-10
[521]. These studies show the importance of natural
immunity and the synergistic action of IL-15 and IL-12
in the process. And in adoptive immunotherapy,
Certain tumors express IL-15R and are candidates for
cytokine-based therapies. An antibody (Mikbeta1) to
the IL-2/IL-15β subunit has been tested in Phase I and
found not beneficial in T cell large granular lymphocyte
leukemia [1363].
Interleukin-16
Chemoattractive; Proinflammatory;
Immunoregulatory; Anti-HIV
IL-16 was originally described in 1982 as the lympho-
cyte chemoattractant factor (LCF) secreted by mitogen-
stimulated peripheral blood mononuclear cells [268,
188
270, 360]. IL-16 is produced mainly by CD8+ T cells. It
is also produced by CD4+ T cells [2205], B cells [921,
1797], fibroblasts [1770], eosinophils [1129], mast cells
[1697], dendritic cells [922] and epithelial cells [70].
IL-16 is also secreted by brain tissue; it may have a role
in the interaction between the immune and nervous
systems [1038].
IL-16 is synthesized as a proprotein. Nascent IL-16
lacks the usual signal peptide found on most secreted
proteins [102]. This lack of a signal peptide is also a
characteristic of IL-1 [89], IL-18 [1488] and FGF [3,
884]. Pro-IL-16 is cleaved to its active form by caspase-3,
an enzyme in the same family as caspase 1 (ICE) which
is responsible for the activation and secretion of IL-1β
and IL-18 [2286].
Regulation of IL-16 release depends on the cell type.
CD8+ T cells produce IL-16 constitutively and store it
in the active form; histamine, serotonin, mitogen and
antigen stimulate IL-16 release [271, 1047, 1048].
CD4+ cells, on the other hand, store inactive pro-IL-16;
T-cell activation increases caspase-3 activity, pro-IL-16
processing and the secretion of active IL-16 [2203]. In
bronchial epithelial cells, histamine, IL-1β and TNF-α
stimulate secretion. Dexamethasone inhibits secretion;
regulation of IL-16 may be part of the glucocorticoid
antiinflammatory effect [70]. In fibroblasts, IL-16
mRNA is produced constitutively. Inflammatory cytok-
ines such IL-1β induce the release of active IL-16 by a
caspase-3 dependent mechanism [1770].
The amino acid sequence for IL-16 is distinct; it has
little or no homology with other known cytokines. The
monomer of IL-16 appears to be inactive; it aggregates
to form a 56,000 mw tetramer, which is the active
cytokine [102, 359, 360, 2286]. The C-terminal region is
particularly well conserved between species and appears
to be the region most critical for cell binding and activity
[102, 941]. Interestingly, the N-terminal prodomain
cleaved from pro-IL-16 by caspase-3 has a cellular func-
tion. It translocates into the nucleus and induces G0/G1
arrest, regulating cell division [269, 1631, 2287].
CD4 appears to be the primary receptor for IL-16.
Cells that respond to IL-16 invariably express CD4.
Anti-CD4 Fab fragments inhibit signaling by IL-16
[270, 358, 359, 360, 361, 1618]. Transfection of CD4
cDNA into CD4−cells enabled the cells to respond to
IL-16 [358, 361]. The absence of CD4 in a mutant clone
eliminated IL-16 response [1191]. IL-16 binds cell sur-
face CD4 and induces signaling [1021, 1700]. A peptide
of IL-16, based on the proposed CD4-binding sequence,
blocked IL-16-induced chemotaxis in cultured spleno-
cytes and decreased airway hyperresponsiveness in
peptide-treated mice [385]. HIV-1 (gp120) binds CD4
Cytokines
and inhibits chemokine signaling. In migrating lympho-
cytes, IL-16 induces protein kinase C; PKC inhibitors
block migration induced by CD4 engagement [1532]. In
macrophages, signaling involved the phosphorylation
of two MAP kinase family members: the stress activated
protein kinase/Jun N-terminal kinase (SAPK/JNK) and
p38 MAP kinase. These two pathways are commonly
activated by environmental stress signals such as UV
irradiation and by certain other proinflammatory cytok-
ines such as IL-1 and TNF-α [1021]. In T cells, the
binding of IL-16 to CD4 resulted in activation of the src
family tyrosine kinase p56 lck, which is associated with
CD4 [1700]. In NK cells that expressed CD4, ligation
induced cytokine secretion and cell migration [141].
The data favoring CD4 as the receptor are strong. But
there is evidence in knockout mice genetically lacking
CD4, that IL-16 was in fact still capable of inducing
cytokine production and lymphocyte migration [1239].
In a mast cell line that lacks CD4, IL-16 mediated
chemotaxis and signaling in a manner that could be
blocked by antibodies to CD9. This suggested that CD9,
a cell surface protein known to be involved in migration
and cell adhesion, serves as an alternative receptor for
IL-16 [1604].
IL-16 influences several cell types. The major effect
is chemoattraction but it also has effects on immune
response and HIV replication. IL-16 responsive cells
are typically CD4 positive [269, 271]. IL-16 sensitive
cells include CD4+ T lymphocytes [270, 357, 359,
1532], monocytes [357, 739, 1240], dendritic cells
[739, 921, 922, 411], eosinophils [1618], mast cells
[1604] and, interestingly, brain cells that are also
CD4+ [1038]. In resting T-cells, IL-16 induced sig-
naling and activation with migration and increased
IL-2 receptor levels [357, 358, 360, 1532, 1700,
1981]. Several chemotactic agents induce migration
in both CD4+ and CD8+ T cells; IL-16 is specific for
CD4+ cells [2271]. It modulates and desensitizes
certain chemokine receptors, thereby orchestrating T
cell recruitment [1613]. Further, IL-16 appears to
prime CD4+ cells for IL-2 and IL-15-induced growth
[357, 1532].
IL-16 has several roles during antigen presentation.
In monocytes and macrophages, IL-16 induced migra-
tion and the secretion of several cytokines including
IL-1β, IL-6, IL-15 and TNF-α [1240]. In DC’s and mac-
rophages, IL-16 stimulated levels of CD25 (IL-2R) and
the costimulatory molecules CD80 and CD83 [739].
Further, IL-16 may act together with thrombopoietin
during DC development to induce tolerogenic dendritic
cells capable of inducing anergy in T cells [411]. And in
lymphnodes, resident B cells and DC’s produce IL-16
Walter M. Lewko and Robert K. Oldham
involved in the trafficking of Th cells and inward
migration of additional dendritic cells [921, 922].
Delayed type hypersensitivity is mediated by antigen
specific Th cells and involves cell movement. The role
of IL-16 in DTH was studied in mice [2255]. IL-16 was
expressed in DTH tissues but not in controls. Extracts
of DTH tissue exhibited chemoattractant activity and
IL-16 neutralizing antibodies inhibited this activity.
When mice were pre-treated with antibodies, swelling,
leukocyte infiltration and chemokine levels associated
with DTH were depressed. These results suggested
IL-16 had an important role in the recruitment of leuko-
cytes and secretion of cytokines associated with DTH
reactions [2255].
While IL-16 stimulates immunity it also has regula-
tory effects. CD4 is part of the TCR/CD3 complex
responsible for antigen-induced activation of T cells.
HIV-1 gp120 and antibodies that bind CD4 tend to inter-
fere with activation [1476]. IL-16 also has immunosup-
pressive effects. It inhibited mixed lymphocyte reactions,
deactivated T cells and lowered IL-2 secretion [1686].
Other studies have shown that skin cells transfected
with IL-16 were immunosuppressive suggesting that
IL-16 might be used to prevent graft rejection [572].
IL-16 induces migration and it also appears to regu-
late migration. When IL-16 bound CD4, there was a
selective loss of chemokine receptor 5 activity. The two
receptors were mutually inactivating. When MIP-1β
bound CCR-5, T cell migration induced by IL-16 was
inhibited. HIV-1 gp120 also appears to inhibit chemokine
receptor signaling [1235].
IL-16 is one of several factors secreted by CD8+ T
cells, which inhibit viruses. (Others antiviral factors
include MIP-1α, MIP-1β and RANTES). Specifically,
IL-16 interferes with HIV-1 replication in T cells [43,
103, 1191, 1734, 2294, 2295], macrophages and den-
dritic cells [2028]. The mechanism of inhibition appears
to involve the interaction of IL-16 with CD4. A factor is
produced which binds the core enhancer DNA, inhibits
HIV-1 promoter activity and as a result blocks viral
replication [1191]. IL-16 also blocks HIV-1 uptake by
the cells [2028]. Decreased virus entry may be due to
IL-16-induced CD-4 downregulation [739]. In a clinical
study, HIV-1 infected patients were followed over an 8
year period. During the asymptomatic phase, serum
IL-16 levels were maintained or increased. With disease
progression, there was a drop in IL-16 [43]. These
results support a natural role and potential therapeutic
benefit of IL-16 in the control of HIV infection.
IL-16 is involved in the development of several
inflammatory diseases. IL-16 levels were elevated in
fluid and tissue samples from patients with systemic
189
lupus erythematosus, asthma, inflammatory bowel
disease and Crohn’s disease [1076, 362, 750, 942, 1236,
1049]. In related animal models, blocking antibodies to
IL-16 ameliorated these diseases. On the other hand,
IL-16 appeared to regulate inflammatory cytokines in a
rheumatoid synovitis mouse model [986]. The synovial
infiltrate contained activated CD4+ T cells secreting the
proinflammatory cytokines IL-1β, IFN-γ, and TNF-α.
CD8+ T cells produced a factor, which lowered the
inflammatory cytokines. Anti IL-16 antibodies blocked
the effects of this factor while treatment with rIL-16
mimicked the factor. It appeared that CD8+ T cells
might have anti-inflammatory effects, which are at least
in part mediated by IL-16 [986].
IL-16 has not been extensively studied in cancer.
Nonetheless, its involvement in the activation and
migration of several cell types including CD4+ T cells,
dendritic cells and macrophages suggests it may have a
role in antigen presentation, the development of tumor
infiltrating lymphocytes and anticancer vaccination. Its
antiviral effects may block HIV-related cancers.
Interleukin-17A
Proinflammatory, Hematopoietic,
Neutrophil Development
The IL-17 cytokine family has six members. IL-17A,
the first discovered, is a 155 amino acid, 20 kDa glyco-
protein [555, 2232]. IL-17 family members have little or
no homology with cytokines outside the family [555,
2232]. The gene is on chromosome 2q31. IL-17A was
originally referred to as CTL-associated antigen-8
(CTLA-8) [1688]. Herpesvirus saimiri encodes a similar
protein [2231]. IL-17 is produced by CD4 T cells, CD8
T cells and γδ T cells [1, 951, 555, 1144, 2232]. Bacterial
endotoxin, IL1β, IL-6, IL-18, IL-23 and TGF-β stimu-
late IL-17A production [852, 1325, 712].
IL-17A is produced primarily by CD4 T-cells that are
now referred to as Th17 cells [718]. These cells along
with Th1, Th2 and Treg cells are major controllers of
immune response. It is now believed that Th17 cells are
responsible for many autoimmune diseases that were
originally classified as Th1 diseases. The discovery of
Th17 cells solved a problem in the observation that mice
lacking the Th1 cytokine IFNγ suffered more, not less,
autoimmunity than controls. The development of Th17
lineage appears to have two phases. In the first phase,
Th0 cells differentiate into Th17 cells in response to
TGFβ and IL-6 (or IL-1β; a proinflammatory signal is
needed) and then there is an expansion/survival phase,
190
which is stimulated by IL-23. Th17 differentiation is
inhibited by IL-2 and IFNγ (type 1 cytokines), IL-4 and
IL-13 (type 2 cytokines), and IL-35 (Treg cytokine)
[149, 718, 985, 1025, 1218, 1451, 1536, 2070].
Receptors for IL-17 have been found in most tissues
tested. There are at least five members of the IL-17 receptor
family. These receptors have little or no homology to
other known cytokine receptors [2231, 2233, 1370].
IL-17 functions in host defenses against infection.
Bacterial products induce IL-17 secretion [852]. IL-17
is required for defence against several types of infec-
tion, for example, bacterial pneumonia [2236]. IL-17 is
involved in hematopoiesis. It appears to promote recovery
in response to radiation damage [1961]. IL-17 is
involved in inflammation. It stimulates production of
molecules such as proinflammatory cytokines, chemok-
ines, CSFs, prostaglandin, MMPs, NOS and antimicro-
bial peptides in a variety of cells including macrophages,
fibroblasts, epithelial cells, keratinocytes, endothelial
cells, mesothelial cells and dendritic cells [231, 513,
555, 1768, 878, 890, 1980, 2178]. IL-17 has particularly
remarkable effects on neutrophil production and tissue
infiltration. IL-17-treated fibroblasts release CSFs that
stimulate neutrophil differentiation [555]. In transgenic
mice that produced excess IL-17, there was enhanced
hematopoiesis in general and granulopoiesis in particu-
lar. Peripheral WBC counts increased fivefold and neu-
trophils increased tenfold [1766]. In gastric mucosa,
infection by H. pylori stimulated production of IL-17.
IL-8 levels were increased and the tissue became infil-
trated. Antibodies to IL-17 inhibited IL-8 secretion and
antibodies to IL-8 blocked PMN leukocyte migration
[1174]. Similar responses occurred in inflamed intesti-
nal and bronchial epithelial cells [93, 1046].
Dysregulation of IL-17 appears to have a role in the
pathology of allergic and autoimmune inflammatory dis-
eases [18, 275, 276, 279, 1011, 1174]. In mice with col-
lagen-induced arthritis, IL-1 and IL-17 levels were
elevated; blocking antibodies for IL-17 alleviated joint
destruction whereas IL-1 blockade had no effect, sug-
gesting IL-17 was the more direct cause of arthritic dam-
age [1169, 1414]. In patients with arthritis, synovial fluid
IL-17 stimulated formation of osteoclasts, which caused
tissue destruction [1011]. IL-17 also increased the pro-
duction of gelatinase in human macrophages [879],
aggrecanase and metalloproteinase in chondrocytes
[230] and nitric oxide in osteoarthritic cartilage [88].
IL-17 has potential in cancer therapy. IL-17 stimulates
bone marrow stromal cells to produce G-CSF, SCF and
IL-8 for the production and mobilization of hematopoi-
etic stem cells [1767]. This may be useful for stem
cell transplantation. IL-17 promotes the maturation of
Cytokines
dendritic cells [57]. This may have a role in cancer
vaccination. It has also been shown that Chinese hamster
ovary cells engineered to produce IL-17 were less inva-
sive and metastatic [764]. There is also evidence that
IL-17-transfected tumor cell vaccines can stimulate the
generation of tumor specific cytotoxic T cells [133]. In a
study on B16 melanoma in mice, tumor antigen specific
CD4 Th cells that had been polarized in culture, were
adoptively transferred into mice with growing tumors;
interestingly, the Th17 cells had superior antitumor activ-
ity compared to Th1 cells, the type traditionally associ-
ated with anticancer activity [1395]. However, there are
reports that by its proinflammatory properties, IL-17
may stimulate development of cancer. It has been shown
that IL-17 increased the growth of cervical tumors trans-
planted in nude mice [1967]. In prostate, normal tissues
produced little IL-17, but most benign prostate hyperpla-
sia specimens and prostate cancers showed high IL-17
expression, mainly in T-cells but also in epithelial cells
[1887]. In lung cancer cell lines, IL-17 had no direct
effect on tumor cell growth but had a remarkable stimu-
latory effect on secretion of several angiogenic CXC
chemokines. In SCID mice, lung cancer cells engineered
to produce IL-17 grew larger and were more vascular
than controls suggesting a role for IL-17-induced
chemokines and angiogenesis in tumor growth [1467].
Interleukin-17B
Proinflammatory Cytokine
IL-17B has 27% amino acid identity with IL-17A. The
gene is on chromosome 5q32–34 [1113]. IL-17B mRNA
is produced by cells in the pancreas, small intestine, and
stomach but not by activated T cells.
IL-17B does not bind the IL-17 receptor. Rather it spe-
cifically binds the protein IL-17Rh1 (IL-17 receptor
homolog 1). At least one additional IL-17 family mem-
ber, IL-25, also binds IL-17Rh1 [1073, 1807]. The mRNA
for this receptor has been detected in pancreas, kidney,
thyroid, liver, brain and intestines. Receptor levels in
intestine were increased during inflammation [1807].
In a monocyte cell line, IL-17B stimulated produc-
tion of IL-1β and TNF-α while IL-17 had little effect. In
activated fibroblasts, IL-17 stimulated production of
IL-6 while IL-17B had no effect [1113]. These results
showed that though the two cytokines are structurally
related, they have distinct activities. In mice, IP injec-
tion of rIL-17B induced influx of neutrophils. This was
probably not a direct effect but due to stimulated release
of chemotactic factors [1807].
Walter M. Lewko and Robert K. Oldham
Interleukin-17C
Proinflammatory Cytokine
IL-17C has approximately 27% homology with IL-17.
The distribution of IL-17C mRNA appears to be rather
restricted. It was found in prostate and fetal kidney.
Activated T cells did not produce it. The gene for
IL-17C is on chromosome 16q24. The receptor used
by IL-17C is not known; it does not appear to bind the
IL-17 receptor [1113].
In a monocyte cell line, IL-17C stimulated produc-
tion of IL-1β and TNF-α while IL-17 had no effect. In
activated fibroblasts, IL-17 stimulated IL-6 secretion
while IL-17C had no effect. These results showed that
while IL-17 and IL-17C are related, they have distinct
activities [1113]. Expression of IL-17C in mouse lung
by adenovirus-IL-17C infection promoted neutrophilia
and inflammatory gene expression such as IL-6 and
IFNγ [825].
Interleukin-17D
Proinflammatory, Inhibitor
of Hematopoiesis
IL-17D has 202 amino acids and as such it is the largest
member of the IL-17 family. IL-17D is most homolo-
gous to IL-17B with 27% identity. The gene is on chro-
mosome 13p11. IL-17D mRNA is expressed in a number
of tissues including skeletal muscle, brain, adipose,
heart, lung and pancreas. It was poorly expressed on
activated CD4 T cells, CD8 T cells, monocytes and
activated B cells [1880].
In cultured endothelial cells, Il-17D stimulated pro-
duction of IL-6, IL-8 and GM-CSF. IL-17D had a sup-
pressive effect of myeloid colony formation in agar
gel assays [1880].
Interleukin-17E: see
Interleukin-25
Interleukin-17F
Proinflammatory, Asthma, Inhibitor
of Angiogenesis
Interleukin-17F is also referred to as ML-1 [930]. It has
high homology with IL-17. The gene is on chromosome
6p. Activated CD4 T cells, monocytes, basophils and
191
mast cells produce IL-17F. It was not expressed by CD8
T cells or B cells [1881]. IL-17F is a homodimer; het-
erodimers with IL17A chain occur in mouse Th17 cells.
Potency was intermediate of the two homodimeric
forms in a chemokine release assay [1122].
The receptor for IL-17F is the protein IL-17RC. This
receptor is related to IL-17R and IL-17A also binds it
[1030].
IL-17F has a remarkable effect on vascular endothe-
lial cells. It stimulated the production of endothelial
IL-2, TGF-β and monocyte chemoattractant protein-1.
In an in vitro assay for angiogenesis, IL-17F inhibited
the formation of tubular, vessel-like structures [1881].
IL-17F may be acting directly or through the induction
of cytokines such as TGF-β, which inhibit angiogenesis
[1219, 1881]. IL-17F also stimulates the production of
IL-6, IL-8 and GM-CSF in bronchial epithelium and it
regulates cartilage matrix turnover [833, 929].
IL-17F is involved in asthma. In mice, expression of
IL-17F in lung by adenovirus-IL-17F infection promoted
neutrophilia and the expression of inflammatory genes
such as IL-6 and IFNγ [825]. In humans, expression of
IL-17F (but not IL-17) was increased in lung biopsies of
asthma patients after allergen challenge. When added to
cultured bronchial epithelial cells, IL-17F stimulated the
production of IL-6 and IL-8 and cell surface ICAM-1; it
recruits neutrophils into bronchial airways [930].
Polymorphism in the IL-17F gene appears to influence
susceptibility to asthma. Japanese subjects with arg sub-
stituted at position 161 were protected from the disease;
this variant blocked the induction of IL-8 by wild type
IL-17F [931].
There are no reports on cancer for this relatively new
cytokine, though IL-17F is of interest for its proinflam-
matory and antiangiogenic effects.
Interleukin-18
Interferon γ, NK and Th1 Response,
Regulation of Inflammation
IL-18 was discovered in mice with severe hepatitis
induced by Propiobacterium acnes and the endotoxin
LPS [1417]. IL-18 was originally called interferon-γ
inducing factor (IGIF) for its remarkable stimulation
IFNγ secretion. It is synthesized as a 192 amino acid pro-
protein, which is processed to the 156 amino acid active
form [2045]. IL-18 is similar to IL-1β in that its nascent
protein lacks the signal peptide usually found on secreted
proteins [120, 1489] and proteolytic activation of the
proprotein is carried out by caspase-1 (ICE) [688].
192
Activation is necessary for secretion; caspase-1-deficient
macrophages do not secrete IL-18 as usual when treated
with LPS [512, 618, 688, 1779].
IL-18 is produced by macrophages, dendritic cells
[1489, 1600] and Kupffer cells [1779, 2045]; levels are
increased by microbial infection or LPS treatment. IL-18
is also produced by adrenal cortex (in the area of cortisol
production) [340], by skin keratinocytes [1895] and by
airway epithelium [234]. In adrenal cortex, expression
was increased by cold stress as a possible link between
the environment and immune response [340]. Treating
mice with contact allergens stimulated IL-18 expression
in keratinocytes [1895]. In airway epithelial cells, IL-18
levels were related to Th1 status [234].
IL-18BP is a binding protein found in circulation,
which has relatively high affinity for this cytokine. As a
feedback loop, IL-18 induces IFNγ and interferon
induces IL-18BP. It competes with the receptor for free
IL-18 and in this way regulates IL-18 activity [963,
1465, 1640]. A relative lack of IL-18BP occurs in
patients with hemophagocytic syndrome, a disease with
elevated free IL-18 and characterized by a strong Th1
response [1260].
The IL-18R contains two subunits [180, 965, 2014].
The α subunit binds IL-18 and the β subunit performs
signal transduction. Although the β subunit does not
bind IL-18, as part of the receptor complex it enhances
affinity for IL-18 [965]. Interestingly, the IL-18Rα sub-
unit was originally discovered and named IL-1 receptor
related protein (IL-1Rrp) for its molecular similarity to
the IL-1 receptor, though this protein did not actually
bind IL-1 [439, 1488, 1538]. At the time, it was referred
to as an orphan receptor for lack of a known ligand; later
it was determined to be the receptor for IL-18 [795,
1986, 2014]. The IL-18Rβ subunit is also a member of
the IL-1R family. It is related to the IL-1R accessory
protein. When IL-18 binds, it activates a pattern of cell
signaling which is similar to that of IL-1 [392, 1000].
In knockout mice genetically defective for the IL-18
receptor, development of Th1 cells was impaired. NK
cells had decreased cytotoxicity and IFN-γ production
[796]. Overexpression of IL-18 in transgenic mice
resulted in high levels of IgE, IgG1, IL-4, IL-5, IL-13 and
IFN-γ, reflecting both Th1 and Th2 cell activation [796].
IL-18 and IL-12 have similar effects on several types
of cells. These effects are often synergistic. In certain
cases, IL-12 appeared to be required for IL-18 response.
This seems to be due to the ability of IL-12 to increase
IL-18 receptor levels. IL-12 upregulates both α and β
subunits. Increased IL-18 receptor levels enhanced the
capacity for response [521, 963, 1724, 2214, 2253]. IL-18
and IL-12 synergistically induce IFN-γ in T cells [998,
Cytokines
1300, 1652, 1894, 2253], B cells [2253], NK cells [1954,
2009], macrophages [1393, 1741] and in DCs [573].
IL-18 stimulates activated Th1 cells to grow and
produce IFN-γ [1489]. It is well known that Th1 cell
differentiation requires IL-12. Il-18 acts synergistically
with IL-12 [1300]. IL-12 initiates differentiation; IL-18
intensifies the response. IL-2 is a costimulator of Th1
differentiation and growth [998]. IL-18 and IL-12 both
increase IL-2 receptor levels. IL-18 stimulates Th1
cells but it did not appear to influence Th2 cell growth
or production of IL-4 and IL-10 [998]. With Th2 dif-
ferentiation, IL-18 receptor expression is lost and with
it capacity to respond to IL-18 [2214].
Although IL-18 and IL-12 have related activities,
there are differences. IL-12 initiates differentiation of
Th1 cells; IL-18 does not initiate but enhances Th1 dif-
ferentiation [1487, 1652]. IL-18 induced Th1 cells to
produce IL-2; IL-12 did not [998]. In mouse activated
PBLs, IL-18 stimulated GM-CSF production and
depressed IL-10; IL-12 did not [2046].
IL-18 is generally considered a proTh1 cytokine, but
IL-18 does stimulate Th2-related responses [995, 1033,
1199, 1684, 2162]. B cells contain receptors for IL-18
and IL-12. These two cytokines synergize in the produc-
tion of immunoglobulin and IFN-γ [13]. IL-18 also
stimulates IL-4 secretion in NKT cells [1085, 2251,
2254] and IL-13 secretion by NK cells [797].
Overexpression of IL-18 by B cells and T cells in trans-
genic mice induced high levels of both Th1 and Th2
cytokines [796].
IL-18 also acts synergistically with interferon-α/β in
the secretion of IFN-γ by T cells. Virus infection induced
macrophages to produce IFN-α/β and IL-18 (but not
IL-12). Conditioned media from these macrophages
induced IFN-γ in T-cells. Neutralizing antibody to IFN-
α/β blocked secretion of IFN-γ; antibody to IL-12 had
no effect. These studies showed that the type I interfer-
ons acted synergistically with IL-18 in the production of
immune interferon [1725]. IFN-α increased the levels
of an adapter molecule (MyD88), which facilitated
IL-18 receptor signaling [1724].
IL-18 has remarkable effects on NK cells. IL-12
and IL-2 are also well known stimulators of NK cells.
IL-18 in combination with IL-12 or IL-2 induced
much higher NK cell proliferation, IFN-γ secretion
and antitumor activity than IL-12 or IL-2 induced
when added alone or together [2009]. These results
suggested an obligatory role for IL-18 in NK prolif-
eration and function [2009].
Cell killing by Fas-mediated apoptosis is stimulated
by IL-18. IL-18 upregulates Fas ligand (discussed
below) in NK cells [2035] and in helper T-cells [381].
Walter M. Lewko and Robert K. Oldham
In NK cells, IL-18 acts together with IL-2 and IL-12.
The IL-18 treated NK cells were positive for perforin
but killed target cells exclusively by FAS engagement
[2035]. By its ability to stimulate interferon-γ secretion,
IL-18 also stimulates Fas levels in many target cells.
IL-18 has a role in the induction and regulation of
inflammation. Neutrophils respond to IL-18 with
increased production of cytokines, CD11b (comple-
ment receptor) and granule release. In a mouse model,
IL-18 neutralizing antibodies suppressed footpad
inflammation caused by carragean injection [1102].
PBLs treated with IL-18 produce pro-inflammatory
TNF-α and in turn IL-1β and the chemokines IL-8 and
MIP-1α [1601]. IL-18 also brings about regulation of
inflammation. In T cells and NK cells, IL-18 together
with IL-2 stimulated the production of IL-13, an inhibitor
of inflammation [797].
IL-18 appears to be involved in rheumatoid arthritis
[652, 1103]. Elevated levels of IL-18 are found in
arthritic synovial fluid and serum [652]. In a collagen-
induced arthritis model, mice lacking IL-18 had a greatly
reduced incidence and severity of arthritis compared
with control mice. T cells from IL-18 deficient mice had
a reduced capacity for antigen-induced cell division and
secretion of proinflammatory cytokines [2131]. IL-18
has also been implicated in other inflammatory diseases
including Crohn’s disease of the bowel [1577] and in
sarcoidosis [662].
IL-18 appears to have a role as both a mediator and
regulator of angiogenesis. In culture, IL-18 stimulated
vascular endothelial cells to migrate and form capil-
lary-like structures. In animals, implants containing
small amounts of IL-18 stimulated the growth of new
vessels [1535]. As a regulator, it has been shown that
IL-18 suppressed angiogenesis and it did this by
blocking bFGF-stimulated cell division in endothelial
cells [240].
IL-18 has anticancer activity in many tumor models.
Elevated IFNγ and NK cells are generally required, but
depending on the system CD4+ or CD8+ T cells may be
involved [1301, 1302, 1509]. Combinations of IL-2 plus
IL-18 [1854, 2156] and IL-12 plus IL-18 [1117, 1509,
1624] generally have synergistic effects. These studies
have been extended to an IL-2/IL-18 fusion protein [4],
which produce NK and T cell responses with lower toxicity.
Additionally, IL-18 is being tested, with some success, in
cancer vaccines [840, 1228, 1852, 1969, 2210, 2224].
Further, it has been reported that NKT (generally consid-
ered to be a regulatory and immunosuppressive cell type)
may have anticancer activity. NKT cells isolated and
cultured with IL-12 plus IL-18 produced activated cells
that inhibited tumors when transferred to mice [118].
193
It has been suggested that IL-18 may be involved in
cytotoxic T cell development by acting as a bridge
between the innate and adoptive immune systems
[1962]. IL-18 stimulates IFN-γ levels and activates NK
cells (Fas L) causing tumor cell apoptosis. This provides
apoptotic bodies for dendritic cell antigen presentation
and the induction of specific CTLs [1962].
IL-18 is being tested in cancer patients. In a phase I
trial, IL-18 was administered to 28 patients, mainly
renal cancer and melanoma. Increases in serum IFNγ,
GM-CSF and other indications of cell activation were
observed. There were two partial responses; further
studies are warranted [1651].
It should be mentioned that while IL-18 has antican-
cer activity, certain cancers may be able to exploit the
IL-18 system. For example, IL-18 binding protein is
naturally secreted in response to IFNγ and it downregu-
lates IL-18 activity. Many tumors secrete IL-18 BP. In
this way the anticancer effect of IL-18 may be inhibited
by the tumor [1545]. In certain cancers, it has been
reported that IL-18 stimulates growth, angiogenesis,
adhesion to endothelium and invasion [246, 962, 1535,
2075]. In these tumors IL-18 may contribute to progres-
sion. Further, IL-18 may have a role in immune escape.
IL-18 upregulates Fas ligand in B16F10 melanoma
cells. It is possible that tumor cell-Fas ligand may induce
apoptotic death of effector cells resulting in immune
suppression [311].
Interleukin-19
Inflammation
IL-19 was discovered in LPS-stimulated monocytes
[582]. It is a member of the IL-10 cytokine family
(IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, IL-29).
The gene for IL-19 is located together with IL-10 on
chromosome 1q31–32. A homodimer appears to be the
active form [582]. LPS and GM-CSF stimulate mono-
cytes to produce IL-19. In LPS activated monocytes,
IL-4 and IL-13 each enhanced [584] and IFNγ depressed
IL-19 secretion [583]. Il-19 is also produced by kerati-
nocytes of psoriasis lesions [1660].
IL-19 binds and signals through the IL-20 receptor
(IL-20Rα/IL20Rβ). IL-19 does not bind the IL-10
receptor. IL-20R is expressed in skin, lungs, reproduc-
tive organs and various glands. Receptor levels are in
inflamed tissues [462, 583, 1540].
Monocytes treated with IL-19 secrete IL-6, TNF-α,
and reactive oxygen species [1125]. In T cells, IL-19
appears to favor Th2 differentiation and IL-4 secretion.
194
[583]. In PBMCs IL-19 induces IL-10 secretion and its
own production. IL-19 production is in turn down-
regulated by IL-10 [902].
IL-19 appears to be involved in asthma [1124] and
psoriasis [1114, 1660]. Asthmatic patients had twice the
serum levels of IL-19 compared with controls; levels of
IL-4 and IL-13 were also elevated. In culture, activated
T cells treated with IL-19 secreted Th2 cytokines IL4,
IL-5, IL-10 and IL-13 [1124].
Interleukin-20
Inflammation, Skin Development
and Immunity
IL-20 is a member of the IL-10 family. The gene for
IL-20 is located on chromosome 1q31–32 together with
the genes for IL-10, IL-19 and IL-24 [582]. IL-20 is pro-
duced by keratinocytes [1641], monocytes [2187] and
glial cells [799].
IL-20 binds two receptors (IL-20Rα/IL20Rβ) and
(IL-22Rα/IL20Rβ) [169, 1540]. IL-20R levels are ele-
vated in psoriatic skin [169]. IL-20 signals by STAT3
activation [462, 463].
IL-20 appears to regulate keratinocyte involvement
in skin inflammation [1641]. Overexpression of IL-20
in transgenic mice resulted in aberrant differentiation
of epidermis, keratinocyte proliferation and abnormal
skin resembling psoriasis. These mice also lack adipose
tissue and their T lymphocytes show high apoptosis;
they die soon after birth [169]. IL-20 stimulates the
growth of CD34+ multipotential progenitors in a unique
way, without imparting any tendency towards a partic-
ular developmental pathway, erythroid, granulocyte-
macrophage or megakaryocyte [1139].
In humans, IL-20 appears to be involved in psoriasis
and skin inflammation [2128]. In lesions from patients,
IL-20 and its receptor were elevated. IL-20-treated T
cells produced increased levels of keratinocyte growth
factor [2128].
Glial cells can produce IL-20 and LPS stimulates
release. This suggests that IL-20 may have a role in
inflammation of the brain [799]. In arthritis patients, syn-
ovial fluid contains elevated IL-20; synovial fibroblasts
treated with IL-20 secreted IL-6 and IL-8. In rat model,
IL-20 blockade decreased severity of arthritis. It appears
that IL-20 is involved in joint inflammation [809].
There is evidence for IL-20 being pro-angiogenic and
anti-angiogenic. Endothelial cells contain IL-20 recep-
tors. When treated with IL-20 these cells proliferate,
migrate and form tubular structures; bFGF, VEGF and
Cytokines
IL-8 are released. However, in studies of lung cancer,
IL-20 inhibited COX-2 and prostaglandin E2 produc-
tion in epithelial and endothelial cells, and it inhibited
PMA-induced angiogenesis [805, 746].
Interleukin-21
NK, T and B Cell Development,
Immunomodulation
IL-21 is a pleiotropic cytokine, which is structurally
related to IL-2, IL-4, and IL-15 [78, 1520, 1540]. IL-21
is produced mainly by Th2, Th17 and follicular T cells
[316]. The receptor for IL-21 has two subunits, IL-21Rα
and IL-2Rγ. IL-21Rα is related to IL-2Rβ and IL-4Rα;
IL-2Rγ is the subunit common to several IL-2 family
receptors. The IL-21 receptor is expressed on T, B, and
NK cells [78, 699, 1520].
T cells are stimulated and regulated by IL-21. Typical
Th2 responses (e.g. to schistosomes, airway inflamma-
tion) depend on IL-21 [570, 1561]. IL-21 also supports
the clonal expansion of virus and tumor antigen spe-
cific effector CD8+ T cells [2059, 1120, 1121]. IL-21
sustains the expression of CD28, an important costimu-
latory receptor [34, 1134]. It increases or decreases
IFN-γ production depending on conditions [1903, 1932,
2208]. But IL-21 also regulates CD8+ T cell expansion
when required by inducing apoptosis [113]. In mice,
plasmid DNA vaccines expressing HIV-1 env glycopro-
tein showed increased T cell response when combined
with IL-21 [177]. At least in part, increased generation
of CD8+ cytotoxic T cells may be the due to a suppres-
sive effect IL-21 has on Foxp3 regulatory T cells (Li
08) or the response of Th cells to Tregs [1550].
IL-21 is involved in NK cell expansion, function and
regulation. In human bone marrow cultures, IL-21 stim-
ulated NK cell proliferation and maturation [1539]. In
cultured mouse cells, IL-21 increased NK function even
as it depressed growth [919]. IL-21 appears to be able to
modulate cytotoxicity towards certain target cells. In
human NK, IL-21 depressed the NKG2D receptors
while enhancing others [227]. When NK cells were
treated with IL-21, human breast cancer cells coated
with Trastuzumab were more effectively killed by
ADCC [1655].
IL-21 influences development of B cells and Ig pro-
duction. IL-21 induces differentiation of naive and
memory B cells into antibody-secreting plasma cells
[501, 1521]. Depending on the conditions, IL-21 may
increase or decrease growth or induce apoptosis [1540,
1285]. IL-21 regulates Ig class switching. It is a switch
Walter M. Lewko and Robert K. Oldham
factor for IgG1 and IgG3; IL-21 depresses IgE [1522,
1551, 1931, 2196].
IL-21 inhibits dendritic cell activation and maturation
[192, 1902]. It is part of a negative feedback loop
between dendritic cells and T cells in which activated
dendritic cells stimulate T cells, inducing them to pro-
duce cytokines including IL-21 which in turn inhibits
dendritic cells [1902].
IL-21 is involved in the Th system and regulation
of inflammation. IL-21 is produced by Th17 cells and
in an autocrine manner IL-21 sustains expression of
the Th17 transcription factor RORγT and secretion of
IL-17 [2129].
IL-21 has been studied in a number of different exper-
imental tumors. It has been shown to have anticancer
activity related to T cells [338, 427, 577, 1361, 1420], B
cells [1835], NK cells [338, 577, 1420, 1849, 1951,
2101] and NKT cells [1849]. Several of these studies
used IL-21 as an adjuvant in vaccines. Addition of IL-15
to IL-21 improved the response [177, 1420, 2275].
Regulatory T cells may limit vaccine immunogenicity. In
one mouse study, use of IL-21 secreting tumor cells
together with anti-CD25 antibodies to block regulatory T
cells increased cure rate from 17% to 70% [338].
IL-21 has direct effects on the growth of some hema-
tologic malignancies. In B cell chronic lymphocytic leu-
kemia, IL-21 is able to induce apoptosis in CpG-treated
[883] or CD40-stimulated cells [387]. On the other
hand, IL-21 is a growth factor for certain adult T-cell
leukemia [2042] and melanoma cells [198].
IL-21 is in clinical studies. Melanoma and renal cell
carcinoma patients, treated with IL-21 in phase I, tolerated
the cytokine reasonably well and showed evidence of anti-
cancer activity [383, 1187]. IL-21 is also being used to
develop more effective CD8+ T cells for adoptive immu-
notherapy [760]
Interleukin-22
Inflammation
IL-22 is a member of the IL-10 cytokine family. It was
originally referred to as IL-10-related T cell-derived
inducible factor (IL-TIF). Activated Th17, Th1 and Th2
cells produce IL-22 [464, 465, 1123, 1186, 2188, 2212].
IL-22 is also expressed by IL-9 stimulated mast cells
[464] and by activated NK cells [2190]
The IL-22 receptor consists of two chains, IL22Rα
and IL-10Rβ. Each chain separated binds IL-22 but both
chains, associated, are necessary for activity. Signaling
is through STAT 1, 3 and 5 [1012, 2212].
195
There is a soluble IL-22 binding protein, which antago-
nizes IL-22 activity. The IL-22BP has homology with
IL-22R, but it lacks the transmembrane cell-binding region.
In effect, the IL-22BP is a decoy receptor [463, 1013].
IL-22 levels rise in LPS-treated animals. IL-22 appears
to function during inflammation and immune response.
IL-22 mainly targets non-circulating cells involved in
innate immunity including epithelium, keratinocytes,
and hepatocytes. In keratinocytes, IL-22 induces hyper-
plasia, migration, antimicrobial proteins and production
of proinflammatory molecules [178, 2191]. In liver cells,
IL-22 increases the production of acute phase proteins
[466]; it also induces LPS binding protein, which neu-
tralizes this proinflammatory and Th17-inducing micro-
bial product [2189]. IL-22 also has a mild inhibitory
effect on IL-4 production by Th2 cells [2212].
IL-22 levels are elevated and it appears to be involved
in certain inflammatory diseases including rheumatoid
arthritis [847], psoriasis [1186, 2191, 2291], inflam-
matory bowel disease and Crohn’s disease [49]. IL-22
does stimulate inflammatory molecules, and may cause
pathology but it also appears to be involved in resolu-
tion of inflammation and tissue regeneration. In mice,
IL-22 has been shown to ameliorate experimental
autoimmune myocarditis [287] and ulcerative colitis
[1922]. In pancreatic acinar cells, IL-22 induces pan-
creatitis-associated protein [11]. IL-22 has a protective
effect during liver injury. IL-22 induced antiapoptotic
factors in hepatocytes and stimulated growth suggest-
ing a role for IL-22 in liver cell survival and tissue
repair [1611].
With regard to cancer, there is a report that IL-22
inhibited the growth of EMT6 breast cancer in treated
mice by cell cycle arrest [2125].
Interleukin-23
T cell Stimulation, Inflammation, Regulator
of Adaptive and Innate Immunity
IL-23 is composed of two subunits. The p19 subunit is
related to p35 of IL-12. And p40 is identical to p40 of
IL-12. Activated dendritic cells, macrophages and kerati-
nocytes produce IL-23 [1502, 1533, 1575]. The IL-23
receptor is composed of IL-23 Rα and IL-12Rβ1. IL-23
activates Jak-STAT signaling characteristic of the IL-12Rβ1
subunit [1573]. The IL-23 receptor is expressed on DC,
macrophages and T cells [1533].
The effects of IL-23 are similar to but distinct form
IL-12. In mouse cells, IL-23 induces proliferation of mem-
ory T cells (CD4+ CD45RB low). IL-12 does not [1502].
196
IL-23 does not stimulate IFN in mouse T cells. In human T
cells, IL-23, like IL-12, increases IFN-γ production, though
to a lesser degree than IL-12. IL-23 also stimulates growth
in human memory CD45RO T cells [1502].
T cells express ICOS (inducible costimulator) protein,
which is a CD28 family member. ICOS is important for
enhancement of Th and CD8+ T cell responses. In human
T cells, ICOS is increased by IL-23 and IL-12; it is inhib-
ited by IL-4 [2122]. This may provide a powerful way
for IL-23 and IL-12 to amplify T cell responses.
IL-23 induces inflammation [1502]. IL-23 appears to
be involved in encephalomyelitis, arthritis and inflam-
matory bowel disease [298, 363, 1399, 2242, 2279].
Transgenic mice overexpressing IL-23 p19, had severe
multiorgan inflammation, impaired growth, infertility
and early death. Blood contained increased levels of
neutrophils, IL-1α, TNF and acute phase proteins [2132].
TGF-β and IL6 induce Th17 differentiation; IL-23 stim-
ulates and maintains the Th17 cells [10, 1058]. The rea-
son for heightened inflammation and pathology may be
explained by the lack of regulatory IL-10 in IL-23-
differentiated T cells. When induced with TGF-β and
IL6 only, T cells secrete IL-10 in addition to IL-17 and
the other proinflammatory cytokines [1267].
Microbial products induce macrophages and DCs to
secrete IL-23. IL-23 is required for neutrophil produc-
tion. Mice that were p40-deficient lacked neutrophils;
treatment with IL-23 (but not IL-12) restored neutrophil
levels [1841].
In dendritic cells, IL-23 stimulated IL-12 production,
as did IL-12 itself. Further, IL-23 treated DC’s promoted
immunogenic antigen presentation of an otherwise tole-
rogenic peptide, which IL-12 did not do [128].
IL-23 has been shown to be an effective adjuvant in
anticancer vaccines for colon carcinoma [1143, 1795,
2112], melanoma [1143, 1497, 1516], and glioma
[2267]. Prior depletion of regulatory T cells enhanced
antitumor response. IL-23 induced IFNγ; CD8+ T cells
mediated antitumor immunity [1497]. There is also
evidence for non-T cell mediated antitumor immune
effects of IL-23 in human pancreatic and esophageal
tumors in nude mice [1794, 2043].
Some human tumors have elevated IL-23 levels. There
is evidence that the proinflammatory nature of IL-23,
compared with IL-12, may actually be responsible for the
tumor growth and progression in certain cancers [1057].
Interleukin-24
IL-24 was discovered in melanoma cells that had been
treated to induce differention, using IFNβ plus mezerein
(a protein kinase c activator) [889]. IL-24 was originally
Cytokines
called mda-7 (melanoma differentiation antigen-7). Its
counterpart in mice is called FISP [1738, 1790]. In
human cancer, Mda-7 is a tumor suppressor; expression
of the gene in melanoma cells inhibited proliferation
and induced apoptosis [889]. Loss of mda-7 expression
was associated with disease progression [488]. Later,
when the cytokine nature of mda-7 became clear, it was
designated IL-24 [261, 278, 810, 1731]. IL-24 is a mem-
ber of the IL-10 family [261]. The gene is located in the
IL-10 cluster on chromosome 1q31–32 [810]. Activated
T cells and macrophages produce IL-24. It is also
expressed by many tumors [1582, 2187]. Expression is
stimulated by IL-1, IL-2 and several other cytokines but
not by Th2-related cytokines [1582]. IL-24 signals
through two receptors IL-20R1/IL-20R2 and IL-22R1/
IL-20R2 [2104]. IL-24 is a pro-inflammatory cytokine;
it stimulates the production of IL-6 and TNF [261].
Overexpression of MDA-7 in tumor cells, by adenovi-
rus-IL-24 gene transfer, suppresses a variety of cancer
types including breast, cervical, colorectal, glioma, lung,
mesothelioma, ovarian, pancreas, prostate and sarcoma
[242, 277, 278, 810, 1071, 1201, 1712, 1731, 1914, 1915,
1916, 2289]. Susceptible tumors exhibit decreased growth
and increased apoptotic death; IL-24 also inhibits angio-
genesis [295, 1617, 1731]. Radiation resistance may be
reversed by IL-24 treatment [1914, 1915, 2218]. Conversely,
IL-24 resistance, as due to overexpression of antiapoptotic
survival factors, may be reversed by irradiation [1914].
There is much interest in IL-24 for the treatment of
cancer [540, 854, 1070]. IL-24 depresses growth,
induces apoptosis, inhibits angiogenesis and increases
radiosensitivity in a variety of tumors, with little toxic-
ity towards normal cells. There is particular interest in
the use of mda-7 for gene therapy [854]. Adenovirus-IL24
infected cells which produce IL-24 are inhibited;
bystander tumor cells are also affected [1913]. Several
experimental adenovirus constructs have been devel-
oped to optimize the process. INGN 241 has success-
fully completed phase I trials [366, 2010].
Interleukin-25
Proinflammatory; Regulation of Th17
IL-25 is a member of the IL-17 family. It is also referred to
as IL-17E. Th2 cells, eosinophils, mast cells, macrophages
and microglial cells produce IL-25 [844, 915, 985, 1807,
2109]. IL-25 binds IL-17Rh1, the same receptor used by
IL-17B [1073, 1807]. There are high levels of this receptor
in kidney and moderate levels in other organs. IL-25 stimu-
lates production of type 2 cytokines that protect against
intestinal helminths [508, 1518, 1519].
Walter M. Lewko and Robert K. Oldham
Transgenic mice that overproduce IL-25 had increased
expression of Th2 cytokines IL-4, IL-5, IL-10 and
IL-13, G-CSF and the Th1 cytokines IFN-γ and TNF-α
as well. The TG mice had eosinophilia, elevated IgE and
IgG1, and immune cell infiltration in many organs.
These mice also suffered epithelial hyperplasia, hyper-
trophy in several organs and jaundice [1526].
IL25 appears to be involved in several inflammatory
diseases. In mice, overexpression of IL-25 in lung by
adenovirus-IL-25 infection induced airway hyperreac-
tivity with Th2-like responses including IL-4, IL-5,
IL-13 and eotaxin production, eosinophil infiltration,
mucus production [825]. In eosinophils, IL-25
enhanced survival, increased expression of surface
adhesion molecules [301] and stimulated production
of proinflammatory IL-6, IL-8, MIP-1α, and MCP-1
[2195]. Lung tissue from asthmatic patients and atopic
skin lesions show elevated expression of IL-25 and its
receptor [2109]. IL-25 induces catabolic activity in
articular cartilage and may be involved in arthritis
[230]. It appears that during inflammation, Th2 cells
and eosinophils/basophils may collaboratively rein-
force one another to cause the disease state [2109].
While IL-25 seems to cause inflammation, IL-25 also
appears to regulate it. For example, IL-25 controls Th17
inflammation. IL-25 −/− mice are much more sensitive
than normal mice to the debilitating effects of experi-
mental autoimmune encephalitis. In the CNS, micro-
glial cells produce IL-25, which in turn induces IL-13 in
Th2 cells. IL-13 downregulates DC activity and lowers
the cytokines IL-1β and IL-6, which drive Th17 differ-
entiation. In this way, IL-25 secretion in brain may be
responsible for alleviation of damage associated with
IL-17 in experimental encephalitis [985].
Interleukin-26
Epithelial Immune Response
IL-26 is a member of the IL-10 family. It is also
referred to as AK155 [532, 988]. IL-26 was discov-
ered as an overexpressed protein in cultured T cells
transformed by herpesvirus saimiri [792, 988]. The
gene for IL-26 is located on chromosome 12q15. T
cells, in particular activated memory T cells, and NK
cells produce IL-26 [2187]. Secreted IL-26 binds hep-
arin where it is stored in an inactive form until it is
released [792].
The receptor for IL-26 is IL20R1/IL-10R2 [792,
1802]. Epithelium is a major target for IL-26. Treatment
with IL-26 enhances expression of ICAM-1 and secre-
tion of IL-8 and IL-10. By targeting colon epithelial
197
cells and skin keratinocytes, IL-26 may have a role in
mucosal and cutaneous immunology [792].
Interleukin-27
Adaptive and Innate Immune Response;
Regulation of Inflammation/Th17
IL-27 is a member of the IL-12 family. This cytokine has
two subunits, p28 and EBI3 (Epstein-Barr virus induced
gene 3). Activated dendritic cells and monocytes produce
IL-27. Microbial and host factors stimulate production.
The IL-27 receptor is WSX-1. The major targets of IL-27
are CD4+ T cells and NK cells [1713]. In naive T cells,
IL-27 supports proliferation and increases IL-12R levels.
IL-27 stimulates IFNγ production in NK cells and in
activated T cells; in naïve T cells, IL-27 induces T-bet,
IL-12R and Th1 differentiation [747, 2258]. Il-27 regu-
lates inflammation. In mice where the IL-27/IL-27R
system was defective, infection resulted in severe
chronic inflammation. IL-17 inflammatory effects were
exaggerated [115, 1663, 1911, 2176]. IL-27 regulates
inflammation at several levels. It stimulates regulatory
cells to secrete IL-10 [115]. IL-27 also appears to act
directly on Th cells to suppress Th17 cell development
[1911, 2258]. IL-27 also acts directly on activated gran-
ulocytes and macrophages to suppress production of
reactive oxygen [2176].
IL-27 has anticancer activity. Expression of IL-27 in
colon cancer cells has been shown to be immunogenic
[309, 772]. Tumors secreting IL-27 were rejected. Mice
developed tumor specific protective immunity. Interferon
γ, CD8+ T cells and NK cells appeared to be involved
[309, 772]. Poorly immunogenic B16F1 melanoma
cells, engineered to secrete IL-27, were also inhibited.
This growth inhibition was due to NK cells; added IL-12
was synergistic [1497]. IL-27 has also been found to be
antiangiogenic [1811].
Interleukin-28A, Interleukin-28B,
Interleukin-29
Type III Interferons; Antiviral, Antitumor
IL-28A (IFN-λ2), IL-28B (IFN-λ3) and IL-29 (IFN-λ1)
are three recently discovered cytokines in a new class of
interferons, referred to as the lambda or Type III inter-
ferons. They are produced by activated (e.g. viral RNA-
treated) dendritic cells, monocytes and macrophages.
IFNα and TNFα enhance the λ interferon response to
198
viruses by increasing expression of toll-like receptors
and signaling molecules. The λ interferons are related to
the type I interferons and to IL-10.
The receptor used by these cytokines is IL-28Rα-
IL-10Rβ. The λ interferons activate typical IFN-
inducible genes including MHC I and II, oligoadenylate
synthetase and MxA. λ interferons inhibit replication of
certain viruses in vitro and stimulate antiviral immune
response in treated mice [54, 1241, 1288, 1514].
IFN-λs are involved in the exquisite cell interac-
tions involved in immune regulation. Monocytes,
macrophages and dendritic cells produce IFN-λs and
these cells are influenced by them, IFN-λ secretion
increases with LPS-induced dendritic cell differentia-
tion and maturation. Keratinocytes may be activated;
Th1/Th2 ratios and cytotoxic T cell activities may be
enhanced [2190]. And it has also been shown that
IFN-λ-treated dendritic cells induce proliferation of
Treg cells, which eventually suppress effector T cell
activity [1292].
IFN-λs also have anticancer activity. In cultured neu-
roendocrine carcinoma cells, IL-28A and IL-29 inhib-
ited cell proliferation and induced apoptosis (2300).
IFN-λ secreting tumor cells implanted in animals stimu-
lated NK and CD8 T cells immune response, resulting
in decreased growth and metastasis in melanoma, fibro-
sarcoma and colon tumor models [1065, 1466, 1728].
Interleukin-31
Pruritis, Allergic Inflammation
IL-31 is a cytokine that is produced by T cells, in
particular, Th2 cells. IL-31 receptor is composed of two
chains, IL-31 Rα and oncostatin M Rβ. The IL-31R is
found in activated monocytes, epithelial cells and in
sensory neuron bodies associated with skin.
IL-31 appears to be responsible for itch sensation.
Transgenic mice overexpressing IL-31 develop itching
skin lesions analogous to human atopic dermatitis. IL-31
is elevated in mouse and human dermatitis lesions, but
not in human psoriatic skin lesions. Pruritis in a mouse
model appeared to be related to IL-31 secretion [431,
1438, 1859, 1952].
IL-31 is involved in asthma. In lung epithelium, IL-31
induced EGF, VEGF and inflammatory chemokines
[855] and inhibited cell proliferation [291].
IL-31 also has regulatory functions. Mice that are
IL31R−/−have exaggerated Th2 responses; when injected
with schistosome eggs, these mice developed severe
lung inflammation, granulomas and fibrosis. Levels of
Cytokines
IL-4, IL-5 and IL-13 were elevated. IL-31 signaling
appears to limit the type 2 inflammatory process [1558].
Interleukin-32
Inflammation
IL-32 was discovered as the transcript NK4 in activated
immune cells. Many types of cells produce IL-33 when
exposed to inflammatory conditions. IL-33 has no
homology with other known cytokines. IL-32 has six
isoforms. Activated NK cells, T cells, and IFNγ-treated
epithelium and monocytes produce IL-32 [438, 900,
964]. IL-32 induces inflammation by way of p38 MAP
kinase and NF-κB signaling [1444].
IL-32 induces monocytes to produce TNFα, IL-8,
PGE2 and other inflammatory factors. It signals
through NFκB and MAP kinase [692]. IL-32 syner-
gizes with the microbe sensing NOD receptor system
for the induction of IL-1 and IL-6 by muramyl dipep-
tide. But IL-32 had no such stimulating effect on the
TLR system [1443].
In addition to inflammation, IL-32 also influences
specific immunity. IL-32 induced differentiation to
macrophages from blood monocytes and, interestingly,
from dendritic cells [1444]. Apoptotic T cells were high
in IL-32. Forced expression of IL-32 induced apopto-
sis. This suggested that IL-32 is involved in AICD. This
effect of IL-32 on T cells may be by a non-secreted,
intracrine mechanism [630]
There is evidence IL-32 is involved in several dis-
eases including rheumatoid arthritis, psoriasis, Crohn’s
disease and chronic obstructive pulmonary disease
[438]. In tissue biopsies from patients with rheumatoid
arthritis compared with osteoarthritis, IL-32 was
increased and levels correlated with disease severity
[900]. Of 171 genes tested, four genes were overex-
pressed in RA fibroblasts, and the gene for IL-32 was
most pronounced [229].
Interleukin-33
Inflammation, Allergy
IL-33 is an IL-1 family member. It is produced by
vascular endothelium and certain fibroblasts. IL-31 is
synthesized as a precursor and cleaved by caspase-1,
in a manner similar to IL-1 processing.
The IL-33 receptor is the IL-1 receptor-related protein
ST2. It signals through NF-κB and MAP kinases.
Walter M. Lewko and Robert K. Oldham
IL-33 enhances expression of Th2 cytokines IL-4,
IL-5 and IL-13. In mast cells, IL-33 stimulates matura-
tion, survival and the production of IL-8 and Th2 cytok-
ines. In treated mice, IL-33 induces severe mucosal
inflammation [28, 434, 839, 1750]. IL-33 and Th2
cytokines were required for expulsion of intestinal
nematodes [821]. In mice, Il-33 has been shown to pro-
tect against the development of atherosclerosis [1310].
In endothelial cells, IL-33 appears to also have an
intracellular role, apart from its receptor. It translocates
into the nucleus where it binds chromatin and as a
nuclear protein it represses transcription [247].
Interleukin-35
Treg Cytokine, Regulation of Th17
IL-35 is a heterodimeric cytokine that is composed of
IL-12α and IL-27β chains. nTreg cells (CD4+ CD25+
Foxp3+) produce IL-35. The secretion of this cytokine
is upregulated when Treg cells are activated by contact
with effector T cells.
IL-35 appears to have a major role in regulation for
IL-27β−/− Treg cells show loss of function. For example,
they do not control homeostatic proliferation as wild
type cells do; they do not cure inflammatory bowel
disease. Recombinant IL-35 has been shown to induce
Treg cell proliferation and production of IL-10. It sup-
pressed the expansion of CD4+ CD25−effector T cells
and it inhibited differentiation and IL-17 production by
Th17 cells. And in treated mice, rIL-35 decreased the
severity of collagen-induced arthritis [335, 1451].
4-1BB Iigand
Costimulation of T Cells, T Cell Memory
4-1BB ligand (4-1BBL) is a 30 kDa transmembrane
glycoprotein. T cells, stromal cells of thymus and spleen,
and antigen presenting cells including monocytes, B
cells and dendritic cells express this ligand [23, 389,
639, 1059, 1584]. The receptor is 4-1BB (CD137). It is
a 30–35 kDa transmembrane protein, which is related to
the TNFR [1585, 1761, 1763]. The human gene is
located on chromosome 1p36 in a cluster of related
genes [1763]. There is a soluble form of this receptor,
the product of alternative mRNA splicing [1787]. 4-1BB
was originally discovered as an activation-induced
antigen on T cells [23, 639, 1044, 1045, 1765]. 4-1BB is
also produced by B cells, dendritic cells, monocytes and
199
NK cells [1761, 2160]. It is also found on epithelial,
endothelial, and neural cells [184, 1622].
In T cells, the engagement of 4-1BB induces prolif-
eration, cytokine secretion, cell survival and the sup-
pression of AICD. 4-1BB stimulates memory response
to second antigen challenges [147, 238, 317, 389, 639,
827, 1585, 1764, 1948]. 4-1BB is one of several costim-
ulatory signaling systems, which may supplement,
modify and, to a degree, replace costimulation through
the B7/CD28 system [213, 389, 390, 391, 715, 828,
833]. 4-1BB is stimulatory but it is also regulatory; it
preferentially induces proliferation of CD8 T cells over
CD4 T cells [1818]. 4-1BB signaling tends to suppress
Th2 responses and humoral immunity [789, 1319]. In
this way, 4-1BB signaling can ameliorate Th2-related
allergy and autoimmunity [312, 1927].
In NK and NKT cells, stimulation of 4-1BB induces
activation and the secretion of cytokines. Mice lacking
4-1BB have depressed NK numbers and activity [1289,
2078]. NK cells are well known as cytotoxic effector
cells but they also appear to help CTL differentiation.
When stimulated with antibodies to 4-1BB, NK cells
produce IL-2 and IFNγ, which increase the growth and
cytotoxicity of antigen-specific T cells [1289, 2160].
4-1BB ligation likewise stimulates dendritic cells to
secrete IL-6 and IL-12, which enhance antigen-specific
T cell response [2159]. In this way 4-1BB costimulates
T cells directly and indirectly by its cytokine stimulat-
ing effects in several types of cells.
On binding its receptor, 4-1BB ligand is capable of
reverse signaling. In T cells, reverse signaling sup-
presses immune response and induces apoptosis [1764].
In monocytes, reverse signaling induces IL-6, IL-8,
TNF-α and ICAM-1 but inhibits the production of
IL-10 [1059].
Several studies show that 4-1BB stimulation serves
as an adjuvant in vaccines against cancer [693, 862,
1029, 1116, 1233, 1290, 1312, 1333, 1718, 2138, 2211,
2215, 2280] and viruses [146, 390, 1958]. For example,
in mice with melanoma, survival was shorter in mice
that lacked 4-1BB compared to normal 4-1BB+ mice.
Treatment of normal mice with agonistic antibodies to
4-1BB further increased survival [906]. Agonistic anti-
bodies to 4-1BB enhanced the efficacy of a tumor lysate-
dendritic cell based vaccination [1866].
4-1BB ligand has been detected in several carcinoma
cells. These tumors cells costimulate T cells and IFNγ
production. Reverse signaling stimulated tumor cell IL-8
production and depressed growth [1717]. Several mouse
studies showed the benefit of injected mabs to 4-1BB
suggesting it may be another receptor for which antibodies
may be useful in the treatment of human cancer.
200
CD27 ligand
Costimulation of T and B Cells; B Cell
Differention; Apoptosis
CD27 ligand (CD27L, CD70) is a cell surface glyco-
protein. It is a member of the TNF family [638].
Activated B cells [1093], T cells [8, 1505], NK cells
[2226], dendritic cells [224] and certain B cell malig-
nancies [1619, 928] produce CD-27L. It is expressed
preferentially on CD45RO memory CD4 T cells [8]. In
dendritic cells, CD27L is induced by CD40L or by TLR
stimulation [224]. Upon binding its receptor, CD27L
may back-signal to increase cytotoxicity in γδ T cells
[1505], antibody production in B cells [1094] and
growth of certain B cell malignancies [928].
CD27 is the receptor for CD27L [187, 638, 763].
CD27 is a 50 kDa transmembrane glycoprotein. It is a
member of the TNFR family [233, 762, 2060]. CD27 is
expressed on naive and memory T cells [8, 155, 1924,
2060], germinal center and circulating memory B cells
[984, 1253, 2015], B cell leukemias [2061] and on NK
cells [1923]. CD27 is considered a memory marker for T
cells [2015] and B cells [985]. Activation of T cells
markedly increases CD27 and induces the release of a
soluble form of the receptor, which can be found in blood
and urine. The soluble form appears to be a proteolytic
product [761, 762, 1146]. Elevated levels are character-
istic of infection, autoimmunity and B cell malignancies.
Soluble recombinant CD27 is an antagonist [87].
The CD27–CD27L system is costimulatory for the
induction of T cells. Stimulating antibodies to CD27
induce T cell activation, proliferation and cytotoxicity
[66, 638, 992, 1924, 2060]. Naive (CD45+) T cells and
CD45RO memory cells respond to CD27 ligation [8,
992]. In NK cells, CD27 expression is upregulated by
IL-2. CD27 ligation stimulates NK activation and
cytotoxicity [1923].
For memory B cells, CD27 appears to be part of the
switch between memory and activated plasma cells.
engagement inhibits differentiation of B cells into
Ig-secreting plasma cells [1616]. It favors the develop-
ment of memory B cells with high antigen affinity
[1615].
CD27 engagement induces apoptosis in activated T and
B cells. The cytoplasmic segment of CD27 is rather short.
Unlike TNF and Fas, CD27 does not have a death domain.
However, this receptor does associate with a cytoplasmic
protein, Siva, which has a death domain segment and Siva
mediates CD27-induced apoptosis [1591].
In the treatment of cancer, there is interest in the
CD27 system for its ability to stimulate T, B, and NK
Cytokines
cells. When vaccine tumor cells are transfected with
CD27L, there is enhanced immune response [348, 1153,
1453]. CD27 ligation supports the clonal outgrowth of
tumor-specific T cells [638, 762]. Further, the CD27
system appears to be important in the production of T
cells for adoptive cell immunotherapy [811, 1589]. IL-2
induces growth of tumor-specific T cells and part of its
stimulatory effect is due to upregulation of T cell
CD27L. Blocking antibodies to CD27L depressed
IL-2-induced growth. It appears that CD27 signaling
mediates IL-2-induced T cell proliferation [811, 1589].
It should be noted, CD27L [928, 1093] and CD27
[2061] are found on B cell malignancies and these mol-
ecules can have a role in tumor progression. Also,
CD27L has been observed on certain non-hematological
tumors including renal cell carcinoma [429] and brain
tumors [732, 2177]. Tumor cell-CD27L causes apopto-
sis in T and B cells and might have a role in immune
escape [429, 2177].
CD30 ligand
T Cell Costimulation, Selection; Apoptosis;
Regulation of Ig Class Switching
CD30 ligand (CD153) is a 40 kDa transmembrane
glycoprotein in the TNF family. It is found on mac-
rophages [1840], B cells [598, 2259, 2260], CD4 and
CD8 T cells [2259], megakaryocytes, neutrophils, eryth-
rocyte precursors [598], eosinophils [1570], mast cells
[541] and many though not all leukemias [599]. On
binding its receptor, CD30L has the capacity to signal
back into its parental cell [2165]. Reverse signaling in
neutrophils stimulates an oxidative burst and IL-8 pro-
duction. In T cells, reverse signaling stimulates prolif-
eration and IL-6 production [2165].
CD30 is the receptor for CD30L. It is a 105–120 kDa
transmembrane protein and a member of the TNFR
family [473, 605, 1840]. The intracellular segment con-
tains two binding sites for TNFR-associated factors
(TRAFs 1, 2, 3) which function in signaling [601]. The
CD30 antigen was originally discovered as Ki-1, a
marker for Reed Sternberg cells in Hodgkin’s lymphoma
[1759]. CD30 is found on activated T, B, and NK cells
[35, 186, 489, 2088]. CD30 expression is transient; in T
cells it depends on activation and CD28 signaling [621].
CD30 is a marker for activated, cytokine secreting
helper T cells (Th0, Th1 and Th2) [704]. In particular,
CD30 expression is associated with Th2 response
[1255]; CD30 is stimulated by IL-4 and inhibited by
IFN-γ [621]. CD30 is expressed on subsets of activated
Walter M. Lewko and Robert K. Oldham
CD45RO+ memory T cells [35]. CD30+ cells produced
IFN-γ and IL-5 while CD30− cells produced more IL-2.
CD30+ T cells showed enhanced ability to provide help
for B cells producing Ig [35].
Soluble CD30 is shed from cells and found in blood.
CD30 shedding is increased by activation of CD30 and
by treating cells with PMA. The protease responsible
is TACE, the enzyme that processes and releases
TNF-α [711]. Elevated levels of sCD30 are found in
the blood of patients with immune disorders such as
lupus erythematosis and rheumatoid arthritis and in
patients with colon cancer and Hodgkin’s disease. In
Hodgkin’s lymphoma patients, sCD30 correlates with
poor prognosis [2256]. Changes in levels may be used
to monitor a disease [711, 868, 904].
The specific biological effects of the CD30L–CD30
system are difficult to discern. Expression of the receptor
is transient and certain effects appear to be opposed.
CD30 engagement is costimulatory for the growth and
differentiation of specific T cells [621, 683, 1840]. But
the CD30L–CD30 system also inhibits function and
induces apoptosis [1407, 1840]. CD30-deficient mice,
have impaired negative selection of thymocytes sug-
gesting a fault in apoptosis [36]. CD30 transgenic mice
overexpressing CD30 in the thymus showed enhanced
thymic negative selection [303]. Moreover, CD30
signaling induced TCR-dependent apoptosis in a T cell
hybridoma cell line [186]. Lack of CD30 expression is
also associated with the development of autoimmune T
cells in experimental diabetes [280, 729, 1041]. On the
other hand, in NOD mice that are genetically prone to
diabetes, neutralizing antibodies to CD30L interfered
with the development of the disease [280]. In a cyto-
toxic large granular lymphocyte cell line, CD30 activa-
tion depressed effector functions including FasL,
perforin and granzyme production while inducing the
effector cell’s own apoptotic program [1407]. CD30
thus appears be able to coordinate gene expression in
NK and T cells to control effector selection, growth and
differentiation and then downregulate cytotoxic func-
tions, inhibit growth and induce effector cell death.
T cells influence B cell antibody production and
class switching by way of the CD30L–CD30 and
CD40L–CD40 systems. CD40 ligation stimulates Ig
class switching. CD30L reverse signaling inhibits class
switching at the level of CD40 signaling [273, 274].
CD30 is also required for the development of B cell
memory responses [593].
Mast cells expressing CD30L appear to have a role in
Hodgkin’s disease. Mast cells were the predominant
CD30L-positive cell type in tumors and these cells were
capable of stimulating growth of Hodgkin and Reed
201
Sternberg cells [1336]. CD30L also appears to have a
role in cutaneous inflammation. In atopic dermatitis and
psoriatic skin lesions, mast cells were the predominant
CD30L-positive cell type. On binding CD30, back
signaling in these mast cells induced the secretion of
IL-8 and other inflammatory chemokines. This mast cell
activation occurred in an IgE-independent manner [539].
Eosinophils express CD30L and they stimulate the
growth of co-cultured CD30-Hodgkin’s lymphoma cells
[1570]. Eosinophils also express CD30; treatment with
agonistic Mabs to CD30 induced eosinophil apoptosis
[142].
A relatively small subset of normal lymphocytes
express CD30 but it is overexpressed in several diseases
making it an attractive target for therapy. In T cells CD30
is increased in autoimmune disease and in allergy [614].
The CD30 antigen is also found on virally transformed
and HIV-infected lymphocytes [729]. It is highly expressed
in Hodgkin’s disease, anaplastic large cell lymphoma
and certain other malignant lymphoid disorders [1759].
In many lymphoid cell lines, CD30 ligation induces
apoptosis but there are cases in which CD30 has no effect
or stimulates growth [683, 1081, 1185]. Where CD30
ligation induces apoptosis in Hodgkin’s lymphoma cells,
the presence of soluble CD30 was found to interfere. This
may explain why sCD30 correlates with poor prognosis
in these patients [2259]. Antibodies are being developed
which bind specifically to cellular CD30 epitopes that are
lacking on the shed receptor [1411].
In patients with Hodgkin’s disease, bispecific
(Anti-CD16/CD30) antibodies have been tested with
some success in a phase I/II trial. This construct was
designed to activate NK cells and target the tumor cells
[721]. SGN-30 is a humanized monoclonal antibody
that inhibits Hodgkin’s disease tumors in nude mice
[2088]; it is currently undergoing clinical trials.
CD40 ligand
Costimulatory, Proinflammatory;
B, and T Cell Stimulation
CD40 ligand (CD154) is a 35 kDa membrane protein. It
is expressed by CD4 T cells [1691] and by CD8 cells
[1707]. The levels of this powerful cytokine are rather
well controlled. It is produced during CD4 cell activa-
tion and down regulated upon binding its receptor [109,
260, 2058, 2240, 2241]. A soluble form of CD40L is
released from CD4 T cells upon activation [653].
Soluble CD40L is biologically active and will substitute
for cell-bound CD40L [955, 1055].
202
CD40 is the receptor for CD40L. It is a 50 kDa mem-
brane protein, constitutively expressed, mainly on B
cells [266], and to a lesser extent on dendritic cells,
macrophages [21], T cells [511], fibroblasts [2241], thy-
mic epithelial cells [585] and endothelial cells [786].
CD40 is also present on some tumor cells. The CD40/
CD40L system stimulates immune response, inflamma-
tion and apoptosis depending on the cell type and the
conditions.
In B cells, CD40 signaling causes activation, matura-
tion and survival [400, 946, 1294]. The triggering of B
cells through CD40 is critical for the induction of Ig
production [72]. In the absence of CD40, B cells were
tolerogenic [221, 952]. Binding Th2 cell CD40L induces
reverse signaling and the secretion of IL-4 that in turn
stimulates B cells [167].
The CD40 system provides help during CTL devel-
opment. CD40L on CD4 T cell interacts with CD40 on
AP cells (DC, B cell, virus-cell or tumor cell) to induce
antigen processing, presentation and cytokine secretion
[136, 756, 1645]. CD40 engagement also increases
DC survival, costimulatory molecules B7.1 and B7.2,
ICAM-1, and cytokines such as IL-12, which stimulate
T cells and NK cells [136, 159, 263, 267, 1645, 1753].
The CD40L–CD40 system is necessary for a good
immune response. CD40L-deficient mice were defective
in antiviral immunity and memory CTL response [181].
Mice defective in the CD40 system were very suscepti-
ble to infection with Leishmania [236, 912, 1861].
Treating mice with stimulatory anti-CD40 Mabs pro-
duced a good T cell immune response to an otherwise
weak Listeria monocytogenes immunogen [1658]. CD40
activation also appears to be required for allograft rejec-
tion; blockade of the CD40 system generally prevents
rejection and induces tolerance [470, 651, 863, 1064].
CD40L is a proinflammatory cytokine. Engagement
of CD40 induces the production of inflammatory cytok-
ines in fibroblasts and macrophages [21, 2241], and the
upregulation of surface adhesion molecules such as
ICAM and VCAM on fibroblasts and endothelial cells
[786, 1062, 2241]. Treating animals with soluble CD40L
intranasally induced lung inflammation with infiltrating
neutrophils and macrophages. IFN-γ intensifies CD40L-
induced inflammation [2163]. Platelets contain both
CD40 and its ligand and may be involved in CD40-
related inflammation and immune responses [377, 953].
CD40 on lung fibroblasts appears to have a role in
pulmonary fibrosis [2241].
CD40–CD40L interaction is also required for a
number of experimental autoimmune diseases. In
animal models for myasthenia gravis [848], multiple
sclerosis [615, 667], arthritis [471] and diabetes [105],
Cytokines
CD40 activation exacerbated the disease while block-
ing CD40 depressed it. Development of autoimmunity
is complex and sometimes paradoxical. The absence of
CD40–CD40L interactions might induce autoimmune
disease [1034]. Patients with rare X-linked mutations
in the CD40L gene develop hyper-IgM syndrome,
show defects in thymus-dependent immune responses,
antibody production and germinal center formation.
These patients suffer recurrent infection, increased
cancer and autoimmune disease [441, 727].
The CD40 system regulates apoptosis, positively or
negatively, depending on the target cells. In dendritic
cells, for example, CD40 ligation on immature DCs
induced survival and maturation, whereas CD40 liga-
tion on LPS-matured DCs induced apoptosis [405].
Where CD40 induces apoptosis, it up-regulates Fas.
Where CD40 inhibits apoptosis, it increases levels of
the survival protein bcl-X [858, 2113].
The CD40–CD40L system has some remarkable
antitumor effects. In normal B cells, CD40 ligation
stimulated growth. In neoplastic B cells CD40 ligation
tended to inhibit growth and induce apoptosis [575].
Certain solid tumors express CD40. When this is the
case, CD40L might act directly on the tumor cells to
induce death by apoptosis [756, 765, 2086]. CD40 liga-
tion stimulates chemokine release. In human cervical
carcinoma cells, CD40L induced macrophage chemoat-
tractant protein-1 (MCP-1) and IFN-γ-inducible protein
10 (IP-10). The addition of IFN-γ had a synergistic
effect [32]. These chemokines are involved in the
recruitment of effector T cells. IP-10 is also angiostatic
[1788]. As mentioned, CD40 ligation activates antigen-
presenting cells and secretion of IL-12 by DCs. Good
cytotoxic T cell responses were observed in animals
treated with stimulating anti-CD40 mabs, or immu-
nized with tumor cells engineered to produce CD40L
[430, 433, 446, 567, 695, 740, 1415, 1863]. CD40
ligation enhanced the outgrowth and longevity of T
cells [1256]. CD40–CD40L interactions were required
for protective anticancer immunity by vaccination
[1196, 695]. When CD40 signaling was blocked, no
systemic antitumor immunity developed [1195].
CD40 signaling stimulates NK activity and related
antitumor effects [245, 1415, 2038]. P815 mastocytoma
cells engineered to produce CD40L were promptly
rejected when injected into mice. NK cells mediated the
anticancer effect. Production of IL-12 was required but
effector CTL cells were not [1415]. In another study,
mice were treated with stimulatory anti-CD40 mabs to
activate signaling [2038]. Three different types of
tumors were tested; growth and metastasis were inhibited.
NK cells were required. The effect of CD40 was not
Walter M. Lewko and Robert K. Oldham
direct but probably through APCs that produced NK cell
stimulating IL-12 [2038].
A phase I trial of rhuCD40L in patients with solid
tumors and NHL, encouraging preliminary results were
seen including one long-term complete response [2087].
In a phase I vaccine trial, patients with CLL were treated
with CD40L-producing autologous tumor cells.
Leukemia cell counts decreased and lymph node size
was reduced again suggesting that CD40L may be thera-
peutically useful [2154].
Chemokines
Cell Activation, Migration,
and Recruitment; Inflammation;
Angiogenesis
Chemokines include a rather large number of proteins,
which have a variety of activities but function primarily
as chemoattractants and activators of leukocytes [1656].
They induce cell migration, recruitment and infiltration
into sites of inflammation, homing to sites of immune
cell differentiation and passage across cellular barriers.
They are involved in T cell activation and the polariza-
tion of Th1/Th2 cells [687, 1656]. Chemokines may
also act on non-hematopoietic cells. They facilitate cell
movement during inflammation, wound repair and
morphogenesis. Chemokines are also involved in
pathology, tumor growth and metastasis. HIV viruses
use certain chemokine receptors (CXCR4) for entry
into cells [524].
Chemokines are relatively small 7–14 kDa proteins.
Most chemokines contain four conserved cysteines in
the amino terminal region. Chemokines are classified
based on the sequence in the region of these cysteines.
1. CXC chemokines (α chemokines): the first two
conserved cysteines are separated by a single non-
conserved amino acid. CXC chemokines may be
classified further based on the presence or absence
of Glu-Leu-Arg (ELR) just preceding the first con-
served cysteine. There are at least five receptors for
CXC chemokines. These receptors are members of
the rhodopsin-like, seven transmembrane domain
receptor family [1147]. As examples of chemokine–
receptor interactions, CXCR1 and CXCR2 bind
IL-8 and GCP-2 (granulocyte chemotactic pro-
tein-2) [5]. CXCR3 binds IP-10 (interferon-induced
protein-10) and Mig (monokine induced by γ inter-
feron) [894]. SDF-1 (Stromal derived factor-1), a
pre-B-cell growth factor as well as a chemokine,
203
appears to be the only ligand for CXCR4 [1409].
ELR CXC chemokines tend to activate migration in
neutrophils, the non-ELR CXC chemokines tend to
activate lymphocytes.
2. CC chemokines (β chemokines): the first two con-
served cysteines are together. There are nine known
receptors for these chemokines termed CCR1, CCR2,
etc. Well studied CC chemokines include MIP-1α
(macrophage inflammatory protein-1α), MIP-1β,
MCP (Monocyte chemoattractant proteins) 1, 2, 3,
RANTES and eotaxin. CC chemokines are chemot-
actic for most types of leukocytes (T, B, DC, NK,
eosinophils, basophils, DC), but not for neutrophils.
[1656].
3. C chemokines: two of the four conserved cysteines
are missing, the first and the third. Lymphotactin is a
C chemokine. NK cells produce it. Lymphotactin is
specifically attractive for lymphocytes [949].
4. CX3C chemokines: the first two conserved cysteines
have three intervening non-conserved amino acids.
There is a specific receptor Cx3CR-1 for these cy-
tokines [849]. Fractalkine (neurotactin) is a Cx3C
chemokine. It has two active forms; one is a mem-
brane bound protein. The other is a soluble protein
released from the membrane by the protease TACE
[2030]. Fractalkine is expressed in neurons, mi-
croglial cells, fibroblasts and endothelial cells. It is
upregulated during inflammation. Fractalkine is a
chemoattractant for T cells, NK cells and monocytes
and it induces adhesion [119, 1530]. In the brain
fractalkine appears to be antiapoptotic, a survival
factor for microglial cells [171].
ELR+ CXC chemokines are angiogenic; they stimulate
endothelial cell migration and tube formation; these
include IL-8, GRO (growth-related oncogene)-α,
GRO-β, GRO-γ, GCP-2 and ENA-78 (epithelial neutro-
phil-activating protein 78) [1906]. The receptor CXCR2
is responsible for ELR+ angiogenesis [5]. Non-ELR
CXC chemokines tend to be inhibitors of angiogenesis.
IP-10 and Mig are angiostatic [1204, 1906]. Both cytok-
ines bind receptor CXCR3 [2142]. The anticancer activ-
ities of several cytokines may involve the downstream
production of angiostatic chemokines such as IP-10 and
Mig [32, 52, 222, 1788].
Ephrins and ephs, their receptors, are also involved
in cell migration. They act as chemodirectants during
morphogenesis, inflammation and immune response.
Ephrins and ephs are found on a wide variety of cells.
In contrast with the chemokines, which mainly stimu-
late migration and adhesion, the ephrins are chemore-
pulsive agents which tend to be transiently expressed
204
at times when the movement of cells needs to be
redirected.
Chemokines are involved in many inflammatory dis-
eases and in the development of cancer. Cell movement
and angiogenesis are hallmarks of malignancy. Elevated
levels of angiogenic chemokines are associated with
tumor progression and metastasis [63, 976, 1167]. IL-8
production has been correlated with tumor vascularity
and progression [976, 2250]. Chemokines are of interest
in the design of therapeutic agents, including antitumor
vaccines. For example, lymphotactin is produced by
NK cells and attracts T cells [949]. A vaccine using
tumor cells co-transfected to produce lymphotactin and
IL-2, induced potent antitumor immunity in mice [432].
In another study, a vaccine with Lewis lung carcinoma
antigen-pulsed dendritic cells, transfected to produce
lymphotactin, induced a much more potent antitumor
response than a control vaccine lacking lymphotactin.
In mice with established tumors, vaccination with the
antigen-pulsed lymphotactin-DCs inhibited metastasis.
Only one relatively small 104 DC dose was required to
immunize the mice [241].
Fas ligand
Inducer of Apoptosis
Fas ligand (FasL) is a transmembrane protein in the
TNF family. It is expressed on activated lymphocytes
and on a variety of other cells including certain tumor
cells. In T cells, FasL is induced by TCR activation. Its
production is inhibited by retinoids and glucocorticoids
[2107, 2228], by inhibitors of protein kinase C and
calcium mobilization [2107], and by cyclosporin [217].
Fas is the receptor for FasL [1410, 1839, 1976]. Fas
(also known as Apo 1, CD95) is expressed on lympho-
cytes and many different types of cells including tumor
cells. There are soluble forms of Fas; they are gener-
ated by alternative mRNA splicing [253]. The FasL–
Fas system is responsible for the induction and
regulation of programmed cell death in the peripheral
immune system. Apoptosis is a way to regulate and ter-
minate an ongoing immune response, or to eliminate
useless or potentially damaging immune cells [1019,
1184].The importance of the FasL/Fas system in the
regulation of lymphocytes can be seen in the autoim-
mune diseases and lymphoproliferative disorders in
mice, associated with mutations in the FasL (gld) and
Fas (lpr) genes [330, 1410, 1698, 1976].
Various types of cancer and tumor cell lines express FasL
[135, 700, 1452, 1469, 1703, 1814, 1897]. The Fas–FasL
Cytokines
system is involved in activation-induced cell death in
lymphocytes. The tumor cells that express FasL are gener-
ally negative for Fas and tend to be resistant to apoptosis
[700, 1468, 1469]. The expression of FasL may explain the
general phenomenon of immune resistance (immune
privilege), by which certain normal tissues (eye chamber,
testis) and tumors are able to evade immune effector cells.
Cells that express surface FasL may induce apoptotic death
in lymphocytes [670, 700]. In tumor biology this idea has
been referred to as “FasL counterattack.” in which infiltrating
lymphocytes may be killed upon contact with FasL+ tumor
cells [135, 1468, 1469]. This theory is interesting but con-
troversial. Certain reports show that tumors expressing FasL
may be rejected [61, 124, 519, 1778]. In transplantation
research, some have tried to engineer graft tissues with
FASL to make them less immunogenic but with they have
met with variable success [31, 916].
Tumor cells may contain Fas. When this is the case,
tumor development and therapy may be influenced by
Fas-induced apoptosis. Dendritic cells express FASL, as
well as other proapoptotic cytokines TNF, LTα1β2,
TRAIL, and may kill tumor cells directly [1166]. CTLs
express FASL. Irradiation was found to increase Fas on
tumor cells and prior irradiation enhanced apoptotic
response to adoptive CTL therapy [281]. Vitamin E
stimulated Fas and Fas ligand levels in certain cultured
breast cancer cells and induced death by apoptosis
[2037]. In some leukemias and solid tumors, drugs such
as adriamycin and methotrexate induce Fas and FasL
causing death [566].
Capillary leak syndrome involves endothelial cell
damage. It is associated with chronic infection and it is
a dose-limiting toxicity in the use of IL-2 and certain
other cytokines to treat cancer patients. Capillary dam-
age appears to be due to cytotoxic lymphocytes [375].
Studies using perforin knockout mice and mice with
defective FasL and Fas genes showed that both perforin
and Fas-FasL were responsible [1612].
FLT-3 ligand
Stimulator of Early Hematopoietic
Stem Cells and Dendritic Cells
Flt-3 ligand is a membrane-bound cytokine. In structure
and activity, it is related to macrophage colony stimulating
factor and stem cell factor [710, 1175]. The human
gene is located on chromosome 19 [1176, 1265]. It is
expressed by a wide variety of hematopoietic and non-
hematopoietic tissues [710, 1176]. Flt-3 ligand is produced
in both membrane bound and soluble forms, both active.
Walter M. Lewko and Robert K. Oldham
Soluble forms are produced by proteolysis [1177] or
alternative mRNA splicing [1176, 1265]. Soluble Flt-3
ligand is elevated in serum of patients with certain ane-
mias [1179]. In patients on myelosuppressive chemo-
therapy, elevated plasma Flt-3 ligand is prognostic for
poor recovery from thrombocytopenia [170].
Flt-3 (fms-like tyrosine kinase-3, CD135) is the
receptor for Flt-3 ligand. It is a transmembrane protein.
Flt-3 was named for its similarity to fms, the receptor
for M-CSF [1679, 1680]. Flt-3 is also referred to as flk-2
(fetal liver kinase 2) [1251] and Stk-1 (stem cell tyrosine
kinase-1) [1837]. It is a 158 kDa glycoprotein. The
human gene is on chromosome 13q12 [1680]. This
receptor is expressed mainly by hematopoietic stem
cells and progenitor cells [1248]; it is low or missing on
most mature cells [370, 1251, 1286, 1837]. Flt-3 ligand
binds and activates the receptor. Dimerization of Flt-3
ligand appears to be required for receptor dimerization
and signaling [710, 1177, 1178].
Flt-3 ligand has a role in the proliferation, survival and
differentiation of hematopoietic progenitor cells [1269].
Mice lacking Flt-3 ligand have reduced numbers of
myeloid progenitors, B-lymphoid progenitors, NK cells,
and dendritic cells [1272]. Mice lacking Flt-3 appear
healthy. However, their stem cells are deficient in their
ability to reconstitute lymphoid and myeloid cells when
transplanted into irradiated mice [1192]. Mice injected
sc with Flt-3 ligand show a remarkable increase in
hematopoietic precursors [193, 194]. Flt-3 ligand stimu-
lates the growth and survival of early progenitor cells
[909, 1298, 1448]. GM-CSF, IL-3, SCF, IL-11 and IL-12
are synergistic [909, 1298] while TNF-α and TGFβ are
inhibitory [2068]. Flt3 ligand acts together with IL-7 or
SCF to stimulate B cell development from progenitors in
bone marrow and thymus [768, 823, 1272, 1424, 2067].
Flt-3 ligand has a remarkable influence on the devel-
opment of dendritic cells. Mice treated with Flt-3 ligand
produce greater numbers of dendritic cells [452, 1224,
1819, 1890]. Spleen cells with markers for dendritic
cells went from less than 1% of total in controls to 20%
in Flt-3 treated mice [1224]. DCs from treated mice
expressed more costimulatory CD80 and CD86 [1890].
Patients treated with Flt-3 ligand, also showed a remark-
able increase in dendritic cells [1221]. Flt-3 ligand and
stem cell factor cooperate to induce recruitment and
expansion of CD34+ CD14+ dendritic cell precursors
(CFU-DC). Flt-3 ligand and SCF are not required for
the differentiation step. Other cytokines, including
GM-CSF, IL-4, TGF-β and TNF-α stimulate maturation
of the expanded precursors [367].
Flt-3 ligand also stimulates the production of NK
cells [194]. Treatment of mice increased the number of
205
NK cells in blood, bone marrow, spleen and liver [1555,
1801]. It appears to do this by stimulating proliferation
of pro-NK cells and the production of mature, non-activated
NK. Flt-3 ligand increases NK cell responsiveness. IL-2
rapidly induces proliferation, cytotoxicity and LAK
activity in Flt3 ligand-treated cells [1801].
Flt3 ligand influences certain non-hematopoietic
cells. Osteoclasts respond to M-CSF with increased
differentiation; Flt-3 ligand can substitute for M-CSF
[1069]. Neural stem cells express Flt-3; Flt-3 ligand
inhibited EGF and FGF-2 induced proliferation. Neurons
are also Flt-3+. In culture, Flt-3 ligand synergized with
nerve growth factor to promote neuron survival [195].
Flt-3 is expressed by many myeloid and lymphocytic
leukemias [157, 370, 1286, 453, 1271, 1678]. When cells
are positive for the receptor, flt-3 ligand may stimulate
growth in culture. In AML, 20–25% of adult patients
have a mutation in flt3. This mutation is an internal
tandem duplication. The presence of this mutation is an
adverse prognostic factor. This is a gain of function muta-
tion that confers growth advantage on the cells [2151];
signaling is constitutively activated [725]. Selective
tyrosine kinase inhibitors are being tested in patients with
flt3+ tumors. In a trial on elderly patients with AML,
responses were observed for lestaurtinib (CEP701) in
both mutated and wild type Flt3 tumors [989].
Tumor bearing mice treated with Flt3 ligand generate
antitumor responses. Several preclinical studies have
shown this in lymphoma, leukemia, and several types of
solid tumors [283, 320, 499, 1183, 2097]. Soluble and
membrane bound forms of Flt-3L were effective.
Dendritic cells and CD8 T cells appeared to be respon-
sible for antitumor activity though NK cells might also
be involved [1546, 1555, 1804, 1826]. In a mouse model
for metastasis, local irradiation plus subsequent Flt-3
ligand treatment enhanced survival. This appeared to be
due to increased presentation of antigen derived from
dying tumor cells. In vivo manipulation of dendritic
cells was used to induce immune response in three
mouse model tumors. Mice were treated with several
daily Flt3 ligand injections to induce DCs, then with CG
oligonucleotide to mature the DCs in the presence of
tumor peptide antigen. Existing tumors were inhibited
and the mice were protected against a new challenge
[1490]. It should be noted again that Flt-3 ligand acts
together with other cytokines. For example, Flt-3 ligand
and CD40 ligand were shown to be synergistic in the
induction of antitumor immunity. Blocking CD40
obviated the effect of Flt3 ligand [179].
Flt3 ligand does not directly influence the growth of
most types of non-hematological tumor cells in culture,
although it has been reported that neural crest tumors
206
express Flt-3 and that cell lines treated with Flt-3 ligand
showed increased growth and decreased apoptosis [1999].
Flt-3 is an apparent site of immune suppression.
Vascular endothelial cell growth factor is produced by
many tumors; it is an angiogenic factor and it is also
immunosuppressive. VEGF interferes with Flt-3-
mediated DC production. It appears to do so by inhibiting
the activation of the transcription factor NF-κB [1483].
There is interest in Flt-3 ligand for the treatment of
cancer patients undergoing stem cell transplants.
Patients may be pre-treated with Flt-3 ligand to mobi-
lize stem cells to increase the number available for har-
vest. Flt-3 may also be used to expand these cells in
culture. Studies in normal volunteers and in cancer
patients showed that Flt-3 ligand is relatively safe. Few
side effects were observed [1098, 1221]. As seen in
mice and other animals, Flt-3 ligand caused a remark-
able increase in stem cells and dendritic cells in blood
and other tissue sites [1221]. Flt-3 ligand is effective
alone but most likely it will be used in combination with
other cytokines such as G-CSF and GM-CSF [193, 194,
1920]. In culture, stem cells and progenitor cells may
proliferate for several weeks when Flt-3 ligand is added
alone or together with other cytokines such as IL-1, Il-3,
Il-6, IL-11, IL-12, erythropoietin, SCF and thrombopoi-
etin [381, 578, 872, 1564, 1568, 1860]. Flt-3 ligand
appears to maintain the stem cells, to sustain long-term
hematopoiesis after reimplantation [381, 1564].
Trials have begun in cancer patients, using Flt-3 ligand
to treat patients or to generate dendritic cells for vaccina-
tion. In a preliminary study, colon cancer patients injected
with Flt3 ligand showed increased lymphocytes in blood,
increased percent dendritic cells in PBMCs and increased
dendritic cells infiltrating tumors [1366]. In another report,
patients were vaccinated with antigen-pulsed autologous
dendritic cells. The patients were pre-treated with Flt-3
ligand to mobilize DCs. The DCs were harvested, loaded
ex vivo with a peptide of carcinoembryonic antigen and
then reinfused. The preliminary results were promising.
Two of 12 patients showed dramatic regressions; one had
a mixed response. Anticancer activity correlated with the
generation of specific CD8 T cells [549].
Leukemia inhibitory factor
T Cell Development, Regulator of
Inflammation, Embryonic Development
Leukemia inhibitory factor (LIF) is a member of the IL-6
cytokine family [2273]. It is a pleiotropic cytokine; many
of its effects overlap with those of IL-6. LIF is produced
by fibroblasts [2008], macrophages [51] endothelial cells
Cytokines
[1168] thymic epithelium [1067] and synoviocytes [395,
249]. The gene for LIF is on chromosome 22q12 where
it colocalizes with the gene of OSM. In immune cells,
secretion of LIF is stimulated by endotoxin, IL-1 and
TNF [1297]. In thymic epithelial cells, EGF and TGF-β
stimulate LIF production [1743].
The receptor for LIF has two subunits, LIFRβ and the
common gp130 utilized by IL-6 family cytokines [602].
LIF has a role in the production of CD4 and CD8 T
cells. LIF is secreted by thymic epithelium [1067]. Too
much or too little LIF causes abnormal T cell develop-
ment [1295]. In transgenic mice that overproduced LIF,
thymic epithelial architecture was disrupted and cortical
thymocytes were decreased [1804]. In LIF deficient
mice, thymocytes were produced but they were insensi-
tive to ConA activation [498].
LIF regulates inflammation. Depending on the set-
ting, LIF may be pro- or anti-inflammatory. LIF knock-
out mice showed abnormal inflammatory response in
nervous tissues [579, 1926, 2003]. Injection of rLIF into
skin and joints induces swelling and infiltration [248,
1277]. LIF is elevated in ulcerative colitis; LIF stimu-
lates the growth of cancer cells and may thus have a role
in the development of colon cancer associated with this
inflammatory disease [622]. LIF is proinflammatory,
but there is also evidence LIF can inhibit inflammation.
In knockout mice lacking LIF, inflammation is exacer-
bated in footpads injected with Freund’s complete adju-
vant. Delivery of LIF using an adenoviral vector
suppressed inflammation [2296]. In psoriasis, LIF and
IL-11 have similar depressive effects on inflammation
and cytokine production [2022].
LIF is involved in septic shock. The blood of patients
had elevated LIF as well as TNF, IL-1 and IL-6; the level
of these cytokines was correlated with the severity of the
disease [2077, 2118, 2119]. LIF and IL-6 appear to be
induced by TNF-α [880]. Blocking antibodies for LIF
depress the lethality of endotoxemia [166]. Interestingly,
treatment of mice with LIF before a lethal dose of endo-
toxin favored survival of the mice [26, 2117]. This may
be due to induction of acute phase proteins that have
antiinflammatory, protective effects [1258].
LIF has growth, differentiation and metabolic effects
in non-hematopoietic tissues [759, 1042, 1295, 1297].
LIF reversibly inhibits the differentiation of embryonic
stem cells [1449]. In fetal pituitary corticotropes, LIF
inhibits cell division, stimulates differention and
increases ACTH production [1884]. LIF regulates neu-
ronal differentiation and increases survival [1345]. It
stimulates bone development and extracellular matrix
metabolism. LIF regulates mammary cell growth and
development [1137]. Maternal expression of LIF is
required for blastocyst implantation [1892].
Walter M. Lewko and Robert K. Oldham
Mice injected with LIF suffer weight loss associ-
ated with cachexia. Weight loss is a characteristic of
several gp130 signaling cytokines, IL-6, IL-11, CNTF
and NNT [1784].
LIF has some remarkable effects in cancer. As men-
tioned, it is required for normal immune and inflammatory
response. In tumor cells, LIF has both negative and posi-
tive direct effects on growth. LIF was discovered based on
its ability to arrest growth and induce differentiation in
murine myeloid leukemia cells [604]. In cultured human
glioma cells, LIF inhibits growth and induces astrocyte-
like differentiation [701]. But LIF can stimulate growth in
many types of tumor cells including human multiple
myeloma [2285] colon cancer [964], breast cancer [425,
500, 948] and prostate cancer [948]. Other cytokines in
the IL-6 family similarly influence tumor cell growth
based on gp130 signaling [2285]. There is interest in
agents that regulate growth by this signaling pathway.
LIGHT
Costimulatory Molecule;
T Cell Activator; Inducer of Apoptosis
LIGHT is a 240 amino acid cytokine that was first dis-
covered in the cDNA library of activated T cells. It is a
member of the TNF family [1044, 1254]. LIGHT is
homotrimeric and it is produced in both soluble and
membrane bound forms, both active. LIGHT is expressed
by T cells, granulocytes, monocytes, immature dendritic
cells and certain tumors [719, 1368, 1957, 1959, 2278].
LIGHT binds two receptors, HVEM (herpes virus
entry mediator) and lymphotoxin β receptor (LTβR)
[2278]. HVEM is a member of the TNFR family [1044,
1352, 1958]. It is the cell receptor for herpes simplex
infection [1344, 1254]. HVEM is also referred to as
TR2 (TNF-related receptor 2) [1044] and ATAR (another
TRAF-associated receptor) [808]. It is a 283 amino acid
transmembrane protein. Signaling involves TRAF bind-
ing and the activation of NF-κB, Jun N-terminal kinase
and AP1 [808, 1231, 1254]. HVEM is localized on T
cells, B cells, NK cells, monocytes, endothelial cells
and immature dendritic cells [720, 1044, 1344, 1958].
Its levels are up regulated by cell activation and down
regulated by LIGHT [1958]. HVEM is also a receptor
for lymphotoxin-α [1254].
In T cells, LIGHT costimulates proliferation, cytokine
secretion and surface protein levels [720, 1044]. Disruption
of LIGHT, interferes with lymphnode formation, depresses
T cell proliferation in a MLR, the secretion of IL-2, IFN-γ,
IL-4 and TNF-α [720, 1739]. Disruption of LIGHT also
prolongs allograft survival [2237].
207
As typical of TNF-family members, LIGHT induces
apoptosis. Normally, only cells that express both HVEM
and LT-β receptors are susceptible to LIGHT-induced
cell death. No apoptosis was observed in cells express-
ing only one of them [719, 2278]. Because lymphocytes
do not express LT-β receptor, LIGHT does not cause
apoptosis in these cells.
TR6 (also called DcR3) is another member of the
TNF receptor family. TR6 is a soluble, non-signaling
protein that binds both LIGHT and FasL and is, there-
fore, a decoy receptor. TR6 appears to have a regulatory
role in LIGHT and FasL mediated cell death [2265].
LIGHT is expressed on immature dendritic cells.
Engagement of LIGHT costimulates T cell proliferation.
Blockade of LIGHT, using soluble recombinant receptors,
inhibits DC-mediated allogeneic T cell response [1957].
LIGHT stimulates inflammatory response. Triggering
HVEM together with toll-like receptors on neutrophils
synergistically stimulates respiratory burst, degranulation,
secretion of IL-8 and opsonization of particles [722].
At an early stage in liver regeneration, lymphocytes
infiltrate the hepatic tissue. Hepatocyte cell division is
stimulated and this depends on the expression of T cell-
LIGHT and liver cell-LTβR [45].
LIGHT costimulates T cell anticancer immunity.
Typically immune cells express LIGHT but it is also
found in melanoma cells and on microvesicles released
from melanoma; co-culture of microvesicles with lym-
phocytes in the presence of IL-2 costimulated CD8 T cell
proliferation and apoptotic death in the melanoma cells
[1368]. Human breast cancer cells transfected with LIGHT
cDNA triggered immune response and regression in an in
vivo model system [2278]. NK cells express HVEM;
tumor cells that express LIGHT activate NK cells to kill
tumors in a manner that requires IFNγ and CD8 cells. The
NK cells provided help (IFN) in the activation of CTLs
[509]. With respect to apoptosis, LIGHT triggers cell
death in tumor cells. Normally, two receptors (HVEM and
LTβR) are required for LIGHT-induced apoptosis but in
tumor cells only one, the LTβR, was necessary [1662].
Lymphotoxin-a (Tumor Necrosis
Factor b)
Lymphoid Organogenesis; Inflammation;
T Cell, B Cell, Bone Cell Development
Lymphotoxin α (LT α) was discovered in the condi-
tioned medium of activated T cells [505, 1078, 1695].
It was named for its lymphocyte origin and its toxicity to
certain cells. The gene for human LTα is on chromosome 6,
208
close to the TNF-α gene. LTα is a TNF family member;
it is also referred to as TNF-β [9, 1553]. LTα is pro-
duced by Th1 cells, CD8 T cells, early B cells, NK cells
and also by astrocytes. LTα is synthesized as a 202
amino acid precursor with a signal peptide and it is
cleaved to 171 and 194 amino acid forms [9]. LTα self-
associates to form homotrimers (LTα3) with a confor-
mation similar to TNF-α. It is a soluble cytokine. The
homotrimer binds both TNFRI and TNFRII [209, 212,
777, 1112]. Because the TNFRII-LTα3 complex does
not signal, for LTα3 TNFRII is a decoy receptor and an
antagonist [1281]. LTα3 also binds herpesvirus entry
mediator (HVEM), a receptor for LIGHT [1254].
Lymphoid organ development depends on LT-α and
other cytokines such as LT-β, TNF-α and LIGHT.
LT-α(−/−) mice, were born without lymph nodes or with
defective nodes. Peyer’s patches were also lacking.
Spleen histology was abnormal [110, 419, 1004, 1706].
Both LT-α and LT-β appeared to be required for normal
lymphoid organogenesis [1249, 1445, 1636].
LT-α and TNF-α are involved in the formation of
B cell follicles, T and B cell segregation, follicular
dendritic cell clustering and the formation of germinal
centers. Mice deficient in either LT-α or TNF-α
failed to form germinal centers [502, 2110].
LT-α (−/−) mice have impaired effector T cells. These
mice were much more susceptible than controls to HSV
infection. CD8 T cells were induced normally but their
effector functions were depressed. The cells failed to
become CTL and did not secrete INF-γ when stimulated
with antigen [1035]. LT-α was also required for the
development of memory B cells and their capacity to
respond to antigen [571].
LT-α has a major part in inflammation. It induces the
expression of leukocyte adhesion molecules VCAM-1
and ICAM-1 on endothelial cells [265, 1581]. In a
mouse model, antibodies to LT-α and TNF-α prevented
allergic encephalitis [1694]. Transgenic mice overex-
pressing LT-α have remarkable inflammation in the sites
of targeted expression. The infiltrate consists of T cells,
B cells, macrophages, and dendritic cells [1020, 1569].
TNF receptors were studied in this system. Mice lacking
TNFRI failed to develop inflammation. TNFRI appears
to be the primary receptor involved in LTα-induced
inflammation. Lack of TNFRII did not did not influence
inflammatory response. Lack of the related cytokine
lymphotoxin β did not prevent inflammation but altered
the response [1705].
LT-α is involved in bone and tooth metabolism.
TNF-α and LT-α stimulate osteoblast and osteoclast
activity [1988]. IL-1 acts synergistically with TNF
Cytokines
to stimulate bone resorption [1883]. In a model for
periodontal disease, soluble recombinant receptors for
IL-1 and LT-α, acted as antagonists and depressed
recruitment of inflammatory cells, osteoclast activity,
bone loss and periodontal destruction [83].
Lymphotoxin-b
Lymphoid Organogenesis; Inflammatory;
NK, Dendritic Cell Development
Lymphotoxin-β (LT-β) is a 33 kDa transmembrane pro-
tein that was originally cloned from a T cell hybridoma
[212, 685]. It is related to LT-α in structure and func-
tion. LT-β is expressed mainly on activated T cells, B
cells and NK cells [2116]. LT-β chains form homotrim-
ers and heterotrimers with LTα; LTα1-LT-β2 is the major
form [209, 212, 777, 2116].
LT-β binds the LT-β receptor (LT-βR). This receptor is
related to the TNF receptors. It binds LT-β in its trimer
and heterotrimeric forms; it does not bind TNF-α or the
LT-α homotrimer [209, 211, 212, 355, 766]. LIGHT is
also a ligand for the LT-βR [2278]. Follicular stromal
cells, monocytes, fibroblasts, smooth muscle and skeletal
muscle cells express the LT-βR. It is also on human mel-
anoma and adenocarcinoma cell lines [211, 402, 2051].
LT-β, LT-α, TNF-α and LIGHT are all critical in the
development of lymphoid organs [1249, 1445, 1636,
1739]. LT-β was also required for the homing of den-
dritic cells to lymph nodes [2206]. LT-β induces IFN-β
and this promoted survival of lymphocytes during
murine cytomegalovirus infection [110].
LT-α and LT-β stimulate the development of NK
cells. LT-β(−/−) mice had fewer NK cells [841, 861,
1846] and LT-βR was required for normal NK develop-
ment [841, 2205]. The effect of LT-β was not dependent
on IL-15, another essential cytokine in NK maturation
[841]. It appeared that an effect of LT-β on marrow
stromal cells was critical for an early step in NK devel-
opment [2205].
LT-α and LT-β are required for the normal develop-
ment of NK T cells. These cells are important in that
they may be stimulated to quickly produce large amounts
of IL-4, IFN-γ, and IL-10. They are involved in certain
types of antitumor activity, the prevention of autoim-
munity and protection against bacterial and parasitic
infections. Mice that are LT-α(−/−) and LT-β(−/−) have
reduced NK T cells and they are low in IL-4 and IL-10.
Both LT-α and LT-β are needed suggesting LT-α1β2
(not the homotrimer) is the active form [483].
Walter M. Lewko and Robert K. Oldham
LT-β has both cell mediated and direct effects on cancer.
It is required for normal production of dendritic, NK,
NK T and LAK cells. Mice lacking the LT-βR showed
enhanced tumor growth and metastasis [861]. Direct LT
effects have also been observed in certain human adeno-
carcinoma cells that have the LT-β receptor. Signaling
through the LT-βR induced cell death in culture and
arrested tumor growth in vivo [211, 1194]. Human mel-
anoma cell lines also express the LT-βR. When the
receptor was activated, growth was inhibited and proin-
flammatory cytokines were secreted [402].
Novel neurotrophin
B Cell Development; Regulator
of Inflammation
Novel neurotrophin (NNT) is a member of the IL-6
cytokine family. It is also referred to as B cell stimulat-
ing factor-3 and cardiotrophin-like cytokine. NNT is a
225 amino acid protein. It is homologous with IL-6 fam-
ily members, in particular cardiotrophin-1 (CT-1) and
ciliary neurotrophic factor (CNTF). The gene for NNT
is on chromosome 11q13 where it is close to the gene
for CNTF (11q12). mRNA for NNT is produced by
lymph nodes and spleen and to a lesser extent by other
tissues [1783, 1808].
The receptor for NNT contains ciliary neurotro-
phin Rα, gp130 and LIFRβ. Signaling, involves the
JAK/STAT pathway and the activation of STAT-3. In
this way NNT resembles other IL-6 family members
[384, 1088].
NNT stimulates B cells. Increased B cell production
and secretion of IgM, IgE, IgG was observed in mice
treated with NNT [1784, 1783]. These mice suffered
weight loss. NNT, like certain other IL-6 family mem-
bers, induces cachexia. As its name implies, this cytokine
is stimulates nerve growth. In culture, NNT increased
the survival of chick embryo neurons [1784]. Gene defi-
ciency studies showed that NNT and the ciliary neu-
rotrophic factor receptor were required for motor neuron
development [396, 1099]. In mice, NNT induced the
production of serum amyloid A protein. It also potenti-
ated IL-1-induced secretion of glucocorticoids. Many of
these effects are shared with certain other members of
the IL-6 family, but there are differences. NNT was
characteristic in that it did not induce hematopoiesis;
NNT induced growth of M1 macrophage cells whereas
IL-6. LIF, OSM and CT-1 inhibited M1 growth and
induced differentiation [1784].
209
Oncostatin M
Lymph Node/T Cell Development;
Inflammation; Growth and Wound Repair
Oncostatin M (OSM) is a 28 kDa cytokine. It was dis-
covered in the conditioned medium of lymphocytes as a
factor capable of inhibiting certain cancer cells [207,
1210, 2274]. Activated T cells, monocytes and neutro-
phils produce OSM [207, 213, 666, 826]. The gene for
OSM is on chromosome 22q12 where it colocalizes
with the gene for LIF. It is a member of the IL-6 cytokine
family that utilizes gp130 as a receptor component
[1664, 2273].
In humans, OSM binds and signals through two
receptors. The first is the receptor for LIF, gp130-LIFRβ.
It is responsible for effects induced in common by OSM
and LIF [601, 602]. The second is gp130-OSMRβ,
which is a specific OSM receptor and is responsible for
the effects particular to OSM [1373, 1984]. OSM recep-
tors are on a variety of cells including tumor cells. The
OSM mechanism of signaling involves MAP kinase,
JAK-STAT and the PIP3-kinase pathways [38, 99,
837, 1009].
Oncostatin M has remarkable effects on lymph node
development and the production of lymphocytes.
Thymus is a key site for T cell processing. But even in
athymic animals a certain amount of T cell produc-
tion occurs in bone marrow, liver, intestine and in
lymphnodes. In transgenic mice overexpressing oncostatin
M, thymus atrophies and T cells (immature and mature)
accumulate in the lymphnodes [175, 325, 1209]. High
OSM endows lymph nodes with the ability to sustain
T cell development. OSM also mobilizes lymphocytes
causing them to move into lymphnodes and to recycle
into circulation.
OSM has multiple effects related to wound repair and
inflammation. It is a growth stimulator for fibroblasts
[793] and vascular smooth muscle cells [679]. OSM is a
fibroblast activator. It stimulates growth of 3T3 cells,
dermal fibroblasts and synoviocytes [694]. OSM
increases the production of extracellular matrix colla-
gen [836, 837] and glycosaminoglycan [469]. It stimu-
lates the production and activation of collagenases that
degrade the extracellular matrix [705, 1644]. Lytic
enzymes and collagen metabolism are part of the growth
process in connective tissue. OSM also increases IL-6
production in fibroblasts [208] which itself stimulates
fibroblast mitosis and collagen production [468].
Transgenic mice that overproduce OSM have develop-
mental abnormalities and visceral fibrosis [1209].
210
OSM has both stimulatory and regulatory effects on
inflammation. It is increased in the synovial fluids of
rheumatoid arthritis patients [817]. In a mouse model
for RA, OSM appeared to suppress inflammation and
tissue damage [2092]. It stimulated the production of
acute phase proteins, which are known to have antiin-
flammatory activity. [1642]. In fibroblasts, OSM inhib-
ited the production of IL-1-stimulated proinflammatory
cytokines IL-8 and GM-CSF [1643]. OSM also increases
fibroblast production of tissue inhibitor of metallopro-
tease (TIMP) [1644], which inhibits collagenase activ-
ity and tissue destruction. On the other hand, OSM has
been shown to have proinflammatory effects. When
injected into joints, it caused cartilage resorption and
inhibited proteoglycan production resulting in tissue
damage [127]. And OSM alone, and together with IL-1
and TNF-α, stimulates production of several matrix
metalloproteinases in cartilage [356, 1009].
As implied by its name, OSM has been shown to
inhibit growth in several cancers including melanoma
[207, 2274], myeloid leukemia [213], glioma cells [702]
and breast cancer [1137]. There is interest in OSM for
these cytostatic effects and for its role in immune
response. But OSM has not been tested clinically. It
should be noted, OSM is a growth factor for Kaposi’s
sarcoma [1308]. Certain of its proinflammatory effects
might exacerbate infiltration and angiogenesis [1605]
Osteopontin
Pro Th1, Nitric Oxide Synthetase
Inhibiting Cytokine
Osteopontin (OPN) is a secreted phosphoglycoprotein.
It is also referred to as early T lymphocyte activation
protein-1 (Eta-1). OPN is produced by activated T cells,
macrophages, osteoblasts, endothelial cells, brain and
certain epithelial cells. Its expression is increased by
IL-1, IFN-γ TNF-α, bFGF, phorbol esters, glucocorti-
coids and 1,25 dihydroxyvitamin D3 [79]. OPN is also
produced by transformed cells [1785].
There are two receptors for osteopontin, integrin
αVβ3 on endothelial cells, fibroblasts and other non-
hematopoietic cells, and CD44 that is on leukocytes [79,
694]. The synthetic peptide GRGDSP blocks integrin
binding and activation [694]. Integrin αVβ3, CD44 and
osteopontin are cell surface extracellular matrix compo-
nents which function structurally and in signaling.
OPN has an important role in bone growth and remod-
eling. OPN binds osteoclasts and functions in osteoclast
recruitment. OPN is a regulator of hydroxyapatite depo-
Cytokines
sition for bone calcification [82, 182, 413]. OPN is not
normally present in soft tissues, but it does accumulate
at sites of abnormal calcium deposition, such as athero-
sclerotic lesions and calcified heart valves [542, 1875].
OSN appears to be controlling calcium deposition, for
in OSN deficient mice, vascular calcification is exacer-
bated [1867].
In bone marrow, OPN appears to control stem cell
migration and the size of the stem cell pool to prevent
excessive expansion [1456, 1893]. Osteopontin controls
the activation of T cells. It favors Th1 immune response
[79]. Mice deficient in OPN fail to develop Th1 immunity
during viral and bacterial infection; production of IL-12
and IFN-γ is decreased while IL-10 is increased [79].
Attenuation of experimental autoimmune encephalitis,
a Th1-related disease, was observed in OPN deficient
mice [881]. OPN appears to stimulate Th1 response at
the level of dendritic cells. OPN promotes migration of
dendritic cells from tissue sites to lymphnodes; it
induces TNF and IL-12 (Th1) cytokines; and it increases
adhesion and costimulatory molecules [1635]. Though
it favors the Th1 pathway, OPN stimulates B cell prolif-
eration and Ig production.
Osteopontin is involved in acute and chronic inflam-
mation. It has chemotactic, pro-inflammatory effects
but it also has prominent regulatory functions. Elevated
OPN levels have been observed in atherosclerosis [87],
glomerulonephritis [816] and in granulomatous diseases
[1430]. During heart failure, increased OPN levels are
observed in myocardial cells [1831]. In a mouse model
for rheumatoid arthritis, OPN appeared to cause joint
destruction by promoting angiogenesis and cartilage
cell apoptosis [2268]. However, OPN depresses nitric
oxide synthetase and NO production in macrophages
[694, 1657], kidney epithelium [814] and endothelial
cells [1771]. Inhibition of nitric oxide appears to be a
major regulatory function of OPN limiting damage dur-
ing inflammation.
In endothelial cells, OPN acts as a survival, cell adhe-
sive and chemotactic factor. It organizes angiogenesis.
Fibroblast growth factor 2 (3, 1068) stimulates OPN
production by endothelial cells. In turn OPN induces
monocyte chemotaxis. Tissue becomes infiltrated with
monocytes. OPN stimulates monocytes to produce
TNF-α and IL-8, which are both angiogenic [1068].
This mechanism could have a role in normal wound
repair or in pathology such as cancer.
Several properties of OPN may contribute to cancer
cell growth and behavior. Variable osteopontin levels
are observed in cancer. Increased OPN secretion was
associated with decreased nitric oxide production and
decreased killing of tumor cells by macrophages and
Walter M. Lewko and Robert K. Oldham
endothelial cells. In this way, OPN may have a role in
immune escape by cancer cells [413]. OPN also induces
cyclooxygenase, and the progression of prostatic cancer
[877]. OPN induces monocyte secretion of IL-1β [1421]
and other growth and proangiogenic factors. OPN is
expressed by normal brain and astrocytoma cells; it
increases astrocytoma cell migration [440]. Hepatocyte
growth factor activates a genetic program in epithelial
cells for cell dissociation, growth and invasion. This
program is upregulated during progression in many
tumors. HGF induces osteopontin; antibody studies sug-
gest that OPN mediates HGF-induced invasive behavior
[1280]. Melanoma produces OPN; in melanocytes OPN
functions as an antiapoptotic survival factor [608].
Osteopontin has been observed in many breast cancers.
Its presence is often associated with calcification.
Several studies have related osteopontin accumulation
in breast cancer with decreased survival [770, 1696,
1834, 2036].
OX40 ligand
Costimulation in T Cell, B Cell
Development/Memory; Inflammation
OX40 ligand (OX40L) is a member of the TNF family.
It is a 32–34 kDa, 183 amino acid, membrane bound
glycoprotein. OX40L is expressed mainly on dendritic
cells [632, 1511], B cells [1908], endothelium and acti-
vated T cells [117, 851, 1571].
OX40 (CD134) is the receptor for OX40L. It is a
48 kDa, 250 amino acid, membrane glycoprotein. OX40
was originally described in CD4 T cells as a surface
antigen, which was similar to the nerve growth factor
receptor [1213, 1542]. It is found only on activated T
cells [232, 2000]. Normally, its levels are very low and
rise following activation [232, 655]. OX40 signaling
involves TRAF-2, TRAF-3 and NF-κB [62].
The OX40L–OX40 system functions as a costimulator
during antigen presentation for the activation and
increased longevity of T cells, in particular CD4+ Th
cells. Mice deficient in OX40 signaling show reduced T
cell response to infection. B cell response appears to be
normal [294, 1006, 1396, 1571, 2000]. OX40L–OX40
interactions occur in addition to B7-CD28 signaling
[111, 660, 386, 887, 2243] between AP and T cells. OX40L
acts synergistically with B7 to costimulate CD4 Th cell
expansion and the secretion of IL-2, IL-4 and IL-5
[655]. OX40L was required for priming [1436] and
sustained later stage CD4 T cell proliferation and
memory [654, 655].
211
OX40L is capable of reverse signaling back into the
cells that express it [1511, 1908]. In intermediate stage
dendritic cells, OX40L ligation, by interaction with T
cells or by agonist Mabs, caused reverse signaling to the
dendritic cell, enhancing maturation and the secretion of
cytokines TNF-α, IL-12, IL-1β and IL-6 [1511].
B cell-deficient mice produce a poor T cell response
[1141]. B cells function during antigen presentation;
they appear to stimulate T cells after their initial contact
with DCs. OX40L has a role in this process. OX40L is
expressed on splenic B cells. When B and T cells inter-
act, OX40 engagement stimulates T cell growth and
IL-2 release [1908, 1909]; reverse signaling through
OX40L on the B cells induces B cell growth and
differentiation.
OX40 ligand is found mainly on antigen presenting
cells. However, OX40L is also present on CD4 T cells
and it appears to have a role in T cell response [1862].
When activated in culture, T cells that were genetically
deficient in OX40L proliferated less than normal T cells
and when they were transferred back into mice, sur-
vival was reduced [1862]. It thus appears that T cell
OX40L provides additional signals to sustain CD4 T
cell longevity.
OX40 ligand has been shown to shut down IL-10 pro-
ducing regulatory T cells. OX40 L inhibited the genera-
tion of the regulatory cells and additionally it inhibited
IL-10 production in differentiated IL-10 producing reg-
ulatory T cells [865].
OX40L is expressed on vascular endothelial cells. It
may function during inflammation in the binding and
extravasation of activated OX40-expressing T cells
[851]. The OX40–OX40L system appears to be involved
in several inflammatory diseases including allergic
encephalomyelitis [1436, 2134], graft versus host dis-
ease [1909, 1910, 2000], inflammatory bowel disease
[751] and rheumatoid arthritis [215]. In certain cases,
amelioration of the disease has been demonstrated when
the animals were treated with anti-OX40 antibodies
[751, 1910, 2134]. Exacerbation of inflammatory dis-
ease has been shown in transgenic animals overexpress-
ing OX40 [1436]. Tumor infiltrating lymphocytes were
also positive suggesting a role for OX40 in host response
to cancer [472, 2073].
There is interest in costimulatory cytokines as adju-
vants during vaccination. OX40 ligation by agonistic
antibodies promoted memory with increased survival of
specific T cells [655]. OX40 costimulation could even
reverse established anergy [112]. It has been shown that
OX40 signaling by several means enhanced immune
response during cancer vaccination [27, 44, 379, 669,
836, 914, 1362, 1529] OX40 costimulation together
212
with GM-CSF was able to produce a strong response to
the naturally occurring Her2/neu tumor antigen, capable
of inducing regression in established tumors [1397].
OX40L and OX40 are on HTLV-infected T cell leu-
kemias and the virus regulates expression. OX40L
and OX40 may be involved in the development and
growth of this cancer [117, 1321].
RANKL/TRANCE
B Cell, DC, Osteoclast Development,
Lymph Node Organogenesis
RANKL (receptor activator of NF-κB ligand) is a mem-
brane bound, TNF-related protein, It functions in the
development of osteoclasts, lymphocytes and in lymph
node organogenesis [450, 1003]. RANKL is also
referred to as TRANCE (TNF-related activation-induced
cytokine), osteoclast differentiation factor [2235] and
osteoprotegerin ligand. RANKL is expressed on T cells
[48, 903, 1003, 2193, 2194], B cells [2269], dendritic
cells [48, 2192] and bone marrow stromal/osteoblast
cells [48, 1950, 2269]. In T cells RANKL levels increase
with TCR activation [2106].
RANK and osteoprotegerin (OPG) are receptors for
RANKL. They are membrane-bound proteins and
members of the TNFR family. RANK is found on a
variety of cell types, in particular, osteoclast precursors
[1003] dendritic cells [48, 1003, 2193, 2221] and B
cells [2269, 2270]. OPG is also referred to as follicular
dendritic cell-derived receptor-1 and osteoclastogene-
sis inhibitory factor [218, 825, 2221, 2235]. OPG has
membrane bound and soluble forms [2221, 2269]. OPG
and RANK are both upregulated by CD40L, an impor-
tant cytokine in germinal center and B cell develop-
ment [2269]. RANK is the active receptor, which
induces response in target cells; OPG is a non-signaling
decoy receptor that competes with RANK [1813, 2270].
Inhibitory activity is mainly associated with soluble
form of OPG. The function of the bound form is not
certain. There is a report that it may be able to induce
apoptosis [2221]. Other investigators were unable to
detect any signaling [2269].
RANKL has several functions in the immune system.
In dendritic cells, it stimulates antigen presentation [48,
903, 1003, 2192], Bcl-XL antiapoptotic activity and sur-
vival [2193]. OPG suppresses this [2270]. The dendritic
cells from OPG-deficient mice are altered in that they
are activated and show increased capacity to stimulate T
cells [2270]. RANKL stimulates normal B cell develop-
ment. OPG also regulates this process. Mice lacking
Cytokines
OPG have altered B cells; transitional B cells are
increased and proB cells are more sensitive to IL-7
growth stimulation [2270]. RANKL is a morphogenesis
factor; it is involved in the early development of
lymphnodes. Mice deficient in RANKL and RANK had
abnormal lymphnode development and B cell production
[416, 1003].
Estrogens tend to inhibit B cell lymphopoiesis during
pregnancy [970]. Estrogens stimulate stromal cell secre-
tion of factors that are inhibitory [1845]. Estrogens also
stimulate the production of OPG [778].
RANKL stimulates osteoclast maturation and bone
development. Osteoprotegerin regulates osteoclast
activity [1050, 1828, 2221]. Macrophages and osteo-
clasts share a common lineage [517]. Peripheral blood
monocytes cultured with RANKL, G-CSF and TGF-β
formed osteoclasts [807, 913, 1793]. B cells may also be
involved in osteoclast development. B cells are a source
of stimulatory RANKL. B-lymphoid progenitor cells
are also a source of osteoclast precursor cells [1215].
Mice lacking the RANKL and RANK have problems
with bone and vascular development. The mice lack
osteoclasts and develop osteoporosis [218]. Mice defi-
cient in OPG are viable but osteoclast activity goes
unchecked and as they age, the mice develop severe
osteoporosis [1328, 2270].
While little has been done with the RANK/RANKL
system in cancer, there is interest in costimulatory mol-
ecules that may enhance anticancer immune response. It
has been shown that DNA vaccines that include the
RANKL gene and dendritic cell vaccines that express
RANK/RANKL produce more potent T cell responses
[1322, 2155]. It may be that controlling OPG will be
useful during immune therapy and vaccination [2270].
Stem cell factor
Early Progenitor and Mast
Cell Growth Factor
Stem cell factor (SCF) was discovered in rat liver as a
protein responsible for the outgrowth of very early pro-
genitor cells in bone marrow [2305]. SCF is also referred
to as kit ligand, steel factor and mast cell growth factor
[47, 345, 2006, 2169]. There are two forms of SCF,
soluble and membrane bound, produced by alternative
splicing of the same pre-mRNA [47, 345, 2006, 2007].
Several types of cells express SCF, including bone mar-
row stroma, fibroblasts, liver and spleen [1244, 1248].
The receptor for SCF turned out to be the product
of the protooncogene c-kit [47, 345, 543, 2169, 2304].
Walter M. Lewko and Robert K. Oldham
This receptor is a protein tyrosine kinase [2219]. It is
found on stem cells, progenitor cells and mast cells
[1947, 2033, 2143]. A ligand-induced dimerization is
part of the receptor activation process. SCF induces
the downregulation of its own receptor by internal-
ization [2238].
SCF stimulates growth of early progenitor cells
(hematopoietic, lymphoid, and myeloid). Other cytok-
ines induce differentiation and SCF may act synergisti-
cally with them [373, 2007, 2033]. SCF has a particularly
remarkable effect on mast cells. Mice genetically defi-
cient for SCF suffer anemia and are very deficient in
tissue mast cells [609, 977, 978]. SCF acts on early
mast cell progenitors to stimulate outgrowth. IL-3 acts
at a later stage to stimulate further growth and antipara-
site activity [2266]. SCF depresses apoptosis and thus
serves as a mast cell survival factor [834]. rhuSCF
injected sc into patients activates mast cell degranula-
tion and proliferation with a wheal and flare response at
the injection site [346].
Mast cells and SCF may be involved in fibrosis.
Fibroblasts are a major source of SCF. Mast cells stim-
ulated by fibroblast SCF release histamine and eotaxin,
the eosinophil chemokine [780]. SCF also stimulates
mast cell adhesion to fibronectin of fibroblasts and
other cells [382]. Mast cells induce fibroblasts to
produce collagen. Protracted inflammation produces
fibrosis [322, 1105]. In this way SCF may be involved
in the development of fibrotic reactions characteristic
of chronic inflammatory disease and tumors. In humans,
IL-4 depresses the growth and survival of mast cells by
down regulating the expression of c-kit [1824].
γδ T cells appear to have a role in innate and adaptive
response to viruses and other parasites [1782]. The pro-
liferation of γδ T cells appears to depend on SCF. Mice
lacking SCF receptor lacked intestinal γδ T cells while
αβ T cells were not affected [1054].
SCF and its receptor are produced by several different
types of tumors [1244, 1427] and may stimulate growth.
Tumors that have tested positive for c-kit and for SCF
include small cell lung carcinoma [748, 1701, 1781],
breast [1426], testicular [1907], uterine, cervical and
ovarian cancer [853] and melanoma [1428]. While it is
not unusual to have the same cell express both the
cytokine and its receptor, SCF will usually influence
tumor growth by a paracrine mechanism. In this way the
normal fibroblast component of a tumor could serve as
the source of SCF for c-kit+ carcinoma cells. For therapy,
it is possible that SCF blockade may inhibit tumor growth
or that presence of this cytokine or c-kit on tumor cells
may be used to target antibodies, drugs or toxins.
Interestingly, loss of c-kit expression has been observed
213
with progression in breast cancer [1426] and melanoma
[1428] reminiscent of the loss of estrogen receptor,
which occurs with progression in breast cancer.
SCF is being tested for its ability to synergize with
G-CSF (filgrastim) to mobilize CD34+ progenitor cells
into peripheral blood. It has been shown that SCF in
combination with G-CSF may be useful to stimulate
hematopoietic recovery after chemotherapy [506, 2124].
SCF with G-CSF also decreased the number of aphere-
ses needed to obtain progenitor cells for autologous
transplantation [562].
Tumor Necrosis Factor a
Inflammation, Immune Regulation,
Apoptosis, Endothelial Damage
TNF-α was first described in 1975 as a factor found in
animals treated with BCG or LPS. It was named for its
ability to induce hemorrhagic necrosis in tumors [250, 661,
2174]. TNF-α was later found to be identical to cachectin,
a factor responsible for metabolic wasting in patients with
advanced cancer or infection [150–153]. Human TNF-α
exists in both soluble and membrane bound forms [1549].
The soluble 17 kDa protein is generated from the 26 kDa
membrane-bound form by a protease, TNF-α converting
enzyme (TACE) [161, 1377]. TNF-α forms a trimer in
solution [75]. The soluble form is more potent and appears
to be responsible for most TNF-α bioactivity. The mem-
brane-bound form is also active; it binds and signals
through its receptor and by reverse signaling back into its
parental cell [91, 714, 753].
TNF-α is produced by a number of different types of
activated cells including macrophages, T cells (CD4+
Th1 and CD8+) [634] and B cells [2029, 2116], den-
dritic cells [2293], neutrophils [2069], adipocytes
[954], keratinocytes [1135], mast cells [158, 644],
mammary epithelium [2049], colon epithelium [907],
pancreatic β cells [2220], osteoblasts [1330], astrocytes
[1077], neurons [1972] and steroid-producing adrenal
cells [637]. Pathogen molecules such as LPS and bacte-
rial DNA stimulate TNF production in macrophages
[241, 1304, 1866, 1876]. TNF-α expression increases
with activation of T cell receptors [634] and B cell
receptors [2029].
TNF-α production is well regulated. In macrophages,
for example, LPS induces TNF-α; then TNF induces
IL-10 [1579, 2114] and IL-10 feeds back on TNF-α
and other inflammatory cytokines [613, 802] in a regu-
latory loop. Release of TNF-α from macrophages is
inhibited by glucocorticoids, progesterone [1309], and
214
by estrogens [288, 1614, 1796]. Glucocorticoids are
well known antiinflammatory agents. Progesterone has
some glucocorticoid activity; antiinflammatory effects
may be beneficial during pregnancy.
There are at least three receptors for TNF-α. TNFRI
(55 kDa) and TNFRII (75 kDa) bind both TNF-α and
LT-α. A third receptor, described in liver, binds TNF-α
but not LT-α [1760]. Nearly all mammalian cells express
TNFRI [659]. It appears to be responsible for most
TNF-α effects [2094]. The cytoplasmic part of TNFRI
contains a death domain that is involved in apoptosis.
TNF-α signaling also involves activation of NF-κB and
p38 MAP kinase pathways, which induce inflammation
and other non-apoptosis responses [2066].
TNFRII preferentially binds membrane bound TNF-α
[665]. TNFRII lacks a death domain though clearly it
stimulates apoptosis [1976, 2288]. It seems that TNFRII
does not induce apoptosis directly but rather it cooper-
ates with TNFRI; a complex is formed which induces
apoptosis (397). Mice lacking TNFRII appeared to
develop normally but were less sensitive to TNF-α and
its toxicity [495].
TNF receptors I and II are shed from cells and found
in tissue fluids [493, 1060]. The soluble receptors are
generated by the protease TNF receptor releasing enzyme
[659, 738, 925, 1389, 1464, 1844]. Production of shed
TNFR is specific and regulated. Activation by LPS or by
TNF-α itself induces TNFR shedding from many types
of cells [131, 344, 1083, 1587, 1889]. In patients with
inflamed livers, soluble TNFR levels were correlated
with disease severity [2184]. Shed receptors bind TNF-α
and act as competitive antagonists. The soluble receptor
thus has an antiinflammatory effect [892, 1889]. IL-10,
an antiinflammatory and immunosuppressive cytokine,
stimulates the release of soluble TNFR from monocytes
as part of its mechanism of action [905].
TNF stimulates the secretion of a number of cytokines
including TNF-α itself, IL-1, IL-6, IL-8, IFN-γ, GM-CSF,
M-CSF, PDGF, IL-10 and NGF. Directly or indirectly, it
increases the expression of several growth factor recep-
tors, adhesion molecules, collagenases and plasminogen
activator. TNF-α and IL-1 are key proinflammatory
cytokines. They share several biological functions [150,
152, 1498] even though they are not related molecularly
nor do they share receptors. It appears that both cytok-
ines, on binding their receptors, activate several down-
stream protein kinases in common [691, 698].
TNF-α has a remarkable ability to induce death by
apoptosis in many different types of cells for its role in
the regulation of morphogenesis, inflammation and
immune response [1726]. When TNF binds RI and
RII, death domains are activated [1965, 2094]. p38
Cytokines
MAPK and NFκB are also activated. Apoptosis is
induced by a caspase protease cascade, starting with
caspase-8. The caspases clip and activate a series of
enzymes and other proteins, producing the morphol-
ogy and DNA fragmentation observed in apoptotic
cells [75, 2094]. TNF-α also induces Fas expression in
cells such as CD4+ T cells, another way in which it
causes cell death by apoptosis [2292].
TNF-α not only induces apoptosis but it also regu-
lates it. Activation of NF-κB by TNF-α induces anti-
apoptotic survival factors that interfere with caspase
activation (691). TNF signaling induces death and
NF-κB, independently. Sensitivity to TNF-induced
apoptosis was enhanced in cells that were not able to
activate NF-κB. Thus TNF-α stimulates apoptosis and
later suppresses it [2050, 2099].
TNF-α influences many processes in immune
response. In dendritic cells, TNF stimulates maturation,
activation and antigen presentation for the induction of
specific T cells [1648]. TNF stimulates IL-12 secretion
in macrophages and other cells; IL-12 enhances T cell
development. In T cells, TNF-α and IL-1 together induce
Th1 development and secretion IFN-γ [1809]. In many
target cells, TNF-α and IFN-γ stimulate MHCI expres-
sion to enhance antigen presentation and susceptibility to
CTL-induced killing [1515]. TNF-α is a major stimula-
tor of outgrowth in γδ T cells. This response was corre-
lated with TNFRII levels [1053]. TNF-α has a major part
in the control of certain infections. For example, TNF-α
works together with γ-IFN and IL-4 on macrophages to
control tuberculosis [545]. In part, this is due to the
induction of nitric oxide synthetase [126, 1729]. TNF-α
also induces apoptosis in the mycobacteria-laden mac-
rophages, sequestering the pathogens in apoptotic bodies
[140, 457]. In neutrophils, TNF-α increased complement
receptor CD11b, adhesion to endothelium, release of
reactive oxygen, degranulation, phagocytosis and ADCC
[586, 983, 1429, 1792]. TNF-α also increased cellular
leukocyte adhesion molecules such as ICAM-1, VCAM-1
and E-Selectin in endothelium [1193], renal tubule epi-
thelium [2209] and in liver [2184]. These adhesion mol-
ecules function during tissue infiltration to direct effector
cell migration and activity.
In capillary endothelium, TNFα is angiogenic and
functions in wound repair [1084]. TNF stimulates fibro-
blast growth and enzyme secretion associated with ves-
sel formation [698]. However TNF-α also causes
damage and it is believed to be responsible for vascular
leak syndrome [458, 1607].
TNF-α is involved in bone metabolism. TNF-α is
produced by osteoblasts; it stimulates osteoblast mitosis
[1330] and bone resorption by osteoclasts [1988].
Walter M. Lewko and Robert K. Oldham
TNF-α is produced by lipocytes and it may have a
role in obesity. TNF-α mRNA levels are increased in the
lipocytes of obese patients; levels decrease with weight
loss [954].
TNF-α is involved in inflammatory and autoimmune
diseases. Most of the deleterious TNF effects appear to
be mediated by TNFRI [37, 1685]. In septic shock, LPS
and bacterial DNA act synergistically to stimulate
TNF-α production [590]. Mice treated with recombi-
nant soluble TNF receptor are protected from toxicity
and death [1100]. Collagen-induced arthritis was atten-
uated in mice treated with antibodies to TNFRI and in
mice genetically deficient for TNFRI [1355, 1991]. A
protease inhibitor which blocks both TACE and matrix
metalloproteases (induced by TNF) produced good
responses in rat arthritis models [343]. Antibodies to
TNF-α (Infliximab, Etanercept) have been approved
for use in patients with rheumatoid arthritis and are
being tested in patients with Crohn’s disease and pso-
riasis [292, 1203, 1454, 1721]. Interestingly, in experi-
mental diabetes and in certain other inflammatory
diseases, TNF-α appears to have a two-part influence.
TNF-α expressed early was required for disease pro-
gression. With prolonged exposure, TNF-α suppressed
inflammatory and autoimmune responses [315, 642,
1609]. Certain autoimmune diseases were exacerbated
in mice lacking TNF and TNFR. The antiinflammatory
effects of long term TNF appear to be due to the sup-
pression of T cell growth and cytokine secretion,
associated with downregulation of the T cell receptor
[668, 730, 870, 1864].
TNF inhibits tumor growth. It does this several ways.
In culture, TNF has a direct effect on many tumor cell
lines [250, 1921]. The addition of IFN-γ often has a syn-
ergistic effect [563, 2174]. In animals, TNF acts sys-
temically to bring about tumor regression [250]. TNF
may act directly on the tumor cells, but its most remark-
able effect is on tumor’s vascular endothelium [1435].
Vessels break down and clotting occurs, shutting off the
tumor’s blood supply. TNF also stimulates the immune
system and upregulates surface antigens involved in
tumor cell recognition. TNF and IL-2 synergistically
increased cytotoxicity in LAK cells [1517], NK cells
[1513] and TIL [1118, 2047, 2111].
TNF may be useful as an adjuvant in tumor vaccines.
In animal models, tumor cells engineered to produce
TNF induced immune response [30, 162, 775]. The
engineered cells were not as tumorigenic; animals with
regressed tumors were immune to subsequent tumor
challenge. Further, TNF-α has been used to produce
dendritic cells for immunization. Dendritic cells pre-
pared from bone marrow were grown in culture with
215
GM-CSF and TNF-α and then pulsed with specific
tumor antigens prior to inoculating mice [262, 1259].
TNF-α generally inhibits tumor growth, however,
there are cases in which it stimulates growth. TNF
appears to be an autocrine and paracrine growth factor
for certain ovarian cancers. TNF-α induced its own
production. IL-1 also stimulated TNF-α levels and
ovarian cancer growth [2207]. In the B16 mouse mela-
noma model, TNF-α stimulated tumor metastasis to
lung. The induction of VCAM-1 by TNF-α may have
been responsible [1486].
TNF-α causes thrombocytopenia. Studies in mice
showed this depended on TNFRI. Since platelets did not
have the TNFR, TNF-α appeared to be stimulating
platelet activation and consumption indirectly, possibly
by increasing thrombin, plasmin or 5-hydroxytryptam-
ine which are platelet agonists [1941].
Clinical studies with tumor necrosis factor in cancer
patients have been extensive. Unfortunately, the results
are disappointing. TNF-α has many functions, perhaps
too many to be useful clinically. At effective doses,
TNF-α causes substantial multiorgan toxicity, hypoten-
sion and flu-like symptoms [1318]. As a single agent, it
has not shown much antitumor effect [220, 522, 1836,
2149]. By administering TNF-α in isolated limb perfu-
sion [559, 1128, 1496], in organ perfusion [25] and
intravesicularly (bladder cancer) [1786], higher doses
may be used while containing the toxicity. In these set-
tings, TNF-α has been given in combination with INFγ
and chemotherapy (e.g. melphalan). This largely avoids
the systemic toxicities. More substantial effects have
been observed. Unfortunately, TNF-α administered
intrapleurally and intraperitoneally have not been very
useful in the control of malignant effusions [771, 1877].
Current strategies with TNF-α emphasize in vitro use to
produce dendritic cells and TNF gene transfected tumor
cells for vaccines. The TNF gene is also being inserted
into activated lymphocytes to exploit the capacity for
tumor-specific T cells to home into the target site where
TNF is released locally.
Recombinant soluble TNF receptor and antibodies to
TNF are being tested for control of TNF-related toxici-
ties during immunotherapy. In one study, soluble recep-
tor was administered in combination with IL-2.
IL-2-related toxicity (due to TNF release) was modu-
lated while the IL-2 antitumor response was preserved
[2020]. Thalidomide, the infamous sedative and terato-
gen [1291], is once again being prescribed as an immu-
nomodulatory agent and it is being tested in cancer
patients. Thalidomide suppresses TNF-α production,
inflammation and certain cell surface adhesion molecules
[1356, 1462, 1719]. It is also antiangiogenic [953].
216
TRAIL
Apoptosis in Neoplastic Cells;
Regulation of Inflammation
Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL, also called apo-2 ligand) is a 281 amino acid
transmembrane protein and a member of the tumor
necrosis factor family [1576, 2166]. Most leukocytes
and certain tumor cells express TRAIL [2166]. In T
cells, TRAIL is induced by activation and by IFN-α/β
[939, 1776]. Cyclosporin and glucocorticoids inhibit its
expression [1406, 2107].
There are at least five receptors for TRAIL; TRAIL-R1
(DR4) [1528], TRAIL-R2 (DR5) [1555, 1751, 1772,
2089], TRAIL-R3 (DcR1) [404, 1341, 1805], TRAIL-R4
(DcR2) [403, 1232, 1527] and osteoprotegerin [490].
TRAIL receptors are found on many cells, often together
with TRAIL. TRAIL-R1 and TRAIL-R2 contain cyto-
plasmic death domains and induce apoptosis in cells,
mainly neoplastic cells. TRAIL may induce apoptosis or
regulate cell growth in normal T cells, mast cells and
hepatocytes [138, 401, 888, 896, 1856].
By comparison with TNF-α and FasL, TRAIL has
little normal tissue toxicity [81, 19]. Two possible
explanations for this lack of toxicity are competitive
antagonism and induction of antiapoptotic proteins.
TRAIL-R3 and TRAIL-R4 bind TRAIL but do not
induce apoptosis. They lack cytoplasmic death domains
and thus serve as decoy receptors [80, 1227, 1555]. In a
similar way, osteoprotegerin is a soluble receptor that
binds TRAIL and competitively antagonizes its apop-
totic activity [490]. Cancer cells may produce OPG and
for these tumors OPG can function as a paracrine
survival factor [785, 1809].
Normal cells and TRAIL-resistant tumor cells gener-
ally express antiapoptotic proteins such as FLIP [671,
856, 2283] and IAP [403, 404, 420, 560, 2100]. These
interfere with caspase activation and function as sur-
vival factors. Drugs that control survival factors potenti-
ate anticancer effects of TRAIL [1140].
TRAIL is responsible for cell contact-induced tumori-
cidal activities of CD4 T cells [1985], NK cells [939],
monocytes [672] and dendritic cells [289, 510]. In CD4
cells, for example, studies using blocking and activating
antibodies showed that TRAIL-induced apoptosis is one
of the ways cytotoxic CD4 T cells kill melanoma [1985].
The upregulation of TRAIL on T cells may be respon-
sible for certain antitumor effects of interferons α and β
[939]. Dendritic cells can kill tumor cells directly by
TRAIL-induced apoptosis. In addition to decreased
tumor burden, DC-TRAIL provides apoptotic bodies
Cytokines
that stimulate dendritic cell maturation [1689] and serve
as antigen for immune response [20, 779].
TRAIL regulates inflammation and autoimmune
disease. This has been studied in mouse models for
autoimmune arthritis [1856] and encephalomyelitis
[757]. Blockade using soluble receptor to TRAIL exac-
erbated these diseases while TRAIL expression dimin-
ished them. TRAIL apparently decreases the activation
of autoimmune T cells.
The anticancer effects of TRAIL are remarkable but
complex. Mice with genetic TRAIL deficiencies had
increased risk for hematological malignancies [2276] and
methylcholanthrene-induced sarcomas [352]. TRAIL-
induces apoptosis in most melanoma cell lines [1985,
2283]. In human colon and breast cancers growing in
nude mice, activation of TRAIL receptors induced apop-
tosis and inhibited growth [81, 319, 2090, 2106]. These
studies show that TRAIL and TRAIL R1 and R2 may
have therapeutic potential in the treatment of cancer. But
not all cancers respond. And in certain tumors TRAIL
induces NF-κB, which promotes survival and growth
[480]. Drugs that inhibited NF-κB signaling sensitized
cells to apoptosis and increased anticancer response to
TRAIL [104, 1140, 1172, 2098].
Clinical trials have been initiated in cancer patients
using apoptosis-inducing TRAIL receptor antibodies
[1580, 2001].
TWEAK
Cell Growth, Survival;
Angiogenesis; Inflammation
TWEAK (tumor necrosis factor-like weak inducer of
apoptosis) is a member of the TNF cytokine family. It is
expressed in many types of cells. Tweak is synthesized
as a membrane-bound protein that is cleaved to release
a soluble, active, trimeric cytokine [304]. The TWEAK
receptor is Fn14. It is a TNF receptor family member.
Fn14 was discovered as a cell surface protein that was
induced by fibroblast growth factor [523, 2164]. Fn14 is
upregulated in injured blood vessels [2164] and in
regenerating liver [523]. The cytoplasmic tail binds and
signals through TNF associated factors-1, 2, 3 and 5
[206, 2164]. TWEAK activates NFκB, which translo-
cates to the nucleus to induce genes; TWEAK activates
caspases 3 and 8 in those cells that undergo Tweak-
induced apoptotic death [206, 2019].
In a cultured monocyte line, TWEAK induced the
cells to differentiate into osteoclasts. Fn14 was not the
receptor for this differentiation since the cell line lacks
Walter M. Lewko and Robert K. Oldham
Fn14 and inactivating mabs to Fn14 did not block the
Tweak effect. This study suggested the existence of a
second TWEAK receptor [1583]. It was recently reported
that CD163 is an additional binding protein for TWEAK.
It is present on monocytes and macrophages where
it is known to function as a scavenger for hemoglobin.
CD163 has been shown to compete with Fn14 for
TWEAK; may serve as an alternative receptor [185].
TWEAK is a remarkably proinflammatory cytokine. It
induces the expression of factors such as IL-6, IL-8,
RANTES, MCP-1, IP-10 and ICAM-1 in a number of
cell types including fibroblasts [305], keratinocytes [891],
synoviocytes [305], bronchial epithelial cells [2216],
macrophages [967], mesangial cells [237], astrocytes
[1702] and endothelial cells [713]. Typically, the addition
of IFN-γ has a synergistic effect. TWEAK levels are ele-
vated in tissues of mice with experimental autoimmune
encephalitis [417] and collagen-induced arthritis [1556].
TWEAK is angiogenic. It induces mitosis and migra-
tion in human endothelial and aortic smooth muscle
cells. FGF-2 and VEGF, which are also angiogenic,
stimulate TWEAK receptor levels. TWEAK induced
strong vessel growth in the rat cornea angiogenesis
assay [449, 713, 1180, 2164].
TWEAK appears to be involved in neoplastic pro-
gression. The Fn14 gene is rapidly induced during liver
regeneration; Fn14 is overexpressed in hepatocellular
carcinomas [523]. When human embryonic kidney cells
were transfected to express TWEAK, these cells formed
larger and more vascular tumors in nude mice [774].
Glioblastoma specimens and glioma cells in culture
overexpress Fn14. And when glioma cells were treated
with TWEAK, the cells became resistant to apoptosis-
based therapy. Tweak activates the NFκB path for
growth and survival. All told, blocking Fn14 may be of
benefit in the management of glioblastoma and other
TWEAK/Fn14 expressing cancers [2018].
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Abbreviations
ADC; antibody dependent cytotoxicity; AICD, activa-
tion-induced cell death; AP, antigen presentation; baso,
basophil; BCGF, B cell growth factor; BCSF, B cell
stimulatory factor; BM, bone marrow; CFR, cytokine
family receptor; CFU, colony forming unit; CNTF, cili-
ary neurotrophic factor; CTL, cytotoxic T lymphocyte;
DC, dendritic cell; DcR, decoy receptor; DR, death
receptor; EAE, experimental autoimmune encephalitis;
EGF, epidermal growth factor; ELR, glutamic acid-leu-
cine-arginine (a chemokine designation); eos, eosino-
phil; EPO, erythropoietin; ETA-1, early T lymphocyte
activation protein (OPN); FISP, IL-4-induced secreted
protein; γc, common gamma chain (IL-2Rγ chain);
G-CSF, granulocyte colony stimulating factor; GM-CSF,
granulocyte macrophage colony stimulating factor; gp,
glycoprotein w/mw (x 10−3); HLA, human leukocyte
antigen; HMW-BCGF, high molec wt-B cell growth
factor (IL-14); HVEM, Herpes virus entry mediator
(LIGHT R); IAP, inhibitor of apoptosis; ICAM, inter-
cellular adhesion molecule; ICE, IL-1β cleavage enzyme
= caspase 1; IFN, interferon; Ig, immunoglobulin; IL,
Cytokines
interleukin; ILA, induced by lymphocyte activation
(4-1BB); IL-1Ra, IL-1 receptor antagonist; IL-1RAcP,
IL-1 receptor accessory protein; IL-1RAK, IL-1 receptor
associated kinase; IL-1Rrp, IL-1 receptor related
protein (IL-18Rα); IL-TIF, interleukin 10-related T
cell-derived inducible factor (IL-22); IP-10, interferon-
induced protein-10; JAK, Janus kinase; -L (as a suffix),
ligand; LAK, lymphokine activated killer; LIF, leuke-
mia inhibitory factor; LIGHT, lymphotoxin-like induc-
ible protein that competes with glycoprotein D for
binding herpesvirus entry mediator on T cells; LIT,
lymphocyte inhibitor of TRAIL; LN, lymphnode; LPS,
lipopolysaccharide; LT-α, lymphotoxin-α (TNF-β),
LT-β, lymphotoxin-β; Macro, macrophage; MAPK,
mitogen-activated protein kinase; MCP, macrophage
chemoattractant protein; M-CSF, macrophage colony
stimulating factor; MHC, major histocompatibility
complex; Mig, monokine induced by IFN-γ; MIP,
macrophage inflammatory protein; MMP, matrix
metalloprotease; Mono, monocyte; neut, neutrophil;
NF-, nuclear (transcription) factor as in NF-κB; NGF,
nerve growth factor; NK, natural killer cell; NNT, novel
neurotrophin; nTreg, natural regulatory T cell; OSM,
oncostatin M; PAMP, pathogen-associated molecular
patterns; OPG, osteoprotegerin; OPN, osteopontin;
PBL, peripheral blood lymphocyte; PDGF, platelet
derived growth factor; PG, prostaglandin; p, protein w/
mw (x10−3); PMA, phorbol myristate acetate; -R (as a
suffix), receptor; RANKL, receptor activator of NF-κB
ligand (TRANCE); RANTES, regulated on activation,
normal T cell expressed and secreted; rhu- (as a prefix)
recombinant human; SAPK, stress activated protein
kinase; s- (as a prefix) soluble (not membrane bound);
SCF, stem cell factor; SCID, severe combined immuno-
deficiency disease; SDF, stromal derived factor; SLE,
systemic lupus erythematosus; SOCS, suppressors of
cytokine signaling; STAT, signal transducers and activa-
tors of transcription; Stk, stem cell tyrosine kinase
(flt-3), TACE, TNF-α converting enzyme; TCR, T cell
receptor; TDAC, tumor derived activated T cell (TIL);
TGFβ, transforming growth factor-β; Th, helper T cell;
TIL, tumor infiltrating lymphocyte; TIMP, tissue inhibi-
tor of metalloprotease; TLR, toll-like receptor; TNF,
tumor necrosis factor; TNFR, TNF receptor; TPO,
thrombopoietin; TRADD, TNF Receptor 1-associated
death domain; TRAF, TNF receptor-associated factor;
TRAIL, TNF-related apoptosis-inducing ligand;
TRANCE, TNF-related activation induced cytokine
(RANKL); Treg, regulatory T cell; TWEAK, Tumor
necrosis factor-like weak inducer of apoptosis; tyk,
tyrosine kinase; VCAM; vascular cell adhesion molecule;
VLS, vascular leak syndrome.
9 Interferons: therapy for cancer
DAVID GOLDSTEIN, ROBERT JONES, RICHARD V. SMALLEY, AND ERNEST C. BORDEN
Stimulate the phagocytes. Drugs are a delusion. Find the germ of
the disease; prepare from it a suitable antitoxin; inject it three times
a day, a quarter of an hour before meals and what is the result? The
phagocytes are stimulated; they devour the disease and the patient
recovers-unless, of course, he’s too far gone.
– Sir Ralph Bloomfield Bonnington in The Doctors Dilemma
George Bernard Shaw, 1902.
Isaacs and Lindemann, in England, first characterised
interferon (IFN) in 1957 and coined the word to signify
a protein, elaborated by virus-infected cells, that func-
tions to prevent their infection by a second virus [108].
However, difficulties with chemical isolation and char-
acterization led to great skepticism about the molecule’s
existence; indeed, “the scientific community dubbed the
discovery ‘imaginon’ ” [192].
Time and effort have proven Isaacs and Lindemann
right. Interferons are now a well-characterized group of
proteins. They are a large family of four major immuno-
logical types, alpha, beta, gamma, and omega [199].
The alpha and beta interferons were collectively known
as the type I interferons while gamma interferon was
referred to as type II interferon. There are multiple natu-
ral alpha interferon molecules and one natural beta
interferon molecule. These type I interferons share a
common receptor which is present on nearly all cells in
the body. The alpha interferons, originally derived from
leucocytes, and beta interferon, originally derived from
fibroblasts, are actually secreted by nearly all mamma-
lian cells. Even though they share a common receptor,
their activation pathway must differ since the transfec-
tion of the human type I receptor gene into murine cells
allows alpha but not beta expression [266]. Gamma
interferon, on the other hand, is produced by T lympho-
cytes upon specific antigen recognition.
All interferon molecules have antiviral and antiprolif-
erative capacity, and to some extent immunomodulatory
activity, although gamma is a stronger immunomodula-
tory molecule than the type I interferons [19]. The enor-
mous amount of clinical study of the interferons in
malignancy established the field of biologic therapy of
cancer. The many new agents currently under investiga-
tion to inhibit angiogenesis and the aberrant growth fac-
tor networks associated with malignancy have all taken
advantage of the insights provided during the clinical
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
study of the role of interferons as anti neoplastic agents.
A review of these interferon studies remains a valuable
resource of guidelines on how to optimise clinical studies
in malignancy using novel non-traditional agents.
Nomenclature
Alpha interferon is produced following viral stimula-
tion. There are at least 14 subtypes of alpha interferon,
each immunologically related but differing by a number
of amino acids [290].
Several nomenclature systems exist for identifying
these subtypes. The two commonly used, but unrelated,
systems use either lettering A, B, C, D, etc., or number-
ing 1, 2, 3, etc. to define the subtypes. A newer pro-
posal, approved by the Nomenclature Committee of the
International Society for Interferon Research, has been
recently published [199]. A corresponding proposal for
nomenclature of the interferon-gene-induced proteins
exists.
Three natural alpha interferon pharmaceutical prod-
ucts are currently available for use in patients in various
countries in the world. The original Finnish Red Cross
partially purified material developed by Cantell and sub-
sequently also produced by others from the buffy coat of
peripheral blood is available as Finnferon® in some
European countries. A highly purified natural alpha inter-
feron produced by virally stimulated human lymphoma
(Namalva) cells has been produced in pharmacologic
quantity by Burroughs Wellcome Co (now Glaxo
Wellcome) and is known by the trade name Wellferon®.
It is marketed in Japan, Canada, Mexico and Europe
but not the United States for treatment of hairy cell leu-
kemia (HCL) and some viral disorders. Alferon N® is an
aqueous formulation of human alpha interferon proteins
with a specific activity of 2 × 108 IU/mg of protein and is
manufactured in the United States by the Purdue
Frederick Company. It has been approved in the United
States for the intralesional treatment of refractory or
recurring external condylomata acuminata in patients
over 18 years of age. These natural alpha interferons
contain most, if not all, of the multiple alpha molecules.
277
278 Interferons: therapy for cancer

In addition to the natural alpha interferon products, found in the native molecule. Betaseron® is approved in
there are several pure single alpha molecules obtained the United States for the treatment of ambulatory patients
by recombinant technology that are available for clini- with relapsing-remitting multiple sclerosis. Biogen, Inc
cal use. Interferon alpha A (alpha 2) has been pharmaco- has produced a recombinant molecule from the natural
logically produced as recombinant alpha interferon by beta interferon gene, which, since it is produced in eukary-
three companies, Hoffman-LaRoche in Nutley, NJ; otic cells, is glycosylated. This product, with the trade
Schering Corporation based in Kenilworth, NJ, and name Avonex®, has been approved in the US for treatment
Boehringer Ingelheim in Germany. These products are of relapsing forms of multiple sclerosis.
known as Roferon A® (rIFN alfa 2a), Intron A® (rIFN Serono also has a beta interferon molecule interferon
alfa 2b) and Berofor® (rIFN alfa 2c), respectively. The beta-1a for multiple sclerosis. Two recombinant gamma
alpha subtype, interferon alpha C (alpha 10) has been interferon molecules have been cloned and were clini-
produced in pharmacologic quantity in Israel. cally developed, one by Genentech, Inc., and one by
Roferon A® is manufactured by Roche Laboratories Biogen, Inc.
using recombinant DNA technology employing a geneti- These will be referred to, respectively, as rIFN gamma
cally engineered E coli bacteria containing an interferon (Genentech) with a trade name of Actimmune® and rIFN
alpha 2 gene obtained from a human myeloid leukemia gamma (Biogen) with a trade name of Immuneron.
cell line; it has an approximate MW of 19,000 daltons Actimmune® is manufactured by bacterial fermentation
and a specific activity of 2 × 108 IU/mg protein and is of a strain of E coli containing the DNA, which encodes
approved for use in the United States for the treatment of for the human gamma interferon molecule. It has a
HCL and AIDS-related Kaposi’s sarcoma in patients 18 specific activity of 30 million units/mg protein, and is
years of age or older. Intron® A is produced by Schering approved for clinical use in the United States for reducing
Corporation and is obtained from the bacterial fermenta- the frequency and severity of serious infections associ-
tion of a strain of E coli which bears a genetically engi- ated with chronic granulomatous disease. Immuneron is
neered plasmid containing an interferon alpha 2 gene not yet approved for prescription use in the US. These
from human leukocytes. It has a specific activity of various products are listed in Table 1. The generic desig-
2 × 108 IU/mg protein and is approved in the United nations will be used throughout this chapter.
States for treatment of patients 18 years of age or older Intracellular signaling in response to binding of type
with HCL and for selected patients with condylomata one and two interferons to their respective receptors has
acuminata, AIDS related Kaposi’s sarcoma, chronic hepatitis been carefully dissected out. Both subtypes of interferon
non-A, non-B/C and with chronic hepatitis B. result in binding and phosphorylation of Janus activated
Pegylated interferon alpha has been extensively stud- kinase molecules (Tyk2, Jak 1 and 2) followed by phos-
ied in hepatitis [140, 152] and initially in renal carci- phorylation of signal transducers and activators of tran-
noma [165] and chronic myeloid leukemia [111]. It scription (Stats) which translocate to the nucleus and
reduces frequency of administration (once/week versus activate interferon stimulated genes [198, 113].
three) and has an improved toxicity profile. A recombi-
nant IFN beta, with a single amino acid substitution
separating it from the natural beta molecule, was devel- Table 1. Interferon nomenclature
oped by Triton Biosciences, Inc., Berkeley, CA, and is
IFN Generic Trade name
currently manufactured by Berlex and marketed by
Chiron Corporation (US) and Schering AG (Germany) Natural alpha interferon
under the trade name Betaseron® for treatment of mul- Cantell IFN IFN alfa (Le) Finnferon®
Lymphoblastoid IFN alfa N1 Wellferon®
tiple sclerosis. It is obtained by bacterial fermentation of Leukocyte derived IFN alfa N3 Alferon N®
a strain of E coli that bears a genetically engineered Recombinant alpha interferon
plasmid containing the altered gene for human inter- Alpha A rIFN alfa 2a Roferon A®
feron beta. The native gene was obtained from human Alpha A rIFN alfa 2b Intron A®
fibroblasts and altered in a way that substitutes serine Alpha A rIFN alfa 2c Berofor®
Alpha C rIFN alfa 10
for the cysteine residue found in position 17 in the native
Recombinant beta interferon
molecule (interferon betaser17). rIFN beta 1B Betaseron®
The recombinant protein is a highly purified molecule rIFN beta (Biogen) Avonex®
with 165 amino acids, a MW of approximately 18,500 Recombinant gamma interferon
daltons and a specific activity of approximately 32 MU/mg rIFN gamma (Genentech) Actimmune®
protein; it does not include the carbohydrate side chains rIFN gamma (Biogen) Immuneron‫‮‬
D. Goldstein et al.
Clinical use
In the early 1970s, work by Kari Cantell and co-workers
led to the production of sufficient quantities of alpha
interferon, made from buffy cell layers, to support lim-
ited clinical trials in patients with several types of malig-
nancies [253, 254, 50]. This work was expanded in
North America by the U.S. National Cancer Institute
(NCI) which sponsored trials beginning in 1975 and by
the American Cancer Society (ACS) which supported
trials beginning in 1978.
Despite very limited clinical information, interferon
was heralded in the popular press at the time as a signifi-
cant new drug for the cure of cancer. The early clinical
trials with this partially purified natural alpha interferon
demonstrated response rates in breast cancer, osteosar-
coma and lymphoma, comparable to those achievable
with chemotherapeutic agents. The wave of enthusiasm
and outpouring of venture capital rapidly led to the
development of DNA recombinant technology and, fol-
lowing the successful cloning in 1979 by Taniguchi et al.
of beta-interferon [259] and the subsequent cloning of
the alpha interferon subtypes and gamma interferon, the
production of recombinant interferon molecules.
Furthermore, the isolation and purification of natural
alpha Interferon from Namalva cell cultures led to far
greater availability of natural alpha interferon and the
number of clinical trials increased dramatically.
The partially purified IFN alfa (Le), used in the early
trials sponsored by the NCI and ACS, had a relatively
low specific activity and, since it was a supernate, was
composed of not only a variety of subtypes of alpha
interferon but multiple other cytokines as well. [95, 18]
working with patients with breast cancer in the US [50]
working with patients with ovarian carcinoma in
Scandinavia, reported response rates of over 20% using
this natural alpha interferon product. These results have
not been reproduced in these tumors using rIFN alfa
2a or 2b, raising the question of what other active
constituent(s) in this interferon preparation may have
induced these responses.
B Cell Malignancy
Several B cell malignancies have been shown to be
therapeutically sensitive to alpha interferon. Multiple
myeloma and non-Hodgkin’s lymphoma (NHL) were
among the first malignancies to respond in the early
trials with the Cantell interferon.
Hairy cell leukemia (HCL) was shown in the early
1980s to be exquisitely sensitive to alpha interferon and
279
this biologic was rapidly approved in most countries for
the treatment of this previously untreatable malignancy.
Hairy Cell Ieukemia
Hairy cell leukemia, a B cell malignancy, is a disease
with an exquisite sensitivity to alpha interferon and was
the first human malignancy to be so identified. Prior to
the development of the interferons, no adequate therapy
existed for this relatively rare disorder. Splenectomy was
the standard approach when treatment was necessary and
was useful for controlling pancytopenia for a period of
time but had no effect on the leukemic process. Quesada
was the first to demonstrate the beneficial effect of alpha
interferon in this disease in 1982 [205].
After finding that a dose of 12 MU/m2 was poorly tol-
erated, he and his colleagues at the MD Anderson
Cancer Center, arbitrarily settled on a regimen of 3 MU
(1 MU = 106 units) administered daily subcutaneously.
Several other investigators followed suit and have
shown that high response rates are attainable with the
potential for a prolonged response duration, even with
lower doses.
Seventy-five percent to 80% of patients will obtain
major clinical benefit with improvement in hematologic
parameters and a decrease in the leukemic (tumor cell)
population. Treatment of several months’ duration is
required for maximal benefit and continued treatment is
necessary to maintain clinical benefit. Equivalent effi-
cacy has been shown with each of the alpha interferon
products and with beta interferon [69, 82, 207, 284, 285,
76, 77, 83, 84, 72, 280, 242]. Gamma interferon is inef-
fective in this disease [209].
Following Quesada’s lead, multiple studies by Golomb
et al. at the University of Chicago, established an objec-
tive anti-leukemic response (CR or PR) rate of 20–25%
and an improvement in hematologic parameters in another
60% for an overall major clinical benefit in 80–85% of
patients [82, 83, 84]. Following cessation of therapy,
patients relapsed but remission could be successfully
reinduced [208, 84].
The studies by the groups at MD Anderson and the
University of Chicago with recombinant alpha inter-
feron led to the approval of alpha interferon in the
United States for the treatment of HCL. A significant
cost benefit associated with interferon treatment was
demonstrated [190] and additional studies with natural
alpha interferon demonstrated its superiority over sple-
nectomy, the inadequate standard of therapy prior to the
development of alpha interferon [244].
During the mid-1980s, investigators in Innsbruck
began to explore the question of dose effect in patients
280
with HCL and determined that a dose as low as 0.5 MU
(500,000 U) was biologically active and immunologi-
cally stimulating, as measured by the production of
neopterin [105, 69]. Subsequent comparative studies
with rIFN alfa 2c [72] and IFN alfa N1, showed the low
dose was as effective as the standard dose in inducing a
return to normal of the platelet count (within 36–38 days
median) and of the neutrophil count (within 90–130
days median), as well as reducing the need for red cell
transfusion support and the incidence of infection. The
standard dose, however, was clinically and statistically
more effective in its antileukemic effect, i.e., reducing
hairy cell infiltration in the marrow [242].
However alpha interferon has been supplanted by
cladribine [289, 26]and pentostatin [112] as the treat-
ment of choice, just 1 decade after it was shown to be the
first effective systemic agent for the treatment of this dis-
ease. Its role as an additional agent has not proven to be
significant [153]. Of interest a new biologic agent – anti
cD22 monoclonal antibody combined with an exotoxin
has shown activity in cladribine resistant disease [128].
Thus the promise shown by interferon that a biologic
agent could have an anti tumour impact and modify the
natural history of a malignant disease has been kept. It
is likely that more targeted biologic therapy will remain
an integral part of the treatment of this disease.
Multiple Myeloma
Interest in the treatment of multiple myeloma with alpha
interferon was initially stimulated by the pilot studies of
Mellstedt et al. in 1979 and the ACS-supported trial
reported in 1980 [160, 187]. Each study demonstrated
objective anti-tumor responses in a small number of
previously heavily treated patients. A number of phase
II trials of both natural and recombinant alpha interferon
were subsequently performed and cumulatively sug-
gested that about 20% of patients with multiple
myeloma, refractory to cytotoxic chemotherapy, might
obtain an objective response to a variety of doses and
schedules [38, 276, 181, 25, 36, 180, 206, 37].
Because of these encouraging results in previously
treated patients, alpha interferon was compared with cyto-
toxic chemotherapy in previously untreated patients but
chemotherapy proved superior in terms of both response
rate and duration of response [4, 147]. Several trials were
organized to evaluate the addition of alpha interferon to
various combinations of cytotoxic chemotherapy in terms
of response induction [35, 227, 114, 182].
One of these a large Swedish trial involving over 300
patients evaluated the addition of IFN alfa (Le) admin-
istered on days 1–5 and days 22–26 of every 4-week
Interferons: therapy for cancer
cycle to melphalan/prednisone which were given on
days 1–4 and demonstrated a benefit in overall response
and survival for patients with either IgA or Bence-Jones
myeloma but not IgG myeloma [188]. This study was
the third in a series of trials by this group to demonstrate
a clinical benefit from this natural alpha interferon in
patients with IgA myeloma. The explanation for benefit
only for patients with IgA myeloma in this series of trials
is not apparent [227].
A study of VMCP with or without alpha interferon
randomised 240 patients, there was no difference in
response rate but fewer patients receiving interferon
progressed and the median duration of response was 6
months longer (12.4 months versus 18.3 months, p < 02)
for patients receiving alpha interferon [146]. A subse-
quent study evaluated the addition of alpha interferon to
a five-drug combination (VBMCP) [182]. In 628
patients the complete response rate was increased 18%
versus 10% and there was an increased time to progres-
sion with rIFN alfa2, 30 months versus 25. Of interest
the best response was seen in the IgA subgroup. A small
Australian study of adding interferon alpha 2a to inten-
sive combined chemotherapy failed to show a benefit in
either response rate or overall survival [114].
Two meta-analyses have now examined the clinical
advantage of adding interferon to chemotherapy. Ludwig
et al. performed a global analysis on 30 trials evaluating
a total of 3,948 randomized patients, 17 of which added
interferon and concluded there was a slight (6.6%)
advantage in response rate and median relapse free sur-
vival (4.8 months) and overall survival 3.1 months
[146]. The subsequent individual patient meta-analysis
of 24 trials with 4,012 patients confirmed the slightly
higher response rates (57.5% versus 53.1%) and pro-
gression free survival – 33% versus 24% at 3 years, a
22% reduction in risk.
The median time to progression was increased by 6
months and the risk of death decreased by 8% [92].
However, any advantage of adding interferon to induc-
tion chemotherapy in this disease is too small to be
clinically meaningful and is probably not worth the
added financial cost (US$42,236.19/life year saved) or
side effects. Open to further exploration however is the
persistent intriguing observation of the possible benefits
in patients with IgA/Bence-Jones myeloma.
Several trials were next established to evaluate the
use of alpha interferon as a maintenance agent in patients
with myeloma. Following the induction of a response
with cytotoxic chemotherapy, various groups random-
ized patients to receive alpha interferon or no further
therapy until the onset of progressive disease [151, 223,
195, 279, 20, 16, 146, 225].
D. Goldstein et al.
In addition, the Nordic Study Group randomized
patients at the start of treatment; those randomized to
receive alpha interferon received it continuously [91].
These studies are summarized in Table 2. The US
cooperative group trial and the trial in Germany showed
no benefit from interferon therapy. The other seven trials
have demonstrated either a prolonged time to progression
or duration of plateau phase in the interferon maintained
group. Ludwig et al. also performed a global analysis for
these maintenance studies also and concluded that alpha
interferon both relapse free survival by 4.4. months and
overall survival by 7.9 months at a cost of US$18,114.95
(Figures 1 and 2) [146]. The individual patient meta
analysis showed an increase in progression free survival
at 3 years of 27% versus 19%, a 34% reduction in risk of
progression and a 12% decreased risk of death [92].
Median survival is increased by 4 months.
Such an effect also needs to be balanced against not
only cost but also quality of life. In that regard a recent
study suggests that after 12 months there is no signifi-
cant difference in quality of life in two Nordic mainte-
nance studies [282]. A recent study with pegylated
interferon suggests it may further improve quality of life
in this setting [240].
A randomised trial of maintenance alpha interferon,
following high dose melphalan consolidation with bone
marrow transplant rescue has also been performed. rIFN
alfa 2b had a beneficial effect on prolonging remission,
especially in those patients who achieve a complete
remission 46 versus 27 months at 52 months of follow
up and in improving survival at 52 months 95% versus
75%. However by 77 months most patients had died and
the benefit was not sustained [40]. In addition the
European marrow transplant registry has reported in a
Table 2. IFN versus CT in CML
IFN effect on Dur
Group/reference Comparison of Resp survival
Italian Alpha IFN versus CT + +
study group
(202)
German study Alpha IFN versus + +
group (75, 76) busulfan
Alpha IFN versus HU + Neg
Japanese study Alpha IFN versus + +
group (128) busulfan
UK study group Maintenance with α + +
(7) IFN versus no
maintenance
HU = Hydroxyurea
281
retrospective analysis of 473 patients that received
maintenance IFN compared to 419 patients who did not
that overall survival and progression free survival were
significantly better – 78 months versus 47 months and
29 versus 20 months. The retrospective nature of the
report requires cautious interpretation [15]. A third study
from the Nordic Group also used maintenance inter-
feron following high dose therapy and showed a benefit
for this program over a historic control but the role of
interferon itself in prolonging survival cannot be deter-
mined [137].
In summary, alpha interferon is an active agent in
myeloma. Current data support its greatest usefulness
when used as an agent capable of prolonging remission
in patients with low tumor burden; in some of these
patients it may also improve survival.
Non-Hodgkin’s lymphoma (NHL)
Several clinical trials conducted in the late 1970s with
the Cantell interferon suggested efficacy in patients with
low and, to a lesser extent, intermediate grade NHL
[161, 95, 144, 104].
Single agent trials in the 1980s with the more highly
purified recombinant and natural alpha interferons con-
firmed the efficacy of alpha interferon in patients with
NHL [64, 177, 136].
Two single institutions and the CALGB, in three sep-
arate trials, combined alpha interferon with an alkylat-
ing agent in patients with low grade NHL and
demonstrated the combination to be relatively non-toxic
and well tolerated. The combination induced a response
in 50–75% of patients, more readily in previously
untreated individuals [29, 28, 189, 30].
The group at MD Anderson, in a single institution
non-randomized study, evaluated the use of IFN alfa N1
as maintenance therapy, inducing a response with CHOP
plus Bleomycin and then treating patients with IFN alfa
N1 until relapse [157]. They concluded, based on his-
torical controls, that alpha interferon prolonged the
duration of response but did not improve survival.
Subsequently a large number of randomised trials have
been reported in complete or abstract form evaluating,
by direct comparison, the addition of alpha interferon to
cytotoxic chemotherapy in the initial treatment of the
disease.
In studies using less intensive regimens such as
chlorambucil or CVP there has been no benefit on over-
all survival and at best a marginal improvement in pro-
gression free survival in one study [197, 96, 216]. There
have been two large prospectively randomized trials
282 Interferons: therapy for cancer

Study start year, Prog’sions/Patients Statistics O.R. & Cl* Odds Redn.
code and name IFN None (O-E) Var. (IFN : None) (SD)

Interferon in Induction:
85G GATLA 3-M-85 13/16 14/15 -2.6 6.3 34% (33): P = 0.5
86I Rome IFN 1 15/21 16/19 -4.2 6.5 45% (29): P = 0.1
86J MGCS 1986 97/105 61/69 -2.9 37.9 -5% (17): P = 0.6

87E EMSG 2 (1) 32/70 37/56 -7.9 16.7 35% (19): P = 0.6
88A EOOG 9486 123/161 121/153 -13.9 59.2 21% (12): P = 0.07
89H KIF, Avicenne 43/97 42/95 1.3 21.2 -7% (22): P = 0.8

90D NMSG 04–90 83/125 89/134 -16.4 44.6 31% (15): P = 0.01
90H ALSG Myeloma II 14/24 21/26 -8.0 7.9 64% (22): P = 0.016

90J Ital NHLSG (I) 11/28 15/21 -4.7 6.0 54% (20): P = 0.04
91B GMM (I), Mexico 20/40 14/40 0.7 6.9 -51% (40): P = 0.6

Subtotal 451/685 440/638 -52.7 213.4 22% (6)

(65.8%) (69.0%) reduction
P = 0.0003
Test for heterogeneity between trails: c20 = 15.8; P = 0.07

Interferon in maintenance:
85D Ital. MMSG M84 35/50 44/51 -14.0 18.0 54% (15): P = 0.001

87C SWOG 8624 78/107 83/104 -8.1 39.9 15% (14): P = 0.2
87D NCI-C MY6 66/89 80/92 -21.1 33.9 45% (15): P = 0.0003
87H MGWS (extended) 45/51 62/64 -23.5 23.6 63% (15): P < 0.0003
89B EMSG 2 (M) 22/46 37/54 -8.8 14.6 45% (15): P = 0.09
89E GMTG MM02 41/52 56/65 -5.0 24.0 10% (16): P = 0.3
89F Royal Marsden 31/42 33/42 -6.0 15.5 20% (21): P = 0.1
89A MRC-MYEL-6e 118/143 116/140 -12.8 57.8 20% (12): P = 0.00
89E CMN (M). Mexico 9/13 7/8 0.6 1.1 -21% (120): P = 0.5

90B PETHEMA 38/50 40/42 -8.7 18.4 39% (19): P = 0.01

90K Ital. NHLSG (M) 30/44 26/48 0.5 14.3 -4% (27): P = 0.9

91E GERM 36/70 46/66 -10.0 19.4 40% (16): P = 0.02

Subtotal 549/787 635/776 -116.7 -260.5 34% (5)

(71.6%) (81.8%) reduction
P < 0.00001
Test for heterogeneity between trials: c211= 21.7; P = 0.03

Total 1000/1452 1075/1414 -169.4 493.9 29% (4)

(68.9%) (76.0%) reduction

95% CI for total 98% CI for individual trials

0.0 0.5 1.0 1.5 2.0
IFN None
better better
Test for heterogeneity (22 trials): c231 = 41.0; P = 0.008 Effect P < 0.0001
Test for heterogeneity between subtotals: c21 = 3.5; P = 0.08

Figure 1. Meta-analysis of impact of interferon on progression free survival (adapted from report in the Br J Haematol, ref.
227)
D. Goldstein et al. 283

0 1 2 3 4 5 6+ years
100

Estimated percentage still progression free

90

80

70

60

50

40
32.6%

30
22.9%
20.0%
20 23.8% 5.3 % SD 2.0
(logrank
- allocated IFN (% ± s.d.) 16.2% 2P < 0.00001)
10 - allocated none (% ± s.d.) 14.7%

0
0 1 2 3 4 5 6+ years
Progressions/period–years
IFN 412/1214 237/770 184/450 57/287 27/176 28/224
None 561/1102 319/558 115/300 45/129 16/113 16/114

Figure 2. Progression free survival for the addition of interferon to standard treatment of multiple myeloma (adapted from
report in the Br J Haematol, ref. 227)

performed by cooperative groups in the United States and an improved overall survival (not reached versus 5.6
and Europe evaluating the effect of alpha interferon years, 10% of patients withdrew because of IFN related
added to four-drug induction cytotoxic chemotherapy toxicity and 28% required dose reduction).
[244, 246]. Both trials added alpha interferon to a The initial ECOG publication reported an improve-
CHOP-like induction regimen; the Group d’Etude des ment in survival but a subsequent analysis a year later,
Lymphomes Folliculaires (GELF) treated only patients although demonstrating a consistent 10% increase in
with follicular lymphoma but with bulky (>7 cm) dis- survival over a 5 year period, did not show statistical
ease while the ECOG in the United States studied significance [8]. A study of quality of life has similar to
patients with bulky or symptomatic low grade and inter- the myeloma study supported a benefit in terms of qual-
mediate grade NHL. The ECOG used cyclophosph- ity adjusted life years for IFN treatment [32].
amide, Oncovin®, prednisone and Adriamycin® (COPA) By contrast a large SWOG study in 571 patients com-
as the chemotherapy regimen, administering alpha inter- paring eight cycles of ProMACE 9 day 1-MOPP (day 8)
feron for 5 days (D22–26) of every 28 day cycle for a with and without interferon alpha as maintenance for 2
total of 8–10 months. years showed no benefit in either progression free or
The GELF used cyclophosphamide, VM 26, predni- overall survival [62].
sone and Adriamycin® monthly for 6 months and then In addition to these reported trials, there have been
every 2 months for an additional 12 months giving alpha two small trials examining the issue of dose, one show-
interferon three times a week for the entire 18 months. ing a dose response [68, Gams, 1990, personal commu-
Although the two trials were very similar in design, nication] the other no statistical difference in response
there were some important differences. Both groups rates. The question of influence of dose and schedule
treated patients with bulky, symptomatic disease, all fol- remains unanswered because of the very small numbers
licular lymphoma in one, and follicular and diffuse in of patients.
the other while the aggressiveness and length of treat- A meta-analysis only reported in abstract form [215]
ment with both chemotherapy and alpha interferon was – summarised eight randomised trails involving 1,756
different. patients. It suggested that maintenance treatment in trials
The GELF demonstrated a better response rate in the with more intense initial therapy showed a 14% survival
group receiving interferon, but had an unusually low advantage at 5 years (74% versus 60%) and 19% at 8
response rate in the chemotherapy-only arm. The GELF years, but no benefit for lower initial intensity treatment.
study demonstrated an improved duration of response Thus Alpha interferon may be an active therapeutic
and time to treatment failure 2.9 years versus 1.5 years agent in patients with NHL, low grade, but its role has
284
yet to be clarified. Optimal dose and schedule have yet
to be defined and the question of whether higher, less
tolerable doses are needed for maximal effect remains
unanswered. Additionally, alpha interferon may be most
active in patients with lower tumor burdens, i.e. with or
following CHOP-like regimens.
In high grade NHL a small randomised study showed
no benefit to 1 year of maintenance therapy following
initial therapy [10].
However a recent survey has shown some evidence
of activity in relapsed high grade disease [9] and trials in
the post transplant setting for relapsed high grade
patients are ongoing. The whole role of IFN is now
uncertain following the approval of anti CD-20 mono-
clonal antibody (mabthera – see chapter).
Its activity and excellent toxicity profile for relapsed
low grade lymphoma both alone and conjugated with
radionucleide and the increasing data on its use as initial
therapy in both low grade and high grade NHL [88]
make it a very attractive biologic agent.
However given that patients may ultimately relapse
following mabthera therapy the role of IFN alpha to
enhance the effectiveness of this therapy requires fur-
ther study [41]. In addition novel directions in combina-
tion to avoid chemotherapy are being examined [120].
Other lymphomas
Several small phase II studies have been performed
in-patients with cutaneous T cell lymphoma (CTCL),
chronic lymphatic leukemia (CLL), and adult T cell leu-
kemia- lymphoma (ATLL). Alpha interferon has been
shown to have an anti-tumor effect in patients with these
disorders. Bunn et al. initially showed that rIFN alfa 2a
induced responses in patients with CTCL. Responses
were seen in both cutaneous and extracutaneous sites
[21, 22].
These encouraging results were followed by a
confirmatory report in a small series from Duke and
Northwestern [185]. Three complete and ten partial
responses (response rate-59%) were induced and the
suggestion of a dose–response relationship was demon-
strated. Investigators from Northwestern University
then combined rIFN alfa 2a with psoralen plus ultraviolet
light irradiation (PUVA) in a Phase I dose escalation
trial in which the dose of alpha interferon was escalated
from 6 to 30 MU IM tiw [132].
This escalation was based on the steep dose–response
relationship observed in the earlier trials. A complete
response was obtained in 12 patients (80%). Several
phase II trials in patients with cutaneous T cell lym-
phoma have confirmed this high response rate [133,
222, 283]. In one long term follow up report the best
Interferons: therapy for cancer
response was seen in earlier stages (Ia, b, Iia), frequency
of complete response was maximal by 6 months. 57%
relapsed within 1 year but 175 had a very prolonged
complete response with a mean of 31 months [115]. One
randomized trial has been reported comparing the rela-
tive value of the combination of PUVA and IFN to retin-
oic acid and IFN [249]. The PUVA combination had a
higher complete response 70% versus 38% and overall
response 80% versus 60%.
Once again the recent reporting of an IL-2 toxin conju-
gate with efficacy in the resistant disease setting, although
at a cost in terms of toxicity [249] suggests that biologic
therapy will have an ongoing role. Combination of IFN
with emerging new therapeutics is continuing [226].
Several small pilot trials have evaluated the use of
alpha interferon in patients with CLL [64, 229, 177,
193]. Alpha interferon appears to have a mild to moder-
ate ability to decrease the population of circulating leu-
kemic cells, especially in patients with early or minimal
disease who have not received prior therapy. However,
none of these studies strongly suggest a clinically sig-
nificant benefit from alpha interferon as a single agent.
ATLL has been etiologically associated with infec-
tion with HTLV-1, a human retrovirus endemic in south-
ern Japan and the Caribbean basin [201]. All three
interferons, alpha, beta, and gamma have been shown to
have an anti-tumor effect occasionally durable, in a
small percentage of patients with the acute form of this
disease. A multi-institutional group studied the two drug
combination of rIFN alfa 2b and AZT in 19 patients and
induced a major response in 11 patients, including a
complete response in five [78]. Some of these patients
had disease resistant to cytotoxic chemotherapy. Several
of the responses were durable.
Chronic Myeloid leukemia (CML)
CML is a triphasic disease, consisting of chronic, accel-
erated, and blastic elements. Management options
include stem cell transplants, alpha-interferon, the
tyrosine kinase inhibitor (STI571) and chemotherapy.
The choice of therapy depends on the phase of the dis-
ease and the characteristics of the patient. Interferon has
most effect in the chronic phase as initially found by
Talpaz et al. at MD Anderson [256]. This group con-
ducted a series of uncontrolled trials, which initially
demonstrated the ability of interferon alphã2a to control
leucocytosis and subsequently demonstrated its ability
to reduce the size of the Ph positive clone and to induce
a clinical and histologic CR. Large multi-institutional
randomized trials have since demonstrated the superior
efficacy of alpha interferon compared to cytotoxic treat-
ment in terms of haematological response and survival
D. Goldstein et al.
and, until recently, it was established as the treatment of
choice for patients with CML who were not eligible for
a bone marrow transplant.
Interferon Monotherapy; Early Studies
Based upon the demonstration of in vitro antiprolifera-
tive activity of alpha interferon against CFUs [183, 174,
210, 12]. Talpaz and co-investigators initiated a series
of trials in the early 1980s initially using partially puri-
fied alpha interferon in patients with CML and subse-
quently with recombinant alpha interferon molecules.
Their initial observations that alpha interferon could
reduce the size of the malignant clone of cells was sub-
sequently confirmed; treatment with alpha interferon
reduced the number of Ph positive cells in over half of
the responding patients.
About 20% of patients obtaining a conversion to a
cytogenetically normal marrow, confirmed by molecular
studies [255, 256, 257, 288]. This is a true pathologic CR.
Such an effect had met with only limited success using
aggressive cytotoxic chemotherapy [241]. It is now
clear that patients gaining a major partial or complete
cytogenetic response have improved survival.
Randomized Studies of Interferon
Monotherapy Versus Chemotherapy
A number of prospectively randomized studies have
confirmed the superiority of interferon monotherapy
over chemotherapy in the treatment of early chronic
phase CML (Table 2).
The Italian Cooperative Group first demonstrated
alpha interferon’s superiority over hydroxyurea with a
karyotypic response rate of 30% in the interferon group
versus 5% in the chemotherapy group and significantly
improved median survival in patients given interferon
[265]. A prospectively randomized study by the German
CML Study Group compared the use of IFN alpha 2a,
given at a daily dose of up to 9 MU SC, with standard
cytotoxic chemotherapy either busulfan or hydroxyurea
[98, 99]. The number of patients with a reduction in the
Ph1 positive clone and the time to progression to accel-
erated or blast crises were both increased in the inter-
feron group compared to either cytotoxic agent.
The overall survival of patients was superior in the
group of patients treated with alpha interferon ther-
apy but statistical significance was demonstrable only
when interferon was compared to busulfan. This high
daily dose was poorly tolerated, however, and 16% of
patients had to discontinue alpha interferon therapy.
The financial cost of interferon therapy was also
substantially higher than for chemotherapy. Further
285
studies from Japan and the UK have reached similar
conclusions [179].
In the Japanese study, newly diagnosed patients with
CML in chronic phase were randomized to receive
either alpha Interferon or busulfan. Although a complete
cytogenetic response occurred in two patients receiving
busulfan (not previously noted with this agent), there
was a statistically and clinically significant difference in
favor of alpha interferon in terms of major cytogenetic
response (13 of 80 patients) and in predicted 5-year sur-
vival rate. The UK study using a different approach
demonstrated superiority of alpha interferon over no
maintenance therapy after induction of a response by
either busulfan or hydroxyurea [6].
Interferon in combination with cytotoxic
drugs in the treatment of CML
Interferon has been used in combination with cytarabine
(ara-C) following work showing ARA-C could selec-
tively suppress CML clones in vitro and also demonstrate
antitumour activity when given as a low dose continuous
infusion in vivo.
The MD Anderson experience of combining inter-
feron alpha with ara-C has been addressed by Kantajaran
et al. They initially reported on a group of 60 patients
with advanced phases of CML in 1992 showing that a
study group receiving daily alpha interferon and inter-
mittent ara-C had a better CHR compared to historical
controls receiving interferon alone. A subsequent report
on 140 patients with Ph positive early chronic phase
CML in 1999 [117] showed that the schedule of ara-C
may also be important. The study group received com-
bination treatment of IFN-alpha (5 × 106 U/m2 daily)
and low dose ara-C (10 mg/m2 daily), compared to his-
torical controls receiving interferon (5 × 106 U/m2 daily).
A significantly improved CHR (92% versus 84%) was
seen with low dose continuous versus intermittent ara-C
and although there was a trend to an improved major
cytogenetic response this was not statistically significant
(50% versus 38%, p = 0.06).
Two randomized studies using a combination of
interferon with cytotoxic drugs have also been reported.
Guilholt et al. (240) reported on 721 patients with previ-
ously untreated early phase CML. Patients received
hydroxyurea (50 mg/kg/day) with interferon (5 × 106 U/
m2 daily) with or without cytarabine (20 mg/m2/day for
10 days each month). There was a significantly improved
CHR (66% versus 55%) and also a survival advantage
at 3 years (86% versus 79%) in the patients given the
ara-C. An Italian study [220] randomized 540 patients
with Ph positive chronic phase CML to receive daily
interferon alone or in combination with ara-C (40 mg/
286
kg/day subcutaneously for 10 days/month). The com-
bined treatment group had a significantly increased
major and complete cytogenetic response rate (28% ver-
sus 19%) and Kaplan Meier calculated survival benefit
of 85% versus 80% at 3 years.
Interferon is clearly highly active in the treatment of
chronic phase CML being able to induce major cytoge-
netic responses reflecting a real survival benefit over
conventional cytotoxic therapy. Combining it with ara-C
appears to increases activity further. However there are
a number of issues that should be considered. Firstly
one should assess the relative benefits of carrying out a
stem cell transplant.
The full details required cannot be covered in the context
of this chapter but in young patients with a good perfor-
mance status a transplant should still be regarded as the
treatment of choice. In addition the dosing and duration of
interferon treatment to achieve maximal anti-tumour effi-
cacy must be balanced with its toxic side effects.
Although their studies were uncontrolled, the MD
Anderson group has amassed a significant amount of
experience and believes that relatively high doses,
administered daily, are required for maximum beneficial
effect [118]. However, a number of their patients require
dose reduction because of lassitude, neurologic problems
and/or thrombocytopenia and neutropenia.
By contrast a subsequent analysis of randomised trials
of the MRC and HOVON showed no benefit for high
dose versus low dose [127]. With regard to duration it
appears that interferon only very rarely elicits a com-
plete molecular response even when a complete cytoge-
netic response is achieved, so there was a real motivation
to obtain a more efficacious but less toxic therapy.
The advent of the tyrosine kinase inhibitor Glivec has
filled that niche [45] reported in patients with chronic
phase CML that had failed interferon therapy showing
that of 54 patients receiving more than 300 mg 53 had
complete haematological responses, with 29 obtaining
cytogenetic responses (17 of which were major or com-
plete) with very little in the way of toxicity. This success
has lead to the rapid FDA approval of Glivec in the
treatment of interferon resistant disease. Subsequent
studies in first line therapy led to its adoption as the
standard of care [176].
However the development of resistance to Glivec
therapy occurs both in chronic phase disease and blast
crisis and the mechanisms of resistance have been anal-
ysed at a molecular level [85]. Despite new targeted
agents, interferon may well still have an important role
as an additional therapeutic option.
Alpha interferon also has clinical utility in patients
with other myeloproliferative disorders including essen-
Interferons: therapy for cancer
tial thrombocytosis and polycythemia rubra vera [238,
79, 94, 135, 258, 261]. Control of the markedly elevated
platelet count, decreasing the risk of resultant life-
threatening complications can be obtained in nearly all
patients with this disorder. The red cell mass, in patients
with PRV, can also be reduced leading to symptomatic
improvement [236]. Pegylated interferon can provide
these benefits with some reduction in toxicity [224].
Solid Tumors
Alpha interferon has a demonstrable beneficial effect in
patients with some solid tumors; patients with malignant
melanoma, renal cell carcinoma, and AIDS related Kaposi’s
sarcoma have benefited by treatment with alpha interferon.
Alpha interferon has been approved in the US for the treat-
ment of two of these tumors, AIDS-related Kaposi’s sar-
coma and for high risk patients with malignant melanoma
following surgical removal of the primary lesion.
Kaposi’s Sarcoma
Trials in patients with Kaposi’s sarcoma (AIDS) have
indicated that about 40% of patients will obtain an
anti-tumor response [130]. There has been the sugges-
tion of a dose–response effect, with better results com-
ing with treatment in the range of 20–50 MU/m2 [43,
90, 212, 270].
Most of these studies indicate benefit is most likely in
patients with stage II and III disease, in patients with the
absence of a history of opportunistic infection, and in
patients with a relatively good lymphocyte (>15,000/
mm3) and helper lymphocyte (CD4) count (>400/mm3).
Patients with advanced stage disease and/or poor
immune status are less likely to respond [73].
An analysis of 364 patients has suggested prolonged
survival for patients with the highest CD4 counts [56].
Studies of interferon in combination with chemotherapy
have not shown any benefit over interferon alone [129].
Studies evaluated the combination of interferon with
AZT and suggested synergy against the HIV virus and
possibly also against the tumor, which is itself virally
induced by HHV-8 [155]. A prospective randomized
trial has shown that moderate doses of IFN, at least
8 mU/m2 are needed [232]. A small randomised trial
against chemotherapy has further supported its use with
an increase in survival of 24 versus 13 months [186].
Unfortunately with the exception of those patients with
high CD4 counts, the length of response is only approxi-
mately 6 months and, a significant beneficial effect on
survival in the majority of patients remains undefined.
D. Goldstein et al.
Once again this indication may be of limited use as
the dramatic impact of effective combination antiretro-
viral therapy has sharply reduced the incidence of kapo-
sis sarcoma and should always be the first approach in a
new patient with Kaposis Sarcoma. Furthermore excel-
lent palliation without the toxicity of IFN is now possi-
ble with liposome encapsulated doxorubicin (Doxil).
Melanoma
The role of interferon 2 alpha in the treatment of mela-
noma has been investigated in the context of metastatic
disease and also as adjuvant therapy following the
removal of lesions at high risk of recurrence.
Although there are a number of trials that have
addressed the role of interferon in metastatic disease most
of the current emphasis has been on defining the exact
role of interferon in the adjuvant setting and this will form
the main point of discussion in this section.
Advanced Disease
Response rates in patients with advanced melanoma
vary from 5% to 27%, with an average of 15% [39, 122]
and an intravenous schedule has allowed higher doses to
be used with less associated toxicity [122]. Metastatic
malignant melanoma is a difficult malignancy to treat.
The response rates for single agent alpha interferon
compare favorably with those of single agent cytotoxic
agents, but response rates with single cytotoxic agent
therapy are low compared to combinations of cytotoxic
drugs [156]. Thus there has been interest in combining
interferon with cytotoxic drugs. An early study random-
izing patients to receive DTIC 200 mg/m2 intravenously
for 5 days every 4 weeks with or without interferon
(given as 15 MU/m2 for 5 days a week over 3 weeks and
then 10 MU/m2 sc three times a week) showed a statisti-
cally significant improvement in response rate and median
survival in the interferon group.
Unfortunately a larger follow up study as well as a
number of other trials have failed to demonstrate a sta-
tistically significant advantage of adding interferon to a
variety of cytotoxic regimens [58].
Considering the additional toxicity afforded by inter-
feron it would therefore appear it has relatively little to
offer in the metastatic setting. However a recent meta-
analysis of 3,273 patients in 20 randomised trials was
recently carried out assessing single-agent DTIC versus
combination chemotherapy with or without immuno-
therapy in metastatic melanoma [107].
This included 926 patients in five trials that utilized
interferon and showed that the combination of DTIC
287
with interferon produced a tumour response rate 53%
greater than with DTIC alone (95% CI 1.10–2.13) but
this was not translated into a survival benefit.
There have also been studies that have used interferon
in combination with interleukin 2 as additional therapy
to conventional cytotoxic drugs. Although there is fre-
quently an increased response with the addition of
Immunotherapy, statistically significant survival advan-
tages are again lacking [119, 217, 55]. This conclusion is
also supported by an additional meta-analysis which also
examined chemotherapy combined with both interferon
and interleukin-2 similarly showing improved response
rates without a survival gain [110].
Thus, considering the considerable toxicity afforded by
such therapy, further studies are unlikely to be of value.
Adjuvant Therapy
A number of trials in the last few years have addressed
the role of alpha interferon as adjuvant therapy after sur-
gical removal of high risk lesions. These have been
designed to test whether interferon improves survival
and if so which disease stage will benefit and how
intense the treatment must be.
The most highly influential of these was the ECOG
E1684 trial which included patients with a T4 lesion,
with or without the presence of local lymph nodes, and
any other primary lesion with proven lymph node
involvement (resected IIB or III) [125]. After surgery
patients were randomized to receive high dose inter-
feron, consisting of an induction phase of 20 MU/m2 for
5 days every week for 4 weeks followed by a mainte-
nance phase of 10 MU/m2 for 3 days a week over 48
weeks in total, or observation. The Kaplan-Meier analy-
sis showed a statistically significant improvement in
disease free survival (26% versus 37%) and overall sur-
vival (37% versus 46%) in the treatment group and led
the FDA to approve the use of interferon alpha in this
patient group. However there was substantial toxicity
experienced in the treatment group and it was hoped
that a less toxic equally efficacious protocol could be
generated.
Thus there was considerable interest in the results of
the WHO melanoma program where lower less toxic
doses were employed. This study included patients with
stage III disease using a 3 MU dose of drug three given
times weekly compared with observation.
Unfortunately, although data is still being generated
from this trial, no global overall or relapse free survival
benefit has yet been demonstrated indicating a low dose
protocol may be ineffective in this patient group [24].
This has been further confirmed in two studies of low
288
dose interferon alone [97, 126] and two in combination
with interleukin-2 and retinoic acid respectively. This
has been reinforced by a further three arm ECOG study,
E1690, evaluating the efficacy of high dose interferon,
as documented in the 1684 study, compared with a lower
dose (3 MU/day three times weekly for 2 years) and
simple observation [123]. This study again showed a
significant improvement in relapse free survival between
the high dose treatment and observation alone.
However there was no relapse free survival advantage
in the low dose treatment compared to observation. Further
support for high dose adjuvant interferon was forthcoming
in the recently published ECOG study, E1694.
This trial was designed to compare a high dose inter-
feron with a markedly less toxic GM2 ganglioside vac-
cine, thought to be active in melanoma, to see if the
vaccine offered a more tolerable alternative. Here the
interferon arm was clearly superior after an interim anal-
ysis thus leading to premature closure of the study [124].
All these studies therefore advocate the use of high dose
interferon in the adjuvant setting for patients with posi-
tive nodal disease. However there are a number of issues
that are cause for discussion. Firstly in the 1690 study
where high dose, low dose and observation arms were
compared there was no statistically significant overall
survival advantage in having the high dose treatment
compared to observation alone. This was is in contrast to
the earlier E1684 study where a clear overall survival
benefit was apparent. Of note the overall survival in the
observation arm in the later E1690 study approached that
of the treatment arm in the E1684 study.
This has not been completely rationalised but one
possibility considers the availability of salvage inter-
feron treatment in those patients relapsing in the obser-
vation arm of E1690. This was not available for patients
relapsing in the E1684 study and thus most of the obser-
vation arm patients in the E1690 study differed from
those in E1684 as they would have received interferon
at some point.
It is possible therefore that any overall survival ben-
efit apparent for the adjuvant treatment in E1690 may
have been diluted. Intuitively this may argue that the use
of interferon could be delayed until relapse considering
the toxicity it causes in the adjuvant setting. However
one must remember that although other studies show
interferon does have activity in metastatic melanoma
this has not been translated into a survival benefit (see
previous section). In addition a follow up study address-
ing quality of life issues in the 1684 trial using Q-TWIST
analysis revealed that in spite of toxicity the adjuvant
treatment group had more quality of life adjusted sur-
vival time than the observation group [33]. This is
Interferons: therapy for cancer
clearly highly persuasive and if such data is also made
available from the E1690 trial it would reinforce the
advantages of adjuvant high dose interferon treatment.
In spite of the above data there are two studies using
low dose interferon have indicated some therapeutic
efficacy in certain patient groups. For example Grob et
al. [89] compared 18 months of low dose interferon with
observation alone in 489 patients with resected disease
of >1.5 cm depth but with no nodal involvement. They
showed a statistically significant improvement in dis-
ease free survival and a trend towards overall survival.
Similar results were also published by Pehamberger et al.
using a year of interferon treatment (196). Those patients
without nodal involvement may therefore represent a group
that could be treated with a less aggressive protocol.
The length of treatment required is uncertain as a
shorter course would clearly improve the toxicity.
Although no randomised trials are testing node positive
patients with very short interferon courses the E1697 is
currently looking at 1 month of HDI versus observation
in T3 N0 resected disease. By contrast a trial – EORTC
18952 comparing two intermediate doses of IFN alpha
2b in high risk melanoma patients showed no benefit
[49a]. Recent data on pegylated interferon alfa-2b
shows similar benefits but with possible reduced toxic-
ity [49b]
A recent meta-analysis concluded that the use of HDI
should be considered in the adjuvant treatment of resected
stage IIB and stage III melanoma because of improve-
ments in disease free and 2 year mortality [268].
The important questions as to whether the full
extended course is required, and whether one may be
able to better select patients who will particularly ben-
efit from treatment remain unanswered. However for
stage IIA disease a low dose protocol may be appropri-
ate. In metastatic disease the additional issue of at best
very modest survival benefits versus additional toxic-
ity mean that treatment should be tailored on an indi-
vidual patient basis and clearly selection must be
careful.
Renal Carcinoma
Alpha interferon role in this malignancy may well now
be only of historical significance. It has an anti-tumor
effect in about 12–15% of patients with metastatic
renal cell carcinoma (RCC) [281, 204] and may be
more effective in patients with less bulky disease and
when given in higher doses [154, 61, 168, 273, 228,
231, 211].
A sufficient number of randomised trials have now
been reported to allow a clear picture of the role of
D. Goldstein et al. 289

single agent interferon. Interferon either alone or in The response rate to interferon of 3% was surpris-
combination with vinblastine is more effective than ingly low however. The benefits of combining interferon
medroxyprogesterone [34] or vinblastine [204]. As a with any other agent appear limited [173, 66, 204, 7,
result a Cochrane Meta analysis [281] analysed 42 164, 46, 165, 166]. The role of interleukin-2 is addressed
studies involving 4,216 patients and showed an average in great detail in another chapter. However it is instruc-
response rate of 10.2% with 3.2 5 CR, a median survival tive to review the results of a recent randomised trial of
time of 11.6 months and 2 year survival of 22%. They interferon alfa versus IL-2 monotherapy versus the
estimated a pooled survival hazard ratio of 0.78 for IFN combination compared to medroxyprogesterone [171].
treated patients. They suggested that IFN alfa 10 MU In 492 patients no survival benefit was shown for either
S.C. three times per week should be the control arm for drug [172].
future studies (Table 3). The recent very impressive results with the antiangio-
A large randomised study of interferon gamma genic agents such as the VEGF inhibitors and their con-
[80] showed that this cytokine is inactive. It also sistent superiority over interferon has made them the
demonstrated that spontaneous remissions do occur standard of care [167, 54, 106]. However at least one
with significant frequency 6% (CI 2.5–13.2%), higher study showed a benefit to the combination of bevaci-
than previously reported and a 15.7 month median zumab and interferon which means it remains an option
survival was noted for this early stage metastatic for exploration but the lack of efficacy of the combination
group [53]. This suggested that the activity of inter- of temsirolimus and interferon compared to temsirolimus
feron alfa in this disease needs to be assessed against alone means that issues of toxicity may differ with each
that standard, at least in good performance early stage drug combination. Other approaches such as dendritic
disease. The role of interferon as adjuvant therapy cell vaccines [103, 131] and autologous transplantation
post nephrectomy showed no survival benefit [203, [27] may still be the subject of combined therapy also.
264, 200].
The benefit of nephrectomy as part of a combined
surgical-biotherapeutic approach has been examined.
Other Solid Tumors
241 patients were randomised to either interferon alone Aside from the three solid tumors listed above, there
5 mU three times per week or nephrectomy plus inter- have been trials in many other types of malignancy.
feron. The survival in patients allocated to nephrectomy There are some encouraging leads. rIFN alfa 2a has been
was superior 12.5 months versus 8.1 months [63]. shown to enhance the cytotoxic effect of 5-FU in vitro
and, in pilot clinical trials, the combination induced a
partial response in over 60% of patients [5, 274, 275].
Table 3. Recent randomised Trials of IFN alpha in renal cell However, a prospective randomized trial evaluating
carcinoma the addition of alpha interferon to 5 FU in patients with
12 advanced colorectal cancer has demonstrated no benefit,
months 24 only added toxicity, from the addition of alpha inter-
RR MST survival months
feron [102].
N (%) (weeks) (%) (%)
By contrast the development of combination therapy
VBL #260 81 2.5 37.8 38 19
with oxaliplatin, irinotecan and the addition of bevaci-
IFN + VBL 79 16.5 67.6 56 38
IFN #121 53 11 34 N/A zumab and cetuximab to them has opened new avenues
IFN + VBL 66 24 24 N/A and will be covered in other chapters. Other trials have
IFN #263 82 12 N/A demonstrated clinical benefit from interferon therapy in
IFN + VBL 83 8 N/A patients with carcinoid tumors [163, 178]. Improvement
MEGACE 176 7 24 32 13
IFN #261 174 14 36 43 22
in symptomatology associated with a decrease in
IFN #269 139 6 88 22 10 5-hydroxyindoleacetic acid levels has been reported in
IFN + 145 12 128 28 19 approximately 50% of patients but objective regression
Cisretinoicacid is much less frequent.
IFN #275 123 4 34 N/A In addition trials using interferon with other agents
IFN + 123 4 50
Nephrectomy such as octreotride have indicated that combined treat-
IFN #267 147 8 56 12 ment may improve efficacy [67]. The role of other tar-
IFN + IL-2 140 19 72 20 geted agents such as bevacizumab is being explored and
MEAN IFN 11 36 combination with these VEGF targeting agents may
Response need to be explored. There has also been encouraging
290
results using interferon with 13-cis retinoic acid as bio-
adjuvant therapy in locally advanced head and neck
squamous cell carcinoma following experimental evi-
dence that these agents may work synergistically in this
tumour [141, 233].
Once again the positive results with cetuximab and
chemotherapy may overtake this work (review else-
where). Anti-tumor activity has also been demonstrated
with a similar drug combination in patients with
squamous cell carcinoma of the cervix [142]. Although
it is relatively ineffective in patients previously treated
with radiation therapy [278].
Mode of Action
Alpha interferon has an antiproliferative effect, antiviral
effect, an immune augmenting effect, and a differentiation
effect and an anti-angiogenic effect. Any one or combina-
tion of these effects may induce an anti-tumor response.
Gamma interferon (Type II IFN) is a more potent
immune stimulator of monocyte function and class II
HLA activity than are either alpha or beta interferon
(Type I IFNs) [269]; however, on a weight basis, the
Type I IFNs are ten times as potent as gamma interferon
in their antiviral effect.
Antiproliferative
In vitro studies and murine models have been used to
demonstrate the anti-proliferative action of interferons
[87, 262]. Decreased tumorigenicity has also been shown
in cells pretreated with human interferon [17, 87].
Whether these models are applicable to human tumors
in vivo remains conjectural.
Cell cycle analysis has shown that interferon causes
extension of all phases of the cell cycle and prolonga-
tion of the overall cell generation time [121, 194, 252].
In some cases, an accumulation of cells in G0 has been
observed, accompanied by a decrease in transition to G1.
This decrease in growth rate may be incompatible with
cell life [262]. The critical antiproliferative mechanisms
operative at the cellular level have not been elucidated,
but it is possible that they may be mediated by the same
inhibitors of DNA and RNA synthesis that are found in
virus-infected cells.
Specifically, induction of 2′5′-oligoadenylate (2,5 A)
synthetase leads to endoribonuclease activation, which
in turn inhibits RNA transcription by degrading mRNA
linked to dsRNA [213, 230]. In addition, a protein kinase
and a phosphodiesterase pathway represent mechanisms
of inhibition of protein synthesis which are parallel to,
Interferons: therapy for cancer
but independent of, 2,5 A synthetase [230]. Whether
these are, in fact, central to antiproliferative as well as
antiviral activity remains speculative [260].
Because of its high degree of sensitivity to the Type I
interferons, HCL would appear to be an ideal disease in
which to determine a mechanism of action for interferon’s
antitumor effect. Type I interferon receptors are present on
hairy cells and are down regulated with therapy [13, 59].
A lack of demonstrable down regulation in vivo has
been associated with lack of response [14]. Immunologic
recovery as manifested by a return of NK activity and
normalization of T and B cell counts has been docu-
mented [175] but responses occur without improvement
in NK activity [70,71]. Further, hairy cells were not sus-
ceptible to NK cytotoxicity in vitro [235]. It has also
been shown that alpha interferon, when cultured with
peripheral blood mononuclear cells of patients with
HCL, gives rise to multi-lineage colonies composed of
myeloid and erythroid progenitors – evidence for the
existence of circulating hematopoietic stem cells respon-
sive to a differentiation effect of interferon [162].
It has also been shown that the mononuclear cells of
HCL are defective in their ability to release tumor necro-
sis factor (TNF) and that interferon overcomes this –
perhaps interferon and TNF then act synergistically as
antiproliferative or cytotoxic agents [2, 3]. The low dose
– standard dose randomized study with IFN alpha N-1
in patients with HCL supports both of these theories;
both doses were comparable in their ability to induce an
increase in platelet and neutrophil counts (differentia-
tion) while the standard dose was more effective as an
antileukemic (antiproliferative) agent [242]. This is the
only prospectively randomized clinical evidence avail-
able to support a dose-response effect. Ford et al. and
subsequently Pagannelli et al. have shown that HCL
proliferation in vitro required B cell growth factor
(BCGF) and Pagannelli et al. have shown that Type I but
not Type II interferon inhibits the effect of BCGF [191,
65]. These observations strongly suggest that interfer-
on’s primary anti-tumor effect is an antiproliferative
one, at least in this tumor setting. Most recently a down
regulation of telomerase activity in human malignant
heamatopoietic cell lines may have identified a novel
antiproliferative mechanism [286].
Antiviral Activity
That interferons have antiviral properties is well estab-
lished and alpha interferon is approved in many coun-
tries for the treatment of several forms of viral hepatitis.
Whether this effect is the basis for interferon’s antitu-
mor effect in tumors possibly related to viral etiologies,
D. Goldstein et al.
such as Kaposi’s sarcoma associated with AIDS and
ATLL, is unknown.
Although other tumors may be of viral origin, most
prominently cervical carcinoma, there has not been a
major investigative effort mounted in this area and cur-
rently the antiviral effect of alpha interferon remains
unexploited in the treatment of malignancy.
Immune Modulation
Support for immune stimulation as a mechanism of
action is found in experiments with animals, in which
tumors known to be interferon-resistant in vitro have
regressed when the animal received systemic treatment
with interferon [42, 87]. A variety of immune changes
have been described, but the most relevant appear to
be the effects on natural killer (NK)-cells and
macrophages.
In vitro work with human lymphocytes shows that
interferon increases the cytotoxic potential of NK-cells
by recruitment of pre-NK-cells, increasing activity of
activated cells, and by augmenting NK-cell-mediated
antibody-dependent cell-mediated cytotoxicity [100,
194]. Alpha and beta interferons also appear to cause NK
activation at a lower dose and over a shorter time interval
than does gamma interferon [263]. In clinical trials, both
the dose and route of interferon administration have been
shown to effect NK-cell activity significantly.
Several trials have suggested that low-dose inter-
feron results in more marked NK-cell activity [51, 134,
47, 48]. This is a confusing issue, however, since oth-
ers have reported widely differing effects on NK-cell
activity, varying from a consistent increase [52, 202,
145] to a consistent decrease [143, 148, 149, 149, 247]
in addition to individual variation without discernable
pattern [170]. Much of the controversy may be related
to the dose of interferon used. Unpublished work from
the NCI by Varesio et al. [267] has suggested a bell-
shaped curve for both the antiproliferative effect and
immune augmentation. These curves do not precisely
overlap.
A few clinical studies have examined the long-term
immunologic effects of alpha interferon treatment;
again, these differ with respect to whether there is an
increase [134, 237] or decrease [148, 149, 150] in NK
activity. A study by Silver et al. [237] (p. 175) showed
that low dose alpha interferon led to an increase in NK
activity within 48 h, which was, however, not sustained
despite continued administration.
Repeated high-dose interferon gave a more sustained
increase of NK activity in the long term, even though it
did not lead to the same initial rise as low-dose inter-
291
feron. However, intravenous administration was used for
the high dose, and intramuscular administration for the
low dose, and the schedule of therapy was also different
(alternate weeks for low-dose versus monthly for high-
dose). Both uncontrolled factors, i.e., route of adminis-
tration and schedule, might have caused the differences
noted. An earlier study with lymphoblastoid interferon
showed a dose-related decrease in NK activity over a
6-week period, after an initial stimulatory effect; but in
that instance the treatment was given three times a week
[134]. This suggests that an intermittent schedule may
result in different patterns of response.
Since interferon consistently stimulates NK-cells in
vitro, the inability to reproduce consistent effects of
interferon on NK-cell activity in clinical practice sug-
gests a need to explore confounding in vivo effects, such
as scheduling, dose, or sampling method (peripheral
blood versus tumor site). Furthermore, both Type I and
Type II interferons have been shown to induce resis-
tance to NK activity in vitro. The controversy surrounding
NK-cell function after interferon treatment suggests that
it may not be a useful immunologic marker.
Macrophages, more consistently than NK-cells, are
affected by gamma interferon; indeed, gamma inter-
feron has all the activities of a macrophage-activation
factor (MAF) [42, 269]. Gamma interferon acts to
increase the number and density of Fc receptors, which
in turn are associated with an increase in antigen-pre-
senting function. Enhancements of phagocytosis and
also of antiviral activity and cytotoxicity occur [194,
252]. Thus, assays of macrophage/monocyte function
may be of more relevance for assessment of gamma
interferon action, whereas NK or antiviral activity may
be the best choice for alpha or beta interferons.
In addition to functional studies, changes in serum
molecules, particularly neopterin, tryptophan and beta 2
microglobulin have been found to be very consistent
markers of biologic response to interferons. Their rela-
tionship to the degree of immune modulation in vivo is
unclear but recent studies with alpha, beta and gamma
interferons have shown the optimal biologic dose to be
well below the maximum tolerated dose [71, 72, 81].
The ability to increase cell-surface antigen expression
presents another aspect of interferon action that may be
important in tumor control. Augmentation has been
demonstrated for the expression of Fc receptors on
lymphocytes and of Class I and II (Ia) MHC antigens
[42, 194, 252] on several other cell types as well.
In addition to augmentation of expression, interferon
has also been shown to induce HLA expression in a
variety of normal cells [219]. In neoplastic cells, inter-
feron has been shown to induce HLA antigen expression
292
and augment tumor-associated antigen (TAA) expres-
sion in several cell lines [74, 86, 75, 23]. This work has
been expanded and augmentation has been demon-
strated in vitro in tumor cells obtained from malignant
effusions and following the intraperitoneal administra-
tion of gamma interferon to patients [81, 93].
The increased expression of TAA occurring concur-
rently with enhancement of macrophage antigen-pre-
senting function, may improve the endogenous antitumor
activity of macrophages.
The actions of interferon in promoting tumor antigen
expression suggest another major line of inquiry, namely,
the combination of interferon with monoclonal antibody
(MoAb) therapy, with or without linkage to a toxin [218].
One of the major stumbling blocks to MoAb therapy has
been modulation or poor expression of tumor antigens; if
pretreatment with interferon can increase antigen expres-
sion, then the effectiveness of subsequent MoAb treat-
ment might be greatly enhanced. However, the risk of
similar expression in nontarget areas must be kept in
mind, since it is often the level of expression rather than
the novel nature of the antigen which characterizes the
tumor cell. At least one early trial has demonstrated
potential therapeutic effect [277].
The group at Stanford University and IDEC Pharma-
ceutical Corporation in California has performed an
interesting trial. These investigators previously reported
the use of anti-idiotype antibodies in patients with B cell
lymphomas, obtaining a response in five of ten patients
including 1 CR [158, 159]. Patients ultimately failed
because of the emergence of a dominant clone of anti-
gen (idiotype) negative cells [158, 159]. An animal
model was developed to evaluate therapeutic modalities
in this setting. Using this model, it was shown that a
synergistic effect was obtained with the combination of
anti-idiotype antibodies and interferon [11]. rIFN alfa
2a, 12 MU/m2 SC tiw, was combined with anti-idiotype
therapy and administered to twelve patients.
Responses were obtained in nine (with 2 CR), not sub-
stantially different from the results with anti-idiotype
therapy alone. Interferon failed to prevent the emergence
of idiotype negative clones. Currently ongoing studies
evaluating the combination of alpha interferon and anti
CD-20 monoclonal antibody may be clinically more
encouraging [41].
Differentiation
Interferons have been shown to have a variety of effects on
differentiation. In vitro differentiation has been enhanced
in mouse myeloid leukemia cells and in erythroblasts of
the Friend leukemia system, whereas conversion of mouse
Interferons: therapy for cancer
3T3-Li cells into adipocytes and of human monocytes to
macrophages has been inhibited [219, 252].
Other evidence supporting an effect on differentiation
includes an increased expression of HLA antigens,
enhanced excitability of nerve cells, and decreased beat-
ing frequency of myocardial cells after interferon treat-
ment [219]. Interestingly, sodium butyrate, a well known
differentiating agent, enhances this effect of interferons in
vitro [219]. An important finding – which may suggest
one cellular mechanism for inducing differentiation – is
the evidence of decreased c-myc and c-Ha-ras gene
expression following interferon treatment, with as much
as a 60% decrease in mRNA formation [31]. With 3T3
fibroblasts, this decrease in mRNA has been associated
with a reversion to normal cell type. Thus, interferon-
induced differentiation appears to be a significant effect
and may be part of the antineoplastic action of interferon.
Anti-Angiogenesis
Inhibition of experimental angiogenesis by interferons
was first demonstrated in a mouse tumor model [234].
This observation was confirmed in infants with life-
threatening hemangiomas [57]. Hemangioma regression
and significant clinical benefit was demonstrated in
more than 80% of seriously effected infants.
Successful angiogenesis is essential for the expansion
of any malignant tumour. It is a complex process involv-
ing extensive interplay between cells, soluble factors
and extracellular matrix component. The mechanism
through which interferons mediate this inhibition is
unclear but inhibition fibroblast growth factor (FGF)
pathways appears to play some part. For example IFN-
alpha can inhibit FGF induced endothelial cell prolifera-
tion and both IFN-alpha and beta can down-regulate the
expression of FGF in human carcinoma cells [101, 239].
In addition experiments in nude mice have shown that
IFN-alpha can lower the expression of FGF and this
correlates with a decreased blood vessel density and an
inhibition of growth of ectopically implanted tumours
in these animals [44].
In the clinical setting interferons have been shown to
be effective in producing antiangiogenic effects in a
number of tumours. These include Kaposi’s sarcoma
[56], bladder carcinoma [248] and giant cell tumour of
the mandible [116]. However the intense interest in
angiogenesis has lead to the development of a number
of newer antiagiogenic agents [139]. Although a num-
ber of these have become standard of care, the pleiotro-
pic activities of interferon in combination may still need
to be understood to add to the overall antineoplastic
effect of some targeted agents [287]
D. Goldstein et al.
Side Effects
While side effects of the Interferons can be debilitating,
most appear to be reversible upon cessation of therapy.
As one might expect, high doses give rise to more severe
manifestations than low doses. The major documented
short term side effects are fevers, headache, and myal-
gia, while fatigue, a long term side effect, can be severe
enough to be dose-limiting.
Gastrointestinal side effects, in particular, anorexia,
nausea, vomiting and/or diarrhea, are neither universal
nor severe, but may lead to weight loss, which can be
profound with high-dose or prolonged moderate-dose
administration. Elevated serum transaminase (but not
clinical hepatitis) has also been noted [134, 245, 237].
Hypotension with higher doses of both alpha and gamma
interferons can be dose limiting. Both granulocytopenia
and thrombocytopenia have occurred, but are rapidly
reversible [237, 15] and rarely dose limiting unless the
patient has had extensive prior radiation. Margination of
leukocytes rather than direct marrow suppression
appears responsible.
Central nervous system toxicities, including paresthe-
sias, weakness, somnolence, decreased attention span,
short-term memory impairment, confusion, depression,
personality change, and even coma at very high doses,
have all been seen. EEG abnormalities, including a
slowing of the dominant alpha rhythm and diffuse slow
waves, have been documented [214, 245, 1, 60].
The potential of depression to limit beneficial therapy
such as adjuvant treatment of melanoma has led to a
randomised study of paroxetine, which substantially
reduced the incidence from 45% to 11%. Although only
a small number of patients were studied, its prophylactic
use may well improve the therapeutic index for higher
dose IFN therapy [169].
Exacerbation of coronary artery disease has been
reported. Although cardiac toxicity may be life-threatening,
serious cardiac toxicity due to interferon is unusual.
A significant history of coronary artery disease is a rela-
tive contraindication for interferon therapy, particularly
at high doses. Life-threatening acute pulmonary toxicity,
renal failure, and unexplained sudden death have also
been noted on rare occasions.
Evidence has increased indicating that long term
treatment with interferons results in the production of
antibodies to interferon. In a study of 51 patients with
hairy cell leukemia, neutralizing antibody was detected
in 16 patients, six of whom developed some degree of
clinical resistance [250].
In a previous review of more than 617 patients who
received intramuscular interferon alfa 2a, 25% were
293
reported to have neutralizing antibody [109]. Antibody
formation is a significantly less frequent phenomenon in
patients treated with either rIFN alfa 2b or IFN alfa N1
[251, 271, 272]. Of the three allelic genes used in the
development of recombinant interferon alfa 2, the gene
used to produce rIFN alfa 2b is by far the more common
allele in North American individuals [138].
Summary
This review of the clinical effects of the interferons indi-
cates that alpha interferon, when used as a single agent,
has antitumor effects in a large number of malignancies,
perhaps more than any single chemotherapeutic agent
does. However, like many cytotoxic agents, the use of
interferons in combination with cytotoxic drugs, other
biologics, or antiviral agents remains under explored.
Clinically, the interferons proved to be a significant addi-
tion to our therapeutic armamentarium and served well
as the prototype for subsequent more targeted biological
therapeutics. Interferon has overcome two misleading
labels at opposite ends of the spectrum: of “imaginon”
when most scientists doubted its existence, and more
recently that of “magic bullet,” when the public was led
to believe it might be the cure-all for cancer. In the con-
text of cancer treatment, interferons have been the proto-
type of the so-called “fourth arm” of therapy; surgery,
radiotherapy, chemotherapy and biotherapy [184].
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10 Monoclonal antibody therapy
ROBERT O. DILLMAN
Abstract Monoclonal antibodies were the first suc-
cessful targeted anti-cancer therapeutics in the modern
era. In the 1970s elucidation of hybridoma and molecular
biology technologies established mechanisms to pro-
duce and modify monoclonal antibodies with therapeu-
tic anti-cancer potential. In the 1980s early investigation
with murine monoclonal antibodies in animal models
and patients established most of the principles of anti-
body therapy including immunologic and regulatory
mechanisms of anti-cancer effects, and the use of immu-
noconjugates in which antibodies act as carriers. These
early studies also showed that most of the toxicities
associated with monoclonal antibodies relate to the
target antigen, and less often immune interactions with
the antibody itself. In 1997 the United States Food and
Drug Administration approved rituximab (Rituxan) for
the treatment of B-cell lymphoma, making it the first
monoclonal antibody to receive regulatory approval for
the treatment of human malignancy. As of early 2008,
there were six commercially available unconjugated
monoclonal antibodies with marketing indications for
treatment of human malignancy. In addition to ritux-
imab for B cell malignancies, this list includes alemtu-
zumab (Campath) for B and T cell malignancies,
trastuzumab (Herceptin) which inhibits Her-2 overex-
pressing breast cancers, anti-epidermal growth factor
receptor (EGFR) antibodies cetuximab (Erbitux) and
panitumumab (Vectibix) which inhibit various epithelial
solid tumors including colorectal cancer, and bevaci-
zumab (Avastin) which binds to vascular endothelial
growth factor (VEGF) and is probably useful to treat
most malignancies, including colorectal, lung, and
breast cancers. These products have been most useful
when combined with chemotherapy because of additive
or synergistic anti-tumor effects.
Keywords Alemtuzumab • bevacizumab • cetuximab
• panitumumab • rituximab • trastuzumab
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
Background and Rationale
for Antibody Therapy
Historical aspects 1900–1980
Monoclonal antibodies (Mab) were the first successful
anti-cancer targeted therapeutics in the modern era
[168]. A chronological history of monoclonal antibody
development is shown in Table 1. At the end of the
nineteenth century antisera and antibodies were discov-
ered as part of the independent work of Emil Behring
and Kitasato Shibegan that led to developing diptheria
antitoxin. By immunizing an animal with foreign anti-
gens contained on cells, early immunologists recog-
nized that they could produce antisera against antigens
on the surface of cells. The potential to utilize antibod-
ies as therapeutic agents was first espoused in the early
twentieth century. The German immunologist, bacteri-
ologist, and 1908 Nobel Prize winner Paul Ehrlich used
the term “passive immunization” to describe the use of
antisera and antibodies in the treatment of disease as
opposed to the “active immunization” using cell or
antigen-based vaccines. Ehrlich, whose 150th birthday
was 14 March 2004, is generally considered the father
of antibody therapy and is credited for the term “magic
bullets” to describe the potential for antibodies to spe-
cifically target bacteria or cancer cells as opposed to
normal cells [201, 644].
Before 1975, it was virtually impossible to isolate a
specific antibody. Initial therapeutic endeavors were
with “antisera”, a collection of heterogeneous anti-
bodies from multiple clones, typically derived from
animal serum. Animals were repeatedly immunized
with preparations of human cells which induced an
immune response. Animal blood was collected several
weeks later at a time when a primary immune response
was estimated to occur, and the serum fraction isolated.
303
304 Monoclonal antibody therapy

Table 1. Milestones in the development of monoclonal antibodies for the treatment of cancer
1908 Paul Ehrlich, German immunologist states concept of “magic bullets” for seeking out cancer cells, awarded Nobel Prize
1927 Publication describing serotherapy of 10 patients with chronic myelogenous leukemia
1975 Kohler and Milstein report “hybridoma” methodology to produce monoclonal antibodies; awarded Nobel Prize in 1986
1980 First publication of treatment of a cancer patient (lymphoma) with a monoclonal antibody
1982 First published report of a complete clinical remission in a patient (lymphoma) treated with a monoclonal antibody,
murine anti-idiotype
1984 First U.S. FDA approval of a therapeutic monoclonal antibody, murine anti-CD3 muromonab-CD3 (Orthoclone OKT3) to
prevent renal allograft rejection
1992 First U.S. FDA approval of a radiolabeled anti-cancer monoclonal antibody, murine anti-B72.3 indium-labeled monoclonal
antibody, 111In-satumomab pendetide (Oncoscint), for radioimmunodetection of colon and ovarian cancer
1995 Approval in Germany of murine monoclonal antibody edrecolomab (Panorex) for the adjuvant treatment of colon cancer
in Germany
1997 U.S. FDA approval of chimeric anti-CD20 monoclonal antibody rituximab (Rituxan) for the treatment of B-cell lymphoma,
first indication to treat human malignancy
1998 FDA approval of humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin) for the treatment of breast cancer,
first indication to treat a solid tumor
2000 U.S. FDA approval of humanized anti-CD33 immunotoxin, gemtuzumab ozgomicin (Mylotarg), for the treatment of acute
myelogenous leukemia, first approval of antibody-targeted immunotoxin
2002 U.S. FDA approval of murine anti-CD20 radiolabeled monoclonal antibody Y-90 ibritumomab tiuxetan (Zevalin) for B-cell
lymphoma, first approval of a radiolabeled antibody for treatment of malignancy
2003 U.S. FDA approval of murine anti-CD20 radiolabeled monoclonal antibody I-131-tositumomab (Bexxar) for B-cell
lymphoma
2004 U.S. FDA approval of bevacizumab (Avastin) for colorectal cancer
2004 U.S. FDA approval of cetuximab (Erbitux) for colorectal cancer
2006 U.S. FDA approval of panitumumab (Vectibix) for colorectal cancer

Such sera were then modified by repeated absorption In 1975 Hans Kohler and Caesar Milstein published a
with normal human tissues that were presumed to not paper in which they described the hybridoma technology,
contain tumor associated antigens. This process which allowed isolation of cloned cells that could
increased the specity of the final product which consti- secrete a single type of antibody [400]. For this work
tuted an antiserum. The degree of reactivity of this they were awarded Nobel Prizes in 1986. Following
immunoglobulin collection was tested in bioassays publication of this paper numerous academic laborato-
using serial dilution titrations, and the product was ries and early biotechnology companies began making
characterized on the basis of the dilutional titers, such Mab by immunizing rodents with human cells and anti-
as 1:64 or 1:256. Typically, large animals such as gens. The potential advantages of Mab over antiserum
horse, sheep, or goats were immunized in order to as a therapeutic product were readily apparent as sum-
yield large volumes of anti-serum. Because each four- marized in Table 2.
legged biofactory was biologically unique, it was
impossible to reproduce a given antiserum exactly,
even in the same animal. There were always issues of
purity and the impossibility of exactly reproducing any
given serum that inevitably consisted of multiple anti- Table 2. Comparative advantages of monoclonal Antibodies
vs. Antisera
bodies with various specificities and affinities. In as
much as antibodies were genetically determined for an Monoclonal Antibody Antisera
individual animal, the supply of any given antiserum Purified antigen to optimize Cell or crude antigen
was limited, and therefore there was no ability to per- satisfactory
Absorptions for specificity Screening for specificity
form large clinical trials with any specific antiserum.
Heterogeneous Homogeneous
Some antisera, such as anti-thymocyte globulin, are Variable lots Reproducible lots
still in clinical use today, but the biotechnology indus- Variable affinities Uniform affinity
try is trying to replace as many as possible with single Variable specificities Unique specificity
or combinations of Mabs. Limited quantities Unlimited production
Robert O. Dillman
Antisera Trials 1925–1980
Between 1925 and 1980 numerous clinical studies were
performed using antisera. These pioneering studies with
heterologous and isologous antisera have been reviewed
in more detail elsewhere [137, 607, 795]. Such trials
were limited by the quantity of purified anti-tumor anti-
sera available, the purity of such preparations, and dif-
ficulties producing similar lots of new antibody.
Nevertheless, some antitumor effects were noted. One
of the earliest reports by Lindstrom in 1927 described
ten chronic myelogenous leukemia (CML) patients
whom he treated with 15 courses of rabbit antisera. A
decline in peripheral blood myeloid cells was noted in
five patients, but there were also significant side effects
that were attributed to impurities in the preparation,
although these may well have been secondary to anti-
body binding to circulating CML cells. In the 1970s
anti-thymocyte globulin (ATG) was used by several
investigators to treat patients with T-cell malignancies
[198, 224, 815]. All eight patients described in these
reports had some improvement in their Sezary syndrome
and/or lymphadenopathy. Therapy was complicated by
hypotension and chills. Prophylaxis with diphenhy-
dramine and steroids seemed to prevent these side
effects during subsequent treatments, but this may have
been because circulating T lymphocytes had already
been eliminated from the circulation.
Interpretation of antisera studies was complicated by
issues of antibody source, purity and specificity. Certain
toxicities were identified, but it was unclear whether
these were due to impurities, hypersensitivity reactions
to foreign proteins, or to antitumor effects. Although
side effects were typically attributed to allergic reaction
to foreign proteins, it is likely that most of the toxicity
observed was actually due to specific interaction with
leukocytes and the release of cytokines [170].
Animal Models and Antibody Therapy
The chapters on antibody therapy in the first three edi-
tions of Principles of Cancer Biotherapy [162], [165],
[169] summarized some of the important animal work
with antisera and Mab that laid the foundation for the
use of such products in man. The demonstration of
safety, tumor targeting, and anti-tumor effects demon-
strated in various animal models justified and guided
human clinical trials that eventually established Mab as
an effective anti-cancer treatment modality [816]. The
following conclusions can be drawn from the heterolo-
gous antisera and studies in animal models. First, anti-
tumor effects can be mediated by Mab in vivo. Second,
305
with some antibodies, antitumor effects are observed in
immunodeficient mice even in the absence of an interac-
tion with complement and/or effector cells, suggesting
that antitumor effects can be mediated by regulatory
effects. Third, immune-mediated antitumor effects vary
to some extent with the Ig subclass and isotype of the
Mab. Fourth, just as with chemotherapy, antitumor
effects are more difficult to achieve in the presence of
large tumor burdens. Fifth, combining antibodies to dif-
ferent antigens is a promising strategy to overcome
problems of heterogeneity [14, 16, 534]. Sixth, animal
models are of little use in predicting toxicities in humans
for Mabs that react with antigens that are more prevalent
in humans or only expressed in humans.
Human Trials with Murine Monoclonal
Antibodies 1980–1989
Clinical trials in man with murine Mab began in 1980 in
hematopoietic malignancies and subsequently in solid
tumors. Most of the clinical investigation during the
1980s involved pilot and phase I trials with mouse
Mab, and relatively few patients were actually studied.
Perhaps the first written report of monoclonal antibody
therapy was that by Nadler et al. who treated a man with
B cell lymphoma with a murine Mab called AB89 [518].
On successive days the patient received slow infusions
of AB89 at doses of 25, 75, and 150 mg; 1 month later a
1.5 gm dose was administered. There was a transient
decrease in the lymphocyte count after each treatment,
but no significant antitumor effect was noted. During
1980 and 1989 a number of clinical studies were con-
ducted with Mab directed against antigens found on
cells from hematologic malignancies [19, 38, 161, 177,
179, 180, 227, 228, 446, 478, 485–488, 518, 569, 600,
666] and solid tumor cancers [109, 161, 205, 240, 262,
263, 328, 375, 443, 513, 535, 625, 651–653, 666, 738,
776]. In 1982, the first report of achieving complete
regression of tumor in a patient with B cell lymphoma
was published [487]. Most of the principles of antibody
therapy, including immunologic and regulatory mecha-
nisms of anti-cancer effects, and the use of immunocon-
jugates in which antibodies act as carriers were
established during these early trials. These early trials
also established that most toxicities and adverse events
associated with monoclonal antibody infusions are the
results of interactions with the target antigens, and less
often due to immune interactions with the antibody
itself. The potential of Mab for the treatment of human
malignancy was clearly recognized [163, 533].
In the early 1990s two radiolabeled Mab received regu-
latory approval for imaging and radioimmunodetection
306
of tumors. In 1995, Germany granted the first approval of
a mouse monoclonal antibody for cancer therapy
(Panorex) for colorectal cancer, although this product
was never approved in the United States. In the early
1990s trials began with murine Mab that had been modi-
fied using molecular biology technologies to produce
antibodies that consisted mostly of human amino acid
sequences. In 1997 the United States Food and Drug
Administration (FDA) approved the first monoclonal
antibody (rituximab) for the treatment of malignancy in
the United States [168]. During the next decade several
unconjugated and conjugated antibody products received
regulatory approval for commercial use.
Antibodies and Antigens
When foreign cells are injected into an animal, they
induce an immune response that results in the produc-
tion of immunoglobulin (Ig) proteins, called antibodies,
each of which binds specifically to certain cell surface
molecules, termed antigens. Collectively these antibod-
ies are antisera. The antigens are characterized as
glycoproteins, glycolipids, polysaccharides, etc. A tumor-
specific antigen is one that is found only on cancer cells.
A tumor-associated antigen is an antigen expressed on
tumor cells, but also known to be present on normal
cells. Antibodies consist of sequences of amino acids
that are linked by disulfide bonds that yield two heavy
chains and two light chains. Each light chain is con-
nected to a heavy chain by a disulfide bridge, and the
two heavy chains also are connected via disulfide
bridges. As shown in Fig. 1, enzymatic cleavage of these
various sites by papain or pepsin yields antibody frag-
ments termed Fab, F(ab’)2, and Fc. The basis for speci-
ficity of antibody binding resides in the variable regions
of the heavy and light chains (Fv), and more specifically
Figure 1. Structure of immunoglobulins and fragments.
Two heavy chains are connected by a disulfide bond, and each
in turn is connected to a light chain by a disulfide bond
Monoclonal antibody therapy
in the sequence of peptides that constitute the hypervariable
region (Fhv) [418, 643]. The Fhv region constitutes the
“idiotype” of the Ig, and the specific peptide sequences
of the idiotype are termed “idiotopes.” The six hyper-
variable subregions of the variable region are also
referred to as the complementarity-determining regions
(CDR). “Lock-and-key” is a popular metaphor for the
unique chemical relationship and structural binding
between a specific antibody and its antigen target. The
affinity of antibody for its antigen is a measure of how
tightly the antibody binds to the antigen.
The diversity of humeral immunity is made possible by
the various combinations and permutations of arrange-
ments and combinations of Fv regions, heavy chains, and
light chains [419, 643]. More specifically there are vari-
able (V), diversity (D), and joining (J) genes that code for
sequences on the heavy chain (V,D, and J), and light chain
(V and J) that provide nearly infinite combinatorial poten-
tial for rearrangement. Based on their chemical composi-
tion, the light chains of Ig are classified as kappa or
lambda, and heavy chains as immunoglobulins IgG, IgM,
IgA, IgD, or IgE. Other differences in heavy chains allow
subclass characterization such as IgG1 IgG2a, IgG2b,
IgG3, IgG4, etc. Human chromosomes 2, 14, and 22 con-
tain various loci for light and heavy chains.
The basis for antibody interaction with other compo-
nents of the immune system, such as complement pro-
teins and effector cells, resides in carbohydrate residues
that are present on the Fc portion of the Ig molecule and
Fc receptors (FcR) on effector cells [250, 524, 746]. The
interaction between Fc and FcR is influenced by several
factors including the affinity between the antibody iso-
type and the specific receptor, the composition of the
sugar side chain on the Fc-portion of the antibody, and
inhibitory characteristics of the FcR. Subclasses of
Fc and receptors have been defined that determine
whether a specific antibody will bind complement
or not. Antibody-dependent cell cytotoxicity (ADCC)
depends in part on the specific N-glycosylation of the Fc
domain of the monoclonal antibody. Cross-linking of
FcR triggers various immune reactions including phago-
cytosis after opsinization, ADCC, and release of various
cytokines that act as inflammatory mediators [117, 701].
FcR that bind human IgG Fc are referred to as FcγR and
these are subclassed based on their binding characteris-
tics and function. From a functional perspective, there
are two main types of FcγR found on the surfaces of
immune effector cells, activating FcγR, and inhibiting
FcγR. Based on genetic polymorphisms the activating
FcγR include the high-affinity receptor CD64 (FcγRI),
and the low affinity receptors CD32a (FcγRIIa) and
CD16 (FcγRIIIa). CD64, which is expressed on monocytes,
Robert O. Dillman 307

macrophages, and dendritic cells, is the only FcγR with scale production of Mab [400]. Various laboratories
a high affinity to Mab monomeric IgG. Cells with a utilized this technology to identify large numbers of
CD64 Fc receptor can bind circulating Mab that is not mouse Mab that reacted with tumor-associated antigens
bound to antigen, while the lower affinity Fc bind better [158]. As shown in Fig. 3, in this methodology mice or
to Mab that is bound to antigen. CD32a is expressed on rats are injected with whole tumor cells or purified
these cells, and also on neutrophils, eosinophils, and tumor associated antigens (such as carcinoembryonic
platelets. CD16a is highly expressed on NK cells and
macrophages. There is some evidence that these poly-
morphisms are predictive of the clinical efficacy of cer-
EFFECTOR
tain antibodies. This is leading to specific strategies to C’
CELL
modify the Fc portion of antibodies in order to optimize COMPLEMENT
the anti-tumor effector functions [667, 695].
ACTIVATED
Theoretically antibody therapy is the most tumor spe-
T-CELL
cific approach to systemic cancer treatment that has been
γ - IFN
discovered. As noted earlier, antibodies directed against a
tumor antigen are tumor specific to the degree to which that
antigen is found only on tumor cells. At one time it was TUMOR
IL-2 CELL RICIN A
believed that all cancers were foreign and that tumor-spe- (Toxin)
cific antigens would be readily discovered. Unfortunately,
TNF
phenotypically and genetically, cancer cells are more like α-, β-IFN
normal cells from that host, and as a result, most tumor DNR
(Drug)
antigens are only relatively tumor specific because they are
found in certain normal tissues as well. However, many Y90
tumor-associated antigens are expressed much more heav- (Isotope)
ily only during embryonic development; and, therefore are
relatively tumor specific. Also, certain viruses produce or Figure 2. Schematic of potential applications of monoclonal
antibodies for cancer therapy. Various antigens, represent-
induce antigens that are relatively tumor specific. The idio- ing the heterogeneity of cancer, are depicted on the tumor
type encoded by B cells of a specific B-cell malignancy is cell surface. Bifunctional antibodies are depicted with one
an example of a tumor specific antigen. Fab directed to a tumor antigen and the other to a biologic
response modifier. One antibody has a Fab directed to a CD2
or CD3 receptor on T cells, which results in their activation
Unconjugated Antibodies as Therapeutic and enhanced cytotoxicity. The Fc portion of the antibodies
Agents are attached to various cytotoxic substances including com-
plement, effector cells (granulocytes, monocytes, killer T
As illustrated in Fig. 2 and Table 3, there are many ways cells) of the immune system, and cytotoxic agents such as
in which antibodies may be used in cancer therapy [161, toxins, radioisotopes, and chemotherapy agents. IFN, inter-
feron; TNF, tumor necrosis factor; IL-2, interleukin-2; DNR,
163, 533]. This chapter covers the rationale for the use daunorubicin. Ann Intern Med 1989, 111:592–603. By per-
of unconjugated (or “naked”) antibodies in cancer ther- mission of the American College of Physicians
apy and the current role of such Mab in the treatment of
human malignancy. In subsequent chapters of this book,
the use of conjugated Mab as carriers of cytotoxic sub-
stances including chemotherapy agents, radioisotopes, Table 3. Strategies for in vivo use of monoclonal antibodies
as anti-cancer therapy
and natural toxins are reviewed.
Antibody alone
Complement mediated cytotoxicity (CMC)
Antibody dependent cell-mediated cytotoxicity (ADCC)
Manufacturing of Monoclonal Regulatory interactions (ligands, receptors)
Anti-idiotype vaccines
Antibodies Immunoconjugates
Radiolabeled antibodies
Hybridoma Technology Immunotoxins
Chemotherapy-antibody conjugates
In 1975 Kohler and Milstein, published a landmark Cytokine immunoconjugates
paper in which they described the “hybridoma technol- Cellular immunoconjugates
ogy,” which opened the doors to reproducible and large- Multi-targeted immunoconjugates
308
HPRT+
Immunization spleen cells
Screening of supernatants for antibody
and selection of clones
Supernatant
(contains 10 to 100
mg /mL MOAb)
Figure 3. Schema for murine monoclonal antibody production. From [176]. By permission of Biomedical Information Corporation
antigen) to induce a polyclonal immune response against
the foreign human proteins. The animal’s immune sys-
tem responds with production of antibodies directed
against the tumor antigens. The blueprints for the anti-
bodies are contained in by B lymphocytes that are found
in large quantities in the spleen. The spleen of an immu-
nized animal is removed and minced into a single-cell
suspension. If placed in tissue cultures, such cells typi-
cally die off in a few days and are unable to proliferate.
For this reason, the B lymphocytes are combined with
other mouse B cells, typically mouse myeloma cells,
that have been specifically selected for certain charac-
teristics, including (1) ability to grow in culture as a
Monoclonal antibody therapy
Nonsecretory
HPRT −
B-cell
culture
Fusion in PEG
Selection in HAT medium
Recloning and subcloning
Purified ascites
(contains 10 to 20
mg/mL MoAb)
single clone, (2) inability to produce and secrete immu-
noglobulin themselves, and (3) inability to grow in the
presence or absence of specific medium constituents. In
the presence of a cell-fusing substance, such as polyeth-
ylene glycol, individual spleen cells with the genetic
machinery for making antibody against the tumor fuse
with cells from the immortal cell line, thus producing
hybrid cells, or hybridomas that retain the ability to
secrete antibodies. By the plating process of limiting
dilution, a single hybridoma clone of cells can be iso-
lated that retains the ability to secrete the antibody
encoded in the DNA of the mouse spleen cells, hence
the term “monoclonal antibody.” The antibodies are isolated
Robert O. Dillman
from culture media and screened for specificity against
various cells and tissues. A major limitation of this
approach is that antigens are defined by the mouse
immune system, and tumor antigens are defined by differ-
ential expression on normal as opposed to cancer cells.
The first large-scale production factories for Mab for
clinical trials were the peritoneal cavities of Balb-C and
athymic mice. Injection of the hybridomas into the peri-
toneal cavity resulted in a malignant ascites that was
rich in the monoclonal antibody. This technology helped
launch the biotechnology industry, and made possible
mass production of promising antibodies [14, 94, 157].
The techniques of mass production included the in vivo
expansion of hybridomas as ascites in mice, and in vitro
expansion of such hybrid cells in various large tissue
culture vessels and bioreactors.
Recombinant Technology
and Engineering Monoclonal Antibodies
Murine hybridomas that produced desirable antibodies
were used as a source for genetic material for mass pro-
duction in unicellular factories, and to make chimeric or
humanized Mab. Recombinant DNA technology was
used to introduce antibody-encoding genes into other
cells to facilitate antibody production by cells such as
Escherichia coli, yeast, and Chinese Hamster Ovary
(CHO) cells [42, 44, 58, 508]. Certain mouse plasmacy-
toma lines also glycosylate proteins in a similar manner.
These unicellular organisms are currently the factories
of choice for Mab production because they produce
human-like N-glycosylation of the proteins, which is
crucial for proper folding of the Ig protein for the stereo-
chemistry that is needed for optimal binding to antigen.
Recombinant DNA technology has displaced hybri-
doma technology as the method of choice for monoclo-
nal antibody production.
Recombinant techniques have made it possible to
engineer a variety of Mab constructs [6, 157, 631].
Furthermore, recombinant DNA biotechnology has made
it possible to modify mouse Mab into partially human-
ized forms. As discussed later in this chapter, this has led
to the production of mouse–human chimeric Mab and
humanized Mab as diagrammed in Fig. 4, as well as anti-
bodies designed to react with more than one antigen as
shown in Fig. 2. As of 2007, of the 18 different therapeu-
tic antibody products approved for human use in various
diseases, ten are manufactured in CHO cell lines and
eight are made in murine plasmacytoma cells. Additional
modifications include engineering the Fc portion to opti-
mize interactions with complement, or binding to FcR to
309
Figure 4. Comparison of different types of monoclonal anti-
bodies mouse antibodies illustrating the component of
murine amino acid sequences in each. Shown are mouse
antibodies (omabs), mouse/human chimeric antibodies
(-ximabs) in which the murine variable region is combined
with a human constant region, “humanized” antibodies
(-zumabs) in which only the hypervariable peptide sequences
are retained from the original mouse antibody, and human
antibodies (umabs)
optimize ADCC. Other genetic engineering modifications
have focused on making smaller antibody molecules as
single chain Fv, Fab, and F(ab’)2 [325, 584].
Human Monoclonal Antibodies
In terms of therapeutic potential, there are several rea-
sons why human Mab offer advantages over those of
other species [21, 258, 416]. First, they are less immuno-
genic and less allergenic than non-human antibodies.
Second, certain subclasses of human antibodies are more
effective than murine antibodies in facilitating comple-
ment and/or antibody-mediated cytotoxicity in vitro.
Third, by dose-to-dose comparisons, human antibodies
have longer half-lives and prolonged serum levels
because there is less non-specific uptake, destruction and
metabolism. There is evidence that antibodies are taken
up by FcR receptors, called neonatal or Brambell recep-
tors (FcRn), which bind the Fc portion of antibodies and
remove them from the circulation [357]; [442]. Human
IgG1, IgG2, and IgG4 have high affinities for FcRn
which transport endocytosed antibodies from the lyso-
some back to the serum, while murine antibodies and
human IgG3 have a low affinity for FcRn. For antibodies
with a low affinity for FcRn, instead of being transported
back to the cell surface, they are catabolized [527, 808].
Several approaches have been used for isolating human
Mab [189, 258, 671]. In one approach, it is presumed that
patients with cancer have developed at least a limited
immune response to their tumor. Regional draining lymph
nodes, which are the first line of regional immunologic
defense against tumor, are excised to obtain a source of
B lymphocytes, some of which make antibodies against
the tumor. Regional lymph nodes that drain known cancer
sites are potential sources of B lymphocytes that may
310
have been programmed to make antibodies against the
primary cancer. Immunization with tumor cells or anti-
gen, followed by excision of draining lymph nodes, espe-
cially sentinel lymph nodes, has also been used to isolate
antibody producing B lymphocytes [282, 299]. Another
source of immune reactive B cells is the peripheral blood,
and the frequency of such cells may be enhanced by
immunization. For example, researchers have injected
irradiated autologous tumor cells into the skin with an
immune stimulant such as Bacillus Calmette-Guerin
(BCG) or granulocyte macrophage colony stimulating
factor (GM-CSF). Rather than harvesting lymph nodes, 2
to 3 weeks after immunization peripheral blood B lym-
phocytes are collected, some of which secrete human
antitumor antibodies
Once antibody-producing B cells have been isolated,
they can be fused with other human cells of B cell lin-
eage that have been selected for similar characteristics
as those immortal mouse B-cell lines described above,
to produce antibody secreting clones [51, 123, 129, 256,
257, 670]. It is also possible to fuse human lymph node
B cells with cells from immortal mouse B-cell lines, and
still get secretion of human antibody, although such
cross-species hybridomas are usually unstable [67, 635,
802]. Other investigators have isolated B lymphocytes
from lymph nodes and then infected these cells with
Epstein-Barr virus to immortalize cells producing
human Ig [96, 113, 189]. Using various techniques,
human Mab have been produced that react with breast
cancer [67, 395, 635], lung cancer [669], colorectal car-
cinoma [299], brain tumors [670], stomach cancer [802]
and melanoma [241, 364, 686]. However, human Mab
were more difficult to develop than murine Mab because
of the instability of human hybridomas and immuno-
globulin secreting cells, low secretion rates, and diffi-
culties in mass producing and screening.
These inherent problems in human Mab production
have been overcome by recent advances in transfection
technology and molecular immunology that have made
it feasible to integrate DNA for human Ig into cells that
can easily produce large quantities of human Mab.
Newer technologies such as polymerase chain reaction
(PCR) [202] and transgenic mice [271] have led to iden-
tification of vast libraries of human antibodies that may
be superior in terms of antigen and antibody selection.
In recent years various laboratories have been able to
clone cDNA for VH and VL contained in hybridomas
and B lymphocytes that were producing low levels of
desirable antibodies. These were then linked to human
hinge and constant genes to produce totally human Mab
in more efficient cellular factories. Today one can take
Ig producing genes from B-lymphocytes and produce
Monoclonal antibody therapy
the entire repertoire of antibodies which can then be
screened for their specificity, and the Fv isolated to pro-
duce fully human recombinatorial products [6, 326,
631]. Using such libraries, fully human Mabs can be
produced by linking Fv from the display library with
human Ig hinge and constant regions. Human Mab can
also be produced by immunizing transgeneic mice whose
immune system has been replaced with a human immune
system engineered to produce human Mab of a specific
class and subclass. Immunization of these animals
results in human B cells that produce human Mab.
Chimeric and Humanized Monoclonal
Antibodies
Because of the difficulties in producing and manufac-
turing human Mab, the extensive characterization of
murine Mab that react against human tumor associ-
ated antigens, and the clinical disadvantages of
murine Mab, other strategies have been developed to
create mouse/human chimeric Mab and genetically
engineered “humanized” Mab (Fig. 4) [1, 44, 58, 124,
325, 386, 440, 508, 510, 788]. One way to improve
the activity of a Mab is to class-switch the Mab so
that it has an Fc portion that is more suited for inter-
action with human complement and/or effector cells
[44, 157, 508]. Human IgG1 is usually considered
most desirable because it effects both CMC and
ADCC. IgG3 also facilitate favorable CMC and
ADCC effects, but these proteins tend to aggregate
which is problematic from a production standpoint. It
has generally been taught that IgG4 is the least use-
ful, especially in terms of CMC, because of a lack of
Fc receptors for this IgG subclass. In the chimeric
approach, the variable domains of the light and heavy
chains of a desirable murine Mab, are linked to hinge
and constant domains of a human Ig. This is most
often done with recombinant techniques in which the
mouse genes for the variable domains of amino acids
are combined with the human domains for the hinge
and constant region amino acids. The genes are then
inserted into bacteria or yeast for large-scale produc-
tion. The bacterial preparations are typically not gly-
cosylated while those grown in yeast are glycosylated,
which is important for interaction via the Fc portion
of the antibody. It is also possible to isolate the genes
specific for the six hypervariable amino acid sequences
on the light and heavy chains (CDR) of a desirable
murine antibody, and these can then be inserted into
the framework of human Ig to create a “humanized”
Mab via what is called “CDR grafting”. In this
Robert O. Dillman 311

construct the only residual mouse amino acid Table 4. Components that precede “mab” in the nomenclature
sequences remaining are those that give the antibody for naming a monoclonal antibody
its three-dimensional binding specificity. All of the Infix Target Inflx Origin
unconjugated monoclonal antibody products approved Tu (m) Tumor -a- Rat
for cancer treatment are either chimeric (cetuximab, Ci (r) Cardiovascular -o- Mouse
rituximab), humanized (alemtuzumab, trastuzumab, Li (m) Immune -xi- Chimeric
bevacizumab) or human (panitumumab). Because of human/mouse
Co (l) Colon tumor -zu- Humanized
the human Fc portion of the Ig molecule, products,
Me (l) Melanoma -u- Human
they possess superior cytotoxic potential based on Go (v) Ovarian tumor -axo- Rat/murine
interactions with complement and/or effector cells, hybrid
and are much less immunogenic, which is associated Pr (o) Prostate
with a longer bioavailability [662]. The availability
of “chimeric,” “humanized,” and fully human Mabs
has greatly reduced the limitations resulting from the
production of endogenous human anti-Ig antibodies regulatory approval. The suffice “mab” indicates that
such as human anti-mouse antibody (HAMA), human the product is a monoclonal antibody. The syllable
anti-chimera antibody (HACA), and human anti- directly in front of mab indicates the source such as
human antibody (HAHA) in man, and facilitated the mouse, chimeric, humanized, or human. The prefix of
use of repeated treatments with specific Mab for the name has no specific meaning. A second syllable
extended periods of time. may be added if there is something else linked or
attached to the product. The key components that appear
before “mab” for names adopted for the United States
Antibodies with Multiple Specificities are shown in Table 4. Most of the Mab are either mouse
and/or Functions (-omab), chimeric (-ximab), humanized (-zumab), or
human (-umab). Thus, rituximab is a chimeric Mab that
It is also possible to make Mab with specificity to more
targets tumor (as is cetuximab). Trastuzumab is a
than one antigen-binding site, and more than one func-
humanized mab that targets tumor (as is alemtuzumab).
tional capability. Some examples of bispecific and
Bevacizumab is a humanized mab that has a cardiovas-
bifunctional Mab, in which the antibody construct
cular target (vascular endothelial growth factor).
reacts with more than one antigen, are illustrated in
Panitumumab is a human mab that targets tumor.
Fig. 2 [124, 223, 711, 772, 793]. This can be done by
Limilimumab and tremelimumab are human mabs that
chemically linking either two different Fab pairs or
that have an immune target (CTLA4) while daclizumab
F(ab’)2 pairs, or two intact antibodies. It is also possi-
is a humanized mab with an immune target. Tositumomab
ble to make quadromas (a hybridoma of two monoclo-
and ibritumomab are murine mab that target tumor
nal antibody-secreting hybridomas), which secrete
while oregovomab is a mouse mab that targets ovary.
Mab that react with two different antigenic binding
Catumaxomab is a rat/mouse hybrid anti-tumor mab.
characteristics [123]. One can then select the appropri-
ate quadroma that is producing antibodies with two
different antigenic binding characteristics. In recent
years these approaches have been replaced by recom-
binant DNA techniques in which the genes for differ-
Mechanisms of Antibody-
ent murine variable or hypervariable regions of interest Mediated Anti-Tumor Effects
are linked to a humanized constant region and then As outlined in Table 1, there are at least three different
manufactured in cells [223]. rationales for using antibodies alone in vivo as cancer
treatment. The first rationale involves two different
immune mediated effects involving the Fc portion of the
Monoclonal Antibody Nomenclature immunoglobulin and interaction with other components
Because of the tremendous number of Mabs that are of the host immune system. The second rationale encom-
under development, a nomenclature system has been passes concepts of regulating tumor cells or their
developed that provides information about the construct microenvironment. The third concept relates to the anti-
and the target. These names are reserved until the prod- idiotype cascade as a means of immunization to induce
uct is being tested in trials that are considered pivotal for endogenous antibodies.
312
Complement Mediated Cytotoxicity
Complement mediated cytotoxicity (CMC) or comple-
ment dependent cytotoxicity (CDC) involves the fixation
of complement via the Fc portion of Ig and activation of
the complement protein cascade resulting in membrane
damage and cell destruction as shown in Fig. 5 [238,
768]. Murine antibodies are usually ineffective in fixing
human complement in vitro. Exceptions to this are IgM
and certain IgG3 murine antibodies [109, 794]. In man,
studies of hemolytic anemia have confirmed that human
IgM is the most efficient human Ig class for complement
fixation followed by IgG1, IgG3, and IgG2. Because
complement fixation occurs at the Fc portion of antibody,
this approach requires whole intact Ig or an Fc segment,
rather than antibody subunits. As noted earlier in this
chapter, in the era of chimeric, humanized, and human
Mab, human Fc constructs can be selected to optimize
for complement binding if that is considered desirable.
Antibody Dependent Cell-Mediated
Cytotoxicity
The second immunological cytotoxic mechanism is
ADCC [304, 305]. Many lymphocytes, monocytes,
COMPLEMENT-DEPENDENT ANTIBODY MEDIATED
Figure 5. Diagram of complement
mediated cytotoxicty (CMC) featuring
antibody binding, fixation of complement, TUMOR CELL
and cell death & ANTIBODY
ANTIBODY-DEPENDENT CELL - MEDIATED
Fc RECEPTOR
Figure 6. Diagram of antibody
dependent cell-mediated cytotoxicity LYMPHOCYTE
featuring antibody binding,
attachment of effector cells via the
Fc receptor to the Fc tail of the
antibody molecule, and cell death
Monoclonal antibody therapy
macrophages, granulocytes and eosinophils have such
receptors and can precipitate tumor cell lysis when
linked via Ig to a cell. Collectively, such cells are referred
to as “effector cells.” The Fc portion of Ig is crucial for
linking antibody to effector cells via their FcR as shown
in Fig. 6; the specific carbohydrates on the Fc are crucial
for this interaction [117, 524]. The numbers of Fc recep-
tors and affinity for Fc predict the degree of ADCC
activity [447, 796]. Antibody may first bind to its cellu-
lar target followed by effector cell binding via the Fc, or
alternatively, effector cells may first bind to the Fc of the
antibody, and then be carried to the tumor target. Ex vivo
incubation of Mab and effector cells followed by in vivo
delivery has produced anti-tumor responses in animal
models [308]. Circulating target cells coated with antibody
are removed in the liver and spleen, presumably because
of Fc receptors on tissue macrophages in these sites. For
human Mab, the ability to bind specifically to macrophages
of the reticuloendothelial (RE) system is restricted to
IgG1 and IgG3 antibodies and is absent from IgM and
other IgG subclasses. There is evidence that mouse Mab
differ in their subclass binding to human effector cells.
Various studies have confirmed that among murine
Mab, the hierarchy for effecting ADCC is IgG2a, followed
by IgG3, apparently because of enhanced Fc receptor
TUMOR CELL LYSIS
Na+
C
K+
H2O
IgG
MEMBRANE TUMOR CELL
DAMAGE LYSIS
CYTOTOXICITY (ADCC)
LYSIS
NOT C’ DEPENDENT
Robert O. Dillman
binding [308, 340]. In addition, different antibodies of
the same subclass and isotype, directed against the
same antigen, exhibit different degrees of Fc binding
[179, 660]. Murine IgG3 anti-disialoganglioside anti-
bodies have been cytotoxic with human effector cells in
vitro [108, 305, 720]. Among commercially available
chimeric and humanized antibodies, most have been
engineered with a human IgG1 Fc portion that can effect
ADCC [691].
For both ADCC and CMC the in vivo anti-tumor
effect is also dependent on the ability of the host
immune system to provide sufficient complement
proteins or effector cells to produce cytotoxicity. The
use of various biological response modifiers may be
useful in augmenting such an immune response. To
maximize efficiency of effector cell-antibody bind-
ing, some investigators have utilized leukopheresis
to harvest leukocytes, and then incubated these with
antibodies to enrich the antibody-effector cell popu-
lation. Various lymphokines, such as interleukins-2, -4,
-12, and -15 may be useful to enhance the cytotoxic
activity of the effector cells. It has been demon-
strated that cytotoxicity, either by ADCC or CMC,
involves a threshold of antigen binding sites.
Lymphokines, such as alpha and gamma interferon,
can enhance target antigen expression in some tis-
sues [180, 274, 275, 432, 519]. Gamma interferon
increases Fc receptors on effector cells and thereby
can enhance ADCC [181, 519, 767, 775]. There are
good examples from animal models suggesting that
various biologicals may be used in combination to
maximize an anti-tumor effect. The agents most
commonly used for this purpose in human trials are
interferon alpha, IL-2 and GM-CSF. These other bio-
logicals might be used systemically, or targeted specifi-
cally by other antibodies.
Regulatory Approaches
Malignancy may result from the overproliferation of
cells, the failure of cells to undergo programmed cell
death (apoptosis), or a combination of these. Antibody
modification of these events constitutes a second mech-
anism of antibody therapy, and is encompassed by the
term “regulatory,” in contrast to “immunologic,” to
characterize this potential mechanism [163]. Such
receptor targets can also be associated with cytotoxic
effects mediated by complement and/or effector cells.
In contrast, ligand or microenvironment targets may
produce anti-tumor effects purely by regulatory biologic
effects. It is known that tumor cells have a variety of
receptors that are important for growth or proliferative
313
advantages and for preventing or delaying apoptosis that
might be targeted for antibody therapy [269, 480, 568,
731]. Advances in understanding of cellular physiology
have led to identification of a number of ligand–recep-
tor–signal transduction systems in which a ligand binds
to a receptor which initiates a series of enzymatic reac-
tions that signals the nucleus of the cell to activate genes,
translation, and transcription to produce proteins that
are important in cell function, differentiation, prolifera-
tion, and apoptosis. Epithelial growth factor (EGF) and
vascular endothelial growth factor (VEGF) and their
receptors are some of the best known examples of such
interactions. Increased levels of these receptors have
been found in increased quantities in cancer cells and/or
other rapidly proliferating cells.
Conceptually, Mab directed against growth factor
receptors may either block or down-regulate the
number of receptors on the cell surface, and thus
impair a cell’s ability to differentiate and/or divide,
ultimately resulting in cell death or apoptosis. Such
antigens may also serve as targets for Mab that can
induce CMC and/or ADCC, or as targets for cytotoxic
immunoconjugates. Many receptors internalize after
their ligand or an antibody bind to them resulting in
what is sometimes referred to as “antigen modula-
ton.” Rapid internalization limits the potential for
CMC or ADCC, but may be an advantage for the
internalization of conjugated cytotoxic chemotherapy
agents or toxins.
Idiotype Network
One of the first targets proposed for a regulatory
approach was the Ig idiotype of B cell lymphoid malig-
nancies based on the cascade of anti-idiotype antibodies
involved in regulating B cell proliferation [65, 246, 247,
351]. Cells from a malignant B-cell clone express and
sometimes secrete a specific antibody with a unique
binding ability. The peptide sequences in the hypervari-
able region of the light and heavy chains of the antibody
are the idiotopes that collectively constitute the idiotype
of that Ig, and account for its specificity. Under normal
conditions memory T cells signal B cells to produce Ig
via surface idiotype receptors. Other B cells may pro-
duce anti-idiotype antibodies that may be important in
negative feedback control of a given B-cell clone that
has been activated. A detailed description of the “net-
work theory” of anti-idiotype regulation is very com-
plex, and beyond the scope of this chapter. In its simplest
construct, infusion of an antibody directed against a
B-cell lymphoma idiotype might suppress (growth reg-
ulate) that clone back to its baseline state by inducing
314
Antigenic
stimulation
B-Cell proliferation
and differentiation
Anti-idiotype
Figure 7. Diagram of anti-idiotype feedback inhibition of B-cell clonal proliferation, based on the specificity of the hypervariable
region of the antibodies produced by that clone of cells
apoptosis and/or limiting proliferation. How this might
relate to B-cell malignancy is depicted simplistically in
Fig. 7. It is also important to note that the idiotype of a
given B-cell tumor is perhaps the most tumor specific
antigen that has been identified.
Idiotype network theory has led to a concept of taking
advantage of idiotype targets as a form of passive immu-
nization [404]. According to the “idiotype network”
theory, the idiotype of an Ig is very immunogenic and
important in negative-feedback self-regulation. Injection
of a mouse antibody (AB1) directed against a patient’s
tumor antigen will lead to production of many different
antibodies, but some will be anti-idiotype antibodies
(AB2) directed against the hypervariable determinants
of the AB1. Some of these AB2 may present the same
three-dimensional antigenic structure as the original
tumor antigen. Subsequently, such AB2 anti-idiotype
antibodies may induce anti-anti-idiotype antibody (AB3),
which because of the “lock-and-key” structural relation-
ship between antigen and antibody, will have the same
binding specificity as the original mouse antibody
(AB1), except that it will be a human antibody (AB3)
[316, 781]. A more direct approach using this same
principle involves simply taking a well-characterized
anti-anti-idiotype antibody (AB2) and directly injecting
it as an immunogen to try to induce a human AB3 that
will react with the tumor antigen. This theoretical
approach has been demonstrated in animals and may
have application in both cancer and autoimmune dis-
eases [314, 378]. Early clinical trials testing this
approach have been associated with successful produc-
tion of sufficient antibodies to produce immunologic
and anti-tumor effect in man. [39, 104, 229, 230, 493,
494]. This concept and results of clinical trials testing
Monoclonal antibody therapy
Ig
Antigenic
stimulatioin
such products are discussed in more detail in this book
in the chapter on vaccines.
Epidermal Growth Factor and its Receptors
(EGF and EGFR)
Another heavily studied regulatory system is the human
epidermal growth factor receptor (EGFR) family of four
transmembrane receptors abbreviated as Her1, Her2,
Her3, and Her 4 or Erb-B1, Erb-B2, Erb-B3, and Erb-
B4 respectively), which are involved in regulation of
cell proliferation and survival. EGFR are overexpressed
on the tumor cells of subsets of patients with most epi-
thelial solid tumors [608, 611]. The prototype for this
family of receptors is Her1 (a.k.a. EGFR or EGFR1)
which binds to a variety of ligand growth factors includ-
ing epidermal growth factor (EGF), transforming growth
factor beta (TGF-β), epiregulin, betacellulin, and amphi-
regulin, which activate the cytoplasmic tyrosine kinase
domain of the receptor, resulting in downstream activa-
tion of various signal transduction intermediates that
result in cell proliferation and other functions associated
with malignancy [471, 480]. This is the target of the
commercially available antibodies cetuximab (Erbitux)
and panitumumab (Vectibix). Her2 (a.k.a. Her-2 neu)
originally attracted interest when it was found to have
oncogenic properties and to be over expressed on tumor
cells from about 25% of breast cancer patients. [632,
673]. Her2 is unique among the EGFR family in that it
has no known ligand, but instead can bind with each
of the other EGFR (a process called “dimerization”)
which also induces signal transduction. When over
expressed, Her2 can dimerize with itself to induce the
tyrosine kinase activity that leads to signal transduction.
Robert O. Dillman
Heregulin is a known ligand for Her3, which unlike the
other heregulin receptors, has no tyrosine kinase activ-
ity. Neuroregulins 1, 3, and 4 and epiregulin are ligands
for Her4.
Vascular Endothelial Growth Factor and its
Receptors
Judah Folkman hypothesized that angiogenesis was
critical for growth and metastasis of tumors. [226].
The angiogenesis-inducing ligand vascular endothe-
lial growth factor (VEGF) and its cell membrane
receptor, vascular endothelial growth factor receptor
(VEGFR) were predicted to be good targets for anti-
cancer therapy [218, 634]. The commercially avail-
able monoclonal antibody bevacizumab (Avastin)
binds VEGF. VEGF (a.k.a. VEGF-A) is one of a fam-
ily of related proteins that induce angiogenesis and/or
lymphoangiogenesis via binding one of several related
receptors.
VEGF is the central mediator of tumor angiogenesis,
which is crucial for proliferation, invasion, and metasta-
sis. It is produced by cells from many tissues and mes-
senger RNA levels of the VEGF gene are over expressed
in most tumors. Elevated levels of VEGF are predictive
of poor prognosis. A variety of experiments suggested
that this ligand would be a useful target for monother-
apy, or in combination with other therapeutic modalities
[12, 55, 248, 420, 681].
Considerations for Clinical use
of Monoclonal Antibodies
as Cancer Therapy
As summarized in Table 5, there are a number of factors
that must be considered, when choosing an antibody for
clinical development, and there are a number of specific
issues associated with therapeutic application. This sec-
tion focuses on the general principles of antibody ther-
apy that were, for the most part, established during
clinical trials conducted between 1980 and 1989 with
Mab directed against antigens found on cells from
hematologic malignancies [19, 38, 161, 177, 179, 180,
227, 328, 446, 478, 485–488, 518, 569, 600], and solid
tumor cancers [109, 161, 205, 240, 262, 263, 375, 439,
513, 535, 625, 651–653, 666, 738, 776]. Many of the
studies used products that reacted with CD5 [38, 177,
179, 227, 228, 488, 486, 488], or anti-idiotype antibod-
ies [446, 478, 487], or the antibody 17-1A against ade-
nocarcinomas [109, 240, 439, 651–653, 738].
315
Table 5. Issues for clinical application of monoclonal antibodies
Affinity and avidity of antibody for tumor antigen
Ability to effect complement and/or antibody-dependent cell
mediated lysis
Biological significance of the antigen to the malignant cell
Biological behavior of the antigen (secreted, internalized, rate
of production)
Density of antigen expression
Dose and schedule of administration
Expression of antigen on normal tissues
Human anti-antibody response (HAMA, HACA, HATA, etc.)
Heterogeneity of antigen expression
Location, size, and vascularity of tumor mass
Therapeutic index: efficacy vs. toxicity
Total body burden of tumor and antigen
Antitumor Effects
In Vivo Binding to Malignant Cells
In association with intravenous (i.v.) infusions of anti-
bodies, several studies used fluorescein conjugated anti-
mouse antibodies to show that many of these products,
especially those resulting from immunizing animals
with malignant hematologic cells, did bind to circulat-
ing blood tumor cells and cells in the bone marrow in
man [19, 38, 161, 177, 488, 518, 600]. Mab binding to
tumor cells located in lymph nodes, tumor masses, and
skin infiltrates were directly demonstrated using immu-
nofluorescence and immunoperoxidase techniques [179,
328, 375, 513, 535], and indirectly with low doses of
Mab conjugated to technetium, indium or iodine, as
radioactive tracers [92, 183, 286–289, 417, 427, 451,
496, 514, 515, 719]. The relative specificity of this
uptake was established by failure to image any “false
positive” lymph node sites, and the successful imaging
of nonpalpable nodes that subsequently were proven to
contain cancer by surgical excision and histopathology
evaluation [183, 719]. Further proof of binding specific-
ity was shown in a patient with T-cell lymphoma, in
whom a radiolabeled anti-CD5 murine Mab was con-
centrated in lymph nodes, but injection of a radiolabeled
murine anti-melanoma Mab of the same subclass was
not associated with lymph node uptake [92]. Various
studies showed that antibody binding was more easily
demonstrated in cutaneous tumors than in solid tumors
located in viscera or in lymph nodes [535]. This may
relate to cutaneous blood supply, but it could also relate
to the relatively small size at which cutaneous tumors
can be recognized. Some investigators have suggested
that direct infusion into the lymphatic system may be
superior to i.v. infusions in certain disease settings
316
[719], but this is not a practical option because of the
technical difficulties associated with such an approach.
In general, after an i.v. infusion of a large quantity of
Mab, the hierarchy of binding to antigen-bearing cells is
circulating blood cells > bone marrow > skin > lymph
nodes > small tumor nodules > large tumor masses.
There has been great interest in finding ways to over-
come the interstitial pressure effects found in larger
tumor lesions, which impair penetration of antibodies
into the core areas of such tumors [52, 345].
Clinical Efficacy and Mechanisms
Significant antitumor responses were relatively uncom-
mon in early trials with mouse Mab, but most of these
involved relatively limited dosing in pilot, phase I, or
limited phase I/II trials, and were not definitive tests of
Mab as cancer treatment. Nevertheless, it was encour-
aging that tumor responses following murine Mab ther-
apy were reported for both hematologic malignancies
and solid tumor cancers. Some of the best responses in
these early trials took place in patients with follicular
(nodular) lymphoma and malignant melanoma, two
diseases in which the frequency of spontaneous regres-
sions had suggested an existing antitumor immune
response [699].
As a general observation, when Mab bind in suffi-
cient number or density to cells in the peripheral blood,
those targeted cells are removed in the reticuloendothe-
lial system. Human trials showed that infusions of 51Cr
or 111I-labeled autologous tumor cells resulted in marked
uptake of the isotope label first in the lung, then in the
liver and spleen [161, 488, 518]. Investigators rarely
could detect associated decreases in complement levels,
although Ritz et al. [600] found deposits of C3 on mono-
clonal antibody-coated cells in one ALL patient, and
complement deposition was demonstrated in tissues in
colon cancer patients receiving KS1/4 [205], and in
melanoma patients receiving the R24 antibody [738].
Changes in the viability of circulating target cells have
been observed following monoclonal antibody binding,
although this is difficult to demonstrate because of tim-
ing and technical reasons [19, 518]. These studies sug-
gest that binding of Mab to peripheral blood cells may
actually damage the cell membrane and that such cells
are then removed in the reticuloendothelial system
rather than lysing in the intravascular compartment. The
best evidence of cell destruction, rather than sequestra-
tion, comes from studies following radiolabeled autolo-
gous tumor cells [177, 488, 518], and the finding of an
increased lactate dehydrogenase (LDH) associated with
decreased AML cells [19].
Monoclonal antibody therapy
In patients in whom tumor regressions have been
seen, the precise mechanism of antibody-mediated
tumor regression is difficult to ascertain. Anti-leukemic
effects are dependent on the Mab used, the rate and dose
of Mab infused, the density of antigen expression, and
whether there is circulating antigen or endogenous anti-
bodies that bind to the therapeutic antibody. In earlier
trials of mouse Mab, given in limited quantities, in most
instances the anti-leukemic responses were relatively
transient, but administration of chimeric and humanized
Mab in large quantities sufficient to sustain a large phar-
macokinetic “area-under-the curve” for prolonged peri-
ods of time, have been associated with meaningful
durable clinical response. In the absence of sustained
levels of Mab, antibody-coated target cells are removed
from the circulation, but are rapidly replaced by cells
from other organs such as bone marrow, lymph nodes,
and possibly spleen. Some cells may survive by traffick-
ing to other sites, and then return to the circulation. For
non-modulating antigens, the leukemia cell count
remains depressed as long as Mab levels persist in the
circulation. However, for modulating (internalizing)
antigens, the cell count begins to recover in association
with entry into the circulation of cells with a lower den-
sity of antigens. Such modulated leukemia cells subse-
quently re-express the target antigen in vitro or in vivo
once the antibody concentration has dropped to a negli-
gible level [639, 663]. Antigen modulation (cycling,
internalizing) has been a limiting factor for many anti-
bodies directed against hematopoietic malignancies, but
is not an issue for Mab that react with stable membrane
antigens, especially when the target antigen is important
as a receptor for cell proliferation, or helps a cell delay
or avoid programmed cell death.
The precise explanation for the responses seen in
B-cell lymphoma patients treated with anti-idiotype
Mab has not been clearly defined. Idiotype is a modulat-
ing target, which led investigators to adopt therapeutic
strategies of repeated low dose therapy so that target
cells would have a sufficient density of antigen to effect
CMC or ADCC. Investigators postulated either a direct
cytotoxic effect or a regulatory effect via the idiotype
network [65, 246, 247, 351]. The most responsive group
was follicular lymphoma. Analyses suggested that those
follicular lymphoma patients who responded had a
greater infiltration with T cells prior to therapy [478].
Responses were more readily achieved in patients with
very low levels of circulating idiotype, which is proba-
bly indicative of dose threshold related to antigen bur-
den. As discussed later in this chapter, even a decade
after its approval, it is still not clear whether immu-
nolgic and/or regulatory effects are more important for
Robert O. Dillman
the clinical effects observed with the anti-CD20 chimeric
Mab rituximab.
Although the best responses in patients with solid
tumors have been achieved with Mab that effect both
ADCC and CMC in vitro, regulatory mechanisms
related to the function of the target antigen (receptor)
may be more important. For example, the mechanism
for the anti-tumor effects that leads to the excellent clin-
ical results seen with antibodies to human EGFR are
probably related to the importance of those receptors in
sustaining proliferation. Secondary immunologic effects
may also be important. Sequential biopsies from mela-
noma patients who exhibited a clinical response to the
R24 mouse antibody revealed increasing infiltration
with CD3+, CD8+, Ia+ T cells in the presence of degranu-
lated tissue mast cells [779]. Complement deposition
was also noted. Even though responses were seen within
2 weeks of beginning therapy, tumors continued to
recede well beyond the treatment period despite the
presence of HAMA. These responses may involve a
more complex interaction with the host immune system,
perhaps triggering a cascade of inflammatory events
including activation of T lymphocytes that persist for a
long period of time [318], or they could be inducing
anti-apopotic and/or anti-proliferative effects. It has
been suggested that mast cells not only have local
inflammatory effects, but also may function as immu-
noregulatory cells in a an immune response [243].
Unfortunately, most advanced cancer patients do not
have tumors that are as easy to analyze sequentially as
melanoma. Selection of patients with primarily lymph
node or soft tissue disease facilitate prospective studies,
but they also result in unintentional bias because soft
tissue and lymph node metastases tend to be more
responsive to any intervention than bone, liver, brain, or
other visceral metastases.
Other interesting observations raise questions regard-
ing the possible mechanisms of antitumor affects against
microscopic disease [183, 287]. A young patient under-
went a left radical neck dissection for regional spread of
melanoma. Subsequently an anti-p97 antibody labeled
with 111Indium illuminated three apparent lesions in the
right neck, although repeated physical examinations and
a computerized tomographic scan of the region were
negative for tumor. No other lesions or lymph nodes
were visualized, but 3 weeks later he developed three
palpable lymph nodes in the right neck in sites consis-
tent with the scan. A neck resection was recommended
but he declined surgery, and the neck lesions subse-
quently resolved. He was known to remain free of dis-
ease for over 3 years. A second male patient had had a
melanoma resected from his upper back and later had a
317
right axillary recurrence. There was uptake of 111Indium
anti-p240 in the right axilla but there was also uptake
in the left axilla, which was clinically negative. Sub-
sequently, a small left axillary node was palpated but
spontaneously regressed. No other lymphadenopathy
was visualized by imaging and no other lymphade-
nopathy was palpated by examination. Both axillae
were explored with confirmation of tumor in the right
axilla, but no tumor was found on the left.
There are at least four possible explanations for these
intriguing observations of what clearly were antigen-spe-
cific interactions. First, there may have been a local anti-
tumor effect against microscopic tumor cells. Second,
there may have been antibody binding to residual antigen
retained in draining regional lymph nodes. Third, there
may have been antibody reaction with regional B lym-
phocytes that were expressing anti-idiotypic antibody to
endogenous anti-p240 or into p97 antibodies. A fourth
possibility is that the uptake was in macrophages or den-
dritic cells that had been loaded with tumor antigen.
The mechanism for the beneficial clinical effects of
anti-VEGF antibodies is also more complex than origi-
nally thought. It was hypothesized that blocking VEGF,
so that it could not bind to VEGFR, would interfere with
tumor vascular supply and starve the tumor of oxygen,
stimulating factors, and other nutrients. There is now
some evidence that changes in afferent and efferent blood
vessels themselves may be important more important in
concentrating chemotherapy at the tumor site than anti-
tumor effects from altered tumor blood supply.
Adverse Events: Toxicity and side Effects
The various adverse events observed with commercially
available anti-cancer Mab are shown in Table 6. These
can be characterized as infusion reactions, acute or
delayed hypersensitivity reactions, or non-immunologic
biologic effects. It should be emphasized that the major
adverse events associated with Mab are secondary to the
biologic effects induced by antibody–antigen binding
rather than immune reactions to the antibody itself [170,
175, 184]. This explains why most of the toxic effects
described for murine Mab are also seen with the chimeric,
humanized, and human forms of Mab directed to the
same antigen. Table 6 clearly shows that alemtuzumab,
is associated with the most toxicity, even though it is a
humanized antibody, because it reacts with circulating T
cells and other mononuclear cells that release large
amounts of cytokines in response to the Mab binding to
CD52 and the destruction of the cells by CMC and/or
ADCC. Rituximab reacts with circulating B cells that
are not as rich in inflammatory cytokines.
318 Monoclonal antibody therapy

Table 6. Percent of patients experiencing specific acute toxicities in clinical trials of monotherapy with a potpourri of murine and
human antibodies (177 patients with 20 different malignancies), rituximab, trastuzumab, alemtuzumab, bevacizumab, cetuximab and
panitumumab
19 Mo 3 Hum Ritux Tras Alem Beva Cetux Panitu
Antibody Mo/Hu Ximab Zumab Zumab Zumab Ximab Humab
Target Many CD20 EGFR-2 CD52 VEGF EGFR1 EGFR1
# of Patients 177 356 352 149 234 420 229
Tumor target Several Lymph Breast CLL Colo Colo Colo
Abdominal Pain <5 14 22 11 8a 26 25
Angioedema – 11 – – – – –
Arthralgia – 10 6 – – – –
Asthenia/malaise – 26 42 13 5a 48 –
Bronchospasm – 8 – 9 – – –
Chills/rigors 13 33 32 86 – 21 <5
Cough – 13 26 25 – 11 14
Diaphoresis 10 15 – 19 – 21 <5
Diarrhea – – – 2a 25 21
Dizziness – 7 13 12 – – –
Dyspnea – 7 22 26 – 17 <5
Fever 15 53 36 85 – 27 <5
Headache – 19 26 24 – 26 <5
Hypertension – – – – 8a – –
Hypotension – 10 – 32 – – <5
Myalgia – 10 – 11 – – –
Nausea 7 23 33 54 6a 29 23
Edema – 8 10 13 – 10 12
Pruritus 12 14 – 24 – 11 57
Rash 12 15 18 40 – 90 90
Rhinitis – 12 14 7 – – –
Throat irritation – 6 12 12 – – –
Urticaria 12 8 – 30 – – –
Vomiting 6 10 23 41 6a 25 19
Ritux = rituximab, Tras = trastuzumab, Alem = alemtuzumab, Bev = bevacizumab, Cetux = cetuximab,
Panitu = panitumumab
a
Bevacizumab data in this table reflects only grade 3 to 5 toxicity.

Because of the individual regulatory development of
these products, it was not always clear which adverse
events were product-related as opposed to target-related,
and this has led physicians to attribute toxicity to the
Mabs themselves, when, in fact, the effects were predict-
able because of the significance of the antigen target, and
the cascade of events that follow CMC and ADCC, many
of which are due to the subsequent release of cytokines,
as opposed to a hypersensitivity reaction to the antibody.
However, there are allergic or hypersensitive differences
as well. For instance, cetuximab and panitumumab both
target EGFR1, and have certain toxicities in common
that relate to biologic effects associated with blocking
EGFR, such as acneiform skin rash and gastrointestinal
symptoms including abdominal pain, nausea, vomiting
and diarrhea. However, the chimeric Mab that is made in
murine plasmacytoma cells has a higher rate of infusion-
associated flu-like symptoms, such as fever, headache,
chills, and dyspnea, compared to the human antibody
that is manufactured in CHO cells.
Infusion Reactions
These include a constellation of symptoms that predict-
ably occur during or shortly after infusion of a Mab.
The typical symptom complex includes fever, chills,
sweats, prostration, nausea, and sometimes dyspnea and
hypotension, which occur within a matter of minutes to
hours after an i.v. antibody infusion is initiated. They
are due to direct effects on tumor and non-tumor cells
which express the antigen, and indirect effects mediated
by the secondary release of various cytokines as a result
Robert O. Dillman
of antibody binding to the target antigen, or formation
of immune complexes with circulating soluble antigen.
Most infusion reactions associated with Mab are non-
allergic in nature, and rather are due to the interaction of
the antibody with circulating cells via antigen or Fc
receptors, and perhaps endothelial cells. However,
because of the effects of cytokines, it can be clinically
difficult to distinguish between allergic and non-allergic
infusion reactions. For many years it has been clear that
most infusion-related adverse events are non-allergic in
nature, and are directly related to antibody-antigen
interactions and the interaction with complement and
effector cells rather than being IgE, mast cell or eosino-
phil mediated reactions [170, 175, 184]. The antigen–
antibody binding to circulating cells in immunocompetent
patients, and subsequent interaction with complement
or effector cells, is predictably followed by the release
of cytokines and/or the removal of those circulating
cells via macrophages in the reticuloendothelial system
[19, 38, 79, 177–179, 180, 227, 228, 435, 446, 478,
485–488, 518, 569, 600, 787]. The onset of these reac-
tions is typically within 15–30 min after initiating an
antibody infusion, but depends on the dose and rate of
infusion, and the distribution of the target antigen.
Typical adverse events associated with binding to cir-
culating cells are the same as those seen with high doses
of various cytokines, such as interferon, interleukin-2,
or high doses of GM-CSF. These include fever, rigors/
chills, sweats, maculopapular erythematous skin rash,
urticaria, pruritus, edema, hypotension, tachycardia,
headache, nausea, vomiting, diarrhea, fatigue, elevated
hepatic transaminases, throat tightness, pain, thrombo-
cytopenia, dyspnea, bronchospasm, anaphylactic shock,
and even death [435]. The severity of infusion reactions
vary greatly depending on the nature of the antibody
(mouse or human), the distribution and density of the
target antigen on circulating cells, and the immunocom-
petence of the patient. Nearly all patients who have
received antilymphocyte and/or antigranulocyte anti-
bodies have experienced such side effects if they had
levels of circulating target cells at the time of infusion.
The reactions are predictable whether a patient is receiv-
ing murine, chimeric, or humanized antibodies, suggest-
ing that the antigenic target is the most important
variable, although differences related to the Fc portion
of the antibody are also important. The only time these
reactions were not seen was in patients repeatedly
treated with murine Mab who had high titers of endog-
enous antimouse antibodies that apparently neutralized
the infused antibody [179]. Thus, the presence of endog-
enous human antimouse antibodies actually prevented
many of these side effects by altering the pharmacology
319
and bioavailability of the murine Mab which limited
binding to the target antigen. The observation that some
F(ab’)2 can cause the same reaction suggests that anti-
body binding to antigen may be sufficient to induce
cytokine release even without interaction of the Fc por-
tion of the antibody in an ADCC reaction [721].
Antibodies that react with circulating T cells, which
are more numerous and contain more cytokines than B
lymphocytes are associated with more severe infusion
reactions. Binding to granulocytes also triggers severe
infusion reactions. As described earlier in this chapter,
studies with radiolabeled cells showed that once anti-
body binds to circulating cells, they are removed in the
reticuloendothelial system including the lung, liver, and
spleen [177, 485, 518]. When large numbers of cells are
removed in the lungs, this may be associated with dysp-
nea and hypotension. For this reason, when it is known
that an antibody will react with circulating cells, the ini-
tial infusion rate needs to be slow, and high-dose bolus
administration is avoided. Once circulating target cells
are no longer present, subsequent infusions may be
safely given at more rapid rates. For instance, the author
has administered full dose rituximab over 30 min during
subsequent weekly treatments once B lymphocytes have
been cleared from the circulation, and others have con-
firmed the safety of this approach in formal trials with
delivery over 45 to 90 min [82, 655].
For patients who are initiating therapy with a mono-
clonal antibody that reacts with circulating cells,
premedication with steroids, acetamenophen and/or
diphenhydramine decrease the severity of symptoms
associated with an infusion reaction, but does not typi-
cally prevent such a reaction [175]. Once the target cells
are gone, there is no need to give such pre-medication.
It is possible that combined prophylactic therapy with
histamine blockers and steroids starting 24 h before
treatment would reduce many of these toxicities, much
as they reduce the immune reactions associated with
infusion of paclitaxel chemotherapy.
Tumor lysis Syndrome
Tumor lysis syndrome is a constellation of symptoms
and complications associated with the rapid cytotoxic
death of rapidly proliferating tumor cells [817, 818]. It
has been well described in association with systemic
chemotherapy for rapidly proliferating malignancies
such as acute leukemia, and patients with bulky tumor
loads of Hodgkin’s disease, large B cell lymphoma, and
testicular cancer in which highly effective systemic
chemotherapy destroys large number of proliferating
cells in a short time period. This is followed by increases
320
in serum potassium, uric acid, and phosphates released
by the destroyed cells, with subsequent hypotension,
renal failure, and other medical complications that can
be life-threatening or lethal. For single-agent unconju-
gated monoclonal antibody therapy, true “tumor lysis
syndrome” occurs when a patient is treated with an anti-
body that targets large numbers of proliferating tumor
cells in the blood stream, but does not happen when
non-replicating cells are removed from the circulation
[170, 175, 184]. This is of particular concern in the set-
ting of acute leukemia, lympholeukemias of mantle cell
and large B cell lymphoma, and prolymphocyte-pre-
dominate chronic lymphocytic leukemia (CLL) in which
large numbers of antigen-positive tumor cells are pres-
ent in the circulation, where the antibody can opsonize
large numbers of cells resulting in their lysis in the cir-
culation and in the reticuloendothelial system [79, 179].
Many of the descriptions of so-called “tumor lysis syn-
drome” in the literature were really predictable antigen–
antibody infusion related reactions rather than true
tumor lysis syndrome with the complications that result
from lysis of large numbers or proliferating cells and
release of intracellular products including high levels of
nucleic acids that lead to hyperuricemia [79, 787, 798].
Antibody-induced tumor lysis syndrome has only been
observed in the setting of large numbers of rapidly pro-
liferating circulating target cells; it has not been observed
in the absence of a large leukemic component of dis-
ease, probably because of the slower penetration of anti-
bodies into tumor tumor masses [345]. Unless the
patient is truly at high risk for tumor lysis syndrome
there is no need for prophylactic measures such as pre-
hydration, sodium bicarbonate to alkalinize the urine,
and anti-uric acid agents such as allopurinol or rasbu-
ricase. For example, in classical CLL, there are primar-
ily mature lymphocytes in the circulation whose
excessive number is more the result of being long-lived
rather than hyperproliferation, and true tumor lysis syn-
drome does not occur in that setting, even with the
removal of hundreds of thousands of cells in just a few
minutes, although, infusion reactions are common, and
can be life threatening in a patient who is medically
compromised by cardiopulmonary, hepatic, or renal dis-
ease. However, a CLL patient with a large number of
prolymphocytes is at risk for tumor lysis syndrome.
Acute Hypersensitivity Reactions
and Anaphylaxis
Because the first Mab were mouse proteins, there was
great concern that infusion of these products would be
associated with acute anaphylactoid reactions. Fortunately,
Monoclonal antibody therapy
such complications were rare, and are even less common
in association with chimeric and humanized antibodies.
Patients with a known history of allergic reaction to
rodents or their by products, were typically excluded
from trials of murine antibodies. Acute allergic reactions
have included anaphylactic shock, less severe anaphy-
lactoid reactions such as bronchospasm, and generalized
pruritus and urticaria [170, 175, 184]. The more severe
reactions can be successfully managed with epinephrine.
Patients who experience full blown anaphylaxis during
or shortly following an antibody infusion should not be
retreated with the agent, but it is important that a predict-
able infusion reaction not be confused with such a severe
allergic reaction. Dermatologic effects including pruritis,
diffuse or focal maculopapular rashes and urticaria may
be seen alone, or as part of a full anaphylactic reaction
with laryngeal edema, hypotension, and bronchospasm.
Pruritus and urticaria alone typically resolve without
treatment, but may be responsive to diphenhydramine or
epinephrine.
Side Effects Secondary to Binding
to Non-Malignant Tissues
Adverse events may also occur as a consequence of
direct effects on noncancerous tissue that also express
the target antigen. This has been especially true for
antibodies that cross-react with adenocarcinomas and
cells of the gastrointestinal tract. Such antibodies have
been associated with a high frequency of diarrhea, nau-
sea and vomiting, abdominal pain, and even large and/
or small bowel mucosal damage in some patients [109,
240, 593]. Other antibodies that are known to cross-
react with antigens on neural tissue have been associ-
ated with specific pain syndromes in some patients
[109, 205, 651, 652]. Cross-reactivity with normal tis-
sue antigens is particularly a concern when antibodies
are conjugated to cytotoxic substances such as radio-
isotopes, chemotherapy agents, or natural toxins. For
instance, the incidence of gastrointestinal toxicity was
much greater when a frequently tested adenocarcinoma
antibody was given conjugated to a vinca chemother-
apy analog, or methotrexate, as compared to adminis-
tration of the naked antibody [205, 636]. Another
adenocarcinoma antibody that cross-reacted with anti-
gens on neural sheaths, produced unacceptable neuro-
toxicity when conjugated to the A chain of the natural
toxin, ricin [541]. Dermatologic toxicities are predict-
able complications of agents that interfere with EGFR
signal transduction, such as cetuximab and panitu-
mumab [343, 551, 650] and the anti-Her2 Mab trastu-
zumab is associated with cardiac toxicity [374, 658, 678].
Robert O. Dillman
Bevacizumab has been associated with hemorrhage,
vascular thrombosis, hypertension, and proteinuria,
presumably all because of effects on blood vessels.
[211, 538, 698]. The complications associated with
these three commercially available antibodies are dis-
cussed in more detail later in this chapter.
Immune complexes
In some instances, Mab are given to patients who are
known to have free or soluble circulating antigen.
Examples include the circulating idiotype in lym-
phoma, and carcinoembryonic antigen (CEA), or
prostate specific antigen (PSA). Immune complexes
may also be formed because of HAMA, HACA, and
HAHA. Only rarely have symptoms been noted in the
presence of the immune complexes formed by the
binding of antibody to circulating antigen, probably
because of the small size of such complexes since the
monoclonal antibody binds to only one determinant
on the circulating antigen. For this reason, the pres-
ence of circulating antigen is not a contraindication to
antibody treatment, although the binding to soluble
antigen greatly alters the pharmacokinetics of the
antibody. However, acute arthralgias, myalgias, nerve
palsies, fever, and skin rashes have occasionally been
seen in this setting and attributed to the acute immune
complex formation [170, 175, 184]. During trials of
murine Mab, the presence of HAMA predictably
decreased the half-life of the antibody, but was rarely
associated with complications that could be attributed
to immune complexes.
Delayed Allergic Reactions and Serum
Sickness
Because of the anticipated production of HAMA, there
was concern that delayed reactions such as serum sick-
ness would be a significant problem following infusion
of murine Mab. Fortunately, immune complex compli-
cations related to HAMA have been uncommon, but
they can occur [170, 175, 184]. Classic serum sickness
has been seen 2 to 3 weeks following exposure to mod-
erate and high doses of murine Mab, and rarely after
chimeric Mab. A typical symptom complex includes
fever, malaise, arthralgias/arthritis, myalgias, maculo-
papular erythematous skin rash, and fatigue. Proteinuria
has been rarely observed in these few patients and renal
insufficiency is extremely rare. Serum sickness can be
managed with non-steroidal, anti-inflammatory agents,
and corticosteroids in more severe cases.
321
Antibody Issues
Immunoglobulin Class and Subclass
In lymphoma, murine anti-idiotype Mab of various sub-
classes produced clinical responses [446, 478, 487]. In
melanoma, only murine IgG3 Mab and humanized
anti-GD2 or anti-GD3 antibodies produced clear-cut
responses [109, 328, 738]. The responses seen with
murine anti-CD5 IgG2a Mab in T-cell lymphoma and
CLL were limited [38, 177, 179, 180, 227, 228, 485,
486, 825]. Even though it appears that mouse IgG3, and
perhaps some mouse IgG2a Mab, have greater potential
to effect ADCC than other subclasses of murine Ig [308,
340, 511, 691], the preferred strategy is to use chimeric,
humanized, or human Ig because of the advantages
associated with the human Fc. Some complement-fixing
IgM Mab would be useful, but their size limits tumor
penetration in solid tumors, and administration of large
doses could lead to hyperviscosity. The issues related to
murine Mab are of decreasing importance since most
investigators and companies have converted their prom-
ising murine Mab into chimeric or humanized Mab.
Most of these conversions have been to a human IgG1
subclass because these are predictably associated with
efficient CDC and ADCC [660]. Class and subclass
switching of antibodies has enabled us to establish more
directly the importance of the binding of antibody to Fc
receptors on effector cells.
Human Anti-Immunoglobulin Response
Because they are foreign proteins, it was not surprising
that murine Mab produce human antimouse antibodies
(HAMA) responses in immunocompetent patients,
especially when large quantities are administered. Trials
involving single exposure and multiple exposure to Mab
confirmed that this is a substantial obstacle to repetitive
therapy, especially in patients with solid tumors, who
typically are less immunosuppressed that patients suf-
fering from hematologic malignancies. [131, 164, 185,
382, 640, 664]. Using radioimmunoassay (RIA) and
enzyme-linked immunosorbent assays (ELISA), inves-
tigators detected HAMA in virtually all patients who
have received mouse antibodies except for those with
CLL, probably because of the immunodeficiency asso-
ciated with that disorder. It also appears that HAMA is
reduced in many B-cell lymphoma patients, and perhaps
the anti-B cell antibodies contribute to this. However,
recent studies involving radiolabeled anti-CD20 murine
Mab as therapy for previously untreated B-cell lym-
phoma patients resulted in 63% of patients exhibiting
322
HAMA within a median of 3.3 weeks of being treated
[362]. This suggests that prior chemotherapy and/or
greater mutations of recurrent and progressive disease
induce a relative immunodeficiency that lowers the rate
of HAMA in more heavily treated patients. Interestingly,
many CTCL patients who developed HAMA had previ-
ously experienced a clinical response to treatment, per-
haps because they are the more immunocompetent
patients, or perhaps because their disease was less resis-
tant because of less exposure to chemotherapy.
Once HAMA is produced, they effectively neutralize
most clinical effects of therapy, although targeting of
tumor cells can still be demonstrated. It appears that a
small percentage of antimouse antibodies react specifi-
cally with the idiotype of the mouse protein rather than
only with specific murine Ig isotype determinants [404,
664]. As discussed earlier in this chapter, it has been sug-
gested that this anti-idiotype cascade may contribute to
the anti-tumor effect as an indirect vaccine [313, 404].
Efforts to block the antimouse immune response
with chemotherapy, radiotherapy, corticosteroids, and
cyclosporine, have not been successful. Some investiga-
tors suggested that infusions of high doses of antibody
would eliminate the antimouse response, but this was
not borne out in other trials. When patients receive high
doses of antibody, they must be evaluated for a pro-
longed period of time because, depending on the assay
used, the excess of mouse antibody may block any evi-
dence of antimouse antibodies during the early phase of
the HAMA, HACA, or HAHA response. Some investi-
gators have suggested that attachment of polyethylene
glycol may decrease the immune response [784], but
this not been confirmed in humans. In the presence of
HAMA, some investigators have combined plasma-
pheresis with administration of higher doses of antibod-
ies, but this was of limited benefit.
Even with modern commercially available antibodies,
there is still the potential for human (HAMA) and/or
human anti-chimeric antibodies (HACA) following
treatment with a chimeric antibody, and even human
anti-human antibodies (HAHA) directed against allo-
typic human antigens on human Mab. Human immune
responses that result in the production of HAMA, HACA,
and HAHA are readily detected by immunoassays that
utilize the therapeutic antibodies in the assays used in the
treatment of patients. As expected, most of the antibod-
ies associated with chimeric antibodies are HAMA
directed to the residual murine determinants. Most of the
antibodies in HACA or HAHA are directed to allotypic
epitopes and carbohydrates on the Fc portion. Fortunately
the various chimeric and humanized Mab that have
entered the clinic have been associated with minimal
Monoclonal antibody therapy
HAMA or HACA, or HAHA. Today there is no need to
try to obtain approval of a therapeutic mouse monoclonal
antibody unless one desires short pharmacokinetics such
as in radioimmunotherapy to reduce the amount of total
body irradiation [174]. These preparations are a signifi-
cant improvement over mouse antibodies, and even when
HACA or HAHA occur, they tend to appear later and
often at lower titers so that repeated intermittent therapy
is possible over several months. Based on the large clini-
cal experiences with current chimeric and humanized
antibodies, it appears that the risk of HACA or HAMA is
only on the order of 1–2% of all patients treated, and this
rarely interferes with treatment.
There is a clinical caveat associated with HAMA
because exposure to such antibodies may sensitize
patients who subsequently will have limited therapeutic
benefit and/or be at risk for allergic reaction if further
antibody doses are given for diagnostic or therapeutic
purposes. The HAMA may also confound the interpre-
tation of serum diagnostic tests such as those for pros-
tate specific antigen (PSA) or carcinoma embryonic
antigen (CEA) etc. that are measured using momabs.
For instance, we have seen a patient develop significant
HAMA that resulted in chronic T cell lymphopenia and
aberrant PSA assays results secondary to exposure to
the OKT3 mouse monoclonal antibody associated with
an adoptive cell therapy in which cells were incubated
in vitro with the OKT3.
Antigen issues
Antigenic Modulation
Antigen modulation is a dynamic process in which sur-
face antigen is decreased in the presence of excess anti-
body. Electron microscopy with autoradiography and
125
I-labeled monoclonal antibody studies showed that
modulation is the result of internalization of the antigen
and the bound antibody [599, 639, 663]. This is pre-
ceded by “capping” of the antibody–antigen complex, a
process during which the complex appears to localize to
one region of the cell surface. Mab directed against
many hematopoietic surface antigens and growth factor
receptors on solid tumors induce modulation in vitro
and in vivo. Modulation may in reality be an example of
antibody acting as a surrogate ligand for a receptor that
normally internalizes after binding to a ligand.
Modulation occurs within minutes of exposure to Mab,
but is reversible once the monoclonal antibody is
removed from the system because of the ongoing pro-
duction or recycling of antigen/receptor. This has been
demonstrated both in vitro and in vivo.
Robert O. Dillman
Antigenic modulation must be differentiated from
immunoselection that might result from the elimination
of antigen-positive cells, thereby leaving only cells that
express no or only low levels of the target antigen. The
conditions for modulation, or antibody-mediated down
regulation of a receptor in vivo include presence of immu-
noreactive Mab in the serum, and persistent existence of
tumor cells that exhibit absent or reduced expression of
the targeted antigen, but continued expression of another
phenotypic marker for the cell in question. For example,
the C22 antigen on malignant and normal B cells modu-
lates after binding of antibody, such that CD22 becomes
undetectable, but CD19 and CD20 are other markers of B
cells that can be measured. In the absence of antibody, the
antigen must be re-expressed to prove there is no immu-
noselection of cells. Thus, if the cells that now appear to
be CD22 negative because of modulation are placed in
cell culture, re-expression of CD22 proves that only mod-
ulation has taken place rather than immunoselection of
for a CD22 negative subclone of cells.
Modulation is a dynamic process that consists of anti-
gen expression, antibody binding, antibody–antigen
internalization, re-expression of surface antigen, addi-
tional antibody binding, etc.; such that at any point in
time there is always at least some amount of residual
surface antigen expression, although this may be diffi-
cult to prove depending on the sensitivity of the assay
being used. This phenomenon has important implica-
tions for passive monoclonal antibody therapy because
during modulation, there are insufficient quantities of
Mab on the cell surface to effect target cell elimination
by complement or effector cells. For circulating cells,
there is a threshold of monoclonal antibody-binding that
is required before cells are eliminated. In fact, in some
cases, cells with a high density of target antigen are rap-
idly eliminated while cells lower in antigen persist and/
or enter the circulation so that total target cell count is
relatively unaffected [177, 179, 180].
Modulation, or down regulation of receptors may be
desirable for antibodies that act through a regulatory
mechanism, perhaps by altering signal transduction.
Continued presence of antibody is needed to sustain this
effect, which has important implications for duration of
therapy. Rapid internalization greatly limits the therapeu-
tic potential of antibodies that effect complement and/or
cell-mediated cytotoxicity, but may be useful or a neces-
sary condition for drug or toxin antibody immunoconju-
gates. As a general rule, modulation is much more common
for hematopoietic cell antigens than solid tumor antigens,
but it appears that some of the best targets for treatment of
solid tumors are modulating/internalizing antigens that
function as growth factor receptors for tumor cells.
323
Free Antigen
Many tumor antigens are either shed in small quantities,
or may be released during apoptosis. Some tumor anti-
gens are shed in large quantities, constituting potential
blocking factors to monoclonal antibody target cell
binding. Such immune complexes might also produce
damage to normal tissues and organs, as well. Most of
the hematopoietic antigens detected by Mab are gener-
ally not shed to an extent that interferes with measure-
ment of monoclonal antibody serum levels. However,
excess circulating antigen has been a practical problem
for anti-idiotype antibodies in the treatment of lym-
phoma. Immediately following treatment, blood antigen
levels decrease precipitously as they complex with anti-
body, but the remainder of the Mab have access to tumor
antigen. Thus, the obstacle of circulating antigen can be
overcome with higher monoclonal antibody doses.
However, immune complex-mediated disease may
occasionally be a complication of this approach.
Antigen Heterogeneity
If a given tumor cell does not express the antigen
detected by a given monoclonal antibody, there is no
basis for monoclonal immune-mediated cytolysis or
receptor inhibition for that cell. For many years there
was great debate as to whether any single Mab could be
clinically efficacious because of the tremendous hetero-
geneity in human cancer cell phenotypes, especially for
solid tumors [220, 307, 522, 637]. Fortunately, in recent
years single antibody products have proven effective
enough as single agents to gain regulatory approval.
With our expanded knowledge of tumor phenotype and
tumor cell antigens, in the future it may be possible to
employ rationale combinations of Mab to overcome this
problem [433, 534]. Ideally, such a combination or
“cocktail” would include Mab in quantities directly
related to specific antigen expression on an individual
patient’s cancer cells rather than fixed proportions being
administered to each patient with a given disease.
However, just as with chemotherapy, a combination
may only be effective if its individual components have
some antitumor effects of their own.
Dose, Schedules, and Pharmacokinetics
Antibody Dose
There is a dose/response relationship at low monoclonal
antibody doses (<10 mg) because of the volume of distri-
bution, nonspecific uptake and metabolism, and the
324
importance of number of antibody molecules on cell sur-
faces for complement, reticuloendothelial, or effector
cell-mediated effects. At higher dose levels that provide
antibody excess, these dose–response relationships are
no longer important, but duration of sustained serum lev-
els (area under the curve) is important for tumors with
non-modulating antigen targets. In the case of antigens
targeted for a regulatory affect (whether modulating or
not), sustained serum levels are needed to continuously
block or down regulate receptors that are continually
being produced by differentiating cells. The cells may
emerge from stem cells or progenitor cells that do not
express the target antigen, and therefore not directly
affected by antibody therapy. Extrapolation from animal
studies, and from studies of biopsies from patients
receiving mouse antibodies, suggested that gram quanti-
ties of antibodies would be needed for a therapeutic
effect because of the importance of tumor penetration
and saturation. Several investigators noted that direct in
vivo tissue binding (as opposed to binding to circulating
cells) could only be demonstrated at doses of 30 to 50 mg
or higher [179, 180, 328, 535]. Some radiolabeled mono-
clonal antibody studies showed that more tumors were
imaged with doses of 10 mg or greater [183, 287, 514].
The clinical successes of recently approved Mab have
been associated with delivery of high doses repeated at
relatively short intervals, such as every 1 to 2 weeks. At
these doses and schedules, continually detectable serum
levels of antibody are assured in most patients. It seems
likely that there are critical thresholds for dose and/or
area under the curve of sustained antibody levels to pro-
duce an anti-tumor effect. Whether the dose/response
relationship is important beyond those threshold levels is
unclear, but sustained serum levels is likely to be related
to duration of response in some settings.
Infusion Rates/Schedules
Rapid infusions of Mab that bind to antigen on circulat-
ing blood cells and/or trigger endogenous immune
responses leading to cytokine release and cell activation
via interactions with Fc receptors, can be life threatening.
[79, 170, 175, 178, 179, 184, 435, 787]. When antibodies
react with circulating cells, infusion rates greater than
25 μg/ml are typically associated with infusion reactions.
Impure preparations that contain microaggregates, pyro-
gen, or other contaminants may also be more toxic if
infused rapidly, but this should not be a problem with
products approved for clinical use. Most investigators
have been satisfied with 2 to 6-h infusions for delivery of
higher doses of antibody, but pure preparations of anti-
bodies which do not react with circulating blood cells can
Monoclonal antibody therapy
be given over a few minutes with no untoward effects.
Prolonged continuous infusions or repeated bolus infu-
sions are probably necessary for Mab that work via regu-
latory mechanisms in order to keep receptors
down-regulated or blocked. This may also be desirable in
order to establish a gradient effect in treating solid tumors
because of tumor penetration problems. Bolus infusions
are well tolerated by most patients if the monoclonal anti-
body preparation is free of aggregates and there is no
cross-reactivity with circulating cells. Intermittent bolus
infusions may be preferred if the down-regulated receptor
needs to be re-expressed in order for the antibody/antigen
(receptor/ligand) interaction to produce an antitumor
effect via CMC or ADCC. In the past, because HAMA
was anticipated, some investigators adopted a strategy to
deliver the maximum dose of murine Mab within 2 weeks,
because HAMA is generally detectable within 2 weeks of
initiating treatment. Modern chimeric and humanized
antibodies are able to be effectively delivered over many
weeks to months because HAMA, HACA, and HAHA
have rarely interfered with serum levels. So far there are
no studies that have rigorously addressed the correlation
between dose, infusion rate, or schedule, even for the
antibodies that are now in widespread clinical use.
Serum Levels and Pharmacokinetics
Because of known antibody–antigen interactions, it has
been easy to devise various enzyme-linked immunosor-
bent assays (ELISA) and radioimmunoassays (RIA) to
measure serum levels of Mab during and following infu-
sions [25, 34, 205, 443, 665, 706]. Serum Mab levels have
been easily detected except during low infusion rates, or in
the presence of high levels of circulating antigens, high
circulating tumor burden, or in the presence of HAMA. In
the leukemias, monoclonal antibody levels tend to fall
rapidly following an infusion because of continuing
absorption by circulating cells and entry of additional cells
into the circulation. However, with some antibodies, with
or without the presence of antigenic modulation, serum
Mab levels are sustained for many days to weeks depend-
ing on the dose given. Using 24- to 48-h infusions, peak
Mab concentrations of several μg/ml have persisted for up
to 2 weeks. The significance of serum levels at any time
point must be viewed in the light of the variables listed
above. In addition to tumor burden, other important vari-
ables include circulating antigen, antigenic modulation,
and the production of antimouse antibodies. With the anti-
CD20 chimeric antibody rituximab, there is a clear asso-
ciation between the area under the curve for the antibody
and tumor response, and return of normal and/or normal
B-lymphocytes does not occur until serum levels of ritux-
Robert O. Dillman 325

imab are negligible [34]. The dose and schedule for the
antibodies in clinical use today are all given at high doses
that sustain levels for several weeks to months. Chimeric,
humanized, and human Mab consistently demonstrate
superior pharmacokinetics, including higher peak levels
and prolonged sustained blood levels, compared to their
mouse counterparts. This is especially true following
repeated administration because of the negative effects of
HAMA on mouse antibody pharmacokinetics. Recent
studies suggest that recycling of IgG after interaction with
Fc gamma receptors may also play a role in sustaining
serum levels [357, 422, 442].
Commercially Available
Unconjugated Mab for Treatment
of Human Malignancy
Although many were disheartened by the early observa-
tions with murine Mab, the experience with these murine
products, and humanization of antibodies through
molecular engineering, suggested that Mab would still
live up to their potential [161, 163, 166, 167, 172, 173,
428, 533]. “Magic bullets” for cancer therapy became a
reality in November 1997 when the anti-CD20 mono-
clonal antibody rituximab (Rituxan) became the first
monoclonal antibody product approved by the US Food
and Drug Administration for the treatment of a malig-
nant disease, namely B-cell lymphoma [168]. Ironically,
this product was produced by the company IDEC
Pharmaceuticals (San Diego, CA), which was originally
founded to develop anti-idiotype antibodies, but instead
gained success on the basis of a highly effective anti-
body that reacts with most normal and malignant
B-lymphocytes. In the following decade, eight addi-
tional monoclonal antibody products obtained regula-
tory approval for the treatment of various malignancies.
The rest of this chapter will focus on the six unconju-
gated Mab that have been approved by the United States
Food and Drug Administration (US FDA) for the treat-
ment of malignant disease; radiolabeled antibodies and
immunotoxins are covered elsewhere in this book.
Rituximab (Rituxan®, Biogen-Idec
Pharmaceuticals, San Diego, CA and
Genentech, South San Francisco, CA)
Rituximab and CD20
The anti-CD20 monoclonal antibody rituximab (Rituxan)
became the first commercially available monoclonal
antibody for the treatment of a human malignancy when
it was approved by the US FDA in November 1997, with
a marketing indication for B-cell lymphoma (Table 7)
[168]. The anti-CD20 murine Mab that originally had
been named 2B8, and later ibritumomab, was converted
to a mouse/human chimeric antibody and named ritux-
imab [782]. Rituximab includes murine variable regions
from the anti-CD20 murine Mab ibritumomab and human
IgG1 kappa constant regions that have been combined
using recombinant DNA technology. The genetically
engineered chimeric monoclonal antibody is manufac-
tured in CHO cells. In vitro it is cytolytic against CD20-
positive cells in the presence of human complement and
human effector cells [260, 441]. Rituxmab’s binding to
CD20 also appears to have a regulatory effect on B cells
by promoting apoptosis [473, 661], and it appears to be
more effective in this regard than other anti-CD20 antibodies

Table 7. Commercially available monoclonal antibodies for the treatment of human malignancies
Year FDA
Generic name Commercial name Antigen Antibody type Fc/Fv Ig type Cell production approved
Rituximab Rituxan CD20 Chimeric IgG1 CHO 1997
Trastuzumab Herceptin EGFR-2 Humanized IgG1 CHO 1998
Daclizumab Zenapax CD25 Humanized IgG1 CHO 1998
Alemtuzumab Campath CD52 Humanized IgG1 CHO 2001
Bevacizumab Avastin VEGF Humanized IgG1 CHO 2004
Cetuximab Erbitux EGFR-1 Chimeric IgG1 MP 2004
Panitumumab Vectibix EGFR-1 Human IgG2 CHO 2006
CD = cluster designation
EGFR = epidermal growth factor receptor
VEGF = vascular endothelial growth factor
CHO = Chinese Hamster Ovary
MP = murine plasmacytoma
326
such as IF5 and anti-B1 [661]. The regulatory conse-
quences of rituximab binding to CD20 may be more
important in vivo than the immunologic-based anti-tumor
effects. In vitro the presence of rituximab was associated
with enhanced sensitivity to chemotherapy which pro-
vided a strong rationale for combined modality treatment
[156]. Rituximab is used alone or in combination with
chemotherapy in indolent B cell malignancies, and is
used in combination with chemotherapy in aggressive B
cell malignancies.
CD20 is a 35 kD antigen with multiple transmem-
brane domains that is expressed on normal and malig-
nant B cells, but not on non-hematopoietic tissue, B-cell
progenitors, plasma cells, T-lymphocytes, monocytes,
dendritic cells, or stem cells [782]. CD20 is neither
internalized nor shed in significant amounts. No ligand
for CD20 has been identified, but its over-expression
appears to be important for resistance to apoptosis of B
lymphocytes [473, 661].
Clinical Trials with Rituximab
Early Clinical Trials with Single-Agent
Rituximab
Maloney et al. conducted the early trials with C2B8
(rituximab) [459–461]. In a dose escalation phase I trial
of single i.v. doses from 10 to 500 mg/m,2 two partial
responses and four minor responses were observed
among 15 patients [459]. In a subsequent phase II trial,
multiple doses of 375–500 mg/m2 were given i.v. weekly
for 4 weeks [461]. There were four complete responses
and 16 partial responses among 34 evaluable patients
for an overall objective response rate of 47%. Most of
the partial responders remained in remission for 4–12
months following treatment.
The pivotal trial for regulatory approval of rituximab
was a multi-institutional study involving 166 patients
with low-grade or indolent lymphoma, mostly follicular,
who had relapsed after prior chemotherapy [474]. This
trial yielded an objective response rate of 50% with a
median duration of response of more than a year; 90% of
responses lasted more than 6 months. Patients in these
trials had already failed a median of four prior chemo-
therapy regimens. The response rate in patients who had
failed high-dose chemotherapy and stem cell transplant
was a remarkable 73% (18/23). None of the responding
patients relapsed in less than 4 months, and 10% remained
in continual remission for 2–5 years. One third of patients
had their initial infusion interrupted because of the clas-
sical infusion reaction complex of fever and chills and
occasional shortness of breath, hypotension, and skin
Monoclonal antibody therapy
rash associated with the binding of antibody to circulat-
ing B lymphocytes. There was very limited toxicity dur-
ing subsequent infusions. There was no evidence of
hypogammaglobulinemia or infections as a consequence
of suppression of humeral immunity.
Retreatment with rituximab at a dose of 375 mg/m2
weekly for 4 weeks resulted in a response rate of 41%
among 40 patients who previously enjoyed a response to
rituximab that had lasted 6 months or longer [149]. The
18-month median duration of response was even longer
than the 13 months noted in the pivotal trial, probably
because most of the patients had a smaller tumor burden
and more favorable pharmacokinetics than the original
population of patients treated with rituximab. There was
no clear explanation as to why the remaining patients
failed to respond to retreatment, but resistance was not
due to loss of CD20 expression or HAMA or HACA
against rituximab. There was no emergence of a CD20-
negative subclone as a consequence of a treatment-related
immunoselection process. While occasional patients do
recur with CD20 negative tumors [145], this has proved
to be an exception, and most patients retain CD20
positivity throughout the course of their disease. A study
of 13 patients in a similar trial conducted in Japan had a
response rate of 38% with rituximab re-treatment but
the median duration of response was only 5.1 months in
this study [339].
Rituximab in Follicular Lymphoma
Rituximab is highly active as a single agent in the treat-
ment of previously treated and previously untreated fol-
licular lymphoma, as shown in Table 8 [15, 56, 97, 125,
147, 231, 251, 277, 460, 461, 474, 562, 790, 791].
In previously treated follicular lymphoma patients,
objective response rates have ranged from 46% to 63%
[15, 97, 145, 231, 461, 474, 562, 790]. In previously
untreated follicular lymphoma patients response rates
are generally higher, ranging from 67% to 78% [125,
251, 277, 791]. Most of these trials utilized the standard
4-week course of treatment at 375 mg/m2, but Piro et al.
used an 8-week treatment schedule [562], Aviles et al.
and Bremer used a 6-week treatment schedule [15, 56],
and Hainsworth et al. utilized additional 4 weeks of
therapy every 6 months for 2 years [277]. Pooling data
from similar trials shows a response rate of 29/37 (78%)
for untreated patients who received more than 4 weeks
of therapy, 100/143 (70%) for untreated patients who
received the standard 4 weeks of therapy, 59/89 (66%)
for relapsed patients who received more than 4 weeks of
therapy, and 238/458 (52%) for relapsed patients who
received the standard 4 weeks of therapy.
Robert O. Dillman 327

Table 8. Rituximab as a single agent in follicular lymphoma Table 9. Rituximab as a single agent in small lymphocytic
lymphoma
Proportion
Citation Clinical setting responding % Response Proportion
Citation Clinical setting responding % Response
[277] Untreated 29/37 78%
[15] Relapsed/ 13/17 76% [277] Initial therapy 15/23 65%
refractory [231] Relapsed/ 4/29 14%
[125] Untreated, low 36/49 73% refractory
tumor burden [562] Relapsed/ 1/7 14%
[791] Untreated 26/37 72% refractory
Grade 1
[474] Relapsed/ 4/32 13%
[562] Relapsed/ 21/30 69%
refractory
refractory
[251] Untreated 38/57 67%
[97] Relapsed/ 24/38 63%
refractory
[56] Relapsed/ 25/42 59%
refractory Table 10. Rituximab as a single agent in marginal zone
[474] Relapsed/ 75/130 58% lymphoma
refractory Proportion
[790] Relapsed/ 36/70 56% Citation Clinical setting responding % Response
refractory
[145, 147] Relapsed/ 12/22 55% [127] No prior 21/24 87%
refractory > chemotherapy
10 cm [469] Relapsed/refractory 20/26 77%
[231] Relapsed/ 32/70 46% gastric
refractory [127] Prior 5/11 45%
[251] Relapsed/ 59/128 46% chemotherapy
refractory

McLaughlin and Foran trials utilized the standard

Long term follow up of the French trial was reported
by Solal-Celigny et al. for 49 untreated follicular lym-
phoma patients with a low tumor burden who were
treated with the standard dose and schedule of ritux-
imab, 375 mg/m2 IV weekly × 4 [683]. Based on the
recent Follicular Lymphoma Internal Prognostic Index
(FLIPI), 45% of these patients would be considered low
risk, 41% intermediate risk, and 14% poor risk. The
eventual response rate was actually 80% with 49%
achieving a CR. Median PFS was 18 months and 34%
had an unmaintained remission that last more than 5
years, and 94% were still living at 5 years.
Rituximab in Small Lymphocytic Lymphoma
Clinical results for rituximab in small lymphocytic lym-
phoma (SLL), another of the indolent lymphomas, are
summarized in Table 9. Objective response rates in the
setting of relapsed or refractory SLL were much lower
than relapsed or refractory follicular lymphoma, but
high response rates were observed when rituximab was
used as initial therapy for previously untreated patients.
These patients are clinically distinguishable from CLL
because they had less than 5,000 lymphocytes/μl. The
4-week course of treatment at 375 mg/m2 [231, 474]
while the Piro trial used an 8-week course of rituximab
[562], and the Hainsworth trial prescribed the standard
therapy followed by planned repeat 4-week courses of
therapy every 6 months for 2 years [277]. Patients in the
relapsed/refractory setting experienced a relatively brief
duration of response, while those who received more
than the standard 4 weeks as initial treatment for SLL
enjoyed a much longer progression free survival.
Rituximab in Marginal Zone Lymphoma
Results of rituximab in mantle zone lymphoma are sum-
marized in Table 10. These indolent lymphomas can
occur anywhere, but when they occur in the gastrointes-
tinal tract, they are referred to as mucosa-associated
lymphoma tissue (MALT). In the Concani study the pri-
mary lymphoma was located in the stomach in 15
patients and was extragastric in 20 [127]. At study entry
12 patients had Ann Arbor stage IE, 3 had stage IIE, and
20 had stage IV disease. The Martinelli trial included 26
evaluable patients presenting with gastric MALT at any
stage, relapsed/refractory to initial treatment or not suit-
able for eradication with antibiotics for anti-Helico-
bacter pylori therapy [469]. The median survival of 36
328 Monoclonal antibody therapy

months reflects the inclusion of previously untreated
patients and exclusion of patients with marginal zone
lymphoma from other sites. In contrast, in the Conconi
trial, the progression free survival was 22 months for
those who had not received prior therapy compared to
12 months for patients who had relapsed after previous
anti-lymphoma therapy [127].
Rituximab in Lymphoplasmacytic
Lymphoma
These tumors are characterized by secretion of IgM
with a range of clinical presentations that include a very
high levels of IgM paraproteinenemia with minimal
lymphadenopathy (Waldenströms macroglobulenemia),
a chronic lymphoid leukemia, or an indolent lymphoma.
These lymphoplasmacytoid lymphomas previously were
a small subset of patients in the A group of the Working
Group Formulation. Unlike the cells in CLL and SLL
and plasma cells, the B-lymphocytes associated with
this disease group have a high-expression of CD20.
Furthermore, although this entity usually does have
increased numbers of circulating B-lymphocytes of the
malignant clone, lymphocyte numbers typically are not
as high as in CLL. Published reports of rituximab in the
treatment of lymphoplasmacytic lymphomas are shown
in Table 11. Response rates from 16% to 65% have been
reported with progression-free survival rates ranging
from 16 to 30 months [187, 231, 249, 726, 728]. In an
earlier trial Treon used the standard 4-week dose and
schedule of rituximab treatment [726], but subsequently
used two 4-week courses of treatment during the first
and third months for all patients. Gertz used a single
standard 4-week course of treatment at 375 mg/m2 [249]
as did Foran [231]. Dimopoulos gave a repeat 4-week
course of treatment 3 months later in patients who had
not progressed [187]. With standard dosing it often took
several months before a response could be declared, and
maximum responses were noted more than 12 months
after starting treatment. In the trials that utilized repeat
dosing, the median time to best response was about 18
months. Pooled data showed that 31/56 (55%) had an
objective response when two cycles of treatment were
used compared to 34/116 (29%) for patients who
received one standard 4-week course of rituximab.
Rituximab in Large B Cell Lymphoma
Results of trials for single-agent rituximab in aggressive
lymphomas are summarized in Table 12. A French trial of
54 patients with aggressive lymphoma included 30 with
large B cell lymphoma [121]. This was designed as a ran-
domized phase II trial in which all patients initially
received 8 weeks of rituximab therapy instead of 4, but
one half were randomized to receive 375 mg/m2 weekly ×
8 weeks, and the other group received 500 mg/m2 on
weeks 2–8 after an initial standard dose. There was no dif-
ference in response rate between the two groups; so, the
response data was grouped together. The overall response
rate was 31% with a median response duration of greater
than 8 months. The response rate was 37% in large cell. In
another European trial a response rate of 25% was
observed in 28 patients with relapsed intermediate grade
lymphomas who were treated with the standard course of
treatment with 375 mg/m2 weekly for 4 weeks [231]. The
median duration of response was about 1 year.
Rituximab in Mantle Cell Lymphoma
The randomized phase II French trial described above
included a 30% response rate for 12 patients with mantle

Table 11. Rituximab as a single agent in lymphoplasmacytic Table 12. Rituximab in large B-cell lymphoma (LBCL)and mantle
lymphoma cell lymphoma
Clinical Proportion Proportion
Citation setting responding Response rate Citation Clinical setting responding % Response
[728] Untreated and 19/29 65% [121] Relapsed large 11/30 37%
relapsed B cell
[726] Relapsed/ 11/22 50% [723] Relapsed large 21/57 37%
refractory B cell
[187] Relapsed/ 6/12 50% [231] Relapsed/ 13/40 33%
refractory refractory
[187] Untreated 6/15 40% mantle cell
[249] Untreated 12/34 35% [121] Relapsed/ 3/10 30%
[249] Relapsed/ 7/35 20% refractory
refractory mantle cell
[231] Relapsed/ 4/25 16% [231] Untreated 10/34 29%
refractory mantle cell
Robert O. Dillman 329

cell lymphoma who were to receive a planned treatment those treated at 1.0 to 1.5 g/m2, and 75% for those treated
course of 8 weeks [121]. Other investigators reported a at the highest dose of 2.25 g/m2, but patients who were
29% response rate in 34 patients with newly diagnosed treated at the higher doses were more often relapsed after,
mantle cell lymphoma, and a 33% response rate in 40 rather than refractory to, fludarabine. Huhn et al. reported
patients with mantle cell lymphoma that was either a 25% response rate using the standard dose and schedule
refractory to or had progressed after chemotherapy of four weekly doses of 375 mg/m2, but they restricted the
[231]. These patients were treated with the standard tumor cell load, as measured by the number of circulating
4-week course of treatment at 375 mg/m2. The median lymphocytes and the spleen size, in the first two cohorts
duration of response was about 1 year. of patients included in the study [333]. Median duration
of response was only 5 months.
A more practical and less expensive strategy in the
Rituximab in Chronic Lymphocytic setting of relapsed CLL is to use Rituximab in combina-
tion with chemotherapy or after a response has been
Leukemia
achieved with chemotherapy. Those strategies reduce
CLL and SLL are morphologically and phenotypically the risk of more intense infusion reactions that are asso-
the same with co-expression of CD19/20 and CD5, but ciated with higher numbers of CD20+ circulating cells,
clinically CLL is characterized by a peripheral blood and would not necessitate repeated or dose of rituximab.
lymphocytes count of greater than 5,000/μl. The density Response rates were much higher in previously untreated
of CD20 expression varies greatly among different CLL who also received more than four weekly doses of
patients. Theoretically higher or repeated doses would be rituximab. Thomas et al. treated for 8 weeks [716], while
needed to optimize the efficacy of rituximab in CLL Hainsworth et al. utilized planned retreatment with
because of the large sink of antigen positive circulating additional 4-week courses of treatment every 6 months
cells that would have to be eliminated before targeting for 2 years [277, 278].
disease in lymph nodes and other tissues. As summarized
in Table 13, in patients with relapsed CLL, standard and
high doses of rituximab have been associated with
Rituximab in Hairy Cell Leukemia
responses of 25–45%, but the duration of response only The circulating lymphocytes of hairy cell leukemia
lasted 5 to 10 months with standard dose rituximab [80, (HCL) typically express very high levels of CD20.
333, 529]. Byrd et al. reported a 45% response rate among Rituximab has been used to treat relapsed HCL using
33 patients who received thrice weekly therapy escalating both the 4 and 8 week treatment schedules. The 4-week
from 100 mg/m2 initially to minimize infusion reactions, schedule produced responses in 5/10 patients in one
up to the standard 375 mg/m2 three times a week [80]. All study [418], and in 6/24 patients who had all relapsed
but one of the responses was partial and the median dura- after cladbribine [819]. The 8-week schedule produced
tion of response was 10 months. O’Brien et al. observed responses in 8/15 relapsed patients [717].
36% response rate among 40 patients, with a median
duration of response of 8 months, in a dose escalation
study in which they administered up to 2.25 g/m2 weekly
Rituximab in Multiple Myeloma
for 4 weeks [529]. Response was correlated with dose: CD20 is rarely expressed on plasma cells, but has been
22% for patients treated at 500 to 825 mg/m2, 43% for reported to be present on malignant cells from up to
20% of patients with multiple myeloma [727]. Rituximab
Table 13. Rituximab as a single agent in chronic lymphocytic has rarely produced objective responses in multiple
leukemia myeloma even in patients whose plasma cells were felt
Clinical Proportion to overexpress CD20. Using the standard dosing of
Citation setting responding Response rate 375 mg/m2, Treon et al. saw objective responses in only
[716] Untreated, low 17/19 90% 1/19 patients [727], and Hussein et al. reported responses
burden only 2/21 patients prior to starting chemotherapy 5
[277] Untreated 18/26 70% weeks later [338]. There has been a much greater unpub-
[278] Untreated 25/43 58% lished experience in the community practice of medi-
[80] Relapsed 15/33 45% cine with anecdotal reports of occasional excellent
[529] Relapsed 14/40 36% responses in refractory myeloma patients whose plasma
[342] Relapsed 8/23 35%
cell overexpressed CD20. Another strategy is to use
[333] Relapsed 7/28 25%
rituximab as an adjuvant treatment after an initial
330 Monoclonal antibody therapy

response to chemotherapy, in an effort to suppress the B specific interactions with the CD20 antigen, the term
cell clone that leads to the malignant plasma cells. Based “maintenance” should be reserved for the use of addi-
on this same rationale, rituximab may be more active tional doses of rituximab to maintain serum levels of
against smoldering myeloma than myeloma with a high rituximab such that cells that express CD20 do not emerge
proliferative index. from pre-CD20-positive B cell clones. Theoretically
this should keep CD20+ clones from appearing, but also
keeps normal B-cell immunity suppressed since normal
Rituximab in Hodgkin’s Disease B cells also express CD20. No clinical trial has ade-
Hodgkin’s disease is a malignancy of B-lymphocytes quately addressed the issue of “maintenance” rituximab
[687]. Lymphocyte-predominant Hodgkin disease in a pure sense.
(LPHD) is characterized by indolent nodal disease that As noted earlier in this section, retreatment with ritux-
tends to relapse after standard radiotherapy or chemo- imab is often still effective at the time of recurrence in
therapy and the malignant cells of LPHD are CD20+. patients who had previously had an objective response to
As shown in Table 14, rituximab alone has been associ- rituximab [148, 339] which suggests that tumor reap-
ated with very high response rates in LPHD, but the peared only because levels of rituximab were insufficient
median progression free survival has usually been no to keep it suppressed. If this is correct, then, continued
more than a year [203, 586, 803]. Rituximab is also treatment with rituximab should prolong progression-
being explored in other Hodgkin’s histologies. In the free survival, but not necessarily overall survival. The
Younes trial patients had to have received at least two real question is, what strategy is best for preventing
prior treatment regimens, and received 6 rather than 4 becoming rituximab-refractory? True refractoriness
weeks of therapy [803]. would be defined by disease progression during treat-
ment with rituximab. For clinical trials purposes it also
has been defined not only as disease progression during
Extended therapy and Maintenance therapy, but also as progression of disease within 6
Rituximab months of treatment in a patient who had stable disease
The term “maintenance” has been used to describe the or a response, which has very different implications. The
use of rituximab therapy after induction therapy for mechanisms of resistance to rituximab appear to relate to
the treatment of B-cell lymphoma. However, much of (1) loss of cellular dependence on CD20 expression in
this usage actually represents adjunctive, sequential terms of regulation of cellular resistance to apoptosis
therapy, or consolidation therapy following an initial or (probable) (2) existence or production of CD20 at a rate
induction therapy that that did not include rituximab. that rapidly consumes standard doses of rituximab (prob-
Since the mechanism of action of rituximab relates to its able), (3) loss of CD20 expression (uncommon), or (4)
induction of neutralizing anti-rituximab antibodies (very
rare), (5) lack of complement (unlikely), or (6) insuffi-
Table 14. Rituximab as a single agent in Hodgkin’s disease cient or inadequate effector cells (unlikely).
Median Several trials suggest that patients who have not expe-
progres- rienced progressive disease while taking single-agent
Clinical Proportion % sion-free rituximab benefit from receiving more than the standard
Citation setting responding Response survival 4 weeks of therapy as summarized in the accompanying
[586] Relapsed, 12/14 86% >12 Table 15. Such approaches have been associated with
LPHD > months higher response rates and longer durations of progres-
30%
sion free survival than were reported with standard ther-
CD20+
[203] Untreated 12/12 100% 10
apy. However, the 95% confidence intervals for response
LPHD months rates overlap with those observed with the 4-week sched-
[203] Relapsed 10/10 100% 10 ule, thus, these results are not clearly better than those
LPHD months seen with 4 weeks of treatment. Randomized trials
[803] Relapsed 5/22 22% 8 months involving patients with follicular lymphoma show that
NSHD, 2 giving additional dose of rituximab after standard dosing
prior
treatments
does prolong PFS [251, 280]. In the Swiss trial the addi-
tion of one additional 375 mg/m2 dose every 2 months
LPH = lymphocyte predominate Hodgkin’s disease for 4 months was associated with a superior progression
NSHD = nodular sclerosing Hodgkin’s disease
free survival of 23 vs. 12 months (p = 0.02) and was even
Robert O. Dillman 331

Table 15. Rituximab treatment schedules utilizing more than the standard 375 mg/m2 weekly × 4 weeks in patients with indolent or
follicular lymphoma (no chemotherapy)
Schedule after 4 weeks of standard
Citation Clinical setting rituximab therapy Patients Response rate PFS
[389] Relapsed indolent 4 doses months 3 if PR or SD 25 76% –
[15] Relapsed follicular 2 more weeks 17 76% >34 months
[277] Relapsed indolent 4 doses months 6,12,18,24 60 74% 34 months
[280] Relapsed indolent 4 doses months 6,12,18,24 35 52% 31 months
[251] Initial & relapsed follicular 1 dose months 3,5,7,9 75 66% 23 months
[264] Relapsed indolent 1 dose monthly if <25 μg/ml 22 63% >25 months
[562] Relapsed follicular 4 more weeks 37 60% >19 months
[56] Relapsed indolent 2 more weeks 68 59% 14 months
PFS = progression free survival

more striking in patients who had not received chemo-
therapy before (36 vs. 19 months, p = 0.009) [251].
To date the only trial that has tried to address the issue of
time to rituximab-refractoriness is the phase II random-
ized trial by Hainsworth et al. [280], in which patients
with relapsed low-grade lymphoma were treated with the
standard 375 mg/m2 weekly × four dosing with ritux-
imab, and then those who did not have progressive dis-
ease were randomized to either planned retreatment with
rituximab every 6 months for 2 years, using the same
dose and schedule, or to delayed treatment until disease
progression occurred, then re-instituted rituximab ther-
apy. This design of “maintenance” is not really “ratio-
nale” since 6 months is too long to sustain serum levels
in some patients, and 4 weeks of therapy is excessive in
many patients in terms of rates of cellular catabolism and
hepatic metabolism vs. interference with appearance of
the CD20 molecule on the cell surface. For these reasons
it is not surprising that there was no difference in the
time to becoming refractory to rituximab between the
retreatment every 6 months compared to retreatment
relapse. However, the additional rituximab therapy was
associated with a higher response rate 52% vs. 35%,
complete response rate (27% vs. 4%). There was a strik-
ing difference in the time to disease progression, medi-
ans of 31 months vs. 8 months (p = 0.007), but no
difference in time to become “rituximab refractory”
which was about 30 months in both arms, nor in overall
survival at a median follow up of 42 months.
However, this was a small trial that was stopped early
because it was originally designed for patients with
indolent lymphoma who had relapsed after chemotherapy
alone, at which time they were treated with rituximab.
Once rituximab became a standard part of initial therapy
for patients with indolent lymphomas, there were no
longer any patients available that met eligibility require-
ments for the study. There is an ongoing cooperative
group trial (RESORT, ECOG 4402) that also attempts to
address this issue in previously untreated low-risk indo-
lent lymphoma patients whose initial therapy is stan-
dard-dose rituximab alone. Patients are then randomized
to “maintenance” utilizing one 375 mg/m2 dose every 3
months until relapse, instead of four doses every 6
months for 2 years, or are randomized to retreatment
with standard-dose rituximab at that time of relapse.
The clinical trial definition of refractoriness is the key
endpoint, but this trial also attempts to address whether
prolonged inhibition of CD20-positive B cells beyond 2
years impairs humoral immunity.
In terms of optimizing progression free survival, the
optimal way to give “maintenance” rituximab would be
based on sustaining serum levels in an effort to prevent
the emergence of CD20+ cells. However, a commercial
assay for serum CD20 levels is not currently available.
The only trial performed to date that utilized serum CD20
levels to guide re-treatment enrolled only 31 patients, and
rituximab levels were only sustained for 1 year; so it did
not adequately address this issue [264]. In this trial ritux-
imab serum levels were measured monthly, and if the
level dropped below 25 μg/ml, an additional 375 mg/m2
dose was administered. For the 22 patients who were
monitored as planned, half of the patients required addi-
tional dosing by 5 months. The response rate for 29 eval-
uable patients was 59%, and was 63% for 22 patients
with indolent histology. If a commercial assay for mea-
suring serum levels of rituximab became available, it
could become the major determinant for optimizing ritux-
imab dosing for individual patients [585].
In practice most physicians have adopted one or more
of the published schedules of maintenance therapy for
their patients with indolent lymphomas, based on the
benefits in terms of response rates and progression free
survival, which is a highly valued endpoint by both
patients and practicing physicians. However, most
332 Monoclonal antibody therapy

patients are not initially treated with rituximab alone,
but rather with rituximab in combination with chemo-
therapy. The issue of maintenance rituximab therapy in
that setting for both indolent and aggressive lymphomas
is discussed latter in this chapter.
Rituximab with Chemotherapy
In vitro studies showed additive and/or synergistic benefit
from combining rituximab with chemotherapy. [156].
Some studies suggest that the presence of rituximab helps
overcome resistance, perhaps because of altered efflux of
chemotherapy drugs, or decreased resistance to apopto-
sis. In the treatment of lymphoma, rituximab has been
combined with virtually every chemotherapy regimen in
common use including cyclophosphamide, vincristine,
and prednisone (CVP) [279, 465], cyclophosphamide,
doxorubicin, vincristine, and prednisone (CHOP) [98,
122, 139, 279, 319, 329, 423, 532, 729, 757], cyclophos-
phamide, doxorubicin, vincristine, prednisone and etopo-
side (CHOEP) [555], fludarabine alone [82, 141],
fludarabine and cyclophosphamide (FC) [373], fludara-
bine and mitoxantrone (FM) [476, 813], cyclophosph-
amide, mitoxantrone, vincristine, and prednisone (CMOP)
[197], and fludarabine, chlorambucil, and mitoxantrone
(FCM) [233], pentostatin alone [193], pentostatin and
cyclophosphamide (PC) [186, 306, 370, 413], pentostatin
and mitoxantrone (PM) [160], cladribine with or without
cyclophosphamide [601], chlorambucil [464], chloram-
bucil, mitoxantrone, and prednisone (CMP) [317], infu-
sional etoposide, platinum, vincristine, cyclophosphamide,
and doxorubicin (EPOCH) [350, 786], cyclophosph-
amide, vincristine, doxorubicin, dexamethasone, metho-
trexate, and cytarabine (hyperCVAD-MA) [605, 718]
ifosfamide, cisplatin, etoposide (ICE) [379], irinotecan,
mitoxantrone, dexamethasone (IMD) [523]. As a gener-
alization, the addition of rituximab does not increase tox-
icity in any significant way, although in randomized trials
there has been a slightly higher rate of neutropenia, but
not febrile neutropenia. There also appear to be no advan-
tages to sequencing chemotherapy and rituximab as
opposed to giving the agents together, since they do not
have any overlapping toxicities. Several trials have been
conducted that confirm the safety and efficacy of admin-
istering Rituximab in combination with chemotherapy in
various B cell lymphoproliferative disorders.
Indolent and Follicular Lymphoma
Combinations of rituximab and a variety of chemothera-
pies have produced high response rates with durable
progression free survival (Table 16). The combinations

Table 16. rials of chemotherapy with concurrent rituximab in patients with indolent lymphoma, mostly patients with follicular lymphoma
Citation Clinical Setting Chemo Regimen Proportion Responding Response Rate
[476] Untreated FMD 76/76 100%
[139,140] Relapsed/untreated CHOP 38/38 100%
[319] Untreated follicular CHOP 214/223 96%
[233] Recurrent follicular FCM 35/37 95%
[532] Untreated CHOP 32/34 94%
[141] Relapsed/Untreated Fld 35/39 90%
[197] Untreated CMOP 38/42 90%
[468] Untreated/relapsed Chl 24/27 89%
[729] Untreated/Relapsed LPCL CHOP 11/13 85%
[160] Untreated PM 20/24 83%
[465] Untreated indolent CVP 131/162 81%
[618] Recurrent follicular FC 37/54 81%
[725] Recurrent follicular FC 60/75 80%
[193] Relapsed Pento 44/57 77%
[306] Untreated/relapsed LPCL PC 10/13 77%
FMD = fludarabine, mitoxantrone, dexamethasone
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
FCM = fludarabine, chlorambucil, mitoxantrone
CMOP = cyclophosphamide, mitoxantrone, vincristine, prednisone
PM = pentostatin, mitoxantrone
CVP = cyclophosphamide, vincristine, prednisone
FC = fludarabine, cyclophosphamide
Clb = chlorambucil
Fld = fludarabine
Pento = pentostatin
C = cyclophosphamide
Robert O. Dillman 333

have not produced additive toxicity, but because of con- As summarized in Table 18, randomized trials have
cerns about the potential for such toxicity, other investi- confirmed a higher response rate and longer progression
gators have utilized rituximab after chemotherapy for free survival when rituximab is added to chemotherapy
patients who were already had a response to therapy. compared to results for the same chemotherapy alone.
This approach of chemotherapy followed by sequential Some studies also suggested there may be an improved
therapy or consolidation rituximab therapy also has overall survival as well. This improved survival has been
been associated with very high response rates and dura- accomplished without any clinically important increase
ble remissions (Table 17). in toxicity. Because of this rituximab with or without che-
Two randomized phase II trials have compared the motherapy has become the treatment of choice for patients
concurrent and sequential approaches but were not with indolent lymphomas who require therapy.
definitive in showing differences [471, 532]. The larger
of the two trials did show a slightly higher response rate
and longer progression free survival for the combined
Chemotherapy and Rituximab in Chronic
approach [471]. Since there has been no evidence of Lymphocytic Leukemia
additive toxicity for the combination regimens, and Although rituximab does not have an FDA regulatory
there is no evidence that the sequential approach offers marketing indication for CLL, numerous phase II trials
any therapeutic advantages, most physicians use the have shown that the combination of chemotherapy plus
concurrent approach. standard doses of rituximab has substantial activity in
Because of the prevalence of follicular lymphoma, the disease as shown in Table 19. This has included
more trials have been conducted with chemotherapy and purine analogs plus rituximab [82, 192, 601, 641], and
rituximab in this B cell malignancy than any other tumor combinations of alkylating agents, purine analogs, plus
type. Longer follow up has established that survival for rituximab [186, 370, 373, 413, 601, 783]. In one ran-
such patients has been improved since the introduction domized phase II trial, concurrent fludarabine plus
of rituximab in the salvage setting, and as part of initial rituximab was compared to the sequence of fludarabine
therapy of patients with follicular lymphoma [141, 327, followed by rituximab in 104 patients [82]. The response
619]. In a trial of 38 patients with indolent lymphomas, rate was higher for concurrent therapy (90% vs. 77%) as
80% of whom had not been previously treated, RCHOP was the complete response rate (47% vs. 28%). A retro-
was associated with a response rate of 100%, a median spective comparison of these patients to a similar popu-
progression-free survival of nearly 7 years, and after 9 lation of patients treated with fludarabine alone at the
years of median follow-up, median survival had not same dose in an earlier randomized trial suggested that
been reached [141]. patients treated with the combination of fludarabine plus
rituximab had much better outcomes, including a higher
response rate (84% vs. 63%), higher complete response
Table 17. Trials of chemotherapy followed by rituximab rate (38% vs. 20%) better progression free survival after
(sequential therapy) in patients with indolent B cell lym- 2 years (67% vs. 45%), and better overall survival at 2
phoma, mostly follicular lymphoma
years (93% vs. 81%) [83].
Citation Clinical Chemo Proportion Response
setting regimen responding rate
[813] Untreated CHOP 67/68 98% Large B Cell Lymphoma
FCL
[532] Untreated CHOP 34/35 97%
The three large randomized trials summarized in Table
[813] Untreated FM 69/782 96% 20 have demonstrated the benefits of combining ritux-
FCL imab with chemotherapy in the treatment of large B cell
[470] Untreated FMD 69/73 95% lymphoma. In France Coiffier et al. compared RCHOP
[754] Untreated FMD 34/36 95% to CHOP in 399 previously untreated patients, ages
[119] Untreated FC 29/33 88%
FCL 60–80 with large B-cell lymphoma, using an eight-
[273] Untreated FM 26/30 87% course schedule that included antibody and chemother-
[462] Untreated CHOP 71/84 84% apy all being given on day one of each course,
FCL prednisone, then Rituximab, then the cyclophosph-
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone amide, doxorubicin, and vincristine [122]. After 1 year
FM = fludarabine, mitoxantrone of follow up and 126 events (disease progression or
FMD = fludarabine, mitoxantrone, dexamethasone death), the CHOP plus Rituximab arm was superior in
FC = fludarabine, cyclophosphamide response rate (76% vs. 60%, p = 0.004), 1-year event-free
334 Monoclonal antibody therapy

Table 18. Randomized trials of chemotherapy ± rituximab in indolent lymphomas

Citation Clinical setting Treatment Regimen Proportion Responding PR Rate CR Rate PFS
[319] Untreated follicular RCHOP 214/223 96%
CHOP 184/205 90%
p = 0.011
[745] Relapsed follicular RCHOP 199/234 85% 30% 33 months
CHOP 166/231 72% 16% 20 months
p < 0.001 p < 0.001 p < 0.001
[465] Untreated follicular RCVP 131/162 81% 41% 32 months
CVP 91/159 57% 10% 15 months
p < 0.0001
[317] Untreated follicular RMCP 166/181 92% 50% >47 months
MCP 133/177 75% 25% 29 months
p = 0.0009 p = 0.004 p < 0.0001
[476] Untreated indolent RFMD 80 100% 88% 76% 3-year
FMD 80 96% 85% 60% 3-year
[233] Relapsed RFCM 52/66 79% 34% –
FCL & Mantle Cell FCM 36/62 58% 12% –
p = 0.01
[75] Untreated RCHOP 36 98%
Lympho- plasmacytoid
CHOP 36 69%
p = 0.01
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
RCVP = rituximab, cyclophosphamide, vincristine, prednisone
CVP = cyclophosphamide, vincristine, prednisone
RMCP = rituximab, mitoxantrone, chlorambucil, prednisolone
MCP = mitoxantrone, chlorambucil, prednisolone
RFMD = rituximab, fludarabine, mitoxantrone, dexamethasone
FMD = fludarabine, mitoxantrone, dexamethasone
RFCM = rituximab, fludarabine, chlorambucil, mitoxantrone
FCM = fludarabine, chlorambucil, mitoxantrone
PR = partial response
PFS = progression free survival
OS = overall survival

Table 19. Phase II trials of chemotherapy plus rituximab in CLL survival (69% vs. 49%, p < 0.0005) and overall survival
% (83% vs. 68%, p < 0.01). Longer follow up at a median
Clinical Median Proportion Resp- of 5-years confirmed the tremendous advantage of
Citation setting age Regimen responding onse RCHOP in terms of PFS and OS, without identification
[373] Untreated 58 years FCR 213/224 95% of any long-term toxicities [219].
[370] Untreated 63 years PCR 58/64 91% A similar trial was conducted by Habermann et al. in
[82] Untreated 63 years FR 46/51 90% a U.S. intergroup trial that enrolled 632 patients over the
[641] Both 59 years FR 27/31 87%
age of 60, except that a different schedule of RCHOP
[186] Untreated 72 years PCR 7/9 78%
[783] Relapsed 59 years FCR 138/179 77% was used, and for the 415 responding patients, there was
[413] Untreated 62 years PCR 26/34 75% a secondary randomization to maintenance rituximab
[601] Relapsed 55 years CCR 14/26 54% vs. observation for 2 years [276]. In this trial the RCHOP
[192] Relapsed 60 years PR 19/59 33% regimen was two cycles of rituximab followed by eight
[186] Relapsed 70 years PCR 2/12 17%
cycles of CHOP with three more cycles of rituximab
FCR = fludarabine, cyclophosphamide, rituximab given before the third, fifth, and seventh doses. Patients
FR = fludarabine, rituximab
PCR = pentostatin, cyclophosphamide, rituximab who had achieved a CR or PR by the end of eight cycles
CCR = cladribine, cyclophosphamide, rituximab were randomized to either observation or to four 4-week
PR = pentostatin, rituximab courses of rituximab at 375 mg/m2 per dose, given every
Robert O. Dillman 335

Table 20. Randomized trials of chemotherapy ± rituximab in large B cell lymphomas

Treatment Patients
Citation Clinical setting regimen enrolled PR rate CR rate PFS OS
[122] Untreated elderly RCHOP 399 86% 76% 53% 3-year 62%
[219] CHOP 71% 63% 35% 3-year 51%
p = 0.007 p = 0.005 p = 0.0008 p = 0.008
[276] Untreated elderly RCHOP 632 or 77% 35% 52% 3-year 67%
a
CHOP 425 76% 35% 39% 3-year 58%
p = 0.003 p = 0.05
[555] Untreated low-risk RCHOP 824 81% 76% 2-year 94%
young
CHOP – 67% 60% 2-year 87%
p = 0.0001 p < 0.0001 p = 0.001
[556] Elderly RCHOP-14 1222 70% 2-year
CHOP-14 57% 2-year
p < 0.0001
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
PR = partial response
CR = complete response
PFS = progression free survival
OS = overall survival
a
Response data are based on all 632 patients, but survival data is limited to 425 patients who did not receive “maintenance” rituximab
therapy, four weekly doses every 6 months for 2 years.

6 months for 2 years. For comparability with the French
study, an analysis was performed that excluded the 207
patients who received maintenance rituximab, which
demonstrated a superiority of RCHOP in terms of PFS and
OS, although there was no difference in response rate.
Both of the above trials dealt with elderly patients.
Pfreundshuh et al. conducted a trial of rituximab and
CHOP-like regimens compared to the same chemotherapy
alone in 824 patients under the age of 60 years who had
was considered low-risk large B cell lymphoma [556].
Low-risk was defined as International Prognostic Index
0–1, stage II–IV disease, or bulky stage I disease.
CHOP-like regimens included the addition of etoposide
(CHOEP) or addition of methotrexate (MACOP). Any
response less than a CR was considered a treatment fail-
ure. This trial also confirmed a substantial advantage for
patients who received rituximab with the chemotherapy.
In elderly patients Pfreundschuh et al. have conducted
a four-arm trial (RICOVER-60) comparing six and eight
cycles of RCHOP-14 and CHOP-14 [557]. RCHOP-14
was clearly superior to CHOP-14, and six cycles of
RCHOP-14 showed a survival advantage over eight
cycles of RCHOP-14. What remains to be done is to
compare RCHOP-14 to RCHOP-21.
Many phase II trials have been conducted with
RCHOP and other rituximab plus chemotherapy regi-
mens which also demonstrated excellent response rates
as initial treatment or at the time of relapse, as shown
in Table 21. One of the earliest was conducted by Vose
et al. in which rituximab was given on day 1 and CHOP
on day 3 of each of six treatment cycles to 33 patients
with intermediate grade lymphomas, about two third
with large B-cell lymphoma (Working Group
Formulation G) and the remainder with large cell fol-
licular (Working Group Formulation D) [757, 758].
The trial by Case et al, in which patients received six
to eight cycles of RCHOP administered on the same
day, followed by 4 cycles of maintenance rituximab
for responders, confirmed the activity of concurrent
RCHOP therapy in the community setting [98]. The
Wohrer trial appears to confirm that RCHOP is highly
active in gastric large B cell lymphoma [792]. The
encouraging results from the REPOCH regimen have
led to randomized trials comparing it to RCHOP [350,
786]. The encouraging results with 14-day RCHOP
regimens, which are administered with prophylactic
filgastrim or pegfilgastrim, has led to phase III trials
using this approach compared to 21-day schedules of
RCHOP [62, 283, 596]. Rituximab plus chemotherapy is
also active in the salvage setting as evidenced by trials
with REPOCH, RICE and RIMD [350, 379, 523].
Mantle Cell lymphoma
Table 22 summarizes some of the trials that have been
conducted with concurrent rituximab and chemotherapy
in patients with mantle cell lymphoma. Howard et al.
336 Monoclonal antibody therapy

Table 21. Phase II trials of chemotherapy with concurrent rituximab in patients with large B cell lymphoma
Citation Clinical Setting Chemo Regimen # Patients Response Rate PFS OS
[757,758] 76% LBCL CHOP 33 94% 82% 5-years 88%-5-years
[792] Gastric LBCL CHOP 15 100%
[98] LBCL CHOP 101 91% 76% 2-years
[283] LBCL CHOP-14 49 >90%? 80% 2-years 90% 2-years
[596] LBCL CHOP-14 26 100% 70% 2-years 79% 2-years
[62] LBCL CHOP-14 50 >90% 68% 2-years 72% 2-years
[786] Initial, aggressive EPOCH 61 92% 82% 5-years 88% 5-years
[350] Relapsed or refractory EPOCH 50 68% 30% 2-years 40% 2-years
[379] Relapsed or refractory ICE 36 78%
[523] Relapsed elderly IMD 30 74% 37% 45%
LBCL = diffuse large B cell lymphoma
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
EPOCH = etoposide, platinum, vincristine, cyclophosphamide, doxorubicin
ICE = ifosfamide, cyclophosphamide, etoposide
IMD = irinotecan, mitoxantrone, dexamethasone
PFS = progression free survival
OS = overall survival

Table 22. Clinical trials of rituximab plus chemotherapy in mantle cell lymphoma
Treatment Proportion
Citation Clinical setting regimen responding PR rate CR rate PFS OS
[424] Untreated RCHOP 58/62 94% 34% 21 months NSD
CHOP 45/60 75% 7% 14 months
p = 0.005 p = 0.0002 p = 0.013
[329] Untreated RCHOP 38/40 96% 48% 17 months
[605] Untreated RHyper- 94/97 97% 87% 64% 82%
CVAD-MA 3-year 3-year
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
RHyper-CVAD-MA = rituximab, cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate,
cytarabine
PR = partial response
CR = complete response
PFS = progression free survival
OS = overall survival

showed that RCHOP was very active in mantle cell
lymphoma, but the PFS was not better than what had
been seen historically with CHOP alone [329]. However,
when Lenz et al. conducted a randomized trial, RCHOP
was superior to CHOP in terms of response rate, CR
rate, and PFS, but not OS [424]. In a single institution
trial, Romaguera et al. found their R-hyperCVAD-MA
regimen to be highly active [605]. Both RCHOP and
R-hyperCVAD-MA are used as initial therapy in
patients with mantle cell lymphoma, and most medi-
cally fit patients go on to consolidation with high dose
chemotherapy and autologous stem cell rescue.
Burkitt’s Lymphoma and ALL
The combination of rituximab and hyper-CVAD with
methotrexate and cytarabine (R-hyperCVAD-MA) has
not only been used for mantle cell lymphoma, but also
for the treatment of adult acute lymphoblastic leukemia
and Burkitt-type lymphomas. Thomas et al. treated 28
evaluable patients with this regimen as initial therapy
[718]. Rituximab was given on days 1 and 11 of the
hyper-CVAD courses and days 1 and 8 of methotrexate
and cytarabine courses. The median age was 46 years,
but 29% were over the age of 60 years. The response
rate was 27/28 with 86% complete responders. The
3-year progression free and overall survival was 88%.
HIV Associated Lymphoma
As summarized in Table 23, the addition of rituximab to
chemotherapy has not been shown to improved outcome
in HIV-associated, non-Hodgkin’s lymphoma (NHL).
These relatively small trials do suggest some survival
Robert O. Dillman
Table 23. Trials of rituximab and chemotherapy in HIV positive patients with B cell lymphoma
Treatment
Citation Clinical setting regimen # Patients
[823] Untreated R-CDE 74
[53] Untreated R-CHOP 52
[365] Untreated R-CHOP 75
CHOP 75
R-CDE = rituximab, cyclophosphamide, dexamethasone, etoposide
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
CHOP = cyclophosphamide, doxorubicin, vincristine, prednisone
CR = complete response
PFS = progression free survival
OS = overall survival
benefit, but also raise issues regarding an increased risk
from infection that was not evident in non-HIV lymphoma
patient populations. Spina et al. used 96-h infusion R-CDE
(cyclophosphamide 187–200 mg/m2/day, doxorubicin
12.5 mg/m2 per day, and etoposide 60 mg/m2 per day) in
74 patients with HIV-associated NHL, most of whom
were receiving concurrent highly active antiretroviral
therapy (HAART) [823]. Ten patients were diagnosed
with opportunistic infections during or within 3 months of
the end of R-CDE, and 17 patients developed non-oppor-
tunistic infections for an infection rate of 37%. Two
patients died of bacterial sepsis and four of opportunistic
infections. In contrast Boue et al. observed a 17% rate of
infection, but they did not address the risk of infection in
the months after completion of therapy [53]. The one ran-
domized trial that has been conducted showed a trend
toward higher response rate and survival with the addition
of rituximab, but a higher rate of infectious death in the
group that received rituximab [802].
Central Nervous System Lymphoma
In the absence of central nervous system (CNS) disease,
negligible levels of rituximab are detected following i.v.
infusions, presumably because of the blood–brain bar-
rier. However, significant levels can be achieved in the
presence of parenchymal disease or other inflammatory
processes that alter the blood–brain barrier, or after
direct injection into the CSF. Rubenstein et al. treated
ten patients with CNS relapse of LBCL with a planned
nine injections of rituximab at 10 mg, 25 mg, or 50 mg
doses via Ommaya reservoir over 5 weeks [613]. The
maximum tolerated dose was determined to be 25 mg
based on patients who received the 50 mg dose experi-
encing dose-limiting hypertension. These patients also
had systemic symptoms including abdominal cramps or
nausea and vomiting. Responses were observed in all
337
Infectious
CR rate PFS OS death
70% 59% 2-years 64% 2-years 8%
77% 75% 2-years 4%
58% 10.4 months 32 months 14%
47% 8.6 months 25 months 2%
p = 0.147 p = 0.035
ten patients including a complete remission in four.
Rituximab has also been used in the treatment of primary
CNS B-cell lymphoma. Limited benefits were observed
in patients with recurrent disease, and rituximab is now
being combined with high dose methotrexate as part of
initial treatment.
Maintenance Rituximab following Chemo-
therapy or Rituximab Plus Chemotherapy
There has been interest in using maintenance rituximab
after an initial response to a combination of rituximab
and chemotherapy. As discussed earlier there also have
been treatment strategies of chemotherapy followed by
rituximab, which is really a consolidation or sequential
treatment approach rather than maintenance. Randomized
trials have failed to show an advantage for the sequential
strategy compared to the combinations of rituximab with
the same chemotherapy [276, 476, 532]. Compared to
observation, randomized trials have shown that adding
rituximab after an initial response to CVP, FCM, or
CHOP is associated with longer PFS in indolent lym-
phoma [233, 322, 745], and adding rituximab after an
initial response to CHOP prolongs PFS in large B cell
lymphoma [276]. However, chemotherapy alone is no
longer considered an appropriate initial option since
rituximab plus chemotherapy is consistently superior to
chemotherapy alone in terms of response rates and pro-
gression free survival for indolent and aggressive lym-
phomas, and overall survival for large B cell lymphoma.
Table 24 shows the results of randomized trials that have
evaluated the effect of maintenance rituximab after ritux-
imab plus chemotherapy, but many were trials in which this
was a secondary randomization limited to patients who
had responded to either chemotherapy alone, or chemo-
therapy plus rituximab after the primary randomization.
338 Monoclonal antibody therapy

Table 24. Randomized trials of rituximab + chemotherapy ± “maintenance” rituximab in B cell lymphoproliferative disorders
Citation Clinical setting Induction regimen Maintenance rituximab # randomized PFS OS
[276] Elderly untreated RCHOP 4 weekly doses q 6 80 77% 2-years NSD
months × 2 years vs. observation
93 75% 2-years
LBCL p = 0.81
[745] Relapsed RCHOP 1 dose q 3 months × 2 years vs. observation 91 52 months NSD
FCL 98 23 months
p = 0.004
[234] Relapsed RFCM 4 doses at 3 months & 9 months vs. 41 36 months NSD
observation
FCL 40 26 months
p = 0.035
LBCL = large B cell lymphoma
FCL = follicular lymphoma
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
RFCM = rituximab, fludarabine, chlorambucil, mitoxantrone
PFS = progression free survival
OS = overall survival

The accompanying table excludes patients who had Table 25. Rituximab in combination with biologicals other
received only chemotherapy as their induction treatment, than antibodies
since this is not longer considered optimal therapy. Clinical Biological Number of Response
Citation setting agent patients rate
[617] Relapsed IFN-α 64 70%
Rituximab with other Biologics indolent
Rituximab with Biologicals other than Monoclonal [149] Relapsed IFN-α 38 45%
indolent
Antibodies [239] Relapsed IL-2 20 55%
To the extent that rituximab’s clinical benefits are due to indolent
CDCC and ADCC, combining rituximab with other bio- [259] Relapsed IL-2 34 26%
logicals may enhance its clinical benefit. Table 25 sum- indolent
marizes several studies that explored such combinations [380] Relapsed IL-2 54 10%
indolent
[8, 9, 148, 239, 259, 368, 380, 475, 609, 617, 743]. [475] Relapsed GM-CSF 12 83%
Thalidomide was given orally and all of the cytokines indolent
were given s.c. Ranges of response were 45–75% for [609] Relapsed GM-CSF 35 60%
interferon-α, 26–55% for IL-2, 60–83% for GM-CSF, indolent
[8] Relapsed IL-12 43 69%
37–69% for IL-12. The large variance in response rates
indolent
for specific combinations is almost certainly due to the [9] Relapsed IL-12 30 37%
specific characteristics of the patient populations in each indolent
small trial. Only randomized trials can establish the [743] Relapsed G-CSF 19 42%
superiority of these responses compared to rituximab indolent
[368] Relapsed Thalidomide 16 81%
alone, which produced response rates from 45% to 85% Mantle Cell
as a single agent in indolent lymphomas. Whether such
IFN-α = interferon alpha
trials will be conducted is questionable since rituximab IL-2 = interleukin-2
plus chemotherapy is considered standard induction GM-CSF = granulocyte macrophage colony stimulating factor
therapy for these malignancies. IL-12 = interleukin-12
G-CSF = granulocyte colony stimulating factor
Rituximab with other monoclonal antibodies
Rituximab has been combined with the anti-CD52 anti-
body alemtuzumab, and the anti-CD22 antibody epratu- et al. and Leonard et al. gave epratuzumab at 360 mg/m2
zumab as shown in Table 26. Faderl et al. gave rituximab i.v.ly over 60 min followed by rituximab at 375 mg/m2,
and alemtuzumab at their standard doses [214]. Strauss weekly for 4 weeks [425, 697].
Robert O. Dillman 339

Table 26. Rituximab in combination with other monoclonal activation of complement, B-lymphocyte cytolysis, and
antibodies TNF-alpha release [40, 787]. The severity of the reaction
Clinical Biological Number of Response depends on the rate of rituximab infusion, the number of
Citation setting agent patients rate CD20+ cells, and the immune competence of the patient.
[214] Relapsed Alemtuzumab 25/48 52% In patients who have high numbers of rapidly proliferat-
CLL ing CD20-positive cells, classic tumor lysis syndrome
[697] Relapsed Epratuzumab 21/33 64% can occur. The rate of penetration and binding of ritux-
FCL
imab to CD20-positive cells located in tumor masses is
[426] Relapsed Epratuzumab 10/15 67%
FCL much slower; therefore in the absence of large numbers
[697] Relapsed Epratuzumab 2/16 13% of circulating tumor cells, tumor lysis syndrome is only
NHL encountered when rituximab is given chemotherapy.
[697] Relapsed Epratuzumab 7/15 47% Once circulating B cells have been eliminated, ritux-
LBCL
[426] Relapsed Epratuzumab 4/6 67%
imab can be infused rapidly with many using an infusion
LBCL time of 45 min for subsequent weekly infusions [82,
CLL = chronic lymphocytic leukemia 170, 175, 655]. If rituximab therapy is being re-started
FCL = follicular lymphoma after being discontinued for several months, and B lym-
NHL = non-Hodgkin’s lymphoma phocytes are again present in the blood stream, then the
LBCL = diffuse large B cell lymphoma slow infusion rate should be used. Once circulating target
cells are no longer present, subsequent infusions may be
safely given at more rapid rates. For instance, the author
Toxicity and side Effects administers full dose rituximab over 30 min during sub-
The toxicities associated with rituximab were summa- sequent weekly treatments once B lymphocytes have
rized earlier in this chapter in Table 6. Rituximab is gen- been cleared from the circulation.
erally well tolerated. The most common toxicities Despite its excellent safety record, rituximab does
observed are the “flu-like” symptom complex associ- carry several “Black Box Warnings” from the US FDA.
ated with i.v. infusion followed by the binding of ritux- These include fatal infusion reactions, tumor lysis syn-
imab to circulating CD20 positive B cells and their drome, severe mucocutaneous reactions, and progres-
subsequent elimination in the reticuloendothelial sys- sive multifocal leukoencepalopathy. As noted above,
tem [170, 175, 184]. In the pivotal trial for rituximab, depending on the clinical setting and rate of rituximab
33% of patients had their first infusion interrupted infusion, patients may experience hypoxia and dyspnea
because of side effects, but toxicities were rarely seen that can be associated with pulmonary infiltrates and
with subsequent infusions after circulating B lympho- acute respiratory distress syndrome, cardiac arrhythmias
cytes were cleared from the circulation [474]. Because including ventricular fibrillation and cardiac shock, and
of the predictably of the initial infusion reaction, cau- myocardial infarction. It is noted that 80% of fatal infu-
tion should be taken with patients who have underlying sion reactions occurred with the first infusion. Tumor
cardiac or respiratory problems who may not tolerate lysis syndrome with renal failure and death can occur
the stress of the cytokines released, and the brief with rituximab alone in the presence of high numbers of
decrease in oxygen saturation associated with the initial rapidly proliferating CD20-positive cells in the circula-
congestion of antibody coated cells in the lungs. Care tion, or when rituximab is given with chemotherapy in
should also be taken when treating patients with high patients who have a high tumor burden of CD20+ cells
circulating B lymphocyte counts, especially if those anywhere in the body. Rare cases of severe mucocutane-
cells strongly express CD20, although most patients ous reactions including Stephens-Johnson syndrome
will tolerate rituximab therapy [79], [363]. and bullous pemphigoid have been reported. Progressive
When rituximab is administered i.v., antibody mole- multifocal leukoencephalopathy associated with the
cules immediately contact CD20+ B cells in the circulation. Creutzfeld-Jacob virus has been rarely observed.
After binding, these are removed in the reticuloendothe-
lial system including the lung, liver, and spleen. Some
Summary
may also be lysed as part of CDC. Both the binding to
CD20 and elimination by CDC and ADCC are associ- The approval and resounding success of rituximab opened
ated with the release of cytokines which produce fever, the doors to other unconjugated or “naked” Mab. In 1998,
chills, sweats, dyspnea, tachycardia, and sometimes the year in which the drug was released, it became the
hypotension. Rituximab infusion is rapidly followed by most successful cancer drug ever launched, surpassing
340
the achievement of the chemotherapeutic paclitaxel
(Taxol), even though the latter had proven useful in mul-
tiple solid tumors including lung, breast and ovarian
cancer. After introduction as an active therapy for relapsed
patients with indolent lymphoma, rituximab quickly
become part of the standard treatment of virtually every
B-cell malignancy. Rituximab has proven to be an out-
standing agent producing high rates of durable responses
in a broad range of B cell lymphoproliferative disorders,
especially when combined with chemotherapy. Rituximab
is appropriate for use either alone or in combination with
chemotherapy as the initial treatment of indolent lympho-
mas. It is appropriate to combine rituximab with chemo-
therapy for the treatment of newly diagnosed aggressive
lymphomas including large B cell and mantle cell. It is
appropriate to combine rituximab with a purine analog
with or without an alkylating agent in the treatment of
newly diagnosed CLL.
Because of the phenomenal impact and success of
rituximab, there are numerous other anti-CD20 antibody
products being developed in an effort to improve on the
existing agent. Most of the rationale for such efforts
relate to the potential to improved on binding to the
CD20 target, or enhancement of immune function
because of alterations in the Fc portion of anti-CD20
antibodies based on several reports suggesting that the
Fc polymorphisms may predict clinical efficacy [95,
385]. However, others have failed to confirm this obser-
vation. [81, 216].
Alemtuzumab (Campath®, Bayer
Healthcare, Tarrytown, NY)
Alemtuzumab and CD52
The second Mab approved by the U.S. FDA for a hemato-
logic malignancy was alemtuzumab (Campath), an anti-
CD52 monoclonal antibody that was approved in May
2001 based on data submitted for the treatment of patients
with CLL that had recurred or been refractory to the purine
analog fludarabine. Development of this product and a
humanized version actually started several years before
rituximab. The original rat antibody Campath-1G was
genetically modified to create Campath1-H, later called
alemtuzumab, by joining the hypervariable region of the
rodent antibody with a human IgG1 kappa variable frame-
work and constant regions [196]. Rat versions of this anti-
body showed limited clinical activity [195], but in vitro
cytotoxicity in the presence of human complement and/or
effector cells was greatly enhanced with the human IgG1
construct, and also showed activity in vivo [284]. Despite
some encouraging clinical results with the humanized
Monoclonal antibody therapy
construct, Burroughs-Wellcome decided to discontinue
trials with this agent [199]. Subsequently, Ilex Oncology
obtained the product and elected to proceed with develop-
ment, and manufactured the humanized derivative in CHO
cells. Alemtuzumab reacts with the CD52 antigen, which
is present on most lymphomas and chronic leukemias, dis-
eases, and is expressed on many cell types including
malignant and normal B and T lymphocytes, natural killer
cells, monocytes, macrophages, dendritic cells, some neu-
trophils [195]. CD52 is also expressed on tissues of the
male reproductive system including epididymis, seminal
vesicles, and mature sperm. CD52 is not internalized after
antibody binding, and it does not appear to be secreted.
Clinical Trials with Alemtuzumab
Early clinical trials of with anti-CD52 Mab
Initial trials with anti-CD52 were conducted in lym-
phoma. There were no significant clinical responses among
nine patients who received the rat IgG2b monoclonal
antibody [195]. In contrast to these results, two of three
patients did respond to a rat/human IgG1 chimeric con-
struct [284]. These two patients had lymphadenopathy,
splenomegaly, and bone marrow involvement that
responded to doses of only 1–20 mg. Tang et al. reported
partial responses in three out of seven patients with recur-
rent indolent lymphoma, who received thrice weekly
treatment with 30 mg of the humanized antibody [708]. In
early trials, the rat version of the antibody was given to
five CLL patients [195, 196]. Three had sustained
decreases in circulating leukemic cells, but only one of
the three had resolution of marrow infiltrating by leuke-
mic cells. A sixth CLL patient received a rat/human chi-
meric IgM CAMPATH antibody and had no response.
Burroughs-Wellcome sponsored clinical trials of the
human IgG1 construct and clinical responses were seen in
patients who had failed fludarabine chemotherapy. One
report described responses in 6/16 patients who received
30 mg thrice weekly for 16 weeks [347]. However, the
company was disappointed in the associated toxicity, and
elected to discontinue the trials [199]. Subsequently ILEX
Pharmaceuticals obtained the rights to the antibody and
proceeded with additional registration trials in CLL patients
who had relapsed or been refractory to fludarabine.
Alemtuzumab in CLL
Table 27 summarizes the results of various clinical trials
of single agent alemtuzumab in patients with CLL [128,
222, 320, 372, 449, 503, 539, 577, 578]. The trials with
30 mg i.v. over 2 h thrice weekly in patients who had
relapsed after or progressed during fludarabine treatment
were used to support the FDA approval of alemtuzumab
Robert O. Dillman 341

Table 27. Alemtuzumab as a single agent in chronic lymphocytic leukemia

Citation Clinical setting I.V. dose and schedule Number of patients Response rate
[449] Untreated 30 mg sc thrice weekly for 18 weeks 38 87%
[320] Untreated 30 mg iv thrice weekly for 12 weeks 149 83%
[503] Relapsed post chemotherapy 30 mg iv thrice weekly for 12 weeks 91 55%
[128] Relapsed post chemotherapy 10 mg sc thrice weekly for 18 weeks 16 50%
[577] Relapsed post fludarabine 30 mg iv thrice weekly for 12 weeks 136 40%
[539] Previously treated 30 mg iv thrice weekly for 12 weeks 29 38%
[372] Relapsed post fludarabine 30 mg iv thrice weekly for 12 weeks 93 33%
[578] Relapsed post fludarabine 30 mg iv thrice weekly for 12 weeks 24 33%
[222] Median 3 prior chemotherapies 30 mg sc or iv thrice weekly for 12 weeks 115 23%

for the treatment fludarabine-resistant CLL [372]. These
trials demonstrated response rates of about 33%, and
durations of response from 7 to 11 months. A high per-
centage of patients also had stable disease. Because of
severe infusion reactions associated with the reactivity
of alemtuzumab with B and T lymphocytes, granulo-
cytes, and monocytes, in these trials the dose of alemtu-
zumab was gradually increased from three to ten and
then to 30 mg thrice weekly.
A more rationale schedule than escalating boluses is
continuous i.v. or subcutaneous (s.c). The s.c. strategy has
worked well with a decrease in the severity of infusion
reactions with similar efficacy, but local skin reactions
occur in almost all patients and can be severe. Based on
an analysis of serum samples from 30 patients who were
treated with i.v. alemtuzumab, and 20 patients treated by
the sc route, blood concentrations were similar and cumu-
lative doses slightly higher using the sc route, but the sc
route appeared to be associated with appearance of anti-
alemtuzumab antibodies in some patients [285]. Other
investigators have prolonged treatment from 12 weeks to
18 to 30 weeks with some increase in response rates and
CR rates with low rates of opportunistic infections in
patients who had received extensive prior chemotherapy
[128, 449, 714]. In a study of 36 CLL patients who were
treated with the standard i.v. dose and schedule, there
was no correlation between response to alemtuzumab and
the high-affinity, FcgammaR receptor polymorphisms
FcgammaR3A and FcgammaR2A [437].
In a European registration trial 297 previously
untreated CLL patients were randomized to oral
chlorambucil 40 mg/m2 monthly vs. standard i.v. alem-
tuzumab [320]. The Mab produced a superior response
rate, 83% vs. 55% (p < 0.0001) and PFS which has
resulted in a marketing indication as initial therapy, even
though in the U.S. purine-analog based therapy has been
considered the treatment of choice ever since random-
ized trials showed that fludarabine was superior to oral
chlorambucil 40 mg/m2 monthly for up to 1 year as initial
therapy, with response rates of 63% vs. 37% and PFS of
20 vs. 14 months [576].
Alemtuzumab has also been explored in other CD52-
positive leukemias. In a small study a response rate of
73% was observed among 15 patients with T-cell pro-
lymphocytic leukemia [545]. In a larger trial a response
rate of 51% and CR rate of 13% was seen in 75 patients
with relapsed T cell prolymphocytic leukemia who were
treated with alemtuzumab at a dose of 30 mg i.v. thrice
weekly [371]. In a small study that included nine patients
with acute myeloid leukemia and six with acute lymphoid
leukemia, a 20% response rate was observed, but 87%
of patients experienced an infection [722].
Because of the efficacy of purine-analog based chemo-
therapy regimens with or without rituximab, many recent
alemtuzumab trials have focused on its use to eradicate
minimal residual disease in responding CLL patients.
Thrice weekly doses of 10 to 30 mg have been adminis-
tered i.v. or s.c. for 4 to 12 weeks as a consolidative therapy
[499, 530, 780]. These studies have shown that consolida-
tion with alemtuzumab does increase clinical, phenotypic,
and molecular complete response rates, but there is an
increased risk of opportunistic infections, even if anti-viral,
anti-bacterial, and anti-Pneumocystis prophylaxis is used.
Alemtuzumab in lymphoma
Alemtuzumab has activity in both B and T cell lymphop-
roliferative disorders including lymphomas. As shown in
Table 28, response rates were highest in patients in the
mycosis fungoides phase of cutaneous T cell lymphoma
(CTCL), especially in patients who were previously
untreated. Infusional complications from cytokine
release were common, especially with the first infusion
[37, 377, 383, 448, 450, 736, 814]. Cytopenias were
common and associated with high rates of infection,
including some deaths, which resulted in early termination
of several trials.
342 Monoclonal antibody therapy

Table 28. Alemtuzumab as a single agent in lymphoma and other lymphoproliferative disorders other than B cell chronic lym-
phocytic leukemia
Citation Clinical setting I.V. dose and schedule Proportion responding Response rate Percent infections
[736] 16 indolent, 2 aggressive NHL 30 mg iv thrice weekly 8/18 44% 67%
[383] Relapsed indolent NHL 30 mg iv thrice weekly 3/18 17% 72%
[448] Relapsed Indolent NHL 30 mg iv over thrice weekly 6/50 14% 32%
[209] PTCL 30 mg iv thrice weekly 5/14 36% 57%
[814] CTCL & PTCL 10 mg iv thrice weekly 6/10 60% 10%
[37] CTCL Mycosis fungoides 10 mg sc thrice weekly 12/14 87% 29%
[450] CTCL Mycosis fungoides 30 mg iv thrice weekly 12/22 55% 50%
[377] CTCL Mycosis fungoides 30 mg iv thrice weekly 3/8 38% >50%
NHL = non-Hodgkin’s lymphoma
CTCL = cutaneous T cell lymphoma
MF = mycosisi fungoides
PTCL = peripheral T cell lymphoma

Alemtuzumab with Chemotherapy in CLL
Alemtuzumab has been combined with fludarabine in
the treatment of CLL. Kennedy et al. reported obtaining
complete response in six patients treated with both
agents concurrently, who were felt to be refractory to
each drug as single agents [377]. Elter et al. reported an
83% response rate among 36 patients who had previ-
ously received a mean of 2.6 chemotherapy regimens
[207]. Two patients developed fungal pneumonias, one
patient died of Escherichia coli sepsis, and two patients
had reactivation of subclinical cytomegalovirus.
Alemtuzumab with other Biologicals in CLL
Alemtuzumab has been combined with rituximab in the
treatment of patients with CLL. In one trial of 41 CLL
patients the response rate for the combination was more
than 50% and median PFS was 6 months, but infections
occurred in more than 50% of patients [214]. In a sec-
ond combination trial, patients were divided into three
cohorts that received alemtuzumab at doses of 3, 10, or
30 mg during therapy, 1/12 patients had a response
[517]. G-CSF was combined with alemtuzumab in the
treatment of 14 CLL patients who had been heavily pre-
treated (median 3.5 chemotherapy regimens, in an effort
to reduce infections complications and perhaps increase
efficacy [436] G-CSF was administered at a dose of
5 μg/kg daily 5 days before and throughout standard i.v.
alemtuzumab therapy. Five patients responded, but nine
patients developed neutropenia and six patients had
reactivation of cytomegalovirus.
warnings for Alemtuzumab include severe pancytope-
nias after higher cumulative doses, infusion reactions,
and infections, including opportunistic infections.
Because it reacts with both B and T cells in the circu-
lation, infusion reactions associated with administration
of alemtuzumab are common and often severe. The high
percentage of significant infusion reactions limits the
ability to deliver high doses i.v. initially, and in trials
about 5% of patients had to discontinue treatment
because of infusion-associated reactions. For this reason,
in the registration trials doses were initially escalated
from 3 to 10 mg to 30 mg during the first week, and then
administered as 2-h infusions of 30 mg thrice weekly for
an additional 11 weeks. Because of the systemic toxicity,
approximately one log lower mg doses of alemtuzumab
are used compared to rituximab. These low doses are
unable to sustain serum levels and promote good pene-
tration into large tumor masses, which is why the best
results with this schedule of therapy have been achieved
in the blood and bone marrow rather than large lymph
nodes. This problem is being overcome by giving s.c.
doses and continuing treatment for longer duration, but
this also prolongs immunosuppression.
A major limitation for clinical use of alemtuzumab is
the immunosuppression that accompanies prolonged
T-lymphopenia and neutropenia, which is associated
with an increased risk of bacterial infections, and oppor-
tunistic infections including Herpes virus, cytomegalo-
virus infections, fungal infections, Pneumocystis carinii,
and mycobacterial infections. The risk of opportunistic
infections can be reduced, but not eliminated, by admin-
istering prophylactic antibiotics and antiviral agents.

Toxicity and Side Effects Summary

The toxicities associated with Alemtuzumab were sum- Alemtuzumab is active in the treatment of both B and T
marized earlier in this chapter in Table 6. Black box cell malignancies, and it widely used as a salvage and
Robert O. Dillman
consolidation agent in CLL. It might have had much
wider use against B cell lymphoid malignancies had
rituximab not been approved and widely adopted before
alemtuzumab became available. Thus, even though
alemtuzumab has a marketing indication in CLL, and
rituximab does not, the latter is more widely used in
CLL. Enthusiasm for alemtuzumab has also been muted
because of its toxicity, especially immunosuppression,
which has greatly limited efforts to combine the agent
with chemotherapy.
Trastuzumab (Herceptin®, Genentech,
South San Francisco, CA)
Trastuzumab and Her2
In September 1998 the U.S. FDA approved trastuzumab
(Herceptin), a humanized Mab that reacts with the sec-
ond component of human epidermal growth factor
receptor (EGFR), known as Her2, for the treatment of
metastatic breast cancer, making it the first Mab
approved for the treatment of a solid tumor [171].
Trastuzumab consists of the idiotopes from the hyper-
variable region of murine antibody 4D5 united with a
human IgG1 kappa antibody that reacts with the
p185HER2/neu receptor [93]. The Mab was humanized
by inserting the complementary-determining regions
(CDRs) of the murine 4D5 into the constant and vari-
able framework of a consensus human IgG1 to produce
rHuMAb 4D5, which was more active in ADCC assays
than its mouse counterpart. Trastuzumab is produced
using recombinant DNA technology with CHO cells
serving as the manufacturing factory.
Her2 is a 185 kD transmembrane receptor that is a
member of the EGFR tyrosine kinase family of recep-
tors and is involved with the autophosphorylation of
specific tyrosine residues after being activated by bind-
ing of the EGF ligand to the receptor. Early analysis
suggested that a subset of about 25% of breast cancer
patients had tumors that overexpressed this receptor
[673]. Overexpression of the erbB-2 proto-oncogene
results in overexpression of the HER-2 receptor on the
cell surface and increased cell proliferation [632].
Binding of trastuzumab to the HER2 receptor results in
internalization (modulation, down regulation) of the
receptor and competitive inhibition to binding of EGF
ligands to the receptor [675]. Such inhibition interferes
with phosphorylation and the subsequent signal trans-
duction that facilitates cell proliferation. The human
IgG1 constant regions on the Fc of the humanized anti-
body mediate ADCC in vitro; so trastuzumab may also
produce anti-tumor effects via the immune system.
343
Indirect support for this comes from a clinical trial in
which breast cancer patients, whose tumor cells had
high expression of Her2 by immunohistochemistry
(IHC), were treated with docetaxel and trastuzumab;
tumor samples contained significantly increased num-
bers of natural killer cells and increased expression of
Granzyme B and TiA1 in lymphocytes compared with
controls [11]. Based on data submitted to the US FDA,
the recommended initial loading dose is 4 mg/kg admin-
istered as a 90-min infusion followed by a weekly main-
tenance dose of 2 mg/kg administered as a 30-min
infusion. A 3 week dosing schedule has also been vali-
dated [25, 431].
Her2 is overexpressed in only about 25% of patients
with breast cancer; so, testing for high expression of
the antigen (receptor) by immunohistochemistry
(IHC), or for overexpression of the Her2-neu gene by
fluorescence in situ hybridization (FISH), is critical
for appropriate patient selection. Large trials have
confirmed that there is poor reproducibility in perfor-
mance and interpretation of IHC assays for HER2 in
laboratories that do not process and analyze large
numbers of samples. Even in the best of hands, there
is not complete concordance between IHC and FISH.
Most centers who rely on IHC perform FISH for
patients who are 2+ by IHC because many trials have
demonstrated lower response rates in IHC 2+ com-
pared to 3+, while benefit is almost always seen in
patients who are FISH positive. The Dako Herceptest
for IHC assay of HER2 was used to determine eligi-
bility in the pivotal trial and is considered the most
reliable IHC test available. However, many questions
have been raised regarding the reliability and repro-
ducibility of this test, especially on paraffin-fixed
rather than fresh tissues [191, 544, 550, 733]. Many
samples were felt to be falsely positive for Her2 based
on negative FISH tests [733]. Comparative studies
confirmed wide variation in interobserver reproduc-
ibility, and confirmed that the two extremes, 0 and 3+
were more reproducible than 1+ and 2+, and there was
poor discrimination between 2+ and 3+ which resulted
in a high rate of false positive tests [544].
Dowsett et al. compared results for 426 breast carci-
nomas from 37 different hospitals for patients that were
being considered for trastuzumab therapy by perform-
ing IHC and FISH in three reference centers [191]. Only
2/270 (0.7%) of IHC 0/1+ tumors were FISH positive
and only 6/102 (5.9%) of IHC 3+ tumors were FISH
negative, which suggested that FISH testing was most
valuable for validating that IHC 2+ tumors were Her2
negative. In a large U.S. adjuvant therapy trial, reference
laboratories confirmed Her2 positivity for 86% of 2,535
344 Monoclonal antibody therapy

registered patients with only 82% concordance between produces objective response rates of 12–33% depending
local and high-volume central laboratories for IHC, and on the specific patient population [23, 25, 68, 118, 755].
88% for FISH when the same methodologies were used In a phase II trial in 46 patients with metastatic breast
[550]. For the discordant cases, the central and refer- cancer in whom at least 25% of tumor cells expressed
ence laboratories had 94% agreement for IHC 0, 1+, and Her2 by IHC, a schedule of 250 mg i.v. followed by
2+ with 95% agreement that these were FISH negative. weekly doses of 100 mg for 9 additional weeks yielded
Compared to the central laboratories, rates of discor- five responses among 43 evaluable patients [23]. Low
dance were 24% for 75 laboratories that interpreted less grade fever was seen in 15% and was believed to be
than 100 HER2 samples per month and only 3% for 29 related to immune complexes formed with shed antigen.
laboratories that interpreted 100 or more HER2 samples The largest trial of trastuzumab as a single-agent,
per month [542]. which was one of the two pivotal trials that led to regu-
latory approval of the agent, was conducted in 222
patients with Her2-positive metastatic breast cancer that
Clinical Trials with Trastuzumab in Breast had recurred after chemotherapy [118]. Trastuzumab
Cancer alone was given at an initial dose of 4 mg/kg followed
Various clinical trials have confirmed the efficacy of by weekly doses of 2 mg/kg. There were eight complete
trastuzumab, especially in combination with chemo- and 26 partial responses with a median duration of
therapy, for the treatment of Her-2 positive breast cancer response of 13 months. In the 222 patients who had
in patients with recurrent metastatic breast cancer, and failed chemotherapy, there were four complete and 21
as the initial treatment for metastatic breast cancer, and partial responses for an overall objective response rate
as adjuvant therapy [331]. At this time most physicians of 12% by intent to treat analysis. The median duration
continue trastuzumab maintenance indefinitely or until of response among the 25 responders was 9 months, and
disease progression since Her2 receptor is continually their median survival was 13 months, which are compa-
being produced by some malignant cells. rable to chemotherapy. The patient population included
66% who had received prior adjuvant chemotherapy;
Trastuzmab Alone 68% had received two or more chemotherapy regimens
Clinical results from various trials with trastuzumab as a for metastatic cancer and 55 patients (25%) had relapsed
single agent in the treatment of metastatic breast cancer after high-dose chemotherapy and autologous stem cell
are summarized in Table 29. In patients with Her2-positive rescue prior to receiving antibody therapy. The response
metastatic breast cancer that has relapsed after chemother- rate was 14% among patients whose tumors were Her2
apy, eHer2available data suggests that trastuzumab alone 2+ or 3+ by IHC, but 20% for patients whose tumors
were FISH+ vs. 0% for FISH−. Treatment was gener-
ally well tolerated with 40% of patients experiencing
Table 29. Clinical trials of trastuzumab alone in Her2-positive
metastatic breast cancer fever during the first infusion. Cardiac dysfunction was
noted in 5% which was usually reversible when treat-
Clinical I.V. dose and Number of Response ment was discontinued.
Citation setting schedule patients rate
Burris et al. gave trastuzumab at twice the standard
[68] Initial 8 mg/kg, then 52 33% doses by the weekly schedule as initial treatment for 8
therapy 4 mg/kg
weekly for 8 weeks for patients with Her2-positive metastatic breast
weeks cancer who had never received chemotherapy [68]. They
[755] Initial 4 or 8 mg/kg, 114 26% reported a response rate of 33% among 52 evaluable
therapy then 2 or patients in a trial that enrolled 61 patients. This response
4 mg/kg rate is the highest reported for single-agent trastuzumab.
weekly
[26] Initial 8 mg/kg, then 105 19% However, in a randomized phase II trial conducted by
therapy 6 mg/kg q 3 Vogel et al, there was no suggestion that this higher dose
weeks produced a higher response rate [755]. In this trial there
[118] Relapsed 4 mg/kg then 222 15% was no significant difference in results for an 8 mg/kg
2 mg/kg
loading dose and 4 mg/kg per week maintenance vs. the
weekly
[26] Relapsed 250 mg, then 43 12% standard 4 mg/kg loading dose and 2 mg/kg per week
100 mg maintenance. The failure to demonstrate any difference
weekly × 10 between these doses is not surprising in view of the sus-
weeks tained serum levels of trastuzumab that were achieved at
Robert O. Dillman 345

the lower dose. In the Vogel trial, trastuzumab alone as
initial treatment for metastatic breast cancer resulted in
seven complete and 23 partial responses, but the response
rates were 35% for patients whose tumors were IHC 3+
compared to 0% for tumors that were IHC 2+, and 34%
among patients whose tumors were FISH+ compared to
7% for tumors that were FISH−. The most common
adverse events attributed to trastuzumab were chills
(25%), asthenia (23%), fever (22%), pain (18%), and
nausea (14%) and two patients (2%) with histories of car-
diac disease experienced reversible cardiac dysfunction.
It would be more convenient for patients if trastu-
zumab were given at higher doses and less frequently
than weekly. Baselga et al. tested an every 3-week
schedule of trastuzumab delivery as initial therapy for
105 patients with Her2-positive metastatic breast cancer
[25]. They observed response rate was 19%, but was
34% for patients whose tumors were IHC 3+ and/or
FISH+ for Her2. As with other trastuzumab schedule,
the most common treatment-related adverse events were
rigors, fever, headache, nausea, and fatigue.
Trastuzumab Plus Single-Agent Chemotherapy
In vitro and animal studies showed that trastuzumab aug-
mented the anti-tumor cytotoxicity of various chemother-
apy agents [24], [547]. Table 30 summarizes published
phase II clinical trials, grouped by the single-agent chemo-
therapy that was combined with trastuzumab for treatment
of patients with Her2-positive breast cancer [11, 22, 70, 71,
103, 112, 155, 212, 235, 267, 344, 431, 498, 537, 543,
546, 604, 630, 657, 710]. Most of the single agents used in
these trials were the taxanes (paclitaxel or docetaxel), or
gemcitabine. The various agents were associated with
response rates between 25% and 75% with median PFS
of 6 to 12 months. Response rates of 50% or greater were
reported in 17 of the 20 trials which appeared to be much
higher than observed with chemotherapy alone. Many of
these reports noted higher response rates in the subsets
of patients who had 3+ IHC Her2 expression by IHC and/
or overexpression by FISH, as opposed to 2+ IHC Her2
or negative FISH. The addition of trastuzumab was not
associated with any additive toxicities, other than cardiac
disease in association with anthracyclines.
Combination Chemotherapy and Trastusumab
Table 31 summarizes published phase II clinical trials
that used combination chemotherapy plus trastuzumab
for treatment of patients with Her2-positive breast cancer
[235, 483, 500, 547, 549, 604, 690, 750]. Most of these
trials utilized chemotherapy combinations of taxanes
and platinum agents. Combination chemotherapy has
consistently produced higher response rates and longer
progression free survival in clinical trials in patients
with metastatic breast cancer, although survival advan-
tages have been harder to establish. As can be seen in
the accompanying table, phase II trials of combination
chemotherapy plus trastuzumab appear to produce
slightly higher response rates (40–92%) than were seen
with single agents plus trastuzumab, and the PFS was
somewhat longer (range 6–16 months).
In a randomized trial, trastuzumab and paclitaxel with or
without carboplatin were compared as first-line therapy

Table 30. Phase II clinical trials of trastuzumab plus concurrent single-agent chemotherapy in metastatic breast cancer
Citation Clinical setting Chemotherapy Number of patients Response rate
[546] Multiple prior treatments Cisplatin 37 24%
[498] 92% prior chemo Docetaxel q 3 weeks 25 70%
[212] 1st or 2nd chemo Docetaxel weekly 30 63%
[11] Neoadjuvant Docetaxel 23 61%
[710] 1st or 2nd chemo Docetaxel weekly 26 50%
[630] Measurable metastatic Docetaxel q 3 weeks 40 65%
[537] 95% prior taxane & anthracycline Gemcitabine 61 38%
[112] Measurable metastatic Liposomal Doxorubicin 29 52%
[235] No prior therapy Paclitaxel weekly 33 62%
[657] 0–3 prior chemo Paclitaxel weekly 88 61%
[431] No prior chemo Paclitaxel q 3 weeks 32 59%
[267] Prior taxane and anthracycline Paclitaxel weekly 25 56%
[604] No prior chemo Paclitaxel q 3 weeks 88 36%
[344] No prior chemo Vinorelbine 37 78%
[69] 82% prior chemo Vinorelbine 40 75%
[71] Initial chemo Vinorelbine 54 68%
[155] 0 or 1 prior chemo for mets Vinorelbine 40 50%
[22] No prior chemo Vinorelbine oral 30 68%
[103] No prior chemo Vinorelbine 62 63%
[543] Prior chemo Vinorelbine 35 51%
346 Monoclonal antibody therapy

for 196 women with HER-2-overexpressing (2+ or 3+ addition of carboplatin based on response rates of 52%
by IHC) metastatic breast cancer [604]. Treatment con- vs. 36% (p = 0.04), and median PFS of 11 vs. 7 months
sisted of six cycles of trastuzumab 4 mg/kg loading dose (hazard ratio 0.66, p = 0.03). In consecutive phase II tri-
and 2 mg/kg weekly thereafter with paclitaxel 175 mg/ als, weekly paclitaxel and carboplatin plus trastuzumab
m2 every 3 weeks, with or without the addition of carbo- appeared superior to an every 3-week schedule of the
platin (AUC = 6) every 3 weeks. Endpoints favored the same agents with response rates of 81% vs. 65%, median
PFS 14 vs. 10 months, and median OS 3.2 vs. 2.3 years,
and was associated with less hematologic toxicity
Table 31. Combination chemotherapy and trastuzumab for [549].
metastatic breast cancer Similar to what has been seen in other trials with
Clinical Number of Response
anthracyclines, the combination of epirubicin plus car-
Citation setting Chemotherapy patients rate boplatin was associated with a high rate of significant
cardiotoxicity (10/45) [750]. The majority of cardiac
[547] 15% prior Docetaxel + 59 58%
adjuvant Carboplatin events occurred late during therapy with trastuzumab
taxane alone. Half of the patients were asymptomatic and all
[547] No prior Docetaxel + 62 79% cases of CHF were resolved using cardiac therapy.
taxane Cisplatin
[750] Initial Docetaxel + 45 67%
therapy Epirubicin
Randomized Trials of Chemotherapy
[549] Initial Paclitaxel + 48 81% With or Without Trastuzumab in Breast Cancer
therapy Carboplatin Randomized trials comparing chemotherapy plus trastu-
weekly zumab to chemotherapy alone are shown in Table 32.
[549] Initial Paclitaxel + 43 65% One of the pivotal trials that led to regulatory approval of
therapy Carboplatin
q 3 weeks trastuzumab compared trastuzumab plus chemotherapy
[604] No prior Paclitaxel + 88 52% to chemotherapy alone in 469 patients with metastatic
chemo- Carboplatin disease who had not received prior chemotherapy for
therapy q 3 weeks metastatic disease [674]. All patients had tumors that
[236] No prior Paclitaxel + 40 52%
overexpressed Her2, which was defined as 2+ or 3+ by
chemo- Gemcitabine
therapy IHC on a scale of 0–3. An initial 4 mg/kg trastuzumab
[483] No prior Paclitaxel + 13 92% dose was followed by 2 mg/kg weekly. The first dose of
chemo Gemcitabine Mab was infused i.v. over 90 min, but in the absence of
[690] 60% Cisplatin + 20 40% significant infusion related toxicity, subsequent doses
taxane, Gemcitabine
90%
were infused i.v. over 30 min. Patients who had not
anthra- received an anthracycline previously were randomized
cylcine to receive trastuzumab alone or with cyclophosphamide
[500] 2nd line Gemcitabine + 30 50% 600 mg/m2 i.v. and doxorubicin 60 mg/m2 or epirubicin
therapy Vinorelbine (AC) i.v. every 3 weeks for six cycles. Patients who had

Table 32. Randomized trials of chemotherapy ± trastuzumab in breast cancer

Citation Clinical setting Chemotherapy Proportion responding Response rate Median PFS
[674] Initial therapy metastatic breast, prior dox Paclitaxel + T 38/92 41% 7 months
Paclitaxel 16/96 17% 3 months
p < 0.001 p < 0.001 p < 0.0001
[674] Initial therapy metastatic breast, no prior dox AC + T 80/143 56% 8 months
AC 58/138 42% 6 months
p = 0.02 p = 0.10 p = 0.002
[470] Initial chemo Docetaxel + T 57/93 61% 12 months
32/93 34% 6 months
Docetaxel p = 0.002 p = 0.0001
T = trastuzumab
AC = doxorubicin or epirubicin plus cyclophosphamide
PFS = progression free survival
Robert O. Dillman
received adjuvant chemotherapy that included an anthra-
cycline were randomized to receive paclitaxel 175 mg/m2
i.v. over 3 h alone every 3 weeks, or with trastuzumab.
The combination of chemotherapy plus trastuzumab was
superior for key end points including: response rate (50%
vs. 32%, p < 0.0001), duration of response (9.1 vs. 6.1
months), progression-free survival (median 7.4 vs. 4.6
months, p < 0.001), and overall survival (death at 1 year,
22% vs. 33%, p = 0.008), and median survival 25.1 vs.
20.3 months, p = 0.046) with a 20% risk reduction of
death. As shown in Table 32, the differences were most
striking for paclitaxel plus trastuzumab vs. paclitaxel
alone. The median OS was 22 months for paclitaxel plus
trastuzumab vs. 18 months for paclitaxel alone (p = 0.17)
and 27 months for AC plus trastuzumab vs. 21 months
for AC alone (p = 0.16).
This trial clearly demonstrated that cardiotoxicity is
associated with trastuzumab. Cardiac dysfunction was
noted in 11/91 (12%) patients who received paclitaxel
plus trastuzumab alone compared to only 1/95 (1%)
who received paclitaxel alone, and in 28/143 (20%) who
received AC plus trastuzumab compared to 7/135 (3%)
who received AC alone. Even though trastuzumab does
enhance the efficacy of anthracycline containing regi-
mens, the increased cardiac toxicity that is seen with
such combinations has made this an inappropriate treat-
ment strategy.
Another randomized trial compared docetaxel plus
trastuzumab to docetaxel alone using in patients with
metastatic breast cancer who had received no prior che-
motherapy. Docetaxel was given every 3 weeks and
trastuzumab was given weekly. This trial confirmed a
similar relative advantage for the addition of trastu-
zumab with roughly a doubling of response rate and
PFS, and better OS 31 vs. 23 months (p = 0.032). The
Table 33. Randomized trials of trastuzumab in the adjuvant treatment of high-risk breast cancer
Citation Clinical setting Treatment arms
[606] Node + or high risk node Trastuzumab no trastuzumab
[560] Node + or high risk node Trastuzumab observation
[677]
[353] Node + or high risk node Trastuzumab observation
DFS = disease free survival
OS = overall survival
HR = hazard ratio
347
trastuzumab arm was associated with more grade 3 and
4 neutropenia (32% vs. 22%), more febrile neutropenia
(23% vs. 17%), and one patient (1%) in the Mab-
containing arm had symptomatic heart failure.
Adjuvant Treatment of Breast Cancer
Based on the positive benefits of trastuzumab in mea-
surable metastatic breast cancer, it was widely assumed
that a similar benefit would be conveyed in the adjuvant
setting against microscopic metastatic disease. Three large
randomized trials have confirmed the expected advan-
tage of this approach as shown in Table 33. Because of
the cardiotoxicity associated with trastuzumab, patients
with active cardiac disease were excluded from these
trials. Based on Her2 biology, it is probable that there is
more of an advantage to give trastuzumab during, rather
than only after chemotherapy, but this has not yet been
established in trials. It is also probable that there is an
advantage for giving trastuzumab indefinitely, even if it
has already been given with chemotherapy, but this also
has not been proven. Finally, completed trials have
attempted to address whether it is better to give adjuvant
trastuzumab for 2 years or 1 year, but it is probable that
it is better to give trastuzumab indefinitely if there is
reason to believe that the breast cancer has not been
completely eliminated.
Three large randomized trials have been reported, at
least in preliminary form [353, 560, 606, 677]. Two U.S.
cooperative group trials were combined for one prelimi-
nary analysis [606]. The National Surgical Adjuvant
Breast and Bowel Project (NSABP) trial B-31 compared
doxorubicin and cyclophosphamide followed by pacli-
taxel every 3 weeks with the same regimen plus 52
weeks of trastuzumab beginning with the first dose of
paclitaxel. The North Central Cancer Treatment Group
Number of patients DFS OS
1,672 87% 3-years 94% 3-years
1,679 75% 3-year 92%-3 years
0.48 HR p = 0.015
p < 0.0001
1,703 86% 2-years
1,698 78% 2-years
0.54 HR 0.66 HR
p < 0.0001 p < 0.0001
116 91% 3-years
116 86% 3-years
0.58 HR p = 0.15
p = 0.005
348
(NCCTG) trial N9831 compared three regimens: doxo-
rubicin and cyclophosphamide followed by weekly
paclitaxel, the same regimen plus 52 weeks of trastu-
zumab initiated concomitantly with paclitaxel, and the
same chemotherapy regimen followed by 52 weeks of
trastuzumab after paclitaxel. For an early analysis at a
median follow up of 2 years, the two similar arms from
each trial were combined and compared: doxorubicin
plus cyclophosphamide followed by paclitaxel with or
without trastuzumab concurrent with paclitaxel [606].
The international, multicenter trial (HERA) random-
ized more than 5,000 women with HER2-positive node
positive or high-risk node negative breast cancer, com-
pared 1 or 2 years of trastuzumab given every 3 weeks
with observation after completion of locoregional ther-
apy and at least four cycles of neoadjuvant or adjuvant
chemotherapy [560, 677]. Initial reports have compared
the observation and 1-year trastuzumab treatment arms
in terms of interim disease free survival [560], and over-
all survival analyses [677]. In the European trial, 1,010
women with node-positive or high risk node-negative
breast cancer were randomized to adjuvant therapy that
consisted of three cycles of either docetaxel or vinorel-
bine, followed by three cycles of fluorouracil, epirubicin,
and cyclophosphamide in both groups, with a secondary
post-chemotherapy randomization for the 232 patients
whose tumors were Her2-positive to either observation
or trastuzumab for 9 weeks [353]. Because of the use of
anthracyclines in these trial designs, patients have been
closely monitored for cardiac toxicity, which has been
higher in the trastuzumab arms. In the NSABP B-31 trial
16% of women discontinued trastuzumab therapy due to
clinical evidence of myocardial dysfunction or signifi-
cant decline in left ventricular function. As summarized
in Table 33, all three of these trials have shown a benefit
for the addition of trastuzumab.
Table 34. Trastuzumab plus chemotherapy as neoadjuvant therapy for locally advanced HER2-positive breast cancer
Citation Clinical setting Neoadjuvant therapy
[495] Locally advanced Trastuzumab × 3 weeks
[77, 78] Locally advanced Chemo alone
Trastuzumab + Chemo × 24 weeks
[130] Stage II or III Trastuzumab + Docetaxel/Carboplatin × 77
18 weeks
[70] Stage II or III Trastuxumab + Paclitaxel q 3 weeks × 40
12 weeks
[334] Locally advanced Trastuzumab + Docetaxel + Cisplatin × 30
12 wks
CR = complete response
Monoclonal antibody therapy
Neo-Adjuvant Treatment of Breast Cancer
As summarized in Table 34 trastuzumab is also being
combined with chemotherapy as part of neo-adjuvant
therapies with pathologic complete response rates rang-
ing from 13% to 65% [70, 77, 78, 130, 334, 495]. After 3
weeks of neoadjuvant treatment with trastuzumab alone,
23% of patients had a PR before going on to receive 12
more weeks of trastuzumab combined with paclitaxel
before surgery [495]. One randomized trial, which was
designed to accrue 164 patients was closed early because
of the marked superiority of the trastuzumab-containing
arm [77]. Chemotherapy consisted of four cycles of pacli-
taxel followed by four cycles of 5FU, epirubicin, and
cyclophosphamide, with weekly trastuzumab for all 24
weeks. After 3 years of follow up, there have been no
recurrences in the patients who received neoadjuvant
therapy, and they have a better PFS (p = 0.041) [78].
Trastuzumab and Hormonal Therapy for Breast
Cancer
About half of Her2-positive patients are hormone recep-
tor positive which has led to interest in the interaction
“cross-talk” between Her2 and hormone receptors which
appears to increase resistance to hormonal therapies.
There is a recent report of a 26% response rate for 31
evaluable patients who were treated with the aromatase
inhibitor letrozole in combination with weekly trastu-
zumab [464]. Her2 positivity was confirmed for 82%
(IHC3+ and/or FISH+) and 82% had previously been
treated with tamoxifen. The median PFS was 6 months.
Trastuzumab in Other Tumor Types
Her2 is over expressed at a low frequency in many other
adenocarcinoma,; so trastuzumab is also being tested
in other tumors in the subsets of patients whose tumors
overexpress Her2 [2, 171]. However, because of the
Number of patients Response rate Pathology CR rate
35 23% –
19 – 26%
23 – 65%
p = 0.016
95% 35%
75% 18%
– 13%
Robert O. Dillman 349

variability in IHC methodology for detecting Her2, many Table 35. Trastuzumab plus chemotherapy in tumor types
of the higher estimates may be fallacious. So far there other than breast
are no tumor types other than breast cancer for which Screened
trastuzumab appears to be efficacious, even in trials patients who
restricted to patients with whose tumors are Her2- Clinical had Her2 + Patients Response
Citation setting tumors treated rate
positive by IHC (2+ or 3+). In ovarian cancer 95/837
(11%) were HER2-positive and only 3/45 (7%) responded [244] Lung non 103/619 51 36%
small (17%)
[48]. In non-small cell lung cancer (NSCLC) 24/209
cell IIIB
(11%) patients were Her2-positive tumors by IHC and & IV
only 1/24 (4%) had a response [116]. In another NSCLC [812] Lung non 77/360 21 38%
trial 13/69 had Her2-positive tumors by IHC, and 0/13 small (21%)
responded to trastuzumab [415]. In hormone-refractory cell IIIB
& IV
prostate cancer 0/18 Her2-positive patients had a [409] Lung non 28/179 64 28%
response [810]. small (16%)
As summarized in Table 35, several exploratory trials cell IIIB
have been conducted in which trastuzumab was com- & IV
bined with chemotherapy in Her2-positive patients with [337] Metastatic 57/109 44 70%
urothe- (49%)
tumor types other than breast cancer [244, 337, 409, 620, lial
621, 812]. Very few of the patients enrolled had tumors [620] Pancreas 16% 34 38%
that were Her2 3+ by IHC. For example, less than 5% of [621] With RT – 19 50% 2-years
patients in two of the NSCLC trials had tumors that were primary OS
Her2 3+ by IHC [244, 812]. Response rates did not esopha-
gus
appear to be higher than anticipated for chemotherapy
alone, but to determine the true contribution of trastu- OS = overall survival
zumab requires randomized trials. Gatzemeier et al. ran-
domized 101 patients with Her2-positive non-small cell
lung cancer to trastuzumab plus gemcitabine and cispla-
tin or the chemotherapy alone, and found no difference
Toxicity and Side Effects
in response rate (36% vs. 41%) or PFS (6.1 vs. 7.0 The toxicities associated with trastuzumab in pivotal
months), but only six patients had tumors that were 3+ trials were summarized earlier in this chapter in Table
Her2 by IHC, and five of them did have an objective 6. Black box warnings accompanying the label indica-
response to treatment [244]. Collectively these studies tion include cardiomyopathy and infusion reactions.
have been disappointing. This does not appear to be a Trastuzumab infusions can result in serious infusion
fruitful area for further investigation given that so few reactions including dyspnea and hypoxia, which rarely
non-breast cancers strongly over express Her2. have been fatal. Most of the time transfusion reactions
are mild, and are probably associated with binding to
Trastuzumab with other Biologicals circulating white blood cells via either cross reactive
Even though the efficacy of trastuzumab might be antigens or Fc receptors, but much milder than seen
enhanced by combining it with other biological response with rituximab. Typically, symptoms occurred during
modifiers, there has been limited exploration of this or within 24 h of trastuzumab infusion. Infusion should
approach because there are so many efficacious chemo- be interrupted for patients experiencing dyspnea or
therapy agents with which to combine trastuzumab. Two clinically significant hypotension, and patients should
trials have combined s.c. IL-2 with trastuzumab [225, be monitored until signs and symptoms completely
589]. In one trial there was a response rate of 9% in 45 resolve. The basis for the minor infusion reactions that
patients, but more than 10% of patients experienced are sometimes seen with initial infusions is not clear,
grade 3 or 4 pulmonary reactions [225]. In the other but may relate to cross reactivity with an antigen expressed
trial, one of ten patients had a clinical response [589]. In on some circulating cells, or a reaction related to binding
both trials the use of IL-2 was associated with an to Fc on some circulating cells.
increase in natural killer cells, but this did not correlate The major toxicity associated with trastuzumab is
with toxicity or clinical benefit. cardiac dysfunction or congestive heart failure, that is
350
generally mild and reversible [374, 548, 658, 678, 712,
730]. Trastuzumab treatment can result in left ventricu-
lar dysfunction and congestive heart failure; therefore
left ventricular function should be evaluated in all
patients prior to and during treatment. The frequency
and severity of left ventricular cardiac dysfunction and/
or clinical congestive heart failure is highest in patients
who receive trastuzuamb concurrently with anthracy-
cline-containing chemotherapy regimens, and is also
higher in patients who have recently received anthra-
cyline-containing regimens. If left ventricular dysfunc-
tion or clinical heart failure occurs, trastuzumab should
be discontinued until cardiac function has improved.
Trastuzumab can often be resumed without recurrence
of left ventricular dysfunction.
As a single agent, trastuzumab alone was associated
with a 4–5% frequency of cardiac dysfunction in patients
with metastatic disease and in the adjuvant setting [118].
The risk of cardiac toxicity was higher in patients with
metastatic breast cancer who received trastuzumab with
chemotherapy. Cardiac dysfunction was noted in 11/91
(12%) patients who received paclitaxel plus trastuzumab
compared to only 1/95 (1%) who received paclitaxel
alone, and in 28/143 (20%) who received AC plus tras-
tuzumab compared to 7/135 (3%) who received AC
alone, 27% for combined therapy vs. 8% for anthracy-
cline-based chemotherapy alone, and 13% combined
therapy vs. 1% for paclitaxel alone [674]. The higher
rate of cardiotoxicity with the doxorubicin plus trastu-
zumab combination may be due in part to increased
drug delivery of doxorubicin to cardiac muscle by loose
binding between doxorubicin and the Mab [182], or
additive or synergistic cardiotoxic effects of both agents.
Other trials have also noted higher rates of cardiotoxic-
ity in patients who have received anthracyclines prior to
or concurrent with trastuzumab [376, 548, 674, 707].
The cardiotoxicity of trastuzumab may relate to HER2
expression on cardiac muscle cells that are involved in
tissue repair, since absence of the HER2/neu gene in
knockout mice is associated with failure to develop an
embryonic heart [421]. Results from clinical trials sug-
gest that cardiotoxicity is increased when trastuzumab
is administered with or following cardiotoxic agents
such as anthracylines. For this reason it is recommended
that trastuzumab not be given in combination with an
anthracycline or any other agent that is known to dam-
age the myocardium. Based on the adjuvant trials, when
trastuzumab is given after an anthracycline, heart failure
rates can be expected to be in the range of 4–5% during
treatment, but there may be an increased risk that
extends beyond treatment.
Monoclonal antibody therapy
Summary
The approval of trastuzumab in September 1998 marked
the first approval by the US FDA of a therapeutic mono-
clonal antibody for the treatment of a solid tumor.
Because trastuzumab has limited clinical benefit as a
single agent, even in patients who overexpress HER2, it
is typically administered with chemotherapy agents that
have proven benefit in breast cancer, other than anthra-
cyclines, which should be avoided because of the increased
risk of cardiotoxicity. A decade after its introduction,
trastuzumab with chemotherapy is now standard ther-
apy in the neoadjuvant, adjuvant, and metastatic disease
settings for breast cancer patients whose tumors are
Her2-positive. It is controversial as to whether trastu-
zumab should be combined with chemotherapy in
patients who have previously progressed during treat-
ment with trastuzumab with or without chemotherapy.
Historically overexpression of HER2 was associated
with a poorer prognosis. Ironically, trastuzumab is so
effective, that the prognosis for HER2-positive breast
cancer patients is now better than for those that are
HER2-negative.
Bevacizumab (Avastin®, Genentech,
South San Francisco, CA)
Bevacizumab and Vascular Endothelial
Growth Factor
The anti-vascular endothelial growth factor (VEGF)
monoclonal antibody bevacizumab (Avastin) was approved
in May 2004 on the strength of randomized trials in which
the agent was combined with 5-FU-based chemotherapy
for the treatment of metastatic colorectal cancer. In June
2006 it was also approved in combination with FOLFOX4
(5-fluorouracil, leucovorin, and oxaliplatin) for the second-
line treatment of metastatic colorectal carcinoma. It also
has an indication combined with paclitaxel and carbopla-
tin chemotherapy for the treatment of metastatic adeno-
carcinoma of the lung. It is also an indicated for first-line
treatment of metastatic breast cancer in combination with
taxane chemotherapy. Bevacizumab is a recombinant
humanized IgG1 Mab that is manufactured in CHO cells
[570]. Unlike other commercially available antibodies
that target antigens, bevacizumab binds to the VEGF
ligand rather than the VEGF receptor. As discussed ear-
lier in this chapter, VEGF is produced by many tumor
cells, is the central mediator of tumor angiogenesis, and
various lines of evidence suggest that VEGF would be a
useful target for anticancer therapy [12, 55, 248, 420, 681].
Robert O. Dillman 351

The binding of bevacizumab to VEGF inhibited its bind-
ing to the VEGF receptor in vitro, and had anti-angiogenic
and anti-tumor activity in various animal model assay
systems [12, 55].
Clinical Trials with Bevacizumab
Exploratory trials with bevacizumab
Because it blocks VEGF, which is also important in normal
angiogenesis, there was concern that the agent might be
associated with bleeding. In phase I trials in 25 patients
there were no grade III or IV toxicities associated with i.v.
doses up to 10 mg/kg [265]. There were no tumor responses,
but many patients had stable disease. Bevacizumab was
subsequently given in combination with a variety of che-
motherapy agents without undo toxicity [466].
Colorectal Cancer
Clinical trials of bevacizumab in colorectal cancer are
summarized in Table 36. A three-arm randomized phase
II trial in metastatic colorectal cancer suggested that
bevacizumab augmented 5-FU based chemotherapy [359].
In this trial 144 previously untreated patients were ran-
domized to 5-fluorouracil and leucovorin (FU/LV) +
low-dose bevacizumab (5 mg/kg q 2 weeks), or to FU/
LV + high-dose bevacizumab (10 mg/kg q 2 weeks) or to
FU (500 mg/m2)/LV (500 mg/m2 alone. FU/LV was given
weekly for the first 6 weeks of each 8-week cycle. Better
results were seen with the addition of bevacizumab, and
there appeared to be an advantage for the lower dose.
In the pivotal trial for regulatory approval, 923 patients
with metastatic colorectal cancer were randomized to
receive irinotecan, 5-FU and leucovorin (IFL) and beva-
cizumab (IFL-BV), IFL and placebo (control), or FL
with bevacizumab (FL-BV) at a dose of 5 mg/kg every 2
weeks [335, 336]. In the first phase of the trial, 313
patients were randomly assigned to these three arms,
then; after a planned initial analysis, the FL-BV arm was
discontinued after enrollment of 110 patients, not because
of inferior results, but because IFL had become the stan-
dard control arm based on other trials in metastatic col-
orectal cancer. IFL-BV was superior to IFL-placebo for
the 813 patients randomized to treatment with one of
these arms, in terms of response rate (p = 0.004), PFS
(HR 0.54, p < 0.001), and OS (HR 0.66, p < 0.001) [335].
The IFL-BV arm was associated with more grade 3 or 4
hypertension (11% vs. 2%). The FL-BV arm also was
superior to the IFL-placebo arm [336].
In a trial of initial therapy for patients with metastatic
colorectal cancer who were not considered optimal can-
didates for first-line irinotecan treatment, 209 patients
were randomized to receive FL + becacizumab at 5 mg/kg
or FL + placebo [361]. The addition of bevacizumab
resulted in a better PFS (HR = 0.50, p < 0.001), higher
response rate (p = 0.055), and a trend toward better sur-
vival (HR = 0.79, p = 0.16). Hypertension was more
frequent with bevacizumab.
Because of the emergence of oxaloplatin as an active
agent in colorectal cancer, the Eastern Cooperative
Oncology Group (ECOG) conducted a three-arm trial in

Table 36. Randomized trials of bevacizumab in the treatment of metastatic colorectal cancer
Citation Clinical setting Treatment arms Number of patients Response rate Median PFS Median OS
[359] Colorectal metastatic untreated FL + BV-5 35 40% 9 months 22 months
FL + BV-10 33 24% 7 months 16 months
FL alone 36 17% 5 months 14 months
[335, 336] Colorectal 1st-line metastatic IFL + BV-5 402 45% 11 months 20 months
IFL + placebo 411 35% 6 months 16 months
FL + BV-5 110 40% 9 months 18 months
[361] Colorectal 1st-line metastatic FL + BV-5 104 26% 9 months 17 months
FL + placebo 105 15% 6 months 13 months
[253] Colorectal relapsed post IFL FOLFOX4 + 290 23% 7 months 13 months
BV-10
FOLFOX 289 9% 5 months 11 months
BV-10 243 3% 3 months 10 months
[391] Pancreas metastatic BV-10 + 602 11% 6 months –
gemcitabine
gemcitabine
10% 6 months –
FL = 5-flurouracil, leucovorin
BV = bevacizumab
FOLFOX = folate (leucovorin), 5-fluroruracil, oxaloplatin
PFS = progression free survival
OS = overall survival
352
which 829 patients whose cancer had recurred after IFL
therapy were randomized to receive oxaliplatin, fluorou-
racil, and leucovorin (FOLFOX4) with bevacizumab or
to FOLFOX4 alone, or to bevacizumab alone [253]. In
this trial bevacizumab was given at 10 mg/kg every 2
weeks. As shown in Table 36, bevacizumab exhibited
limited activity as a single agent, and FOLFOX4 plus
bevacizumab was superior to FOLFOX alone for all key
endpoints, including OS (HR 0.75, p = 0.001), PFS (HR
0.61, p < 0.0001) and response rate (p < 0.0001). There
were higher rates of hypertension and bleeding in the
FOLFOX plus bevacizumab arm.
A metanalysis was performed using the raw data from
three randomized trials that included 5-FU plus leuco-
vorin and bevacizumab (FL-BV) as initial treatment for
patients with metastatic colorectal cancer [360]. For the
combined data, FL-BV (n = 249) was superior to FL (n
= 241) in terms of response rate (34% vs. 24%, p =
0.019), PFS (9 vs. 6 months, HR = 0.63, p < 0.001), and
OS (18 vs. 15 months, HR 0.74, p = 0.008). In contrast
to these excellent results in previously untreated patients,
in an expanded access trial 350 patients with metastatic
colorectal cancer, who had relapsed after or progressed
during both irinotecan and oxaloplatin based therapy,
the response rate was only 4% in the first 100 patients
enrolled by investigator analysis, and only 1% based on
blinded central review [106].
ECOG conducted a trial with IFL and bevacizumab in
87 patients with untreated advanced colorectal cancer
[252]. The response rate among 81 evaluable patients
was 49%, but the dose of irinotecan and 5FU both had
to be reduced by 20 to 25% because of vomiting, diar-
rhea and neutropenia. Bleeding occurred in 37 patients
(46%) and nine patients (11%) had grade 3 or 4 throm-
boembolic events.
FOLFOX4 and bevacizumab was used to treat 53
patients with previously untreated metastatic colorectal
cancer [208]. The response rate was 68%. Hemorrhage
was not a problem in this trial, and only one patient had
a severe thromboembolic event.
Non-Small Cell Lung
Randomized clinical trials involving bevacizumab in
tumor types other than colorectal cancer are summa-
rized in Table 37. In a three-arm randomized phase II in
non-small cell lung cancer (NSCLC), 99 previously
untreated patients were randomized to treatment every 3
weeks with paclitaxel 200 mg/m2 and carboplatin (AUC
= 6) (PC) alone or in combination with bevacizumab at
7.5 or 15 mg/kg [354]. The response rate and PFS were
higher for the 15 mg/kg bevacizumab arm compared to
PC alone with a trend toward better survival. Bleeding
Monoclonal antibody therapy
was the most significant complication associated with
bevacizumab and included minor mucocutaneous hem-
orrhage, and major hemoptysis associated with centrally
located squamous cell cancers accompanied by tumor
necrosis and cavitation.
In a large two-arm randomized trial, 878 patients with
previously untreated non-squamous NSCLC were ran-
domized to PC + bevacizumab (15 mg/kg) or PC alone
on a 3 week schedule [629]. The bevacizumab arm was
associated with a higher response rate (p < 0.001), better
OS (HR = 0.79, p = 0.003), and PFS (HR 0.66, p <
0.001). Even though patients with squamous cell histol-
ogy were not enrolled in this trial, there were still 5
hemorrhagic deaths in the bevacizumab arm (1.2%) and
significant bleeding was more frequent in the bevaci-
zumab arm (4.4% vs. 0.7%, p < 0.001).
In a large European trial 1,043 patients with previ-
ously untreated non-squamous NSCLC were random-
ized to gemcitabine plus cisplatin with bevacizumab at
10 mg/kg or 5 mg/kg or placebo [463]. Results favored
the bevacizumab arms, although the benefit was not as
impressive as in the U.S. trial with PC.
In a phase I/II trial, 40 patients with metastatic non-
squamous NSCLC who had relapsed after previous
chemotherapy were treated with bevacizumab 15 mg/kg
i.v. every 2 weeks and erlotinib 150 mg orally daily
[309]. Eight patients had a partial response.
Breast
Miller et al. randomized 462 patients to p.o. capecit-
abine 2,500 mg/m2 twice daily days 1 through 14 every
3 weeks, with or without i.v. bevacizumab 15 mg/kg on
day 1 [484]. There was more grade 3 or 4 hypertension
requiring treatment (18% v 0.5%) in patients receiving
bevacizumab. The response rate was twice as high for
the combination therapy as capecitabine alone, but this
did not result in superior survival, which is not surpris-
ing since most of these patients had failed at least three
chemotherapy regimens prior to entering the trial.
In a randomized trial bevacizumab plus paclitaxel
was compared to paclitaxel alone as initial therapy for
722 patients with metastatic breast cancer [482]. There
was an improvement in response rate and PFS, but not
in OS, and there was an increased risk of complications
in the bevacizumab arm. The addition of bevacizumab
was associated with a longer prolonged progression-free
survival (median, 12 vs. 6 months; hazard ratio for pro-
gression, 0.60; p < 0.001) and increased the objective
response rate (37% vs. 21%, p < 0.001). The overall
survival rate, however, was similar in the two groups
(median, 27 vs. 25 months; hazard ratio, 0.88; p = 0.16).
Grade 3 or 4 hypertension (15% vs. 0%, p < 0.001),
Robert O. Dillman 353

Table 37. Randomized trials of bevacizumab in the treatment of metastatic cancers other than colorectal cancer
Citation Clinical setting Treatment arms Number of patients Response rate Median PFS Median OS
[354] NSCLC IIIB or IV NSCLC PC + BV-15 35 32% 7 months 18 months
PC + BV-7.5 32 28% 4 months 12 months
PC 32 19% 4 months 15 months
[629] NSCLC IIIB or IV PC + BV-15 434 35% 6 months 12 months
non-squamous
PC 444 15% 4 months 10 months
[463] NSCLC IIIB or IV GC + BV-15 351 34% 6.7 months –
non-squamous
GC + BV-7.5 345 30% 6.5 months –
GC + Placebo 347 20% 6.1 months –
[484] Breast metastatic Cap + BV-15 231 19% 4.9 months 15 months
Capecitabine 231 9% 4.2 months 14 months
p = 0.001 NSD NSD
[482] Breast metastatic Pac + BV-10 368 37% 12 months
Pac 354 21% 6 months
[799] Renal cell metastatic BV-10 39 10% 4.8 months
BV-3 37 0% 3.0 months
Placebo 40 0% 2.5 months
[210] Renal cell metastatic IFN + BV-10 327 31% 10 months
IFN + Placebo 322 12% 5 months
p < 0.001 p > 0.001 p = 0.067
[391] Pancreas metastatic Gem + BV-10 301 11% 6 months
Gecitabine 301 10% 6 months
NSCLC = non small cell lung cancer
BV = bevacizumab, numbers refer to mg/kg
PC = paclitaxel, carboplatin
GC = gemcitabine, carboplatin
IFN = interferon-α
Gem = gemcitabine
Pac = paclitaxel
NSD = no significant difference
PFS = progression free survival
OS = overall survival
NSD = No significant difference

proteinuria (4% vs. 0%, p < 0.001), headache (2% vs.
0%, p = 0.008), cerebrovascular ischemia (2% vs. 0%,
p = 0.02), and infection (9% vs. 3%, p < 0.001), were
more frequent in patients receiving paclitaxel plus
bevacizumab.
The combination bevacizumab 10 mg/kg on days 1
and 15 of a 28-day cycle in combination with docetaxel
35 mg/m2 on days 1, 8, and 15 was associated with a
52% response rate in patients with metastatic breast
cancer who had not been heavily treated with prior che-
motherapy [579].
Renal Cell
Hypervascularity has long been noted as a feature of
renal cell carcinoma. Most cases of sporadic clear cell
carcinoma of the kidney are associated with loss of
heterozygosity von Hippel-Lindau (VHL) gene on chro-
mosome 3, and inactivation of the VHL allele. This is
associated with a loss of suppressor gene function and
induction of genes that are regulated by hypoxia, one of
which is the gene associated with production of VEGF.
For this reason, renal cell carcinoma was considered an
excellent target for bevacizumab trials. In a randomized
double-blind, phase II trial, bevacizumab at 3 and 10 mg/kg
every 2 weeks was compared to placebo in patients with
metastatic renal cell cancer [799]. After randomization
of 116 patients, the trial was stopped early because there
was a significant prolongation of progression of free
survival in the 10 mg/kg bevacizumab arm compared
with placebo (hazard ratio, 2.55; p < 0.001). At the time
the study was discontinued, the 5 mg/kg bevacizumab
arm also had a slightly longer PFS compared to placebo
that did not quite reach statistical significance (hazard
ratio, 1.26; p = 0.053). There was no difference in sur-
vival, but this analysis was of uncertain significance
since cross-over to bevacizumab was permitted in the
354 Monoclonal antibody therapy

placebo group. Hypertension and asymptomatic protei-
nuria were the main side effects noted.
Large randomized phase III trials comparing inter-
feron-alpha (IFN-α) plus bevacizumab with IFN-α
alone in patients with metastatic renal cell cancer
have shown an advantage for the addition of the anti-
VEGF antibody. IFN-α was given at a dose of 9 MIU
s.c. every other day thrice weekly and bevacizumab
10 mg/kg i.v. every 2 weeks in a large European trial
[210]. A similar comparison is ongoing in the US [597].
Hypertension and proteniuria were much more common
in the bevacizumab arm.
In a phase II trial patients with metastatic clear-cell
renal carcinoma were treated with bevacizumab 10 mg/
kg i.v. every 2 weeks and erlotinib 150 mg orally daily
[280]. The objective response rate was 20%. The most
common adverse events were mild to moderate rash and
diarrhea which were attributed to erlotonib, and protei-
nuria which was attributed to bevacizumab.
In dose-finding phase I–II trials, thalidomide, which
also has anti-antigenesis properties was combined with
bevacizumab in patients with metastatic renal cell cancer
whose disease had progressed after receiving placebo
in another trial [204]. Sequential cohorts of 10 to 12
patients were treated with bevacizumab 3 mg/kg alone
or bevacizumab plus the maximum tolerated dose of
thalidomide per dose escalation. The combination was
well-tolerated with more than 50% of patients able to
escalate their thalidomide dose to at least 500 mg/day.
Toxicities were similar for both treatments and there
were no objective responses. Progression free survival was
similar between groups, 3.0 months for bevacizumab plus
thalidomide and 2.4 months for bevacizumab alone.
Gastric and Gastroesophageal Junction
Adenocarcinoma
Non-randomized trials involving bevacizumab in various
tumor types are summarized in Table 38. Forty-seven
patients with metastatic or unresectable adenocarcinoma
of the stomach or gastroesophageal junction were treated
every 3 weeks with bevacizumab 15 mg/kg on day 1,
irinotecan 65 mg/m2, and cisplatin 30 mg/m2 on days 1
and 8 [659]. Possible bevacizumab-related toxicity
included grade 3 hypertension (28%), grade 3 to 4
thromboembolic events (25%) grade 3 to 4 thromboem-
bolic events (25%), gastric perforation (6%), myocar-
dial infarction (2%), and significant upper gastrointestinal
bleed (2%). The primary tumor was unresected in 40
patients, but only one patient had a significant upper
gastrointestinal bleed.

Table 38. Single arm phase II trials of bevacizumab with or without chemotherapy
Citation Clinical setting I.V. dose and schedule Number of patients Response rate
[106] Colorectal: metastatic refractory BV-5 q 2 weeks + bolus or 100 4%
to irinotecan & oxaloplatin continuous FL
[252] Colorectal: advanced, untreated BV-10 q 2 weeks + IFL 81 49%
[208] Colorectal: chemo-naïve metastatic BV-5 q 2 weeks + FOLFOX 53 68%
[579] Breast: metastatic breast 1st BV-10 q 2 weeks = docetaxel 27 52%
or 2nd line
[310] Lung: NSCLC non squamous, BV-15 q 3 weeks + erlotonib 40 20%
previously treated, recurrent
[659] Gastric and GE: adenocarcinoma BV-15 q 3 weeks + irinotecan + 34 65%
cisplatin
[809] Hepatocellular BV-10 q 2 week + GemOx 30 20%
[132] Pancreas: locally advanced, BV 2.5–10 q 2 weeks Capecitabine + 48 20%
inoperable RT
[66] Ovarian relapsed BV-15 q 3 weeks 62 21%
[87] Ovarian relapsed BV-15 q 3 weeks 44 16%
[390] Pancreas: metastatic BV-10 day 1 & 15 + Gemcitabine 52 21%
[281] Renal cell:clear cell, metastatic BV-10 q 2 weeks + erlotonib 59 25%
[143] Sarcoma: soft tissue BV-15 q 3 weeks + Doxorubicin 17 12%
[759] Glioblastoma BV-10 q 2 weeks + 23 61%
Anaplastic Astrocytoma Irinotecan 9 67%
BV = bevacizumab
IFL = irinotecan, 5-flurouracil, leucovorin
FL = 5-flurouracil, leucovorin
GemOx = gemcitabine, oxaloplatin
RT = radiation therapy
Robert O. Dillman
Hepatocellular
Bevacizumab has been given to patients with measur-
able unresectable or metastatic HCC [809]. For cycle
1 (14 days), bevacizumab 10 mg/kg was administered
alone i.v. on day 1. For cycle 2 and beyond (28 days/
cycle), bevacizumab 10 mg/kg was administered on
days 1 and 15, gemcitabine 1,000 mg/m2 was admin-
istered as a dose rate infusion at 10 mg/m2/min fol-
lowed by oxaliplatin at 85 mg/m2 on days 2 and 16.
The most common treatment-related grade 3 to 4 tox-
icities included leukopenia/neutropenia, transient ele-
vation of aminotransferases, hypertension, and fatigue.
Pancreas
Bevacizumab has been a disappointment in cancer of the
pancreas. In one trial, 48 patients with inoperable pancre-
atic adenocarcinoma received bevacizumab 2 weeks
before radiotherapy, then every 2 weeks during radiother-
apy, and then after radiotherapy until disease progression
[132]. Each cohort included 12 patients at doses of 2.5,
5.0, 7.5, and 10 mg/kg). Capecitabine was administered
on days 14 through 52. Four had ulceration and bleeding
in the radiation field possibly related to bevacizumab.
Three patients had tumor-associated bleeding duodenal
ulcers, and one had a duodenal perforation. [132]. In
another trial, patients with previously untreated meta-
static pancreatic cancer received gemcitabine 1,000 mg/
m2 IV over 30 min on days 1, 8, and 15 every 28 days and
bevacizumab, 10 mg/kg IV after gemcitabine on days 1
and 15 [390]. Grade 3 and 4 toxicities included hyperten-
sion in 19% of the patients, thrombosis in 13%, visceral
perforation in 8%, and bleeding in 2%.
Cancer and Leukemia Group B conducted a random-
ized phase III trial of gemcitabine plus bevacizumab vs.
gemcitabine plus placebo [391]. There was no advantage
for the addition of bevacizumab in terms of response,
rate, PFS or OS.
Ovarian Cancer
In contrast to reported results for other tumor types,
single-agent bevacizumab is quite active in the treatment
of ovarian cancer with response rates of 15–20% in
patients who had progressive disease after two or more
courses of chemotherapy [66, 87, 292]. The most frequent
serious adverse events that were potentially related to
bevacizumab and noted by Burger et al. were hypertension
(10%) and thromboembolic events (3%) [66]. Cannistra
et al. treated reported a 16% response rate in 44 patients,
but serious toxicities included proteinuria (16%), GI per-
foration (11%) hypertension (9%), arterial thromboembolic
355
events (7%), bleeding (2%), and wound-healing complica-
tions (2%) [87]. There were three treatment-related
deaths (7%). The rate of bowel perforation was unusually
high in this trial, but occurred in 24% of patients who had
received three prior chemotherapy regimens, compared to
0% in patients who received less than three.
It seems likely that the high rate of small bowel per-
foration is the result of rapid elimination of bowel wall
tumor implants, and/or and additional insult to bowel
that has been damaged in some manner by chemotherapy
[87]. However, a broader experience based on multiple
trials suggests that this complication is observed in only
about 5% of all ovarian patients [292]. The risk of this
complication appears to be increased in more heavily
treated patients. A three-arm trial has been started which
compares paclitaxel plus carboplatin (PC) with placebo
followed placebo, PC with bevacizumab followed by
placebo, and PC with bevacizumab followed by bevaci-
zumab for 15 months.
Prostate
Bevacizumab was combined with APC8015 (sipuleucel-
T) in the treatment of 22 patients with hormone-refractory
prostate cancer [598]. Sipuleucel-T is a cellular prostate
cancer vaccine containing T-lymphocytes and dendritic
cells loaded with a recombinant prostatic acid phosphatase
and granulocyte-macrophage-colony-stimulating factor
fusion protein. Bevacizumab was given at a dose of 10 mg/
kg i.v. on weeks 0, 2, 4, and every 2 weeks thereafter while
sipuleucel-T was given i.v. weeks 0, 2, and 4. One patient
had a >50% decrease in PSA.
Gliomas
In a phase II trial adult patients with recurrent anaplastic
astrocytoma or glioblastoma (grade III or IV glioma)
received bevacizumab at 10 mg/kg and irinotecan i.v.
every 2 weeks of a 6-week cycle [759]. The dose of irino-
tecan was determined based on whether patients were taking
antiepileptic drugs that induced hepatic enzymes that
accelerate metabolism of irinotecan. Patients taking
enzyme-inducing antiepileptic drugs received irinotecan
at 340 mg/m2, while other patients received irinotecan at
125 mg/m2. Although the significance of radiographic
changes in treated gliomas is questionable, based on study
design the authors alleged an overall response rate of 63%
for all patients. In other trials of bevacizumab patients
with brain metastases were excluded because of concerns
that there might be an increased risk of intracerebral
hemorrhage. However, in this trial there were no instances
of central nervous system hemorrhage, but four patients
(12%) experienced thromboemboic complications.
356
Sarcomas
Seventeen patients with metastatic soft tissue sarcoma
were treated with doxorubicin at 75 mg/m2 i.v. followed
by bevacizumab 15 mg/kg i.v. every 3 weeks [143].
Dexrazoxane was given as a cardio-protectant once the
total doxorubicin dose exceeded 300 mg/m2. Six patients
developed cardiac toxicity grade 2 or greater despite
close monitoring and standard use of dexrazoxane.
Mechanisms of Anti-Tumor Activity
Bevacizumab probably promotes anti-tumor effects by a
number of mechanisms. The original concept for bevaci-
zumab activity was that it would decrease tumor angio-
genesis by blocking VEGF, resulting in a decrease in
tumor blood supply which in turn would lead to cancer
cell death. However, it appears that bevacizumab treat-
ment is also associated with afferent vascular dilatation
and efferent vascular constriction of tumor vessels, which
may help concentrate chemotherapy at the tumor site.
One study in patients with rectal cancer showed that the
bevacizumab decreased tumor perfusion, vascular volume,
interstitial fluid pressure, microvascular density and
circulating endothelial cells while increasing the propor-
tion of blood vessels with percytes [785]. The decrease in
interstial fluid pressure might allow easier penetration for
the Mab itself, which might carry some chemotherapy
molecules with it. Another study in patients with inflam-
matory breast cancer demonstrated a decrease of in phos-
phorylated VEGFR2 in tumor cells, a decrease in vascular
permeability, and an increase in tumor cell apoptosis, but
no significant changes in microvessel density or VEGF-A
expression [771]. In the pivotal trial of IFL and bevaci-
zumab as initial treatment for metastatic colon cancer,
levels of epithelial and stromal VEGF, stromal thrombos-
pondin-2, and microvessel density failed to predict clinical
benefit associated with the addition of bevacizumab as
opposed to placebo [356].
Toxicities and Adverse Events
The toxicities associated with bevacizumab are summa-
rized earlier in this chapter in Table 6. The package insert
for the product warns of hemorrhage, hypertension, pro-
teinuria, impaired wound healing, GI perforation, and
congestive heart failure. The most serious, and sometimes
fatal, bevacizumab associated toxicities are gastroin-
testinal perforation, wound-healing complications, hem-
orrhage, arterial thromboembolic events, hypertensive
crisis, nephrotic syndrome, and congestive heart failure
[211]. The impaired wound-healing has led to sugges-
tions of waiting at least 4 to 6 weeks after bevacizumab
has been discontinued before taking a patient to surgery,
Monoclonal antibody therapy
and waiting at least 2 to 3 weeks after surgery before
instituting bevacizumab therapy. Bowel perforation has
been a problem in the setting of gastrointestinal and ovarian
cancers that are prone to produce tumor implants in the
bowel wall, and may be a result of anti-tumor effect.
Interestingly, the adverse events have varied somewhat
by tumor types, and some may reflect anti-tumor effects
rather than non-specific vascular effects. For instance,
massive hemoptysis has typically been limited to patients
with large centrally located lung cancers, and bowel per-
foration has been more common in colon cancer and
especially ovarian cancer, entities often associated with
metastatic implants on bowel surfaces. Although patients
with brain metastases have been excluded from many
studies, in part because of a fear of intracranial hemor-
rhage, bleeding has not been a problem in patients with
glioblastoma who have received bevacizumab.
Hypertension and proteinuria occur in all settings,
and may be due to effects on small renal vessels since
VEGF is involved in repair of glomeruli endothelial
cells. Hypertension is usually readily controlled with a
single anti-hypertensive agent. Protenuria can progress
to full-blown nepthrotic syndrome. Patients should be
monitored for protenuria and treatment discontinued if
protenuria exceeds 2 g/24 h. Increased rates of expistaxis
have been noted in many trials, but increased rates of
vascular thrombosis have also been noted.
Summary
Despite some safety concerns, bevacizumab has been a
tremendous addition to our therapeutic armamenterar-
ium. Unlike most targeted therapies, it is potentially
useful in every type of solid tumor because of the impor-
tance of tumor angiogenesis. Bevacizumab seems to
enhance chemotherapy in virtually every tumor type in
which chemotherapy is active, and is already in wide-
spread use in cancers of the colon, lung, breast, brain,
ovary, and kidney. Although its single-agent activity
appears limited, bevacizumab alone may be useful in
the adjuvant setting, or following complete remission
after systemic therapy, because of the inhibition of
neoangiogenesis required to develop metastatic tumors.
Cetuximab (Erbitux®), Bristol-Myers
Squibb
Cetuximab and the Epidermal Growth
Factor Receptor
The rationale for targeting the epidermal growth factor
receptor (EGFR), which is expressed to some extent on
all epithelial tumors, was addressed earlier in this chapter.
The human epidermal growth factor receptor (EGFR),
Robert O. Dillman 357

and its ligands, such as transforming growth factor alpha
(TGF-α, have long been recognized as a potential targets
for antibody-based therapy. EGFR is associated with
tyrosine kinase activity, and is frequently overexpressed
in cancers of the breast, ovary, bladder, head and neck,
prostate, pancreas, and colon. EGFR are expressed at
high levels in about one third of all epithelial cancers, and
are associated with accelerated growth [480]. Autocrine
activation and overexpression of EGFR appears to be
crucial for the accelerated growth of many cancers and is
associated with increased expression of VEGF.
The murine antibody to EGFR-1 called 225 was
shown to block receptor function and inhibit cell growth
in cultures in nude mouse xenografts [471]. The Mab
bound to the extracellular domain of EGFR causing
internalization of the receptor and antibody and “down-
regulation” of the receptor. For this reason most early
development with anti-EGFR antibodies focused on
immunoconjugates, especially ricin immunotoxins [472].
Cetuximab, the chimeric form of the antibody, was actu-
ally more effective in the mouse tumor models than the
murine antibody, apparently because of a higher affinity
for the EGFR target [571]. Internalization of the recep-
tor is slow enough that cetuximab is cytotoxic in vitro in
the presence of human effector cells and complement,
but its major anti-tumor mechanism is believed to relate
to the receptor blockade preventing signal transduction
that is associated with cellular proliferation. In labora-
tory tests against tumor cells and in animal models with
human tumor xenografts, cetuximab enhanced the chemo-
therapy effects of many different chemotherapy agents
including platinums, campothecins, taxanes, fluoropy-
rimidines,, and gemcitabine.
The anti-EGFR chimeric Mab cetuximab (Erbitux)
was approved in 2004 based on randomized trials in
which the agent was combined with the chemotherapy
agent irinotecan in the treatment of metastatic colorectal
cancer. It is a recombinant chimeric monoclonal anti-
body that includes a murine Fv and human IgG1 kappa
constant regions. Unlike most monoclonal antibody
products, cetuximab is manufactured in murine plasma-
cytoma cells. This cellular factory results in murine
glycosylation which may increase the risks of allergic
reactions in some patients.
Clinical Trials with Cetuximab
Colorectal cancer
Many of the initial trials with cetuximab, as a single
agent and in combination with chemotherapy, were con-
ducted in patients with colorectal cancer, as summarized
in Table 39.
Trial designs included cetuximab alone, and cetuximab
in combination with irinotecan in patients with metastatic
colorectal cancer that had previously progressed in the
face of irinotecan. In these trials cetuximab was given at
an initial loading dose of 400 mg/m2 i.v. over 2 h, then
250 mg/m2 i.v. over 1 h weekly.
Saltz et al. treated 57 patients whose metstatic cancer
had not responded to prior irinotecan alone or as part of
combination chemotherapy [628]. Patients were required
to have EGFR demonstrated on formalin-fixed paraffin-
embedded tumor tissue by IHC, but it is now known that
quantitative expression of EGFR is not predictive of
response to cetuximab treatment. The most common
adverse events were an acne-like skin rash, predominantly

Table 39. Trials of cetuximab in patients with metastatic colorectal cancer

Median time to Median overall
Citation Regimen Clinical setting # of patients Response rate progression survival
[628] Cetuximab Prior irinotecan 57 9% 1.4 months 6.4 months
[424] Cetuximab Prior 5FU, irinotecan, 346 12% 1.4 months 6.6 months
oxaloplatin
[355] Cetuximab vs. Prior 5FU, irinotecan, 287 8% 41% 3 months 6.1 months
supportive care oxaloplatin
285 0% 24% 3 months 4.6 months
p = 0.001 p = 0.005
[136] Cetuximab Irinotecan refractory 111 11% 1.5 months 8.6 months
Cetuximab + irinotecan 218 23% 4.1 months 6.9 months
[245] Cetuximab + irinotecan Prior 5FU, irinotecan, 60 20% 3.1 6 months
oxaloplatin
[752] Cetuximab + irinotecan Refractory to irinotecan 55 25% 4.7 months 9.8 months
or oxaloplatin
[703] Cetuximab + FOLFOX First-line 43 72% 12.3 months 30 months
5FU = 5-fluorouracil
FOLFOX = folate (leucovorin), 5-fluororuracil, oxaloplatin
358
on the face and upper chest (86%) and the constellation
of asthenia, fatigue, malaise, or lethargy (56%). Three
patients were reported to have had a grade 3 allergic reac-
tion; two were withdrawn.
Lenz et al. treated 346 patients whose metastatic
cancer was considered refractory to fluoropyrimidines,
irinotecan and oxaliplatin [424]. EGFR positivity by
IHC was an eligibility requirement, but degree of posi-
tivity did not correlate with clinical benefit. The most
prevalent toxicity was the acneiform rash which was
noted in 83% of patients, which was predictive of clin-
ical benefit.
In a randomized trial Jonker et al. randomized 572
patients who had progressed despite 5FU, irinotecan,
and oxaloplatin, to standard dose cetuximab or obser-
vation [355]. Although the objective response rate was
only 8%, OS and PFS were better in the cetuximab arm.
Severe adverse events of rash and infection were more
common in the cetuximab arm. Hypomagnesemia was
also more common with cetuximab. Eleven patients
discontinued cetuximab because of infusion reactions.
In a regulatory pivotal trial Cunningham et al. ran-
domized 329 patients with colorectal cancer, that had
progressed during or within 3 months after discontinua-
tion of irinotecan, to receive irinotecan plus cetuximab
or cetuximab alone using a 2: 1 randomization [136].
Protocol prescribed irinotecan was to be the same as
used prestudy. The response rate was higher for the com-
bination therapy (p = 0.007), as was PFS (p < 0.001).
Gebbia et al. treated 60 patients who had received at
least two prior therapies, and whose metastatic cancer was
considered refractory to oxaliplatin and irinotecan [245].
Standard cetuximab dosing was combined with irinotecan
120 mg/m2 weekly for 4 out of 6 weeks. Responses were
not predicted by EGFR expression. The main grade 3 and
4 toxicities were mostly attributable to the chemotherapy,
and included nausea (33%), diarrhea (27%), leukopenia
(18%), asthenia (13%), and acneiform skin rash (13%).
Vincenzi et al. treated 55 patients whose metastatic
cancer was considered refractory to oxaliplatin and iri-
notecan [752]. Standard cetuximab dosing was combined
with irinotecan 90 mg/m2 weekly. Skin toxicity was
observed in 89% of patients. The most common grade 3
to 4 adverse events were dermatologic (33%), diarrhea
(16%), fatigue (13%) and stomatitis (7%). Fever was
noted 25%, typically in association with the first infusion
of cetuximab, but no allergic reactions were recorded.
More recently cetuximab has been combined with
modern combination therapies such as FOLFOX and
FOLFIRI as the initial treatment of patients with meta-
static colorectal cancer. Tabernero et al. used FOLFOX
Monoclonal antibody therapy
and cetuximab as the initial treatment for 43 patients
with metastatic colorectal cancer [703]. Cetuximab was
given day 1 at a dose of 400 mg/m2 during week 1, and
then 250 mg/m2 weekly thereafter. Treatment was well-
tolerated and the response rate was 72%. In a randomized
trial of 337 patients, a preliminary report showed that
cetuximab plus FOLFOX was superior to FOLFOX
alone in terms of response rate (46% vs. 36%, p = 0.064)
[46]. In a randomized trial of 1,198 patients, an initial
report showed that cetuximab plus FOLFIRI was
superior to FOLFIRI alone in terms of response rate
(47% vs. 39%, p = 0.004) and PFS (8.9 vs. 8.0 months,
p = 0.048) [741].
Cetuximab is also being explored in rectal cancer.
Based on a trial in 40 patients with rectal cancer, pre-
operative radiotherapy in combination with capecit-
abine and cetuximab is feasible [452]. Cetuximab in
combination with capecitabine, weekly irinotecan, and
radiotherapy is being evaluated as neoadjuvant therapy
for rectal cancer [324].
Squamous Cell Cancers of the Head and Neck
Cetuximab has also been extensively evaluated in
squamous cell cancers of the head and neck as shown in
Table 40. Vermorken et al. gave single-agent cetuximab
at the standard dose and schedule to 109 patients who
had recurrent and/or metastatic disease that progressed
during platinum chemotherapy [751]. Acneiform skin
rash, which occurred in 49%, was the most common
toxicity There was one death due to an infusion-related
allergic reaction. The response rate was similar to that
of single-agent cetuximab in colorectal cancer.
Phase I trials suggested that cetuximab could be
safely given with radiation therapy and might enhance
therapeutic benefit [602]. Bonner et al. conducted a
multinational trial comparing cetuximab plus radiother-
apy to radiotherapy alone in 424 patients with locore-
gionally advanced disease, that showed a benefit for the
addition of cetuximab including a 26% reduction in
death with an increase in survival from 29 to 49 months
(p = 0.03) [47]. There was no increase in toxicity other
than the acneiform rash.
Pfister et al. treated 22 patients locoregionally advanced
disease with cisplatin, cetuximab, and 70 Gy of radiother-
apy [553]. This trial was closed early because of several
adverse events related to infection and cardiac events.
Grade 3 or 4 cetuximab-related toxicities included acnei-
form rash in 10% and hypersensitivity in 5%. With a
median follow-up of 52 months, the 3-year overall sur-
vival rate is 76%, the 3-year progression-free survival rate
is 56%, and the 3-year locoregional control rate is 71%.
Robert O. Dillman 359

Table 40. Trials of cetuximab in patients with squamous cell cancer of the head and neck
Citation Treatment regimen Clinical setting Number of patients Response rate Median PFS
[751] Cetuximab Prior platinum, recurrent, 103 13% 1.6 months
metastatic
[47] Cetuximab or Placebo + 70 Gy RT Loco-regional advanced 211 – 24 months
213 – 15 months
p = 0.005
[553] Cetuximab + Cisplatin + RT Stage III or IV, M0 22 – 56% 3-year
[309] Cetuximab + cisplatin Refractory to cisplatin + 5FU 130 13% –
or paclitaxel
[26] Cetuximab + cisplatin refractory to cisplatin 96 10% 2.8 months
[72] Cisplatin + cetuximab Recurrent or metastatic 117 26% 4.2 months
Cisplatin + placebo 10% 2.7 months
p = 0.03
[54] Cetuximab + 5FU + Cis- Recurrent or metastatic 53 36%
or carboplatin
[102] Carboplatin Nasopharynx recurrent 59 12% 3.2 months
or metastatic
5FU = 5-fluoruracil
PFS = progression free survival

Herbst et al. treated 130 patients, who did not have
an objective response or relapsed within 90 days of
completing two cycles of cisplatin/paclitaxel or cispla-
tin/fluorouracil, with standard cetuximab and cisplatin
(75 or 100 mg/m2 i.v.) every 3 weeks [310]. The most
common toxicities were anemia, acneiform skin rash,
leukopenia, fatigue/malaise, and nausea/vomiting.
Seven patients (5%) experienced a grade 3 or 4 hyper-
sensitivity reaction to cetuximab.
Baselga et al. treated 96 patients, who were refractory
to cisplatin, with standard cetuximab and cisplatin at the
same dose and schedule during which progressive dis-
ease had occurred [26]. Acneiform rash was the most
common toxicity.
Burtness et al. randomized 117 patients who had
recurrent or metastatic disease to receive cisplatin every
4 weeks with weekly cetuximab or placebo [72].
Response rate was higher with the addition of cetux-
imab, but PFS and OS were not improved. Survival was
better for those patients who developed the skin rash.
Bourhis et al. treated 53 patients who had recurrent or
metastatic disease with a combination of cetuximab,
cisplatin or carboplatin, and escalating doses of 5FU as
initial therapy [54]. Dermatologic toxicity was the most
common adverse event, but the most common grade 3 or
4 adverse events were leucopenia (38%), asthenia
(25%), thrombocytopenia (15%), vomiting (14%).
Chan et al. treated 59 patients who had recurrent or
metastatic nasopharyngeal cancer that had recurred
after cisplatin, with cetuximab and carboplatin [102].
Six patients (10%) experienced serious treatment-related
adverse events during cetuximab.
Cancers other than Colorectal and Head and Neck
Exploratory trials with cetuximab have been conducted
in a variety of other tumor types as shown in Table 41.
Pinto et al. treated 34 patients who had advanced gastric
or gastroesophageal junction adenocarcinoma with
cetuximab and the FOLFIRI regimen as first-line therapy
for up to 24 weeks, with cetuximab alone continued for
patients whose tumors had not progressed [561]. Median
survival was 12 months. Grade 3 to 4 toxicity included
neutropenia (42%), acneiform rash (21%), diarrhea
(8%), asthenia (5), stomatitis (5), and hypertransami-
nasemia (5%) with one treatment-related death.
In pancreatic cancer, Xiong et al. treated 41 untreated
patients who had measurable locally advanced or meta-
static pancreatic cancer and whose tumors had EGFR
expression by IHC, with cetuximab and gemcitabine
[797]. Median survival was 7.1 months and 1-year sur-
vival was 32%. The most common grade 3 to 4 adverse
events were neutropenia (39.0%), asthenia (22.0%),
abdominal pain (22.0%), and thrombocytopenia (17.1%).
Despite the encouraging results of this phase II trial,
preliminary results of a randomized trial conducted by
the Southwest Oncology Group (SWOG) in 735 patients
with advanced pancreatic cancer showed no advantage
for the combination therapy over gemcitabine alone
with a response rate of only 12%, and the same median
survival of approximately 6 months that has been
360 Monoclonal antibody therapy

Table 41. Clinical trials of cetuximab in patients with other tumor types other than colorectal or head and neck
Number of patients Response rate Median PFS Median OS
[561] Cetuximab FOLFIRI Gastric and GE junction 34 44% 8 months 12 months
untreated
[797] Gemcitabine Pancreas advanced 41 12% 3.8 months 7.1 months
[559] Cetuximab + Pancreas advanced 352 12% 3.5 months 6.5 months
Gemcitabine
[293] Cetuximab alone NSCLC previously treated 66 5% 2.3 months 8.9 months
[715] Paclitaxel + NSCLC untreated metastatic 31 26% 5 months 11 months
Carboplatin
[603] Gemcitabine & NSCLC untreated metastatic 35 29% 5.4 months 10.2 months
Carboplatin
[509] Cetuximab Renal cell metastatic 55 0% 1 month 49 months
29 months
p = 0.03
FOLFIRI = folate (leucovorin), 5-fluorouracil, irinotecan
GE = gastroesophogeal
NSCLC = non small cell lung cancer
PFS = progression free survival
OS = overall survival

observed in virtually all randomized trials involving Mechanisms of Anti-Tumor Activity

such patients [559].
In NSCLC Hanna et al. observed a response rate of
only 5% for cetuximab alone in 66 patients with meta-
static disease who had progressed after previous systemic
therapy [293]. Grade 3 to 4 toxicities were uncommon,
but included acneiform rash (6%), anaphylactic reactions
(2%), and diarrhea (2%). Other studies have combined
cetuximab with chemotherapy.
Thienelt et al. treated 31 previously untreated patients
with paclitaxel, carboplatin, and cetuximab [715]. Over
80% of patients experienced dermatologic toxicity, with
13% grade 3 or 4. Robert et al. treated 35 patients with
gemcitabine and carboplatin [603]. The most common
toxicities attributed to cetuximab were acneiform rash
(89%), asthenia (31), fever/chills (20%), and nausea/
vomiting (17.1%).
In metastatic renal cell cancer, Motzer et al. observed
no responses with single-agent cetuximab in 55 patients
[509]. Grade 3 to 4 skin toxicity was noted in 17%.
These results were so discouraging that cetuximab has
not been evaluated further in kidney cancer.
A number of trials are in progress that combine cetux-
imab with combined modality chemotherapy plus radia-
tion therapy regimens. A trial combining cetuximab
with gemcitabine and radiation therapy was being
conducted in patients with locally advanced pancreatic
cancer [408]. A trial of cetuximab with temozolomide
and radiation therapy is being conducted in patients
with previously untreated glioblastoma [126]. Cetuximab
plus intensity modulated radiation therapy in non small
cell lung cancer [349].
and Toxicity
Sustained blocking of EFGR is believed to be critical
for the anti-tumor effects of cetuximab. Based on ran-
domized trials exploring single doses of 50, 100, 250,
400, or 500 mg/m2, a dose of 250 mg/m2 was predicted
to nearly saturate epidermal growth factor receptors
[237, 706]. Skin biopsies in 39 patients, before and after
i.v. infusions of cetuximab, confirmed a dose-dependent
relationship with suppression of EGFR expression [237].
In these trials there was no association between dose and
cutaneous rash, but rash predicted for clinical benefit.
Pharmacokinetic trials of cetuximab with irinotecan
revealed no change in cetuximab clearance or metabolism
when combined with irinotecan compared to infusions
of cetuximab alone [154, 213].
Because all preclinical testing of cetuximab and its
murine precursors were conducted in animals bearing
tumors that strongly expressed EGFR, most early trials
restricted patient eligibility to those whose tumors had
documentable expression of EGFR by IHC. However, in
clinical practice it is now generally accepted that IHC
testing for EGFR expression is not necessary for deter-
mining appropriateness of cetuximab-based therapy,
because of inherent limitations in the assays and poten-
tial for sampling error, and because responses have been
described in patients whose tumors were EGFR-negative
by IHC. In eight colorectal cancer patients who were
refractory to irinotecan, and whose tumors were nega-
tive for EGFR by IHC, four responded to cetuximab
plus irinotecan therapy [301]. A small trial of 31 patients
Robert O. Dillman
suggested that EGFR gene copy number might predict
benefit [504]. However, in a much larger prospective
trial, cell surface expression of EGFR, EGFR kinase
domain mutations, and EGFR gene amplification did
not predict clinical benefit, probably because of the het-
erogeneity of tumors [355, 424]. A pilot study involving
tumors from 39 colorectal cancer patients suggested that
cyclin D1 A870G and EGF A61G polymorphisms may
predict efficacy of single-agent Cetuximab [806].
The most common side effect associated with cetux-
imab and other anti-EGFR therapies is a characteristic
acneiform rash, which is most prominent on face, chest
and upper back [74]. In many trials the prevalence of this
side effect was over 80%. The rash usually appears within
a week of starting treatment. Serial punch biopsies in
patients revealed two main reaction patterns: a superficial
dermal inflammatory cell infiltrate, and a superficial
folliculitis. The rash can be severe and should be treated
to decrease the risk of secondary infection and cellulites.
Grade 3 to 4 dermatitis was reported in 5–15% of patients
in most trials. Prophylactic topical lubricants are suffi-
cient in many patients, but others require treatment with
tetracycline antibiotics or clindamycin. Other common
side effects include asthenia and diarrhea. Cetuximab
does not appear to increase the toxicity of any of the che-
motherapy agents with which it has been combined.
Black box warnings for cetuximab include infusion
reactions and cardiac arrest. The somewhat high rate of
severe infusion related reactions, many of which have
been characterized as anaphylactic reactions, is of con-
cern. At this time it is not clear whether this is actually
do to cross-reactivity with antigens on circulating cells
in some patients, effects of Fc interactions with effector
cells, or a true allergic reaction to murine amino acids
and/or glycosylation because of the murine plasmacy-
toma cell line used to produce the product. There was a
2% risk of sudden death in 208 patients with squamous
cell cancer of the head and neck who received cetux-
imab with radiation therapy [47].
Summary
Cetuximab is widely used in combination with chemo-
therapy for patients who have relapsed after primary
treatment for colorectal cancer. However, based on the
comparative strengths and differences of clinical trials,
bevacizumab with combination chemotherapy is usually
preferred as the initial treatment. Cetuximab is increas-
ingly being used with radiation therapy in patients who
are considered poor candidates to receive combined
modality treatment with chemotherapy and radiation
therapy, which is currently standard for treatment of
361
locoregionally advanced squamous cell cancers of the
head and neck. It will be interesting to see randomized
comparisons of chemotherapy plus radiation vs. cetux-
imab plus radiation, but there are also trial comparing
cetuximab plus chemoradiotherapy to chemoradiother-
apy alone. There may be a role for cetuximab with che-
motherapy in NSCLC, but again bevacizumab with
chemotherapy is currently the standard initial therapy.
Panitumumab (Vectibix™) Amgen,
Thousand Oaks, California
Panitumumab and EGFR
The epidermal growth factor receptor (EGFR) mono-
clonal antibody panitumumab (Vectibix) was approved
in 2006 based on randomized trials in which the agent
was superior to placebo in patients with relapsed, refrac-
tory, metastatic colorectal cancer. Formerly known as
ABX-EGF, panitumumab was the first fully human
monoclonal antibody to receive regulatory approval and
the first totally human Mab approved for cancer therapy
[115, 120]. It is a human IgG2. Like the chimeric Mab
cetuximab, the human Mab panitumumab binds to the
extracellular domain of EGFR causing internalization
of the receptor and antibody and “down-regulation” of
the receptor with disruption of potential downstream
signal transduction that enhances proliferation and
resistance to apopotosis in normal and malignant cells.
Panitumumab was produced with the hope that its clini-
cal efficacy might be even greater than cetuximab
because it is a totally human construct, which may offer
advantages in terms of pharmacokinetics, decreased
allergenic potential, and possibly enhanced ADCC. The
anti-tumor activity of panitumumab was confirmed in vitro
and in vivo, against many cancers, including lung, kidney
and colorectal. In these models it was well tolerated and
clinically active both as monotherapy and in combination
chemotherapy agents. Panitumumab was originally made
in transgenic mice, but is manufactured in CHO cells.
Clinical Trials with Panitumumab
The few clinical trials published for panitumumab are
summarized in Table 42. A pivotal trial for regulatory
approval compared panitumumab at 6 mg/kg i.v. every
2 weeks to best supportive care in patients with meta-
static colorectal cancer whose disease had progressed
during or after standard therapy with fluoropyrimidine-,
oxaliplatin-, and irinotecan-containing chemotherapy
regimens [254, 255, 741, 742]. All patients had epider-
mal growth factor receptor (EGFR)-expressing tumors.
362
Table 42. Trials of panitumumab in patients with metastatic colorectal cancer
Citation Clinical setting Treatment
[741] Progressed after prior chemo Panitumumab
Supportive care
[302] Progressed after prior chemo Panitumumab
[35] No prior therapy IFL + panitumumab 19
[35] No prior therapy FOLFIRI +
panitumumab
IFL = irinotecan, 5-fluorouracil, and leucovorin
FOLFIRI = folate (leucovorin), 5-fluorouracil, irinotecan
Panitumumab produced a 10% objective response rate
and a longer progression free survival compared to
supportive care alone. There was no difference in
survival, but approximately 75% of patients in the
supportive care alone arm crossed over to receive panitu-
mumab after disease progression. Quality of life was also
better in the patients who received panitumumab [668].
For 176 patients who progressed on the supportive care
arm, and then subsequently did receive panitumumab,
12% had an objective response and two patients had
complete responses [742].
Hecht et al. treated 148 patients whose metastatic col-
orectal cancer had progressed on chemotherapy that
included a fluoropyrimidine and irinotecan or oxalipla-
tin, or both [302]. Panitumumab was given i.v. at a dose
of 2.5 mg/kg weekly for 8 of each 9 weeks until disease
progression or excessive toxicity. Skin toxicity occurred
in 95% and 5% were grade 3 or 4. Four patients discon-
tinued therapy because of toxicity and one patient had
an infusion reaction, but was able to resume treatment.
EGFR of at least 1+ by IHC was an eligibility require-
ment. There was no difference in response for 105
patients who were judged as having high EGFR by IHC
compared to 43 patients who were characterized as
having a low EGFR.
Berlin et al. tested the combination of irinotecan, 5-FU
and leucovorin, and panitumumab as initial therapy in
patients with metastatic colorectal cancer [35]. This was
a two-part, multicenter, phase II study of panitumumab
2.5 mg/kg weekly with bolus 5-FU (IFL) in the first part
of the trial, and infusional 5-FU (FOLFIRI) in the second
part. Grade 3 to 4 diarrhea occurred in 11 patients (58%)
in part 1 and six patients (25%) in part 2. All patients had
dermatologic toxicity, but none was grade 4. The authors
concluded that panitumumab + FOLFIRI was better tol-
erated than panitumumab + IFL.
Monoclonal antibody therapy
Median time to Median overall
# of patients Response rate progression survival
231 10% 8 weeks –
232 0% 7.3 weeks –
p < 0.0001
148 9% 14 weeks 9 months
46% 5.6 months 17 months
24 42% 10.9 months 22 months
Toxicities
The toxicities and adverse events associated with pani-
tumumab are summarized earlier in this chapter in Table 6.
Panitumumab black box warnings include dermatologic
toxicities and infusion reactions. Panitumumab was
developed in the belief that it would be less toxic and
more active than cetuximab or other less than com-
pletely human antibodies. Panitumumab has been well
tolerated whether administered alone or in combination
with chemotherapy. However, there was a 1% rate of
anaphylaxis in trials submitted for registration of the
agent. During eight clinical trials, a very sensitive assay
detected anti-panitumumab responses in 25 of 604
(4.1%) subjects, and eight developed neutralizing anti-
bodies [445]. There is a report of at least one patient
who had a severe infusion reaction during treatment
with cetuximab, who was then successfully treated with
panitumumab [332], but because most infusion reac-
tions are seen only with a first infusion, it is not clear
that decreased toxicity will be an important benefit,
since most infusion reactions are due to antigen–antibody
interactions rather than antibody reactions to murine
components of the chimeric or humanized Mab.
Similar to other agents targeting the epidermal growth
factor receptor pathway, skin rash has been the primary
toxicity recognized in association with panitumumab
therapy, and has occurred in 100% of patients receiving
doses of 2.5 mg/kg or higher. Hypomagnesaemia and
diarrhea were also most commonly reported. No grade
severe or life-threatening reactions were noted in the
large randomized trial in patients with colorectal cancer
[741, 742]. Other common adverse events noted were
paronychia, fatigue, abdominal pain and nausea. The
most serious adverse events were severe dermatologic
toxicity complicated by infectious sequelae and septic
Robert O. Dillman
death, infusion reactions, pulmonary fibrosis, hypomag-
nesemia, and gastrointestinal problems including abdom-
inal pain, nausea, vomiting, diarrhea, and constipation.
Summary
Panitumumab is the first fully human antibody to receive
regulatory approval, and joins cetuximab as an anti
EGFR Mab. It remains to be seen whether the to totally
human construct offers any meaningful advantages of
the chimeric construct.
Conclusions
“Magic bullets” are now part of the standard anti-cancer
therapeutic armamentarium [168]. It has now been more
than a decade since rituximab and trastuzumab became
the first Mabs approved for the treatment of cancer, and
both have become well-established “blockbuster drugs.”
Alemtuzumab would be more widely used in the treat-
ment of hematologic malignancies if its target was more
specific, and rituximab was not so safe and effective in
the treatment of B cell malignancies. While five of these
agents are limited in their scope of malignant targets
because of their specificity for surface antigens, bevaci-
zumab has the potential to be useful in every cancer set-
ting because of the importance of the VEGF ligand for
tumor angiogenesis. Cetuximab and panitumumab are
potentially useful in the same patient populations, and it
will be interesting to see whether the totally human Ig
has any meaningful clinical advantages over the chime-
ric product. It is interesting that other than rituximab,
these agents have rather limited anti-tumor effects, but
all dramatically increase the effects of chemotherapy,
which is what has led to their rapid and widespread adop-
tion in cancer therapy. The potential for synergistic and
additive effects resulting from the use of Mab in combi-
nation with other biological response modifiers has not
been established, and certainly is not superior to the
common chemotherapy plus Mab combinations.
Other Antibodies and Antigens
This section is organized by antigenic targets and covers
many other Mab that have investigated but are not
approved for clinical use as anti-cancer therapy, although
some are clinically available based on other indications.
Some trials that involved tracer quantities of radiola-
beled Mabs and are also included if in fact patients
received at least several mg of unmodified antibody. The
information described is based on publication in the
363
medical literature and cannot be considered all incon-
clusive because of the volume of unpublished results
known only to certain companies who have pursued
commercial development of certain antibodies, and the
US FDA.
Anti-Idiotype Antibodies
The idiotype of malignant B-cell clones is perhaps the
best example of a tumor-specific antigen. More than 15
years before the first approval of a Mab for the treatment
of malignancy, the first antibody-mediated CR was
reported following administration of a murine anti-
idiotype antibody [487]. The patient had follicular B
cell lymphoma that had progressed after prior treatment
with cyclophosphamide–vincristine–prednisone che-
motherapy, and interferon-alpha biotherapy. The complete
response persisted for over 7 years. Subsequent publica-
tions established a 66% response rates among 45 patients
with indolent lymphoma who received anti-idiotype
antibodies alone, or administered with single-agent
chlorambucil, interferon-alpha, or interleukin-2 [146].
Despite the promising early results with anti-idiotype
antibodies, commercial development in this area has not
evolved because of the need to develop a specific anti-
idiotype antibody for each patient. Interest for commercial
development increased when several investigators
suggested that combinations of anti-idiotype antibodies
commonly expressed on B-cell lymphomas might be a
useful clinical product [16, 394, 489, 614, 692, 702], but
enthusiasm dwindled when the frequency of these
“shared” or “cross reactive” idiotypes was found to be
much lower than originally suggested.. The company
IDEC of San Diego, CA, which was originally founded
as an anti-idiotype company, subsequently acquired
Analytical Biosystems of Sunnyvale, California, estab-
lished by Levy and Miller of Stanford, which was the
first anti-idiotype company to pursue the anti-idiotype
approach. IDEC eventually dropped development of a
composite anti-idiotype preparation called Specifid® in
order to focus its resources on a product called C2B8, a
chimeric anti-CD20 antibody that became the block-
buster product rituximab [168].
The largest experience with anti-idiotype antibodies
was in lymphoma as initially reported by Meeker et al.
[474] and subsequently by others at Stanford who were
working with Ron Levy [60, 61, 146, 446, 458]. Among 17
patients receiving individualized anti-idiotype antibod-
ies at doses ranging from 400 mg to over 9 g, there were
nine partial responses including the sustained, complete
remission in the first patient treated, and others lasting
from 1 to 6 months [478, 487]. Favorable responses were
364
associated with increased T-cell infiltration of involved
lymph nodes [446]. In some cases, resistance to therapy
was shown to result from emergence of idiotype-variant
clones [478]. Stanford investigators combined IFN-α
with anti-idiotype antibodies based on the rationale that
IFN-α might upregulate the idiotype antigen expression,
and IFN-α is known to have antiproliferative effects on
follicular B-cell lymphoma cells. Of 12 patients treated
in this manner, who received total antibody doses rang-
ing from 1.7 to 8.0 g, there were two CRs and seven PRs
[61]. Another trial combined chlorambucil with anti-
idiotype antibodies and one CR and seven PR were
described in 13 patients [458]. It was not clear that either
of these combination therapies was more effective than
anti-idiotype antibodies alone because of changes in
patient selection and the known efficacy of both IFN-α
or chlorambucil as single agents in the treatment of fol-
licular lymphoma. Other groups have reported on lim-
ited trials with anti-idiotype antibodies with limited
success [99, 291, 581]. Rankin et al. noted minimal anti-
tumor effects in two lymphoma patients who received
murine anti-idiotype Mab at individual doses from 5 to
160 mg and total doses of 3.8 and 5.8 g respectively
[581]. Another report relates an initial experience with a
mouse/human chimeric anti-idiotype in a patient with
B-cell lymphoma who had no tumor response [291].
The brief and limited responses observed in CLL may
have been due to the relatively low doses of Mab admin-
istered in those trials [99].
Antigens Associated
with Hematopoietic Cells
Because of the ready availability of clinical material for
testing, many of the first murine Mab were developed
after immunization with hematopoietic cells. The vari-
ous antigens identified on blood cells were classified by
a cluster designation (CD) numbering system [36].
Many of these antibodies were utilized in early pilot,
phase I, and phase I/II trials.
CD3
CD3 is a pan-T cell antigen and an important receptor for
activation of T lymphocytes. The first monoclonal anti-
body approved for any use, was the murine antibody
OKT3 which now has the generic name muromonab-CD3
[638], approved to prevent kidney transplant rejection in
1984. Theoretically, CD3 might be a target as a tumor
associated antigen in T cell lymphoma, or to activate
normal T cells to modulate or induce an indirect antitumor
action through other components of the immune system.
Monoclonal antibody therapy
Urba et al. treated 36 patients with OKT3 antibody in
an effort to activate T cells in the hope of promoting an
antitumor effect [737]. Five patients received a 30 ug
dose by i.v. bolus and the other 23 received 3-h infu-
sions of 1, 10, 30, or 100 ug. An additional 8 patients
received the anti-CD3 daily for 14 days by either bolus
3-h infusion, or 24-h infusion. The dose-limiting toxic-
ity in this study was headache, accompanied by signs
and symptoms of meningeal irritation. Eight of the 16
patients tested exhibited HAMA. No tumor responses
were observed.
Richards et al. treated 13 patients with OKT3. Six
patients received 50 ug, and 7 received 100 ug [593]. A
partial response was described in one patient with meta-
static renal cell carcinoma. Again, neurotoxicity was a
significant problem and was observed in 11/13 patients
after the first treatment. Headache and confusion were
noted. In all patients, neurotoxicity was transient and
interestingly, did not recur with re-treatment. In both of
these this study and that by Urba et al, a cerebral spinal
fluid lymphocytosis was noted in patients who under-
went lumbar puncture, and headache was a frequent
complaint [593, 737]. Because of the evidence that
OKT3 had stimulated an immune response in the central
nervous system, Wiseman et al. conducted a trial with
OKT3 in patients with gliomas who had failed conven-
tional therapy and evidence of progressive disease [789].
Patients received 25 to 75 ug of OKT3 over 1-h, fol-
lowed a day later by 300 mg/m2 of cyclophosphamide as
a dose intended to reduce effects of suppressor or regu-
latory T cells. Three of nine patients were reported to
have had objective tumor regressions, based on brain
studies with magnetic resonance imaging. Anticipated
side effects included headache, fever, stupor, nausea,
emesis, and transient decreases in T-lymphocyte counts,
but no severe or life-threatening toxicity.
Several investigators have explored the use of the
T-lymphocyte cytokine interleukin-2 (IL-2) in combination
with OKT3 because of the potential for synergistic or
additive T-cell stimulation. Sosman et al. treated 54
patients with doses of OKT3, ranging between 75 to
600 ug/m2 followed by high-dose bolus IL-2 therapy
[684]. The numbers of circulating T cells expressing the
IL-2 receptor were not increased, and the tumor response
rate was no better than had been observed with IL-2
alone. Buter et al. gave 50 to 400 ug OKT3 with low-
dose s.c. IL-2 to eight patients [76]. Neurotoxicity was
the limiting toxicity at the highest dose. There was no
enhancement of activated lymphocytes and no responses.
Another approach involves the production of antibodies
that are genetically fused to various cytokines to func-
tion as targeted biological response modifiers [388].
Robert O. Dillman
These multifunctional products are covered in other
parts of this section based on the tumor antigen that is
being targeted.
A humanized anti-CD3 antibody, initially called
HuM291, and now named visilizumab, has been studied
in a variety of disorders to suppress activated T lympho-
cytes, including ulcerative colitis, kidney rejection, and
graft vs. host disease following allogeneic stem cell
transplants [91, 367, 525, 563, 592]. Visilizumab is a
mutated IgG2 isotype whose Fc tail does not bind to Fc
gamma-receptors, but can induce apoptosis of activated
T lymphocytes. Doses from 0.1 to 15 mg/kg have been
infused in various trials, and infusion related toxicities
and CNS toxicities seem to be much less than were
observed with muromonab. To date there are no reports
of its use in cancer patients.
CD4
CD4 is typically associated with helper T cells, and is
expressed on many T cell malignancies. Knox et al.
treated seven T-cell lymphoma patients with an anti-
CD4 chimeric Mab [397]. Doses of 10, 20, 40, and
80 mg were given i.v. twice a week for 3 consecutive
weeks. Circulating cells were coated with antibody, but
there was no significant change in the number of T cells.
Some transient benefit was seen in all patients, but these
clinical results were not clearly better than those seen
with anti-CD5 murine Mab.
Zanolimumab is a human IgG1 antibody against CD4
which may exert anti-tumor effects by several mecha-
nisms [594]. Two are regulatory mechanisms that
include rapid inhibition of signal transduction mediated
by the CD4-associated tyrosine kinase p56lck, and then
chronic down-regulation of CD4 molecules. The third is
direct Fc-dependent lysis of circulating CD4+ T cells;
CD45RO+ cells are more sensitive to ADCC killing
than CD45RA+ cells. Zanolimumab is being evaluated
in cutaneous T-cell lymphoma (CTCL) and peripheral
T-cell lymphomas (PTCL) Kim et al. reported results of
two phase two trials of zanolimumab in 37 patients with
refractory CTCL, nine of whom were still in the myco-
sis fungoides (MF) stage of disease [387]. Patients
received up to 4 months of weekly infusions with 280 to
980 mg per dose. The objective response rate was 32%.
Adverse events included infusion reactions with the
initial depletion of peripheral T cells, and low-grade
infections including eczematous dermatitis, especially
in MF patients. Based on these encouraging results,
additional studies are being conducted in hopes of regu-
latory approval.
365
CD5
CD5 is a pan T cell antigen that is also co-expressed on
the B-lymphocytes of CLL and small B-cell lymphoma
(well-differentiated diffuse B-cell lymphoma), and the
B-lymphoctyes of mantle cell lymphoma [188]. Normal
CD5+ B-cells are prevalent during fetal development, but
their number decrease after birth and with age, but increase
again in the elderly. In the early years of Mab develop-
ment, several laboratories produced anti-CD5 antibodies
in response to immunization of mice with lymphocytes.
CD5 is expressed on most T cells. A number of the
early trials with mouse Mabs were conducted in patients
with T cell malignancies because of the early availabil-
ity of antibodies that reacted with the CD-5 antigen on
T lymphocytes. Levy and colleagues treated eight T-cell
leukemia patients with the anti-CD5 momab Leu1, that
induces antigenic modulation, and two anti-leukemia
antibodies that did not induce antigenic modulation
[429]. Patients received 1 to 92 mg of Leu-1 alone or in
combination with the other Mab. Transient reductions in
circulating leukemia cell counts were observed, but
there were no sustained antitumor effects. There was no
obvious advantage to treating with a combination of
antibodies in these trials.
Miller et al. reported an early success in a patient with
CTCL who received 17 infusions of 1 to 20 mg of anti-
CD5 antibody Leu1 that was administered over a
10-week period [485, 486]. He experienced a partial
response including marked shrinkage of lymph nodes,
skin lesions, and a neck mass. There was also a transient
decrease in circulating T lymphocytes after each treat-
ment. These same investigators treated five additional
CTCL patients, and one with a large PTCL [488].
Individual patients received four to seven treatments
over 2 to 10 weeks at doses of 250 ug to 100 mg infused
over 4–6 h for total doses of 13–761 mg. Four patients
had brief tumor regressions lasting from 1 to 4 months.
The IgG2a murine Mab T101, which reacts with the
CD5 lymphocyte antigen, was one of the first mouse anti-
bodies to be extensively tested in human trials. The CD5
antigen rapidly internalizes after T101 binding, and the
antibody was not cytotoxic in in vitro assays of CMC or
ADCC with human effector cells or human complement
[181]. Dillman et al. treated two CTCL patients with mul-
tiple 2-h infusions of the anti-CD5 momab T101 [179],
and subsequently treated an additional 10 CTCL patients
with T-101 at doses from 10–500 mg, given over 24 h
[180]. With the prolonged infusions, brief antitumor
responses lasting from days to weeks were seen in four
patients. One of the most interesting responses was the
366
dramatic remission of rheumatoid arthritis in a patient
with CTCL who was bedridden because of arthritis, and
became ambulatory within 24 h of treatment with T101. In
other trials with T101, Foon et al. administered the anti-
body to several CTCL patients. Some patients had mini-
mal improvement in skin lesions [227]. Bertram et al. gave
T101 to eight patients with CTCL and to four other
patients with various T-cell lymphoproliferative disorders
[38]. One patient with CTCL had a partial response of his
cutaneous lesions that persisted for 3 months. Another
patient with convoluted T-cell lymphoma was alleged to
have had a complete remission based on a decrease in
bone marrow blasts from 6% to 3% which persisted for 2
months following doses of 1–100 mg T101.
Dillman et al. treated 10 CLL patients with T101
[177]. Two patients received 1 to 10 mg over 15 min,
two received weekly doses of 10 mg infused over 2 h,
and 6 received 24-h infusions of T101 at doses of 10, 50,
100, 150, or 500 mg. Limited unsustained clinical ben-
efit was noted despite confirmation of in vivo binding to
CLL cells in blood, lymph nodes, and bone marrow. The
same T101 antibody was studied by Foon et al. in 13
patients with CLL [228]. Successive three-patient
cohorts received doses of 1, 10, 50, 100 mg and one
patient received 140 mg. At all doses there binding to
circulating and bone marrow cells could be demon-
strated, and a rapid but transient decline in circulating
leukemia cell counts. Durable responses were not seen.
CD10
CD10 was originally known as the common acute lym-
phoblastic leukemia antigen (CALLA). CD10. Ritz and
colleagues explored the mouse mab J-5 that reacts with
CD 10, in four patients with acute lymphocytic leukemia
(ALL) [600]. Doses of 1 to170 mg of J-5 were infused
over 15 min to 2 h. In those patients with circulating
blasts, treatment was followed by a rapid decrease in cir-
culating CALLA+ blasts, but a large number of CALLA-
negative lymphoblasts persisted. However, once J-5 was
no longer detectable in the serum, the lymphoblasts reex-
pressed CALLA, consistent with antigenic modulation or
internalization of the surface antigen in the presence of
the antibody. There was no decrease in marrow blasts
even though J-5 binding was demonstrated. There was no
significant toxicity noted in these patients although all
three patients who had circulating blast cells had tempera-
ture elevations in association with therapy.
CD15
CD15 is a granulocyte-associated antigen. In prelimi-
nary trials Ball et al. had treated three AML patients
Monoclonal antibody therapy
with one or more of the IgM anti-glycolipid momabs
PMN-81, PMN-29, and PM-81 and a fourth antibody,
an IgG2b called AML-2-23, which reacts with a protein
antigen [19]. None of these antibodies induced antigenic
modulation in vitro. Multiple 8 to 12 h infusions of 20 to
70 mg of the various antibodies were associated with
rapid, but transient decreases in circulating blasts and
elevations of serum lactic dehydrogenase suggesting
that leukemia cells had been destroyed, although sus-
tained remission of leukemia did not occur. Ball et al.
treated another 16 acute myeloid leukemia (AML)
patients with 24-h continuous infusion of anti-CD20
Mab PM81 (also called MDX-11) at doses from 0.5 to
1.5 mg/kg in a dose escalation study [20]. Transient
reductions in circulating blasts were noted, but there did
not appear to be an effect on marrow blasts at these rela-
tively low doses. Circulating CD15 antigen presented a
problem in terms of immune complexes. The toxicities
observed in this trial (fever, chills, hypotension, tachy-
cardia) were the same as observed with other Mab that
bind to circulating cells.
CD19
CD 19 is a 90 kD glycoprotein differentiation antigen
expressed on normal and malignant B cells that might be
a useful regulatory target on B cells [753]. The CD19
molecule acts as an internalizing receptor that is physi-
cally and functionally associated with certain protoonco-
gene phosphokinases. Hekman et al. treated 6 lymphoma
patients with an IgG2a murine Mab against the B-cell
CD19 [303]. Total doses from 225 to 1,000 mg were
given over 4 h without major toxicity. One patient was
felt to have had a partial remission that lasted 8 months
following his first treatment, and then 9 months follow-
ing a second treatment. Because of the internalization
(modulation) of this antigen, recent studies have focused
more on immunoconjugate approaches.
CD20
This is the target for the chimeric Mab rituximab, which
was discussed in detail in this chapter in the section on
commercially available antibodies. It is also the target
of the commercially available radiolabeleled antibodies
I-131 tositumomab (Bexxar) and Y-90 ibritumomab
tiuxetan (Zevalin) [174]. The earliest evidence of the
therapeutic potential of targeting CD20 was reported by
Press et al. [569] who used the anti-CD20 momab 1F5
to treat four patients with refractory B-cell lymphoma.
Total treatment was given over 5 to 10 days. Two
patients had a 90% reduction in circulating B cells. The
total doses delivered were 52 mg in a patient who had
Robert O. Dillman
progressive disease, 104 mg in a patient who had stable
disease, 1,032 mg in a patient who had a minor response,
and 2,380 mg in a patient who had a partial remission
including a 90% reduction in lymph nodes that persisted
for 6 weeks.
This same antibody, which is now called tositu-
momab, is given as part of I-131 radioimmunotherapy.
In one randomized trial that included a control arm in
which 36 patients received two 450 mg doses of tositu-
momab over 1 to 2 weeks, there was a 19% objective
response rate [150]. In a similarly designed randomized
trial of Y-90 radioimmunotherapy, 4 weeks of standard
dose rituximab had an objective response rate of 56%
[790].
Because of the tremendous clinical success of ritux-
imab, a number of companies have made new CD20
Mab with variations in Fv and Fc in efforts to enhance
therapeutic benefit. It will be interesting to see how
many of the products are tested in randomized trials
against rituximab as opposed to going for a unique
indication in a subset of CD20 positive malignancy.
CD22
CD22 is a modulating antigen widely expressed on B
lymphocytes. Epratuzumab, a humanized IgG1 antibody
that targets CD22, was originally developed for radioim-
munotherapy, although preliminary work with anti-
CD22 antibodies focused on immunoconjugates because
of the internalization of the antigen after antibody bind-
ing. Leonard et al. treated 55 patients who had recurrent
indolent or large B-cell lymphoma with epratuzumab at
doses of 120 to 1,000 mg/m2 over 30 to 60 min weekly
for four treatments [425]. There was an 18% response
rate among 51 evaluable patients with a median duration
of response or more than a year. All nine responses were
in patients with follicular lymphoma. One complete
response persisted for more than a year in a lymphoma
patient who was considered refractory to the anti CD20
ximab rituximab. The treatment with this agent has been
well-tolerated with no surprising toxicities described.
Subsequently clinical trials were launched in both folli-
cular and large B cell lymphoma using four weekly doses
of 360 mg/m2, but these were not completed.
CD23
CD23 has been characterized as a low-affinity IgE
receptor that mediates allergic responses, but it is also
widely expressed on CLL cells and its expression is
used to help differentiated CLL and SLL from mantle
cell lymphoma. Lumiliximab is an IgG1 anti-CD23 chi-
meric Mab that is in clinical trials in CLL [587]. O’Brien
367
et al. conducted a phase I trial involving 46 patients who
had relapsed or refractory CLL [84]. Lumiliximab was
given i.v. at 125, 250, 375 mg/m2 or 500 mg/m2 weekly
for 4 weeks or 500 mg/m2 thrice during week 1 then
500 mg/m2 weekly for the next 3 weeks, or 500 mg/m2
thrice a week for 4 weeks. Little toxicity was reported at
any dose level, but there were no objective responses.
CD25
Daclizumab (Zenapax®, Roche Laboratories,
Nutley, NJ)
The interleukin-2 receptor, CD25, is overexpressed in
activated T-cells and many T cell malignancies. It is com-
posed of 3 subunits including an alpha chain (p55), a beta
chain (p75), and a gamma chain (p65) which combine
noncovalently to bind the important lymphokine interleu-
kin-2 (IL-2) which is not expressed on resting lympho-
cytes, but is often overexpressed in activated and
malignant lymphocytes, especially T-lymphocytes [762].
The anti-CD25 Mab daclizumab (Zenapax™, Roche
Laboratories, Nutley, NJ) was approved by the US FDA
in 1998 for the marketing indication of kidney transplant
rejection [766], but there is a strong rationale for it having
clinical activity in certain hematopoietic malignancies
[761, 764]. It is a humanized Mab derived by genetic
engineering modification of a murine anti-Tac (55 kD
subunit of the IL-2 receptor, IL-2Rα) Mab. The human-
ized form of this antibody was originally called anti-
TAC-H. Later daclizumab was constructed using a human
IgG1 constant framework [358]. The murine version was
unable to effect ADCC with human effector cells, but the
humanized construct was cytolytic in ADCC assays. As
expected, daclizumab was much less immunogenic in
monkeys than the mouse Mab. Daclizumab was approved
as part of combination prophylaxis regimens for the pre-
vention of renal allograft rejection. These trials utilized
five weekly 1 mg/kg doses of daclizumab. Daclizumab
was well-tolerated at that dose in kidney transplant
patients who were also receiving other immunosuppres-
sive agents (cyclosporine, cyclosporine plus corticoster-
oids, or those two agents plus azathioprine). Because of
its immunosuppressive effects, daclizumab is also being
tested in other transplant settings for the treatment of graft
vs. host disease (GVHD) [572].
Daclizumab and T Cell Malignancy
The expression of CD25 is increased in malignant cells
of adult T-cell leukemia which is induced by the human
T-cell lymphotrophic virus I (HTLV-I) [272]. Since very
few normal cells express IL-2Rα, but abnormal T cells
in patients with lymphoid malignancies do, targeting the
IL-2Rα with daclizumab might be of therapeutic benefit.
368
Waldmann et al. used the original murine anti-Tac
(T-activated cells) Mab (predecessor to the humanized
daclizumab to treat 19 patients with adult T-cell leuke-
mia [765]. Patients received 20, 40, 50, 60, or 100 mg
doses over 8 to 445 days with variable total doses ranging
from 220 mg over 9 days, 490 mg over 51 days, and
220 mg over 445 days. The Mab was given as 3–11 infu-
sions per treatment course. One patient, who had previ-
ously failed aggressive combination chemotherapy had
a complete response that lasted for 5 months, and then a
partial response which lasted 6 months after retreatment.
There were two complete and four partial responses
observed in this trial as well as one mixed response. The
duration of remissions ranged from 2 months to 3 years.
Another patient had a reduction in peripheral leukemia
cells, but had an increase in malignant lymphadenopa-
thy at the same time. Despite the reactivity with circu-
lating T cells, these patients reportedly had no significant
toxicity other than low-grade fever in two patients, and
hives in one patient. However, these patients were
already markedly immunosuppressed, and they may
have been further immunosuppressed by the anti-T-cell
antibody. While on study, one patient developed
Kaposi’s sarcoma, one developed Pneumocystis carinii
pneumonia, and a third developed Staphylococcal-A
septicemia. Further evidence of immunosuppression
was the observation that only one of the nine patients
treated developed HAMA. The CD25 IL-2 receptor is
also sometimes expressed in Hodgkin’s disease; so,
antibodies such as daclizumab might have activity in
selected patients.
Few formal studies of daclizumab in T cell malignan-
cies have been published, therefore most hematologists
prefer to use other products that target CD25, such as
denileukin diftitox (Ontak), the fusion product of IL-2
and diptheria A chain as treatment for CD25 positive
malignancies [627]. CD25 is probably a better target for
immunoconjugates therapy, such as immunotoxins,
because binding leads to internalization of CD25.
Theoretically daclizumab therapy may only eliminate
circulating cells that express large numbers of CD25
molecules, since CDC and ADCC correlate with the
extent of antibody binding. This limitation is supported
by the study by Koon et al. who treated 10 CD25+ leu-
kemia patients with daclizumab in a study focused on
pharmacokinetics of the antibody and pharmacodynamics
of interaction with the CD25 target. [403]. They confirmed
that high numbers of CD25+ circulating tumor cells
were predictive of accelerated pharmacokinetics, and
high levels of CD25 antigen expression correlated with
the extent of target cell clearance. Despite clearance of
Monoclonal antibody therapy
peripheral leukemic cells, and schedules that sustained
daclizumab in the blood, durable responses were not
obtained. There are other anti-CD25 Mabs being inves-
tigated, including the chimeric Mab basiliximab, that
could be investigated in lymphoid malignancy [352].
CD30
CD30 is an excellent target for immunotherapy of
Hodgkin’s lymphoma (HL) because it is overexpressed on
Hodgkin’s and Reed-Sternberg cells and the lymphocytes
of some anaplastic lymphomas, but has limited expression
on normal tissue, which makes it a potential target for
Mab therapy [402]. Like many targets on hematologic
cells, it may not only be a target for CDC and ADCC, but
there is also evidence CD30 may be a regulatory target
involved in signal transduction. There are a number of dif-
ferent anti-CD30 products under investigation including
unmodified Mab, bispecific Mab in which CD30 is one of
the targets, and various immunoconjugates. MDX-060 is
a human IgG1 kappa anti-CD30 Mab that has exhibited
anti-tumor effects in preclinical models. Ansell et al.
treated 72 patients who had CD30+ tumors including 63
with Hodgkin’s lymphoma, seven with anaplastic large-
cell lymphoma, and two with CD30+ T-cell lymphoma
[10]. Treatment was well-tolerated during treatment with
escalating i.v. doses of 1, 5, 10, and 15 mg/kg, with only
7% of patients experienced grade 3 or 4 treatment-related
adverse events. Clinical responses were observed in six
patients (8.3%) and more than one third of the patients had
a long duration of stable disease.
The bifunctional antibody HRS-3/A9 (BiMAb) targets
the Fc gamma receptor CD16 that is expressed on
various effector cells including NK cells, and CD30
antigen on Hodgkin’s cells. Fifteen patients with refrac-
tory Hodgkin’s disease were treated with this antibody
in a phase I/II trial [295]. The maximum tolerated dose
was 16 mg/m2 and the major side effects were fever,
rash and painful lymph nodes. One complete, one
partial, one mixed and three minor responses were
noted. Nine patients developed HAMA. In a subsequent
trial 16 patients with refractory Hodgkins disease were
randomized to either 25 mg by continuous i.v. infusion
daily for 4 days, or 25 mg i.v. over 1 h every other day
for four doses [296]. There was a 25% response rate
including one CR.
Another bifunctional product consists of F(ab’) frag-
ments derived from the murine anti-CD30 Mab Ki-4 and
the humanized CD64-specific Mab H22. Ten patients
were treated with escalating doses of 1, 2.5, 5, 10, and
20 mg/m2 per day i.v. on days 1, 3, 5, and 7 [49]. All five
Robert O. Dillman
doses were well tolerated. Mild infusion reactions were
observed and four patients had a partial response.
CD33
M195 is a murine IgG2a monoclonal antibody that
reacts with the CD-33 antigen that is expressed on early
myeloid precursors. This receptor is rapidly modulated
or down regulated in the presence of M195. Scheinberg
et al. treated 10 AML patients using escalating doses of
M195 (1, 5, and 10 mg/m2) up to a total dose of 76 mg
[633]. Radioisotope tracer studies and bone marrow
biopsies demonstrated binding to bone marrow cells,
but rapid antigenic modulation took place. Sustained
antitumor effects were not seen.
Because of the theoretical limitations of HAMA, a
CDR-grafted human IgG1 version of M195 (HuM195,
lintuzumab) was developed that is able to effect ADCC
with human effector cells in vitro [89]. Thirteen AML
patients received six i.v. doses over 3 weeks along with a
radiolabelled tracer dose. A decrease in marrow blasts
was only noted in one patient. Intermittent dosing with
3 mg/m2/day was found to saturate available binding sites
[88]. In a subsequent trial 10 patients with refractory
AML were given higher doses of HuM195 as daily infu-
sions of 12, 24, and 36 mg/m2 on days 1 to 4 and 15 to 18
[90]. One patient had a complete remission that lasted
more than 2.5 years. Mild infusion reactions including
fever and rigors were noted. In a successor trial, 50 refrac-
tory AML patients were given HuM195 as first salvage
therapy in 24 patients and as second or subsequent sal-
vage therapy in 26 patients [217]. Patients were random-
ized to receive 12 or 36 mg/m2 on days 1 to 4 and 15 to 18.
Three responses were recorded, all at the 12 mg/m2 dose.
Infusion-related fevers and chills were the predominant
toxicities, and were similar for the two doses.
Feldman et al. randomized 191 patients with primary
refractory or initial relapsed AML to combination che-
motherapy consisting of mitoxantrone, etoposide, and
cytarabine (MEC) with or without lintuzumab. There
was no significant difference in response rate, 36% for
MEC plus lintuzumab vs. 28% for MEC alone (p = 0.28)
nor in median survival. Mild antibody infusion-related
toxicities including fever, chills, and hypotension were
common in the lintuzumab arm.
HuM195 was preceded by low-dose s.c. IL-2 in 13
patients with relapsed or refractory, followed AML
[406]. After 5 weeks of s.c. IL-2 patients received i.v.
infusions of HuM195 at a dose of 3 mg/m2 twice a week
for 3 weeks. Infusion reactions that included nausea,
rigors, and fever were frequently observed. Two patients
369
had significant decreases in bone marrow blasts and one
was considered a CR.
CD38
CD38 antigen is present on the majority of neoplastic
plasma cells and some CLL, and other B cell popula-
tions. Stevenson et al. developed a mouse/human chi-
meric antibody to CD38 that consisted of human IgG1
chemically linked to mouse Fab was able to mediate
ADCC in laboratory studies [693] The same group has
also developed a CDR-grafted humanized IgG1 from
the same murine antibody [206], but noted little differ-
ence between the two in various in vitro assays, including
down regulation of the receptor. Clinical results with
these products have been disappointing [694].
CD40
Interaction with CD40 activates antigen-presenting cells
and enhances immune responses, but targeting CD40 that
is expressed on solid tumors induces apoptosis. CP-870,893
is a fully human anti-CD40 Mab that was given to 29
patients in a phase I trial [756]. Weekly doses from 0.01 to
0.3 mg/kg were associated with grade 1 to 2 infusion reac-
tions that included fever, chills and rigors as well as brief
decreases in lymphocytes, monocytes and platelets, but
there was also evidence of B cell activation. Abnormalities
in D-dimer and liver function tests were noted 24 to 48 h
after infusion, and one patient experienced a deep venous
thrombosis. Despite these low doses and the cross reactiv-
ity with circulating cells, four metastatic melanoma patients
were judged to have had a PR (14%).
CD45
The CD45 antigen is expressed on all hematopoietic
cells. Anti-CD45 Mabs cause marrow suppressive
effects that range from leucopenia to marrow aplasia in
some rodent models, so CD45 may be a useful therapeu-
tic target for hematoproliferative malignancies. Krance
et al. gave the rat anti-human CD45 MAbs, YTH25.4
and YTH54.12, which bind to different epitopes on
CD45, to 14 patients with hematologic malignancies
who were scheduled to undergo stem cell transplantion
[407]. Escalating doses were given; the maximum toler-
ated dose was 400 μg/kg/day for 4 days. Treatment was
followed by marked reduction in circulating lymphoid
and myeloid cells, but had little impact on normal mar-
row cells despite reducing the percentage of leukemic
blasts in two of three patients who a leukemic marrow.
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CD74
Milatuzumab (hLL1 or IMMU-115) is a humanized
anti-CD74 monoclonal antibody [689]. CD74 is a surface
membrane protein that functions as a survival receptor
in that it enhances cell proliferation and survival [685].
It also functions as an MHC class II chaperone. It is
expressed on lymphoid cells, especially malignant B
cells, and carcinomas of the kidney, lung, and stomach,
and by certain sarcomas, but has limited expression on
other normal tissues. In vitro milatuzumab does not
induce ADCC or CDC, probably because of the rapid
internalization of this receptor molecule. However,
despite the rapid internalization of milatuzumab after
binding to CD74, preclinical models have suggested
anti-tumor activity with unconjugated antibody, in addi-
tion to immunoconjugates [688]. Unlike rituximab,
CD74 is also highly expressed on the malignant plasma
cells of multiple myeloma [73]. CD74 is also expressed
on T cells and NK cells, and therapy is associated with
depletion of lymphocytes in primate models. The product
is being explored in B cell lymphoma, multiple myeloma,
and CLL.
CD80
Galiximab reacts with CD80, which is expressed on a
variety of lymphoid and myeloid cells. Czuczman et al.
treated 37 patients suffering from relapsed or refractory B
cell lymphoma with four weekly i.v. infusions of galix-
imab at doses of 125, 250, 375, or 500 mg/m2 [142]. Four
of 35 evaluable patients had an objective response (11%).
Treatment was well-tolerated with mild nausea, head-
ache, and fatigue the most common toxicities noted.
CD122
CD122 is the beta-subunit shared by the IL-2 and IL-15
receptors [763]. Both IL-2 and IL-15 activate T cells,
but IL-2 also induces apoptosis of self-reactive T cells
while IL-15 stimulates their persistence. Mikbeta1 and
its humanized version (HuMikbeta1) bind to CD122.
In some cell models HuMikbeta1 is more effective in
blocking IL-2 stimulation then IL-15 stimulation. The
murine Mikbeta 1 was given to 12 cytopenic patients
with T cell large granular lymphocyte leukemia (T-LGL)
in a phase I dose-escalation trial in which 0.1, 0.5 or
1.5 mg/kg i.v. doses were infused on days 1, 4, 7, and 10
[505]. At these doses there were no significant toxicities
and HAMA was not detected, but such patients are
already cytopenic and immunosuppressed. The investi-
gators were able to demonstrate saturation and down-
Monoclonal antibody therapy
modulation of the CD122 IL-2/IL-15 beta receptor on
the surfaces of the TLGL cells, but there was no sustained
decrease in leukemia cell numbers. Clinical trials with
the huMikbeta1 are planned. Higher doses of the human-
ized Mab may be associated with more clinical activity,
but perhaps also toxicity.
HLA DR
LYM-1 is an IgG2a murine Mab that reacts with a polymor-
phic HLA-Dr antigen on all B cells, but the target antigen is
not shed nor does it modulate in the presence of antibody.
Ten patients with refractory B cell lymphomas received
weekly i.v. infusions of escalating doses of LYM-1 over 4
weeks without any objective tumor responses [330].
Apolizumab (hu1D10) is a humanized Mab against a
polymorphic epitope on HLA DRbeta. Rech et al. treated
six patients who had relapsed or refractory 1D10-positive
non-Hodgkin’s lymphoma with granulocyte colony
stimulating factor (GCSF) and apolizumab at doses rang-
ing from 0.15 to 1.5 mg/m2 [582]. There were no clear
anti-tumor effects. One patient had skin rash, one throm-
bocytopenia, and one auto-immune hemolytic anemia.
HM1.24
HM1.24 has been touted as a human plasma cell specific
antigen that is over expressed on myeloma cells.
Anti-HM1.24 inhibits proliferation of plasma cells that
overexpress HM1.24. A humanized IgG1/kappa anti-
HM1.24 (AHM) has been developed that exhibits strong
ADCC activity in vitro with human effector cells against
myeloma KPMM2 and ARH77 human myeloma cells
in the presence of human PBMCs myeloma cells [540].
It also shows promising activity in animal xenograft
models that is diminished by pretreating animals with
an anti-Fc gamma receptor II/III antibody [369].
Anti CTLA4 (Cytotoxic T Lymphocyte
Antigen 4)
CTL-associated antigen 4 (CTLA-4) can inhibit T-cell acti-
vation and helps maintain peripheral self-tolerance of anti-
gens expressed on both normal and tumor cells. CTLA-4
is expressed on the surface of activated T-lymphocytes
where it suppresses the induction of immune responses
that normally follow the interaction between T-cell recep-
tors and HLA molecules on the antigen-presenting cells.
Mouse experiments showed that blockade of CTLA4
could yield anti-tumor effects. The two anti-CTLA 4 Mabs,
ipilimumab (MDX-010) and tremelimumab (CP-675, 206;
Robert O. Dillman
formerly known as ticilimumab), have been extensively
tested with promising activity [414]. However, both have
been associated with significant immune-related adverse
events (IRAE) including dermatitis, inflammatory bowel
disease, uveitis, arthritis, hypophysitis, and others.
Generally, these IRAE have reversed after cessation of
therapy and use of i.v. or topical corticosteroids. However,
colectomy has been required in several severe cases of
inflammatory bowel disease, and several IRAE-associated
deaths have been reported [31]. Despite this toxicity pro-
file, it is likely that one or both of these agents may receive
regulatory approval.
Ipilimumab
Striking anti-tumor activity has been seen in melanoma,
but mostly in conjunction with substantial autoimmune
toxicity [133, 531]. Downey et al. reported a 17%
response rate among 139 metastatic melanoma patients
who were treated with ipilimumab, mostly in conjunction
with peptide vaccines [190]. The details of the dose
escalations used in these trials had been previously
reported [455, 558]. IRAE, including enterocolitis,
arthritis and uveitis, were documented in 62% of
patients and were highly correlated with a measurable
anti-tumor effect. The response rate was 36% among
24 patients who had colitis compared to 11% among
113 patients who did not (p = 0.006) [31]. The three
patients who experienced a CR had grade 4 toxicities.
Anti-tumor activity has been demonstrated in renal cell
cancer. Yang et al. treated 61 metastatic renal cell patients
with either 3 mg/kg followed by 1 mg/kg every 3 weeks (n
= 21) or 3 mg/kg every 3 weeks [800]. Partial responses
were noted for 6/61 patients (10%), five at the higher dose.
Grade 3 to 4 toxicity was documented in 33% of patients,
most of which appeared to be auto-immune mediated,
especially enteritis and endocrine deficiencies. The
response rate was 35% among 17 patients who experi-
enced colitis and only 2% among 44 patients who did not
(p = 0.002) [31].
A small number of prostate cancer patients have been
treated with ipilimumab [676]. Fourteen patients with
metastatic hormone-refractory prostate cancer received
ipilimumab at a dose of 3 mg/kg i.v. dose. Two patients
had a 50% decline in PSA level. Treatment was gener-
ally well tolerated and only one patient developed grade
3 dermatititis that required systemic corticosteroids.
Tremelimumab (CP-675,206, Ticilimumab)
Striking anti-tumor activity has been seen in melanoma,
but mostly in conjunction with substantial autoimmune
toxicity [85, 709]. Ribas et al. conducted a phase I dose
escalation trial of seven different dose levels of tremeli-
371
mumab in 39 patients, including 34 with melanoma and
four with renal cell cancer [591]. Among 29 melanoma
patients who had measurable disease, there were two
durable CR and two durable PR lasting more than 2
years. Dose-limiting toxicities were IRAE including
autoimmune phenomena such as diarrhea, dermatitis,
vitiligo, panhypopituitarism and hyperthyroidism. The
maximum tolerated dose for a single i.v. infusion was
15 mg/kg. Among 30 melanoma patients who received
tremelimumab at a dose of 10 mg/kg monthly (n = 20) or
15 mg/kg every 3 months (n = 10) 4/12 patients with
IRAE had an objective tumor responses compared to
only 1/18 without IRAE (p = 0.046) [590]. The IRAE
and responses were associated with increased production
of IL-2 and lack of these was associated with increased
production of IL-10. IRAE attributed dermatitis, colitis,
hepatitis, and hypophysitis with panhypopituitarism. In a
preliminary report on 20 patients, there was uveitis or
vision changes noted during extensive opthalamalogic
evaluations during and after treatment [696].
Tumor Necrosis Factor-Related Apoptosis-
Inducing Ligand (Trail) Receptor
Tumor necrosis factor (TNR)-related apoptosis-inducing
ligand (TRAIL) is a TNF family member capable of induc-
ing apoptosis by binding to its two death receptors (DR)
DR4 (TRAIL-R1) and DR5 (TRAIL-R2) [64]. There are at
least two Mab being investigated that target the tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)
receptors. Both are fully human Mabs. Mapatumumab
(HGS-ETR1, TRM-1) targets DR4 (TRAIL-R1) [760].
Lexatumumab (HGS-ETR2), targets DR2 (TRAIL-R2)
[467]. TRAIL receptors are upregulated by chemotherapy.
DR4 and DR5 may be useful targets for both hematologic
malignancies and solid tumors.
Mapatumumab (HGS-ETR1, TRM-1)
Tolcher et al. treated 49 solid tumor patients with 158
courses of mapatumumab as i.v. doses ranging from
0.01 to 10 mg/kg infused over 30 to 120 min [724].
Initially mapatumumab was given as a single dose, then
every 28 days, and then 10 mg/kg every 14 days.
TRAIL-R1 was expressed in 68% of patients whose
tumors were assayed. No objective tumor responses
were seen. There was minimal toxicity with mild infu-
sion reactions being the most common adverse event
reported, including fatigue, fever, and myalgia.
Lexatmumab (HGS-ETR2)
Plummer et al. treated 37 solid tumor patients with 120
cycles of lexatumumab at doses ranging from 0.1 to
372
20 mg/kg every 21 days [564]. The maximum tolerated
dose was 10 mg/kg based on dose-limiting toxicities
that included asymptomatic elevations of serum amy-
lase, transaminases, and bilirubin in three of seven
patients treated with 20 mg/kg. There were no tumor
responses.
Nuclear Factor-Kappa B Ligand
Nuclear factor-kappa B (NF-ΚB) is a family of ubiqui-
tous transcription factors that are believed to control
inflammation. NF-KB normally resides in the cytoplasm
of cells, but in response to a variety of stimuli, including
inflammatory cytokines, growth factors, carcinogens
and ionizing radiation, NF-ΚB translocates to the
nucleus where it upregulates the expression of over 400
different gene products associated with cell survival,
proliferation, invasion, and angiogenesis [5, 194].
Activation of NF-ΚB has been linked to oncogenesis
and the biologic behavior of cancer cells, including
resistance to chemotherapy and radiation therapy via
transcription of anti-apoptotic proteins, leading to
increased proliferation of cells and tumour growth.
Activation of the NF-ΚB signal pathway follows the
interaction of NF-ΚB ligand and receptor (RANKL).
Denosumab is a fully human monoclonal antibody to
the receptor activator of nuclear factor-kappa B (NF-ΚB)
ligand [430]. Lipton et al. gave denosumab to 255 women
who had breast cancer-related bone metastases not previ-
ously treated with i.v. bisphosphonates [439]. Denosumab
was administered s.c. every 4 weeks (30, 120, or 180 mg)
or every 12 weeks (60 or 180 mg). Treatment was well
tolerated and there was evidence of suppression of bone
resorption. Anti-cancer activity was not noted.
Body et al. conducted a small randomized, double-
blind trial of single doses of s.c. denosumab (0.1, 0.3,
1.0, or 3.0 mg/kg) or i.v. pamidronate (90 mg), in 29
patients with breast cancer and 25 with multiple
myeloma [45]. Patients received a single dose of either
agent and a placebo of the other. Treatment was well-
tolerated. The magnitude of decrease in bone resorption
markers was similar to pamidronate, but the effects
lasted longer with denosumab.
Integrins
Integrins are cell adhesion receptors that mediate inter-
cellular communication. Integrins are implicated in
tumor cell survival, angiogenesis, cancer invasiveness,
and metastasis, and therefore are a potential target of
cancer therapy [520]. The integrin alphaV-beta3 is an
adhesion molecule expressed by proliferating endothelial
Monoclonal antibody therapy
cells and antibodies blocking this integrin inhibit angio-
genesis in preclinical models.
Mullamitha et al. treated 24 patients with CNTO 905,
a fully human Mab to anti-alphaV integrins that inhibits
angiogenesis and tumor growth in preclinical models
[512]. CNTO 95 (0.1, 0.3, 1.0, 3.0, and 10.0 mg/kg) was
infused on days 0, 28, 35, and 42. CNTO 95 was gener-
ally well tolerated with only infusion-related fever being
the most common adverse event. At the 10.0.mg dose, a
patient with cutaneous angiosarcoma had a 9-month
partial response and a patient with an ovarian carcino-
sarcoma had a lesion that became negative by positron
emission tomography, although a stable lesion persisted
by computerized axial tomogragphy.
McNeel et al. treated 25 solid tumor patients with
MEDI-522, a second generation humanized anti-alphaV-
beta3 antibody designed for antiangiogenic therapy, in a
phase I study in which patients received doses ranging
from 2 to 10 mg/kg/wk [477]. Treatment was well toler-
ated and no dose-limiting toxicities were seen in this dose
range. The major adverse events observed were grade 1
and 2 infusion-related reactions (fever, rigors, flushing,
and tachycardia), and low-grade constitutional and gas-
trointestinal symptoms (fatigue, myalgias, and nausea),
and asymptomatic hypophosphatemia. There were no
objective responses observed, but three patients with
metastatic renal cell cancer had prolonged stable disease
Serial biopsies of skin showed detected MEDI-522 both
in quiescent and in angiogenically active skin blood
vessels as well as in the dermal interstitial space [805].
Posey et al. treated metastatic cancer patients with
vitaxin, a humanized monoclonal antibody, which has
specificity for the integrin alphaV-beta 3 (vitronectin
receptor) which inhibits tumor cell mediated angiogen-
esis in pre-clinical animal models [565]. Vitaxin was
given i.v. at doses of 10, 50 or 200 mg in cohorts of three
patients. There was no significant toxicity noted in these
three dose levels. There were no objective anti-tumor
responses. An every 3 week schedule of 200 mg appeared
to sustain serum levels.
Human Epidermal Growth Factor Receptor
Antigens (EGFR1)
Cetuximab and panitumumab, the two FDA-approved
products that target EGFR1, were discussed earlier in
this chapter. A number of other Mabs have been devel-
oped that target this receptor. Mab ch806 is a chimeric
antibody that detects a unique epitope on EGFR that is
exposed only on mutanted or ligand-activated forms of
the receptor on cancer cells in which it is overexpressed
[648, 649]. In preclinical studies murine 806 blocked
Robert O. Dillman
EGFR-mediated signal transduction resulting in anti-
tumor effects on human tumor xenografts, while in vivo
studies in human showed tumor specific targeting of
ch806. ABX-EGF is a fully human anti-EGFR Mab that
was given to 88 patients with metastatic renal cell can-
cer at doses of 1.0, 1.5, 2.0, or 2.5 mg/kg weekly [612].
Three patients had an objective response and the majority
of patients had stable disease for at least 2 months. A dose-
related acneiform rash was the most common toxicity,
and occurred 100% of patients who received at least
three doses of ABX-EGF at 2.5 mg/kg/wk.
The humanized anti-EGFR Mab h-R3 was given to
29 patients with newly diagnosed malignant gliomas
including 16 glioblastoma, 12 anaplastic astrocytoma and
one anaplastic oligodendroglioma (AO) [580]. Following
debulking surgery, patients received six 200 mg infu-
sions of h-R3 weekly in during radiotherapy. Interestingly,
no patients developed acneiform rash, and there were no
allergic reactions. Median survival was 17months for
the glioblastoma patients. Mab h-R3 was given at four
dose levels weekly for 6 weeks in combination with
radiotherapy, to 24 patients with advanced head and
neck cancer [135]. In this trial there wee some infusion
reactions, but no dermatologic or allergic toxicities
were noted. The overall survival of patients was consid-
ered encouraging.
HuMax-EGFr is a fully human Mab that was given in
escalating doses of up to 8 mg/kg to 28 patients with
squamous cell cancer of the head and neck [28]. Most
frequently reported adverse event was rash. There were
seven responses noted among 11 patients who were treated
at the higher doses.
Matuzumab is a humanized anti-EGFR Mab formerly
known as EMD 72000 that has been explored in patients
with a variety of cancers. Nine patients with squamous
cell cancer of the head and neck were treated in a phase
I trial in which two doses were given before surgery and
three doses after surgery [41]. Successive patient groups
received a single dose of 100, 200 or 400 followed by
four weekly doses of 50, 100, and 200 mg, respectively.
Treatment was well-tolerated. The most frequent adverse
events attributed to the antibody were fever and a tran-
sient elevation of liver enzymes, but there was no significant
skin toxicity. In another dose-escalation trial 22 patients
with various solid tumors received matuzumab at five
different dose levels randing from 400 to 2,000 mg/wk
[744]. The maximum-tolerated dose was 1,600 mg/wk
because of severe headaches at the 2,000 mg dose.
At these dose levels mild acneiform rash was the most
common toxicity. Matuzumab was given to 37 women
with intra-abdominal platinum-resistant ovarian cancer at
doses of 1,600 mg/wk [656]. Therapy was well tolerated,
373
but at this dose dermatologic toxicities were common,
including acne, papillary rash, dry skin, and paronychia,
but also headache, fatigue, and diarrhea, and case of pan-
creatitis that was possibly treatment related. There were
no objective tumor responses. Matuzumab was given at
weekly doses of matuzumab (100, 200, 400 or 800 mg)
concurrently with paclitaxel at 175 mg/m2 every 3 weeks
in 18 patients with NSCLC [401]. No additive toxicity was
noted even at the 800 mg dose. Low grade acneiform
skin rash occurred in 14 patients. There were five objec-
tive tumor responses. Escalating doses of matuzumab
(400 mg weekly, 800 mg biweekly, or 800 mg weekly)
were given with gemcitabine to three groups of patients
with advanced pancreatic adenocarcinoma [270]. Skin
toxicity was the most frequent side effect. There were
three partial responses in patients who received 800 mg
of Mab weekly.
A bispecific antibody called MDX-447 was constructed
by cross-linking antigen binding F(ab’) fragments) of a
humanized Fab anti-EGFR to a humanized Fab which
binds to CD64, the high-affinity immunoglobulin G
receptor known as Fc gamma receptor I. Escalating
weekly doses from 1 to 15 mg/m2 were administered to
36 patients with various refractory EGFR-positive can-
cers including cancers of the kidney, bladder, prostate,
head and neck, and skin bladder, ovarian, prostate cancer
and skin cancer, with and without G-CSF [138]. MDX-
447 infusions were associated with decreases in mono-
cytes and increases in plasma cytokines in association
with fever, chills, hypotension, and myalgias. Fury et al.
reported on 41 patients treated with MDX-447 at doses
from 1 to 40 mg/m2 and 23 patients who received 1 to
15 mg/m2 of MDX-447 in combination with GCSF [242].
Hypotension was the major dose-limiting toxicity in both
cohorts, and accrual to MDX-447 plus GCSF was stopped
because of excessive toxicity in association with higher
levels of TNF-α and IL-6. The maximum tolerated dose
for MDX-447 alone was 30 mg/m2 i.v. weekly. There were
no objective responses.
Human Epidermal Growth Factor Receptor
Antigens (EGFR2)
Trastuzumab, the only FDA-approved product that tar-
gets EGFR2, was discussed earlier in this chapter. Other
antibodies have been developed that also target HER3.
Pertuzumab (rhuMAb 2C4) is a humanized construct
of the murine 2C4 Mab that binds to the HER-2 receptor
and blocks its ability to dimerize with other HER recep-
tors. In a phase I trial pertuzumab was given at doses of
(0.5 to 15 mg/kg) i.v. every 3 weeks to 21 patients with
various advanced solid malignancies [3]. Treatment was
374
well-tolerated. Two objective responses were recorded.
Patients with relapsed ovarian cancer were treated with
pertuzumab, including 61who received a loading dose
of 840 mg followed by 420 mg every 3 weeks, and 62
patients who received 1,050 mg every 3 weeks [266].
Pertuzumab was well tolerated, but diarrhea occurred in
69% (11% grade 3, no grade 4). Five patients had
asymptomatic left ventricular ejection fraction decreases
to less than 50%. There were five PRs. Herbst et al. gave
pertuzumab once every 3 weeks to 43 previously treated
NSCLC patients [311]. Treatment was well-tolerated,
but there were no objective responses. Agus et al. gave
pertuzumab as a loading dose of 840 mg followed by
420 mg for subsequent cycles, to 41 patients with hor-
mone refractory prostate cancer who had also experi-
enced disease progression after taxane chemotherapy
[4]. Pertuzumab was well tolerated; grade 1 to 3 diar-
rhea was the most common toxicity. There were no
tumor responses. A trial using the same doses of pertu-
zumab was conducted in 35 patients with hormone
refractory prostate cancer who had not been treated with
chemotherapy, and another 33 were treated with a fixed
dose of 1,050 mg [153]. Treatment was well tolerated,
but there were no tumor responses. Attard et al. com-
bined pertuzumab (1,050 mg fixed dose and later the
840 mg loading dose, then 420 mg) with docetaxel che-
motherapy with both agents given together every 3
weeks [13]. Based on toxicity they recommended further
evaluation with docetaxel 75 mg m2 and 420 mg pertu-
zumab following a loading dose of 840 mg.
Ertumaxomab (2B1) is a bispecific murine monoclonal
antibody that binds to the extracellular domains of HER2/
neu and FcgammaRIII. Twenty women with metastatic
breast cancer, all but one of whom had received prior
chemotherapy were treated in a phase I trial [50]. The
initial dose of 2.5 mg/m/day was associated with dose-
limiting toxicities in three of the first eight; so the remaining
12 patients received 1 mg/m/day. Objective antitumor
responses were not seen. A trifunctional version of this
antibody which also binds to CD3 on T lymphocytes was
tested at doses of 10–200 μg weekly for up to three doses
in 17 patients with various malignancies [384]. The maxi-
mum tolerated dose was 100 μg because of a systemic
inflammatory response syndrome and acute renal failure
in a patient treated with 150 μg and severe hypotension
and respiratory distress syndrome in one patient and
exacerbation of heart failure in two patients who received
200 μg. The most common drug-related adverse events
were mild, transient infusion reactions including fever,
rigors, headache, nausea, and vomiting, but grade 3 to 4
lymphocytopenia occurred in 76% of patients and ele-
vated hepatic enzymes in 47%. Elevated levels of IL-2,
Monoclonal antibody therapy
IL-6, TNF-α, and IFN-γ were seen in association with the
infusion reactions. Objective tumor responses were seen
in 5 patients.
The bispecific antibody MDX-H210 was constructed
by cross-linking antigen binding F(ab’) fragments of
Mab H22 to CD64, the high-affinity immunoglobulin G
receptor FcgammaRI) and mAb 520C9 to HER2 [770].
In a dose escalation trial of single i.v. infusions in
patients with breast and ovarian cancer, one partial
response was observed in a patient with breast cancer
[740]. Elevated plasma levels of TNF-α, IL-6, G-CSF
and neopterin were detected at higher doses of the anti-
body. Seven patients with HER2-positive prostate can-
cer received 1 to 8 mg/m2 of MDX-H210 as 2-h infusions
three times a week [642]. Over this dose range MDX-
H210 was well tolerated other than mild infusion-related
fever, chills, myalgias and malaise, in association with
increased plasma levels of TNF-α and IL-6. No dose-
limiting toxicicty was noted. In another study, 25
patients with HER2+ advanced prostate cancer were
treated with s.c. GM-CSF at a dose of 5 μg/m2 for 4 days
of each week along with MDX-H210 at 15 μg/m2 weekly
for 6 weeks [346]. Toxicities leading to withdrawal from
the trial included grade 3 heart failure, dyspnea and
allergic reactions, but 35% of patients had at least a 50%
decrease in serum PSA levels.
Insulin-Like Growth Factor Receptor
The anti insulin-like growth factor-I receptor Mab
CP-751,871 was given in doses up to 20 mg every 3
weeks to 24 patients with various malignancies [290].
A maximum tolerated dose was not identified over this
dose range. Mild adverse events included hyperglycemia,
hyperuricemia, elevated hepatic enzymes, anorexia,
nausea, diarrhea, and fatigue. There were no objective
responses.
Transferrin Receptor
The murine Mab 42/6 reacts with the human transferrin
receptor thereby blocking the iron transport protein
transferrin, which is crucial for iron transport by cells
[732]. In vitro studies showed that 42/6 had inhibitory
effects on a various types of cancer cells [704], [705]. In
a phase I trial 33 treatments at doses of 2.5 to 300 mg/m2
were given as 24-h infusions to 27 patients [57]. 42/6
was well tolerated, although one patient experienced an
allergic-type response associated with a HAMA
response during a second course of treatment. Limited
mixed tumor responses were observed in three patients
with hematological malignancies, but there were no
Robert O. Dillman
partial or complete remissions. HAMA was detected
33% of patients.
Vascular Ligands and Receptors
Bevacizumab, which reacts with the VEGF ligand, is the
only FDA-approved product in this group. Bevacizumab
was reviewed in detail earlier in this chapter. Several
other Mab products that target VEGF or VEGFR are
under development.
IMC-1C11 is a chimeric Mab that has anti-angiogenesis
properties based on its binding to the VEGF receptor 2
rather than the VEGF ligand. In a dose escalation study,
14 patients with colorectal carcinoma and hepatic metas-
tases received weekly doses at 0.2, 0.6, 2.0, or 4.0 mg/
kg for 4 weeks [566]. There were no serious toxicities,
although minor bleeding occurred in four patients
treated at the two lowest dose levels. HACA was
detected in half the patients and altered pharmacokinetics
of IMC-1C11 in two patients. There were no objective
tumor responses.
HuMV833 is a humanized IgG4 anti-VEGF Mab that
was given to 20 patients who had advanced solid tumors
by i.v. infusion on days 1, 15, 22, and 29 [348]. Treatment
was well-tolerated over the dose range of 0.1 to 10 mg/
kg per dose. One patient with ovarian cancer experi-
enced a partial response. Dose of 1 and 3 mg/kg were
selected for further investigation.
J591 targets the external domain of prostate-specific
membrane antigen (PSMA), but is also expressed on the
neovasculature of various solid tumors. J591 was given
every 3 weeks in doses of 5, 10, 20, 40, 60, or 100 mg,
along with a 2 mg tracer dose of Mab conjugated to
10 mCi of indium-111 to 20 patients with a variety of
malignancies [507]. In a second trial 27 patients received
doses of 5, 10, 20, 40, or 80 mg and some patients
received weekly therapy for up to 6 weeks [490]. Tumor
localization was confirmed in both studies and there
were no dose limiting adverse events. No tumor responses
were observed in either study.
Carcinoembryonic Antigen (CEA)
Numerous clinical studies with antibodies directed
against carcinoembryonic antigen (CEA) and other anti-
gens of gastrointestinal mucosa have been reported.
Several different murine IgG1 anti-CEA antibodies at
doses from 1 to 80 mg were infused into 30 colorectal
cancer patients utilizing single 1- to 2-h infusions with
tracer doses of 111-Indium labeled antibody [183]. With
one such Mab three patients had dramatic infusion reac-
tions with fever and rigors which turned out to be due to
375
cross-reactivity with granulocytes, and associated with
transient decreases in total leukocyte counts [178].
There were no tumor responses. Other trials utilizing
small dose of murine anti-CEA Mab that did not cross-
react with granulocytes were associated with a mini-
mum of side effects, and were taken up into tumors, but
not associated with tumor responses [451], [30], [288].
In December 1992 the 111-indium conjugate of a
murine Mab B-72.3 (OncoScint™) became the first Mab
approved specifically for use in cancer patients, albeit as
an imaging agent for patients with ovarian and colon
cancer rather than a therapeutic agent. approved for clin-
ical use for radioimmunodetection of cancer in patients
with ovarian cancer and colorectal cancer in Numerous
patients received doses of 10 to 40 mg of antibody along
with 1 mg doses of radiolabelled antibody [454]. Tumor
regressions were not noted in the trials in patients with
known colorectal cancer. However, humanized forms of
this antibody appeared promising based on in vitro assays
of ADCC. Khazaeli et al. treated 12 patients with 3 to
7 mg of B-72.3 chimeric IgG4 antibody [381]. No toxicities
or tumor effects were seen at these low doses.
Haisma et al. treated 20 colorectal cancer patients
with the human IgM Mab 16.88, labeled with 131iodine
for tumor localization [282]. Patients received an 8 mg
dose followed 1 week later by 200, 500, or 1,000 mg of
antibody. Tumor uptake was seen in at least one tumor
site in 80% of the patients, but no tumor regressions
were reported.
Epithelial Cell Adhesion Molecule (EpCAM)
One of the targets for many murine Mab produced in
different laboratories by immunizations with epithelial
cancer cells was a surface glycoprotein called 17-1A,
GA733-2, or KSA. This molecule was expressed on
most malignant epithelial cells and some normal human
cells. It was subsequently recognized that this antigen
is what is now called epithelial cell adhesion molecule
(EpCAM) [820]. Loss of membranous EpCAM is associ-
ated with nuclear beta-catenin localization, reduced
cell-cell adhesion, and increased migratory potential of
cells, which suggests that blocking this molecule could
be associated with anti-cancer effects.
10.6.2.27.1
Edrecolomab (17-1A, Panorex®), was the most heavily
studied anti-EpCAM Mab [268]. This was actually the
first therapeutic Mab approved for the treatment of
cancer internationally, but it never received approval in
the U.S. Edrecolomab is a murine IgG2a that reacts with
a 37–40 kD glycoprotein found on various adenocarci-
376
nomas and on normal epithelial tissues [315]. The
EpCAM antigen is not shed into the circulation nor is it
internalized. Edrecolomab may produce anti-cancer
effects by various mechanisms [404], [316]. It was one
of the few murine Mabs that could effect ADCC with
human mononuclear cells [312]. Edrecolomab induced
a high frequency of HAMA in humans [651], [653].
Some of these HAMA (Ab2) react with the idiotype of
the 17-1A mouse antibody (Ab1). As part of the idiotype
network control of B-cell proliferation, the Ab2 induces
additional antibodies against itself, including antibodies
(Ab3) that react with the idiotype of the Ab2. Such an
Ab3 is actually an endogenous human antibody with the
same reactivity against the tumor antigen as Ab1. This
phenomenon has been observed in patients who have
received edrecolomab [316], [215]. Single injections of
edrecolomab at doses of 15–1,000 mg were well toler-
ated, but 50% of patients developed HAMA after a
single injection [651], [653]. Uptake of 17-1A into
tumors was demonstrated by radiolabeled antibodies
and by immunohistochemistry [666, 496]. There were
no objective responses among 40 patients who received
single infusions of 15–1,000 mg of 17-1A during phase
I trials in patients with various gastrointestinal adeno-
carcinomas [651], [653]. In a phase II trial of 20 patients
who received 200–850 mg of 17-1A, one response was
claimed in a patient with recurrent rectal cancer [240].
In a trial of 25 patients who received one to four doses
of 400 mg of 17-1A, one patient was interpreted as hav-
ing a complete response [443]. Eleven patients experi-
enced mild gastrointestinal toxicity during treatment.
Subsequently, this group treated eight patients with a
17-1A chimeric antibody and confirmed the longer
serum half-life and reduced rate of HAMA, but there
were no responders even though the IgG4 human subclass
heavy chain enhanced ADCC [444].
Additional trials with 17-1A were conducted in
patients with colorectal carcinoma by Mellstedt and col-
leagues who reported one objective response among 52
patients who were treated in various ways [240], [821].
There were no responses among 10 patients who
received a single 200 to 400 mg dose of 17-1A every
4–6 weeks. There was one minor response among 10
patients who received 200 to 400 mg of 17-1A preceded
by 400 mg/m2 of the chemotherapy agent cyclophosph-
amide, which was given in an effort decrease the
frequency of HAMA. There was one CR and two minor
responses among 14 patients who received the same
dose/schedule of Mab preincubated with autologous
peripheral blood mononuclear cells. There were no
responses in five patients who received a total dose of
3.6 g of 17-1A given as 400 mg daily on days 1, 3 and 6,
Monoclonal antibody therapy
at 3-week intervals for 2 treatment courses. There were
no responses among seven patients who received 200 to
400 mg every other day up to a total dose of 4.8 to 7.6 g.
There were no responses among six patients who
received 500 mg 3 days a week, to a total dose of 12 g.
Enthusiasm for edrecolomab as a potentially useful
anti-cancer agent peaked after publication of the results
of a multicenter randomized German trial of adjuvant
therapy that was conducted during 1990–1992 in
patients with resected Dukes C colorectal cancer [822].
There were 90 patients randomized to observation,
and 99 to a post-operative regimen of edrecolomab
500 mg i.v., then 100 mg i.v. once a month for 4 months.
Mild side effects were observed in association with the
infusions, including fever, chills, abdominal pain,
nausea, and diarrhea, but there were four episodes of
anaphylaxis during 371 infusions (1.1%). After a
median follow up of 5 years, there was a statistically
significant difference in disease-free and overall sur-
vival curves with a 30% reduction in death rate and
27% reduction in tumor recurrence in the 17-1A group.
The magnitude of improvements in key endpoints were
similar to those observed in U.S. trials of 5-fluououra-
cil and levamisole that had led to acceptance of 5-FU
based chemotherapy in the adjuvant treatment of colon
cancer. After a median follow up of 7 years, the appar-
ent benefits of edrecolomab persisted with a 23%
reduction in recurrence and 32% reduction of death in
the antibody group [595]. On the basis of this trial,
Edrecolomab was approved for the treatment of Dukes
C colorectal cancer in Germany in 1995.
These promising results were followed by four addi-
tional phase III trials in the United States and Europe.
Trials in North America compared 5-FU + levamisole, or
5-FU + leucovorin to the same agents plus edrecolomab in
Dukes C colon cancer using the same dose and schedule
used in the German trial. The European trials involve
comparisons among 17-1A antibody alone, 5-FU + leuco-
vorin, and 5-FU + leucovorin + 17-1A. Edrecolomab was
also studied in combination with chemotherapy and radio-
therapy in Dukes B and C rectal cancer. Results of the
large randomized European adjuvant trial did not demon-
strate a benefit for the use of edrecolomab as adjuvant
therapy [574]. There were 2,761 patients randomized in
the three-arm trial. The major toxicities observed in the
edrecolomab-alone arm were diarrhea in 32%, although
severe or life-threatening diarrhea was noted in only 2%.
Various types of hypersensitivity reactions to edrecolomab
were documented in 25% of patients. Results in the
edrecolomab-alone arm were inferior to the 5FU-containing
arms (p < 0.05), and there was no evidence of better results
when the antibody was combined with 5FU (p = 0.53).
Robert O. Dillman
Three-year survival rates were 76% for 5FU/leuovorin,
75% for the antibody/chemotherapy combination, and
70% for the antibody alone. The differences in 3-year
event free survival also revealed inferior results for anti-
body alone, and no advantage to the chemotherapy plus
antibody combination.
In another trial 377 patients with stage 2 colon cancer,
stratified according by whether the primary tumor was
T3 or T4, were randomized post-operatively to treat-
ment with 900 mg edrecolomab or observation [297].
The study was stopped because of discontinuation of
drug supply in Germany after negative results from
other trials became public. After a median follow-up of
3.5 years there was no difference in overall survival or
disease-free survival.
Several trials have explored edrecolomab in combina-
tion with other biologicals with or without chemotherapy.
Edrecolomab was combined with IFN-γ in an effort to
increase Fc receptors on effector cells and increase the
expression of the antigen. Weiner et al. gave 150 mg of
17-1A on days 2 to 4 in combination with 1.0 MIU/m2 of
IFN-γ on days 1 to 4, to 19 colorectal cancer patients, but
no antitumor responses were noted [774]. In a second trial
27 patients with colon or pancreatic cancer were given
IFN-γ at doses up to 40 MIU/day for 4 days followed by
400 mg 17-1A on day 5 [773]. No objective tumor
responses were not seen and the authors concluded that
the low dose of IFN-γ was as effective as higher doses..
Saleh et al. treated 15 colorectal cancer patients with
0.1 mg/m2 IFN-γ on days 1 to 15 and 400 mg of 17-1A at a
dose of 400 mg on days 5, 7, 9 and 22 [622]. No significant
objective tumor responses were described.
Weiner et al. conducted a phase II multicenter trial of
edrecolomab in patients with unresectable pancreatic
carcinoma [776]. A dose of 500 mg was given i.v. thrice
weekly for 8 weeks. Because of rapid progression of
disease in several patients, only 16/28 patients received
the planned total dose of 12 grams. There was one dura-
ble PR. Tempero et al. treated 30 patients who had
advanced, measurable pancreatic cancer with IFN-γ at a
dose of 1 MIU/m2 daily for 4 days and 150 mg of 17-1A
admixed with autologous leukocytes on days 2, 3 and 4
[713]. One patient had a CR that persisted for 4 months.
The median survival for this group of patients was only
5 months. There was no evidence of increased HLA-DR
expression on monocytes or lymphocytes following the
administration of IFN-γ.
GM-CSF and IL-2 have also been combined with
edrecolomab in an effort to enhance the immune
response. In a trial of 20 patients who received 17-1A
plus GM-CSF to increase effector cells and the expres-
sion of Fc receptors, there were two CRs [575]. In a
377
subsequent trial 20 patients with advanced colorectal
cancer received edrecolomab 400 mg at day 3 of a
10-day treatment cycle with the simultaneous adminis-
tration of s.c 250 μg/m2 GM-CSF once daily and 2.4
million U/m2 IL-2 twice daily for 10 days in a planned
monthly treatment cycle [321]. Doses of the various
drugs had to be decreased because of infusion/allergic
type reactions. There was one PR. In a trial of similar
design, 12 colorectal cancer patients were treated with
300 μg GM-CSF and 6 million units IL-2 s.c. daily from
day 1 to 10 with 400 mg edrecolomab on day 3 of the
first cycle, and reduced to150 mg on subsequent cycles
up to a maximum of four cycles [221]. There were no
tumor responses. Edrecolomab was combined with
chemotherapy, GM-CSF, and IFN-α for 27 patients
with metastatic colorectal cancer in one trial [434],
and with capecitabine alone in 27 patients with various
metastatic adenocarcinomas [457]. It was unclear whether
the clinical activity seen was any better than what might
have been expected with the same chemotherapy alone.
Other anti EpCAM antibodies
Elias et al. treated NSCLC patients with the IgG1
momab KS1/4 that reacts EpCAM [205]. Five patients
received sequential doses of 1, 10, 60, 100, and 1,000 mg
over 2 weeks for a total of 1,661 mg. Minor upper
gastrointestinal toxicity was seen in some patients. No
antitumor responses were seen.
The anti-EpCAM human-engineered Mab ING-1 was
given to 25 advanced adenocarcinoma patients at doses
of 0.03 mg/kg, 0.10 mg/kg, and 0.30 mg/kg [151]. Two
patients experienced reversible, but dose limiting pan-
creatitis; so, 0.10 mg/kg was felt to be the maximum
tolerated dose. There were no responses observed in the
first 25 patients, or seven additional patients who were
treated with repeated weekly doses of 0.10 mg/kg.
Adecatumumab (MT201) is a human recombinant
IgG1 Mab that binds EpCAM. Twenty patients with
hormone refractory prostate cancer were treated with two
adecatumumab infusions on days 0 and 14 in over a dose
range of 10 to 262 mg/m2 [528]. Mild infusion reactions
including fever and nausea were the most common
adverse events. No tumor responses were reported.
Catumaxomab is a trifunctional Mab that binds
human EpCAM and CD3. Escalating doses of catumax-
omab from 2 to 7.5 μg were infused over 8 h in patients
with advanced NSCLC who were premedicated with
corticosteroids and antihistamines to reduce infusion
reactions [654]. Hepatic transaminasemia was the dose-
limiting toxicity. The MTD was determined to be 40 mg
of dexamethasone followed by 5 μg of catumaxomab.
No tumor responses were reported.
378
SK-1 is a human Mab generated using hybridoma
fusion methods with lymphocytes from regional lymph
nodes of colon cancer patients. that recognizes a glyco-
protein expressed on the majority of colon cancer tissues
that appears to be in the EpCAM family of molecules
[398], [801]. In the initial dose escalation study colon
cancer patients received 2, 4, or 10 mg given three times.
Treatment was well-tolerated; no tumor response were
reported [399].
CA125
Oregovomab (B43.13, OvaRex) is a murine Mab that
targets CA125 [32]. Because CA125 is shed into the cir-
culation, infusion of oregovomab is associated with
immune complexes which probably would complicate a
direct therapeutic approach. However, in patients who
received low doses of oregovmab, it was possible to
demonstrate antibody and T cell responses to CA125
that were not present before injection of the antibody; so
it has been hypothesized that these immune complexes
are taken up by dendritic cells leading to an anti-CA125
immune response that may be therapeutic [526]. In a
phase II pilot study 13 heavily pretreated, platinum-re-
sistant ovarian cancer patients CA125 > 35 U/mL, were
given 2 mg of oregovomab i.v. at weeks 0, 2, 4, 8, and
12, followed by additional dose every 3 months for up
to 2 years [200]. More than half the patients exhibited
enhanced anti-CA125 immune responses, four patients
showed decreases in CA125 levels, and three patients
had progression-free disease for more than 2 years. In a
phase III trial 145 patients with stage 3 or 4 ovarian can-
cer, who had achieved a complete response following
other therapy, were randomized to oregovomab or
placebo administered at weeks 0, 4, and 8, and then
every 12 weeks for up to 2 years [33]. There was no dif-
ference between the two arms in time to recurrence,
13.3 months for oregovomab and 10.3 months for pla-
cebo (p = 0.71).
Abagovomab (formerly ACA125) is a murine anti-
idiotypic Mab antibody to a Mab that reacts with CA125
(Ab2). It is being explored as a vaccine in ovarian can-
cer and appeared promising in phase I trials based on the
induction of a human anti-CA125 antibody (Ab3)
response [554, 616]. This approach is discussed in more
detail in the vaccine chapter of this text.
MOv18 is a chimeric Mab that reacts with a folate
receptor on ovarian cancer cells. In a phase I trial 15
ovarian cancer patients received single i.v. doses rang-
ing from 5 to 75 mg [497]. At doses of 50 mg and above
all patients experienced side effects that included fever,
headache and nausea of mild degree. No responses were
Monoclonal antibody therapy
reported. A bifunctional momab variant of MOv18
called OC/TR combines the anti-ovarian binding of the
MOv18 with anti-CD3 to target T lymphocytes. The
product was given as an F(ab’)2 in an effort to avoid sys-
temic toxicity related to removal of T cells in the reticu-
loendothelial system [721]. However, significant
infusion reactions still occurred except at the lowest i.v.
doses among five patients, suggesting that the binding to
CD3 itself led to the acute release of cytokines that
mediated the side effects. This Mab has also been given
i.p. as one or two 5-day cycles with IL-2 to 28 patients
who had residual limited disease after surgical debulk-
ing and chemotherapy [86]. Responses were confirmed
in seven patients based on surgical restaging, although
one had concurrent progression in retroperitoneal nodes.
Most of the responders subsequently progressed outside
the peritoneal cavity, but three of the CRs lasted 18 to 24
months. HAMA was detected in 21/25 patients. Elevated
plasma levels of TNF-α, IL-6, G-CSF and neopterin
were detected following infusions of higher doses of
Mov18. A chimeric version of this Mab has been devel-
oped and given to small numbers of patients [748]. An
IgE construct has also been engineered [366].
Other Anti-Adenocarcinoma
Monoclonal Antibodies
L6
L6 is a mouse Mab that reacts with a 24 kD epithelial
antigen with expression on most adenocarcinomas. It
mediates CMC with human complement and ADCC
with human NK cells and macrophages. The L6 antigen
is now believed to be a distant member of the transmem-
brane-4 superfamily (TM4SF). Goodman et al. treated
five metastatic breast cancer patients with daily doses
from 5 to 400 mg/m2 of murine L6 for 7 days to a total
dose of 35 to 2,800 mg [262]. One patient with hormone
receptor negative cancer, which had progressed on the
chest wall despite chemotherapy and radiation therapy,
had a CR after receiving a 400 mg dose. The response
did not become apparent until about 5 weeks after treat-
ment, and it took about 14 weeks before the CR was
attained. Doses from Doses from 5 to 400 mg/m2 were
given daily for 7 days to treat nine patients with advanced
ovarian cancer [262]. No responses were seen. No evi-
dence of anti-tumor effects in three patients with NSCLC
who received L6 [262]. Among five colorectal cancer
patients, one patient had a partial response following
seven daily 2-h infusions of L6 followed by 1 week of
rest, and then 4 days of s.c. IL-2 [811].
A humanized chimeric form of L6 was developed and
administered as single infusions ranging from 350 to
Robert O. Dillman
750 mg/m2, but no responses were seen in a phase II trial
of 21 patients [263]. Subsequent work with this Mab
focused on radioimmunotherapy and a chemotherapy
immunoconjugate.
Monoclonal antibody 494/32
Buchler et al. conducted a prospective randomized
trial of observation or adjuvant therapy with a 10-day
course of 370 mg of the murine IgG1 Mab 494/32 in 61
patients who had undergone a Whipple resection for
pancreatic cancer [63]. Analysis after 10 months
showed no significant difference in survival between
the two treatment groups who had median survivals of
428 days for the treatment group, compared to 386
days for the control group.
Ryan et al. administered three different IgM anti-
breast human Mabs, selected based on their patterns of
tissue reactivity, to 10 patients with metastatic breast
cancer [615]. One patient each was treated at dose levels
of 1, 2, 4, and 11 mg, and then 3 patients were treated at
20 mg, and three other patients received 22 mg in addi-
tion to tracer doses of 111-Indium labeled antibody.
No tumor regressions were seen in this pilot study or
relatively low doses of antibody.
Saleh et al. investigated momab D612 in a dose esca-
lation trial of multiple doses of 10 to 180 mg/m2 over 8
days in patients with various gastrointestinal malignan-
cies [625]. The dose limiting toxicity was a secretory
diarrhea. HAMA was detected in 18/21 patients. The
dose of 40 mg/m2 was selected for phase II trials. No
responses were reported. In a subsequent study, 14
patients with metastatic gastrointestinal cancer received
the D612, 40 mg/m2, days 4, 7, and 11 along with 80 μg/kg/
day of recombinant human monocyte colony-stimulating
factor [626]. Secretory diarrhea occurred in 10 of 14
patients. There were no anti-tumor responses.
Mucin Proteins and Human Milk Fat Globulin
MUC1 is a member of a family of mucin transmem-
brane glycoproteins expressed on most glandular epi-
thelia [672]. Many adenocarcinomas over-express and
shed MUC1, and elevated MUC1 serum levels can serve
as a marker of tumor burden and progressive disease.
Human milk fat globulin (HMFG1) antigen, which is
over-expressed in more than 90% of breast cancers and
serous ovarian cancers in an underglycosylated form, is
the same as MUC1. Mab HMFG1 is a mouse IgG1 that
reacts with epitopes of MUC1 on high molecular weight
human milk fat globulin glycoprotein antigen. Kosmos
et al. treated 15 ovarian cancer patients with intraperito-
neal HMFG1 rather than i.v. based on evidence that high
circulating levels of antigen result in immune complexes
379
and altered biodistribution [405]. A dose-dependent in
vitro T-cell proliferation was observed in 13/15 patients,
but no tumor responses were seen. A single-chain Fv
(scFv) and Fab fragment from this antibody have been
engineered that retain their specificity for MUC1 [552].
The huHMFG-1 (AS1402) antibody is a humanized
IgG1 directed against MUC1 and is currently in clinical
trials for the treatment of breast carcinoma [502].
Seventeen patients with MUC1-positive cancers were
treated with the murine anti-MUC1 BrevaRex mAb-
AR20.5, which was given i.v. in doses of 1, 2 or 4 mg
every 2 to 4 weeks for a total of six doses, with the intent
of forming immunogenic complexes as a vaccination
approach [152]. MUC1-specific anti-T cell responses
were confirmed in five patients.
c30.6 anti-colorectal cancer antibody
The mouse Mab m30.6, which had been used as a diag-
nostic tool to detect colorectal cancer cells, was engi-
neered to create the chimeric Mab c30.6. Clinical trials
were initiated after demonstration of in vitro and in vivo
anti-tumor effects in preclinical models. Four patients
with metastatic colorectal cancer received 3 mg of
123I-labeled c30.6, and the subsequent 13 patients were
treated with single doses up to 50 mg/m2 [769]. The
most frequent side effect was infusion-related erythema
and a severe burning sensation on the face, neck, ears,
chest, palms, soles, and genitalia, which was reduced by
premedication with blockade of type I and II histamine
receptors. No anti-tumor responses were seen.
A33
The A33 antigen is a cell membrane surface glycopro-
tein that is sole or predominantly expressed on human
gastrointestinal epithelium and on nearly all colorectal
cancers [300]. HuA33, is a CDR-grafted humanized
Mab against the A33 antigen. In an initial phase I study
11 patients with metastatic, chemotherapy-resistant
colorectal cancer were given huA33 at dose of 10, 25, or
50 mg/m2/week every 4 weeks [778]. Significant toxicity
occurred in four patients who had developed a strong
HAHA response against the Mab. Two patients experi-
enced infusion reactions that included fevers, rigors,
facial flushing, and blood pressure changes, while the
other two patients had serum-sickness type reactions
associated with skin rash, fever, and myalgia. One
patient, who was HAHA negative, had a PR. In a second
phase I trial 12 colorectal cancer patients were given
lower doses of huA33, 0.25, 1.0, 5.0, and 10 mg/m2
along with a tracer dose of radiolabeled Mab prior to
surgery [647]. At these doses there was no significant
toxicity and tumor localization was confirmed.
380
Lewis Y Antigen
Lewis Y antigen is a blood group antigen that is highly
expressed on the surface of epithelial tumors. Hu3S193,
a humanized Mab that binds to the Le(y) antigen, was
given as four weekly doses to eight colorectal cancer
patients, six breast cancer patients, and one NSCLC
patient, at doses of 5, 10, 20, and 40 mg/m2 [648, 649].
All patients had tumors that were Le (y)-positive. Mild
transient nausea and vomiting was observed following
the 40 mg/m2 dose. Indium-111 tracer studies confirmed
uptake in metastatic sites. No objective tumor responses
were recorded. A similar trial with huS193 was carried
out in 10 patients with Le (y)-positive small cell lung
cancer [410]. Tumor uptake of radiolabeled Mab was
confirmed. Toxicities, which again were mild, and only
seen at the 40 mg/m2 dose, included two patients with
nausea/vomiting, and one who developed urticaria.
G250 (CAIX, MN/CA9) Renal Cell
Cancer Antigen
The monoclonal antibody G250 (mAbG250) was pro-
duced by immunizing against human renal cell carci-
noma (RCC). The G250 antigen was isolated and
determined to be the same as the MN/CA9 antigen of
HeLa cells which has carbonic anhydrase activity. The
G250 antigen, which is also referred to as G250MN pro-
tein, and carbonic anhydrase IX (CAIX), is found on
95% of clear cell RCC, is rarely expressed on other types
of kidney cancer or cancers from other organs [412]. The
associated carbonic anhydrase activity has been impli-
cated in regulation of cell proliferation in response to
hypoxia. For many years it has been proposed as a good
target for Mab-directed therapy [734].
Initial studies were done with the murine Mab G250.
In a phase I study, 16 RCC patients received five differ-
ent doses of 131-I labeled G250 a week prior to nephre-
ctomy [536]. Good tumor localization was confirmed
and no significant side effects. The murine IgG2a G250
was converted to a mouse kappa light chain/human IgG1
chimeric Mab, cG250 [700]. In a dose escalation trial,
13 patients received doses of 5, 10, 25, or 50 mg/m2
given weekly i.v. for 6 weeks [144]. There were no seri-
ous toxicities and one patient had a CR. In a phase II
trial 36 RCC patients, 21 of whom were pre-treated with
IL-2 and or IFN-α, were given weekly doses of 50 mg
cG250 was i.v. for 12 weeks [43, 749]. There were no
serious adverse events. There were no objective
responses by the end of treatment, but one of ten patients
who continued treatment for an additional 8 weeks
eventually developed a CR. Doses of 5, 10, 25, or 50 mg/m2
were given weekly by i.v. infusion for 6 weeks.
Monoclonal antibody therapy
Prostate Cancer Associated Antigens
Low doses of IgG1 murine Mabs reactive with prostatic
acid phosphatase (PAP) or prostate specific antigen
(PSA) were given to 19 patients with metastatic prostate
cancer [183, 286]. Tracer doses of 111Indium-labeled
antibodies showed uptake in some sites of tumor. There
were no significant toxicities associated with treatment,
but nearly half of patients developed HAMA. No tumor
responses were observed. J591 is a humanized IgG1
Mab that targets the extracellular domain of the trans-
membrane prostate-specific membrane antigen (PSMA).
Fourteen patients with hormone refractory progressive
metastatic prostate cancer received 10, 25, 50, and
100 mg of J591 with 2 mg of Indium-111 tracer [506].
There were no significant toxicities and tumor uptake
was confirmed. Investigators have reported results for
more than 2,000 prostate cancer patients who received
111-In CYT-356 (Capromab, Prostascint™) [298, 680].
Uptake in regional lymph nodes has been confirmed in
some patients, but there have not been reports of anti-
tumor effects. but tumor responses could not be mea-
sured in this trial design.
Melanoma and Neuroectodermal
Associated Antigens
Melanoma Glycoprotein Antigens
Many of the first Mab developed against solid tumors were
to melanoma-associated antigens, including melanotrans-
ferrin (p97, p96.5 or gp95 antigen) and high molecular
weight chondroitin sulfated proteoglycan core protein
(p240, p280). None of the trials utilizing anti-p97 and anti-
p240 murine Mab resulted in objective tumor responses,
but this may be due to the limited evidence in vitro for
CMC or ADCC with these murine Mabs and/or lack of
regulatory significance for these antigens, and the rela-
tively small doses that were delivered in these trials.
Dillman and Halpern infused 1 to 50 mg of mouse Mabs
into melanoma patients as single 2-h infusions, with or
without a 1 mg tracer dose of 111Indium radiolabeled Mab
[183, 287]. Twenty-four patients received IgG1 Mabs
directed against the p97 antigen [287, 679], and another 28
received IgG2a Mabs directed against the p240 antigen
[183, 288]. No definitive tumor responses were seen in
patients with measurable disease. Similar experiences were
reported by other investigators utilizing relatively low
quantities of these same Mabs with radiolabeled tracers
[392, 514, 515]. Oldham et al. treated eight patients with
9.2.27, an IgG2a murine Mab that reacts with the p240
glycoprotein antigen [535]. Patients received twice weekly
escalating doses of 1, 10, 50, 100, and 200 mg. There was a
Robert O. Dillman
relationship between dose and tumor penetration and anti-
gen saturation based on immunohistochemical staining of
tumor biopsies which showed in vivo localization in s.c.
tumors in 6/8 patients following doses of 50 mg or greater.
No tumor responses were seen. Goodman treated four
melanoma patients with a combination of two mouse
Mabs, an IgG1 anti-P97 and an IgG1 anti-p240, and a fifth
patient received anti-p97 alone [261]. Escalating antibody
doses were administered as 6-h infusions daily for 10 days
to a maximum dose of 50 mg per infusion. No objective
tumor regressions were observed.
Melanoma Disialoganglioside Antigens
Many early murine Mabs derived from immunization
with melanoma cells reacted with disialoganglioside
glycolipid antigens, such as GD2 and GD3, which are
commonly expressed on neuroectodermal tissues [573].
Anti-GD3
The murine anti-GD3 Mab R24 was given to 21 patients
at doses of 1, 10, 30, or 50 mg/m2 every other day for 2
weeks for a totals of 8, 80, 240, or 400 mg/m2 [328,
738]. At higher doses all patients developed urticaria
and pruritus that typically occurred within 2 to 4 h
following treatment and often appeared around tumor
or at sites of previous tumor excision. In patients who
received 30 and 50 mg/m2, uptake of Mab in tumor was
readily demonstrated using biopsies and immunohis-
tochemical analysis. Significant tumor regressions were
seen in four patients: 2/6, 1/6, 1/6, and 0/3 for each
successive dose level. Responses were first noted within
2 weeks of completing treatment, but continued to
increase for several months.
An effort was made to intensify the administration
of mouse R24 since treatment was associated with a
high rate of urticaria for patients receiving cumulative
doses of 400 mg/m2 over 2 weeks [18]. Eight patients
received R24 at doses of 800 and 1,200 mg/m2 over 1
week by continuous i.v. infusion along with prophy-
lactic histamine blockers. Severe toxicity was noted.
One patient developed anaphylaxis. All patients devel-
oped lymphopenia and decreases in serum complement.
A cytokine induced vascular leak syndrome occurred
in seven patients during the first 2 days of therapy.
Serum sickness was observed in six patients within a
week, which coincided with the ability to detect
HAMA. The maximum tolerated dose was felt to be
800 mg/m2 based on dose-limiting toxicities that
included hypertension, chest pain, and vision changes
at the 1,200 mg/m2.
In another phase I trial in 11 patients with advanced
melanoma, R24 was given i.v. daily for 5 days at doses
381
of 10 mg/m2, 30 mg/m2 or 50 mg/m2 [453]. Toxicity was
substantial and included hypoalbumenemia in all cases,
and a death at the highest dose. There was one mixed
tumor response.
In another phase I trial with murine R24, 37 patients
with advanced melanoma received 1, 10, 20, 40, or
80 mg/m2 with five to six patients at each dose [393].
Urticaria was the most frequent adverse event. A dose-
limiting toxicity of pulmonary capillary leak syndrome
occurred in three of five patients at the highest dose.
Hepatic enzyme elevation and chest pain were noted
even at lower doses. Two responses were seen at the
lowest dose.
R24 has been given in combination with a variety of
different immunostimulatory cytokines in the hopes of
enhancing antitumor effects. Caulfield et al. gave five
daily 6-h infusions of escalating dose of R24 in combi-
nation with intramuscular with IFN-α2a to 15 mela-
noma patients [100]. Toxicities were similar to those
seen in other trials of R24. No tumor regressions were
reported, which was disappointing in view of the appar-
ent activity of R24 and IFN-α as single agents.
Several trials of R24 and IL-2 have been conducted.
Bajorin et al. evaluated the combination of IL-2 and
murine R24 in 20 patients with metastatic melanoma in
a phase I trial in which IL-2 was given at a dose of 6
MIU/m2 i.v. over 6 h on days 1–5 and 8–12, while the
anti-GD3 antibody R24 was given on days 8–12 at 1, 3,
8, or 12 mg/m2 per day with five patients evaluated at
each dose level [17]. Some in vitro T-cell activation was
demonstrated and one patient had a PR in soft tissue
sites lasting 6 months, and two other patients had minor
responses [779]. R24 combined with low-dose continu-
ous infusion IL-2 resulted in one PR and two minor
responses in 28 melanoma patients who were treated in
a dose escalation trial [682]. At higher doses two patients
experienced chest and abdominal discomfort that neces-
sitated dose reductions. In a separate trial, Creekmore et
al. gave a higher dose of continuous infusion IL-2 fol-
lowed by R24 [134]. This sequencing of the agents was
associated with responses in 10/28 patients while con-
current administration was associated with responses in
only 1/17 patients. However, in the sequential trial, there
were also five patients who never received R24 because
of the severity of IL-2-related toxicity.
R24 has also been given to melanoma patients in com-
bination with GM-CSF, M-CSF, or TNF-α. Chachoua et al.
gave s.c. GM-CSF for 21 days at a dose of 150 ug/m2/day,
and gave R24 by continuous i.v. infusion on days 8–15 at
doses of 0, 10, or 50 mg/m2 [101]. There were no tumor
responses observed with GM-CSF alone in five patients,
or the lower R24 dose in six patients, but two responses
382
were seen at the 50 mg/m2 dose in nine patients. However,
4/9 were unable to complete this single course of therapy
because of toxicity. Minasian et al. treated 19 metastatic
melanoma patients with a 14-day continuous infusion of
recombinant human M-CSF at a dose of 80 ug/kg/day in
combination with R24 which was administered daily by
i.v. infusion at doses of 1, 3, 10, 0 and 50 ug/m2/day on days
6 to 10 [492]. There were no objective tumor responses,
although three patients did have a mixed response with
regression of some lesions. In a pilot study R24 was
combined with two different doses of TNF in eight
patients with melanoma [491]. One patient had a dra-
matic tumor lysis syndrome associated with hemorrhagic
tumor necrosis in multiple visceral sites of disease.
Combining data from 11 R24 trials cited above reveals
a cumulative response rate of 6/77 for R24 alone, and
6/127 for R24 in combination with other biologicals.
Anti-GD2
The IgG3 anti-GD2 mouse Mab 3F8 was used by
Cheung et al. to treat melanoma patients who received
5, 20, 50, or 100 mg/m2 as 8-h infusions given daily for
2 to 4 days [109]. The study was closed at the 100 mg/m2
dose because all patients treated at that dose level devel-
oped hypertension. Treatments were associated with focal
pain at tumor sites and pain especially over the abdomen,
back, and extremities which was attributed to reactivity
with neural tissue. Urticaria, fever, nausea, vomiting
and sweats were also noted. Inflammatory actions were
observed around tumors, and partial responses were
reported for 2/9 patients and two others had a mixed
response. Six melanoma patients were given a combina-
tion of R24 and 3F8 at doses of only 1–10 mg/m2; but no
tumor regressions were seen.
Saleh et al. conducted a phase I trial of a different
murine anti-GD2 Mab 14G2a, an IgG2a switch variant
of the IgG3 anti-GD2 Mab 14.18, in which 12 patients
with melanoma received single i.v. infusions of doses
from 10 to 120 mg [623]. Treatment was complicated by
abdominal and pelvic pain, which necessitated narcotic
analgesia for control. All 12 patients developed HAMA.
There was one PR. Murray et al. gave Mab 14G2a to 11
patients with metastatic melanoma as part of a phase I
trial [516]. There were no objective remissions although
two patients exhibited mixed responses to the antibody.
Significant adverse events were noted including gener-
alized pain, fever, rash, paresthesias, weakness, hypona-
tremia and postural hypotension were the significant
toxicities observed. The investigators suggested 100 mg/
m2 as the maximum tolerated dose of this Mab.
A chimeric version of the anti-GD2 antibody (ch14.18)
with a constant region of human IgG1κ has been tested
Monoclonal antibody therapy
in a phase I trial in 13 melanoma patient, who received
5 to 100 mg of Mab [624]. As has been seen in other trials
of anti-GD2 antibodies in adult patients, the major tox-
icity associated with this therapy was abdominal/pelvic
pain syndrome that necessitated use of narcotic analgesics.
No tumor responses were seen in this cohort of patients.
KM871 is an IgG1 kappa chimeric Mab to GD3, that
was given to 17 metastatic melanoma patients at dose
levels of 1, 5, 10, 20, or 40 mg/m2 at 2-week intervals for
three doses [645]. Specific targeting of melanoma tissue
was demonstrated using indium-111 tracer Mab. No
dose-limiting toxicity was observed although three
patients experienced pain and/or erythema at tumor sites
that was not related to dose. One patient had a partial
response that lasted 11 months. KW-2871 is another
IgG1 kappa chimeric Mab to GD3 that was given to 17
metastatic melanoma patients who received an initial
10 mg/m2 and then 2 weeks later were stratified into four
cohorts to receive four doses of KW-2871 at 2-week
intervals at doses of 20, 40, 60, and 80 mg/m2 [232]. The
maximum tolerated dose for this schedule was 40 mg/m2
based on grade three dose-limiting toxicities of laryn-
gospasm and chest tightness at doses of 80 and 60 mg/
m2. Urticaria were noted in all 16 patients who were not
premedicated with antihistamines.
Neuroblastomas also express disialogangliosides.
Treatment of eight children with the 3F8 anti-GD2
mouse Mab produced two CRs, one at 5 mg/m2, and the
other at 20 mg/m2 [109]). The abdominal/pelvic pain
syndrome was not as problematic in these pediatric
patients as it had been in adults with melanoma. 3F8
was used to treat 16 neuroblastoma patients who had
stage 4 disease [111]. Adverse reactions included pain,
fever, urticaria, hypertension, hypotension and anaphy-
lactoid reactions. Three patients survived more than 6
years. In an effort to eradicate minimal residual disease,
3F8 was given after completion of chemotherapy, to 34
neuroblastoma patients, most of whom had bone and/or
bone marrow metastases [110]. Only 23 were in CR at
the time 3F8 was given. Pain, fever, and urticaria were
the most frequent adverse events noted. Fourteen
patients were still alive after 3 to 10 years of follow up.
Murray et al. reported two PR among five neuroblas-
toma patients who were treated with the 14.18 Mab as
part of a phase I trial [516]. They also noted that pediat-
ric patients tolerated the agent much better than adults.
The anti-GD2 Mabs have been combined with vari-
ous cytokines in an effort to enhance anti-tumor effi-
cace. Yu and colleagues treated 17 neuroblastoma
patients, aged 2 to 8 years, with GM-CSF and the chi-
meric Mab ch14.18, which was engineered with a
human IgG1κ modification of the murine 14.18 [804].
Robert O. Dillman
Toxicity was minimal in these children as opposed to
the experience in adult patients with melanoma.
Significant tumor responses were noted in eight patients,
including three complete remissions. 3F8 and GM-CSF
were given to 19 neuroblastoma patients whose disease
was resistant to initial therapy [411]. One cycle consisted
of s.c. GM-CSF alone for 5 days, and then GM-CSF i.v.
followed hour later by i.v. 3F8 on days 6–10, and 13–17
along with additional s.c. doses of GM-CSF on days 11
and 12. The treatment was given in an outpatient setting.
Side effects were mild and included manageable pain
and occasional rash. Complete responses were recorded
for 12/19 patients.
Choi et al. treated 23 melanoma and four sarcoma
patients with a combination of ch14.18 and R24 anti-
bodies together with continuous IL-2 [114]. When com-
bined with IL-2, the maximum tolerated dose for both
Mab was 5 mg/m2/day because of dose-limiting toxici-
ties including severe allergic reactions and pain with
both products at the 7.5 mg/m2/day dose. Two melanoma
patients had PRs.
L612 HuMAb is a human IgM that binds to ganglio-
side GM3 and effects CDC with human complement.
Nine patients with measurable metastatic melanoma were
given a continuous infusion of L612 at a dose of 960 mg,
1,440 mg, or 1,920 mg over 48 h [341]. Mild pruritus and
skin rash were noted. No objective responses occurred,
but one patient reported pain in s.c. tumor sites.
Other Ligand Targets
Interleukin-6
Interleukin-6 (IL-6) is a growth factor for plasma cells
that is involved in autocrine/paracrine growth regulation
[107]. Soluble IL-6 receptors and L-6 are typically ele-
vated in the advanced stages of myeloma. Klein et al.
treated a patient whose primary plasma cell leukemia
whose disease was refractory to chemotherapy, with
daily i.v. anti-IL-6 Mab [396]. Serial monitoring during
the 2 months of treatment demonstrated a decrease in
serum calcium, serum paraprotein, C-reactive protein,
and the percentage of cells in S-phase in the bone mar-
row, but an objective tumor response could not be
claimed. There were no major toxicities but decreases in
both platelet and white blood cell counts were noted. A
chimeric anti-IL-6 Mab was given to 12 patients with
myeloma at daily doses of 5, 10, 20, 40, and total doses
of 140, 280, 560 mg, and 1,120 mg given as two 14-day
treatment cycles [747]. There were no responses, and
much of the anti-IL-6 antibody was complexed to circu-
lating IL-6. Bataille et al. treated 10 patients with
383
extramedullary myeloma with a murine anti-IL6 Mab
[29]. Three patients had an objective antiproliferative
effect based on labeling index, and one had a minor
regression of a tumor mass. Most patients had some
worsening of neutropenia and thrombocytopenia.
Moreau et al. have used a combination of a murine anti-
IL-6 monoclonal antibody with dexamethasone and
high dose melphalan in the autologous transplant setting
[501]. A major obstacle to treatment with anti-IL-6 is
the very high rates of IL-6 production in some patients
with myeloma, which would need to be reduced by
another therapeutic modality [610]
IL-15
Interleukin-15 (IL-15) is a proinflammatory cytokine that
stimulates T lymphocytes and NK cells. HuMax-IL15 is
a human IgG1 anti-IL-15 Mab that suppresses T cell
proliferation and induces apoptosis in vitro. HuMax-IL15
was given for 12 weeks to patients with rheumatoid
arthritis in a double-blinded phase I/II dose-escalation,
placebo-controlled trial that enrolled 30 patients [27].
At the doses used in this trial there, HuMax-IL15 was
well tolerated and there were no significant effects on
subsets of T lymphocytes. This product may be devel-
oped mostly for the treatment of auto-immune disease,
but could also be considered for certain hematologic malig-
nancies in which IL-15 stimulation may be important.
Gastrin Releasing Peptide (Bombesin)
Peptides such as human gastrin-releasing peptide (GRP)
and the peptide bombesin, are sometimes produced by
lung cancers. Mulshine et al. infused the anti-GRP
murine Mab 2A11 into 12 patients with NSCLC, at dose
of 1, 10, or 100 mg/m2 i.v. thrice weekly for 4 weeks
[513]. No tumor regressions were noted. In a phase II
trial, 12 patients with small cell carcinoma of the lung
were given 2A11 at dose and schedule of 250 mg/m2
thrice weekly for 4 weeks [375]. In this study one patient
achieved a complete radiographic remission which
lasted 5 months, and four patients had stable disease. No
significant toxicities were observed. In an accompany-
ing radioimmunodetection study, tumor uptake of
111-Indium conjugated 2A111 was detected in 11/12
patients [105]. The receptor for GRP/bombesin could
also be a target for Mab therapy [807].
Fibroblast Activation Protein
Sibrotuzumab (BIBH 1) is a humanised version of the
murine anti-FAP mAb F19 which reacts with fibroblast
activation protein (FAP). FAP is highly expressed by acti-
vated fibroblasts of the tumor stroma in many malignan-
cies including breast, colon and lung carcinomas [476].
384
In a phase I trial, 26 patients, including 20 with metastatic
colorectal cancer and six with NSCLC, were given sibrotu-
zumab at doses of 5, 10, 25, or 50 mg/m2 weekly for 12
weeks, with trace 131-I labeled Mab during weeks 1, 5, and
9 [646, 323]. There was minimal toxicity during 218 infu-
sions of sibrotuzumab administered during the first 12
weeks and only one dose limiting toxicity was observed.
Tumor uptake of the Mab was confirmed. There were no
tumor responses.
In a phase II trial 25 metastatic colorectal cancer
patients were given i.v. weekly 100 mg doses of sibrotu-
zumab for 12 scheduled weeks [777]. Five patients
experienced infusion reactions that included rigors or
chills, flushing, nausea, and one episode of bronchos-
pasm. HACA were detected in three patients after four
to 12 infusions. There were no tumor responses.
Tumor Necrosis Factor Alpha (TNF-α)
Infliximab (Remicade®, Centocore, Malvern, PA)
Infliximab is a chimeric IgG1 kappa that neutralizes
tumor necrois factor alpha (TNF-α) by blocking binding
of the ligand to its receptor because of its own high affin-
ity binding to the ligand. It is used in the treatment of a
variety of chronic inflammatory autoimmune disorders
including Crohn’s disease, ulcerative colitis, rheumatoid
and psoriatic arthritis, uveitis, and ankylosing sondylitis
[739]. Not surprisingly, sepsis and opportunistic infec-
tions are significant complications of TNF-α inhibition.
Infliximab was given to 19 patients with cytokine-
resistant renal cell carcinoma (RCC) at doses of 5 mg/kg
at weeks 0, 2, and 6, and then every 8 weeks and then to
18 RCC patients at doses of 10 mg/kg at weeks 0, 2, and
6, and then every 4 weeks [294]. Severe adverse events
included a grade 3 hypersensitivity reaction and a death
from pulmonary infection with sepsis. Clinical activity
was promising with partial responses observed in 3/19
patients using the first dose/schedule and 11/18 using the
second dose schedule. In a trial that enrolled patients
with a variety of advanced malignancies, 21 patients
received infliximab at 5 mg/kg and 20 patients were
treated at 10 mg/kg at 0 and 2 weeks and then every 4
weeks [59]. These doses and schedule were not associ-
ated with any dose-limiting toxic effects. There were no
objective responses in this heterogeneous patient popu-
lation. In both these trials neutralization of plasma
TNF-α was demonstrated shortly after the first infusion.
Adalimumab (Humira®, Abbott, North Chicago, IL)
Adalimumab is a recombinant human IgG1 that blocks the
binding of TNF-α to its cell surface receptors. Like inflix-
imab, it is also used in a variety of chronic inflammatory
Monoclonal antibody therapy
autoimmune diseases and associated with similar risks for
infection. The specific marketing indications for adali-
mumab are rheumatoid and psoriatic arthritis, ankylosing
spondylitis, and Chron’s disease [739]. At this time there
are no published clinical trials in which adalimumab has
been used in the treatment of human malignancy.
Anti-Idiotype Vaccines
As discussed earlier in this chapter, the anti-idiotype
cascade provides a rationale for developing Ab2 Mab
products as a vaccine to induce an endogenous Ab3
response against the target antigen.
In the nomenclature that is now in use, the generic
names of such products have “ab” at the beginning of
their name.
Anti-Idiotype Vaccines in Adenocarcinomas
After clinical trials suggested that murine Mab 17-1A
could induce an anti-idiotype cascade, Herlyn et al. tried
to induce endogenous human anti-tumor antibodies in
patients with colorectal cancer [314]. They gave patients
repeated s.c. injections of a goat antibody against the
idiotype of Mab 17-1A. The goat antibody induced
human antibodies against the same antigen targeted by
17-1A. A significant tumor response was seen in 1/30
patients so treated.
Foon et al. treated 32 patients following resection of
locoregionally advanced colorectal cancer, with 2 mg
s.c. injections of CeaVac, an anti-idiotype antibody to
an anti CEA antibody, every other week for four injec-
tions and then monthly until tumor recurrence or pro-
gression [229]. CeaVac induced a potent anti-CEA
humoral and cellular immune response in all 32 patients,
including 15 patients who received 5-FU chemotherapy
during vaccination.
The anti-idiotype Mab, an anti-HMFG antibody, vac-
cine 11D10 (TriAb), was given to 45 patients with meta-
static breast cancer that had responded to standard
chemotherapy, before and after high dose chemotherapy
and autologous stem cell rescue [583]. Idiotype-specific
humoral and T-cell proliferative responses were demon-
strated in most patients despite the immunosuppressive
effects of high-dose chemotherapy. The 48% 3-year
overall survival rate is similar to what has been reported
in other series of stem cell transplant in patients with
advanced breast cancer.
In a multicenter cooperative group trial, 56 patients
with colorectal cancer who had undergone curative
resection of their liver metastases, received a combina-
tion of two anti-idiotype monoclonal antibody vaccines,
Robert O. Dillman
one to CEA(CeaVac) and the other to HMFG (TriAb)
[567]. About 40% of patients were progression-free
after 2 years, which is similar to what is seen with
surgery alone.
The anti-idiotypic antibody vaccine ACA125, that is
designed to induce endogenous immune responses against
tumor antigen CA125, was given to 119 women with
advanced ovarian carcinoma [588]. A specific anti-anti-
idiotypic antibody (Ab3) response was detected in 68%
and endogenous CA125 antibodies were detected in 50%.
Ab3-positive patients had a much longer survival than
Ab3-negative patients (23 vs. 5 month, p < 0.0001).
In a randomized phase II trial, prior to surgery 67
patients with colorectal cancer were randomized to
receive the human anti-idiotypic Mab 105AD7 with or
without BCG or to no treatment [735]. Vaccinations
were initiated in 32 patients prior to surgery and were
planned to be continued for up to 2 years. Most of the
immunized patients exhibited immune responses, but
specific immune data and comparative data have not
been reported.
Anti-Idiotype Vaccines in Melanoma
Mittelman and colleagues gave anti-idiotype antibodies
directed Mab that targeted the gp240 glycoprotein anti-
gen, in effort to induce endogenous antibodies against
gp240 on melanoma cells. In a phase I trial involving 16
patients, s.c. injections of MAF11-39 from 0.5 mg to
4 mg were well tolerated; so a phase II trial was carried
out in which an additional 21 patients were given 2 mg
on days 0,7, and 28 [493]. At the 2 mg dose 16 patients
exhibited a response to gp240 and one patient had a CR.
In another trial the MK-23 anti-idiotype antibody was
conjugated to keyhole limpet hemocyanin (KLH) and
coadministered with BCG in 25 patients [494]. Three
PRs were observed, and 14 of 23 patients developed
endogenous human antibodies against gp240.
Foon et al. treated 47 melanoma patients with TriGem,
an anti-idiotype antibody to a Mab that binds to disialo-
ganglioside GD2, using weekly s.c. injections of 1, 2, 4,
or 8 mg in combination with QS-21 as an adjuvant [230].
An anti-anti-idiotype (AB3) response was detected in
40/47 patients. One patient had a complete response.
1E10 Anti-Idiotype Vaccines
to Gangliosides
A series of trials have been conducted with aluminum
hydroxide-precipitated 1E10, which is an anti-idiotype
murine Mab (Ab2) specific to an Ab1 Mab that reacts
385
with NeuGc-containing ganglioside that is present on
various solid tumors. Up to six i.d. injections of 1 to
2 mg of anti-Id mAb were given every 2 weeks. In 20
patients with advanced melanoma, the treatment was
well tolerated and 16 patients exhibited strong Ab3
responses against GM3 [7]. In locally advanced or met-
astatic breast cancer, eight out of nine patients who
received four or more injections exhibited strong Ab3
responses against GM3 [159]. Similar reactions were
seen among nine patients with advanced NSCLC who
received injections of 1E10 following a good response
to chemotherapy and/or radiotherapy [521].
Summary
The hybridoma and recombinant DNA technologies,
combined with advances in our understanding of immu-
nology, tumor biology, and ligand–receptor–signal trans-
duction cell physiology, laid the foundation for monoclonal
antibody therapy of cancer. During1980–1990 exploratory
clinical trials confirmed many of the points to consider
when developing Mab therapy. During 1990–1995 many
murine mab were engineered to produce chimeric and
humanized antibodies. During 1997–2007 six unconju-
gated Mab that have clinical indications specifically for
the treatment of human malignancy became available for
widespread clinical use. Several of these have revolution-
ized cancer therapy and have become the highest reve-
nue-generating cancer drugs ever. Hundreds of other
Mab are in development and/or clinical trials, including
several totally human Mab. Many of these may be in
wide-spread clinical use by the time this chapter is pub-
lished. The next several years will see the introduction of
many more Mab into the clinic and continued evolution of
their integration into cancer therapy, and perhaps eventu-
ally replacement of many current forms of therapy.
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11 Immunotoxins
ARTHUR E. FRANKEL, JUNG-HEE WOO, AND DAVID M. NEVILLE
Introduction
Immunotoxins are protein molecules composed of a cell
surface-directed ligand covalently linked to a peptide
cytotoxin. This definition excludes a number of impor-
tant therapeutic compounds with distinct pharmacologic
properties which react with intracellular targets. Such
molecules would be unlikely to find and react with their
intracellular targets due to the permeability barrier of the
plasma membrane. Thus, the ligands must bind cell sur-
face receptors or antigens. Ligands which have been
used include monoclonal antibodies and antibody frag-
ments, adhesion molecules, growth factors, and cytok-
ines. The toxophore must be peptide in nature. This
excludes radiolabels such as 90Y, 213Bi, or 131I and small
molecular weight drugs such as calicheamicin and doxo-
rubicin [1, 2]. These immunoconjugates are the subjects
of other chapters in this book. We have further restricted
the toxin moieties to cytotoxins. Thus, peptides which
modify coagulation [3], complement [4], or immune
responses [5] are not considered here. Conjugates with
these compounds are important potential therapeutics
and are considered separately in chapters discussing
anti-angiogenesis and cell-directed therapies. There are
three major classes of peptide cytotoxins [6, 7]. Class I
toxins are proteins which are intracellular enzymes. They
catalytically modify critical intracellular functions. Class
II toxins bind to cell surfaces and trigger intracellular
signal pathways. Class III toxins are pore-forming pep-
tides which cause leaks in the plasma membrane.
Immunotoxins have been prepared with toxins of each
class, as listed in Table 1 and discussed below. We will
first review the structure and molecular mechanisms of
cell intoxication for the peptide cytotoxins used in prepa-
ration of immunoconjugates.
Peptide Cytotoxins
The DNA sequence, amino acid sequence, and three-
dimensional structures for most of the toxins used in
conjugate synthesis have been defined. In most cases
separate domains contribute to different toxin functions.
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
The information on toxin structure has been extremely
useful in immunotoxin design.
Type I plant A-B Toxins
The type I A-B toxins are produced by plants and bacte-
ria. The plant toxins – ricin, abrin, and viscu-min (mistle-
toe lectin I) – are encoded by single exons. The sequence
from the N-terminus for each includes a signal peptide, a
catalytic domain, and an alkaline protease-sensitive
disulfide loop, followed by a cell binding domain [40–42].
The linker is cleaved by plant vesicle endoproteases,
N-linked glycosyl groups are added, and the signal
peptide is removed by a signal peptidase prior to toxin
secretion [43]. Thus, the fully processed, native proteins
are hetero-dimers of approximately 60,000 Mr, with a
30,000 Mr catalytic subunit (A chain) and a 30,000 Mr
cell binding subunit (B chain). The B chains have Ω-loop
structures (Fig. 1a) [44–46]. There are three lectin binding
sites/subunit which bind (3-galactosyl residues on mam-
malian cell surface glycoproteins (Fig. 2a) [47, 48]).
There are also extensive salt and hydrophobic bonds
between the A and B chains which stabilize the heterodi-
meric structures [44]. After cell binding, the heterodimers
internalize into endosomes using both clathrin-dependent
and clathrin independent pathways [49], and, via a Rab9-
independent process, reach the transreticular Golgi [50].
In the Golgi, the toxins bind endoplasmic reticulum
shuttle proteins which undergo retrograde transport via
COP-I-independent Rab6 regulated vesicles [51]. The
toxins are then transported to the endoplasmic reticulum
(ER) [52]. In the ER, the toxin is reduced, the A chain
unfolds with the help of chaperones, and the A chain
translocates to the cytosol using the Sec61p transposon
[53]. In the cytosol the A chain refolds. The A chain has
an α-helical/β-sheet open sandwich tertiary structure
with a large open catalytic cleft. The A chains function as
RNA N-glycosidases removing a critical adenine base
from the 28S ribosomal RNA (Fig. 3) [54]. This alters the
interaction of elongation factor 2 (EF2) with the ribosome
and prevents the structural modifications leading the GTP
hydrolysis (Fig. 4). Protein elongation is blocked irre-
versibly (Fig. 5). Cell death follows.
407
408 Immunotoxins

Table 1. Peptide cytotoxins used in immunotoxin synthesis

Protein Type Structure Order Source (species) Reference
Ricin I A-B Plant Ricinus communis [8]
Abrin I A-B Plant Abrus precatorias [9]
Viscumin I A-B Plant Viscumalbum [10]
Nigrin b I A-B Plant Sambucus nigra [607]
Gelonin I A Plant Geloniummulfiflorum [11]
Saporin I A Plant Saponaria officinalis [12]
Pokeweed antiviral protein I A Plant Phytolacca americana [13]
Luffin I A Plant Luffaa egyptiaca [14]
Bouganin I A Plant Bougainvillea spectabilis [15]
Trichokirin I A Plant Tiichosantheskifilowfi [16]
Trichosanthin I A Plant Tiichosantheskifilowfi [349]
BRIP I A Plant Hordeum vulgare [17]
Ebulin I A Plant Sambucus ebulus [558]
Bryodin I A Plant Bryonia dimca [18, 21]
Momordin I A Plant Momordica charanfia [19]
Momorcochin I A Plant Momorfica coohinchinensis [20]
Moschatin I A Plant Cucurbita moschata [571]
Dianthin 30 I A Plant Dianthus caryophyllus [22]
Ocymoidine I A Plant Saponaria ocymoides [23]
Pyramidatine I A Plant Vaccaria pyramidata [23]
Colocin 1 I A Plant Citrullus colocynthis [440]
Botulinum neurotype type C I A-B Bacteria Clostridia botulinum [560]
Diphtheria toxin I A-B Bacteria Corynbacterium diphthefiae [24]
Pseudomonas exotoxin I A-B Bacteria Pseudomonas aeruginosa [25]
Anthrax toxins I Binary Bacteria Bacillus anthracis [26]
Seminal ribonuclease I A Cow Bos taurus [27]
Bosinophil neurotoxin I A Human Homo sapiens [28]
Angiogenin I A Human Homo sapiens [29]
Pancreatic ribonuclease I A Human Homo sapiens [30]
RibonucleaseA I A Cow BosTaurus [31]
Ranpirnase I A Frog Rana pipiens [32]
Barnase I A Bacteria Bacillus amyloliquefaciens [579]
a-Sarcin I A Fungi Aspergillus giganteus [33]
Restrictocin I A Fungi Aspergillus restfictus [34]
Clavin I A Fungi Aspergillus clavatus [35]
Mitogillin I A Fungi Aspergillus fumigatus [93]
Bax I A Human Homo sapiens [167]
D-(KLAKLAK)2 I A Synthetic [563]
Phospholipase C II Bacteria Clostfidium perfringens [36]
TNF II Human Homo sapiens [574]
sTRAIL II Human Homos sapiens [590]
Hemolytic toxin III Sea anemone Stoichactis helianthus [37]
Cecropin/Shiva III Insect Hyalophora ceaopia [38]
Pyrularia thionin III Plant Pyrulafia pubera [39]
Proaerolysin III Bacteria Aeromonas hydrophila [548]
Cyt1Aa Toxin III Bacteria Bacillus thuringiensis [559]

targeting sequence of 25–29 amino acid residues. The

Type I plant A toxins
The type I plant A toxins are 25–31 kDa M, proteins.
They have similar primary amino acid sequences [55–
57], and similar three-dimensional structures [55, 56,
58–60], and are all ribosomal RNA N-glycosidases
which remove the same adenine base from the 28S
ribosomal RNA of the mammalian ribosomes [61].
However, they do not crossreact immunologically. The
preproproteins have a signal peptide, the catalytic
domain, and a C-terminal extension with a vacuolar
signal peptide and C-terminal extension are processed
on secretion, thus protecting the cytosolic plant ribo-
somes from intoxication. The three-dimensional struc-
ture contains eight α-helices and a β-sheet composed
of six strands. The sequence, structure, and enzyme
activity are homologous to the catalytic A subunit of
type I plant A-B toxins. There is an open cleft contain-
ing a conserved glutamic acid residue, an arginine resi-
due, and a tyrosine and a tryptophan residue. These
residues function to bind and stabilize the transition
Arthur E. Frankel et al. 409

state for the C-N bond cleavage [62]. There are three
conserved lysyl residues in the C-terminus of the
mature proteins which likely are involved in ribosomal
substrate recognition [56].
Type I Bacterial A-B Toxins
The type I bacterial A-B toxins which have been modi-
fied for targeted therapy include diphtheria toxin (DT)
and Pseudomonas exotoxin (PE). These toxins will be
discussed separately.
DT has a 25-residue leader sequence followed by 535
amino acid residues [63, 64]. The mature protein has
three domains (Fig. 1b) [65, 66]. There is an N-terminal
catalytic domain (amino acid residues 186), also called
the A fragment. This domain is followed by a 14 amino
acid loop bordered by Cys-186 and Cys-201. The loop is
arginine-rich and is a substrate for endosomal furin endo-
protease. The second domain is the translocation domain
(amino acid residues 187–381) and contains multiple
amphipathic helices with two negatively charged amino
acid residues (Glu-349 and Asp-352) in a helical hairpin
between two of the helices (TH8 and TH9). The translo-

Figure 1. Drawings of peptide toxin a-carbon backbone structures. (a) ricin; (b) DT; (c) PA; (d) RNase; (e) restrictocin; (f)
Clostridia perfringens PLC; (g) cecropin (similar to Shiva-1); (h) crambin (similar to PT). Based on coordinates determined
from references described in the text. Note that the type I A-B and binary toxins have multiple domains which may subserve
distinct functions (see Fig. 2). Cylinders are α-helices and ribbons are β-strands. In the ricin structure (a), red tubes are RTB
subunit S2-loops, and yellowblue structures are RTA enzymatic subunit. In the DT structure (b), red is β-sheet binding domain,
yellow is translocation domain with amphipathic helices, and green in enzymatic domain. In PA (c), blue is receptor binding
β-sheet domain, green is the heptamerforming and membrane-inserting domain, and red is 20 kDa protease-sensitive frag-
ment. EDN (d) has an α + β structure with two α-helices, a β-sheet, a third α-helix and four additional β-sheets separated by
loops. There are binding sites for bases and phosphates of RNA. The catalytic histidine residues are at the two ends
410 Immunotoxins

Figure 1. (continued) Restrictocin (e) shows structural homology to Rnases. It has catalytic histidines similar to RNase. There
is a three-turn α-helix packed against a five-stranded antiparallel β-sheet. PLC (f) has an N-terminal domain (residues 1–246)
composed of six stacked α-helices. It has the phospholipase C active site. There is a flexible linker (residues 247–255) followed
by a C-terminal domain (residues 256–370) with an eight β-strand jelly-roll topology with Ca++-dependent phospholipid-binding
function. The cecropins (g) are small 36 amino acid residue peptides with a strongly basic N-terminal amphipathic helix linked
via a flexible Gly-Pro linker to a neutral C-terminal amphipathic helix. These stack in membranes as shown creating pores. PT
toxin has two amphipathic Ochelices connected to two antiparallel β-sheets. The related crambin (h) lacks β-sheets, but the
loops serve the same function (Fig. 1 h)
 a Ricin
c PE

Galactosyl-terminated Alpa2-macroglobulin
oligosaccharides
Endosomes

KDEL-receptor Endosome

N-glycosidase reaction on
Trans-reticular ribosomes
Golgi
Trans-recticular
Golgi
37κD PE fragment
ER
ER
Translocation
EF2
of RTA
Ribosomes

b DT
d
Heparin-binding 20κDa LF
EGF fragment EF
63κDa
PA fragment
Protease

Endosome
PA receptor

DTA
EF2

Endosome

Ribosomes MAPKK

cAMP

Figure 2. Steps in peptide toxin intoxication of cells. (a) ricin; (b) DT; (c) PE; (d) anthrax toxins. Note for all toxins there is
an initial cell binding event. For the shown type I toxins (a–d), cell binding is followed by internalization. After intracellular
processing and transport the type I toxins escape to the cytosol where they catalytically inactivate key cellular processes. For
type II and III toxins the membrane perturbations lead to altered cell physiology or cell death

Figure 3. Atomic stick model of the S/R stem loop (red) portion of 28S rRNA (black). In addition to elongation factor binding
to the ribosome A site which displaces the peptidyl-tRNA (see Figs. 4 and 5), there is also an rRNA conformation change which
occurs spontaneously to drive elongation. This is catalyzed by other EF2 (EFG) domains (II-III) in which the GTPase activity
modifies ribosome structure. Ricin and type I plant A toxins remove an adenine base (rRNA N-glycosidase activity) from the
large rRNA at a specific location on the S/R conserved stem-loop (S = α-sarcin site and R = ricin site). This stem-loop normally
switches between two states which change overall rRNA and ribosome structure in the large subunit. Once the site is chemically
modified, EF2 or EFG can no longer bind properly and elongation stops. Note that EF2 or EFG interacts with the ribosome at
least two sites in domain IV and II-III in addition to its interaction with the mRNA at the anticodon loop and each of these two
sites are affected separately by type I peptide toxins
412 Immunotoxins

c
TOXIN

O
His O
H C OPO AMP
N
H O
N O

H
OH OH

Glu
N
R N Diphthamide
H
EF2

Figure 4. (a) Structure of EF-G and EF-Tu-Phe-tRNA-GTP ternary complex. Note that domain IV (the lower tip of EF-G and EF2)
resembles the tRNA portion of the EF-Tu-Phe-tRNA-GTP complex. Additional data show that this region is adjacent to the anti-
codon loop and binds the A site of the ribosome. (b) Model of EF2 α-carbon backbone showing in yellow the location of diph-
thamide residue at the tip of domain IV. The diphthamide residue is the target for DT and PE ADP-ribosylation. (c) The enzymatic
reaction catalyzed by DT and PE. The His and Arg residues of the catalytic site of DT and PE interact with the nicotinamide ring
and stabilize the position of the NAD molecule in such a way that the electrophilic carbon atom of the ribose group can interact
with the nucleophilic residues of diphthamide on EF2

aa-tRNA-EF-Tu-GTP cation domain ends in a flexible spacer (amino acid resi-

dues 382–390) which is then connected to a β-sheet rich
50S cell-binding domain (amino acid residues 391–535). DT
EF-Tu-GDP binds to cells via amino acid residues in the cell-binding
A-site
P-site domain to cell-surface-expressed heparin-binding epider-
30S mal growth factor-like growth factor (HB-EGF) precursor
[67, 68]. The amino acid residues of the DT cell-binding
domain, in particular Lys-516, binds a crevice in the
extracellular EGF-like domain of the HB-EGF precursor
(amino acid residues 122–148). There is a critical salt
bridge interaction between Lys-516 of DT and Glu-141
EF-G-GDP of HB-EGF precursor. The HB-EGF precursor associ-
ates on the plasma membrane with CD9 and heparan
EF-G-GDP
sulfate proteoglycan [69, 70]. This complex has a higher
Figure 5. Protein synthesis steps affinity for DT. Animals and cell lines with absent or
Arthur E. Frankel et al.
low-affinity receptors for DT are insensitive to the toxin
[67]. After cell binding the DT-HB-EGF precursor com-
plex undergoes clathrin-dependent endocytosis [71].
Interestingly, the cytoplasmic domain of the HB-EGF
precursor lacks an internalization motif (although it has
a membrane proximal Tyr-192) [72]. The complex
internalizes at 1%/min, and dynamin is required to com-
plete vesicle formation [73]. Once internalized into early
endosomes [74], DT u Glu zndergoes furin cleavage at
the arginine-rich loop [75], low pH-induced protonation
of the helical hairpin aspartate and glutamate [76], inser-
tion of the TH8 and TH9 amphipathic helices of the trans-
location domain into the vesicle membrane [77], unfolding
of the catalytic domain [78], reduction of the disulfide
bridge linking the A fragment with the remainder of DT
[79, 80], and transfer of the A fragment through the mem-
brane to the cytosol (Fig. 2b) [81]. The DT A fragment
then catalytically ADP-ribosylates elongation factor 2
(EF2) on the diphthamide residue in EF2 domain IV [82,
83, 92]. A detailed molecular mechanism for the enzy-
matic activity has been proposed. First, residues 39–46 of
the A fragment ‘active site’ loop bind NAD [84]. The dis-
lodged loop then binds EF2 in combination with the
exposed NAD. A fragment Glu-148 then attacks the
strained N-glycosidic bond of the NAD. The Glu-148
carboxylate group activates the diphthamide imidazole of
EF2 for a nucleophilic attack on Cl’N in an SN2-type
displacement reaction. This leads to addition of ADP to
the domain IV diphthamide residue. This irreversible
modification prevents the elongation factor from displac-
ing the tRNA petidyl complex from the A site to the P site
[85, 86]. Protein synthesis is inactivated, and cells die by
lysis or programmed cell death [87, 88]. The prolonged
action of a single A fragment molecule in the cytosol is
sufficient to inhibit protein synthesis and prevent cell
growth and division [81, 89]. Cellular resistance to DT
can occur due to altered DT receptors [67], lack of inter-
nalization [73], failure of arginine-rich loop proteolysis
[75], lack of endosomal acidification [90], too great a dis-
tance between the DT translocation domain and the vesi-
cle membrane [71], incomplete inter-domain disulfide
reduction [79], incomplete A fragment unfolding [78], too
rapid cytosolic proteolysis of the A fragment [81], and
resistant EF2 [91].
PE is a 66 kDa Mr, protein produced by Pseudomonas
aeruginosa. It consists of 638 amino acid residues
including a 25 amino acid hydrophobic leader peptide
[95]. The molecule has three distinct domains [96].
Domain I is an antiparallel β-structure with 17 β-strands.
It includes amino acid residues 1–252 (Ia) and 365–404
(Ib). The first 13 strands form an elongated β-barrel.
Domain Ia, is the cell-binding domain. Domain II consists
of residues 253–364 and has six consecutive α-helices.
413
Domain II is the translocation domain. Domain III is the
carboxylterminal third of the molecule and includes
residues 405–613. It has an extended catalytic cleft.
Domain III is the enzymatic domain. PE in the blood
reacts with plasma carboxypeptidase, and the terminal
lysine residue is cleaved [97]. PE612 then binds the
α2-macroglobulin receptor/low density lipoprotein
receptor-related protein (α2MR/LRP) on the surface of
mammalian cells including fibroblasts and hepatocytes
[98]. PE612 domain Ia, residue Lys-57 is important in
this binding reaction [99]. The PE612, α2 MR/LRP
complex undergoes receptor-mediated endocytosis
[100]. Once reaching the endosomes the PE612 under-
goes furin cleavage between Arg-279 and Gly-280 [101,
102], a pH-dependent conformation change [103] fol-
lowed by reduction of the disulfide bond between Cys-
265 and Cys-287 [104]. This creates a 28 kDa N-terminal
fragment and a 37 kDa C-terminal fragment. The
C-terminal REDL of the 37 kDa fragment then binds to
the KDEL receptor and the complex is routed to the
endoplasmic reticulum (ER) [105, 106]. In the ER the
37 kDa fragment utilizes residues in domain II to trans-
locate the cytosol [107]. Thirty-four amino acid residues
at the N-terminus of the 37 kDa fragment appear critical
for translocation, including Trp-281, Leu-284, and Tyr-
289 [108, 109]. There is an unidentified saturable pro-
tein which binds the fragment and facilitates transfer.
Most of the translocation domain helices, with the
exception of the first and last helix, are essential for the
translocation process [110]. The translocon may be part
of the machinery for PE transfer to the cytosol, since the
translocation occurs in the ER (Fig. 2c). The cytosolic
C-terminal fragment then catalyzes the ADP-ribosylation
of EF2 similar to DT [111]. Glu-553 is important for
NAD binding. The NAD is cleaved and the ADP moiety
transferred to the diphthamide residue of bound EF2
[112]. Cell death follows by a process indistinguishable
from DT [81, 89]. Cell resistance to PE has been seen
secondary to alterations of individual steps in the intoxi-
cation process. Cells may lack receptors [113]; cell
intracellular trafficking may be disrupted [114–115];
endosomal acidification may not occur [116]; furin-
mediated cleavage may not occur due to low or abnor-
mal furin [117]; EF2 may be mutated so that the
diphthamide residue is absent [91]; finally, cells may be
resistant to PE-induced apoptosis [87].
Type I Bacterial Binary Toxin
Anthrax toxin produced by Bacillus anthracis is a binary
toxin. The catalytic domain and binding domains are on
completely separate proteins. Anthrax toxins have two
catalytic proteins – edema factor (EF) and lethal factor
414
(LF). Either of these proteins can interact with the cell-
binding protein, protective antigen (PA).
PA has 764 amino acid residues with a 29 residue sig-
nal peptide. Mature PA is a 735 amino acid residue,
83 kDa secreted protein [118]. It has four domains [119].
PA domain 4 (residues 596–735) has an initial hairpin
and helix which serves as a flexible linker followed by an
immunoglobulin-like fold which functions in receptor
binding (Fig. 1c) [120, 121]. The nature of the cell sur-
face receptor is unknown, but there are approximately
30,000 receptors/macrophage [122]. After cell binding,
cell surface furin or PACE4 nicks PA in the middle of
domain 1 (residues 1–258) at residues 164–167 (Fig. 2d)
[123]. This releases the N-terminal 20 kDa Mr fragment,
PA20. The remainder of PA, PA63, remains on the cell
surface. A large hydrophobic surface on the residual
domain 1 of PA63 serves as a site for EF or LF binding.
EF and LF are 90 kDa Mr, 770 amino acid residue pro-
teins with a similar N-terminal 250 amino acid domain
which mediates binding to PA63 [124, 125]. These pro-
teins have distinct C-terminal domains which have ade-
nylate cyclase and Zn2+-dependent metalloprotease
activity, respectively [124–132]. The EF/LF-PA63 and
PA63 oligomerize on the cell surface to heptamers facili-
tated by the new N-terminal domain 1 and domain 2
(residues 259–487) of PA63 [133]. There can be up to
seven LF or EF molecules bound per heptamer. Both
monomers and heptamers then undergo endocytosis
[134]. The low pH of the endosomes triggers protonation
of critical histidines and unfolding of a large amphip-
athic hairpin loop in PA domain 2 which is hypothesized
to insert into the membrane and, together with those of
other PA molecules in the heptamer, form a 14-strand
β-barrel [135]. EF or LF then unfolds and translocates to
the cytosol through the barrel pore [136, 137]. Once in
the cytosol, EF and LF refold. EF is a calcium-depen-
dent-calmodulin-dependent adenylate cyclase that raises
the cytosolic cyclic AMP (cAMP). Elevated cAMP leads
to paralysis of several macrophage physiological path-
ways [138]. LF proteolytically cleaves members of the
mitogen-activated protein kinase kinase (MAPKK or
MEK) family. In the case of MEKs I and 2, this cleavage
results in their inactivation. Consequently, they cannot
activate their substrate, MAPK. Depending on the cell
type, this may have a variety of consequences including
apoptosis or altered physiology [139].
Type I Vertebrate A Toxins
Several members of the vertebrate ribonuclease (RNase)
superfamily have been linked to tumor selective ligands.
These ribonucleic acid-degrading proteins are small
Immunotoxins
(14–16 kDa Mr) proteins with an α + β structure (Fig.
1d) [140]. From the N-terminus there are two α-helices,
a β-sheet, a third α-helix, and four additional β-sheets
separated by loops. There are binding sites for bases and
phosphates of RNA. The catalytic residues include His-
12, Lys-41, Thr45, and His-119. His-12 and His-119 act
as the base and acid, respectively, in the transphospho-
rylation step that generates the cyclic phosphate inter-
mediate. The roles of His-12 and His-119 are reversed
in the subsequent hydrolysis step (Fig. 6) [141]. Lys-41
and Thr-45 bind the substrate phosphate and pyrimidine
base, respectively, to stabilize the transition state
intermediate.
The conjugated vertebrate RNases include bovine
seminal RNase (BS-RNase), human eosinophil derived
neurotoxin (EDN), human angiogenin (ang), frog ranpir-
nase, bovine pancreatic RNase A, and human pancreatic
ribonuclease I (hRNase 1). They each have distinct prop-
erties. BS-RNase is a dimeric protein (32 kDa Mr) with
strong and selective cytotoxicity to tumor cells, germ
cells, and T cells [142]. EDN is a 15 kDa, 134 amino acid
residue, highly basic protein which has selective toxicity
to Purkinje cells, parasites, and tumor cells [28, 143]. Ang
is a 123 amino acid residue, 14 kDa Mr protein that spe-
cifically degrades tRNA. It binds 170 kDa and 42 kDa Mr
receptors on endothelial and vascular smooth muscle cell
surfaces and induces cell proliferation, cell adhesion, pro-
His119
R
His12
HN NH
O
O
P
O
N NH
O O H
B
O
His12
His119
H
NH
HN N H O O O HN
P
O O
O B
Figure 6. Steps in RNase hydrolysis. This is a two-step pro-
cess in which initially a cyclic phosphate intermediate is formed.
His-12 acts as a general-base catalyst and His-119 acts as a
general acid to protonate the leaving group (the RNA fragment
designated R). The second step is a hydrolysis step in which
His-119 activates the attack of water by general base catalysis
and His-12 is the acid catalyst, protonating the leaving group
Arthur E. Frankel et al.
duction of cell-associated proteases, and cell migration
and invasion. The protein’s ribonucleolytic activity is
essential in these activities. The protein is internalized by
receptor-mediated endocytosis, translocated to the cytosol
and then the nucleus [29, 144]. Ranpirnase is a basic
14 kDa Mr protein which binds tumor cells and vascular
endothelium and degrades tRNA selectively [32, 145].
Unlike ang, ranpirnase triggers cell apoptosis. Neither
BS-RNase nor ranpirnase is bound by cytosolic ribonu-
clease inhibitor protein (RI), and, since they are not inac-
tivated in the cell, have potent cell toxicity. RI constitutes
>0.01% of cytosolic proteins and inactivates ribonu-
cleases by forming tight complexes that prevent RNA
substrates from entering the active site. Human pancre-
atic RNase 1 and bovine pancreatic RNase A are not toxic
to cells, lack cell-binding functions, and are good sub-
strates for RI. Interestingly, a variant of RNasel – des.
1-7hpRNase 1 – is not a substrate for RI and is 100-fold
more cytotoxic when delivered to cells by conjugation to
epidermal growth factor (EGF) [30]. The nature of the
cell-binding and internalization functions of BS-RNase,
EDN, ang, and ranpirnase are unknown.
Type I Fungal A Toxins
The fungal cytotoxins include α-sarcin, mitogillin, clavin,
and restrictocin. These are 17 kDa Mr, basic proteins with 20
lysines, four arginines, and eight histidines. α-Sarcin, restric-
tocin, clavin, and mitogillin share 86–99% amino acid
homology [94]. They have four cysteines forming two disul-
fide bridges between residues 6 and 148 and residues 76 and
132. The first disulfide bond is not essential for the catalytic
activity. The proteins show structural homology to RNase
T1 and have catalytic histidines similar to RNase T1 and
other RNases (Fig. 1f). There is a three-turn α-helix packed
against a five-stranded antiparallel β-sheet [146, 147]. Large
positively charged peripheral loops near the active site con-
struct a platform with a concave surface for RNA binding. A
large 39-residue loop L3 may contribute to membrane bind-
ing. They have acquired a mechanism to enter cells – inter-
acting with negatively charged phospholipids in membranes
and translocating across membranes. Further, in the cytosol,
they cleave a single phosphodiester bond (3′ to guanosine
residue 4325) in a highly conserved stem-loop structure of
28S ribosomal RNA. This target is two bases removed from
the target for depurination by the plant rRNA N-glycosidases
[148]. This stemloop rRNA structure is a binding site for
elongation factors, and its modification irreversibly blocks
protein synthesis (Fig. 3). These toxins share antigenicity
with Aspergillus fumigatus allergen I, and produce hyper-
sensitivity reactions in patients. These fungal toxins are
cytotoxic to tumor cells [33–35, 93].
415
Type II Toxins
Type II toxins interact with cell surfaces and produce
changes leading to secondary intracellular signals and
often cell death. Clostridia perfringens α-toxin is a 370
residue, zinc metalloenzyme with phospholipase C
activity [149]. It is the key virulence determinant in gas
gangrene. It binds to membranes in the presence of cal-
cium. It has two domains (Fig. 1f). The N-terminal
domain (residues 1–246) is composed of six stacked
a-helices. It has the phospholipase C active site. There is
a flexible linker (residues 247–255) followed by a
C-terminal domain (residues 256–370) with an eight
(three-strand jelly roll topology). The C-terminal domain
resembles eukaryotic C2 domains and has Ca Z′-dependent
phospholipid binding function. Membrane phospholipid
hydrolysis results in the perturbation of cell metabolism
and activation of the arachidonic acid cascade and
protein kinase C. Cell death and altered physiology such
as endothelial cell integrin and cytokine expression and
platelet aggregation ensues.
Type III Toxins
Type III toxins produce pores in cell membranes leading
to cell death. Sticholysin II from the tentacle nemato-
cysts of the sea anemone Stichodactpla helianthus is a
member of the actinoporin family. It is a 175 amino acid,
19 kDa Mr basic protein containing five tryptophans, 12
tyrosines, and no cysteines [150]. This water-soluble
protein binds membrane lipid (principally sphingomy-
elin) and partially unfolds [151]. The protein’s β-sheets
facilitate tetramer formation [152]. This is followed by
membrane insertion of the N-terminal amphipathic helices.
One nanometer pores are produced, which causes colloid
osmotic shock and cell death.
Cecropins are peptides found in the hemolymph of the
giant silk moth Hpalophora cecropia [153]. The synthetic
cecropin analog, Shiva-1, is a 36 residue basic peptide
devoid of cysteine with a strongly basic N-terminal
amphipathic helix linked via a flexible Gly-Pro linker to
a neutral C-terminal amphipathic helix (Fig. 1g) [154].
The peptide is toxic to bacteria, parasites, and tumor
cells. The peptide binds cell surface acidic lipids and
forms channels in the membranes followed by cell lysis.
Pyrularia thionin (PT), from the parasitic plant
Pyrularia pubera, is a 47 amino acid basic peptide with
five lysines, four arginines, and eight cysteines (which
form four disulfide bonds) [155]. The molecule consists
of two amphipathic α-helices connected to two anti-
parallel β-sheets (Fig. 1h). PT has a hydrophobic and
hydrophilic surface. It binds to cell membrane phos-
416
phatidylserine with its hydrophilic side and interacts with
phospholipid hydrocarbon tails through the hydrophobic
domain. This destabilizes the membrane bilayer with
formation of inverted micelles [156]. The altered
membrane organization can lead to cell death. The mem-
brane depolarization also produces an influx of calcium
and induction of phospholipaseA2 [157].
Proaerolysin from Aeromonas hydrophila is a 53 kDa
Mr protein. It exists as a dimer and binds glycophos-
phatidylinositol-anchored proteins found on the surface
of most mammalian cells. It contains a C-terminal inhib-
itory domain that is cleaved by membrane proteases such
as furin. The N-terminal fragment, aerolysin, oligomerizes
and enters plasma membranes to form highly stable pores
causing cell death [549].
Toxin Modification and Ligand
Conjugation
To prepare immunotoxins, three tasks must be com-
pleted. The toxin normal tissue binding and toxicity
functions must be removed or modified; target cell
selective peptide ligands must be identified; and the
modified toxin and ligand must be covalently linked in
such a manner that there are no alterations to the toxin
translocation and enzymatic functions or the ligand-
binding or internalization functions. An alternative
approach can be used to deliver peptide toxins to cells.
Bispecific monoclonal antibodies that have one binding
domain for target cell surfaces and the other binding
domain to react with peptide toxins have been synthe-
sized. These molecules have shown potent antitumor
activity in vitro and in animal models [480]. Because of
space limitations we have focused our review only on
covalently linked ligandtoxin conjugates.
Toxin Modification
The type I A-B, type I binary toxin, type II, and type III
toxins may react with normal vital tissues and, conse-
quently, produce serious side-effects. The structure/
function studies described above suggest solutions for
the type I and one of the type III toxins.
The lectin subunit of the plant type I A-B toxins may
be removed either by reduction and purification of the A
subunit [8] or production of the A subunit by heterolo-
gous expression [158]. A third approach is to use affinity
crosslinkers to block the B subunit lectin sites [159].
Genetic engineering has been used to produce B subunit
variants with modifications of amino acid residues at the
lectin sites [160].
The normal tissue receptor-binding domains of the bac-
terial type I A-B toxins and the binary toxins have been
Immunotoxins
approached in a similar way to the plant toxins. Trypsin
cleavage followed by reduction and purification have been
used to isolate DT A fragment [161]. DT and PE mutants
with altered amino acid residues important in receptor
binding have been used [24, 162]. The most common
approach for the bacterial A-B-toxins has been to geneti-
cally delete the receptor binding domain. This has been
accomplished successfully for DT with the generation of
DT385, DT388, DAB389, DT390 and DT486 and for PE
with synthesis of PE35, PE38, and PE40 [163, 164].
The type I binary toxin, anthrax toxin, has been modi-
fied both by removing critical receptor-binding residues
of PA domain 4 and by changing the protease cleavage
sequence in PA domain 1 so that the toxin is processed
only on tumor cells [165, 166]. Further, the anthrax
toxin lethal factor has been genetically modified to
attach the PE catalytic domain to the lethal factor PA
binding domain to produce FP59 [550].
Addition of sphingomyelin to type III actinoporin
conjugates, including sticholysin and equinatoxin II,
blocks normal tissue binding [151, 234]. The type III
toxin, proaerolysin, has been modified to replace the
furin site with the prostate cancer specific cleavage pro-
tease, PSA, cleavage sequence [548, 549].
The type I A toxins lack normal tissue-binding func-
tion and hence do not require structural modification for
reducing toxicities.
Ligand Selection
The peptide ligand must bind all the target cells and, in
the case of type I toxins, undergo internalization. In addi-
tion, the ligand should have minimal or no binding to
vital normal tissues. The identification of selective
ligands for immunotoxin synthesis is one of the most
important steps. Table 2 shows the variety of ligand-
receptor systems which have been used for immunotoxin
targeting. Antibodies, antibody fragments, cytokines,
growth factors, antigens, and receptor fragments have
been linked to toxins. Only a subset of the synthesized
immunotoxins have shown selective cytotoxicity to target
cells. Problems with constructs have included steric
hindrance or misfolding of the ligand, insufficient
internalization for type I toxin conjugates, and unex-
pected normal tissue reactivities. Ligands which make
inactive conjugates with one toxin may be highly effica-
cious when either (a) combined with another toxin, (b)
conjugated or fused with a different linker, or (c)
expressed using a different vector or host. These will be
discussed in greater detail in the next section. Recently,
bispecific immunotoxins have been made by Vallera
employing two ligands with one toxin (e.g., EGF and
IL3, sFv anti-ErbB2 with sFv anti-EpCAM, IL13 with
Arthur E. Frankel et al. 417

Table 2. Ligands used to prepare immuntoxins

Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
UCHT1F(ab)’2 Fab’2 fragment CD3ε C UCHT1F(ab’)2-RTA T-cell ALL [401]
BisFv Bivalent sFv CD3ε G A-dmDT390-bisFv T-cell ALL [168]
SFv-Cys SFv-Cys mCD3ε G (DT390-sFv-Cys)2 T-cell ALL [169]
sFv sFv mCD3ε G DT390anti-CD3sFv T-cell ALL [170]
SPV-T3a MAb mCD3 C SPV-T3a-RTA T-cell ALL [411]
UCHT1 MAb CD3ε C UCHT1-CRM9, UCHT1-DTA, T-cell ALL [171,
UCHT1-MSPSA, UCHT1-DT, 172, 312]
UCHT1-ricin
WT32 MAb CD3 C WT32-RTA T-cell ALL [329]
64.1 MAb CD3 C 64.1-RTA T-cell ALL [420]
F(ab’)2 sheep Polyclonal CD3-anti-CD3 C F(ab )2-dianthin, F(ab’)2-saporin, T-cell ALL [20]
anti-mouse Ig F(ab )2-PAP, F(ab’)2-bryodin,
F(ab )2-momordin, F(ab’)2-
momorcochin, F(ab’)2-trichokirin
FN18 MAb rCD3 C FN18-CRM9, DT390-C207 T-cell ALL [173,
diabody 613]
898112-6-15 MAb pCD3 C pCD3-CRM9 T-cell ALL [24]
HB-EGF Growth factor EGFR G HBEGF-saporin Lung cancer [437]
EGF Growth factor EGFR G, C DAB339EGF, DAB486EGF, Lung cancer [30, 175,
EGF-DTA, EGF-RTA, EGF- 176, 477,
hpRNasel, EGF-ang, SAP-EGF 600]
EGF Growth factor EGFR, IL13R G DTEGF13 Prostate cancer, [551,
glioma 556]
TGFα Growth factor EGFR G TGFα-PE40, PEα53L/TGFα/KDEL Pancreas cancer [264,
295]
Mints MAb EGFR C Mint5-ocymoidine, Mint5- Breast cancer [23]
pyramidatine
425.3 MAb EGFR C 425.3-PE Breast cancer [283]
425 sFv EGFR G 425(scFv)-ETA’ Pancreas cancer [584]
528 MAb EGFR C 528-RTA Lung cancer [425]
r3 Mab EGFR C egf/r3Mab-HT Lung [557]
adenocarcinoma
B4G7 MAb EGFR C B4G7-gelonin Lung cancer [472]
14171(dsFv) dsFv EGFRvIII G sFv(14E1)-ETA Gliomas [291]
14171(Fv) sFv EGFRvIII G dsFv(14E1)-ETA Gliomas [291]
Anti- sFv EGFRvIII G Anti-EGFRvIII(Fv)-PE40 Gliomas [262]
EGFRvIII(Fv)
MR1-1 dsFv mEGFRvIII G MR1-1(dsFv)PE38KDEL Gliomas [608]
L2 Growth factor IL2R G DAB389IL2, DAB486IL2, Lymphomas [160, 162,
IL2-PE66GIu4, IL2-Bax, 167, 177,
IL2-ricin, IL2-PE40, IL2-PAP, 178, 413,
IL2-pancRNase1 476]
Mik-betal(Fv) sFv UR G Mik-beta1(Fv)-PE40 Lymphomas [253]
Bell 0 MAb IL2R C BB10-saporin Lymphomas [447]
RFT5 MAb IL2R C RFT5-RTA Lymphomas [382]
Anti-Tac MAb IL2R C Anti-Tac-RTA, Anti-CD25-MLA Lymphomas [314,
413, 10]
Anti-Tac(Fab) Fab IL2R G AntiTac(Fab)-PE40, Anti- Lymphomas [36, 260]
Tac(Fab)-phospholipase C
Anti-Tac(Fv) sFv IL2R G Anti-Tac(Fv)-PE40, Anti- Lymphomas [260,
Tac(Fv)-PE38 279]
RTF5(Fv) sFv IL2R G RFT5(Fv)-ETA’ Lymphomas [285]
GMCSF Growth factor GMCSFR, uPAR G DTU2GMCSF AML [555]
GMCSF Cytokine GMCSFR G DT388GMCSF, DT385-L-GMCSF, AML [179, 180,
GMCSF-PE40, GMCSF-ricin 181]
mGMCSF Cytokine mGMCSFR G DT390mGMCSF, DT388mGMCSF AML [182, 183]
IL3 Cytokine IL3R G DT388IL3, DT388K116W AML [184, 558]
(continued)
418 Immunotoxins

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
26292 sFv CD123 G 26292(Fv)-PE38-KDEL AML [605]
mIL3 CytoKne mIL3R G DT390mIL3, DT389 L-mIL3 AML [185, 186]
Transferrin Growth factor Transferrin C Tf-CRM9, Tf-CRM107, Gliomas, [187, 188,
receptor/CD71 Tf-equinatoxin II, Tf-PE, T-mellALL 234, 259,
Tf-Saporin, Tf-gelonin, 402, 412,
Tf-RNase, Tf-KFT25-RTA 421, 426]
anti-TfR(Fv) sFv Transferrin G DT388-anti-TfR(Fv),anti- AML [28, 29,
receptor/CD71 TfR(Fv)-PE40, anti-TfR(Fv)-EDN, 34, 179,
Anti-TfR(Fv)-pancRNase, 407]
Anti-TfR(Fv)-angL2, Antii-TfR(Fv)
L1, Anti-TfR(Fv) restrictocin
454A12 MAb Transferrin C 454A12-CRM107, 454A12-RTA, Gliomas, breast [188–191]
receptor/CD71 454A12-rRTA, 454A12-RNase cancer
HB21 MAb Transferrin C HB21-PE, HB21-RTA, HB21- Breast cancer [14, 33,
receptor/CD71 gelonin, HB21-bryodin, 191,
HB21-luffin, HB21-α-sarcin 360]
mIL18 Growth factor mIL18R G mIL18-PE38 T cell leukemia [585]
B7 Modulator CD28 G B7-2-L-PE40KDEL GVHD [602]
IP-10 Chemokine CXCR3 G DT390-IP-10-Sralpha Multiple sclerosis [604]
CCL17 Chemokine CCR4 G CCL17-EDN, CCL17-PE38 T lymphoma [606]
HB21 (Fv) sFv Transferrin G HB21(Fv)-PE40 Breast cancer [287]
receptor/CD71
51E9 MAb Transferrin C 5E9-gelonin Breast cancer [414]
receptor/CD71
B2/25 MAb Transferrin C B2/25-saporin Breast cancer [442]
receptor/CD71
OKT9 MAb Transferrin C OKT9-gelonin Breast cancer [459]
receptor/CD71
R17217 MAb Murine transferrin C R17217-rRTA Breast cancer [237]
receptor
Anti-Ly2.1 MAb mTcell antigen C Anti-Ly2.1-ricin T-cell ALL [330]
IL7 Cytokine IL7R G DAB389IL7 Pre-B cell ALL [200]
ASF Lectin Asialogly C ASF-DTA Hepatocarcinoma [201]
coprotein
receptor
IL4 Cytokine IL4R G DAB389IL4, IL-4(38-37)- Gliomas, KS [202, 203,
PE38KDEL, cpIL-4(13D)- 587]
PE38KDEL
mIL4 Cytokine mIL4R G DAB389mIL4, DT390mIL4, AML, [204–206]
mIL4-PE40 mastocytosis
IL13 Cytokine IL 13R G IL 13-PE38QQR, DT388IL 13, Renal cell cancer, [207, 247,
DTEGF13, DTAT13 gliomas 551, 553,
556]
αMSH Growth factor MSHR G DAB389αMSH, DAB486αMSH Melanoma [208, 209]
GRP Hormone GRPR/bb2 G DAB389GRP Adenocarcinoma [210]
NT-4 Growth factor TrkB G DAB389NT4 Neuroblastoma [211]
CD4 T cell HIVgp120 G DAB389CD4, CD4-PE40, HIV + tumors [212, 213,
co-receptor CD4-RTA 337]
3B3(Fv) sFv HIVgp120 G 3B3(Fv)-PE38 HIV + tumors [235]
Anti-gp120 MAb HIVgp120 C Anti-gp120-RTA HIV + tumors [236]
0.5beta MAb HIVgp120 C 0.5beta-RTA HIV + tumors [336]
Anti-gp120 Polyclonal HIVgp120 C Anti-gp120-RTA HIV +tumors [355]
CyanovirinN Antiviral protein HIVgp120 G FLAG-CV-N-PE38 HIV +tumors [274]
7B2 MAb HIVgp41 C Anti-gp41-RTA HIV + tumors [236, 318,
564]
HRG13 Growth factor HER4 G HRG13-PE38KDEL Breast cancer [299]
(continued)
Arthur E. Frankel et al. 419

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
HRGbetal Growth factor HER4 G HRGbeta2-PE38KDEL Breast cancer [299]
HRGbeta2 Growth factor HER4 G HRGbetal-PE38KDEL Breast cancer [299]
Betacellulin Growth factor HER4 G BTC-TX50, BTC-TX48 Breast cancer [300]
48–50
Hrg Growth factor HER4 G DT389hrg, hrg-PE38KDEL, Breast cancer [214, 248,
hrg-PE40 277]
HERB sFv HER2 G hERB-hRNase Breast cancer [582]
FRP5 Mab HER2 G chFRP5-ZZ-PE38 Breast cancer [614]
FRP5(SFv) SFv HER2 G SFv(FRP5)-ETA Prostate cancer [281]
e23(dsFv)2 (dsFv)2 HER2 G e23(dsFv)2-PE38 Lung cancer [290]
e23(dsFv) dsFv HER2 G e23(dsFv)-PE38 Lung cancer [290]
e23(sFv) sFv HER2 G e23(Fv)-PE38KDEL Prostate cancer [292]
520C9 MAb HER2 C 520C9-RTA Breast cancer [316]
741 F8 MAb HER2 C 741 F8-RTA Breast cancer [422]
454C11 MAb HER2 C 454C11-RTA Breast cancer [422]
Mgr6 MAb HER2 C Mgr6-clavin Breast cancer [35]
BACH250 Humanized MAlk HER2 C BACH250-gelonin Breast cancer [11]
Anti-HER2 MAb HER2 C Anti-HER2-saporin Breast cancer [444]
ConA Lectin Mannose C ConA-DTA, ConA-RTA Carcinoma [215, 216]
TRH Hormone TRHR C TRH-CRM45, TRH-CRM26 Pituitary tumors [217]
Ricin Lectin Galactose C Ricin-DTA Carcinoma [218]
sIL 15 Cytokine IL 15R G DAB389IL 15 Lymphoma [219]
D3 MAb p148 C D3-DTA Hepatocarcinoma [220]
GCSF Cytokine GMCSFR G DAB389GCSF, GCSF-PE40 AML [223, 224]
H65 MAb CD5 C H65-RTA, H65-mitogillin, T-cell ALL [93, 394,
H65-gelonin 466]
STI MAb CD5 C STI-RTA T-mellALL [395]
T101 MAb CD5 C Anti-CD5-CRM9, T101-RTA, T-cell ALL [187, 190,
T101-RNase, T101-ricin, 228, 312,
T101-ricin-125l, anti-CD5-MLA, 327, 328,
T101 Fab-RTA, T101 F(ab’)2- 10, 19,
RTA, anti-CD5-momordin 25]
anti-CD5-Pyrularia thionin
OKT1 MAb CD5 C OKT1-sap or in T-cell ALL [451]
SM5-1 sFv gp230 G SMFv-PE38KDELmut1 Hepatocellular [612]
cancer
MEL sFv gp240 G MELsFv-rGel, scFvMEL/TNF Melanoma [569, 574]
ZME01 8 MAb Proteoglycan, C ZME018-RTA, ZME018-gelonin Melanoma [353]
p250
9.2.27 MAb Proteoglycan, C 9.2.27-DTA, 9.2.27-abrin, Melanoma [225, 254,
p250 9.2.27-PE, 9.2 27-gelonin, 307, 311]
9.2.27-RTA, 9.2.27-PAP
NR-ML-05 MAb Proteoglycan, C NR-ML-05-abrin, NR-ML-05-PE Melanoma [254]
p250
SV10016 MAb Proteoglycan, C SV10016-CRM103 Melanoma [187]
p250
Ep2 MAb Proteoglycan, C Ep2-saporin Melanoma [448]
p250
BrE3 MAb Mucin, MUC1 C BrE3-RTA SCLC [375]
Anti-MUC1 sFv MUC1 G sFv(MUC1)-ETA Breast cancer [603]
Mc5 MAb Mucin C Mc5-DTA Breast cancer [226]
BM7 MAb Mucin C BM7-PE Breast cancer [283]
C242 MAb Mucin C C242-PE, C242-NlysPE40, Breast cancer [255, 367]
C242-RTA
C242rF(ab’) Antibody Mucin C C242F(ab’)-PE38QQR Colon cancer [268]
fragment
MBR1 MAb Mumn C MBR1-restrictocin Breast cancer [322]
(continued)
420 Immunotoxins

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
260F9 MAb p55 C 260F9-RTA, 260F9-rRTA Breast cancer [158, 191]
171A MAb Ep-CAM C 171A-DTA, 171A-RTA Breast cancer [227]
Anti-EpCAM sFv EpCAM G DTEpCAM23 Colon cancer [552]
4D5MoCB sFv EpCAM G 4D5MoCB-ETA Colon cancer [568]
A9(Fab)’ Fab fragment p100 C A9(Fab)’-DTA A.castellani [229]
Anti-SV40 Polyclonal SV40 antigens C Anti-SV40-DT SV40 + tumors [230]
Cholera toxin B Toxin fragment GM1 ganglioside C Cholera B-DTA Gliomas [231]
Anti-ConA Polyclonal ConA C Anti-ConA-DTA ConA-treated [232]
tumors
RTB Toxin fragment Galactose C RTB-DTA, RTB-MLA, RTB- Carcinomas [233, 387,
momordin 417]
TEClgM MAb IgM Fc C TEClgM-saporin Myeloma [455]
Protein A Toxin fragment ImmunoglobulinFc C Protein A-RTA Ig + tumors [238]
74124 MAb pCD4 C 74124-PAP Transplants [431]
MT151 MAb CD4 C Anti-CD4-PAP, MT151-RTA, HIV + tumors [239–241,
MT151-blocked ricin, 453]
anti-CD4-saporin
OVB3 MAb Ovarian antigen C OVB3-PE Ovarian cancer [242]
NR-LU-10 MAb Breast cancer C NR-LU-10-PE Breast cancer [243]
antigen
ATF Protease Urokinase PA G, C ATF-PE38, ATF-PE38KDEL, AML, gliomas [244–246,
fragment receptor uPA-SAP, DT388N-termURO, 434]
ATF-saporin
11A8 MAh bFGFR C 11 A8-saporin Breast cancer [435]
bFGF Growth factor bFGFR G bFGF-PE40, hFGF-PE66Glu4, Breast cancer [249, 435,
hFGF-saporin, DT389bFGF 599]
aFGF Growth factor aFGFR G aFGF-PE40, aFGF-PE66Glu4KDEL Breast cancer [249, 252]
PR1(Fv) sFv PR1 antigen G PR1(Fv)-PE38KDEL Prostate cancer [250]
MRK16 MAb P-glycoprotein C MRK16-PE, MRK-RTA, Renal cancer [251, 365,
MRK-saporin 450]
PA Toxin peptide PA receptor G FP59 + PA, FP59 + PA(MMP) Gliomas, [256, 286]
carcinomas
IGF-I Growth factor IGF-IR G IGF-I-PE40 Breast cancer [257]
Anti-CD8 MAb CDS C Anti-CD8-ricin, Anti-CDs-saporin Tcell lymphoma [331, 453]
Lym-1 MAb HLA-DR C Lym-l-gelonin Lymphoma [463]
2G5 MAb HLA-DR C 2G5-RTA Lymphoma [361]
HB55 MAb HLA-DR C HB55-ricin Lymphoma [343]
KM231 MAb Sialyl-Lea-antigen C KM231-RTA Colon cancer [356]
84.1C MAb mlgE C 84.1c-RTA Allergies [396]
Phlp56 Allergan Ant-P5 G P5-ETA’ Allergies [593]
B3 MAb Lewisy antigen G B3-LysPE38 Breast cancer [266]
B3(Fab) Fab fragment Lewisy antigen G B3(Fab)-PE38 Breast cancer [297]
B3(dsFv) dsFv Lewisy antigen G B3(dsFv)-PE38, dsFvB3-gran- Breast cancer [297, 580]
zyme B
B3(Fv) sFv Lewisy antigen G B3(Fv)-PE38 Breast cancer [258]
B1(Fv) sFv Lewisy antigen G B1(Fv)-PE38 Breast cancer [267]
B1(dsFv) dsFv) Lewisy antigen G B1(dsFv)-PE38 Breast cancer [267]
BR96(sFv) sFv Lewisy antigen G BR96sFv-PE40 (SGN10), Breast cancer [21, 278]
BR96sFv-bryodin
Folate Vitamin Folate receptor C Folate-momordin, Folate- KS [261]
LysPE38, Folate-Cys-PE35
Anti-FRbeta Mab Folate receptor C anti-FRbeta-PEA RA [592]
IL9 Cytokine IL9R G rhIL9-ETA’ Hodgkin’s disease [263]
55.1(Fv) sFv Colon antigen G 55.1(Fv)PE38, 55.1(Fv)PE38KDEL Colon cancer [265]
55.1 (dsFv) dsFv Colon antigen G 55.1(dsFv)-PE38, 55.1(dsFv)- Colon cancer [265]
PE38KDEL
ME20 MAh p105 C ME20-lysPE40 Melanoma [269]
(continued)
Arthur E. Frankel et al. 421

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
G28- sFv CD40 G G28-5sFv(VL-VH)-PE40, Lymphoma [18, 270]
5sFv(VL-VH) G28-5sFv(VL-VH)-hryodin
GnRH Hormone GnRHR C GnRH-PE66, GnRH-PE40, Breast cancer [271, 293,
GnRH-PAP 13]
MOC31 MAb EGP-2 C MOC31-ETA252-61 3 SCLC [272]
WF lectin Lectin N-acetyl- C WF-DTA Carcinoma [221]
Galactosamine
hPL Hormone hPLR C hPL-DTA Breast cancer [222]
AntiCD30sFv sFv CD30 G Anti-CD30(sFv)-PE38, Anti- Hodgkin’s [280]
CD30(Fv)-PE38KDEL disease
Ki4 MAb CD30 C Ki4-RTA Hodgkin’s [398]
disease
Ki4(sFv) sFv CD30 G Ki(sFv)-ETA’, scFvKit4- Hodgkin’s [294, 565]
angiogenin disease
BerH2 sFv CD30 G BerH2-scFv-hpRNase Hodgkin’s [609]
disease
BerH2 MAb CD30 C BerH2-saporin, BerH2-RTA, Hodgkin’s [22, 373,
BerH2-momordin, BerH2- disease 416, 428]
dianthin, BerH2-PAP
HRS3 MAb CD30 C HRS3-RTA Hodgkin’s [338]
disease
HRS3Fab’ Fab’ fragment CD30 C HRS3Fah’-RTA Hodgkin’s [338]
disease
TP3 Mab p80 C TP3-PAP Osteosarcoma [174]
TP3(sFv) sFv Osteosarcoma G TP-3(sFv)-PE38 Osteosarcoma [26]
antigen
TP3(dsFv) dsFv Osteosarcoma G TP-3(dsFv)-PE38 Osteosarcoma [26]
antigen
RANTES Chemokine CCR5 G RANTES-PE40, DT390-RANTES Rheumatoid [282, 561]
arthritis
HD6 MAb CD22 C HD6-saporin Lymphomas [452]
HD39 MAb CD22 C HD39-saporin Lymphomas [452]
OM124 MAb CD22 C OM124-PAP, OM124-saporin Lymphomas [348]
RFB4 MAb CD22 C RFB4-RTA Lymphomas [332]
RFB4(dsFv) dsFv CD22 G RFB4(dsFv)PE38, RFB4(dsFv) Lymphomas [288, 562]
PE38KDEL, HA22
LL2 MAb CD22 C LL2-onconase Lymphomas [32]
RFB4 sFv CD22 G DT2219ARL B-cell ALL, CLL [554]
AchRα Antigen Anti-AchR G AchRα-gelonin Myasthenia [594]
gravis
Dsg3ΔNI Antigenic Anti-desmoglen3 G DsgΔN1-PE40KDEL, PE37- Pemphigus [289]
peptide Dsg3ΔN1-KDEL vulgaris
hMN14sFv sFv CEA G hMN14(Fv)-PE40 Colon cancer [296]
C110 MAb CEA C C110-RTA-s°Y Colon cancer [357]
C19 MAb CEA C C19-RTA Colon cancer [304]
CB-CEA-1 MAb CEA C CB-CEA-1-hemolytic toxin Colon cancer [37]
I-1 MAb CEA C I-1-blocked ricin Colon cancer [391]
C27 MAb CEA C C27-ahrin A Colon cancer [9]
E4 MAb Prostate antigen C E4-PE35KDEL Prostate cancer [298]
E4(Fv) sFv Prostate antigen G E4Fv-PE38KDEL Prostate cancer [298]
M6 MAb Idiotype C M6-ricin, M6-RTA Lymphoma [302]
38.13 MAb Pk antigen C 38.13-RTA, 38.13-gelonin, Burkitt’s [306]
38.13-PAP lymphoma
Fab’anti-L3T4 Fab’ fragment Murine C Fab’anti-L3T4-RTA Lymphoma [308]
Tcellantigen
486P3121 MAb Bladder cancer C 486P-RTA + 486P-RTB Bladder cancer [310]
antigen
RFT11 MAb CD2 C RFT11-RTA T-cell ALL [326]
(continued)
422 Immunotoxins

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
35.1 MAb CD2 C 35.1-ricin, 35.1-RTA T-cell ALL [312, 326]
OKT11 MAb CD2 C Anti-CD2-RTA, Anti-CD2-saporin, T-cell ALL [393, 473]
OKT11-gelonin
452D9 MAh p74 C 452D9-RTA Ha-ras + tumors [313]
Thyroglobulin Antigen Ig anti- C Thyroglobulin-RTA Thyroiditis [305]
thyroglobulin
Anti-CD64 Mab CD64 C Anti-CD64-RTA Rheumatoid [598]
arthritis
H2-D(d)tet Tetramer MHC C H2-D(d)tetramer-biotin-avidin- GVHD [601]
saporin
2F8 Mab SR-A/CD163 C 2F8-Saporin Ovarian cancer [610]
E9 sFv FCRL1 G E9(Fv)-PE38 CLL [615]
96.5 MAb p97 C 96.5-RTA Melanoma [424]
HB2 MAb CD7 C HB2-saporin T-cell ALL [434]
TXU MAb CD7 C TXU-PAP T-cell ALL [390]
3A1e(Fv) Fv-Cys CD7 C 3A1e(Fv)Cys-RTA T-cell ALL [408]
3A1f sFv CD7 G Rnase-TRHRQPRGWEQL-anti- T-cell ALL [588]
CD7-sFv
Anti-CD7 sFv CD7 G scFvCD7:sTRAIL T-cell lymphoma [590]
3A1 e MAb CD7 C 3A1e-RTA T-cell ALL [427]
WTI MAb CD7 C WT1-RTA T-cell ALL [317]
791T/36 MAb p72 C 791T/36-RTA Colon cancer [319]
8A MAb Myeloma antigen C 8A-momordin, 8A-saporin Myeloma [320, 321]
IORT6 MAb T-cell antigen C IORT6-hemolytic toxin T-cell ALL [323]
SN5d MAb CD10 C SN5d-RTA Pre-Bcell ALL [324]
Anti-CALLA MAb CD10 C Anti-CALLA-RTA Pre-B cell ALL [380]
AntiGE2 MAb GE2 C Anti-GE2-ricin, AntiGE2-RTA Gliomas [325]
AR3 MAb CAR-3 C AR3-RTA, AR3-ricin, AR3-gelonin Gastric cancer [333, 461]
8C MAb Ovarian cancer C 8C-RTA Ovarian cancer [334]
antigen
My9 MAb CD33 C My9-blocked ricin AML [346]
p67.7 MAb CD33 C p67.7-RTA AML [352]
HuM195 Humanized CD33 C HuM195-gelonin AML [460]
MAlk
Anti-CD33 sFv CD33 G Anti-CD33(sFv)-ETA AML [597]
Anti- MAb Vasopressin C Anti-vasopressin-RTA Pituitary tumor [347]
vasopressin
Hepama-1 MAb Hepatoma antigen C Hepama-l-trichosanthin Hepatoma [349]
Cluster 2 MAb MAb Cluster 2 C Cluster 2 MAb-RTA SOLO [350]
antigen-SCLC
SOKTI MAb T-cell antigen C SOKT1-RTA T-cell antigen [351]
Fib75 MAb Bladder cancer C Fih75-RTA, Fih75-a-sarmn, Bladder cancer [354, 471]
antigen Fih75-gelonin
MAb2 MAb Gastric antigen C MAh2-RTA Gastric cancer [358]
16 MAb Oncofetal antigen C 16-RTA, 16-MLA Lymphoma [359, 374]
Anti-laryngeal MAb Laryngeal cancer C Anti-laryngeal cancer-RTA Laryngeal cancer [362]
cancer antigen
W3 MAb ICAM C W3-RTA Myeloma [363]
Bispecific Bispecific MAlk CD4 and CD26 C Bispecific (CD4/CD26)-blocked Tissue allografts [364]
MAb(CD4/ ricin
CD26)
SWAII MAb CD24 C SWAII-RTA Lymphoma [366]
HAE9 MAb Erythroblast C HAE9-RTA Eyrtholeukemia [368]
antigen
HAE3 MAb Glycophorin C HAE3-RTA Erythroleukemia [368]
(continued)
Arthur E. Frankel et al. 423

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
Bispecific Bispecific MAlk CD4 and CD29 C Bispecific (CD4/CD29)-blocked Tissue allografts [369]
MAb(CD4/ ricin
CD29)
CLL2m MAb CLLantigen C CLL2m-RTA CLL [370]
SEN31 MAb Cluster 5a SOLO C SEN31-blocked ricin SCLC [371]
antigen
317G5 MAb p42 C 317G5-RTA Breast cancer [372]
SEN36 MAb NCAWCD56 C SEN36-RTA SCLC [376]
SEN7 MAb NCAWCD56 C SEN7-PE, SEN7-blocked ricin SCLC [383]
N901 MAb NCAWCD56 C N901-blocked ricin SCLC [400]
BDI-1 MAb Bladder antigen C BDI-1-ricin Bladder cancer [377]
Anti-mu MAb IgM Fc C Anti-mu-RTA Myeloma [378]
Anti-CD6 MAb CD6 C Anti-CD6-blocked ricin GVHD [379]
Anti-CRF MAb Corticotropin- C Anti-CRF-RTA Pituitary tumor [381]
releasing factor
CRF Hormone CRFreceptor C CRF-saporin Pituitary tumor [436]
ScFvH17 sFv Placental alkaline C scFvH17-BSRNase Germ cell tumor [27, 384]
phosphatase
Anti-la MAb la C Anti-la-RTA Carcinoma [385]
Ng76 Mab Melanoma antigen C Ng76-luffin, Ng76-moschatin Melanoma [571, 572]
E6 Mab PSMA C E6-dgA Prostate cancer [583]
A5 sFv PSMA G A5-PE40 Prostate cancer [595]
7E4B11 Mab RPTPβ C 7E4B11-saporin Gliomas [589]
IB4 Mab CD38 C IB4-saporin-S6 Myeloma [596]
HB7 MAb CD38 C HB7-blocked ricin Myeìoma [386]
Anti- MAb Asialo-GM2 C AntiasialoGM2-RTA Carcinoma [388]
asialoGM2
Anti-vbeta6 MAb vbeta6 T cell C Antivbeta6-RTA Myasthenia [389]
receptor gravis
14G2a MAb Disialoganglioside C 14G2a-RTA Neumblastomm [392]
Peanut Lectin D-glucosyl moiety C Peanut agglutinin-RTA Lymphoma [397]
agglutinin
BU12 MAb CD19 C BU12-saporin Lymphoma [441]
B43 MAb CD19 C B43-PAP Lymphoma [432]
HD37 MAb CD19 C HD37-RTA, HD37-saporin Lymphoma [399, 452]
B4 MAb CD19 C B4-blocked ricin Lymphoma [415]
B-c3 Mab CD19 C Anti-CD19:K CLL [563]
Anti-CD19 sFv CD19 G CD19-ETA’ CLL [611]
Anti-CD19 sFv CD19 G DT2219ARL ALL [554]
Fv5191/cys sFv-Cys CD19 C Fvs191-Cys-RTA Lymphoma [406]
Rituximab Mab CD20 C Rituximab/Saporin-S6, Lymphoma [581, 586]
Rituximab-allinase
ONSM21(Fv) sFv Medulloblastoma C ONSM21 (Fv)-RTA Medulloblastoma [419]
antigen
ONSM21 MAb Medulloblastoma C ONSM21-RTA Medulloblastoma [403]
antigen
35 MAb Nicotinic C 35-ricin Strabismus [404]
acetylcholine
receptor
K42C10 MAb Endoglin/CD105 C K42C10-RTA Breast cancer [405]
44G4 Mab CD105 C 44G4-ebulin, 44G4-nigrin b Breast cancer [558, 607]
SN6j MAb Endoglin/CD105 C SN6j-RTA Breast cancer [418]
8OG MAb Alphafetoprotein C 8OG-gelonin Hepatoma [471]
15A8 MAb Breast cancer C 15A8-gelonin Breast cancer [467]
antigen
HB5 MAb C3d receptor C HB5-gelonin EBV infection [474]
Anti-Lyt2.2 MAb Lyt2.2 antigen C Anti-Lyt2.2-gel on in T-cell lymphoma [475]
(continued)
424 Immunotoxins

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
SN7 MAb B cell antigen C SN7-RTA B-cell ALL [409]
Anti-VIP MAb Vasoactive C Anti-VIP-RTA Pituitary tumor [410]
intestinal peptide
Anti-CMV MAb mCMV antigen C Anti-mCMV-RTA, Anti-mCMV- CMV infection [411, 462]
gelonin
Anti-CMV Polyclonal Human CMV C Anti-huCMV-gelonin CMV infection [464]
antigens
Anti-d(beta)h MAb Dopamine beta- C Antid(beta)h-saporin Pitirflary tumors [12]
hydroxlase
M24 MAb CD80 C M24-bouganin, M24-gelonin, Hodkgin’s [15]
M24-saporin disease
1G10 MAb CD86 C IG10-bouganin, IG10-gelonin, Hodkgin’s [15, 570]
IG10-saporin, IG10-gelonin disease
LHRH Hormone LHRHR C LHRH-bovineRNase Breast cancer [31]
Anti-Pbs21(Fv) sFv Pbs21 G Anti-Pbs21(Fv)-Shiva-1 Malaria [38]
Anti- MAb Melanoma antigen C Anti-melanoma-BRIP Melanoma [17]
melanoma
48127 MAb gp54 C 48127-PAP, 48127-saporin Bladder cancer [429]
J3109 MAb CD72 C J3109-PAP B-mellALL [430]
0×7 MAb mCD90 C 0 × 7-DT, 0 × 7-saporin T-cell ALL [199, 315]
O × 7F(ab’)2 F(ab )2 mCD90 C O × 7F(ab’)-saporin T-mellALL [315]
fragment
Anti-Thy1.2 MAb mCD90 C Anti-Thy1-trichokirin, Anti-Thy1- T-mellALL [16, 458]
gelonin
Anti-Thy1.2 Polyclonal mCD90 C Anti-Thy1-RTA T-mellALL [342]
mFclgE Fcfragment mFcεRI receptor G Fc(2′-3)-PE40 Allergies [273]
IR162 MAb rIgEFc receptor C IR162-RTA, IR162-ricin Allergies [303, 309]
mSCF Cytokine mc-kit G mSCF-PE40 SCLC, AML [275]
K1 MAb Mesothelin p40 G K1-LysPE38QQR Mesothelioma [284]
GPI-anchored
K1(Fv) sFv Menothelin p40 G Anti-mesothelin(Fv)-PE38 Menothelioma [276]
GPI-anchored
SS1(dsFv) dsFv Mesothelin p40 G SS1(dsFv)-PE38, D-SS1P Mesothelioma [543, 616]
GPI-anchored
Anti-0X40 MAb T cell antigen C Anti-OX40-RTA Multiple sclerosis [423]
MBP(66-88) Antigenic Anti-MBP Ig G MBP(66-88)-PE40 Multiple sclerosis [301]
peptide
MBPp8799 Peptide anti-MBP G, C MBPp87-99-cyt1Aa Multiple sclerosis [559]
CD64 Mab CD64 C CD64-riA Rheumatoid [566]
arthritis
m22 sFv CD64 G m22(scFv)-ETA’ AML [573]
anti-JL1 Mab JL1 C Anti-JL1-gelonin Leukemias [567]
Campath-1 MAb CD52 C Campath-l-saporin Lymphoma [335]
L6 MAb Lung cancer C L6-ricin Lung cancer [339]
antigen
Anti-CTLA4 Mab CTLA-4 C Anti-CTLA4-saporin Tolerance [575]
Anti-CTLA4 sFv CTLA-4 G scFvCTLA4-perforin Tolerance [576]
8H9 dsFv glycoprotein G 8H9(dsFv)-PE38 Osteosarcoma [577]
LL1 Fusion L2-H2 CD74 G 2L-Rap-hLL1-gamma4P Myeloma [591]
Anti-mFcD MAb msIgD C Anti-mFcD-RTA Lymphoma [340]
SWA11 MAb SCLC antigen C SWA11-RTA SCLC [341]
Anti-T.cruzi MAb T. muzi antigen C Anti-T. cruziRTA, anti-T. Chagas disease [344]
cruzi-abrin A
Anti-CD45RO Mab CD45RO C Anti-CD45RO-RTA HIV [578]
BMAC1 MAb CD45 C BMAC1-RTA, MBAC1-MLA Renal transplant [345]
OX1 MAb rCD45 C OX1-RTA, OX1-MLA Renal transplant [345]
(continued)
Arthur E. Frankel et al. 425

Table 2. (continued)
Type
Ligand Type Receptor/antigen linkage Compounds Disease Reference
M20.4 MAb primate NGFR p75 C M20.4-saporin Neuromas [437]
1921g MAb rat NGFR p75 C 192Ig-saporin, Neuromas [433]
192Ig-trichosanthin
OKT10 MAb CD38 C OKT10-saporin Myeloma [434]
Insulin Hormone Insulin receptors C Insulin-saporin Breast cancer [438]
BB2 MAb Myeloma antigen C BB2-saporin Myeloma [439]
BB4 MAb Myeloma antigen C BB4-saporin Myeloma [439]
Anti-epithelial MAb Epithelial antigen C Anti-epithelial Lung cancer [440]
antigen antigen-colocin 1
Anti-Ab1 MAb Idiotype anti-DNA C Anti-Ab1-saporin SLE [443]
Anti-Id MAb Idiotype C Anti-Id-saporin Lymphoma [456]
lymphoma
Anti-CD37 MAb CD37 C Anti-CD37-saporin Lymphoma [445]
ML30 MAb Heat shock protein C ML30-saporin AML [446]
Anti-AML MAb AML-M5 antigen C Anti-AML-saporin AML [449]
8A MAb Myeloma antigen C 8A-saporin Myeloma [454]
62B1 MAb Myeloma antigen C 62B1-saporin Myeloma [454]
TTC Toxin fragment p15 G DAB389TTC Neuromas [192]
SP Hormone Neurokinin-1 G DAB389SP CML, neuromas [193]
IL6 Cytokine IL6R G DAB389IL6, IL6-PE40, Myelomas, KS [194, 195]
IL6-PE66GIu4
VEGF165 Growth factor flk-1, flk-1/KDR G, C DT390VEGF165, KS [196, 197]
VEGF165-DT385
VEGF121 Growth factor flk-1/KDR G, C DT390VEGF121, KS, breast cancer [196–198]
VEGF121-DT385,
VEGF121/rGel

oLH Hormone Ovine LHR C oLH-gelonin Prostate cancer [457]

gp330 Antigen Anti-gp330 Ig C gp330-gelonin Heyman’s [465]
nephritis
Anti-pichinde MAb, Pichinde virus C Anti-Pichinde virus-gelonin Pichinde virus [468]
virus polyclonal antigens
NDA4 MAb NDA4 Tcell antigen C NDA4-gelonin T-cell ALL [469]
14G2a MAb GD2 ganglioside C 14G2a-gelonin Melanoma [470]
MSN-1 MAb Endometrial Ca C MSN-1-gelonin Endometrial [547]
antigen cancer

RTA, ricin toxin A chain; sFv, single chain antibody; Cys, cysteine; DT, diphtheria toxin; DTA, diphtheria toxin A fragment; CRM9, binding
site DT mutant; HBEGF, heparin-binding epidermal growth factor; ETA, Pseudomonas exotoxin; PE, Pseudomonas exotoxin; PE40,
40 kDa PE fragment; GMCSF, granulocyte-macrophage colony-stimulating factor; IL3, interleukin-3; CRM107, binding site DT mutant;
rRTA, recombinant RTA; mIL3, mouse IL3; mGMCSF, mouse GMCSF; EGF, epidermal growth factor; TGFα, tumor growth factor-alpha;
dsFv, disuffide-stabilized sFv; MAb, monoclonal antibody; R, receptor; G, genetic fusion; C, chemical linkage; RNase, ribonuclease;
AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; CA, cancer; IL7, interleukin-7; IL4, interleukin-4; mIL4, murine IL4;
IL13, interleukin-13; aMSH, alpha-melanocyte-stimulating hormone; NT-4, neurotrophin-4; GRP, gastrin-releasing peptide; HRG,
heregulin; TRH, thyroid releasing hormone; hPL, human prolactin; Wf, Wistaria floribunda; ConA, concanavalin A; HIV, human immu-
nodeficiency virus; SCLC, small cell lung cancer; IL15, interleukin-15; Ep-CAM, epithelial cell adhesion molecule; RTB, ricin toxin B
chain; MLA, mistletoe lectin A chain; KS, Kaposi’s sarcoma; LH, luteinizing hormone; VEGF, vascular endothelial growth factor; CML,
chronic myeloid leukemia; IL6, interleukin-6; SLE, systemic lupus erythematosis; Id, idiotype; SP, substance P; TTC, tetanus toxin
C-terminal domain fragment; T. cruzi, trypanosoma cruzi; SCF, stem cell factor; Gel, gelonin; PAP, pokeweed antiviral protein; Ig, immu-
noglobulin; NGF, nerve growth factor.MBP, myelin basic protein; VIP, vasoactive intestinal peptide; CMV, cytomegalovirus; LHRH,
luteinizing hormone releasing hormone; CRF, corticotropin releasing factor; GVHD, graft-versus host disease; CLL, chronic ymphocytic
leukemia; GnRH, gonadotropin-releasing hormone; IL9, interleukin-9; Tf, transferrin; GCSF, granulocyte colony-stimulating facor; ASF,
asialofetuin; IGF, insulin-like growth factor; FGF, fibroblast growth factor; a, acidic; b, basic; o, ovine
426
the N-terminal fragment of urokinase, sFv anti-CD19
with sFv anti-CD22) [551–554]. Dual specific immuno-
toxins have also been tested by Frankel (urokinase
activated DT with GMCSF ligand) [555].
Conjugation of Toxin and Ligand
The linkage of the modified toxin with the ligand must be
covalent and stable, so that metabolism in the bloodstream
or interstitial tissues will not occur. Both amide linkage
by genetic engineering and chemical crosslinking using
bifunctional reagents such as thiolating compounds –
3-(2-pyridyldithio)proprionicacid N-hydroxysuccinimide
ester (SPDP) [8] or 3-maleimidobenzoic acid N-hydroxy-
succinimide ester (MBS) [24] – have been used. The
coupling must not severely alter the ligand affinity for
its receptor or impair the ability of the toxin domains to
translocate to the cytosol and enzymatically inactivate
cell functions (type I toxins) or interact and alter cell
membrane functions (type II and III cytotoxins). Genetic
fusions are well defined and controlled, and generally
have less effect on toxin functions than chemical conju-
gation. Further, the fusion can be designed so that the
linkage is remote from ligand and toxin domains critical
for their normal functions. Chemical conjugation meth-
ods yield a heterogeneous product due to the reaction of
the derivatizing reagent with multiple amino acid resi-
dues in the ligand and/or toxin. Cysteines and lysines
are the most frequent substrates for bifunctional
reagents. Both chemical conjugates and genetic fusions
have been developed and used on patients with benefi-
cial results. Successful clinical development has been
done with both chemical conjugates and genetic
fusions.
Preclinical Studies with
Immunotoxins
Over a 1,000 immunotoxins have been synthesized in
the past 3 decades, but most have been evaluated with
markedly different tissue culture and animal model
assays. Thus, comparisons are fraught with potential
errors. Nevertheless, in the spirit of a review, we will
attempt to draw some general observations.
The more potent immunotoxins show higher affinity
binding to their cell surface receptors and have more
receptors/cell [189]. Immunotoxin efficacy is also influ-
enced by the location of interaction of the ligand and the
receptor. Antibodies which bind different epitopes on
receptors can vary greatly in conjugate cytotoxicity
[326, 422]. The ability of the ligand-receptor complex
Immunotoxins
to internalize is also a critical parameter, at least for
conjugates with type I toxins [329]. While chemical
conjugates represent the vast majority of reported immu-
notoxins, recombinant toxin conjugates offer advantages
for production [164]. The molecules are homogeneous
with a single site of linkage between the haptophore and
toxophore. Furthermore, the synthesis does not require
manipulations of different chemical components. The
specificity of the immunotoxin is important, since
crossreaction with vital normal tissues can produce
severe toxicities (see the next section). Both normal
human tissue immunostaining and testing in nonhuman
primate models have been used to identify normal
tissues likely to be damaged by systemic immunotoxin
administration [478, 479]. The size of the immunotoxin
appears to influence both efficacy and safety. Larger
conjugates have greater difficulty in reaching extravas-
cular sites of disease in most cases [228]. In one report,
a larger immunotoxin targeting HER2 was more effective
in vivo [614]. The larger molecules have prolonged
circulating half-life, leading to increased vascular
endothelial injury and vascular leak syndrome (VLS)
[8]. However, with conjugates much smaller than 58 kDa
Mr, the immunotoxin will be rapidly cleared by the
kidneys, leading to very short half-life and insufficient
antitumor effect. Further, renal tubular injury is more
likely [169]. Immunotoxins made with human ligands
and toxins are likely to be far less immunogenic than
those made with non-human components [28, 29, 32,
190, 407, 426]. Some toxins perform better for particular
classes of tumors or target cells. DT conjugates are more
toxic to myeloid malignancies than PE, ricin, or other
type I conjugates. This is likely due to the requirement
for routing to the endoplasmic reticulum for the type I
toxins other than DT and anthrax toxins. Since most
myeloid cells rapidly traffic internalized materials to the
lysosome for degradation, conjugates requiring trans-
port to the endoplasmic reticulum do not intoxicate
these cells well [179, 181]. Consequently, improved
efficacy has been observed with DT conjugates for
myeloid disorders. Stability in the bloodstream is also
important. Disulfide-stabilized single chain immuno-
toxins or tandem or dimeric single chain immunotoxins
appear to be more stable in vivo than single-chain Fv
fragment conjugates [163, 168]. These molecules also
have a better in vivo therapeutic index.
None of the immunotoxins which have been tested in
clinical trials to date is perfect. The ligands are generally not
tumor-specific. Many of the conjugates are not recombinant.
The protein components are frequently immunogenic.
The size is often too large. In some cases the target cell
may not always endocytose the immunotoxin-receptor
Arthur E. Frankel et al.
complex. The patients have often not been optimally
selected for the individual agent. However, many of the
newly described immunotoxins which have entered clin-
ical trials in the past few years, or will enter clinical trials
in the near future, have improved characteristics, and
better patient selection is being used. The recent results
in the clinic are very exciting and will be described in
detail in the next section.
Clinical Experience
with Immunotoxins
Clinical trials have been conducted with only several
dozen immunotoxins over the past 3 decades. The com-
plexities in synthesis and purification of these multi-
domain polypeptides, and the necessary but extensive
safety and regulatory hurdles, have limited the rapid
application of this technology. Direct comparisons are
difficult due to the different conjugate constructions; the
different patient populations; and the different routes,
doses, and schedules of drug used in the various trials.
Nevertheless, we will address the efficacy, pharmacoki-
netics, immune responses, and toxicities for each agent
and try to present common principles. Excitingly, sev-
eral of these drugs are showing significant anticancer
activity in phase I and II clinical studies in patients with
chemotherapy-refractory cancers.
Efficacy
Immunotoxins with the highest response rate in clinical
studies have been active in tissue culture in the picomo-
lar range, have been targeted to hematologic malignan-
cies or diseases, and have been fully recombinant
molecules. The only exceptions in which good response
rates were seen in non-hematologic diseases were in
cases in which immunotoxins were administered locally
by intracavitary or interstitial infusions.
BL22 consists of a disulfide-stabilized anti-CD22 sFv
fused to PE38. When given at doses of 3–50 μg/kg i.v.
over 30 min every other day for three doses every month
for up to 14 cycles to patients with chemotherapy refrac-
tory B cell malignancies, there were 61% CR and 19%
PR in purine-analog refractory hairy cell leukemia
(HCL) [481, 482, 617]. The remissions were durable
with only 27% responders relapsing after 10–23 months.
Retreatment again produced CRs. Responses were dose
dependent and the MTD was 40 μg/kg/dose. Neutralizing
antibodies developed in 24% of patients. A reversible
hemolyticuremic syndrome requiring plasmapheresis
was observed. Other side effects were hypoalbuminemia,
427
transaminasemia, fatigue, edema. Thus, BL22 immuno-
toxin appears to be the current best salvage treatment
for relapsed HCL patients. An improved higher affinity
variant, HA22, has begun clinical testing in patients
with refractory B-cell malignancies [562].
LMB2 is composed of an anti-CD25 sFv fused to
PE38. LMB2 has been administered to 35 patients with
lymphomas at doses of 2–63 μg/kg i.v. over 30 min q.o.d.
× 3 [279]. A complete remission was observed in HCL
which was ongoing at 20 months. There were seven partial
remissions in patients with HCL (three patients),
CTCL (one patient), CLL (one patient), HD (one patient),
and ATL (one patient). Responders received at least
60 μg/kg total dose of LMB-2 per cycle. The durations of
remissions have not been determined, but exceed 20 and
6 months for two patients and 1 month for the remaining
patients. Responding patients had clearance of circulat-
ing malignant cells, improvement in skin lesions, and
regression of lymph nodes and splenomegaly. DLT was
reversible transaminase elevations, diarrhea and cardio-
myopathy. MTD was 40 μg/kg/dose. Other side effect
was fever. 17% of patients developed neutralizing anti-
bodies after the first cycle. Drug half-life was 4 h.
Responders received at least 60 μg/kg total dose of
LMB-2 per cycle. LMB-2 was also administered with
MART-1/gp-100 vaccine to patients with metastatic mel-
anoma in an effort to augment anti-melanoma immunity
via Treg depletion [618].
Denileukin diftitox is a fusion protein composed of
the catalytic and translocation domains of DT fused to
human IL-2. Among CTCL patients with stage IB to
IVA disease refractory to other therapies and treated
with 9 or 18 μg/kg/day for 5 days every 3 weeks, there
were 10% CRs and 20% PRs lasting a median of 6
months [486, 488, 489]. Denileukin diftitox produced a
durable complete remission in a patient with transplant-
refractory large cell lymphoma that was ongoing after 5
years [485]. Two patients with low-grade lymphoma
obtained partial remissions. Based on the above results,
in 1999 the FDA made denileukin diftitox the first
approved immunotoxin [539]. Drug half-life was 30 min,
and side effects included an acute cytokine reaction
(fever, chills, nausea, vomiting, myalgias, chest pain,
arthralgias, back pain), transient liver enzyme transami-
nasemia, and a vascular leak syndrome (hypotension,
hypoalbuminemia, dyspnea, edema). Most patients
developed an immune response to the agent, but this did
not correlate with toxicities or response. In patients with
non-CTCL relapsed/refractory T-cell non-Hodgkin’s
lymphoma, the agent produced a 48% remission rate
with 22% CRs and 26% PRs with median response
duration of 6 months [619]. Denileukin diftitox also
428
produced remissions in CLL for a total of 27% overall
response (4% CRs and 23% PRs) and median response
duration of 6 months [620]. Denileukin diftitox given
for patients with relapsed or refractory B cell non-
Hodgkin’s lymphoma yielded 7% CRs and 18% PRs
with median response duration of 7 months [621]. There
are case reports of denileukin diftitox-induced remis-
sions in patients with systemic mastocytosis [622],
extranodal natural killer/T-cell lymphoma [623], periph-
eral T-cell lymphoma [490], subcutaneous panniculitis-
like T cell lymphoma [624], and adult T-cell leukemia
[625]. Among non-malignant conditions, denileukin
diftitox depletes autoimmune activated T cells yielding
a 71% response rate after two to six doses at 9 μg/kg in
patients with steroid refractory acute GVHD [626] and
yielding remissions in patients with psoriasis [491–493].
Additional studies evaluated the role of expression of
the individual IL2R subunits in CTCL response. The
alpha subunit expression was measured by immunohis-
tochemistry [627]. Denileukin diftitox responses
occurred in both CD25 positive and negative tumors,
although patients with strongly positive tumors were
more likely to respond. Responses were also more likely
at 18 μg/kg versus 9 μg/kg [486]. Rare toxicities included
thyrotoxicosis and optic neuritis [628, 629].
Combinations of denileukin diftitox with other agents
including bexarotene, rituximab and hyper-CVAD
enhanced response rates in CTCL, B-cell non-Hodk-
gin’s lymphoma, and adult T-cell leukemia, respectively
[487, 630, 631]. The effects of denileukin diftitox on
IL2R expressing T-regulatory cells has been used to
augment immune responses to poxvirus vaccine and
melanomas [632, 633].
TransMid is a chemical conjugate of transferrin and the
DT-binding site mutant, CRM107. Forty-four patients
with recurrent high-grade gliomas had placement of one
to two catheters into the tumor beds [483, 484]. Forty
milliliters of TransMid (0.66 μg/ml) was delivered by
convection over 5 days. There were 12 responders
including four complete responsers by magnetic reso-
nance imaging (MRI) with median survival of >71
weeks. Toxicity was brain injury. TransMid is being
tested in low grade gliomas and metastatic cancer to
brain [634].
NBI-3001, IL4(38-37)PE38KDEL, was administered
by one to three stereotactic catheters at 0.2–6 μg/mL in
30–185 mL over 4–8 days to nine patients with recurrent
high-grade gliomas [517]. Six of nine patients showed
tumor necrosis by MRI and histopathology. There was
one unmaintained CR lasting >18 months. NB-3001
given at 6 μg/mL × 40 mL to 9 μg/mL × 100 mL intratu-
morally via one to three catheters over 4–8 days resulted
Immunotoxins
in central nervous system toxicity in 22% of patients at
the MTD of 6 μg/mL × 40 mL [635]. MRI showed tumor
necrosis following treatment. Median survival was 7
months. NBI-3001 was administered IV at 0.008–
0.27 mg/m2 days × 5 every 28 days to patients with renal
cancer and non-small cell lung cancer [636]. DLT was
transient transaminasemia. Neutralizing antibodies were
detected in 71% of patients after two cycles. No responses
were seen.
The binding site deleted Pseudomonas exotoxin PE38
fragment used to construct BL22 and LMB-2 was fused
to an anti-mesothelin disulfide stabilized Fv to create
the recombinant immunotoxin, SS1P. SS1P was given
as 30 min IV infusions every other day for three to six
doses to patients with advanced mesothelioma, ovarian
cancer and pancreatic cancer at 18–45 μg/kg/dose [637].
DLT was pleuritis, and the MTD was 45 μg/kg × 3 doses.
There were a few minor responses and over half of
patients had stable disease. Phase II clinical studies are
underway. Combinations with gemcitabine and pacli-
taxel are planned [638, 639]. Novel constructs with
releasable PEGylation have been tested in animals and
they maintain efficacy with reduced immunogenicity
and toxicity [616].
PRX302 is a furin cleavage site modified proaerolysin.
The new site is selectively cleaved by PSA. It was
administered intratumorally to 24 patients with locally
recurrent prostate cancer after primary radiotherapy
failure [640]. The drug was well tolerated without DLT
at doses of 0.03–3 μg/g prostate. Delivery was by a single
multi-deposit, transrectal ultrasound-guided transperineal
intraprostatic injection using a modified brachytherapy
technique. PSA levels decreased in 63% of patients. The
percentage of positive prostate biopsy cores post-therapy
revealed a decrease in 75% of patients with three patients
showing no positive biopsy cores at 30 days post-therapy.
A phase IB study has been initiated at multiple institutions
to optimize the dose and delivery method.
IL13-PEQQR or cintredekin besudotox is composed
of interleukin-13 fused to PE38 with a C-terminal QQR.
Cintredekin besudotox was administered by convection-
enhanced delivery to recurrent glioblastoma multiforme
tumors for up to 6 days [641]. Optimal dose was
0.4 mL/h at 0.5 μg/mL over 50 h through two catheters
with systemic corticosteroids. Toxicity was tumor
necrosis and cerebral edema. 33% of patients had remis-
sions lasting 7–117+ weeks. Response and survival
depended upon optimal catheter placement. Unique
characteristics of patient brain and tumor growth caused
complex fluid distribution patterns after catheter place-
ment and convection delivery. Both 123I-labeled human
serum albumin PET imaging and MR diffusion tensor
Arthur E. Frankel et al.
imaging predicted patient-specific drug distribution by
convection-enhanced delivery and may improve pro-
spective selection of catheter trajectories and targeted
toxin efficacy [642].
The catalytic and translocation domains of diphtheria
toxin (DT388) were fused via a Met-His linker to human
interleukin-3 to make DT388IL3. DT388IL3 was admin-
istered IV over 15 min every other day for up to six doses
to 45 patients with poor-risk, relapsed or refractory AML
or MDS [643]. Half-life was 30 min, and peak levels
were dose dependent. An inter-patient dose escalation
schema was used with doses from 4 to 12.5 μg/kg/dose.
DLT was not observed, but side effects included tran-
sient transaminasemia, hypoalbuminemia, fever, chills,
nausea and vomiting. Antibodies to drug developed
between day 15 and day 30 in most patients. Responses
included one CR lasting 8 months. And two PRs of 3 and
4 months. Duration. A phase IB inter-patient dose escala-
tion study in AML and MDS patients with 10–40% mar-
row blast index (cellularity fraction times percent blasts)
with five daily doses is ongoing. A higher affinity vari-
ant, DT388K116W, is in final stages of testing and is
expected to begin clinical trials in late 2008 [644].
ScFv(FRP5)-ETA was prepared by fusing the scFv of
the anti-erbB2 monoclonal antibody FRP5 to domains II
and III of Pseudomonas exotoxin. ScFv(FRP5)-ETA was
injected intratumorally once daily for 7–10 days with
daily doses of 60–900 μg in patients with subcutaneous
tumor nodules of metastatic breast cancer, colorectal can-
cer and malignant melanoma [645]. Adverse reactions
were local injection site pain and inflammation. Partial or
complete regression of injected nodules was seen in 20%
and 40% of patients, respectively. Next, patients with
metastatic breast cancer, prostate cancer, head and neck
cancer, non-small cell lung cancer and transitional cell
carcinoma were treated on an inter-patient dose escala-
tion schedule systemically with 2–20 μg/kg bolus infu-
sion daily for 5 days of each wk for 2 weeks [646].
The DLT was transient transaminasemia. The MTD
was 12.5 μg/kg, and the peak drug level was 100 ng/mL.
Most patients developed anti-scFv(FRP5)-ETA antibodies
after 8 days. There were no responses. Phase II studies
are ongoing.
Responses have been observed with other immuno-
toxins including Fab’(antiCD22)-dgA, HerH2-saporin,
TP40, DAB486IL2, LMB-1, IgG-HD37-dgA, DA7,
DAB389EGF, antiB4-blocked ricin, T101-RTA, H65-
RTA, 260F9-rA, XMMME001-RTA, N901-blocked
ricin, IgRFT5-dgA, SPV-T3a-dgA/WT1-dgA, DT388
GMCSF, B43-PAP, IgRFB4-dgA, 791/T36-RTA, anti-
TAC-PE, OVB3-PE, 454A12-rA, erb38, and LMB-7
but these molecules have not undergone further
429
development [158, 159, 228, 242, 266, 319, 415, 428,
494–538].
Pharmacokinetics and Tissue Distribution
Larger molecules have longer half-lives in the circulation
and poorer tissue penetration. The half-life of monoclonal
antibody conjugates with RTA, ricin, PAP, and PE ranged
from 9 to 24 h [266, 427, 494, 497, 499]. The molecular
weights were all around 200,000 Mr. There was likely
little clearance of these molecules via renal glomeruli,
and penetration into extravascular sites such as nodes,
marrow, or skin nodules was poor [158, 228, 536]. In con-
trast, the smaller recombinant immunotoxins (60,000–
70,000 Mr) have had shorter half-lives of 1–5 h [279, 481,
485, 501, 531] and should have better penetration into
extravascular sites of disease. However, there are no reports
to date regarding the saturation of extravascular tumor
deposits in clinical trials with recombinant immuno-
toxins. The shorter circulation times and increased vascular
permeability of the smaller recombinant immunotoxins
may contribute to their greater antitumor efficacy and
reduced endothelial toxicity (see below).
Immune Responses
Because most individuals in the US are immunized with
diphtheria toxoid as children, almost half of adults have
anti-DT antibody titers pretreatment with DT conjugates
[540]. Further, between 10% and 20% of patients have
had previous Pseudomonas infections (often subclinical)
yielding pretreatment anti-PE antibody titers [541].
Patients exposed to castor oil may show anti-ricin anti-
bodies. These circulating antibodies reduce the half-life
and AUC for immunotoxins and, in some cases, neutralize
cell cytotoxicity [266, 486, 542]. Even if patients lack pre-
treatment immunity, after administration of the foreign
protein most patients develop anti-immunotoxin antibod-
ies. The exceptions include CLL patients and some
patients with severe immunosuppression. PEGylation or
use of human ligands and toxins may reduce the immune
response [582, 616].
Toxicities
There are two general classes of side-effects due to
immunotoxins. In some instances the targeted toxin
receptor/antigen is present on normal tissues. This can
lead to significant toxicities. LMB-1 and LMB-7 bind the
Lewisy antigen which is overexpressed on many carci-
nomas. The Lewisy antigen is also expressed on normal
stomach mucosa. Initial DLTs for these immunotoxins
430
were nausea, vomiting, and diarrhea with endoscopic
biopsy evidence of gastritis [266, 530]. After prophy-
laxis with omeprozole, antiemetics, and loperamide,
this side effect was alleviated and dose escalation could
proceed. 260F9-rA binds a 55 kDa Mr antigen found on
breast carcinomas and normal Schwann cells [158]. The
clinical trial was stopped after the occurrence of revers-
ible, but prolonged, peripheral neuropathies with
biopsy-confirmed axonal loss. Similarly, clinical trials
with OVB3-PE and 454A12-rA were discontinued after
CNS toxicity and ligand crossreactivity with normal
CNS tissue antigens (an antigen in the pons for OVB3
and transferrin receptors on normal brain capillaries for
454A12) were found [242, 524]. DAB389EGF and erb-
38 reacted with EGFR and HER-2 receptors present on
normal hepatocytes and produced dose-limiting liver
injury [527, 532]. DT388GMCSF reacts with normal
liver macrophages – Kupffer cells. Subsequent cytokine
release triggers dose-limiting hepatocyte damage [531].
Tf-CRM107 produced focal brain injury in the peritu-
moral normal cortex [483]. Histopathology showed
crossreactivity and damage to normal brain capillaries.
Toxicities that are independent of ligand have been
observed in almost every immunotoxin clinical study.
The similarity of the toxicities observed with different
immunotoxin trials suggests that the lesions are due to
the toxin moieties. The five classes of side effects are:
(a) transient constitutional symptoms with fever, chills,
nausea, vomiting, myalgias, arthralgias, asthenia and
hypotension; (b) transient hepatotoxicity with elevated
transaminases and, rarely, other liver enzymes; and (c)
transient vascular leak syndrome (VLS) consisting of
edema, hypoalbuminemia, weight gain, and, rarely, dys-
pnea and aphasia; (d) assorted syndromes including
rhabdomyolysis, hemolytic uremic syndrome, and renal
insufficiency with proteinuria and renal tubular acido-
sis; and (e) allergic reactions. The toxicities may be due
to binding of the toxin moieties to normal tissues.
Reaction of immunotoxins with macrophages, lympho-
cytes, or endothelium may lead to cytokine release with
consequent constitutional symptoms and elevated circu-
lating cytokines. Elevations of interleukin-6 and tumor
necrosis factor alpha have been reported in some but not
all cases [481, 495, 497, 531]. Where studied, corticos-
teroids or infiiximab + rofecoxib appeared to dampen or
eliminate this side-effect [481, 487].
The hepatotoxicity may be directly due to immunotoxin
binding affected by the pI of the ligand in the conjugate
or secondary release of cytokine from macrophages
may damage the liver [543, 544]. In most cases, impair-
ments of hepatic function such as factor VII production
or bilirubin clearance were not observed, and the liver
Immunotoxins
injury was reversible. VLS is a syndrome observed with
interleukin-2, monoclonal antibodies, some chemotherapy
drugs such as taxotere, and many immunotoxins. The
syndrome usually has onset of 4–6 days after initiating
therapy and last 410 days. ‘Leaky’ capillaries throughout
the body produce low serum albumin, edema, weight
gain, dyspnea, aphasia, rhabdomyolysis, and proteinuria.
The abnormal vascular endothelium may be intoxicated
by the immunotoxin directly or may be ‘activated’ by
elevated circulating cytokines. Evidence from tissue
culture and animal models has been reported for a direct
binding site of toxins on vascular endothelial cells and
for an indirect toxic effect of immunotoxin-triggered
inflammatory cytokines on endothelial cells [545, 546].
There do not appear to be effective methods of prophy-
laxis, although paradoxically active hydration appears
to reduce the incidence and severity of VLS. Further,
smaller recombinant immunotoxins appear to have a
lower incidence, possibly due to their shorter circulating
half-life. Excluding patients with prior radiotherapy
may also lower the incidence or severity of the VLS
syndrome [8]. The rare cases of HUS and rhabdomyolysis
may be patient-specific types of vascular injury or due to
ligand (particularly anti-CD22 antibodies). Allergic or
anaphylactoid reactions occur very rarely, are IgE-
mediated, and are treated in a similar way to allergic
reactions to other foreign proteins such as L-asparaginase.
Fluids, cardiac monitoring, oxygen, corticosteroids,
diphenhydramine, theophyllines, and rarely epinephrine
can be given. Patients with anaphylactoid reactions
should not be retreated with the same immunotoxin.
Ongoing and Future Clinical
Studies
The current and near-future applications of immunotoxins
are focused on expanding the use of some of the active,
established agents described above, as well as testing
novel compounds. Further protocols are being performed
with denileukin diftitox, TransMID, NBI-3001, DT388IL3,
DT388K116W,LMB-2,Cintredekinbesudotox, AdmDT390-
bisFv(UCHT1), BL22, HA22, PRX302, scFv(FRP5)-ETA,
PrAgU2/FP59, and DT2219ARL.
These studies should help define the niche for immu-
notoxins in the eventual multi-agent armamentarium for
therapy of cancer. The past 2 decades are finally showing
the original promise for these ‘magic bullets’. Discoveries
in the next few years of methods to modulate cell surface
receptor number and physiology, as well as methods to
ameliorate normal tissue toxicities of these agents,
Arthur E. Frankel et al.
should refine the applications of these chimeric proteins
in the management of cancer and autoimmune disorders.
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receptor and more potent leukemia cell cytotoxicity. Exp Hematol
2004; 32: 277–81.
645. Azemar M, Djahansouzi S, Jager E et al. Regression of cutaneous
tumor lesions in patients intratumorally injected with a recombi-
nant single-chain antibody-toxin targeted to ErbB2/HER2. Breast
Cancer Res Treat 2003; 82: 155–64.
646. von Minckwitz G, Harder S, Hovelmann S et al. Phase I clinical
study of the recombinant antibody toxin scFv(FRP5)-ETA spe-
cific for the ErbB2/HER2 receptor in patients with advanced
solid malignancies. Breast Cancer Res 2005; 7:R617–26.
12 Drug Immunoconjugates
MALEK SAFA, KENNETH A. FOON, AND ROBERT K. OLDHAM
Monoclonal antibodies and their immunoconjugates
represent one of the first practical methods for the selec-
tive treatment of cancer [49, 96]. Monoclonal antibody
technology now allows for the generation of antibodies
or “cocktails” of antibodies that have some selectivity
for cancer tissue as compared with the normal tissue of
origin. These antibodies can be tested as unconjugated
antibody alone or in conjunction with effector cells. The
“signal strength” of the antibody may be made more
powerful by conjugating antibody to drugs, toxins, bio-
logicals, and radioisotopes with different mechanisms
of action and different levels of potency. This chapter
will focus on the use of drug immunoconjugates for
cancer treatment.
The Problem of Heterogeneity:
Antibody-Based Therapeutics as
a Solution
The problem of tumor-cell heterogeneity (Fig. 1) and
the implications for therapy have been reviewed in
Chapter 2. Curiously, although tumor-cell heterogeneity
has been broadly recognized for decades, clinicians
continue to take the simplistic view that treatment need
not reflect a specific approach to heterogeneity [90,
113–115]. Thus, treatment is still designed as if there are
singular underlying common principles useful in cancer
therapy. Single modalities or fixed combinations aimed
at eradicating cancer without a strategy designed to
approach the problem of tumor-cell heterogeneity still
dominate cancer research and treatment [60, 67, 68, 71,
74, 89].
Current data on tumor-cell heterogeneity and the bio-
logic basis of that heterogeneity is covered in Chapter 2
and have been previously reviewed [29]. The basic ten-
ant of these studies is that each primary tumor is com-
posed of a smaller or larger number of clones, each of
which has its own genotypic and phenotypic character-
istics. Metastasis tends to occur from single cells or
clumps of cells from within the primary tumor. By virtue
of a series of phenotypic analyses, as well as certain
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
kinds of genotypic inferences, these authors and others
have demonstrated beyond doubt that heterogeneity is
characteristic of both animal and human tumors (Fig. 1).
Recently, there have been references to the implications of
this tumor-cell heterogeneity for treatment, and proposals
have been made to approach the problem clinically [58,
59, 61–63, 74, 89].
There are two types of tumor-cell heterogeneity [73].
There are differences between patients, with heterogene-
ity being apparent among tumors of the same histological
class. This is macroheterogeneity. Tumors such as breast
cancer can be very similar by histological examination,
and yet lethality can occur in 7 months or 17 years.
Clinical observations have made it clear that what is seen
under the microscope bears little relationship to the
behavior of the cancer in the patient. This sort of “pheno-
typic analysis” has led to further investigations, and it is
now recognized by most cancer biologists that consider-
able differences exist among patients with respect to the
phenotypic characteristics of the cancer. In addition, there
is the problem of heterogeneity within each tumor in each
patient; microheterogeneity. Thus, many studies have
demonstrated that multiple clones may exist within the
primary tumor and that these clones may have different
metastatic capabilities, giving rise to heterogeneity even
among different deposits in a single patient.
Many still believe that even with heterogeneity
between individuals and within each patient’s cancer,
underlying common features still exist and will allow a
general treatment to be developed that might be useful
for all cancer, or all of a histological type of cancer. This
view supports the “drug development paradigm,” wherein
drugs are developed as broadly active agents to be tested
in different histological types of cancer (see Chapter 3).
Another view is that each cancer and its antigenic phe-
notype (and behavior) are unique. This view, currently
held by a minority of investigators, would suggest the
need to individualize treatment for each patient and may
even require individual therapeutic manipulations for
a single patient over the clinical course of his or her
disease based on these differences [65, 68, 71–74].
Acceptance of this hypothesis would dramatically change
cancer treatment and would require a laboratory-clinic
451
452
Figure 1. Illustration of tumor cell heterogeneity
interface of a type not often used in cancer therapeutics.
With the advent of the “new biology” using genetically
engineered biologicals and monoclonal antibodies, one
has diverse mechanisms for the generation of biological
responses, which may match the diversity implicit in
tumor cell populations. For the first time, there is hope
that one can truly approach the heterogeneity of each
patient’s cancer and the other biologic differences of each
patient in a rational manner [36, 58, 59, 60, 62, 63, 65, 74].
Rationale for
Immunoconjugates
Most of the early monoclonal antibody clinical trials
focused on the use of unconjugated single antibodies
in order to determine toxicity, tolerance, the localization
of antibody in solid tumor deposits, and the distribution
of antibody in normal and neoplastic tissues [21, 31,
53–55, 81, 83, 93–95, 96]. Although the clinical responses
to unconjugated antibody have not been consistent, there
is unequivocal evidence that antibody does selectively localize
in tumor deposits and on individual cancer cells after
Drug Immunoconjugates
intravenous injection [62, 63, 93]. Biodistribution
studies utilizing antibody conjugated to tracer quantities
of isotope have demonstrated excellent tumor imaging,
but a considerable amount of the antibody is distributed
to the liver, spleen, and other organs of the reticuloen-
dothelial system [2, 12, 13, 35, 43, 46, 113]. The infre-
quency of response to unconjugated antibody and the
ability of conjugates to induce regression of bulky cancer
in animal models support the concept of immunoconju-
gate therapy.
When considering the development of a conjugate–
monoclonal antibody complex for drug targeting, the
following factors are of importance:
1. The recognition site for the monoclonal antibody should be
located on the surface of the cell.
2. The antibodies should have sufficient tumor tissue specificity.
3. The extent of localization of the antibody at the target site.
4. Biodistribution of the conjugate–antibody conjugate in the
body relative to that of the parent antibody.
5. Stability of the conjugate–antibody complex in blood.
6. The toxicity of the conjugate.
7. The conjugate must be non-immunogenic and biodegradable.
8. The conjugate must be released upon interaction between
the carrier molecule and the cell and the toxic component
must be active at the surface or be internalized into the cell.
Malek Safa et al.
Rationale for Antitumor
Cocktails
There already exist many different monoclonal antibod-
ies to be assessed in clinical trials in patients with solid
tumors [18, 19, 65, 74]. A large number of antibodies for
leukemia, lymphoma, melanoma, lung, breast, and
colon cancer have been described and characterized.
Antibody preparations are available as immunoglobulin
M (IgM), IgA, and various subclasses of IgG. There are
several monoclonal antibodies which have been engi-
neered to be predominantly human. These are: Rituximab
(Rituxan) directed against CD-20 antigen, Campath
(Alemtuzumab) against CD52, Trastuzumab (Herceptin)
against HER-2/neu receptor, Bevacizumab (Avastin)
against VEGF, Cetuximab (Erbitux) and Panitumumab
(Vectibix) against EGFR receptor. These antibodies
have been approved and are in clinical use for the treat-
ment of non-Hodgkin’s lymphoma, CLL, breast, lung
and colon cancer [52, 74, 112]. In addition, the use of
murine monoclonal antibody MAb-17–1A has resulted
in improved survival of patients with Dukes’ C colorec-
tal cancer [82]. A large variety of new monoclonal anti-
bodies, both murine and human, continue to be
discovered and developed. The recent success in the
humanization process has further improved the bio-
availability and reduced the immunogenicity of these
antibodies. Furthermore, the technological evolution
from murine-based therapeutic monoclonal antibodies
to chimeric (part murine part human protein such as
cetuximab), humanized (e.g. trastuzumab) and fully
humanized antibodies (bevacizumab, panitumumab)
has led to reduction in immune-mediated clearance and
hypersensitivity, improved the safety and feasibility of
repeated administration and thereby created a viable
therapeutic strategy. Thus, it is apparent that the limita-
tion for the use of monoclonal antibody preparations in
treatment will not be due to a shortage of antibody prep-
arations [58, 74].
Although the “perfect” antibody for use as an unconju-
gated antibody or as a targeting agent has not been iden-
tified for any human cancer, there are a variety of selective
antibodies available for clinical investigation. Methods
exist to manufacture high-purity (>99%), homogeneous
preparations of monoclonal antibodies. Characterization
as to antibody isotype, level of purity, degree of
contamination by other substances, stability, and other
pertinent physical/chemical characteristics is possible.
Thus, no insurmountable obstacles exist with respect to
testing a wide variety of monoclonal antibodies in
patients (Table 1) [74].
453
Table 1. Optimization of monoclonal antibody therapy
ANTIBODY SPECIFICITY
Immunoperoxidase
Immunofluorescence
Radiolocalization
ANTIGEN CHARACTERIZATION
Biochemical nature
Topography and density
Epitopes
Heterogeneity
ANTIBODY–ANTIGEN INTERACTION
Turnover of antibody bound to tumor cells
Degree of antibody internalization
Antigen affinity
Antigen levels in serum
ANTIBODY DELIVERY
Dose
Regimen
Route
Pharmacokinetics
Comparison of various cytotoxic agents conjugated to the
same antibody
Given a wide choice of antibody preparations and the
ability to prepare them for the clinic, the next consider-
ation is, how does one select preparations and patients
for clinical trials? It seems unlikely that firm rules can
be made on the selection of antibody preparations for all
solid tumors. Obviously, the distribution of the tumor,
its vascularity, its sensitivity to drugs and radiation, and
the quantitative expression of antigens on the tumor cell
surfaces are all significant factors. Using conjugates of
isotopes, drugs, biologicals, and toxins, the issue may
be essentially one of biodistribution within the tumor
bed and access of the toxic agent to the tumor cell within
each tumor nodule. Thus, it is apparent that while cer-
tain principles in the use of monoclonal antibody for
human solid tumors may be important, each tumor type
may have to be individually evaluated for the optimal
use of a monoclonal antibody preparation [73, 74].
Antibodies presumably must reach the tumor bed to
be effective [58, 62, 63]. One general principle has been
that antibody fragments may more quickly diffuse from
the vascular compartment to the tumor bed. Data have
indicated that the more antibody one infuses into the
vascular compartment, the more antibody one delivers
to the tumor cell bed [58, 62, 63]. Although access to the
tumor bed is clearly quite important, retention within
the tumor bed may be equally or more important. The
preparations of antibody fragments may diffuse more
quickly into the tumor nodule [2], but larger molecules
may be retained for a longer time within that same nod-
ule [58]. Therefore, at the level of these elementary
principles, there is much to be learned about the use of
454 Drug Immunoconjugates

CB4F10.11+AC4C2 99%

CB4F10.11+AC4C3 99%

MC6G10+140.72 97%

MC6G10+6PL4 93%

MC6G10+AC4C2 87%

MC6G10+AC4C6 92%

MC6G10+AC4C3 74%

MC6G10+DC3.24 40%

MC6G10+CD4F10.15 96.6%

MC6G10+CD4F10.11 49.2%

Figure 2. Antibody preparations to a single patient’s melanoma as seen on flow cytometry

BREAST TUMOR

C
O BR3
U 97% MPC 105 BR1 + BR3
N 100% MPC 185
T

BR-1
94% MPC 98

1 200
LOG GREEN FLUORESCENCE (VIABLE)

Figure 3. A two-antibody cocktail for breast cancer

these antibody preparations in clinical trials, and these
clinical trials should not yet be subject to hard and fast
rules [58, 74]. On the basis of early assessments of the
heterogeneity of malignant melanoma [45], including
studies of both melanoma cell lines and fresh biopsies
of melanoma nodules, it became obvious that consider-
able antigenic heterogeneity exists. When the data were
analyzed in detail with a panel of antimelanoma mono-
clonal antibodies, each patient’s tumor had its own pattern
of reactivity. As an extension of this approach, we have
generated antibodies for individual patients, making up
to ten antibodies per patient to analyze heterogeneity
[4, 50, 73]. As shown in Fig. 2, the flow cytometry patterns
for each antibody may differ considerably when tested
against the patient’s cancer. By additive testing of the
individual antibodies, a cocktail of antibodies, consisting
of two to five components, can be created to cover all of
the cells in the tumor as assessed by flow cytometry and
immunoperioxidase staining (Fig. 3).
Our data support the concept that tumor-cell heterogene-
ity calls for the generation of antibodies and/or the use
of typing panels to prepare antibody cocktails containing
Malek Safa et al.
multiple components in an attempt to deliver antibody and its
conjugated toxic substance to all the replicating cells in the
cancer [73]. This approach should substantially improve the
ability to target toxic substances selectively to the cancer of
an individual patient. Further analysis is needed with respect
to heterogeneity within the individual. Our preliminary data
appear to indicate that this microheterogeneity is not strik-
ing, and it may be possible to cover it with a sufficiently
complex cocktail. However, the possibility of somatic
mutation and/or selective pressure in treatment leading to
further tumor-cell diversity in patients with metastatic dis-
ease is real [23]. In lymphoma, there is evidence to indicate
a multiclonality; while it is not completely clear, some of
this multiclonality may result from selective pressures
induced by treatment with anti-idiotypic antibody [33, 54].
Clinical-Laboratory Integration
for Drug Immunoconjugate Trials
There are certain important principles in the design and
execution of clinical trials using monoclonal antibodies
[62–64, 72, 74]. To learn from these early trials it is
necessary to have an active and competent laboratory
available for in vitro assessments. It is essential to dem-
onstrate that the antibody reaches the tumor nodule. For
this, the technique of immunohistology using biopsy
specimens subsequent to the infusion of antibody has
been quite valuable [36, 62, 92–94]. The distribution of
antibody within the tumor nodule in contrast to surround-
ing normal tissues can be assessed. This technique also gives
information on heterogeneity of antigen expression.
In vitro overlay with the treatment antibody can assess
saturation, and the use of other antibodies can define
heterogeneity of antigen expression. Isotope-labeled
antibody preparations may also be used to evaluate the
biodistribution of antibody [12, 13, 35, 43, 46].
In addition to these techniques using “fixed” tissue,
cytofluorometry can be used to visualize individual cells
with respect to antigen distribution, antibody localization,
and saturation. When the tumor nodule is disaggregated
after in vivo treatment, one can determine with great preci-
sion the level of antibody localization on individual cells
and, by adding exogenous antibody, the degree of satura-
tion on these individual cells [62, 92–94]. Thus, we have
techniques to assess the delivery of antibody conjugates to
the tumor, as well as the degree of saturation of membrane
antigens. In addition, actual determinations of heterogene-
ity within individual tumor biopsies and between patients
can easily be made from these studies [73].
The ability to measure antigen and antigen–antibody
complexes before and after treatment, and circulating
455
antibody after injection, is critical to an understanding
of the pharmacokinetics in these antibody trials [3, 5, 73].
Further, it is important to measure antiglobulin levels
initially and after treatment in conducting these clinical
trials, since such antiglobulin responses may ultimately
be important in the biodistribution, toxicity and efficacy
of an infused antibody [20, 62, 91–94].
Chemotherapeutic Drug
Immunoconjugates
The rationale for conjugating antibody to chemothera-
peutic drugs is fairly straightforward. These drugs have
been used for many years in clinical studies. The spec-
trum of activity and the toxicology of these agents are
well understood. In addition, there is considerable evidence
to support the belief that dose–response curves exist
with these drugs in the killing of tumor cells. Their lack
of selectivity for cancer and the major problem of nor-
mal tissue toxicity are well known. All of these factors
constitute a firm basis for attempting antibody delivery
to target the chemotherapeutic agent to the tumor site.
Surprisingly, very few clinical studies have been done
using antibody-targeted chemotherapy. Perhaps the reason
relates to the lower specific activity seen in vitro with these
conjugates. Compared with immunotoxins, drug conju-
gates have been less active in preclinical studies, and this
finding has discouraged investigators with respect to the
possible in vivo activity of such preparations [23, 33].
Pre-Clinical Studies
Preclinical studies using chemotherapeutic agents conju-
gated to antibody have been reported for a variety of
drugs [10, 19, 22, 33, 40, 44, 47, 57, 79, 97, 100, 101,
102]. These studies have indicated that drug conjugates
can have in vitro and in vivo activity. The drugs most
often used have been vinca alkaloids, methotrexate,
daunorubicin, and doxorubicin. In one study methotrex-
ate was coupled to an IgM monoclonal antibody targeted
to a variety of tumors, including mouse teratocarcinoma
[9]. Deposition of immunoconjugate in viable tumor and
lack of binding to antigen negative normal tissue was
demonstrated in an in vivo animal model. Also, metho-
trexate conjugated to monoclonal antibody has been com-
pared with methotrexate-IgG (nonspecific antibody) and
free methotrexate against EL4 lymphoma [34]. The
methotrexate-antibody conjugate was three and seven
times more effective against EL4 lymphoma than metho-
trexate-IgG and free methotrexate, respectively. Similarly,
paclitaxel–antibody conjugates afford selective toxicity
456
and are more cytotoxic in vitro than equimolar concentra-
tions of free paclitaxel or free paclitaxel plus free anti-
body [37]. In an in vivo model of xenografted tumors,
systemic administration of paclitaxel–antibody conju-
gates prevented tumor growth and prolonged survival of
mice better than free drugs [37]. In another study, amin-
opterin which is a more potent antifolate than methotrex-
ate, was coupled to a monoclonal antibody [84]. These
investigators demonstrated that administering leucovorin
48–72 h following a sublethal dose of the aminopterin–
antibody conjugate resulted in maintenance of the anti-
tumor efficacy of the immunoconjugate and a significant
reduction in toxicity. Vindesine (a vinca alkaloid) has
been conjugated with different anti-tumor antibodies,
such as anti-CEA, melanoma, osteosarcoma and others
[78, 86–88]. Studies with vindesine-anti-CEA conjugates
[88] indicate that they increase the therapeutic index of
vindesine by decreasing the toxicity and increasing the
specificity to tumor. Furthermore, evaluation of the con-
jugate against non-CEA producing colon carcinoma
xenograft and CEA-producing xenograft showed that sig-
nificant retardation in the growth of tumor was observed
only in CEA-producing tumor [101]. The anthracycline
family of antitumor antibiotics, most notably doxorubicin
(DOX) and daunorubicin, has been used extensively for
drug targeting applications (102). BR96-DOX, which is
chimeric with human IgG1, binds to Ley-related tumor-
associated antigen expressed on most human carcinomas
and on normal cells of the gastrointestinal tract of humans,
dogs, and rats [107]. BR96-DOX induced cures of human
lung, breast and colon carcinomas in athymic mice and
rats [38, 107, 108], and syngeneic colon tumors in immu-
nocompetent rats [100, 101]. In another study [116], the
monoclonal antibody MSN-1 (IgM), which reacts with
endometrial adenocarcinomas, was combined with DOX,
and the complex showed significant antitumor activity in
athymic mice bearing endometrial carcinoma cell tumors.
In one study, IMMU-110, an anti-CD74-DOX immuno-
conjugate was cytotoxic to non-Burkitt and in Burkitt
non-Hodgkin lymphoma cell lines. In addition, IMMU-
110 was therapeutic in drug-sensitive and drug-resistant
non-Hodgkin lymphoma animal models, suggesting that
antibody targeting can bypass the multi-drug resistant
(MDR) drug efflux system that prevents free doxorubicin
from being therapeutic [11]. Anti-CD74-DOX immuno-
conjugate (IMMU-110) is cytotoxic in non-Hodgkin’s
lymphoma models and overcomes MDR [11]. In another
study, IMMU-110 showed significant activity against
multiple myeloma cell lines and in SCID mouse models
of disseminated multiple myeloma [103]. A two-step
method has been developed for targeting cytotoxic drugs
into tumor cells [14]. It first involves the binding to tumor
Drug Immunoconjugates
cells of antibody-phospholipase C (PLC) immunoconju-
gates. Then, liposomes containing daunorubicin are intro-
duced which are specifically lysed at the tumor site by
PLC to release their cytotoxic contents in the vicinity of
the tumor cells. For tumor xenografts in mice, the anti-
body-PLC/liposome approach resulted in an inhibition of
tumor growth without eradication of tumors [14]. In
another report [85], idarubicin, an analogue of daunorubi-
cin, was coupled to a CD19 antibody. A human pre-B-cell
acute lymphoblastic leukemia cell line (NALM-6) was
implanted into nude mice and this immunoconjugate
was demonstrated to have substantial anti-tumor activity,
was non-toxic and was stable in vivo. It was proposed as
an excellent immunoconjugate for clinical trials.
Another anti-tumor antibiotic agent, DU-257 which is
a duocarmycin derivative, was conjugated to a monoclonal
antibody (KM231) specifically reactive to GD3 antigen
which is highly expressed on the surface of many malig-
nant tumors. The conjugate showed significant growth
inhibition of a human colorectal carcinoma cell line
(SW1116) [106].
Recently, two new antibody–drug conjugates have
been developed as potential therapies for colon cancer
and lung cancer. The antibodies were hMN-14 (labetu-
zumab), a non-internalizing humanized antibody that
binds to the carcinoembryonic antigen (CEA) expressed
by many solid cancers, and hRS7, a rapidly internalizing
humanized antibody targeting epithelial glycoprotein-1
(EGP-1) expressed on high amounts in certain human
cancers. The drug selected for conjugation was SN-38,
the active metabolite of irinotecan, a chemotherapeutic
agent used for the treatment of colorectal, lung, and other
cancers. SN-38 cannot be systemically administered to
patients with cancer due to its toxicity and poor solubility.
In an animal model of colon cancer with lung metastases,
therapy with labetuzumab-SN-38 increased median sur-
vival time two-fold to 73.5 days compared to non-treated
and non-targeting conjugate controls. Similar results
were observed in animals bearing human non-small cell
lung cancer treated with hRS-SN-38 [28].
In a different approach [17] investigators studied
maytansinoids. These drugs are 100–1,000 times more
cytotoxic than common anticancer drugs. These investiga-
tors felt that the limitation of drug immunoconjugates is
that they act stoichiometrically, requiring much higher
intracellular concentrations to achieve the comparable
cytotoxicity as compared to a protein toxin where one mol-
ecule in the cytoplasm leads to cell death. Maytansine was
coupled to a monoclonal antibody via disulfide containing
linkers that are cleaved intracellularly to release the drug.
They demonstrated antigen specific cytotoxicity of cul-
tured human cells and minimal systemic toxicity in mice
Malek Safa et al.
with an excellent pharmacokinetic profile. In one study, the
immunoconjugate of b-76-8, a monoclonal antibody
against HM1.24/BST-2 (CD317) which is a cell surface
antigen overexpressed on multiple myeloma cells, with the
analog of maytansine was shown to be active in a xenograft
model of human multiple myeloma [76]. Similarly,
SAR3419, a novel humanized anti-CD19 antibody (huB4)
conjugated to a maytansine derivative (DM4) produced
100% cures in two non-Hodgkin lymphoma cell lines [1].
These studies and many others in preclinical models using
antibody conjugated to chemotherapeutic agents lend sup-
port to the idea that drug-conjugated antibodies may allow
better targeting of chemotherapeutic agents to the tumor
site [7, 16, 19, 23, 30, 41, 42, 64–68, 77, 105, 109].
Src proto-oncogene family protein tyrosine kinases
(PTKs) play a key role in cell function and attempts have
been made to develop agents that specifically inhibit Src-
family PTKs. In one report [110], a general PTK inhibi-
tor, Genistein, that inhibits all members of the Src PTK
family was used to target to cancer cells. Genistein is an
isoflavone derived from fermentation broth of
Pseudomonas. It is also a natural occurring tyrosine
kinase inhibitor present in soybeans. Genistein inhibits
purified Lck kinase from human lymphoid cells at micro-
molar concentrations. These investigators conjugated
Genistein to an anti-CD19 monoclonal antibody. An anti-
CD19 antibody was chosen because the CD19 receptor is
not shed from cells, it is internalized upon association
with antibody and is physically and functionally associ-
ated with the Src protooncogene family PTKs including
Lck. Human acute lymphoblastic leukemia of the pre-B
cell variety was treated in a severe combined immunode-
ficient mouse model with this immunoconjugate. These
investigators demonstrated that the immunoconjugate
bound with high affinity to the leukemia cells, inhibited
selectively the CD19-associated tyrosine kinases, and
triggered rapid apoptotic cell death. Furthermore, at less
than one tenth the maximum tolerated dose, greater than
99.99% of the leukemia cells were killed, which led to
100% long-term event-free survival in these animals. In
another study [55], LL2, an anti-CD22 monoclonal anti-
body against B-cell lymphoma, was covalently linked to
the amphibian ribonuclease, onconase, a member of the
pancreatic Rnase A superfamily. The LL2-onconase con-
jugate showed significant antitumor activity against dis-
seminated Daudi lymphoma in mice with severe combined
immunodeficiency disease. Furthermore, the life span of
these mice was increased 135% as compared to controls.
Similarly, CMC-544, an IgG4 CD-22-humanized tar-
geted immunoconjugate of the antitumor antibiotic cali-
cheamicin, showed activity against a panel of non-Hodgkin
lymphoma cell lines. In the same study, lymphoma-bearing
457
mice treated with rituximab in combination with CMC-
544 had the longest median survival as compared to con-
trol mice [39]. Targeting CD20 and CD22 with rituximab
in combination with CMC-544 results in improved anti-
tumor activity against non-Hodgkin’s lymphoma pre-
clinical models. In another study, antibody–drug
conjugates to CD79 (anti-CD79-MCCDM1 and anti-
CD79b-MC-MMAF) showed activity in non-hodgkin
lymphoma cell lines [80].
Clinical Trials
One of the first reports using drug-labeled antibody used
antibody 791T/36 conjugated to methotrexate (MTX) [7].
Initial studies with this conjugate demonstrated that meth-
otrexate conjugation did not alter the pharmacokinetics
or tumor localization indices for the antibody labeled with
131
I [25]. In a different study [8], 16 patients with primary
colorectal cancer were injected intravenously with I131-
labeled 791T-36-MTX and imaged using a gamma cam-
era after 48–72 h. Assays of tissue radioactivity showed
high tumor uptake of drug-antibody conjugate in 13 of 15
tumor specimens. Also, the monoclonal antibody KS1/4,
which targets an antigen expressed on epithelial malig-
nancies and some normal epithelial cells were conjugated
to methotrexate [26]. Eleven patients with advanced
non-small cell carcinoma of the lung were treated up to a
maximum tolerated antibody dose of 1,750 mg/m2 with a
cumulative dose of methotrexate of 40 mg/m2. Toxicities
in this study included fever, anorexia, nausea, vomiting,
diarrhea, abdominal pain, guaiac positive stools and
hypoalbuminemia. Two patients had episodes of aseptic
meningitis. One patient had a greater than 50% decrease
of two lung nodules. Post-treatment tumor biopsies
demonstrated binding of the immunoconjugate to tumor
epithelial cells. Some of the gastrointestinal toxicities
may also have been secondary to binding of the immuno-
conjugate to a similar antigen on gastrointestinal epithe-
lial cells.
Mylotarg (gemtuzumab ozogamicin, previously known
as CMA-676) is an FDA approved antibody-targeted
chemotherapy agent consisting of the humanized murine
CD33 antibody to which the calicheamicin γ1 derivative
is attached [111]. It is assumed that binding of Mylotarg
to the CD33 antigen results in internalization followed
by the release of the potent antitumor antibiotic cali-
cheamicin and subsequent induction of cell death. In one
study [111], 122 patients with relapsed acute myeloge-
nous leukemia (AML) were treated with Mylotarg as a
single infusion at a dose of 9 mg/m2. Each patient received
two Mylotarg doses with at least 14 days between the
doses. Within 3–6 h after infusion, near complete satura-
458
tion of CD33 antigen sited by Mylotarg was reached for
AML blasts. Furthermore, Mylotarg induced dose-
dependent apoptosis in myeloid cells in vitro. In a multi-
center trial [98], 142 patients with CD-33 positive AML
in first relapse were treated with two doses of Mylotarg.
Thirty percent of patients achieved remission with a
favorable safety profile. Based on these results, Mylotarg
was approved for the treatment of relapsed CD-33 posi-
tive acute myeloid leukemia.
In another study, 48 patients with follicular (FL) or
diffuse large B-cell lymphoma (DLBCL) were treated
with CMC-544 (antiCD22 conjugated to calicheamicin).
The objective response rate was 69% and 33% for patients
with FL and DLBCL, respectively [27]. In another dose
escalation study of AVE9633, an antiCD33-Maytansinoid
immunoconjugate, antileukemia activity was observed in
patients with relapsed or refractory CD-33 positive
acute myeloid leukemia [48]. In a phase I study, huN9-
01-DM1 (BB-10901) which is a humanized monoclonal
antibody–maytansinoid immunoconjugate that binds
with high affinity to CD56, was shown to be safe and
clinically active in patients with relapsed or refractory
CD56 positive multiple myeloma [15]. In addition, in a
phase II trial, huN901-DM1 showed activity in patients
with CD-56 positive relapsed small cell cancer [51].
Other studies further support the possibility of clini-
cal trials with drug immunoconjugates [18, 19, 25, 30,
32–34, 104]. The rationale for such studies has been
previously described [61–67, 72–74] and the dose-
delivery curves for tissue penetration and antigen satu-
ration have been fully described [62, 69, 73–75]. Issues
of antibody specificity, antigen heterogeneity, optimal
linkers, drug selection, and specific activity of the
immunoconjugate all remain under active investigation.
We have utilized cocktails of antibodies custom tai-
lored to individual patient tumors [68, 71, 73, 75]. These
studies have utilized a biopsy of the cancer from each
patient and either the generation of new antibodies to
that patient’s tumor or the use of existing panels of anti-
bodies to type the patient’s tumor [3, 50]. From these
two methods, cocktails of two to six antibodies were
generated and conjugated to doxorubicin [56, 68–71] or
mitomycin-C [75]. The immunoglobulin-to-doxorubicin
ratios were in the range of 1:40 to 1:60. This level of
coupling did not alter the antibody’s in vitro immunore-
activity, and excellent in vitro cytotoxicity was observed
on antigen-positive cell lines compared to antigen-nega-
tive controls. The spectrum of clinical toxicities was
strikingly different for doxorubicin conjugated to anti-
body as compared with free doxorubicin [68, 70]. More
than 1 g of doxorubicin on up to 5 g of antibody was
Drug Immunoconjugates
administered over a 3-week period without alopecia or
severe bone marrow depression. In several of these
patients, antibody delivery was confirmed by biopsy of
skin and analysis of cytologically positive pleural fluid.
Persistence of antibody conjugates for up to 1 week
post-treatment was noted with doxorubicin doses of
50 mg borne on approximately 300 mg of antibody.
These doxorubicin–monoclonal antibody cocktail
immunoconjugates were used in 24 patients. Mild toxic
reactions were seen in 17/24 (fever, chills, pruritis, and
skin rash) after the administration of these drug immu-
noconjugates. In several patients there was limited anti-
genic drift among the sequential biopsies within the
same patient over time. Five minor responses were seen
with these conjugates. Two patients with breast cancer
had isolated improvement in skin ulcerations, and three
additional patients with other types of cancers had minor
responses. None of these responses were sufficient to
reach a partial response (50% reduction in mean tumor
diameters) by standard oncological criteria.
Toward the end of these studies and at the higher
doses of doxorubicin, toxicities were noted in these
patients. Urine color turned red, and alopecia with bone
marrow suppression was noted. On reexamination of the
doxorubicin procured from Adria Laboratories, it was
noted that the preparation method for the doxorubicin
had changed. A stabilizer (methylparaben) had been
added to the preparation (RDF-Adriamycin), which
likely changed the stability of the doxorubicin immuno-
conjugates. This change occurred near the end of the
study, and the clinical studies were terminated to deter-
mine a better method for manufacturing doxorubicin
immunoconjugates in light of the changed chemistry
[23, 24, 56, 69, 70, 73].
A subsequent study was carried out using mitomycin-
C immunoconjugates [75]. Nineteen patients were
treated with mitomycin-C conjugated to similar cock-
tails of monoclonal antibodies. Thrombocytopenia at
the 60 mg (mitomycin) dose in this protocol was dose-
limiting. The anti-mouse antibody titers were lower in
the mitomycin-C compared to the doxorubicin-treated
patients [5]. No responses were seen with mitomycin-C
immunoconjugates, although several patients had less
tumor-related pain after treatment [73].
The role of drug resistance in cancer treatment is
another issue to be considered. The current widely held
opinion is that tumor cells, once resistant, cannot effec-
tively be treated with the same drug. A variety of
approaches using mechanisms to overcome membrane
resistance have been suggested. However, antibody-
mediated delivery may be another such mechanism, and
Malek Safa et al. 459

it is unclear whether doxorubicin-resistant tumor cells
will be resistant to doxorubicin conjugates if delivered in
appropriate quantities. In vitro and animal model data
have confirmed sensitivity to doxorubicin conjugate
where the cell line was resistant to free drug [19, 22–24].
These and many other issues will be approachable through
the use of drug–antibody conjugates. Other studies com-
paring drug and toxin conjugates are of interest in regard
to the issue of specific activity [23, 25]. While the immu-
notoxins had a higher specific cytolytic capability in vitro,
and may be most useful where target cells express low
levels of antigens, drug conjugates may be preferable
when target cells express a high density of antigens or
when large doses of conjugate are needed to penetrate
tumors. In these situations, higher-specific-activity conju-
gates (toxins) may be too toxic for clinical use.
Conclusions
Our studies, as well as many similar studies, clearly
indicate the potential for the new selective approach to
cancer treatment. Selective and effective new anti-can-
cer approaches are needed in the systemic treatment of
human malignancies. Many very toxic molecules are
well known to medical science (Table 2). Of key impor-
tance is the delivery of these toxic substances to tumor
cells in ways that will avoid the undesirable side effects
on normal tissues. Given these considerations, it is prob-
ably no longer necessary to distinguish between drugs
and toxins. Perhaps we should speak of strong and weak
drugs (chemicals), in that toxin molecules are simply
biological chemicals with a higher degree of toxicity
than the chemicals we commonly identify as drugs. This
may, however, only partially be the case, since most tox-
ins are complex biologicals and most chemotherapeutic
drugs are relatively simple chemicals. Such differences in
size and structure may profoundly affect in vivo trafficking
and immunogenicity.
With the advent of monoclonal antibodies and the
possibility of delivering toxic substances selectively to
cancer cells, many toxic chemicals previously rejected
as chemotherapeutic agents may now be recognized for
use in association with antibody in an immunoconjugate
[99]. Pro-drug strategies and methods of in vivo activation
may add further selectivity [6]. Thus, cancer treatment
can now enter a new era, since selective delivery offers
the hope of increased specific activity against the cancer
with less toxicity to the patient. With FDA approval of
Mylotarg, clinical proof of principle for drug immuno-
conjugates was achieved [74]. We are now in an era
where costs [58, 68, 73], not technology, will now be the
limiting factor in developmental therapeutics [59].

Table 2. Immunoconjugates with cytotoxic agents

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13 Targeted radionuclide therapy of cancer
JOHN M. PAGEL, OTTO C. BOERMAN, HAZEL B. BREITZ AND RUBY F. MEREDITH
This chapter is an update of the former Chapter 13
“Radiolabeled Monoclonal Antibodies for Management
of Metastatic Cancer”. The emphasis on the update is
use of antibody as well as non-antibody radionuclide
conjugates in treatment of cancer.
Introduction
Radiolabeled monoclonal antibodies and peptide based
radiopharmaceuticals have been evaluated as vehicles to
selectively target radioactivity directly to tumor cells for
more than a decade. Success in achieving significant
remissions in patients with hematologic malignancies
such as Non-Hodgkin Lymphoma (NHL) and acute
myeloid leukemia (AML) has encouraged investigators
to continue working to optimize radioimmunotherapy
(RIT) approaches. RIT has thus become the third stan-
dard modality of treatment in hematologic malignan-
cies, while promising results have been obtained in
some solid tumors. Until the past few years, these thera-
pies have focused almost entirely on using beta emitting
radionuclides, carried directly by antibodies. In NHL,
for example, response rates have varied from 50% to >80%,
with best responses seen using non-myeloablative doses
of therapeutic radionuclide as first-line treatment in
radiosensitive tumors [1–4]. RIT has also been employed
with success as a boost to the radiation dose in combina-
tion with other treatments, particularly in association
with stem cell rescue, as adjuvant therapy, or for treating
small volume disease.
Despite the enormous potential for targeted therapy,
problems were identified soon after the early clinical
RIT trials were completed. Initially it was thought that
any tumor could be targeted efficiently with monoclonal
antibodies. However, the long circulatory half-life of the
blood-borne radiolabeled antibody causes prolonged
high background activity levels leading to non-specific
normal organ exposure to radiation. After the detection of
peptide receptor expression by tumors, peptide analogues
began to be investigated for specific tumor targeting.
Peptides used for tumor targeting show several advantages
over antibodies: peptides are small and show rapid diffu-
sion into (target) tissues resulting in rapid pharmacokinetics.
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
Their fast blood clearance could lead to high tumor-
to-background ratios shortly after administration of the
radiopeptide.
Considerable expansion has been accomplished with
use of pretargeted RIT and other methods to enhance
tumor-to-normal organ tissue ratios such as the use of
alpha emitting radionuclides. This chapter will focus on
these clinical therapeutic aspects of targeted radionu-
clides for diagnosis and therapy of tumors. The current
state of clinical use of radiolabeled antibodies and radi-
opeptides, as well as the state of development of new
compounds and future innovations are reviewed.
Discussion of pre-clinical studies will be limited to
those that offer insights into future direction for clinical
study. The radioisotopes appropriate for diagnosis and
therapy and the radiolabeling methods that are currently
in use are briefly described.
Radionuclides for Radioimmunotherapy
The choice of radionuclide with which to label antibodies
or other targeting entities is governed by several consid-
erations [5, 6]. These include the clinical indication as
well as physical properties such as mode of decay,
energy, and abundance of the emissions and half-life, as
well as chemical properties affecting protein attachment
and in vivo handling; and finally production aspects
including specific activity, availability at needed scale, and
cost. Recognition of the advantages and disadvantages
of the available choices is important since different
strategies are required to maximize their potential.
Dose rate is a specific factor modifying therapeutic
efficacy, particularly for beta emitting radionuclides.
Traditional external beam radiation is given at a much
higher rate than internally administered radionuclides.
In RIT, radiation is delivered in the range of 10–30 cGy/h
and continuously decreasing because of decay.
Generally, effectiveness of cell killing goes down as the
dose rate lowers because more time is available for
repair of sublethal damage [7]. Considering the dose
rate effect, some have suggested that 20–30% more dose
is needed to sterilize tumors compared to fractionated
external beam treatment [7, 8], although a review of RIT
studies in animal xenografts by Wessels suggests dose
463
464
effects from RIT are comparable to external beam [9].
An inverse effect resulting in enhancement of low-dose
rate effects has been observed where cells accumulate in
the radiosensitive G2M stage of the cell cycle, which
may contribute to efficacy of the low-dose rate radiation
of RIT. The type of malignancy may also be a factor as
lymphomas have been especially sensitive to inverse
dose rate effects [10].
The physical half-life of the radionuclide, as well as
the biological half-life for tumor uptake, retention, and
elimination from normal tissues of the carrying vehicle
must be considered for radionuclide selection. In par-
ticular, radionuclide half-life considerations include
retention time for antibody or another targeting agent
such as a peptide in tumor in order to deliver a dose
commensurate with the fraction of injected activity that
localizes to target tissue. Thus, with conventionally
radiolabeled antibodies or peptides (i.e., injected with
radionuclide bound to targeting agent, the half-life must
be long enough for tumor uptake as well as tumor irra-
diation during time of useful tumor to normal tissue
ratios). As examples, 131I (8 day t1/2), 186Re (3.7 day t1/2)
and 90Y (2.7 day t1/2) are suitable for whole antibody
pharmacokinetics where maximum tumor uptake of
intact antibodies requires 24–48 h, but tumor retention
can persist for several days [11, 12]. Conversely 188Re
(17 h t½) would be more compatible with short-lived
small molecule targeting.
Another general limitation of radiotherapy is that the
maximum rate of decay, hence therapeutic efficacy,
occurs at the time of injection. For any process that
involves slow accumulation in tumor, typical for anti-
body protein, being a large molecule, much of the radia-
tion decay affects non-tumor tissue before selective
tumor to non-tumor ratios are achieved. Therefore, much
effort has gone into engineering of various antibody and
peptide forms over the last several years to further over-
come targeting limitations. Intact IgG antibodies are
approximately 150 kD molecular weight and thus are
relatively large molecules for their role as targeting vehi-
cles. The whole antibody characteristics of slow disap-
pearance from the blood and the slow tumor uptake
kinetics have limited their ability to achieve high tumor-
to-normal tissue and blood ratios, and have resulted in
high radiation exposure of the radiosensitive marrow
cells. Early efforts to improve on the pharmacokinetic
limitations of whole antibodies included removing the
Fc portion to yield F(ab’)2 fragments of 100 kD size.
These have been further split into Fab’ (50 kD), or in
some cases, Fab fragments of similar size. In general,
blood disappearance rates, and tumor-to-normal tissue
Targeted radionuclide therapy of cancer
ratios increased with use of these smaller constructs, but
absolute tumor uptake and retention was decreased. In
the case of Fab and Fab’ fragments, increased kidney
localization also occurred because the efficiency of fil-
tration for proteins of this size increases and tubular
reabsorption occurs. Improved penetration and retention
in tumor has been further explained with the use of a
variety of novel molecular antibody forms with different
sizes in both solid and hematologic malignancies. These
include chimeric and humanized whole antibody forms,
CH2 deletion constructs, single-chain forms, diabodies,
minibodies, and fusion proteins [6, 13–18]. The CH2
deletion constructs are derived from the constant region
of about 125 kD, while single chain Fv of 25 kD are from
the variable region. Diabodies are divalent forms of Fvs
(scFv dimer) whereas minibodies are scFv-CH3 dimers of
80 kD [13, 14, 19]. Evaluation of these immunotargeting
forms has indicated gains in tumor-to-normal tissue
ratios, but limitations still exist in kidney and liver uptake
as well as tumor retention.
Types of emissions considered are beta particles
(electrons emitted with a wide range of energies), alpha
decay (in which helium + two particles are emitted),
and low energy Auger electron emissions, a byproduct
of electron capture decay. While the particulate property
of the radiation decay mode determines the therapeutic
potential, gamma ray emission often associated with the
radiation decay, provides the ability to image the biodis-
tribution in vivo, thus indicating tumor localization and
non-target uptake and retention. Ideally, gamma radia-
tion should be of low abundance such that contribution
to non-target organ irradiation is minimized.
Beta Particle Decay
A wide range of beta emitter energies of emission and
half-life are available for RIT (Table 1). Adelstein [20],
Howell [21], Humm [22], and Wheldon [23] have consid-
ered the application of beta emitters with respect to emis-
sion energy and penetration, number of cell reversals, and
size of tumor targets. Beta emitters have the unique
advantage of exerting their cytotoxicity by a crossfire
Table 1. Selected beta emitting radionuclides for RIT
Beta emitting Path-length Energy delivered
isotopes t1/2 (mm) (MeV)
Iodine-131 8.1 days 0.8 0.6
Yttrium-90 64 h 2.7 2.3
Rhenium-188 17 h 2.4 2.1
Copper-67 62 days 0.05–2.1 0.6
Lutetium-177 6.7 days 0.04–1.8 0.5
John M. Pagel et al.
effect as only occasional beta particles will achieve lethal
double strand DNA breaks. When a sufficient concentra-
tion of emission occurs in a tissue volume, the probability
of lethal hits increases, predominantly from sources
bound to other cells. This crossfire killing property obvi-
ates the need for targeting every cancer cell in contrast to
antibody targeted delivery of drug or toxin conjugates.
The crossfire effect is efficient for tumor masses larger in
diameter than the average beta path length.
Relatively few beta emitters have been studied in clini-
cal trials for RIT. These include the low energy emitter
131
I that has a maximum energy of 0.61 MeV and rhenium
(186Re) with a moderate energy of 1.07 MeV. The higher
energy beta emitter that is currently most widely used
radionuclide in clinical trials and clinical practice is 90Y
(2.3 MeV). Comparing isotopes, Humm [22] estimated
54% absorption of energy from particles of 131I in a 1 mm
cluster but only 10% absorption of 90Y in this small vol-
ume. Further analysis by Wheldon and coworkers suggest
that moderate or lower energy emitters should be used for
clusters of up to [107] cells while an emitter such as 90Y
should be used for tumors of [108] cells or greater [23].
Alpha Particle Decay
There has been significant interest in targeting alpha
radiation for RIT over the past several years (Table 2).
With the large He particle emitted, alpha decay results
in high linear energy transfer (LET) or energy delivery
over a distance of only several cell diameters. This
results in the advantages of high potency and lack of
oxygen dependence with correspondingly no shoulder
on the cell survival curve. The short path length, how-
ever, results in the need for much more homogeneous
targeting for complete tumor cell kill. Alpha emitters
that have been studied for antibody mediated tumor
targeting include 212Bi (t1/2 1.06 h), 213Bi (t1/2 0.76 h), 211At
(t1/2 7.2 h), and 225Ac (t1/2 10 days).
As these are short-lived radionuclides, applications
have been mainly in leukemia and lymphoma and non-
systemic administration for other tumors such as intraperi-
toneal injection. Macklis and coworkers evaluated physical
characteristics of 212Bi labeled antibody in lymphoma [24].
Table 2. Selected alpha emitting radionuclides for RIT
Alpha emitting Path-length Energy delivered
isotopes t1/2 (μm) (MeV)
Bismuth-213 46 min 84 6.0
Actinium-225 10 days 50–80 8
Astatine-211 7.2 h 60 6 rated into a new generation of novel treatment approaches
465
Only 27 212Bi atoms and 4 alpha-particle tracks (“hits”) of
212
Bi were required for a log of target cell killing. Satisfying
the requirement of homogeneous targeting of alpha emit-
ters is more difficult with solid tumors which are often
poorly vascularized and may be access-limited by high
interstitial pressure due to poor lymphatic drainage [25].
Toxicity to normal tissue via antibody cross-reactivity can
be high over a short range due to the potency of alpha
radiation and lack of repair potential of double-stranded
DNA lesions. As expected, good efficacy has been seen in
micrometastatic models, and in established solid tumors
little more than a delay in tumor growth has been observed.
Alpha particle radiotherapy studies have been reviewed in
detail by Hassfjell and Brechbiel [26].
Emerging Radionuclides for use in RIT
Additional pre-clinical investigations have focused on a
wider range of beta and alpha emitting radionuclides with
different energies of emission and half-lives to improve
the efficacy of RIT. Two promising beta emitting radio-
nuclides are 177Lu (0.5 MeV) and 67Cu (0.6 MeV).
Lutetium-177 has a moderate beta energy that is similar
to 131I and possesses a 6.7 day half-life. An advantage for
the use of 177Lu is that this lanthanide has similar chela-
tion properties to 90Y, suggesting that use of standard
chelating agents will lead to stable delivery of 177Lu to
targeted cells. Moreover, 177Lu can be produced in high
specific activities relatively inexpensively, making the
use of this agent in clinical trials probable for the near
future. Two copper radionuclides have emerged with con-
siderable promise for RIT of hematologic malignancies.
Copper-67 has a t1/2 greater than 2 days and emits beta
particles with reasonable energy for therapeutic benefit.
Copper-64 is also a beta emitting isotope, but in addition
64
Cu emits positrons, making this radionuclide appealing
for use with positron emission tomography to allow for
accurate estimations of the absorbed doses of therapeutic
67
Cu. Readily accessible quantities of these copper radio-
metals have been limited, however, and must be more
widely available at affordable cost before these agents
gain widespread application in RIT protocols.
Antibody-Based
Radiopharmaceuticals
Extensive work exploiting the safety and efficacy of
radiolabeled monoclonal antibodies that target specific
surface antigens on malignant cells has been incorpo-
466
for cancer. The characteristics of the antibodies that
must be considered for RIT are the same as those for
immunotherapy, and are listed in Table 3.
Beierwaltes’ successful treatment of metastatic mela-
noma in a single patient with 131I-labeled polyclonal anti-
body raised to the patient’s own tumor was the first
indication that RIT might be feasible [27]. Order et al.
reported initial trials of 131I and 90Y-labeled antiferritin
polyclonal antibodies [28–30]. These studies showed that
131
I-labeled antiferritin polyclonal antiserum could pro-
duce regressions of bulky hepatomas [29]. Four of 24
patients had partial responses (PR) and one had a com-
plete response (CR). Single doses as high as 150 mCi were
given with acceptable hematologic toxicity. Subsequent
studies combined cytotoxic agents (5-FU and adriamycin)
and external beam radiation with lower doses (50 mCi in
divided doses) of antibody-labeled radiation and achieved
a response rate of 48% (7% CR; 41% PR). Monoclonal
anti-ferritin was found to be inferior to polyclonal antisera
because of high localization in the liver, and 90Y-labeled
anti-ferritin to be superior to 131I-anti-ferritin.
Responses following up to 30 mCi 90Y-anti-ferritin
antibodies were also seen in patients with Hodgkin
disease when used in conjunction with chemotherapy [30].
A response rate of 62% was reported in 27 patients with
advanced Hodgkin lymphoma with 90Y-anti-ferritin.
Marrow toxicity at 30 mCi limited further dose escalation,
although multiple infusions were administered in some
patients. In a separate approach polyclonal radiolabeled
anti-ferritin antibody was delivered by dose fractionation.
At low doses, this did not improve response rates, yet may
Table 3. Considerations for radiolabeled antibody studies
Antibody Antigen – Location, cellular density,
modulation, circulating
Affinity
Specificity
Mass
Molecular form – intact, fragments
Form-murine, chimeric, humanized, human
Radionuclide Half-life
Emissions-type, energy, abundance
Chemistry
Specific activity
Preparation Percent protein bound
Immunoreactivity
Purity
Patient Pharmacokinetics
Images, normal organ biodistribution,
tumor uptake
Radiation absorbed dose estimates
Antiglobulin response
Tumor response following RIT
Targeted radionuclide therapy of cancer
need to be evaluated at doses requiring marrow rescue to
achieve optimal results [31].
Bierman et al. reported that 30 mCi 90Y-polyclonal
anti-ferritin combined with high-dose chemotherapy in
patients with Hodgkin disease undergoing bone marrow
transplantation did not cause any additional adverse
effects [32]. The marrow irradiation from the 90Y did
not cause increased toxicity nor did it interfere with
re-engraftment in this study. Although no definite
improvement in outcome could be attributed to the RIT,
the lack of additional toxicities with the additional low-
dose rate irradiation suggested that further similar studies
in patients with better performance status were warranted.
Overall those early radiolocalization studies per-
formed with polyclonal antibodies, however, suggested
that more homogeneous antibodies were required in
order to be clinically useful. Hybridoma technology
permitted careful selection of monoclonal antibodies
directed to particular tumors, such that antibodies with
the highest localization and binding potential and with
the lowest cross-reactivity with normal tissue are identi-
fied. The majority of the clinical trials have utilized
radiolabeled murine monoclonal antibodies either with
or without other concomitant therapy, combined with
131 90
I, Y and less frequently, 186Re as the radiolabel.
Radioimmunotherapy of Hematologic
Tumors
Lymphoma
The most impressive results of RIT to date have been
achieved in NHL. Several monoclonal antibodies conju-
gated with radioactivity have been evaluated in the treat-
ment of NHL. RIT has proven to be particularly effective
in the treatment of patients with NHL because of the
sensitivity of lymphocytes to radiation, the large num-
ber of target antigens on the surface of lymphocytes and
the vascular accessibility of the malignancies. The first
RIT trials for B-cell lymphomas were using 131I-labeled
Lym-1 antibody. Lym-1 is a novel, murine, IgG2a mono-
clonal antibody that recognizes a 31–35 kD membrane
antigen expressed on malignant B cells characterized as
HLA-DR10 [33].
The DeNardo group has generally taken the approach
of administering lower doses of fractionated RIT, con-
sistent with standard oncological practice using chemo-
therapy and external beam therapy, to deliver higher
overall dosages with lower toxicity. In a study using low
dose, fractionated therapy 30 patients with B-cell malig-
nancies received either 30 or 60 mCi 131I-labeled Lym-1
antibody delivered every 2–6 weeks up to a total dose of
John M. Pagel et al.
300 mCi. Acute toxicity (e.g., fever, rash) was mild and
transient. Dose limiting toxicity was thrombocytopenia,
particularly in patients with low platelet counts at base-
line and in patients with lymphomatous marrow involve-
ment. Eleven of the 30 patients received the intended
dose of 300 mCi (thought to be the maximum-tolerated
dose (MTD) based on marrow dose estimates). The
overall response rate (ORR) was 57% for all patients
and 94% of the 18 patients who received at least
180 mCi. Ten percent of patients achieved a CR lasting
10–44 months [34].
A second study was designed to define the MTD and
efficacy of at least the first two, of a maximum of four,
high doses of 131I-Lym-1 given 4 weeks apart [35].
Dosages studied were 40 to 100 mCi/m2 administered
every 4 weeks. Twenty patients with different advanced
NHL, histologic subtypes that were resistant to standard
therapy were treated. Dose-limiting toxicity was found
to be thrombocytopenia at an MTD of 100 mCi/m2. The
ORR was 52% with seven patients (33%) achieving a
CR, persisting for median duration of 14 months. All
three patients that received the MTD (100 mCi/m2 × 2)
had a CR. The median duration of survival was 19
months in responding patients and 1.9 months in non-
responders. Considering both studies, responses were
observed in 30 of 45 patients including 95% of patients
who received more than 200 mCi of 131I Lym-1. Of note,
the high dose approach achieved a higher CR rate than
the low dose approach.
The Lym-1 antibody has also been labeled with 67Cu,
in four patients to compare the biodistribution and
dosimetry to 131I-Lym-1 [36]. Both the uptake in tumor
and the retention time in tumor were found to be higher
with the 67Cu-Lym-1 than the 131I-Lym-1. Marrow dose
estimates, however, were lower with 67Cu and liver dose
estimates were higher. Copper-67-Lym-1 was further
administered to 12 patients in a Phase I/II dose escala-
tion trial. Up to four doses of 25 or 50–60 mCi/m2 were
administered, the lower dose when marrow involvement
was present. Dose limiting toxicity was hematologic
and the ORR was 58% [37]. Although the supply of
67
Cu is at present uncertain, this remains an isotope worthy
of further study.
The majority of RIT investigators have aimed however
to deliver the MTD in one injection in order to deliver the
highest dose rate possible to the patient. This has been, at
least in part, due to the concern of immunoglobulin devel-
opment toward the therapeutic antibody, which could
potentially limit the number of infusions that can be safely
administered. Results have been reported by various inves-
tigators using single non-myeloablative doses of radiola-
beled antibodies directed against a variety of lymphoid
467
differentiation antigens, including HLA class II variant
molecules, idiotypic immunoglobulins, the CD 5, 20, 21,
22 and 37 antigens, and the IL2 receptor. Targeting radia-
tion to the CD19, CD20, and CD22 antigens has proven,
however, the most effective in clinical NHL trials. These
target antigens are expressed in high density with >95% of
all B-cell lymphomas expressing CD19 and CD20, and
70% expressing CD22. Two anti-CD20 radiopharmaceu-
ticals (131I-tositumomab and 90Y-ibritumomab tiuxetan)
have now been approved for use in patients with relapsed
or refractory indolent disease.
131
I-Tositumomab
Kaminski et al. first described the use of low-dose
131
I-labeled anti-CD20 antibody (tositumomab) to treat
patients with relapsed indolent NHL [39]. Patients
receiving this therapy first received tositumomab labeled
with a trace amount of 131I (5 mCi) followed by a thera-
peutic infusion based on a predicted estimate of the
whole body radiation absorbed dose. Each dose of radi-
olabeled antibody was preceded by an infusion of unla-
beled anti-tositumomab antibody to reduce normal,
antigen-specific binding. A Phase I dose-escalation trial
was subsequently conducted to assess the toxicity and
efficacy of non-myeloablative doses of 131I-tositumomab
[40] Patients were treated with whole-body radiation
doses escalating in 10 cGy increments from 25 to 85 cGy.
Twenty-eight of the 34 patients enrolled completed
treatment. The overall response rate was 79% and 50%
achieved a CR. The median duration of response was 12
months for all patients and 16 months for those patients
who reached a CR. Sixteen of 17 patients who achieved
a response of 6 months or more in duration remained
alive 6 years after treatment. The whole body clearance
was found to be variable between patients, most likely
dependent on the B-cell load and tumor bulk, thus the
tracer study was deemed necessary to administer the
MTD to each patient. Hematologic toxicity was dose
limiting at a whole-body radiation absorbed dose of
75 cGy. These early studies led to development of a
diagnostic dose to determine pharmacokinetics for
selecting a therapy dose that will deliver a whole body
dose of 75 cGy, the MTD for patients without compro-
mised marrow function (in patients with lower baseline
counts, 65 cGy is the target whole body dose).
Based on the low toxicity and the clinical results
found in the Phase I trials, a trial was designed to evalu-
ate 131I-tositumomab with the primary clinical endpoint
being the comparison between the patient’s duration of
remission following 131I-tositumomab therapy and the
duration of remission on the patient’s last chemotherapy
[42]. The study included 60 indolent or transformed
468
NHL patients who were refractory prior to chemother-
apy. Seventy-four percent of the patients with indolent
NHL experienced a longer duration of response to
131
I-tositumomab compared to 26% who experienced a
longer duration of response to prior chemotherapy (p <
0.001). The median duration of remission after
131
I-labeled antibody was 6.5 months, approximately
doubling the 3.4-month median duration of remission
patients experienced on their last chemotherapy regi-
men. In addition, a response was observed in only 28%
of patients following their last chemotherapy regimen
compared to 65% of patients following a regimen of
131
I-tositumomab (p < 0.001).
Updated and long-term data on 53 chemotherapy-
relapsed/refractory patients, including patients who had
received a prior autologous stem cell transplant (ASCT),
that were treated with iodine 131I-tositumomab have been
provided [43]. Dose-escalations were conducted sepa-
rately in patients who had or had not undergone a prior
ASCT until a non-myeloablative maximally tolerated
whole body dose was established (non-ASCT = 75 cGy,
post-ASCT = 45 cGy) and then 14 additional non-ASCT
patients were treated with 75 cGy. Forty-two of 59
patients (71%) responded and 20 patients (34%) had a
CR. Thirty-five of 42 patients (83%) with low-grade or
transformed NHL responded versus 7 of 17 (41%) with
de novo intermediate-grade NHL (p = 0.005). For all 42
responders, the median progression-free survival was 12
months and 20.3 for those with CR. Seven patients
remained in CR 3 to 5.7 years. Sixteen patients were
retreated after progression; nine responded and five had
a CR. Reversible hematologic toxicity was dose limiting.
Only ten patients (17%) had human anti-mouse antibod-
ies detected. Long-term, five patients developed elevated
thyroid-stimulating hormone levels, five were diagnosed
with myelodysplasia and three with solid tumors. An
expanded access study of 359 patients with relapsed or
refractory low grade or transformed low grade NHL was
carried out. In 273 patients evaluable for response, the
ORR was 58% and the CRR was 27%. Follow up at 17
months showed that the median duration of response had
not yet been reached [44]. A single, well-tolerated treat-
ment with iodine 131I-tositumomab can, therefore, pro-
duce frequent and durable responses in NHL, especially
low-grade or transformed NHL.
As a first line therapy in follicular lymphoma,
131
I-tositumomab is currently showing promising results.
Kaminski et al. reported a 95% overall response rate
(75% complete response) in 76 previously untreated fol-
licular lymphoma patients. Molecular remissions were
demonstrated in 80% of patients who achieved a com-
plete response [45]. Actuarial 5-year survival was 59%
Targeted radionuclide therapy of cancer
with a median PFS of 6.1 years. Analysis according to
the Follicular Lymphoma International Prognostic Index
risk categories demonstrated that 85% of the patients
were intermediate or high risk [46].
Phase II studies of RIT as consolidation following
upfront therapy with a variety of chemotherapy regimens
including CHOP and fludarabine have resulted in high
overall response rates and excellent PFS [2, 3]. In a phase
II trial conducted by the Southwest Oncology Group, the
complete response/unconfirmed complete response rate
improved from 39% after CHOP to 69% following con-
solidative 131I-tositumomab with an estimated 5-year
PFS and overall survival of 67 and 87%, respectively [2].
Based on these promising results, an ongoing phase III
trial compares consolidation with 131I-tositumomab fol-
lowing CHOP chemotherapy to observation in untreated
follicular lymphoma patients. The current US Intergroup
phase III trial randomizes untreated follicular lymphoma
patients to either six cycles of CHOP followed by
131
I-tositumomab or to rituximab plus CHOP providing a
valuable comparison between two contemporary treat-
ment options for the frontline therapy of follicular
lymphoma. Whether upfront cytoreduction with chemo-
therapy benefits previously untreated patients receiving
131
I-tositumomab is unknown. Clinical outcomes follow-
ing RIT alone were similar to those achieved with che-
motherapy followed by RIT and a randomized trial will
be required to address this issue.
90
Y-ibritumomab tiuxetan
Ibritumomab (IDEC-Y2B8, Zevalin®) is a murine IgG1
kappa monoclonal antibody that covalently binds
MX-DTPA (tiuxetan), which chelates therapeutic 90Y.
A multi-institution Phase I/II study evaluated the safety
and efficacy of treatment with 90Y-ibritumomab tiuxetan
in 58 patients with low or intermediate grade and mantle-
cell NHL [47]. The amount of 90Y-labeled antibody was
dose-escalated from 0.2 mCi/kg to 0.4 mCi/kg. Prior to
90
Y treatment, biodistribution studies were conducted
using a surrogate 111In-labeled antibody conjugate to
allow for gamma imaging. Rituximab was pre-adminis-
tered as the unlabeled blocking antibody (250 mg/m2) to
allow for known sites of disease to be more easily visu-
alized and this also decreased the projected dose of radi-
ation to this spleen and marrow. The MTD was 0.4 mCi/
kg (0.3 mCi/kg for patients with baseline platelet counts
100 to 149,000/ml). The only significant toxicity was
myelosuppression. The ORR for the intent-to-treat pop-
ulation (n = 51) was 67% (26% CR; 41% PR); for low-
grade disease (n = 34), 82% (26% CR; 56% PR); for
intermediate-grade disease (n = 14), 43%. Responses
occurred in patients with bulky disease (≥7 cm; 41%)
John M. Pagel et al.
and splenomegaly (50%). The median time to progres-
sion for patients who responded was 12.7 months. The
phase I/II trials were followed by a phase III trial that
randomized 143 eligible patients to either rituximab or
90
Y-ibritumomab tiuxetan radioimmunoconjugate to
demonstrate that the combination of the 90Y radioiso-
tope to the murine anti-CD20 antibody provided addi-
tional efficacy over the unconjugated (“cold”) rituximab
alone [48]. A planned interim analysis of the first 90
patients demonstrated an ORR of 80% with
90Y-ibritumomab tiuxetan versus 44% for rituximab (P
< 0.05). To provide additional evidence of the benefit of
90
Y radioimmunotherapy over rituximab immunother-
apy, patients who were nonresponsive or refractory to
rituximab were enrolled in an additional trial and treated
with 90Y-ibritumomab tiuxetan 0.4 mCi/kg. An ORR of
46% was achieved in these rituximab-refractory patients.
These results provide further evidence of the added
value of 90Y [49].
More recently a phase II trial combining six cycles of
chemotherapy with cyclophosphamide, doxorubicin, vin-
cristine, and prednisone (CHOP) followed 6–10 weeks
later by 90Y-ibritumomab tiuxetan was conducted to eval-
uate the efficacy and safety in untreated elderly diffuse
large B-cell lymphoma (DLBCL) patients. In 20 eligible
elderly (age ≥ 60 years) patients with previously untreated
DLBCL using this novel regimen the ORR to the entire
treatment regimen was 100%, including 95% CRs and
5% PRs. Four (80%) of the five patients who achieved
less than a CR with CHOP improved their remission sta-
tus after 90Y-ibritumomab tiuxetan RIT. With a median
follow-up of 15 months, the 2-year PFS was estimated to
be 75%, with a 2-year OS of 95%. This study therefore
has demonstrated the feasibility and tolerability of this
regimen for elderly patients with DLBCL [4].
High-dose Radioimmunotherapy with Stem Cell
Support for Lymphoma
Data from prior studies showing an inverse relationship
of recurrence rates to radiation dosage led investigators
to hypothesize that if the radiation dose could be further
safely escalated to tumor sites, relapse rates would be
reduced without incurring additional toxicity. Press et
al. explored the use of myeloablative doses of 131I-labeled
monoclonal antibodies with ASCT support in 43 patients
with B cell lymphomas who had failed conventional
chemotherapy [50]. Two anti-CD20 antibodies (tositu-
momab and 1F5) and one anti-CD37 antibody (MB-1)
were evaluated. Patients were selected for treatment
after tracer studies with increasing mass doses of anti-
body that determined that the absorbed dose to tumor
would be greater than that to normal organs. Sixty-four
469
percent of patients screened were eligible for thera-
peutic doses of radiolabeled antibody. Patients received
58–1,168 mg antibody labeled with 234–777 mCi of 131I.
At absorbed doses above 27.25 Gy to lungs, two of four
patients experienced cardiopulmonary toxicity. Sixteen
of 19 patients achieved a CR and 2 of 19 patients
achieved a PR. Mean response duration was in excess of
19 months. A phase II study repeated these results and
obtained responses in 15 of 19 patients. At 6 years, the
projected overall survival was 78% for patients with
indolent lymphoma and 43% for patients with aggressive
histologies [51].
Based on the results above, a study to evaluate treat-
ment with the MTD of radioactivity was conducted [52].
Twenty-two of 25 patients evaluated with trace labeled
doses achieved biodistributions considered adequate to
receive a therapeutic infusion. Twenty-one patients were
treated with therapeutic infusions of 131I-tositumomab
antibody calculated to deliver not more than 27 Gy to
normal organs followed by autologous hematopoietic
stem cell reinfusion. Seventeen (81%) achieved CR with
a median duration of response of 38 months.
The relative long-term efficacy of this high-dose RIT
strategy was evaluated via a multivariable cohort analy-
sis of 125 patients with relapsed or refractory follicular
lymphoma treated with either myeloablative I-131 tosi-
tumomab followed by autologous HCT or conventional
high dose therapy followed by autologous HCT. In this
study, the estimated 5-year OS for high-dose RIT was
67% and for conventional high-dose therapy was 53%
(p = 0.004) [53]. Likewise, 5-year PFS was 48% for the
high-dose RIT group and 29% for the conventional
transplant group (p = 0.03). Furthermore, 100-day treat-
ment-related mortality was lower in the high-dose RIT
group than conventional HCT group (3.7% versus
11.2%) with no evidence of increased MDS/AML at 8
years of follow-up. Based on the exceedingly low non-
hematopoietic toxicity of single-agent HD-RIT followed
by autologous HCT, this strategy was evaluated in older
adults with relapsed B-NHL. Older patients with refrac-
tory or relapsed NHL typically have limited therapeutic
options largely due to the excessive toxicity associated
with potentially curative dose intense regimens and
co-morbidities [53]. In this study, 24 patients ≥60 years
of age with relapsed or refractory B-NHL received high-
dose RIT targeted to ≤25–27 Gy to normal organs fol-
lowed by ASCT. Notable findings from this series
included low rates of non-hematopoietic toxicity (<10%
grade 4), no treatment-related deaths, and an estimated
51% 3 year PFS.
These results, although encouraging, led to relapse in the
majority of patients. Therefore, Press et al. combined
470
the MTD of 131I-tositumomab with high-dose etoposide
and cyclophosphamide prior to autologous HCT [54].
While the toxicities were significantly greater for the
combination regimen than for single-agent therapy, the
estimated overall survival at 2 years was 83% and the
progression free survival is 68%. Non-randomized con-
trols treated with identical doses of chemotherapy but
with total body irradiation had inferior PFS (p < 0.002)
and OS (p < 0.004) compared to those who received
radiolabeled antibody based regimen. More recently, a
subset of 16 patients with refractory mantle cell lym-
phoma who had been treated with this approach were
reported and showed 3-year PFS and OS from trans-
plantation estimated at 61% and 93%, respectively [55].
Extended phase II studies using this combined high-
dose chemoimmunotherapy regimen in various B-NHL
histologies are ongoing to better define the safety and
efficacy of this regimen.
Leukemia
Abs directed against the CD33, CD45, and CD66
hematopoietic differentiation antigens have been most
extensively explored for RIT of leukemia. CD33 is an
attractive target for RIT of myeloid leukemias, since
CD33 is expressed on most myeloid leukemic blasts, but
not on hematopoietic stem cells [56]. Anti-CD33 Abs
have been shown to rapidly saturate antigenic targets and
the Ab-antigen complexes are rapidly internalized into
the cell. Other alternative targets for RIT that do not
modulate upon Ab interaction have also been explored.
CD45 is stably expressed at a high density on the surface
of virtually all hematopoietic cells and does not undergo
significant modulation upon Ab binding. Most hemato-
logic malignancies, including 85–90% of acute lymphoid
and myeloid leukemias express CD45 [57, 58]. The
CD66 antigen is another target that also does not inter-
nalize or shed into the circulation. CD66 is expressed at
high density on maturing hematopoietic cells but is not
found on AML blasts [59]. Therefore, cytotoxic radia-
tion is delivered to bystander leukemic cells by targeting
normal CD66 expressing myeloid cells.
Beta emitting Radionuclides for RIT of
Leukemia
A relatively small number of beta emitters have been
studied in clinical trials for RIT of leukemia. Radioiodine
was first used for RIT of AML because of its well-estab-
lished experience as a medical radiotherapeutic. Two
groups of investigators demonstrated that 131I-labeled
monoclonal antibodies (p67 and M195) reactive with
Targeted radionuclide therapy of cancer
the CD33 antigen could be used to treat patients with
acute myeloid leukemia (AML) [60, 61]. In about half
of the patients radiotherapy was delivered with relative
specificity to the marrow. The liver was the only other
organ showing localization of the radioimmunoconju-
gate and was considered to be the dose-limiting normal
organ. Success appeared limited by low CD33 expres-
sion on target cells and by internalization of the anti-
body–antigen complex, leading to deiodination and
release of 131I. Jurcic et al. studied a humanized anti-CD33
antibody (HuM195) radiolabeled with 90Y. Anti-leukemic
activity was seen, but with prolonged myelosuppression,
suggesting the utility of this approach as a pretransplant
preparative agent [62].
Matthews et al. treated patients with leukemia using
BC8, a murine anti-CD45 IgG1 antibody that does not
internalize but is reactive with 85–90% of cases of acute
leukemia, and the majority of nucleated cells in normal
marrow, but not with cells outside the lymphoid or
hematopoietic lineages. A phase I trial in 44 patients
with AML, or acute lymphoblastic leukemia (ALL)
beyond first remission or advanced myelodysplastic
syndrome (MDS) was carried out in conjunction with
cyclophosphamide, 12 Gy TBI, and stem cell rescue. In
this dose escalation trial patients were selected for ther-
apy only when a diagnostic dose indicated that the bone
marrow and spleen would receive more radiation than
the other normal organs. Eighty-four percent of patients
had favorable biodistributions. This improved biodistri-
bution rate was likely due to the characteristics of the
anti-CD45 antibody, with longer retention of the radia-
tion at the tumor. Liver dose from a tracer study was
used to limit the dose escalation. The MTD was 10.5 Gy
to the liver. This trial demonstrated that 131I-labeled anti-
CD45 antibody could deliver two to five times more
radiation to the marrow and spleen compared to normal
organs. This approach at the MTD resulted in 24 Gy
more radiation delivered to the marrow and approxi-
mately 50 Gy to the spleen, without excessive toxicity in
a setting of conventional cyclophosphamide/TBI [63].
Seven of 25 patients with AML/MDS were disease free
for a median of 26–100 months at the time of the report,
and three of nine patients with ALL were disease free
for 34–82 months post transplant.
The same preparative regimen was used in a later
study for patients with AML beyond first remission with
relapsed/primary refractory AML, or advanced MDS.
Results suggested that a greater radiation absorbed dose
delivered to marrow may lead to lower post-transplant
relapse rates for patients with high-risk AML/MDS [64].
Half of the patients treated received a dose to marrow
<7 cGy/mCi while the remaining half of patients received
John M. Pagel et al.
>7 cGy/mCi to marrow. Although the number of patients
was limited, only one of nine patients relapsed in the
high marrow-absorbed dose group while six of nine
patients relapsed in the lower dose cohort. The hazard of
relapse in the group that received <7 cGy/mCi to
marrow was more than six-times higher than that seen
in the group that received the higher marrow dose. Since
greater estimated radiation doses could be delivered
safely by an 131I-anti-CD45 antibody to marrow and
spleen compared to non-target organs, a trial for patients
with AML in first remission was conducted [65]. Forty-
nine patients received high dose targeted irradiation
delivered by 131I-anti-CD45 antibody combined with
busulfan and cyclophosphamide. The radiolabeled anti-
body delivered an average of 11 Gy to marrow and
almost 30 Gy to spleen. These first remission patients
had a low relapse rate (20%) and a 62% estimate of
3-year disease-free survival after transplantation. These
data appear promising since at least 30% of patients
with AML in first remission would be expected to
develop leukemic relapse after being treated with stan-
dard busulfan and cyclophosphamide conditioning and
stem cell transplant [66]. Nevertheless, carefully con-
trolled randomized trials will be necessary to defini-
tively assess the contribution of the 131I-anti-CD45
antibody to standard transplant regimens.
These encouraging results suggested that this
approach might be applied to older patients. Given the
reduced toxicity of a low-intensity conditioning regi-
men in high-risk older patients, the anti-leukemic effect
of a non-ablative approach might also be improved by
the addition of targeted hematopoietic irradiation deliv-
ered by a radiolabeled antibody. An allogeneic trans-
plant trial using targeted hematopoietic irradiation
delivered by an 131I-anti-CD45 antibody (BC8) com-
bined with fludarabine and 200 cGy TBI has been initi-
ated for a particularly poor risk population of advanced
older AML and high-risk MDS patients. This study has
demonstrated that at least an average of 27 Gy of tar-
geted radiotherapy can be delivered to bone marrow and
an average of 81 Gy to the spleen, in addition to a stan-
dard reduced intensity transplant regimen, without a
marked increase in day 100 mortality [67]. Whether use
of a radiolabeled antibody combined with a non-
myeloablative transplant will reduce post-transplant
relapse rates for these older patients with high-risk
AML/MDS, however, remains to be determined.
Rhenium radioisotopes have also been investigated as
part of conditioning regimens prior to HCT. In particu-
lar, 188Re labeled anti-CD66 antibody has been used in
patients with high-risk AML [68]. At a follow-up of 18
months in 36 relatively younger patients who underwent
471
allogeneic transplantation after 188Re-anti-CD66 condi-
tioning, 45% were disease free; in addition, significantly
more patients remaining disease free if they were in
remission at the time of transplant, compared to those
patients who were not in remission at the time of trans-
plant (67% versus 31%). For older high-risk AML or
MDS patients, RIT using an anti-CD66 antibody labeled
with either 188Re or 90Y was also combined with a
reduced intensity conditioning regimen followed by
transplant using T-cell-depleted allografts [69]. Despite
the low level of toxicities seen in older patients using
this approach, the cumulative incidence of relapse
remained high (55% at 30 months post-transplant). The
risk of relapse was almost 20% higher for patients not in
remission compared to those in either first or second
remission. These studies highlight the difficulty of tar-
geting multiple low-energy beta emissions to neighboring
leukemic blasts by relying on crossfire from normal
targeted CD66 positive granulopoietic cells.
Alpha Emitting Radionuclides for RIT of
Leukemia
The feasibility of using alpha emitting radionuclides
was established in an initial study that employed
213
Bi-HuM195 for patients with refractory or relapsed
AML or MDS [70]. In 18 patients with refractory or
relapsed AML or MDS, 13 patients had decreases in the
percentage of marrow blasts and 10 of 12 evaluable
patients had reduced peripheral blood leukemia cells
[62]. 213Bi-HuM195 was given in three to six fractions
over 2–4 days. Uptake of antibody in marrow, spleen,
and liver was seen within 10 min. There were no signifi-
cant acute toxicities, however, transient elevated liver
function abnormalities occurred. Myelosuppression
lasted 34–50 days. Since alpha-particle RIT also has
promise to be effective in the treatment of minimal leu-
kemia states, a follow-up study has been initiated using
chemotherapy to first achieve a significant degree of
cytoreduction prior to the delivery of 213Bi-HuM195.
Clinical use of 213Bi to date has been otherwise limited,
however, since this radionuclide is relatively short lived,
which may result in a potentially short marrow resi-
dence time of the radionuclide. Recognizing this limita-
tion and that 225Ac has a 10 day half-life, a phase I trial
investigating the use of 225Ac-HuM195 for advanced
myeloid leukemias has been initiated at Memorial Sloan
Kettering Cancer Center [71]. This study holds consid-
erable promise for advancing the field of alpha-particle
RIT because of the longer half-life and the generation of
short-lived daughter therapeutic alpha-particles (221Fr,
217
At, 213Bi) from 225Ac inside a targeted leukemia cell,
472
which largely accounts for the increased potency of
225
Ac-atomic nanogenerators over 213Bi constructs [72].
CD25 and CD30 have recently been explored as
rational targets for RIT of leukemia as these antigens are
expressed by a high proportion of disease specific neo-
plastic cells and not by normal resting cells. Recently, a
pre-clinical model of leukemia labeled with the alpha
emitter 211At has been studied as a potential radioimmu-
notherapeutic for patients with CD25 and CD30 posi-
tive leukemias [73, 74]. These findings have set the
foundation for progression to clinical studies using radi-
olabeled Abs for the treatment of patients with either
CD25 or CD30 expressing leukemias.
RIT of Non-Hematologic Tumors
A variety of antibody based proteins have been studied
for RIT of solid tumors including whole antibodies and
various fragments derived from them, as well as chime-
ric and humanized modifications to minimize immuno-
genicity. Fractionated as well as single doses have been
administered, marrow rescue has been used for high-
dose studies, and the targeted radionuclide has been
selected from 131I, 90Y, 186Re, or the Auger emitting iso-
tope, 125I. Internalizing and non-internalizing antibodies
have also been evaluated. Most of the antibodies react
with several tumor types, but we have chosen to catego-
rize these studies according to site of primary disease.
Gastrointestinal Carcinomas
Patients with gastrointestinal malignancies have been
widely studied in Phase I trials. In general these patients
have not been exposed to intensive chemotherapy from
myelosuppressive drugs and do not have rapidly progres-
sive disease, and thus they are relatively ideal patients for
proof of concept and Phase I studies. A review of several
RIT trials for treating gastrointestinal cancer revealed an
extremely low response rate [41, 75–89].
Of almost 300 patients studied in these phase I trials,
tumor responses included only one CR, 4 PR and
although stable disease or minimal responses were not
always reported, this may have been present in 10% of
patients or more. Marrow toxicity invariably limited
dose escalation, even in these patients with often rela-
tively little prior myelosuppressive chemotherapy.
Additional strategies will have to be implemented for
RIT in solid tumor patients to achieve higher response
rates. A potential complicating factor in several Phase I
trials for colon cancer has been the use of anti-CEA
antibodies that interact with circulating CEA antigen to
alter the pharmacokinetics of the therapeutic moiety.
Targeted radionuclide therapy of cancer
For example, in both NP-4 and COL-1 studies, complexes
were detected within 5 min of injection, indicating that
antibodies directed against circulating antigen are sub-
ject to variable pharmacokinetics related to the level of
circulating antigen [77, 90]. In other anti-CEA antibody
studies (e.g., c84.66 antibody), however, tumor burden
was noted to alter the pharmacokinetics probably even
more so than in patients when antibodies targeting
non-shedding antigens were used [83].
Other CEA-Expressing Cancers
Phase I studies with anti-CEA antibodies have also been
carried out in patients with ovarian cancer and unre-
spectable or metastatic medullary thyroid cancer (MTC).
Juweid et al. reported on a dose escalation trial in which
131
I-MN-14 was administered to 11 patients with
advanced ovarian cancer, but a clinical response was
seen in only one patient [77] Juweid reported a study
with non-myeloablative doses of 131I-labeled NP-4 and
MN-14 intact antibodies and their bivalent fragments in
17 patients with MTC [91]. Dosages of 131I ranged from
46 to 268 mCi, depending on whether a fragment was
administered. Minor responses were observed in 5 of 11
evaluable patients, mostly with the F(ab’)2 fragments of
both antibodies. Juweid also reported on the toxicity
and therapeutic potential of high-dose 131I-MN-14
F(ab’)2 combined with ASCT in patients with rapidly
progressing metastatic MTC [92]. Twelve patients were
entered into the study and dose escalation was based on
prescribed radiation doses to critical normal organs. The
highest dose level reported was 12 Gy to critical organs
and was not dose limiting. One patient had a partial
remission for 1 year, another had a minor response for 3
months, and 10 patients had stabilization of disease last-
ing between 1 and 16 months. The authors of this study
suggested that the anti-tumor responses seen in these
patients with aggressive, rapidly progressing disease
was encouraging and warranted further study.
Prostate Cancer
CYT-356, Prostascint™, uses an antibody directed against
a prostate specific antigen and has been studied exten-
sively as an imaging agent [93]. This antibody has also
been radiolabeled with 90Y for a phase I therapy study in
prostate cancer patients. Twelve patients were studied,
the MTD was below 12 mCi/m2, limited by marrow toxic-
ity. The authors believed marrow toxicity to be related to
extensive bony involvement and prior marrow irradiation
to more than half the marrow. No objective responses
were seen, but improvement in symptoms were reported.
John M. Pagel et al.
KC4, is a pancarcinoma antibody directed against a
high molecular weight membrane and cytoplasmic gly-
coprotein. In a dose escalation study, calcium disodium
EDTA was administered for 72 h in conjunction with
escalating doses of 90Y-KC4 to patients with refractory
prostate cancer [94]. Again here, symptomatic improve-
ment was reported, but at the low dose of 9 mCi/m2
severe marrow toxicity was observed.
Meredith et al. has reported on several phase I and II
trials using 131I-CC49 in prostate cancer patients [95, 96].
A phase II study with 75 mCi/m2 131I-CC49 showed
symptomatic improvement in two thirds of patients but
all patients developed human anti-mouse antibody
(HAMA) within 4 weeks of therapy [95, 96]. A trial with
a low-dose of cyclosporin and the dose of 131I-CC49 frac-
tionated to 38 mCi/m2 at 15-day intervals was unsuccess-
ful because HAMA still developed in the majority of
patients. The single-dose approach was adapted, with the
addition of alpha interferon and the higher 75 mCi/m2 131I
dose. Again, no objective anti-tumor responses were
seen, but there was substantial bone pain relief seen in
several patients. Marrow toxicity was not related to the
extent of bony involvement. In a high-dose therapy study,
three patients were treated at the initial dose level of
100 mCi/m2 131I-CC49, followed by 13.2 Gy TBI given
over 4 days, beginning 8 days following RIT. All three
patients treated at the first dose level achieved objective
tumor response [96].
Breast Cancer
The radiosensitivity of breast cancer, and the results
reported from the breast cancer trials so far suggest that
RIT may play a clinical role, particularly with some of the
additional strategies to improve response rates that are
still under investigation. Although non-myeloablative
doses have produced disappointing results, ASCT studies
have shown that 100–300 Gy can be delivered to meta-
static sites with non-toxic doses to normal organs [97].
L-6 is an antibody directed toward an antigen expressed
on ∼50% breast adenocarcinomas, with biological activ-
ity that has been evaluated using 131I and [90]Y. Preliminary
work by DeNardo et al. demonstrated the importance of
administering unlabeled antibody prior to radiolabeled
antibody to saturate the antigenic sites on normal tissues,
in this case the vascular endothelium [98]. In a phase I
study, 200 mg cold blocker was administered with 131I
murine L-6. The cold blocker reduced lung activity from
19% to 4%, allowing the radiolabeled antibody to be
visualized at the tumor sites. It appeared that a transient
inflammatory reaction increased the delivery of subse-
quently administered radiolabeled antibody to tumor.
473
Three to 15 mg L-6 was administered with 10 mCi 131I per
mg antibody and the MTD was 60 mCi/m2. One patient
achieved a CR, but 40% of patients also developed a
HAMA response [98]. In a multiple-dose protocol, ten
heavily pretreated patients were treated with monthly
injections of 131I-chimeric L-6. The MTD was 60 mCi/m2
twice limited by myelotoxicity. In five of the ten patients,
clinically measurable tumor responses were seen [99].
In this study, serum levels of IL-2 receptors were mea-
sured and the increase in serum IL-2 was greatest in the
patients who responded. Immunoglobulin developed in
eight of ten patients at varying times, and did prevent
further therapy doses in four patients. The authors specu-
lated that the reason for success with the L-6 antibody is
a combination of the increased vascular permeability
from the unlabeled biologically active antibody with
these relatively low doses of radiation, and the activated
effector cell mechanism. On other multiple dose proto-
cols, patients received G-CSF 7 to 20 days following
infusion, or patients underwent immunopheresis using a
goat anti-mouse antibody to reduce the non-bound circu-
lating antibody [100]. A high dose study using 150 mCi/
m2 given at eight weekly intervals was initiated. Patients
had PBSC reinfused when circulating radioactivity reached
an acceptable level. Thrombocytopenia was successfully
managed with this regimen. Multiple doses were limited
by HAMA for the first two patients. The third patient was
given cyclosporin which prevented HAMA formation
even after three doses, and she showed evidence of tumor
response [101]. A study now underway with 90Y-DOTA-
peptide-ChL6 appears to provide enhanced therapeutic
index [102].
BrE-3 is an IgG1 antibody directed against the peptide
epitope of the MUC-1 antigen, present on 95% of breast
cancers that have been evaluated for RIT labeled with 90Y.
DeNardo reported objective responses in three of six
patients treated at single low doses. High tumor doses
were estimated, however HAMA developed in five of six
patients [103]. Schrier treated nine heavily treated patients
with stage IV breast cancer with 90Y-labeled BrE-3 anti-
body [104]. Harvested autologous marrow or PBSC were
reinfused at 15 days in conjunction with G-CSF. Fifteen
and 20 mCi/m2 90Y was administered. Pharmacokinetic
data indicated that the 90Y biodistribution was not identi-
cal to the 111In biodistribution. Six patients developed
transient grade 4 marrow toxicity but all recovered and no
other toxicities occurred. The 90Y conjugated with
MX-DTPA dissociated from the antibody and deposited
in the bone, causing the high incidence of marrow toxic-
ity, bone biopsies confirmed this localization of 90Y in the
bone. Based on 111In imaging, tumor doses 35–56 cGy/
mCi were reported. These values in terms of cGy per mCi
474
are higher than with other radioimmunoconjugates, but
the total dose that could be administered was limited by
the marrow toxicity. Four of eight evaluable patients
achieved a PR, noted in lymph nodes, skin and marrow
lesions, and one achieved a clinical CR. Because of
immunogenicity, a human BrE was developed, and nine
patients received increasing doses of 90Y-hBRe with
peripheral blood stem cell support. Although the initial
response rates are less encouraging, this study may define
the second organ of toxicity [105].
CC49 is an antibody that targets the TAG-72 antigen,
which is expressed in 90% of breast carcinomas.
Mulligan studied nine patients with 177Lu-DOTA-CC49
(five of whom were breast cancer patients). Luticium-177
was considered an attractive alternative radionuclide
with a lower energy beta emission and longer half life
than 90Y [106]. However, 177Lu accumulated in the cells
of the reticuloendothelial system and was retained there
for a prolonged period. Images demonstrated the tumor
uptake as well as activity in the bone marrow. This
resulted in myelosuppression at low doses and the MTD
was 15 mCi/m2. Reduction in the RES uptake would be
required for this to become a useful radioimmunoconju-
gate. Nine breast cancer patients were also treated with
131
I-CC49 in conjunction with Thiotepa and total body
radiation, followed by stem cell rescue. No life threaten-
ing toxicities were noted and two patients had a partial
response by imaging criteria [107].
The biological response modifier alpha interferon is
able to increase the antigenic expression of TAG-72 and
increase antibody targeting in tumors [81]. Murray
tested this in 15 patients with breast cancer, assessing
TAG-72 expression and 131I-CC49 uptake [108]. After 3
days of alpha interferon, 3 million units daily, 10 or
20 mCi 131I-CC49 was administered and 48 h later a
biopsy was taken and compared with a pre-study biopsy.
The TAG-72 expression was increased by 45% follow-
ing the alpha interferon (p < 0.05), and there was an
apparent increased 131I-CC49 localization in tumor.
However, because of intra- and inter-patient variability
in percentage of tumor cells in the biopsy specimens as
well as heterogeneity of TAG-72 expression, this was
difficult to evaluate with certainty. Pharmacokinetics
and biodistribution were changed with administration of
the alpha interferon. RIT at the MTD with concurrent
alpha interferon administration is under investigation
and in a preliminary report, 1 of 15 patients had achieved
a PR at the expense of increased marrow toxicity, from
a more prolonged serum clearance at the higher doses.
Wong et al. evaluated the high affinity, anti-CEA chi-
meric antibody, cT84.66 in 22 breast cancer patients, in
a dose escalation trial with 90Y-DTPA. Dose limiting
Targeted radionuclide therapy of cancer
marrow toxicity was observed at 22 mCi/m2, and a study
with stem cell support is ongoing. Results are prelimi-
nary but appear encouraging in the six first patients
studied [109].
Glioma
The high-grade malignant gliomas (anaplastic astrocy-
tomas and glioblastoma) have a very poor prognosis
with current surgery, radiotherapy and chemotherapy
approaches. The use of specific monoclonal antibodies
labeled with a suitable isotope (131I or 90Y) represents an
effective approach to reduce tumor regrowth. Because
of the difficulty in interpreting diagnostic imaging scans,
and the short survival time in these patients (median 10
months) survival has been used as an endpoint.
Radiation, following surgical resection, offers a several
month advantage in survival time, but has been limited
by normal brain tolerance. Targeted radiation is there-
fore an attractive approach to increase radiation dose to
tumor and potentially contribute to increased survival.
Some investigators have injected the antibodies intrave-
nously, or have tried to increase the tumor/background
ratio with the avidin/biotin system. In several studies the
labeled monoclonal antibodies were injected directly
into the tumoral bed after surgery.
Systemic Injection
Kalofonos reported a study with 131I-anti-epidermal
growth factor receptor antibody (EGFr) [110]. Ten
patients with recurrent gliomas received intravenous or
intra-arterial administration of the radioimmunoconju-
gate and six showed clinical improvement. Brady treated
101 patients with high grade gliomas by intravenous or
intra-arterial injections of 125I-labeled anti-epidermal
growth factor receptor antibody, EGF-425 [111].
Patients with both primary and recurrent astrocytomas
and glioblastoma multiforme were treated with an aver-
age total dose of 139 mCi 125I, administered divided into
three weekly infusions. Brady reported 1 CR and 2 PR
of short duration following 125I EGF-425 intravenously.
The median survival in both groups was improved with
these relatively radioresistent tumors. Fifteen of these
patients had recurrent malignant astrocytomas and were
treated with 25 to 130 mCi intra-arterially. The median
survival was 8 ± 7 months [111].
Twenty-five patients with malignant astrocytomas were
then treated in a Phase II adjunctive therapy trial [112].
Four to 6 weeks following surgical debulking (2) or biopsy
(13), and external beam radiation, one to three doses
of 125I-labeled antibody 425 was administered intravenously
John M. Pagel et al.
or intra-arterially at 7–14 day intervals. Cumulative doses
ranged from 40 to 224 mCi. HAMA did not develop in any
of these patients. The 1 year survival was 60% with a
projected median survival of 15.6 months.
Compartmental Injection
Malignant gliomas tend to spread by local invasion
rather than metastases. Thus intralesional, intra cavitary
or intrathecal administration of the therapeutic radiola-
beled antibody may be useful to deliver the therapeutic
radiation directly to these solid tumors.
Intralesional
One approach is to inject the radioimmunoconjugate
through an Ommaya reservoir into the surgical cavity
following surgical resection, or directly into a cystic
tumor when the lesion has a predominantly cystic com-
ponent. Here the antibody binding to antigen serves to
prolong retention of the radiation at the target, while
sparing normal brain tissue. Initial studies with 131I anti-
bodies into the surgical cavity demonstrated the feasi-
bility of an intralesional or intracavity approach
following surgical resection. Riva et al. [113] reported
their experience with intralesional administration of
131
I-labeled antibodies for treatment of both newly diag-
nosed and recurrent glioma. Anti-tenascin antibodies,
BC-2 and BC-4 were radiolabeled with up to 65 mCi of
131
I, and injected into the resection cavity through a
catheter. The radioactivity remained at the target site for
an effective half time of 60 h and thus could deliver high
doses to the tumor, on average 42,000 rad/cycle. Up to
four cycles were given to 50 patients. Although HAMA
developed in some patients, it did not interfere with
tumor targeting in subsequent cycles. In all there were 3
CRs, 6 PRs, 11 tumor stabilizations and 19 tumor pro-
gressions recorded. Eleven patients with no radiological
evidence of disease at the time of treatment remained
disease free. In 26 patients the median time to progres-
sion was 3 months, and in the group of newly diagnosed
patients who were treated, time to progression was 7
months. The median survival was 20 months; 17 months
in patients with bulky disease, and 23 months in patients
with minimal or microscopic disease. The median sur-
vival of these patients is usually 10 months; thus this
study suggests an improved outcome for these patients.
In addition, Riva has reported an improved survival in
the patients with bulky glioblastoma, 30 months [114].
Another anti-tenascin antibody, 81C6, radiolabeled
with up to 100 mCi 131I has been studied in patients with
recurrent cystic gliomas and in patients with a surgically
475
created resection cavity from primary or metastatic
brain tumor [115] High retention in the cysts with little
systemic dissemination resulted in absorbed dose esti-
mates of 127–703 Gy. No hematological or neurotoxic-
ity was observed. In preliminary results, all five patients
with recurrent cystic glioma appeared to benefit from
the treatment with an increased survival. In the majority
of patients with administration into the surgically cre-
ated cavity, stabilization of disease was observed [116]
Chimeric 81C6 carrying the alpha emitter 211At is also
under study for injection into the surgical cavity of
recurrent gliomas. Dose limiting toxicity has not yet
been established [117].
ERIC-1 antibody was radiolabeled with up to 60 mCi
131
I and administered to nine patients with relapsed
glioma who had a cyst or cavity following prior resec-
tion [118]. In two patients with cystic lesions, the need
for aspiration was markedly reduced. The short range of
the 131I beta particles delivers a high dose to only a rim
of tissue approximately 1 mm thick, around the surgical
cavity. In a study with 90Y-labeled ERIC-1, 15 patients
were treated with up to 18 mCi and the cerebral edema
that developed was managed successfully with dexam-
ethasone [119]. Dose estimates from this treatment
ranged from 55 to 351 Gy, depending on whether com-
plete binding on antibody occurred or not. Again in this
study, the two patients with cystic tumors required less
frequent aspirations. While the overall benefit of the
RIT could not be easily determined from this group of
patients, median survival was 6 months from treatment.
Intrathecal
In neoplastic meningitis, external beam therapy is lim-
ited by dose to the normal nervous system. The average
survival of patients with neoplastic meningitis is 3
months from diagnosis. Tumor cells are found floating
freely in the CSF or in sheets lining the meninges. Thus
an intrathecal route of administration bypasses the prob-
lems of access to tumor antigen that are present when
radioimmunoconjugates are administered systemically.
Papanastassiou treated patients with diffuse leptome-
ningeal deposits with intrathecal radiolabeled antibod-
ies administered through an Ommaya reservoir. Patients
included those with carcinomatous meningitis (n = 7),
primitive neuroectodermal tumors (n = 18), and CNS
leukemia (n = 22), with the antibody depending on the
disease [120]. The radioimmunoconjugate leaves the
intrathecal space relatively quickly, peak blood flow lev-
els of 6–48% of the injected dose reached the vascular
system at 24–56 h following injection. Dosage of 131I
ranged from 17 to 90 mCi on 1.7 to 9 mg antibody.
476
Transient aseptic mengitis was common following
intrathecal RIT, occurring in approximately 60% of
patients. Occasional patients developed seizures. No
long term sequelae have been observed. Significant
myelosuppression occurred in several patients who
received more than 54 mCi. Response was difficult to
assess and was assessed by clearance of cells from the
CNS and clinical improvement. Papanastassiou reported
a 33% overall response rate. This was poorest in the car-
cinoma patients. The mean time to relapse in 37% of
PNET patients was 10 months, In patients with leuke-
mia, marked responses of clearance of leukemic cells
from the CSF were observed, but for only 4–8 weeks.
Brown et al. reported on patients with leptomeningeal
disease and brain tumor resection cavities with subarach-
noid communication. Patients with anaplastic gliomas,
ependyomas, medullablastoma and anaplastic astrocy-
toma received a single dose of up to 100 mCi of 131I 81C6
antibody administered intrathecally through an Omaya
reservoir [121] No nonhematological toxicity occurred
and the MTD was 80 mCi, limited by hematological tox-
icity. Ten of 24 evaluable patient developed HAMA, but
in two patients who were retreated, there was no altera-
tion in biodistribution of radiolabeled antibody. In 31
patients with malignant gliomas, one patient showed a
radiographic PR and disease stabilization occurred in
42% of patients, representing an apparent increased sur-
vival rate although this was difficult to evaluate.
Bigner used the (Fab’)2 fragment of Mel-14, anti-
melanoma antibody directed against anti-proteoglycan
chondroitin sulfate associated protein, to treat a patient
with a brain metastasis from melanoma [116]. The
patient received Mel-14 F(ab’)2 labeled with 37 mCi 131I
injected into the surgical resection cavity, and achieved
a complete local response. Eleven patients were treated
with up to 80 mCi 131I-Mel-14 (Fab’)2 and 81C6 intrath-
ecally via lumbar puncture. They had melanoma (n = 8),
melanosis (n = 1), oligodendroglioma (n = 1) and glio-
blastoma (n = 1). One patient at 80 mCi had hematologi-
cal toxicity. After treatment, three patients had complete
CSF responses, two had partial responses radiologically
and survival ranged from 1 to 11 months, an apparent
improvement on the 3 month average survival.
Peptide-Based
Radiopharmaceuticals
After the detection of peptide receptor expression by
tumors, somatostatin analogues became the first radiola-
beled regulatory peptides used for specific tumor targeting.
Targeted radionuclide therapy of cancer
Peptides used for tumor targeting show several advan-
tages over antibodies: peptides are small and show rapid
diffusion into (target) tissues resulting in rapid pharma-
cokinetics. Their fast blood clearance could lead to high
tumor-to-background ratios shortly after administration
of the radiopeptide [122–126]. As exemplified by soma-
tostatin, analogs of the peptide that are stable in blood
show a better tumor uptake than the natural peptides
although both are degraded after internalization [124].
To prevent internalized radionuclides from externaliza-
tion, a so-called residualizing label consisting of a che-
lator linked to the peptide and a radiometal such as 111In,
177
Lu or 90Y is used. These radiometal/chelator com-
plexes, are retained in the lysosomes and thus they are
trapped inside the cell [126].
To date, the 111In-labeled somatostatin analog oct-
reotide (OctreoScan®) is the most successful radiopep-
tide for tumor imaging and has been the first to be
approved for diagnostic use. It is considered the diag-
nostic gold-standard in several types of gastrointestinal
and other neuroendocrine tumors (NET) and plays a
crucial role in the diagnosis and follow-up of patients
with these tumors [127–129]. Furthermore, somatosta-
tin receptor scintigraphy (SRS) also contributes signifi-
cantly to decision making in the management of patients
with NET [130–133]. Currently, octreotide-based soma-
tostatin analogs labeled with the ß-emitters 90Y or 177Lu
are undergoing clinical trials for peptide receptor radio-
nuclide therapy (PRRT) of NET.
Other peptides for tumor targeting in development or
undergoing clinical trials are CCK2 receptor-binding
peptides, bombesin, substance P, neurotensin, glucagon-
like peptide-1, and RGD-peptides for imaging of ανβ3
expression in tumors [126, 133–136].
Regulatory Peptides and their Receptors
Regulatory peptides are potent small messenger mole-
cules binding to specific receptors. Usually, they are not
larger than 30–40 amino acids and are able to rapidly
extravasate and penetrate tissues. Regulatory peptides
are synthesized mainly in the brain and gastrointestinal
tract. They may also play a role in the peripheral ner-
vous system or in the immune system [125, 126, 137].
Due to their hydrophilicity, they do not cross the blood–
brain-barrier in either direction. Therefore, the central
nervous system and the periphery (e.g., the gastrointes-
tinal tract) form two independent regulatory systems
which thus use the same messenger molecules without
any danger of confusing interaction of both. All regula-
tory peptides bind to and act through transmembrane G
protein-coupled receptors. The signal transduction of
John M. Pagel et al.
these receptors is triggered by the binding of the respec-
tive peptide to the extracellular domain of its receptor
which will, in turn, activate the intracellular guanine
nucleotide binding proteins (G proteins). An important
inactivation pathway is the internalization of the ligand–
receptor complex, physiologically leading to either deg-
radation or recycling of the internalized receptor to the
cell surface [125]. This inactivation pathway plays a
crucial role for the use of radiopeptides in scintigraphy
and PRRT.
Regulatory peptides influence many aspects of mam-
malian physiology. Their role as flexible messenger
molecules requires rapid degradation in the blood to
prevent prolonged action of secreted peptides.
Ubiquitously occurring peptidases are responsible for
rapid deactivation of regulatory peptides. For radiopep-
tides, rapid degradation in the circulation or in other tis-
sues would be a major obstacle for reaching the target.
Thus, peptides used for tumor targeting should be meta-
bolically stable.
Radiolabeling of Peptides
The somatostatin analog octreotide was the first peptide
used for the scintigraphic detection of NET. Somatostatin
itself has a very short half-life in serum. Thus, octreotide
with its higher stability was used for scintigraphic pur-
poses. As the easiest method of radiolabeling peptides is
radioiodination using 125I, 123I, or 131I [138] a tyrosyl moi-
ety was introduced into the stabilized peptide in position 3,
replacing phenylalanine in the natural sequence.
Iodination of the tyrosine residue is relatively easy. Thus,
123
I-Tyr3-octreotide was the first radiopeptide used for
imaging of NET in a patient [123]. If an amino-acid is to
be replaced by tyrosine for radioiodination, it should not
be involved in receptor-binding or activation [126].
However, after internalization, peptides will undergo
proteolytic lysosomal degradation. Mono- or di-iodi-
nated tyrosine will be formed and these metabolites will
rapidly be excreted from the cells. Mono- or di-iodinated
tyrosine may then be deiodinated, preferably in the liver.
Excretion of the radioiodine label from the targeted cells
is the major drawback of the use of this so-called “non-
internalizing” radiolabel in peptide receptor targeting.
Thus, the tumor-to-background ratios that can be
obtained using this approach are suboptimal and the sen-
sitivity of 131I-octreotide for detecting somatostatin
receptor expressing tumors was inferior to that of pente-
treotide labeled with the radiometal 111In [124].
In contrast to radioiodinated amino acids, following
internationalization by the target cells, radiometal chela-
tor conjugated amino acids are trapped in the lysosomes
477
and thus are retained inside the cells. The most widely
used radiometal chelators are DTPA (diethylene-triamin
e-pentaacetate) and DOTA (tetra-azacyclododecane-
N,N’,N”,N” ’-tetra-acetate). For diagnostic purposes,
the DTPA can stably chelate 111In. This chelator is used
in the commercially available somatostatin analog
OctreoScan®. However, chelation of other radiometals
by DTPA used for therapeutic purposes is not suffi-
ciently stable in vivo. In contrast to DTPA, the cyclic
chelator DOTA demonstrates sufficient stability for a
broad range of other metallic ions, such as 90Y, 177Lu,
153
Sm, 212/213Bi, or 212Pb [139, 140]. Therefore, it is the most
frequently used chelator in PRRT.
Radiometal chelators such as DTPA or DOTA may be
covalently linked to the N-terminus of a given peptide or
to the ε-amino group of a lysine moiety. If the N-terminus
is essential for receptor-binding or internalization, a
lysine moiety within the primary structure has to be used
for linking the chelator. Even if lysine is already part of
the peptide, it may be crucial for binding or internaliza-
tion. Thus, introduction of an additional lysine may be
applied to conjugate the chelator to the peptide. Thus, the
problem of development of stable analogs of (regulatory)
peptides not only consists of the labeling alone, but also
of changing the amino acid sequence to obtain a site for
linking the chelator without decreasing the receptor
binding affinity. Furthermore, after modification some
peptides will still bind, but will no longer internalize any
more. Internalization of the peptide/receptor complex is
considered a crucial step in the process of in vivo recep-
tor targeting with peptides [141], although it has recently
been shown that noninternalizing antagonists could also
lead to efficient receptor targeting [142].
As stated above, peptides for receptor targeting should
be metabolically stable. In contrast to other drugs, stabi-
lization by binding to serum proteins is not helpful in
this context – due to higher blood retention and liver
clearance. Although high serum stability is warranted,
high tumor-to-background ratios will only be obtained
if there is rapid clearance from the blood. Thus, amino
acids may be replaced by D-isomers so that serum pep-
tidases will not affect these modified peptides. Other
typical modifications for stabilization are the use of
unnatural amino acids, N-terminal acetylation, or
C-terminal amidation [143]. Only after internalization,
lysosomal degradation will occur. For rapid renal excre-
tion, hydrophilicity is required. Lipophilic peptides will
show a higher background activity possibly masking
tumors in highly perfused organs such as the lungs or
the liver in early images. Furthermore, higher liver
extraction will even increase liver activity which is not
warranted in abdominal tumors.
478
Clinical Experience with Somatostatin
Analogs
As the physical half-life of 111In is 2.8 days, whole body
images should be acquired not only 4h but also 24-h
after injection of 200 MBq of the 111In-radiopeptide.
Due to continuous clearance from non-target tissues, the
tumor-to-background ratio will increase over time so
that the 24-h images show a higher diagnostic yield than
the 4-h images. Twenty-four-hour scans show a mark-
edly higher sensitivity [144]. However, image acquisi-
tion at both time points might help to differentiate
specific from non-specific uptake. Although 99mTc is the
preferred radionuclide in diagnostic nuclear medicine
(ideal γ-energy of 140 keV, high photon flux, relatively
low radiation burden, unlimited availability), 111In is
also quite suitable. Obviously, gamma cameras should
be equipped with medium energy collimators, because
of the higher γ-energy of 111In in comparison to 99mTc.
The scanning speed should not exceed 5–6 cm per min-
ute to obtain high quality images with high count rates.
Additional three-dimensional SPECT images (single
photon emission computed tomography) may further
increase the sensitivity and should thus be acquired rou-
tinely [144, 145]. If bowel activity obscures abdominal
tumor manifestations, additional 48-h scanning can be
performed, possibly combined with the use of laxatives
to accelerate the passage of intestinal radioactivity.
Organs with physiologic octreotide-uptake are kidney,
liver (later gall bladder and bowel because of biliary
excretion), spleen, thyroid, and pituitary [126].
Lymphocytes (and activated leukocytes) and epithel-
oid cells in granulomas express somatostatin receptors
so that scar tissue and granulomas could show up in
SRS [146, 147]. Lymphocytes are also responsible for
the splenic uptake. After irradiation of the mediastinum,
“non-specific” uptake may also be observed. However,
this uptake is caused by receptors on accumulated
leukocytes and epitheloid cells [148].
Cold somatostatin may also decrease tumor uptake
thus possibly leading to false-negative results. If a patient
is on medication with somatostatin for inhibition of
tumor growth [149], receptors may be blocked by cold
somatostatin so that the splenic uptake drops in compari-
son to the liver uptake. This is an important finding in the
evaluation of somatostatin scans [132, 145].
Octreotide, the metabolically stable somatostatin ana-
log, has high affinity for the sst2 and sst5 receptor sub-
types, while it has no affinity for subtype 1 and 4 and
affinity to subtype 3 is low [125, 150]. Thus, clinical
somatostatin receptor scintigraphy with 111In-DTPA-
octreotide mainly visualizes sst2 and sst5 receptor-posi-
Targeted radionuclide therapy of cancer
tive tumors, but not sst1 and sst4 receptor-positive tumors.
A broad variety of tumors express the somatostatin receptor
subtypes 2 and 5, which includes most neuroendocrine
tumors, lung cancer, breast cancer, differentiated thyroid
cancers, but also meningiomas, well-differentiated astro-
cytomas, pituitary tumors, or malignant lymphomas and
several others [124, 151–154]. SRS plays a major role in
the diagnosis of neuroendocrine tumors and their metasta-
ses. In some gastrointestinal NET, it is considered the
diagnostic gold-standard [127–129]. In endocrine tumors
of the pancreas, SRS is positive in 50–90% of the cases,
dependent on the tumor type. As many insulinomas do not
express the subtypes 2 and 5 of the somatostatin receptor,
the sensitivity of SRS is only 40–60% [127]. On the other
hand, virtually all gastrinomas express somatostatin recep-
tors in a high density and indeed, SRS has a sensitivity of
up to 90% in these tumors. Carcinoid tumors of the intes-
tine may also be detected using SRS with sensitivities
>80% in comparison to only ∼50% for morphologic imag-
ing modalities. Even if CT, MRI, and ultrasonography are
combined, they will not reach the sensitivity of SRS in
patients with gastrointestinal NET [127, 145]. Only endo-
scopic ultrasonography will perform better in the detec-
tion of pancreatic NET. Figure 1 shows an example of a
SRS in a patient with an ileal carcinoid.
The sensitivity of SRS is mainly dependent on the
expression of the sstr2 and 5 subtypes of the somatosta-
tin receptor. Thus, insulinomas will be visualized in
only a limited number of patients with a sensitivity
around 50%. Gastrinomas will be visualized with higher
sensitivities of at least 60–90%, dependent on their size
[155–157]. It has been shown that the scanning tech-
nique has a strong influence on the results of SRS in
pancreatic tumors as they may be small and undetect-
able on planar images [145]. Thus, using SPECT, the
sensitivity can be expected to be closer to 90% than to
60%. Endoscopic ultrasonography is the only imaging
technique that shows results equivalent to SRS or even
better [127].
Active and inactive carcinoid tumors of the gastroin-
testinal tract may be detected by SRS with high sensi-
tivities exceeding 80% [124, 126, 127, 145] Especially
metastatic disease may even be detected by SRS when
morphologic imaging modalities fail to show tumors in
unexpected locations or outside their anatomical range.
Furthermore, SRS can provide information about the
somatostatin receptor status of tumors in vivo. This is
relevant for the decision whether treatment with cold
somatostatin is potentially useful or not. In summary, in
patients with carcinoid tumors of the intestine, SRS
should be used first in the diagnostic process together
with abdominal ultrasound [128, 129].
John M. Pagel et al.
Figure 1. SRS (24-h scans) of a patient with a carcinoid of the ileum with metastases mainly to liver and bone. Anterior
view on the left, posterior view on the right. Uptake in the liver metastases is high, uptake in the tumor in the left shoulder is
comparable to that in normal liver tissue
Other Somatostatin Analogs
Besides octreotide, other somatostatin analogs have
been approved for clinical use or are under clinical evalu-
ation. Depreotide, a 99mTc-labeled somatostatin analog
(Neotect®) has been registered in the US for the charac-
terization of solitary pulmonary nodules. Depreotide is a
synthetic peptide with a cyclic, 6 amino-acid sequence
[158, 159]. It is a cost-effective alternative to FDG-PET.
In comparison to 111In-labeled somatostatin analogs, the
shorter physical half-life of 99mTc allows a simplification
of the imaging protocol. A higher activity is injected
(700–800 MBq) and diagnostic images can already be
obtained 90–240 min post injection [158, 160] including
SPECT. In comparison to CT-guided biopsy, 99mTc-dep-
reotide shows a comparable sensitivity while the specific-
ity is lower [159], due to uptake into inflammatory tissue,
such as granulomas. The specificity of 99mTc-depreotide
scintigraphy is clearly higher than that of CT [161].
Another interesting 99mTc-labeled somatostatin ana-
log is 99mTc-HYNIC-D-Phe1-Tyr3-octreotide. It shows
favorable results as compared to the commercially
479
available OctreoScan® (111In-DTPA-D-Phe1-octreotide).
Further clinical studies are necessary to finally deter-
mine whether this promising compound may be able to
replace 111In-labeled somatostatin analogs in clinical
routine [162, 163].
Lanreotide is an 111In-labeled somatostatin analog that
may also be used for cold somatostatin therapy as an
alternative to cold octreotide (Sandostatin®) [149].
Although it was reported that lanreotide has a higher
affinity for sstr3 as compared octreotide [164], these data
could not be confirmed by others [165]. As lanreotide is
more lipophilic than octroetide, tumor-to-background
ratios may be lower than with octreotide [166] Lanreotide
does not show clear advantages over octreotide.
Other Peptides for Tumor Targeting
Gastrin
Although SRS is a very potent tool in the diagnosis of
NET, it is of only limited value in patients with medul-
lary thyroid carcinoma (MTC) due to limited sstr2
480
expression of these tumors. Given the outstanding sensi-
tivity of the pentagastrin test in the detection of persist-
ing or recurrent MTC, the expression of the corresponding
receptor type in human MTC was postulated [167–169].
Indeed, more then 90% of medullary thyroid carcino-
mas (as well as other tumors) were found to express
CCK2 receptors [170, 171]. After initial evaluation of
several radioiodinated gastrin-derived compounds, a
DTPA-derivatized compound with the residualizing
111
In label was developed [126]. First clinical studies
show that 111In-DTPA-D-Glu1-Minigastrin is able to
Figure 2. Patient with a bronchial carcinoid with metastases to bone, liver, and mediastinum. Gastrin scan on the left, somatostatin
scan on the right. Both anterior view, obtained 24 h after injection
Targeted radionuclide therapy of cancer
demonstrate metastatic MTC with the highest sensitiv-
ity in comparison to PET, CT, and SRS [172]. Gastrin
receptors are also expressed in many gastrointestinal
NET [173]. A study comparing SRS with gastrin recep-
tor scintigaphy (GRS) showed that GRS was positive in
about half of the patients in whom octreotide uptake was
missing [172]. Furthermore, more than 20% of all
patients had higher uptake of gastrin than of octreotide
into the tumor lesions. Figure 2 shows a gastrin scan in
comparison to a somatostatin scan in a patient with a
bronchial carcinoid.
John M. Pagel et al.
Vasoactive Intestinal Peptide (VIP)
VIP receptors are expressed by most of human epithe-
lial cancers lacking expression of somatostatin recep-
tors or of the subtypes SSTR 2 or 5 detectable by SRS
[125]. Thus, VIP receptor scintigraphy was thought to
have great potential for diagnosis and possibly even
treatment of a wide range of tumors with high incidence,
especially colorectal cancer, gastric cancer, and exo-
crine pancreatic cancers. In comparison to somatostatin
analogs, biodistribution of VIP shows high pulmonary
uptake due to high levels of physiologic VIP receptor
expression in the lungs. Abdominal uptake is lower in
comparison to octreotide [174]. In clinical studies,
Virgolini and co-workers showed that primary tumors
and metastases of intestinal adenocarcinomas as well as
NET could be detected with 123I-labeled VIP [175, 176].
However, in a study of Hessenius et al. no adequate 123I-
VIP uptake into the tumors was observed [177]. These
findings were confirmed by autoradiographic studies of
resected tumors demonstrating lower VIP receptor
expression in the tumors in comparison to physiologic
VIP receptor expression in the surrounding tissues.
Neurotensin
Pre-clinical data suggest that 111In-DTPA and 111In-DOTA
labeled neurotensin analogs may be of high value in the
management of patients with exocrine pancreatic cancer
[134]. Buchegger et al. have studied the use of Tc-99 m
labeled neurotensin analog NT-XI in patients with ductal
pancreatic adenocarcinoma. Tumor was only visualized
in one out of four patients [178].
Bombesin
Receptors for bombesin are expressed on colon
cancer, glioblastoma, prostate cancer, and SCLC [126].
Radiolabeled bombesin potentially could be used to
visualize prostate cancer. After the development of radio-
metal labeled metabolically stable bombesin analogs
with high receptor affinity, clinical studies are currently
undertaken [179, 180]. A new 99mTc labeled bombesin
analog (demobesin 1) showed very favorable results in a
human prostate cancer mouse model with high prolonged
tumor uptake [181]. Clinical evaluation of this com-
pound will have to shows its value.
Glucagon-like Peptide-1 (GLP-1)
GLP-1 is an incretin hormone that induces postprandial
insulin secretion from pancreatic ß-cells. Thus, receptors
for this peptide are expressed by insulinomas (deriving
481
from pancreatic ß-cells), but also by other NET like gas-
trointestinal carcinoids [173]. For scintigraphic imaging,
GLP-1 and its analogs exendin 3 and exendin 4 seem to
have potential [135]. Especially for diagnosis and treat-
ment of insulinoma, GLP-1 might offer new possibilities.
Exendin-4 was modified C-terminally and conjugated
with DTPA. This Exendin-4 analog labeled with
111
In-had extremely high uptake in insulinoma lesions in
mice and even showed therapeutic efficacy when higher
activity doses were administered [182, 183].
RGD peptides: RGD peptides are a particularly inter-
esting entity of tumor-targeting agents. RGD-peptides
contain the amino acid sequence Arg-Gly-Asp and bind
to ανß3 integrin. ανß3 integrin is a heterodimeric trans-
membrane proteins involved in cell-cell interaction and
mediating cellular adhesion to extracellular matrix pro-
teins such as vitronectin, fibronectin, a.o. [184, 185]
RGD peptides inhibit neoangiogenesis in growing
tumors [186]. Specific tumor targeting of radiolabeled
RGD peptides could be demonstrated in several animal
models for melanoma, breast cancer, osteosarcoma,
pancreatic tumors, and ovarian cancer [136, 187–190].
RGD peptides clearing via kidney instead of hepatobil-
iary secretion have been developed. These peptides were
labeled with 99mTc-HYNIC and 111In-DOTA for tumor
imaging and evaluation of biokinetics in a mouse model
of a human ovarian cancer xenograft. Target-to-
background ratios as high as 92:1 and 26:1 were
achieved using this model [136]. Furthermore, the
90
Y-labeled compound showed inhibition of tumor pro-
gression in an in vivo mouse model. It has been pointed
out that RGD peptides seem to be an interesting more
general approach to the diagnosis and treatment of a
variety of tumors as neoangiogenesis is not limited to
one specific tumor entity only. However, one has to keep
in mind that the most effective targeting with radiola-
beled RGD peptides has been demonstrated in mouse/
tumor models in which the tumor cells themselves
express the ανß3 receptor on their cell surface.
Peptide Receptor Radionuclide Therapy
(PRRT)
For PRRT, radionuclides with high cytotoxic potential
that can be stably chelated are used. The ß-emitter 90Y
has been the first radionuclide to be applied for PRRT in
humans. Alternative radionuclides are the ß-emitter 177Lu
and the auger-electron emitter 111In (which is usually
used at lower activities for diagnostic purposes because
of its γ-emission). Other interesting candidates are
α-emitters such as 213/212Bi, or 211At. However, no stable
and secure labels with α-emitters are available for clinical
application in PRRT at the moment [126, 191, 192].
482
So far, most experience with PRRT is based on the
use of 90Y and 177Lu-labeled somatostatin analogs.
Dependent on the inclusion criteria, these studies show
that the rate of minor/partial responses is between
10–35%, while the rate of stabilizations of previously
progressive disease is higher. Interestingly, some
patients show a response several months after treatment
[193–202]. There is evidence that application of the
same cumulative activity results in a better therapeutic
response if the number of therapeutic cycles is lower
[198]. In general, PRRT is very well tolerated and only
a limited number of patients shows relevant haemato-
toxicity above grade 3. Nephrotoxicity is one of the
major problems of this therapeutic concept [203, 204],
but may be reduced by co-infusion of basic amino acids
[205, 206]. In comparison to the toxicity associated with
chemotherapy regimens for NET [207], PRRT is toler-
ated extremely well by the patients while the overall
response rates seem to be higher.
As the therapeutic success with 111In-labeled oct-
reotide is rather low, 90Y was preferred as a radionuclide
[208–211]. The data on the use of 177Lu labeled soma-
tostatin analogs are extremely promising as the rate of
complete and partial remissions rises to over 50% in
previously progressive patients. Thus, 177Lu-labeled
somatostatin analogs seem to be a real progress in the
treatment of NET [212].
Strategies to Improve Outcome
of RIT
Targeting radiolabeled Abs and peptides to malignant
cells has led to the establishment of noteworthy princi-
ples and encouraging results, yet obstacles still remain
that must be overcome to achieve optimal targeting and
elimination of cancer cells by radioimmunoconjugates.
For example, the above review indicates clearly that a
single dose of conventionally radiolabeled murine
monoclonal antibody administered at the MTD will not
provide sufficient radiation to tumor to be effective as
therapy for patients with solid tumors. In Phase I studies
with radiolabeled monoclonal antibodies, bone marrow
toxicity has been dose limiting and few complete
responses have been seen, even at the MTD. An impor-
tant reason for the poor results with systemic therapy
using radiolabeled antibodies has been the low accretion
of antibody in tumors and the fact that antibodies are not
ideal carriers of radiation to a tumor target. In general,
less than 0.01% of the injected dose localizes to each
gram of tumor. Explanations for this low accretion
Targeted radionuclide therapy of cancer
include restricted antigen expression on tumor cells,
the small fraction of the cardiac output which reaches
the tumor (it is estimated that it takes 400 h for all the
blood to pass through a 1 g tumor), and poor vascular
permeability because of vascular spasm and high inter-
stitial pressure from decreased lymphatic drainage that
results in a high pressure gradient compromising transport
towards the center [25]. Therefore several challenges asso-
ciated with RIT exist that limit the radiation absorbed
dose, and thus limit the therapeutic ratio. The dose is
inadequate because of poor penetration of the antibody
for reasons just mentioned, and the amount of radioac-
tivity that can be injected is limited because of marrow
toxicity. Although dose fractionation is used in chemo-
therapy and with external beam radiation therapy, the
development of HAMA precluded multiple doses on
antibody, particularly with murine antibody. RIT may be
most effective for small tumors which are more vascular,
but further work is still required to identify the role of
RIT in the management of cancer.
In order to increase the radiation absorbed dose to the
tumor, one can approach the issue of the therapeutic
ratio by either increasing the dose intensity or increas-
ing the radiation sensitivity of the tumor, or alternately
reducing the toxicity and thereby allowing higher doses
of radioactivity to be administered (Table 4).
Table 4. Strategies to improve outcome of RIT
Increasing dose Increase radioactivity administered
intensity Single high dose
Reduce immunogenicity to enable
multiple dosing
Improve the effectiveness of the
administered dose
Increase tumor localization
Compartmental administration
Intraperitoneal
Intra-lesional
Intrathecal
Increasing vascular permeability
Hyperthermia
External beam radiation
Interleukin-2
Integrin antagonists
Biological response modifiers
Increasing the dose rate
Smaller molecules
Pretargeting
Increasing radiation Chemotherapy agents
sensitivity Halogenated pyrimidines
Reducing the exposure Dose fractionation
to normal tissues Immunoadsorption columns
Pretargeting
John M. Pagel et al.
Increasing Dose Intensity
Increase Radioactivity Administered
The initial approach to increase the dose intensity was to
administer a single dose of more radioactivity. The dose
limiting toxicity is hematological and can be managed
with transfusions, cytokines and autologous marrow or
peripheral blood stem cell rescue, with reinfusion when
the total body radioactivity is at low levels, usually within
10 days. Blumenthal showed that IL-1 and GM-CSF can
allow increased doses of radioactivity to be administered
in mice [213]. The addition of hematopoietic growth fac-
tors has aided the recovery of patients undergoing these
procedures [214]. In one study, 90 mCi/m2 of 186Re was
the MTD for a pancarcinoma antibody, and with PBSC
rescue, doses up to 300 mCi/m2, were used, which was
not yet the MTD [215]. Two of three patients with ovarian
cancer treated with peripheral blood HCT achieved a PR,
whereas there were no responses at non-myeloabaltive
doses [216]. Similarly, 300 mCi/m2 of 131I-CC49 anti-
body was the MTD with marrow rescue [82], compared
with 75 mCi/m2 of 131I for this antibody without marrow
rescue. This approach has increased the MTD, however,
but has not resulted in significantly increased response
rates, and thus additional tactics to achieve tumor regres-
sion remain necessary.
The ability to administer more than one dose of murine
monoclonal antibody has been limited by HAMA [96,
217–219]. Immunosuppressive agents have been inves-
tigated to reduce HAMA. Lederman et al. [79] and Lane
et al. [78] administered Cyclosporin A to suppress the
development of HAMA in studies involving radiola-
beled anti-CEA murine monoclonal antibodies with
modest success. While this was successful in suppress-
ing HAMA to a F(ab’)2 fragment, this approach has not
been universally successful with an intact antibody
[220]. Low-dose cyclosporin, as used by Meredith with
a highly immunogenic antibody, was unable to signifi-
cantly reduce HAMA following murine CC49 delivery
[95]. Thus, cyclosporin may have some efficacy in
reducing immunogenicity of murine antibodies in
patients, but does not appear to be sufficient to permit
administration of multiple doses in all patients.
With modern genetic engineering, chimeric antibod-
ies have been produced to in an attempt to overcome
HAMA [219]. The immunogenicity of the chimeric
antibodies in generally less than murine antibodies, but
has not been sufficiently reduced to uniformly adminis-
ter multiple dose of RIT [41, 80]. Several humanized
antibodies, with only the hypervariable region of the
antibody molecule being murine, have now been studied
483
in clinical trials and have established that repetitive
doses of human monoclonal antibodies can be adminis-
tered without evidence of alloimmunization [221]. Thus,
it appears that the problem of HAMA may be solved
with human antibodies and dose fractionation may be
becoming more feasible.
Improving the Effect of the Administered
dose
Increasing Tumor Localization
Because of the physical limitation of accessibility of
antibodies to tumors, strategies have been developed to
increase tumor localization. Initially these included
changing the labeling procedures, changing the radio-
isotope, increasing the mass amount of antibody and
using an intra-arterial administration, none of which had
a significant impact. More recently other approaches
have been more successful.
Compartmental Administration
Intraperitoneal administration of the immunoconjugates
for RIT was shown to be feasible, relatively safe, and has
demonstrated anti-tumor effects as direct tumor cell
exposure results in improved antibody–antigen binding
[222]. Several trials have been undertaken in patients
with ovarian cancer using intraperitoneal administration.
131
I-, 90Y-, 186Re- and 177Lu-labeled antibodies have been
used for this purpose [223–228]. Encouraging tumor
responses were observed in patients with Stage III ovar-
ian carcinoma who had tumor nodules less than 2 cm in
diameter. Stewart initially reported 5/21 PR following
delivery of 131I-OC125 antibodies [228]. However,
another study with this radioimmunoconjugate showed
no benefit [229]. Following intraperitoneal 186Re-labeled
NR-LU-10, 5 of 12 patients with minimal ovarian cancer
achieved a PR [226]. The MTD following intraperitoneal
infusion of this radioimmunoconjugate, 150 mCi/m2 was
higher than following intravenous infusion (90 mCi/m2)
because of the reduced blood radioactivity accounting
for less marrow exposure [76]. Kavanagh reported anti-
tumor effects in patients treated with 90Y-B72.3, and
increased tolerance to 90Y with administration of EDTA
[230]. Iodine-131-DOTA-CC49 has also been successful
in achieving tumor responses in patients with <1 cm nod-
ules or extended progression-free survival in patients
with occult disease or microscopic disease [223]. 131I
MOV-18 was similarly shown to improve survival in
minimal disease [231].
Hird reported anti-tumor activity following 90Y-HFMG
but marrow toxicity limited the administered dose to
484
<30 mCi, even with the addition of intravenous EDTA to
chelate the unbound 90Y [225], and in patients with sub-
clinical disease there was a prolongation of survival fol-
lowing one IP dose of 90Y-HFMG [232]. Kosmos
reported that immunoglobulin against the chelate pre-
cluded more than one treatment using a DOTA chelate
to 90Y [233]. In a group of 21 patients who had achieved
CR following surgery and conventional chemotherapy,
one administration of intraperitoneal HMFG1 monoclo-
nal antibody labeled with 18 mCi/m2 90Y improved sur-
vival. The median survival had not been reached with a
maximum follow-up of 12 years and survival at greater
than 10 years was 78% [224].
Riva reported results in patients with peritoneal gas-
trointestinal carcinomatosis who received 100 mCi
131
I-FO23C5 via the intraperitoneal and intravenous
route [114]. Therapy was administered in conjunction
with Cyclosporin A every 3 months with up to four
injections. Three complete and six partial responses
were observed in 34 patients. Seventeen patients
received alpha-interferon to increase the expression of
CEA, with an increased the response rate to 59% com-
pared with 29% without alpha-interferon.
Increasing Vascular Permeability
RIT may be most effective for small tumors that are more
vascular, but manipulations to improve tumor blood flow
to larger tumors may be worthwhile. The influence of
vascular permeability on antibody uptake and distribu-
tion in tumors has been well documented [234]. Studies
to increase vascular flow or permeability at the tumor
using hyperthermia have been carried out by Stickney
et al. [235]. Hyperthermia reduces interstitial fluid pressure
and may improve tumor-associated antigen expression.
Local hyperthermia can also exert direct cytotoxic
effects, particularly on radioresistant and hypoxic cells
which are nutritionally deprived and acutely acidic, and
also cells in the S phase of the cell cycle. The degree of
cell killing depends on the degree and duration of hyper-
thermia. The results of the preclinical studies to assess
vascular permeability from external beam radiation prior
to administering immunoconjugate have varied with
tumor types and location [236, 237]. A three-fold
increase in antibody uptake was reported when antibody
was administered 1 day following low dose external
beam therapy in patients with hepatoma, without affect-
ing normal liver uptake [96]. Combined RIT/external
beam therapy clinical studies are underway for both
hepatic tumors [238] and head and neck cancer [239].
Interleukin-2 alone, modified with polyethylene glycol
or conjugated with antibody augments vascular permea-
bility and antibody uptake in tumor by a reaction with
Targeted radionuclide therapy of cancer
endothelial cells, either directly or via the activated
lymphocytes [214]. DeNardo et al. reported that modified
IL-2, PEGIL-2, administered before radiolabeled anti-
body enhanced antibody localization by a factor of two in
mice by increasing vascular permeability to the antibody
[240]. DeNardo also showed the rIL-2 increased the
response rate in xenografts treated with 67Cu-Lym-1 by
about 20% [241]. In a pre-clinical study, when IL-2 was
conjugated to an anti-CEA antibody, ZCE025, Nakamura
found that the cytokine function of IL-2 was destroyed,
but that vascular permeability specific to the tumor was
increased [242]. Epstein used IL-2 conjugated to TV-1,
an antibody to fibronectin that targets the basement mem-
brane and causes increased vascular permeability [243].
Followed by a radioimmunoconjugate directed against a
tumor-associated antigen, this showed three- to fourfold
increased tumor targeting. Increasing the vascular perme-
ability of newly formed vessels also appears to increase
antibody accumulation. DeNardo et al. used RGB penta-
peptide, which is a αvβ3 integrin antagonist and showed
a transient increase of 45–50% deposition of 111In-ChL6
after 24 h in a xenograft study [244].
Biological Response Modifiers
IL-2 and alpha interferon appear to be successful in
upregulating the antigen expression of CEA and TAG-
72. However, definitive results on the work with alpha
interferon and with IL-2 in clinical trials have yet to be
published. In 1990, Murray et al. showed altered biodis-
tribution and an improved relative tumor uptake of
111
In-anti-melanoma antibody in patients who had
received alpha-interferon [108]. In other clinical studies
evaluating the effects of alpha interferon, Greiner et al.
showed increased serum levels of TAG-72 and CEA
antigen [245]. Greiner et al. also demonstrated increased
levels of both antigens in ascites cells following intrap-
eritoneal injection of gamma interferon to patients with
ovarian cancer and increasing targeting in tumors, but
whether this will translate into increased tumor response
rate is still under investigation [246]. Increased response
rates have been noted in patients with breast cancer
receiving alpha interferon in conjunction with 131I-labeled
CC49 RIT, and IL-2 have been studied further [81].
Increasing the Dose Rate
Increasing the dose rate may be able to be achieved by
using radiolabeled antibody fragments or small molecules
which penetrate the tumor more rapidly. Early preclinical
studies supported the theory that total dose delivered may
be of primary importance. Thus, the belief that greater
exposure of tumor to radioactivity as determined by area
John M. Pagel et al.
under the curve led to the acceptance of intact antibod-
ies as the vehicle to carry the therapeutic radiation.
Dose rates effects have been examined by assessing
whether radioactivity delivered on an antibody frag-
ment that reaches the target earlier than using an intact
antibody. Fragments may provide an advantage compared
with the slower localization of intact radiolabeled anti-
bodies, even though the retention of the intact antibody at
the tumor is longer. Behr et al. compared response rates
of patients with CEA expressing tumors who received the
intact antibodies and the F(ab’)2 fragment of NP-4 and
MN-14 [75]. The data also showed that more frequent
responses occurred in patients receiving the radiolabeled
fragments, although only minor responses were reported.
Recently studies of 90Y on Octreotide peptide have shown
responses in phase I RIT studies for patients with neu-
roendocrine tumors [105].
Increasing Radiation Sensitivity
Radiation sensitizing chemotherapeutic agents such as
5-fluorouracil (5-FU) and cis-platinum have been used
in combination with 30 Gy external beam therapy and
have resulted in improved response rates in carcinoma
of the esophagus, colon, anus, cervix, bladder, and head
and neck compared with chemotherapy alone [247].
Radiation enhancement with these drugs has also been
seen in vitro with low dose-rate radiation. [248, 249]
Drugs to increase radiation sensitivity to RIT have been
studied in mouse xenograft models. Paclitaxel and 90Y-
chL6 [250], topotecan and 90Y-huBrE [251], cisplatin
with 131I-323/A3 [252] and 131A33 [249], gemcitabine
and B72.3 [253] have been studied. In pre-clinical stud-
ies, combinations of RIT with paclitaxel have induced
cures without increased toxicity [250] and topotecan
with RIT has shown an increased survival [251]. Clinical
studies with taxol are in progress. The effects of these
combination approaches are dependent on the timing
and dosing of the chemotherapeutic agents, and will
require considerable study in clinical trials.
Another approach to increase radiation sensitization is
to use halogenated pyrimidines as radiation sensitizers
[254]; 5-iododeoxyuridine [255, 256], fluorodeoxyuri-
dine (FUdR) and bromodeoxyuridine (BudR) [257–259]
have been most widely studied [255–257, 260]. These
act as a thymidine analogue that is incorporated into
DNA via the thymidine salvage pathway. This appears to
increase DNA susceptibility to radiation and inhibits
DNA repair. In vitro studies and xenograft studies of
these pyrimidines in conjunction with RIT have shown
increased tumor growth delay [256]. Clinical studies
using BudR and external beam therapy have shown a
485
survival advantage in patients with anaplastic astrocy-
toma [260] and responses have been reported in patients
with unresectable liver metastases from colon cancer and
FudR with external beam hepatic irradiation [261].
Reducing the Exposure to Normal Tissues
Several strategies have been proposed to reduce the
exposure of the marrow to radiation. Dose fractionation
appears to have an impact on reducing marrow toxicity,
but repeated fractions are limited by the formation of
HAMA with murine antibodies as discussed above [34,
35, 99, 104, 262]. An attempt to reduce toxicity was
done by fractionating the intraperitoneal 186Re NR-LU-
10 dose and administering a second dose of intraperito-
neal 186Re NR-LU-10 7 days after the first dose [227,
263]. At the highest dose level of 90 mCi/m2 twice (i.e.,
180 mCi/m2) there was severe marrow toxicity in one of
three patients. In contrast, similar toxicity was seen in
two of three patients in the single dose study at only
150 mCi/m2.
Meredith et al. have incorporated this strategy into sev-
eral clinical trials with B72.3 in patients with colorectal
cancer and with CC49 in patients with colon and prostate
cancer, and have confirmed a reduction in myelotoxicity
but the number of infusions was still limited by HAMA
[80, 95] Another approach is to remove the circulating
radioactivity that has not localized at the tumor site by
a clearing agent. Such a clearing agent could either be
administered intravenously or be extracorporeal, i.e., part
of an immunoabsorption column [264]. Candidate clear-
ing agents include anti-antibodies directed at the Fc por-
tion of the immunoconjugate or the avidin/biotin system
with one component linked to the immunoconjugate and
the other to the clearing agent [265].
The extracorporeal approach is being evaluated with
external immunoadsorption columns [264]. In this pro-
cedure, the patient’s blood is separated into cells and
plasma by a cell separator, phereis machine, and the
plasma is then circulated through a column which spe-
cifically removes the radiolabeled antibody, either by
another antibody, e.g., goat anti-mouse antibody or by
avidin removing a biotinylated radiolabeled antibody.
DeNardo et al. have employed some of these approaches
and have reported responses in patients with advanced
breast cancer with multiple doses of 131I-labeled chime-
ric antibody, L6 [264]. Some patients received G-CSF
7–20 days following infusion while others underwent
immunophoresis using a goat anti-mouse antibody to
reduce the non-bound circulating antibody. Four of nine
patients who were able to receive more than one dose
achieved a PR [101].
486
Pretargeted RIT
Conventionally radiolabeled antibodies or other large
molecules remain in the circulation for long periods of
time exposing the normal organs, especially the radio-
sensitive bone marrow, to radiation. The prolonged cir-
culating radiation limits the dose that can be safely
administered and ultimately limits the efficacy of the
approach. An alternative approach, pretargeted RIT, has
been used to administer the antibody and the therapeutic
radioisotope separately with some means of bringing
them together at the tumor. This has been an attractive
approach since the targeting specificity of the antibody
is retained, but the whole body radiation dose is sub-
stantially decreased because the radioisotope is admin-
istered on a small molecule, which is rapidly is removed
from the body through the kidneys if it does not bind
(Fig. 3). The potential advantages and disadvantages of
pretargeted RIT systems are summarized in Table 5.
Goodwin et al. pioneered the use of pretargeting, ini-
tially studying bispecific antibodies. The first step
involved pre-localizing an unlabeled antibody in tumors
that recognized both the tumor associated antigen and a
hapten, and then administering a radiolabeled hapten
that disappeared from the blood stream rapidly [266]
With a moderate affinity between the hapten and the
antibody, tumor uptake and radiation were sub-optimal
and a strategy with a higher affinity system, the affinity
enhancement system (AES) was developed [267, 268].
Here a bivalent hapten is used, that can cross-link the
bispecific antibody bound to the tumor, thus improving
tumor localization of the radioactivity. More recent
bispecific agents developed include a “universal” mole-
cule where one arm’s specificity reacts with the chelator
used for various radionuclides [269]. The AES system
has been evaluated for patients with small cell lung can-
cer. An anti-CEA x anti-DTPA-indium bispecific anti-
body was injected to assess feasibility, and this was
1st Step
Tumor localization
Blood clearance
mAb
Streptavidin
Figure 3. Pretargeted RIT example to remove unbound Ab constructs from the bloodstream prior to the administration of a
therapeutic radionuclide. Divalent Ab-SA conjugate followed by delivery of radiolabeled biotin [278]
Targeted radionuclide therapy of cancer
Table 5. Potential advantages and disadvantages of
pretargeted RIT systems
Potential advantages Potential disadvantages
• Favorable biodistribution • Ab constructs may be
and blood clearance difficult to manufacture
• Achieves high • Monovalent (to tumor antigen)
target-to-non-target Ab fragment constructs may
organ ratios decrease tumor residence time
• Flexibility with respect • Tetravalent constructs (e.g.,
to isotope and antigen SA) may cross-link
target Ab-antigens and internalize
• Immunogenicity (e.g., SA)
• Hapten release from
processed Ab may poison
tumor-associated conjugate
• Perceived as complex with
respect to dosing and timing
followed by the bivalent hapten, labeled with 40 to
200 mCi 131I. The MTD without stem cell rescue was
100 mCi. For patients receiving >100 mCi, 4 of the 15
patients had grade III/IV thrombocytopenia. At 12
months, there were 3 PR, one stable disease and nine
progressions in 13 patients evaluated. The pretargeted
approach with bifunctional antibodies and AES was
also studied in 26 patients with MTC. Biodistribution
was variable among patients, and dose limiting myelo-
toxicity was reached at 60 mCi/m2 131I. Anti-tumor,
minor responses and stablilization of disease were also
observed [267], but this preliminary study showed no
improvement over the conventionally labeled antibody
as regards tumor response or MTD.
Another popular approach takes advantage of the
ultra-high affinity avidin-biotin system (Kd = 10.15 M).
Avidin-biotin has been widely used in in vitro applica-
tions but was first translated into in vivo localizing strat-
egies by Goodwin, Meares and McCall [266]. Both
avidin and its bacterial counterpart streptavidin have
been used in pretargeting applications. Two and three
2nd step
Radio-delivery
Radiolabeled Biotin
John M. Pagel et al.
step pretargeting approaches were described with the
antibody carrying either the avidin/streptavidin or biotin
for imaging and therapy [270].
Imaging studies using bifunctional antibodies and the
avidin-biotin approach demonstrated the concept and
that improved tumor to background activity ratios could
be achieved [270–273]. A variety of tumors and anti-
bodies have been studied using this technique. The pro-
cedure has been safe and well tolerated, with improved
sensitivity due to the higher tumor-to-background
activity ratios in comparison to conventional radioim-
munoscintigraphy. The multistep approach, often requiring
72–96 h for completion has not been a practical
approach for imaging purposes. Rather, the potential of
this approach may be greater for therapy applications.
A number of approaches have been under study for ther-
apeutic applications. These include the reverse avidin-
biotin approach for targeted radionuclide therapy where
the antibody is conjugated to streptavadin rather than
biotin, and the biotin is linked to the radionuclide. The
preclinical performance of this system has been detailed
by Axworthy and coworkers, who reported 80–100%
cures of subcutaneous small cell, breast and colon carci-
noma xenografts (250 mm3) at 90Y doses of up to
800 mCi/mouse with negligible hematologic toxicity
[274]. With conventionally labeled antibodies, 200 uCi
was dose limiting because of marrow toxicity. An early
clinical study evaluated whether an improved tumor-to-
red marrow therapeutic ratio could be achieved using
pretargeted RIT compared with conventional RIT, and
at the same time preserve the efficiency of tumor target-
ing [275]. Forty-three patients with adenocarcinomas
reactive to an anti-EPCAM adenocarcinoma murine
monoclonal antibody, NR-LU-10 were studied. The
antibody–streptavidin conjugate was injected, followed
by a biotin-galactose-human serum albumin clearing
agent, followed by 90Y-DOTA-biotin as the final step for
therapy. In some patients, the conjugate was radiola-
beled with 186Re as an imaging tracer to assess biodistri-
bution of the conjugate and effectiveness of the clearing
agent. Indium-111-DOTA-biotin was co-injected with
90
Y-DOTA-biotin to assess the biodistribution, pharma-
cokinetics and dosimetry of the 90Y-DOTA-biotin. No
significant adverse events were initially observed after
administration of any of the components. The non-
tumor-bound antibody-conjugate was efficiently cleared
from the circulation by the clearing agent. The mean
tumor-to-marrow absorbed dose ratio when using the
optimized pretargeting schema was 63:1, compared
with a 6:1 ratio reported previously for conventional
RIT. This initial study confirmed that the pretargeted
RIT approach appeared to be safe and feasible, and
487
achieved a higher therapeutic ratio than that achieved
with conventional RIT using the same antibody.
Clinical data with both avidin-biotin approaches indi-
cate a substantial improvement in the tumor-to-marrow
absorbed dose ratios, with acceptable radiation dose to
other normal organs. A significant reduction in marrow
toxicity using the pretargeting approach to treat patients
with Grades III or IV glioma has been noted by Paganelli
and coworkers. They used doses of 150 mCi 90Y-DOTA-
biotin, about six to ten times the MTD by the conven-
tional approach, with negligible hematologic toxicity
and no acute or delayed side effects [270, 276]. At 2
months, in 45 patients there was a tumor effect in 20%,
55% of patients had disease stabilization and 25%
progressed. The tumor responses were maintained in
11% of patients at 12 months. Grade III/IV marrow tox-
icity occurred in two of five patients treated twice at the
highest dose level. Antibody developed to the streptavidin,
and only after repeated administrations to the biotiny-
lated antibody.
Pretargeted RIT for Lymphoma and
Leukemia
Pretargeted RIT was evaluated for treatment of patients
with relapsed or refractory NHL. In the first study, the ten
patients enrolled had received prior therapy, including
high-dose chemotherapy and peripheral stem cell trans-
plant (n = 3); 131I-tositumomab antibody therapy (n = 1);
and prior rituximab (n = 6). In this preliminary study, chi-
meric, anti-CD20 antibody (C2B8, rituximab) was chemi-
cally conjugated to streptavidin. Thirty-four hours after
the antibody conjugate was administered, a clearing agent
(synthetic biotin-N-acetyl-galactosamine) was adminis-
tered to remove non-localized conjugate from the circula-
tion. A DOTA-biotin ligand, labeled with 111In for imaging
and/or 90Y for therapy was administered 18 h later [277].
In three patients, the C2B8/SA conjugate was radiolabeled
with a trace amount of 186Re in order to assess pharma-
cokinetics and biodistribution using gamma camera imag-
ing. Rhenium-186 C2B8/SA images confirmed that the
conjugate localized to known tumor sites and that the
clearing agent removed >95% of the conjugate from the
circulation. The images demonstrated tumor targeting of
the DOTA-biotin within 10 min after the injection and
identified previously unknown disease in several patients.
Localization of radioactivity in normal organs was low
and unbound radiobiotin was rapidly excreted from the
whole body and normal organs. The median tumor-to-
whole body dose ratio of 35:1 was higher than previously
reported with conventional radiolabeled antibodies.
Seven patients received 30 or 50 mCi/m2 90Y-DOTA-biotin.
488
The regimen was safe and well tolerated; only grade I/II
non-hematologic toxicity was observed, the most preva-
lent toxicity was primarily fatigue. Hematologic toxicity
was also not severe; five of the seven patients who received
30 or 50 mCi/m2 of 90Y-DOTA-biotin experienced only
transient grade III (but no grade IV) toxicity. Although six
of ten patients developed humoral immune responses to
the streptavidin, these were delayed and transient. Six of
seven patients who received 30 or 50 mCi/m2 90Y (51 to
109 mCi total dose) achieved objective tumor regression,
including 3 CR and 1 PR. Although these results are pre-
liminary, they suggested that even heavily treated patients
with NHL can tolerate high doses of radioactivity with the
pretargeted RIT approach without requiring stem cell
transplantation.
Recent murine studies using human leukemia xeno-
grafts have demonstrated that a single treatment of pre-
targeted 90Y-DOTA-biotin after delivery of an anti-CD45
antibody-strepavidin conjugate results in a minimal level
of toxicity, and tumor-to-blood ratios of absorbed radio-
activity improved by as much as 30-fold over those seen
with a directly radiolabeled Ab [278]. It is estimated that
at least twice as much absorbed radiation can be deliv-
ered to the marrow and five-times more to the spleen,
using pretargeted anti-CD45 RIT as compared to doses
estimated to be delivered by a directly labeled antibody.
Improved reagents for pretargeting have been devel-
oped, including a synthetic clearing agent and fusion
proteins that include single-chain Fv fused to streptavidin
monomers (scFv)4−SA [14, 279]. The synthetic clearing
agent consists of a defined dendrimeric type synthetic
molecule with multiple galactosamine units and a biotin
for binding circulating streptavidin containing protein and
transferring it to the liver via the Ashwell receptors [280].
Advantages of the synthetic clearing agent are defined by
reproducible in vivo behavior, lack of biotin release fol-
lowing liver uptake and lack of competition for tumor
targeting by radiolabeled DOTA-biotin. The fusion protein
improvement that has been validated for several different
antibodies also results in expression of a defined protein
by E. coli, retained binding for biotin, comparable tumor
targeting compared to whole IgG antibodies and economic
advantages for protein production. These results have
prompted large-scale cGMP production of the anti-CD45
fusion protein for pretargeted RIT trials to be conducted in
patients with advanced myeloid leukemias.
A preliminary clinical study using 90Y-DOTA-biotin
pretargeted with either an anti-CD20 fusion protein
(B9E9FP) yielded encouraging results in 12 patients
[281]. The ratio of average tumor to whole-body radiation
dose was 49:1 in this study and no significant hemato-
logic toxicities related to the pretargeted RIT and with
Targeted radionuclide therapy of cancer
objective clinical remissions. With continued research,
RIT has the potential to find a place as first-line treatment
in radiosensitive tumors, as treatment for small volume
disease, or as a radiation boost in combination with other
treatments, such as with stem cell rescue or as adjuvant
therapy. Investigators are optimistic that these approaches
may improve the cure rates of the thousands of patients
with acute leukemia who are diagnosed each year.
Summary
RIT is a complex, multidisciplinary effort that still faces
many challenges. We have come to understand the obsta-
cles and have realized that a single high-dose of a radio-
labeled moiety administered systemically is unlikely to
be successful in curing most cancers. In most of the RIT
trials, radiation absorbed doses higher than 30 Gy have
only been infrequently reported, and responses cannot be
typically induced in the relatively resistant solid tumors.
However, these doses may provide clinical benefit in cer-
tain circumstances, in small volume or subclinical dis-
ease or with additional manipulations. The tumor
regressions observed in pre-clinical studies and in patients
with B-cell lymphoma and leukemia have encouraged
investigators to pursue antibodies and peptides as vehicles
for targeted therapy. In the hematological malignancies,
response rates are high enough and of long enough duration,
that two radioimmunoconjugates for NHL have been
approved for use in the United States. For hematological
malignancies, there may well be a place for RIT as first
line treatment with current technology. Although it seems
that the field of RIT has been moving very slowly, because
of the multiple obstacles that had to be overcome, there is
still much optimism for targeted therapy as the newer
approaches are being investigated.
The inadequate delivery of radiation to tumor has been
the major problem compromising RIT and increasing the
fraction of the radioimmunoconjugate that localizes in
the target tumor tissue has been difficult. Pretargeting the
antibody prior to injection of the radioisotope may be
one strategy that offers promise for lowering marrow
exposure to radioactivity and increasing the peak dose
rate to the tumor site. The dilemma of the proper posi-
tioning of RIT and the design of future clinical trials
remains. It appears that RIT may be used to treat small
disease or as adjuvant therapy for solid tumors, unless
the approaches described above are shown to be success-
ful in markedly improving the amount of radiation
deposited at the tumor. Despite the use of new peptides
in diagnosis and therapy of tumors and non-malignant
John M. Pagel et al.
diseases that have already been described, the most excit-
ing development in the near future may be the combina-
tion of different peptides and radionuclides in PRRT.
New developments include the development of multiva-
lent peptides that have a higher uptake in the target cell
[173], and the development of new peptide analogs with
affinity to more somatostatin receptor subtypes [282].
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14 Stem cell/bone marrow transplantation
as biotherapy
ROBERT K. OLDHAM
Autologous Bone Marrow
Transplantation
It is clear that bone marrow transplantation has become
a major technique in the treatment of metastatic cancer.
The use of autologous bone marrow transplantation has
allowed for much higher doses of chemotherapy to be
given in an attempt to eliminate all of the cancer cells
from the patient with the sacrifice of normal bone mar-
row function in the process. Autologous, cryopreserved
bone marrow or peripheral blood stem cells (PBSC) can
be reinfused to reconstitute stem cells and bone marrow
function. This process is still dose limited by damage to
the gastrointestinal tract, liver, lung, heart and other
critical organs [25, 42]. Some progress has been made
using this technique to allow dose escalation with che-
motherapeutic agents. Escalation of doses to the level
causing damage to secondary target organs is the current
limitation of this technique.
The problems of graft versus host disease (GVHD)
limits the use of allogeneic bone marrow transplantation
(ALBMT), although matching techniques and immuno-
suppression have improved the results for allogeneic
grafts [87, 92]. By contrast, autologous bone marrow
transplantation (ABMT) is a technique that does not
involve GVHD and allows for significant drug dose esca-
lation. Unfortunately, residual tumor cells can exist in
autologous bone marrow. Techniques must be developed
to effectively eliminate all replicating tumor cells from
these specimens such that the cancer is never reinitiated
in the patient after cure by high dose therapy [68].
Considerable progress has been made in leukemias
and lymphomas, and there is an ongoing and increasing
effort with BMT in solid tumors [59, 72, 82, 86].
Histocompatible donors can be selected leading to
successful allogeneic transplants [24, 59, 84]. The per-
fect transplant only occurs with identical twins (available
in less than 1 in 300 transplants), but matching of donors
at the HLA-A, -B, -C, and -D loci and selecting for those
individuals with negative mixed lymphocyte cultures
have improved the results of ALBMT. Engraftment of a
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
stable chimeric state can be demonstrated [84], but
GVHD (acute and chronic) results in the recognition of
recipient tissues by transplanted donor T lymphocytes
and occurs in more than 50% of ALBMT patients. More
than half of these individuals will have a fatal outcome
[35, 47, 84]. A variety of techniques have been used to
attempt to eliminate T cells from these marrow grafts.
These have included monoclonal antibodies [63, 72],
monoclonal antibody with complement [8, 33, 40, 51,
56, 64, 74, 80, 95], immunotoxins [23, 52, 65, 90], and
physical methods [6, 36, 62] to eliminate T cells from the
donor graft. Allogeneic BMT is covered later in this
chapter, but various techniques to eliminate T cells have
been reviewed [37]. Many of the techniques to deplete
the T cells for ALBMT are similar to techniques being
used to eliminate residual tumor cells in ABMT.
Therefore, a review of this literature is critical to the
reader who wants to understand all the current techniques
available for elimination of subsets of cells from bone
marrow prior to transplantation.
ABMT is the most popular current method of bone
marrow reconstitution and offers the advantage of avoid-
ing GVHD. Bone marrow cells in numbers (1 − 5 × 1010)
sufficient to reconstitute the individual’s marrow function,
through the stem-cell transplant, can easily be obtained
by multiple punctures of the bone with marrow aspira-
tion and then storage of the separated cells in liquid
nitrogen. These cells can be thawed and reinfused after
high-dose chemotherapy with consistent reconstitution
of the bone marrow. In fact, bone marrows can now be
separated and divided in such a way as to prepare one to
three grafts from a single bone marrow donor.
More recent application of this reconstituting tech-
nique has involved the use of PBSC harvests through
leukapheresis. This technique allows for stem cells to be
harvested by high volume leukapheresis and represents
a selection method for stem cells that avoids many of the
problems of tumor cell infiltration in the bone marrow.
Although peripheral blood may contain circulating
tumor cells, the evidence thus far indicates that consid-
erable positive selection of stem cells can be effected by
a peripheral blood harvest. This may obviate purging of
497
498
bone marrow or at least make the purging process easier
by virtue of having fewer tumor cells in the stem-cell
preparations. The techniques for removing tumor cells
from autologous marrow are numerous and have been
extensively discussed elsewhere [48, 61, 65, 89, 94].
Two comprehensive reviews can be found in the work of
McIntyre [53] and Gross and Gee [37].
The Preparation of Bone Marrow
for Transplantation in ABMT
Marrow manipulation must be carried out to preserve
the pluripotent hematopoietic stem cell in sufficient
numbers to insure engraftment. All of the procedures to
eliminate tumor cells discussed in this chapter must be
done in such a way to preserve adequate stem-cell activ-
ity. Clonogenic assays can be used to determine the
number and function of stem cells by assaying these
preparations for granulocyte/macrophage colony form-
ing cells (GM-CFC), burst forming unit-erythroid
(BFU-E), and granulocyte, erythroid, macrophage, and
megakaryocyte colony-forming cells (GEMM-CFC).
Long-term bone marrow cultures may also be used with
the Dexter culture system to measure cell renewal in
these systems [2, 22, 28, 32, 54]. Recently, simple flow
cytometry assessment of CD-34 lymphocyte numbers
has been used to predict stem cell numbers. While these
assays are useful to measure progenitor proliferation
and differentiation, the critical test is marrow engraft-
ment in the patient treated by the ABMT technique.
ABMT hematopoietic recovery is usually seen in the
20- to 40-day period after bone marrow infusion. White
blood cell recovery generally precedes platelet recov-
ery; and delayed engraftment can lead to fatal complica-
tions. These patients are often isolated from potential
surrounding infectious risk and must be carefully moni-
tored for viral, bacterial, and fungal infections during
the course of ABMT. Once the white count recovers to
the level of 500 granulocytes/mm3 and the platelet count
exceeds 30,000/mm3, the risk of infection and bleeding
markedly decreases. ABMT impairs immune function
but much less so than ALBMT. Immune recovery may
take months, and in some patients immune competence
is never really fully reconstituted [27, 31, 49, 66, 99].
To prepare marrow aspirates for transplantation, some
positive selection of the appropriate reconstituting cells
by physical means is commonly done. This may include
the simple task of cryopreservation, which eliminates a
large number of red blood cells, but may also include
separation procedures to obtain mononuclear cells from
the marrow [9, 21, 43]. Isopyknic centrifugation of
Stem cell/bone marrow transplantation as biotherapy
Ficoll-Hypaque gradients, isopyknic sedimentation on
discontinuous Percoll gradients, and the use of centrifu-
gation in blood-cell separators without a gradient are all
techniques that have been used to separate mononuclear
cells containing the stem-cell fraction [9, 21, 29, 43].
These techniques have eliminated the problems of clump-
ing and cell lysis, yield high numbers of progenitor stem
cells, and routinely give marrow engraftment in patients.
The technique of removal, manipulation, and preparation
for ABMT and the technique of peripheral stem-cell iso-
lation by leukapheresis in an outpatient setting have
gained wide clinical acceptance. These techniques are
now available and being utilized broadly in the clinical
practice of oncology, such that ABMT has become a
major treatment technique in patients with previously
refractory solid tumors [4, 10, 30, 32, 46, 76, 78, 84].
Techniques to Eliminate Specific
Subpopulations of Cells (Tumor
Cells and Lymphocytes) from
Marrow Specimens
There has been a huge number of specific techniques
utilized to attempt to eliminate either tumor cells and/or
T cells from bone marrow. Elimination of the former is
essential in ABMT, and elimination of the latter is desir-
able in allogeneic BMT. Some of these techniques
depend on a biological agent and thus represent a form
of ex vivo biotherapy, and others utilize chemotherapy,
radiotherapy, or physical techniques to effect marrow
purging. Each of these techniques will be reviewed with
an emphasis on biotherapeutic techniques for bone
marrow purging.
Although the techniques of purging tumor cells in
ABMT studies will be reviewed, it has been reported
that the purging of autologous marrow has no impact on
survival in patients with lymphoma. Data from the
European Blood and Marrow Transplant Lymphoma
Registry (EBMT) demonstrated that the purging of bone
marrow for tumor cells did not affect the rate of hemato-
logic engraftment or the risk of procedure-related death.
There was no significant difference in survival for
patients whose bone marrow was purged vs. case con-
trols left unpurged [83, 96].
Ex vivo Purging with Chemotherapy
Various agents have been utilized to exploit the differen-
tial toxicity for a specific chemotherapy drug on tumor
cells in contrast to the activity on the marrow stem
Robert K. Oldham
cells. Preclinical studies have demonstrated the effect
of corticosteroids [44], VP-16 and Verapamil [11],
4-hydroperoxycyclophosphamide (4-HC) and platinum
[60], 1-β-d-arabinofuranosylcytosine (Ara-C) and deoxycy-
tidine [34], VP-16 and corticosteroids [79], and alkyl
lysophospholipids (ALP) [94]. These studies indicate that
each of these approaches can eliminate tumor cells and/or
T cells (glucocorticoids) from marrows to be reinfused in
experimental systems and some in clinical trials [41].
Mafosfamide [69] and 4-HC, both relatives of cyclo-
phosphamide, have been utilized in clinical studies to
eliminate residual tumor cells. These cyclophosphamide
derivatives, as well as other chemotherapy agents,
appear to be promising in the elimination of residual
tumor cells from these marrow grafts [37].
Biophysical and Physical Approaches
A variety of biophysical methods, including photoradia-
tion [38], laser photoradiation [39], and isotope-medi-
ated purging [50] have been used to purge marrow of
unwanted tumor cells. These studies, sometimes with
chemotherapy purging, have primarily been used in pre-
clinical studies.
Physical separation methods such as elutriation [58]
can be used to positively select bone marrow stem cells
for ABMT, and this technique can be used as a negative
selection technique against tumor cells in ABMT and T
cells in ALBMT. Clinical studies of this technique are
underway and appear promising. Cell separators can be
utilized for the separation of mononuclear cells for mar-
row [1] or peripheral blood stem cells [97] for transplan-
tation. Various other physical approaches have been
used, but the most promising and straightforward
approaches involve machines that can carry out such
processing in an automated format.
Biotherapeutic Approaches
A variety of biotherapeutic approaches have been used to
treat bone marrow prior to reinfusion. Lymphokine acti-
vated killer (LAK) cells induced by interleukin 2 [18]
have been used to purge bone marrow of residual tumor
cells with interesting preclinical results. Antibody alone
and antibody plus complement have been used by a vari-
ety of investigators for the elimination of residual tumor
cells [70]. The Campath series of antibodies may have
the dual use of purging lymphoma or leukemia cells
from the marrow and may also be useful in the in vivo
elimination of residual tumor cells after ABMT. More
than 520 patients have been treated in various European
transplant centers in an approach to deplete T cells and
lessen GVHD in allogeneic BMT [16]. These results cer-
499
tainly demonstrate the usefulness of this approach in
eliminating T cells. The application of this technique to
ABMT for purging malignant lymphocytes carrying the
antigens recognized by the Campath antibodies is the next
logical step. In multiple myeloma, antibody plus com-
plement [88] can eliminate residual myeloma cells from
the marrow. Antibody plus complement has been used in
preclinical and clinical studies [55] to purge myelocytic
leukemia cells from marrow prior to ABMT. An inter-
esting study of an antibody conjugated to Adriamycin,
where the antibody also fixed complement, has been
reported, but the preclinical activities demonstrated
insufficient selectivity for clinical application [98].
In addition to antibody alone and antibody-fixing
complement, there has been a series of studies using
antibody conjugated to toxins, usually ricin, to prepare
immunotoxins that can be used in bone marrow purging
[67, 93]. These immunotoxins have been used in ABMT
for T-cell acute lymphoblastic leukemia [91] and to
eliminate T cells to decrease GVHD in ALBMT. These
ricin immunotoxins have been utilized in clinical stud-
ies in marrow purging of solid tumors such as neuro-
blastoma [13] and breast cancer [77].
Combination Techniques
Investigators are pursuing combination techniques with
antibody tied to magnetic spheres (biotherapy plus
physical separation) to eliminate residual marrow tumor
cells. These studies are very well reviewed by Gross and
Gee [37] and will not be covered in detail here. Suffice
it to say that various physical techniques are being
developed where bone marrow is being purged by the
dispersion of magnetic beads coated with antibody.
These antibody-coated beads attach to tumor cells and
can be extracted from bone marrow by passing the prep-
aration over a magnet. By pulling out the residual tumor
cells, one can prepare a bone marrow free of residual
tumor cells for reinfusion. These studies certainly appear
interesting, and further preclinical studies and early
clinical applications are being pursued [37].
Stem-cell Selection
There is now increasing evidence that biological tech-
niques to select stem cells by positive methods will result
in enhanced marrow preparations for ABMT. Antibody to
CD-34 can be used to select stem cells from bone marrow
or peripheral blood [5, 15]. This technique has mainly
been applied to bone marrow, but with the development
of PBSC collection techniques [97], the same positive
stem-cell selection process could be applied to peripheral
blood. High speed clinical cell sorters and antibody-based
500
positive selection systems are both used in clinical trials as
methods to select and purify peripheral blood stem cells
for ABMT.
A technique with far-reaching implications is to cul-
ture stem cells continuously in vitro [17, 57]. This
approach may allow for the repeated infusion of autolo-
gous bone marrow stem cells free of any residual tumor
cells and without the presence of any immunologically
active T cells [3]. The potential for the broad application
of culture stem cells is obvious, even to the point of cryo-
preserving such stem cells for each interested individual,
while living and healthy, in anticipation of their use later
in life in the presence of disease. Preserving cord blood or
fetal tissues for use later in life as stem cells is also being
explored. Cord blood is now routinely used for reconsti-
tution of marrow function after marrow ablative therapy
[20]. Such techniques, while interesting, bring up the
whole question of how far technology should take us in
anticipating disease and dealing with the potential for
developing specific diseases. There are various social,
political, and economic consequences of techniques that
may predict or be useful in the treatment of future dis-
eases. In fact, the whole area of genetic therapy is now
coming forth as a potential major form of biotherapy. Not
only can stem cells be grown in vitro for eventual use in
reconstituting bone marrow, gene therapy with electropo-
ration and other techniques can be used to insert specific
genes in normal or defective human cells [45]. Very prim-
itive stem cells may carry few transplantation antigens
and after long-term culture may be useful broadly in
reconstituting stem cells. It is clear that biotherapy and
gene therapy will lead to tremendous advances and oppor-
tunities but also bring forth major considerations of “who
pays for clinical research” and “who should have access
to these new techniques.” (See Chapter 3)
Allogeneic Bone Marrow
Transplantation
Allogeneic BMT has been successful in treating certain
lymphoid and hemopoietic neoplasms. Initially, the total
body irradiation plus high dose chemotherapy followed
by ALBMT was felt to work primarily through the cytore-
ductive effects of the chemoradiotherapy. Innovative sug-
gestions by Professor Georges Mathé as early as 1965
and follow-up evidence reviewed by George Santos in
1972 suggested that there was an anti-tumor effect
independent of the chemoradiotherapy from ALBMT.
This has more recently been clarified as a form of graft-
versus-tumor (GVT) effect similar to the concept of
Stem cell/bone marrow transplantation as biotherapy
GVHD as has been well understood in allogeneic bone
marrow transplantation since the early days of this tech-
nique [71]. GVHD has been well characterized for many
years. It is known to have an acute and more chronic vari-
ety, primarily manifest by a clinical syndrome of skin
rash, liver and gastrointestinal dysfunction as well as a
variety of autoimmune type effects [71]. A variety of
strategies, primarily total body irradiation and/or sys-
temic chemotherapy has been used to reduce the number
of T cells from the graft responsible for GVHD. These
strategies have been more or less effective, usually requir-
ing lifelong treatment of patients who have experienced
allogeneic GVHD.
Of particular interest has been the concept of autolo-
gous GVHD which was initially quite a controversial
theory [73]. The autoreactive cells found in autologous
GVHD can cause damage to a variety of organs, most of
which are less severe than that seen in allogeneic BMT.
Histocompatibility differences cannot explain this phe-
nomenon since these autologous transplants cannot have
histocompatibility differences. Therefore, immune dis-
regulation is felt to play the major role in autologous
GVHD. Treatment is usually not necessary since the
syndrome is normally benign and symptomatic care is
usually sufficient for patients with manifestations of
autologous GVHD [19].
Graft-versus-tumor Effects
Early evidence that bone marrow cells of allogeneic
BMT producing a graft-versus-leukemia effect has
prompted the more general idea of graft-versus-tumor
(GVT) effect of both ALBMT and ABMT. Most stud-
ies found a correlation between GVH and reduced
relapse rate for patients with leukemia treated by
ALBMT. Similar observations have been seen, though
less striking, in ABMT [73]. The presence of autoreac-
tive T cells related to Ia antigens in many of these syn-
dromes and also noting that Ia antigens are expressed
on leukemias and lymphomas with perhaps similar
antigens being present on certain solid tumors. The
potential for identifying these cells and using them after
ex vivo application continues to intrigue investigators in
this area. Whether these cells may be purely T cells or
be some form of LAK or NK cell is currently under
investigation. The ability to grow large numbers of acti-
vated T cells opens the possibility of utilizing cells
identified by transplantation techniques as useful for
their GVT activities [19].
The idea of using ABMT as primary therapy to reduce
tumor load and then to exploit the immunosuppression
Robert K. Oldham
that follows to administer partially or unmatched donor
lymphocyte infusions(DLI) for their further biothera-
peutic effect until the host eliminates them is being
tested in several European centers and less frequently in
the USA. Complete responses of persistent disease post
ABMT has caused considerable enthusiasm for this
approach in some circles [19].
Recent evidence has provided further credence to
the concept that the immune system is active in treating
leukemia post allogeneic BMT. This information has
come from studies in patients with chronic myelogenous
leukemia and includes the following factors.
1. Marrow grafts that have been T-cell depleted have a
higher rate of leukemic relapse and less GVHD.
2. An inverse relationship has been seen in HLA identical
BMT between the occurrence of GVHD and leukemia
relapse.
3. The GVT effect arises from a reduction in leukemic
recurrences post allogeneic BMT compared to
homozygous twin transplants.
4. Relapsed allogeneic BMT CML patients can be rein-
duced into complete remission simply by infusing
donor leukocytes [85].
Conclusion
ABMT and ALBMT offer major opportunities in cancer
biotherapy [19]. With ABMT the major question relates
to the sensitivity of cancer to a dose escalation strategy of
chemotherapy and radiotherapy. If these modalities can
eliminate cancer from the patient at doses between the
marrow lethal dose and the maximum tolerated dose to
the next organ system (gut, liver, lung, heart, etc.), then
the technique of ABMT with high-dose therapy may be
broadly applicable and curative in certain solid tumors.
This technique will always have major, inherent toxicity
because of the toxicities of the therapeutic agents.
However, short-term toxicity and even some risk of long-
term effects would be tolerated by patients with lethal
diseases if complete remissions and prolonged remission
duration can be engendered. On the other hand, if the
dose-response curve for human solid tumors is insuffi-
ciently steep to allow for the induction of complete
response and improved survival with these techniques,
then the broader application of this approach may not be
warranted, given the significant toxicities involved.
Recent results, summarized above, using allogenic
peripheral blood mononuclear cells are particularly
encouraging, with clear GVT effects now being seen in
CML and other leukemias/lymphomas, confirming the
501
earlier, more intuitive observations of Mathé and Santos
[71]. As summarized by Bishop [7], the non-ablative
(mini) allogenic bone marrow transplant is being inves-
tigated as a potentially less toxic transplant where the
donor cells are functioning as a form of adoptive cellu-
lar therapy using the GVT effect as biotherapy.
Studies of ALBMT comparing myeloablative condi-
tioning regimens with non-myeloablative treatment in
lymphoma and chronic lymphocytic leukemia has shown
that the outcomes were similar and that the nonablative
regimens could be applied to patients with more comor-
bidities and in older age groups [81], however, the greater
power of myeloablative ALBMT has also been demon-
strated when used after failure of ABMT in lymphoma.
Studies from the International Bone Marrow Transplant
Registry have demonstrated complete remission in
selected patients who have failed ABMT and then treated
with myeloablative ALBMT [26].
Biotherapeutic approaches toward the elimination of
residual tumor cells appear to be the most promising
with regard to ABMT. Similar approaches to eliminate T
cells from allogenic bone marrow may broaden its appli-
cation. The two most promising techniques appear to
rest in the use of antibody to negatively or positively
select appropriate cell populations in marrow transplan-
tation and the use of physical separation methods, such
as leukapheresis, to positively select for replicating stem
cells. Most specifically, the technique of peripheral blood
leukapheresis to select for stem cells, perhaps with their
eventual long-term culture for repetitive use, does appear
to be a highly promising technique to provide stem-cell
reconstitution in patients lethally damaged by high-dose
chemotherapy and radiotherapy. These studies are at an
early stage but they are rapidly evolving, with the tech-
nique of ABMT being already widespread in the clinical
practice of oncology [10, 75]. The availability of colony-
stimulating factors (see Chapters 17 and 18) provides a
further mechanism for the manipulation of stem-cell
numbers and activity. The outpatient use of autologous
bone marrow transplantation through leukapheresis to
select the stem cells and the use of colony-stimulating
factors to support stem cell growth in vitro and in vivo
appear to be promising approaches in the treatment of
advanced solid tumors [12, 14, 76].
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15 Cellular immunotherapy (CI), where have
we been and where are we going?
JOHN R. YANNELLI
Abbreviations used CI, Cellular Immunotherapy; NSCLC,
Non-small cell lung cancer; DC, dendritic cell; TIL,
tumor infiltrating lymphocyte; LAK, lymphokine activated
killer cell; MHC, major histocompatibility complex;
IL-2, Interleukin-2; GM-CSF, granulocyte macrophage
colony stimulating factor; TCR, T cell receptor; LDTA,
lymphocyte defined tumor antigens; MLTC, Mixed
lymphocyte tumor cell culture
Overview
Previous reviews have detailed the development of
cellular therapies using immunologically nonspecific
lymphokine activated killer cells (LAK), activated killer
monocytes (AKM), and eventually tumor specific tumor
infiltrating lymphocytes (TIL). The innovations in large
scale cell culture methodologies and the developments
in immune assessment approaches have made the thera-
pies more widespread as well as more effective against
a variety of tumor histologies.
While surgery, radiation therapy, and chemotherapy
remain the accepted conventional treatments for cancer;
immunotherapy continues to make a case as a therapy to
be used in the adjuvant setting. Surgery reduces tumor
burden, radiation therapy kills residual tumor cells in
the surgical field, and chemotherapy kills residual and
micrometastatic disease both systemically and also in
the surgical field. However, neither radiation nor che-
motherapy provide tumor specificity and both can inca-
pacitate patients due to their effects on normal cells,
particularly the bone marrow. Cellular immunotherapy
(CI), a potential fourth modality of cancer treatment,
can target tumor cells sparing normal cells and tissues.
The difficulty that is now faced, in 2008, focuses on
the overall effectiveness of CI and justifying its use
based on the cost which includes: cell culture reagents,
technician time and recombinant cytokines. In the
current funding environment, these costs have mounted
to levels which make it difficult for researchers at aca-
demic NIH supported centers to seriously utilize CI, at
least in the way it has been practiced in the past. I have
a colleague who scoffs at me when I talk about this
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
subject; he says if the field were successful it would
bankrupt America. While being accurate to some degree,
this concept has forced a re-evaluation and a forced
reduction in the in vitro approaches. A new focus has
arisen which is on generating the effector cells in the
patients, using vaccine therapies as stimulus. The majority
of newer CI protocols to date reflect this approach.
Vaccines coupled with pre-treatment conditioning using
chemotherapeutic agents is the way of the future, at
least for the next few years.
The current chapter will serve as a historical review
and present some of the new and exciting approaches
being utilized in CI which is nearing the 25th anniver-
sary of Steve Rosenberg’s report on LAK cells in the
New England Journal of Medicine [1]. The Author’s
experience since this time should serve to enlighten and
hopefully be comprehensive enough to allow the Reader
to consider CI and realize its value to cancer treatment.
A Recap of where the Field
has been
In the early 1980s, focus of CI was on the use of the
innate arm (Natural killer cells, NK cells; Lymphokine
activated killer cells, LAK cells; and macrophages) [2]
of the cellular immune response to treat primary and
metastatic cancer. The idea that immune effector cells
such as LAK could be activated to recognize and kill
tumor cells across a range of tumor histologies with little
consideration for immune specificity drove the laboratory
and clinical studies [1, 3, 4]. Interestingly, pre-clinical
and murine studies being conducted at this time at the
NCI ([5–7], began to lay the framework for the future
direction that CI would take. Their studies on lymphocytes
that infiltrate tumors (tumor infiltrating lymphocytes, TIL)
were fundamental to current CI approaches which now
focus on antigen targeted therapies.
In some cases, especially in melanoma, TIL were
shown to have functional reactivity against tumor cells
and were shown to traffic to tumor sites [8–11]. This
ability to home to tumor is considered critical for
505
506 Cellular immunotherapy (CI), where have we been and where are we going?
clinical effectiveness [8]. The discovery of the T cell
receptor (TCR) and details regarding the three dimensional
structure formed by the interaction of antigenic peptides
and self-MHC (either class I or class II; major histo-
compatibility complex, MHC) furthered our under-
standing and provided more rationale for their clinical
use [12–15]. On a practical level, improved and stream-
lined cell culture techniques, the bulk production of
newly identified growth and/or differentiation inducing
cytokines such as IL-2 and the identification of lympho-
cyte defined tumor antigens (LDTAs) facilitated large
scale growth of these tumor reactive T cells (TIL) for
clinical trials.
TIL populations are comprised of varying numbers of
CD4 helper and/or CD8 cytotoxic T cells (CTL). CD4
helper cells recognize self MHC class II and 18–23
length amino acid peptides derived from tumor associ-
ated antigens. Specific CD4 cells help to orchestrate
immune responses through cytokine production. In some
cases, certain populations of CD4+ cells, co-expressing
CD25, have been shown to suppress immune responses
to tumor [16, 17]. This population will be further
discussed below. CD8+ CTL recognize MHC class I
antigens and eight to ten amino acid length peptides.
Following recognition, CTL are capable of cytokine
production and killing of tumor cells by direct granule
mediated cytotoxicity as well as delivering death sig-
nals mediated by FAS (See Reviews) [18–20]. These
two mechanisms may actually be preferentially used in
different anatomic sites. Thus, a means to tailor CTL
therapies is suggested [21]. Much of what was learned
concerning lymphocyte recognition of tumors was
gained from studies using TIL derived from melanoma
patients. In addition to tumor biopsies, melanoma spe-
cific T cells have also been derived from both patient
and normal donor peripheral blood [5, 6, 22–42]. Both
melanoma specific TIL and specific T cells derived from
peripheral blood have been utilized as tools to identify
LDTAs using a variety of cellular and molecular tech-
niques (see reviews: [37], [43–45].
As mentioned above, in some cases, CI can target
tumor cells and spare normal cells. With our greater
understanding of the nature of LDTAs, however, it is
now clear that a number of tumor antigens are normal
cell proteins with altered expression patterns. For exam-
ple, in TIL trials it was shown that melanoma specific
TIL, following reinfusion, were also capable of killing
normal melanocytes [46]. Thus, it has become impor-
tant to understand T cell specificity for tumors within
the context of mechanisms which control peripheral
tolerance. Emphasis now is to better understand toler-
ance, apply principals of tolerance to tumor models and
alter tolerigenic mechanisms to allow the destruction of
tumor but spare normal tissue. But, considering expres-
sion patterns of some of the LDTAs, the overall benefit
of immediate tumor cell destruction and the potential
for memory against tumor recurrence probably outweigh
some of these concerns.
Our expertise and initial successes in immunotherapy
were gained in melanoma patients [47]. While the
majority of LDTAs have been described in melanoma, a
highly immunogenic tumor, LDTAs have also been
identified in other solid (breast, colon and lung for
example) and liquid tumors (lymphomas and leukemias)
(See Reviews [48–51]). This provided the rationale
to try CI in these tumors, once considered non-
immunogenic and not candidates for experimental
therapies. The goal is to improve CI in melanoma and
apply the concepts and improvements to existing thera-
pies for other tumor histologies. All this considered,
with the extensive knowledge and experiences gained
thus far, investigators are now able to do what was origi-
nally intended in the early 1980s. That is to use defined
tumor antigens in vitro to generate more homogeneous
anti-tumor T cell populations. The relative monetary
savings over earlier bulk T cell approaches using these
selected T cell populations made the therapy attractive
for clinical investigators, laboratory personnel, and
regulatory agencies. Extending these advances to the
current use of vaccines to educate the immune system
with these antigen preparations in vivo against specific
tumor antigen targets has become a more logical recent
focus. It may be more advantageous to generate anti-tumor
immunity in vivo, an active form of immunotherapy,
and spare the uncertainties associated with large scale
cell culture. Using sound immunologic based principals
learned in viral and bacterial systems, these vaccines
should be capable of boosting existing anti-tumor
immune responses and help to generate new responses
against tumor antigens that have escaped recognition.
A point to consider, however, is whether immuniza-
tion strategies will be effective against bulky metastatic
disease. It is possible that vaccines may only play a role
in the adjuvant setting. Currently, the therapies are
structured in the following way. Surgery remains the
first line of therapy as a de-bulking procedure. Radiation
and/or chemotherapy are used for the removal of residual
and micro-metastatic disease. Concurrently, construct
vaccines and perform safety testing and deliver the
vaccines in prime and boost strategies when the patient’s
immune system has recovered. This approach should
generate “sentinel” lymphocytes which can seek out and
destroy small pockets of metastatic tumor which escape
recognition or become resistant to conventional therapies.
John R. Yannelli
The inherent nature of specific lymphocytes also
predicts the development of memory to protect against
future tumor recurrence. Thus, the combination of
standard therapies with immunotherapies may provide
valuable data and an exciting future in cancer therapies
[52–57]. This strategy of combined chemotherapy and
immunotherapy is being investigated by our group
(Hirschowitz and Yannelli, 2008 unpublished observa-
tions). A clinical trial of maintenance chemotherapy
in non small cell lung cancer using Pemetrexed + or −
allogeneic tumor cell vaccine is being tested in our
center. It is early in the study but using stage III and IV
NSCLC patients who possess bulky disease; we are
monitoring time to progression as well as immunologic
responses to the vaccine. Both clinical evaluation and
immunologic monitoring taken together will provide
more impetus for moving forward with vaccines and a
better understanding of much needed improvements.
Finally, it also remains to be seen, however, whether
vaccines can be used as a defense against the initial
development of tumors. These studies are best tested in
murine models of spontaneous disease. Once we gain
experience with these models and novel vaccines are
proven efficacious, populations at risk for certain forms
of cancer due to environmental factors or genetic dispo-
sition should be considered as potential candidates.
Use of LAK and TIL
in the Treatment of Cancer,
are they gone Forever?
In the early 1990s, frustration arose concerning CI trials
such as LAK [1, 3, 4] and TIL [22, 58–60]. These stud-
ies had been ongoing since 1984 but examination of the
clinical responses made investigators consider more
closely the cost to benefit ratio. While LAK was work-
ing in some melanoma patients, it was a difficult therapy
to deliver. The priming steps with large doses of recom-
binant IL-2, the repeated leukapheresis procedures, the
large-scale cell culture, and the requirement for large
numbers (>1011) of LAK cells in multiple cycles made
this a challenging therapy. In addition, the large doses of
IL-2 required for LAK cell maintenance made this clini-
cally difficult for physicians, support staff and patients.
The requirement for hospitalization, many times care in
the ICU, made LAK therapy a challenge and doomed its
spread to the general practice of Oncology. In addition,
complete and durable responses were not the norm.
The financial costs associated with this therapy were
also daunting for both the clinicians and the patients.
507
Unfortunately, the therapy utilizing a short exposure of
leukapheresis products to high concentrations of IL-2
(Pulsed-LAK; [61]) did not engender the enthusiasm
which was originally intended. Thus, LAK cell therapy
overall was considered too cumbersome a therapy to
routinely deliver. Today, with the renewed interest in the
innate immune response to cancer, an improved form of
this therapy may receive more attention and be utilized
in combination with more specific cellular approaches.
Whether this approach is successful remains to be seen.
In the mid 1980s, the TIL trials began with the hope
that more durable clinical successes in a wider range of
tumor types could be realized. However, the enthusiasm
that greeted the Science report by Rosenberg et al. in
1986 [60] detailing the murine TIL studies was not real-
ized in the early human clinical trials of TIL. In hind-
sight, the reason for this can be attributed to a number of
issues. Immunogenicity of the tumors being studied,
clinical use of bulk TIL populations containing nonspe-
cific T cell populations, and the failure to co-deliver
effective cytoreductive and/or immunosuppressive
chemotherapy were some of the reasons for the disap-
pointing results. Additionally, TIL were being used to
treat bulky disease. We now realize the tumor burden
contributes to a significant tumor and/or host derived
suppressive environment. In retrospect, all of these
factors served to dampen the initial enthusiasm.
A breakthrough occurred in the early 1990s following
a retrospective analysis of the accumulated clinical and
laboratory data. A 1991 report by Aebersold et al. [24]
discussed characteristics of effector cells that were
important for clinical responses. For instance, age of the
TIL, doubling time of TIL, and the ability of the TIL to
immunologically recognize autologous or MHC
matched tumor all proved to be predictors of clinical
responses. In two follow-up reports by this group, both
Rosenberg et al. [60] and Schwartzentruber et al. [62]
extended the observations following an analysis of 5
years of laboratory and clinical data. Interestingly, the
tumor most responsive to immunotherapy was mela-
noma. Other tumors such as breast, colon and lung can-
cer for instance did not appear to be responsive to TIL
therapy. The 30% or so of melanoma patients that
showed clinical responses all demonstrated the ability
of their TIL to either kill in chromium release assays or
specifically secrete cytokines following incubation with
autologous or MHC matched melanoma tumor cells.
The concept of shared tumor associated antigens pre-
sented by common MHC antigens had already been
described in reports by Topalian et al. [6], and Muul
et al. [5, 39]. These investigators had shown that mela-
noma TIL could recognize autologous tumor cells and
508 Cellular immunotherapy (CI), where have we been and where are we going?
in many cases melanoma tumor cell lines expressing
shared MHC class I antigens (HLA-A2 for example).
These observations were later extended to MHC class II
antigens [28, 29, 34].
The relatively simple melanoma-TIL culture conditions
and the frequency of clinical responses make melanoma
an ideal tumor model to study CI. While culture of enzy-
matically digested tumor biopsies in high concentrations
of IL-2 resulted in tumor specific CD4 and/or CD8 T
cells from about 40% of melanoma biopsies tested [35],
it is also one of the easier 1tumors from which to derive
stable long term tumor cell lines [47, 63]. Stable TC
lines make detailed immunologic analysis easier.
Interestingly, in the early studies, site of melanoma
tumor biopsy appeared to predict whether specific T
cells were obtained. That was, cutaneous and visceral
lesions were more likely to provide anti-tumor specific-
ity than were TIL derived from tumor involved lymph
nodes [35]. A recent report however, showed melanoma
reactive TIL could be derived from 39% of tumor
involved lymph nodes tested [64].
A recent TIL report reiterates the enormous potential
of this therapy, particularly in melanoma. With improve-
ments to both laboratory TIL procedures and pre-treat-
ment of patients with targeted chemotherapy, increased
clinical effectiveness of the approach was shown [46,
65–67]. Use of highly selected, antigen specific mela-
noma reactive TIL following pretreatment of patients
with the immuno-depleting drugs fludarabine and cyclo-
phosphamide resulted in 6 of 13 objective clinical
responses with 4 additional mixed responses. The
infused TIL were shown to proliferate in vivo, traffic to
tumor sites, and displayed marked functional reactivity.
Compared to the early TIL trials, these results were
markedly improved. Thus, in melanoma, TIL remains a
very viable treatment option and continues to provide
the most interesting clinical results of all immunothera-
py’s tested to date. This group continues to lead in the
laboratory aspects of CI with a study published online.
In the most recent report, evidence is presented that very
early TIL cultures are the most efficacious in terms of
anti-tumor reactivity. These T cells contained in early
culture, while possessing the largest number of anti-
tumor T cell clones, do not suffer from long periods of
time in cell culture where important clones are over-
grown or critical cell surface receptors and costimula-
tory molecules are down-regulated [68].
Examples of specific TIL derived from other tumor
histologies include: colon cancer [69, 70]; esophageal
cancer [71]; breast cancer [72–75]; follicular lymphomas
[76]; ovarian cancer [77, 78]; renal cell cancer [79–82];
and non small cell lung cancer [83, 84] to name a few.
The frequency of successes, however, using standard TIL
culture conditions was far less than seen in melanoma. In
the majority of cases, nonspecific TIL or TIL with no
functional reactivity were derived. This result may reflect
an inherent inability to lyse fresh tumor cells other than
melanoma. But, even when tested against stable MHC
matched cell lines, no specific lysis or cytokine release
was usually observed. Based on the predictions made by
the Investigators at the NCI [24, 60, 62], the infrequent
generation of specific T cells from non-melanoma tumor
biopsies could explain the overall disappointing clinical
results obtained in the early TIL trials. This assessment is
strengthened by other published reports [81, 85].
Despite these difficulties, novel cell culture method-
ologies have been developed since the early TIL trials
were begun and improved results in non-melanoma T
cell cultures. These studies showed that tumor specific T
cells could be derived from the peripheral blood of can-
cer patients with a variety of cancers. Specific immuno-
logic reagents were then made available for clinical and
further immunologic studies. Interestingly, however, the
proliferative potential of these T cells is not the same as
T cells derived from solid tumor. TIL appear to grow
more consistently and to higher levels than specific T
cells obtained from peripheral blood. TIL can prolifer-
ate in the presence of IL-2 alone and often do not need
antigen restimulation. In the TIL studies, over 1011 T
cells were often derived from less than 1 × 106 cells [23,
35]. In our own laboratory, we generated over 1011 effec-
tor T cells from as few as 100 T cells. It is now evident
that the growth and differentiation signals vary for T
cells derived from different anatomic sites including
peripheral blood, solid and liquid tumors, and tumor
involved or tumor free lymph nodes. These differences
reflect the maturational state of the T cells that are con-
tained in these sites including differences in the expres-
sion of the chemokine receptor CCR7 and the isoforms
of CD45 [86–88]. Thus, concerning peripheral blood,
factors such as when the blood sample is obtained may
be critical. There are likely differences in what propor-
tions of immature versus mature T cells are contained in
the sample which is most probably influenced by the
overall tumor status and trafficking patterns of T cells. It
is then unknown what is required for optimal expansion
including: antigenic stimuli, frequency of restimulation,
growth factors, and costimulation. With this said, the
removal of the T cells from the potential suppressive
environment of a tumor bearing host [89–95] for in vitro
expansion should be advantageous.
Autologous or allogeneic melanoma tumor cells were
used as stimulators in mixed lymphocyte tumor cell cul-
tures (MLTCs) and MHC class I restricted specific CTL
John R. Yannelli
were sometimes observed [96–99]. Peripheral blood
from both normal donors and cancer patients were used
in these studies. Celis et al. [100] derived CTL from
the peripheral blood of normal donors using antigenic
peptides derived from melanoma LDTAs. The culture
conditions in these studies relied on low concentrations
of IL-2 and in some cases IL-7 provided at multiple
intervals during the stimulation and growth phases. More
recent methodologies used gene transfer of costimulatory
molecules such as CD80 into fresh or cultured tumor
cell lines. In these studies, tumor specific T cells against
melanoma [38, 101] and other solid tumors were
observed including NSCLC [102], and renal cell cancer
[103–105]. Interestingly, our studies (Bixby and Yannelli
[102] and [84] have shown that NSCLC specific CTL
could only be generated following exposure of T cells to
CD80 gene modified tumor cells confirming a require-
ment for costimulation.
Finally, gene transfer as a tool to improve T cell reac-
tivity has gained recent interest after a series of early
problems [106–110]. Our group at the NCI performed
early studies using retroviral mediated gene transfer of
TIL with genes encoding the cytokines IL-2 and TNF-
alpha [111, 112]. The novelty of using IL-2 was associ-
ated with the desire of infusing anti-tumor T cells that
had an inherent capacity to release IL-2 in order to pro-
vide growth stimulus to the T cells in vivo. Of course an
issue with IL-2 release is controlling the endogenous
IL-2 production thus preventing the growth of T cells
with other specificities, particularly self reactive T lym-
phocytes. TNF-alpha, however, was very intriguing
during the early days of gene therapy. While we produced
autologous melanoma tumor cell vaccines secreting
both IL-2 and TNF-alpha [63], much effort was made to
transduce T cells with the same retroviral vectors. In
murine models TNF-alpha delivered at the tumor site
was observed to be a very potent modulator of tumor
response, speculated to be the result of tumor vascular
destruction. These results led to limited human trials
which were eventually halted by the FDA due to safety
issues encountered early on [113]. Coupled with the
understanding of lymphocyte defined tumor antigens
(LDTAs) and the characterization of tumor reactive T
cell receptors {Cole, 1994 #28}{Shilyansky, 1997 #26;
Cole, 1997 #25; Cole, 1995 #27; Shilyansky, 1994 #30;
Nishimura, 1994 #29}{Roszkowski, 2005 #9}, most
recent attempts are proving fruitful in the clinical setting
[106–110]. Unfortunately, the laboratory requirements
in expertise and molecular probes along with regulatory
issues make this approach less applicable to the general
academic and oncologic setting. None the less, the
knowledge of TCRs and particularly LDTAs is critical
509
to the future improvement of CI. Acknowledging the
critical importance of the discovery of LDTAs;
(Reviewed in: [43–45, 114]), the remainder of this
Chapter will focus on the new approaches to CI utilizing
defined LDTAs as targets for therapy.
Lymphocyte Defined Tumor
Associated Antigens (LDTAs)
Lymphocyte defined tumor antigens (LDTAs) have been
identified using a variety of molecular techniques (See
Reviews: [37, 43–45, 115]. There have been more than
70 identified at the writing of this Chapter. The tech-
niques used to identify antigens include: (1) cDNA
cloning (described below) [26, 116, 117]; (2) a “reverse
immunology” approach which generate specific T cells
against peptides derived from sequences of serologically
defined tumor antigens [118–123]; (3) peptide stripping
and HPLC [124–127]; and the newer more advanced
molecular techniques including SEREX [128–130],
Proteomics [48, 131, 132], and Microarray analysis
[133, 134]. The technique initially used to describe a
number of antigens in melanoma and other histologies
which is still in use is cDNA-cloning [25–27, 30–33, 37,
116, 135–137]. A distinct advantage of this technique is
that it is based on the availability of already defined
tumor specific T cells. It does not speculate as to their
existence as some of the newer techniques do, thus
bringing into question the relevance of the LDTA. The
reagents required are tumor specific CTL, cDNA gener-
ated from a tumor cell line recognized in an MHC
restricted fashion by the T cells, and an indicator cell
line which can be transfected with both MHC antigens
and pools of cDNAs. A cDNA library is prepared, pools
of cDNA are transfected into 293 human kidney cells or
COS cells which express the restriction element recog-
nized by the T cells. The CTL must be tumor specific as
defined by extensive specificity analysis, and should
specifically release cytokines which can be measured
using standard ELISA assays. In addition, a reasonable
number of T cells are required (generally 5 × 108 to 1 ×
109). When pools of cDNA are identified which confer
recognition of the indicator cells by the CTL, the experi-
ments are repeated by screening dilutions of the cDNA
pools until a single cDNA is identified. The cDNA is
sequenced, the protein identified and characterized for
expression on tumors of the same histology, tumors of
different histologies, and normal cells.
The antigens discovered thus far by all these techniques
are classified into three categories based on histologic
510 Cellular immunotherapy (CI), where have we been and where are we going?
expression of the genes. The first group identified are
termed cancer testes antigens and are expressed in
normal testes, melanoma, and other tumor histologies
including breast, lung, bladder, and squamous carcino-
mas. These antigens include the MAGE gene family:
MAGE-1 [116], MAGE-2 [138] MAGE-3 [139] and
BAGE [140]. Additional cancer testes antigens identified
include MAGE-12 [141], GAGE [142], and NY-ESO-1
[130]. The second group includes melanocyte lineage
proteins which are expressed on normal melanocytes as
well as tumor cells. These antigens include gp100 [26],
MART-1/Melan-A [25], tyrosinase [143] tyrosinase related
protein-1 (TRP-1) [33], tyrosinase related protein-2 [32]
and melanocyte-stimulating hormone receptor (MC1R)
[144]. The third group are mutated normal proteins which
generate unique epitopes, such as the case with Beta-
catenin [137] and others including MUM-1 [145], CDK4
[146], caspase-8 [147], and KIA0205 [148]. Certain of
these melanoma antigens such as tyrosinase, have also
been shown to be recognized by class II restricted T cells,
one such antigen is tyrosinase [29, 34]. The questions
now which remain unanswered are which antigens are
most biologically relevant to the cancer patient and which
peptides derived from each protein are the most immuno-
genic? Once these answers are obtained, it is possible that
combinations of peptides, both CD4 and CD8 specific,
and/or antigens will be shown to be more efficacious as
targets for vaccine strategies.
There are also a number of antigens, fewer defined at
the moment, which have been identified in other cancers
such as MUC-1 [149, 150], CEA [151], p53 [152] Her-2/
neu [153, 154], SART-1 [155], ART-1 [156] and ART-4
[157]. As populations of specific T cells are being iden-
tified, so are the antigens they recognize. Our lab, utilizing
specific T cells generated against CD80 gene modified
NSCLC lines, identified two antigens which include a
myoinositol monophosphatase protein (IMPA) [158,
159]and a GTP binding protein termed GNAS [160].
Unfortunately, based on normal tissue distribution, both
appear to be minor histocompatibility antigens which
limit their eventual clinical use [161]. However, cDNA
cloning strategy remains a very powerful tool for LDTA
identification.
Cancer Vaccines
The major thrust of CI over the past 10 years, has been
the development of vaccines for cancer treatment (See
Reviews [162–169]. Regardless of what constitutes the
vaccine, the desired result is to provide tumor antigen in
a stimulatory manner which allows the in vivo generation
of a potent anti-tumor immune response. Vaccines con-
stitute an active form of immunotherapy since they are
not dependent on the injection of large numbers of in
vitro derived effector cells. Rather, in most cases, the
vaccine is delivered in hopes that the patient’s own
immune system generates the effector cells in vivo.
Thus, patients enrolled in vaccine trials must be immuno-
competent. Ideally, a cancer vaccine should: (1) exhibit
minimal toxicity, (2) induce the appropriate tumor-
specific immune responses against primary tumor and
metastatic lesions, (3) induce a memory response to
protect against tumor recurrence and (4) be designed so
that it is affordable by cancer patients. The overall goal
of course is to achieve a measurable therapeutic benefit,
most likely derived from both the cellular and humoral
arms of the immune system. In a Review, Bodey et al.
[170] summarizes the potential pitfalls to the approach.
Since vaccines rely on specific B and T cell immunity,
many factors must be considered including: (1) loss of
MHC expression by tumor cells, (2) low immunogenicity
of many of the LDTAs, (3) LDTA down-regulation, (4)
secretion of immuno-regulatory molecules by tumor
and infiltrating host cells, and (5) impaired antigen
presentation by Dendritic cells (DCs). These points
taken alone or in combination can predispose many
vaccines to failure. However, in light of these difficul-
ties, successes have still been reported.
In general, cancer vaccines can be divided into three
categories. These include purified LDTAs, whole
autologous or allogeneic tumor cell vaccines, and anti-
gen pulsed dendritic cells (DCs). Each has its advan-
tages and disadvantages and will not be discussed in
any particular order of importance. The first category
is based on recently identified LDTAs and utilizes
these molecules as immunizing agents. This includes
natural or synthetic proteins, protein derived peptides,
RNA, “naked” DNA, or DNA encoded in delivery
vehicles such as recombinant viruses, plasmids, or
recombinant bacteria (See Reviews above). Liposomes
have also been used for the delivery of a variety of
immunogens [171]. In the development of this type of
vaccine, melanoma again has served as the prototype
tumor. Similar advantages have been taken of the
established immunogenicity, thus, melanoma vaccine
development has proceeded rapidly in the past 10 years.
Vaccines have been generated using the defined LDTAs
including MART-1, gp100, gp75, MAGE-1, MAGE-3,
BAGE, GAGE, tyrosinase, GM2 and GD2 [162]. In
addition, as antigens are being discovered in other
histologies, vaccine development is also proceeding
rapidly. This is evidenced by the use of carcinoembryonic
antigen (CEA), MUC-1, Her-2neu and Prostate specific
John R. Yannelli
membrane antigen (PSMA) as immunogens in clinical
trials [163, 164]. Overall, this approach is promising
for many cancer patients since it is based on sound
immunologic principals we have learned in bacterial
and viral systems.
The ability to apply LDTAs as a vaccine strategy is
very dependent on the knowledge of the patient’s tumor
antigen expression. This can only easily be determined
by the availability of autologous tumor. Unfortunately,
this is not always possible since some patients are unre-
sectable, or previously resected tumor lesions are
unavailable at the time of the trial. In addition, since the
vaccine relies on priming the patient against tumor pro-
teins and MHC presented peptide antigens, MHC and
LDTA expression by the tumor cells is critical. Decreased
expression of these proteins by the tumor will hamper
the effectiveness of the vaccine. There is much evidence
in the literature for the decreased expression of MHC
antigens, particularly HLA-A locus antigens by tumor
cells [172–174]. One solution is combining the vaccine
with agents designed to maintain or increase MHC
expression, such as gamma interferon. This will work
assuming that genetic changes have not occurred
affecting MHC class I or Beta-2 microglobulin produc-
tion, assembly or transport to the cell surface. Secondly,
the inclusion of multiple antigens in the vaccine, a
multivalent approach, increases the expression of at
least one of the antigens if the other is lost by mutations
or mechanisms of protein down-regulation exercised by
selected cells in the tumor. Monitoring expression is
also important during the course of treatment to insure
that changes do not occur affecting the potential long
term success of the vaccine.
A second vaccine strategy is a whole cellular vaccine
using autologous or allogeneic fresh tumor cells or
tumor cell lines. Autologous tumor cell vaccines attempt
in a controlled fashion to immunize the patient against
all the antigens that are endogenously expressed by their
tumor. The use of autologous tumor cells to make indi-
vidualized vaccines is certainly optimal; however, it
does have its drawbacks. To accommodate multiple or
even a single vaccination requires a reasonable amount
of fresh tumor biopsy. This in itself selects against a
number of patients. Some patients have small lesions,
the tumor is inaccessible or the patient is inoperable.
This is a particular problem in patients with NSCLC.
Widespread tumors in the thoracic cavity are many
times too difficult to obtain and it becomes an issue of
cost versus benefit to the patient. In some cases the
tumor cell content of the biopsy obtained is variable or
the tumor cells are necrotic. An alternative strategy
is the development of an autologous tumor cell line but
511
that can be a challenge depending upon the histology
being studied. Some histologies such as melanoma are
easier to derive a cell line from than other histologies.
The development of a stable tumor cell line requires
careful attention to growth medium, serum concentra-
tion, growth factors, and substrate making some histolo-
gies more difficult but certainly not impossible to derive
stable long term lines [63, 175–178] (Yannelli et al.,
Lung Cancer, submitted 2009 still in review). As a solu-
tion, short term tumor lines are often attempted and used
for study [179–181]. Sometimes, however, one has to
question the integrity of the lines in regard to tumor cell
content. The difficulty is that tumor biopsies are not
100% tumor cells. The tumor cell content is variable
and biopsies are mixed with fibroblasts and host infil-
trating leukocytes. In addition, the viability of tumor
cells can be a problem. Often, viable tumor cells exist
on the periphery surrounding dead necrotic tissue in the
center of the lesion. Following dissection and enzyme
digestion the tumor cells often die in the processing.
Hence, what appears to be a short term tumor cell
line is in many cases normal cells such as fibroblasts
and/or macrophages. In the vaccine, what has the
patient actually been exposed to, tumor cells or adher-
ent normal cells? Many of these studies fail to fully
characterize the short term lines before use. Thus, clin-
ical results can be misleading. It is critical to evaluate
the line for tumor antigen expression, MHC expression
and certainly cell viability. In our own studies with
NSCLC, we observe tumor colonies forming early in the
cell culture period. With time, however, these colonies
fail to increase in size and are overgrown by fibroblasts
(Yannelli et al., Submitted to Lung Cancer, 2008).
None the less, clinical studies conducted by Berd et al.
[182] utilized autologous whole-cell tumor vaccines in
stage IV melanoma patients. Their nonrandomized trial
demonstrated a 12.5% response rate, as measured by the
development of delayed-type hypersensitivity against
autologous melanoma tumor cells. In a subsequent
study, they conjugated the hapten dinitrophenyl to the
autologous vaccine and observed striking inflammatory
responses in metastatic lesions of patients. In addition,
an increase in disease-free survival was achieved [183].
Use of autologous tumor cell vaccines has also been
attempted in other tumor histologies with some degree
of success. Vaccination in renal cell cancer also demon-
strated an anti-tumor immune response and a survival
benefit in treated patients [184]. More recently, Dillman
et al. [181] showed that short term lines could be gener-
ated 43% of the time in a study including 125 cancer
patients of varying histologies. In this study, patients
who developed positive delayed type hypersensitivity
512 Cellular immunotherapy (CI), where have we been and where are we going?
(DTH) responses to autologous tumor cell vaccines
showed improved survival.
For the difficulties described using autologous tumor,
many investigators have chosen to study stable long
term allogeneic tumor cell lines as vaccines due to their
availability and the presence of shared tumor antigens.
Allogeneic lines are normally standardized in terms of
growth conditions and are amenable to manipulations like
gene transfer. Use of these lines decreases the variability
in vaccine preparations used for multiple immunizations.
These lines also reduce the variability between patients.
Again, however, it is critical to characterize antigen
expression by patient’s tumor and be sure the antigen or
antigens are expressed by the allogeneic line chosen for
the vaccine. It is also important to match at least one or
more MHC class I antigens between the cell line and
patient to insure the tumor derived peptides are pre-
sented properly. The availability of over 30 NSCLC
lines in our lab makes an allogeneic tumor cell vaccine
very plausible. We have characterized: (1) the growth
characteristics of the lines, (2) tumor antigen expression
of the lines, and (3) MHC expression of the lines [185].
These are critical parameters in the development of
allogeneic tumor cell vaccines. In a published study, a
polyvalent whole cell melanoma vaccine, prepared
with three allogeneic cell lines, produced a higher
median survival than historical controls and resulted in
both cellular and humoral immune responses against
melanoma antigens [186, 187]. Evaluation of a vaccine
(Melacine) consisting of lysates from two allogeneic
cell lines in the adjuvant Detox found it comparable to
chemotherapy in prolonging survival while causing
much less toxicity [188–190]. However, while the
clinical results appear promising in melanoma [191],
there should be caution when using allogeneic reagents
for immunotherapy. These tumor cells, while potentially
expressing shared tumor antigens, also express alloge-
neic MHC class I and in some cases class II antigens
which can give rise to potent allogeneic immune responses.
These vaccines may be quickly rejected before the
tumor response is allowed to develop (direct response
involving T cell recognition of allogeneic MHC antigens
on tumor cells). Alternatively, a slower developing
allogeneic response (indirect response involving pro-
cessing and presentation of allogeneic MHC antigens
by host DCs), may develop along with the anti-tumor
response. The response to allogeneic antigens may
provide additional levels of critical cytokines necessary
for the overall development of the anti-tumor response
(bystander effect). It is important, however, to have the
reagents available to decipher the response and deter-
mine if it is an anti-tumor, an allogeneic, or a mix of the
two. It is possible to be misled. What appears to be an
anti-tumor response may actually have simply been an
allo response. The vaccine was rejected with little to no
impact on the patient’s tumor. Clinical ineffectiveness
may be due to a faulty vaccine. On the other hand, there
has been literature over the years suggesting that the
generation of a third party allo-immune response can be
beneficial to the overall development of an anti-tumor
response [192].
An additional approach to tumor cell vaccines has
been to genetically modify the autologous or allogeneic
tumor cells to express agents designed to enhance the
anti-tumor immune response [63, 193–196]. Immune
stimulating molecules such as the cytokines TNF-alpha,
GM-CSF, gamma interferon or IL-2 for example have
been used to aid in the development of immune
responses. In some cases the tumor cells have been
gene-modified to deliver lymphocyte co-stimulatory
molecules such as CD80 [197–199]. Allogeneic mela-
noma cell lines engineered to secrete IL-2 were admin-
istered to melanoma patients in separate studies [200,
201] and each produced clinical responses, albeit weak
ones. Haight et al. [202]demonstrated an antibody
response to IL-2 expressing allogeneic neuroblastoma
cell lines in six patients with neuroblastoma. Additional
studies have tested a large number of cytokine genes for
efficacy. Using a murine melanoma model, Dranoff
et al. [203]compared ten genes for their ability to enhance
tumor cell immunogenicity. Of all the genes tested,
retroviral transduction with GM-CSF produced potent,
long-lasting specific anti-tumor immunity. GM-CSF
was first recognized for its ability to stimulate the growth
and differentiation of myeloid lineage hematopoietic
progenitor cells. GMCSF is produced by T cells, mac-
rophages, endothelial cells and fibroblasts in response to
immune stimuli, which acts in a paracrine fashion to
recruit neutrophils, monocytes and lymphocytes for
host defense [24–26]. Central to its use as an immune
adjuvant in vaccines, GMCSF is known to enhance
macrophage activity and increase dendritic cell (DC)
maturation and function, thereby augmenting antigen
processing and presentation [204]. Subsequent studies
revealed this immunostimulatory cytokine exerts pleio-
tropic effects on the immune system. Importantly, it
promotes the differentiation and stimulation of dendritic
cells (DCs) which are professional antigen presenting
cells capable of sensitizing naïve T lymphocytes and
eliciting a primary T cell response. Interestingly, early
TIL trials in melanoma demonstrated that the specific
release of GM-CSF by TIL often correlated with significant
clinical responses [62]. One can speculate that the CD14+
cells recruited to the tumor site through inflammatory
John R. Yannelli
mechanisms become stimulated by the GM-CSF,
undergo differentiation and accomplish tumor antigen
presentation to T cells in draining lymph nodes. Thus,
GM-CSF may be an especially promising cytokine for
gene-modified vaccines.
Synergy between antigenic protein and GMCSF has
been shown in several clinical studies [205–209]. In
NSCLC, Nemunatis et al. evaluated the effects of
GM-CSF gene-modified autologous cancer cells with
encouraging biologic and clinical results [210, 211].
Similarly, an autologous melanoma vaccine secreting
GMCSF reported in 1997 produced anti-tumor immune
responses associated with a partial but temporary
clinical benefit [197, 212]. A Phase I clinical trial inves-
tigating the biologic activity of these vaccines found
infiltration of T cells, DCs, macrophages and eosino-
phils at the immunization sites in all 21 patients evalu-
ated [213, 214]. In addition, autologous tumor-reactive
CTL were detected following vaccination. GM-CSF-
secreting autologous tumor vaccines have also been uti-
lized in clinical trials for renal cell cancer and prostate
cancer [195, 215–217], and similar anti-tumor immune
responses were observed. Together, these results dem-
onstrate the feasibility and safety of this vaccination
strategy, the low level of toxicity to patients, and
confirm the bioactivity of this reagent.
Thirdly and the most recent approach is the use of
antigen-loaded DCs as immunogens [218]. While all the
results are not in at the time of the preparation of this
Chapter, we would speculate that in a future review the
clinical use of DCs will have been proven in one appli-
cation or another. We anticipate that DCs will constitute
a critical component of vaccines delivered to not only
cancer patients but also to many patients having a vari-
ety of immunologic disorders. DCs are the most potent
antigen-presenting cells in the body. It is speculated in
many cases that an inherent problem in tumor immunol-
ogy is the inadequate presentation of tumor antigens to
lymphocyte precursors. This vaccine approach is
designed to remove DC precursors from the patient, dif-
ferentiate them in an environment free of the tumor-
induced suppression, and pulse them with relevant
tumor antigen before injection. The high expression of
costimulatory molecules (CD80, 86, 40) and MHC class
I and II molecules; the DCs ability to process and
present relevant tumor derived peptides; and their abil-
ity to secrete IL-12p70 makes them very attractive as
immunogens. Interestingly, DC based vaccines do not
require that tumor antigens be fully characterized. This
approach can be utilized in the treatment of tumors with
primarily undefined antigens. If autologous tumor cell
preparations are used to pulse the DCs, the patient is
513
receiving his or her tumor-derived peptides in the
context of self-MHC. The primary disadvantage of
utilizing DCs as a vaccine, however, is the potential
development of immune responses against “self” anti-
gens shared by tumor and normal cells. Ludewig et al.
[219]reported on the induction of severe autoimmune
disease with dendritic cell (DC) immunotherapy. With
that stated, however, DCs may still be the most power-
ful tool in immunotherapy to date.
Dendritic Cells and Vaccines
DCs can be derived from normal donors and patients
with cancer including: B cell lymphoma [220, 221];
breast [222]; CML [223]; colon [224]; gliomas [225];
gynecologic malignancies [226]; medullary thyroid
carcinoma [227]; melanoma [10, 228–230]; myeloma
[231]; NSCLC [232]; prostate [233–235]; renal cell
cancer [236, 237]; and various leukemias [238];
(additional references in [218]). Dendritic cells are
capable of presenting peptides processed from tumor
protein antigens to CD4 helper and CD8 cytotoxic T
cells. In addition, through cytokine release DCs can
regulate the function of NK and NKT cells.
In the laboratory, DCs are routinely generated from
CD14+ adherent monocytes obtained from either
peripheral blood draws or leukapheresis products. In
some cases, CD34+ precursors are used and are obtained
following mobilization using G-CSF (granulocyte
colony stimulating factor) infusions. The potency of
DCs to orchestrate T cell immune responses and the
technologies recently developed to generate large numbers
has made them an attractive component of immuno-
therapy protocols. Therefore it is not surprising that
researchers have focused on their utilization in the
development of novel vaccine strategies for the treat-
ment of cancer [218, 239].
CD34+ DC progenitors differentiate into CD14+/
HLA-DR+/CD11+ intermediates that circulate systemi-
cally and come to reside in peripheral tissues [240, 241].
The differentiation of DCs from CD14+ precursors is a
complex process which results in a heterogeneous popu-
lation of cells characterized by phenotypically defined
subtypes. In addition, depending upon the mode of
cellular and/or cytokine stimulation, these DC subtypes
may acquire distinct and opposing functions in regard to
the cellular immune response (stimulatory, DC1; inhibi-
tory, DC2; See Reviews [218, 242–244]. This is the
topic of much debate when considering which differ-
entiation agents to use in the generation of DCs for
clinical trials.
514 Cellular immunotherapy (CI), where have we been and where are we going?
CD14+ cells removed from peripheral blood are
less than stimulatory for cellular immune responses
due to the large amounts of IL-10 secreted following
stimulation with soluble agents (LPS for instance)
(Yannelli et al., unpublished observation). Thus,
proper differentiation of CD14+ cells to DCs is criti-
cal for T cell stimulation. The differentiation steps
most likely begin at the site of inflammation.
Interestingly, it is the final maturation of DCs which
appears to be critical to the development of a potent
Th1 cellular immune response. Immature DCs are
considerably less stimulatory and may actually be
inhibitory toward the development of the desired cell
mediated immune response [245]. These maturation
steps can be monitored using appropriate in vitro
analysis.
CD14+ cells begin the differentiation process to
“immature DCs”, that is, cells that are highly phago-
cytic, express moderate levels of MHC class I and II
antigens and relatively low levels of adhesion and T cell
costimulatory molecules, including CD40, CD80, and
CD86 (See Review [243, 244]). At the site of inflamma-
tion (bacterial or viral infection, growing tumor nodule),
the immature DCs phagocytize particulate matter
derived from damaged tissue including viral infected
cells, bacteria, and necrotic or apoptotic tumor cell bod-
ies. At this point, presumably in response to local
cytokine signals, the DCs begin the final differentiation
to mature DCs. These signals can be replicated in vitro
as will be discussed below. In vivo, it is thought that
passage across an endothelial cell layer and a basement
membrane, serves to further differentiate the DCs. These
mature DCs traffic to the paracortical region of draining
lymph nodes via afferent lymphatics. “Mature DCs”,
having stopped phagocytosis, process the proteins into
peptides which are presented in both MHC class I and/
or II molecules at the cell surface. The DCs change their
cytokine secretion pattern from IL-10 production char-
acteristic of CD14+ cells to the release of IL-12p70
(Th1 supporting cytokine). At this point, the mature
DCs up regulate the proteins necessary for antigen pre-
sentation. In the paracortical regions of the lymph nodes,
the DCs present peptides derived from tumor antigen,
for instance, to both CD4 and CD8 precursor T cells
which circulate through the node. The antigen stimu-
lated T cells express IL-2 receptors, proliferate and
mature to effector cells. These antigen specific T cells
leave the node and circulate to the site of inflammation
or the tumor bed to provide effector function. Randolf
et al. [246] details what may occur in vivo with the mat-
uration of DCs from CD14+ precursors. In this elegant
report the investigators demonstrated that mature DCs
do not rely on proliferation since irradiated cells were
capable of the differentiation process. These investigators
showed that passage across an endothelial cell layer
induced the expression of DC phenotypic characteristics
including costimulatory molecule expression, ingestion
of particulate matter, and expression of CD83, considered
by many to be a marker of DC maturation.
DCs can be matured from CD14+ precursors in vitro
in order to study DC biology and provide cells for clini-
cal studies. The clinical trials currently on-going use
DCs pulsed with antigen in a variety of forms ranging
from purified peptides, crude cell extracts, necrotic or
apoptotic tumor cells, and RNA or DNA through gene
transfer techniques. There are a number of good refer-
ences detailing the generation of DCs for clinical trials
[218, 247–249]. The initial cytokine requirements for
in vitro derivation of DCs from CD14+ cells was reported
by Romani et al. [250] and Sallusto and Lanzavecchia
[247]. These Investigators demonstrated that the culture
of adherent CD14+ cells in GM-CSF and IL-4 resulted
in cells that lost CD14 expression, were CD1a+ and
capable of phagocytosis or antigen uptake. The matura-
tion of DCs in vitro following 5–7 days of culture can be
accomplished by exposing the DCs to either: (1) monocyte
conditioned medium (MCM) [248]; (2) T cell conditioned
medium [251]; (3) CD4s bearing CD40L or anti-CD40
mAb [252]; (4) exposure to combinations of cytokines
including IL-1Beta/IL-6/TNF-alpha and PGE-2 [223];
or (5) TNF-alpha/Poly (IC) [253]. In considering which
maturation factor to use for immunotherapy trials one
must consider (1) the need to recover a high percentage of
viable DCs, (2) the secretion of high amounts of IL-12p70
with lesser amounts of IL-10, (3) the expression of high
levels of T cell costimulatory molecules, and (4) insure
at a minimum that the DCs are stimulatory for T cells in
allogeneic mixed DC lymphocyte cultures (MLDCC).
More importantly, studies should be conducted to dem-
onstrate that the mature DCs can indeed present tumor
antigen to T cell precursors.
A number of clinical trials are currently underway
studying DCs in a variety of tumor histologies (Reviewed
in Steinman and Dhodapkar [218, 254]. One of the first
reported DC clinical trials involved the vaccination of
autologous antigen pulsed DCs into patients with malig-
nant B cell lymphoma [220]. A total of ten patients were
treated and a majority developed T cell mediated anti-
idiotype proliferative responses. Moreover, two patients
experienced complete tumor regression, one had a partial
response, and three each stable disease or disease progres-
sion. This group subsequently initiated trials in patients
with multiple myeloma [231] and prostate cancer
[255, 256]. One study examined the effects of different
routes of DC administration on the ability to induce
immune responses in patients, and antigen-specific
John R. Yannelli
T cell responses were observed in all patients regardless
of the route utilized [256]. However, the quality of the
responses was variable. A second group has been utiliz-
ing autologous DCs pulsed with HLA-A2 binding
peptides derived from prostate-specific membrane
antigen (PSMA). In the initial Phase I study, seven par-
tial responders out of 51 participants had decreased serum
PSA levels. In a subsequent Phase II trial, GM-CSF was
included as an adjuvant to DC vaccination and 31% of
the evaluable patient’s experienced partial or complete
responses [232, 257–259]. Another trial reported the
immunization of patients with advanced ovarian or
breast carcinoma with Her-2/neu or muc-1 peptide-
pulsed DCs. Peptide-specific CTL responses were
detected in five of ten patients, as assessed by intracel-
lular gamma-IFN staining and in microcytotoxicity
assays [150]. Kugler et al. [260] reported responses in 7
of 17 patients with metastatic renal cell carcinoma
following vaccination with tumor cell-dendritic cell
hybrids. There has been a recent study in patients with
advanced colorectal cancer [224]. Of 15 patients vacci-
nated with autologous DCs pulsed with tumor RNA and
keyhole limpet hemocyanin (KLH), 11 developed a
positive skin test reaction to KLH and a decrease in
CEA levels were observed in seven of these patients.
DC vaccination approaches have been examined in
the setting of melanoma as well. In one study, patients
received DCs pulsed with either tumor cell lysate or a
cocktail composed of different peptides [228]. Delayed-
type hypersensitivity reactions to peptide loaded DCs
were observed in 11/16 patients and peptide-specific
CTL were identified, indicating the vaccination was
successful in generating an antigen-specific immune
response. Additionally, 5/16 patients exhibited demon-
strable regression of organ metastases yielding two
complete and three partial responses. A recent Phase I
clinical trial administered intravenously, tyrosinase and
gp100 peptide-pulsed DCs derived from PBMC to
melanoma patients [261]. In the 16 patients treated, one
patient had a complete remission of metastatic disease,
two had stable disease and two had mixed responses.
In another trial, DCs obtained from CD34+ progenitor
cells were pulsed with melanoma peptides derived from
Melan-A/MART-1, tyrosinase, MAGE-3 and gp100, as
well as control antigens, and administered subcutane-
ously to 18 patients [262]. An immune response was
generated against the control antigens in 16/18 patients.
Ten of these patients exhibited responses to more than
two of the melanoma antigens, and seven of these had
regression of metastatic disease.
In our own Center at the University of Kentucky,
Markey Cancer Center, we have concluded our clinical
trial of antigen pulsed DCs in NSCLC [263, 264].
515
In these studies, autologous DCs were generated from
adherent or Miltenyi bead purified CD14+ cells obtained
from leukapheresis products. The DCs were pulsed with
apoptotic bodies derived from an allogeneic NSCLC
line, 1650-TC. Following safety testing, DCs were used
in prime and boost doses consisting of 100 million anti-
gen pulsed DCs per indradermal/subcutaneous injec-
tion. Our results, focused on immune assessment,
showed that an immune responses was observed in
24/35 patients receiving the vaccine (69%). Importantly,
while the sample size was small, we showed that
immune responses were generated regardless of prior
conventional therapy or stage of the disease treated,
although the final evaluation of the data indicated that
early stage patients responded more favorably. None the
less, even stages III and IV benefited. In our Center,
attention has now shifted from the DC trial to a new
vaccine we have constructed consisting of the apoptotic
bodies derived from 1650-TC along with recombinant
GMCSF. The simplicity of this approach has allowed
the immunization of patients in four clinical centers
across Kentucky, strengthening the value of the approach
in that it is a transferable technology. An evaluation of
the pilot data from ten patients has shown that similar
immunologic results are being obtained as determined
using gamma interferon ELISPOT assay. Thus, our
present focus, combining vaccines with chemotherapy,
utilize this approach.
All in all, these clinical trials verify: (1) the ability to
generate significant numbers of antigen pulsed DCs for
clinical trials, (2) the route of administration needed
for DC vaccination, and (3) in some cases significant
clinical responses. Thus, DCs have already proven their
worth as a treatment option for patients with cancer.
A major question that remains, similar to the LAK and
TIL trials, is whether these cells can be routinely generated
in small clinical centers. If these cells are only available
at the NCI and large Cancer Centers, many patients will
not benefit because they will never gain access to the
therapy. As we have demonstrated in our approach,
making DCs is a cumbersome expensive process which
requires FDA oversite. As discussed above, while our
results were fruitful and consistent with others in the
literature, focus is shifting to easier tumor cell based
vaccines.
Treg Cells in Cancer: Biology and Potential
Role in Tumor Immunity
A review of the immunologic literature over the past 4
decades has suggested the role of immune cells in
controlling the immune response. Early on, the focus
was on suppression. It was suggested that the immune
516 Cellular immunotherapy (CI), where have we been and where are we going?
response to tumors which appear to grow uncontrolla-
bly even in the presence of an apparent immune response
was greatly affected by their presence and function. In
the past decade, the biology of suppressor cells, and
their increased role in maintaining immunologic homeo-
stasis, has been uncovered.
The control of immune responses, that is limiting
hyper-responsiveness and maintaining tolerance, has
been shown in part to be controlled by a subset of CD4
helper cells called regulatory T cells or Tregs [265].
Early work by Sakaguchi [266, 267] demonstrated that
thymectomized newborn mice developed autoimmu-
nity. The transfer of CD4+CD25+ T cells reversed
the effects. In rodent models, Treg depletions lead to
autoimmunity and increased allo and tumor reactivity
leading to rejection of transplanted tumors [268, 269].
Since tumor antigens are in most cases normal antigens,
increased numbers of Tregs during anti-tumor responses
could inhibit anti-tumor reactivity.
There are different phenotypes reported for human
Tregs. A naturally occurring form (nTreg) co-expresses
CD4, CD25, CTLA4, the newly described glucocor-
ticoid induced receptor (GITR) and FoxP3 [270, 271].
It is difficult to assess suppressive activity to all
CD4+CD25+ cells since CD25 is expressed by activated
CD4s. However, FoxP3, a transcriptional regulator,
appears to be the more attractive marker since induction
of FoxP3 on non-regulatory T cells by gene transfer
induces a suppressor phenotype [270, 271]. Mutations
in the FoxP3 gene causes the absence of Treg cells in
both mice and humans [272]. Most recently, it was
reported that Foxp3 acts as both a transcriptional acti-
vator and repressor [273] and that FoxP3 binds to the
promoters of well characterized regulators of T cell
activation and function [274].
Phenotypic analysis of normal donor peripheral blood
reveals that 5–10% [275]of all peripheral CD4 cells are
Tregs. Cancer patients can have as high as 20–25% [276]
Tregs in circulation. O’Garra and Vieira in Nature
Medicine [277], described two types of Tregs, the natu-
rally occurring nTregs (CD4+CD25+) and Treg produc-
ing IL-10 (IL-10Treg, also secrete TGF-Beta). These two
populations can take different names including natural
(CD4+CD25+ arising in the thymus) and inducible
(CD4+CD25− arising in the periphery, iTregs). A third
population has also been described which is CD25− but
still expresses FoxP3 [277] Tregs have complex func-
tions which result in both nonspecific or specific immune
suppression. Tregs act on naïve T cells as well as acti-
vated T cells and function depends on both cell:cell
contact or through cytokine release. Tregs can inhibit
IL-2 production and may have inhibitory effects on CTL
effector function through surface or secreted TGF-beta
[278]. While Tregs act directly on lymphocytes, it is
believed their activation may follow interaction with DCs
[279] or in some cases tumor cells. Elegant studies by
Rong Fu Wang have been the first to document antigen
specificity of Tregs in a human TIL system, suggesting
that tumor peptides may be directly recognized [280].
It is well documented that the percentage of Tregs is
higher in cancer patients tumor tissue, blood, pleural effu-
sions, and in draining lymph nodes [16, 275, 281–283].
Interestingly, CD4+CD25+FOXP3+ cells were found to
be selectively drawn to tumor sites (tumor Tregs) by the
expression of CCL22 by tumor macrophages and tumor
cells [284]. This accumulation was correlated with poor
clinical outcome. While murine studies have shown that
the depletion of Tregs results in maximal tumor rejection
[285] and, depletion of human Tregs enhances CD8 T cell
responses following DNA immunization [286], there are
limited reports on the effects of tumor antigen vaccines
on levels of Treg cells before and after vaccination. We
have been examining this question in our DC vaccine
study and are summarizing results for publication at this
time (Yannelli et al., 2008).
Immunological Monitoring
In all the discussion presented, the key to understanding
the immune response which is a direct result of the
immune intervention is the development of immune
assessment assays which are reliable, reproducible, and
can be utilized from site to site to insure comparative
results. Prior to the immunologic assay, however, is the
handling, preparation and cryopreservation of PBMC.
These issues will be discussed below.
In terms of collecting and handling of PBMC for fur-
ther immunologic testing, it is standard to subject the
peripheral blood product or leukapheresis product to
Ficoll Hypaque gradient separation in order to obtain
enriched mononuclear cells including monocytes, T
cells, B cells and NK cells. Within the T cell population
of course are contained the cytolytic cells, the helper
cells and the regulatory T cells or Tregs. It is from these
cell types that many of the immune effector cells can be
routinely generated including CTL, DCs, and Tregs. In
most cases, immune assessment studies are not done
immediately. It is beneficial to wait until the patients
have undergone all of the immunizations and a reason-
able follow up time has passed.
The issue of cryopreparation and eventual thaw is a
critical point that does not always appear to receive the
attention it should. Recently the NCI has requested
John R. Yannelli
uniformity in this approach and has funded a number of
studies to consolidate approaches and provide a mean-
ingful, easy technique for cryopreservation. In our studies,
we were struck by the use of human serum in cryo-
preservation and its cost. We determined that the fluid
replacement medium Plasmalyte A could be substituted
for either human serum or fetal bovine serum in the
freezing of human PBMC [287]. The average cost of a
liter of Plasmalyte A is around $7. This provides signifi-
cant savings over conventional serum.
In order to assess immune function following vaccine
administration, most investigators examine cytotoxicity,
proliferation and cytokine production; all of which
monitor antigen specific responsiveness of T cells.
Unfortunately, as detailed in Keilholz et al., 2002, short-
comings exist for each of the analyses mentioned [288].
In most cases, one is interested in the increase in spe-
cific lymphocyte precursor frequency following immune
intervention compared to that found before the vaccine
is delivered. An issue of course is using peripheral blood
as a source of T cells which may not represent the
immune response in lymph nodes and tumor tissue.
However, because of its universal availability, periph-
eral blood remains an accepted anatomic site for study.
Cytotoxicity as performed using chromium release is
not sensitive and relies on expansion of T cells from
the original sample. Proliferation assays are highly
variable and cytokine release assays also lack sensitivity.
Interestingly, an assay which has received much favor has
been the enzyme linked immunospot assay or ELISPOT.
Many investigators utilize this approach and if stan-
dardization of immune assessment is to be made, it is
likely in this assay. Lymphocytes to be analyzed are cul-
tured with antigen presenting cells on membranes con-
taining cytokine capture antibodies in a 96 well format.
Captured cytokine is visualized using color reagents
and quantitated as spots indicating single cytokine pro-
ducing cells. The results indicate an increase in “spots”
which correlate an increase in the relative number of
cytokine producing cells. In our own studies, we have
observed increases in gamma interferon producing cells
following vaccine therapy, usually at multiple time
points correlating with the prime and the boost vaccine
[262, 263]. The single cell cytokine assays can also be
performed using flow cytometry with considerable sen-
sitivity. This assay can be sued to further delineate T cell
subsets secreting cytokine. An issue with this assay is
the routine availability of a flow cytometer, although
ELISPOT analysis requires a reader system which can
also be very expensive to purchase.
Finally, tetramer staining is a non-functional, quantita-
tive measure of antigen specific T cell frequency in the
517
peripheral blood. Fluorescent labeled tetramers are con-
structed to bind a unique MHC-peptide complex to the
TCR, the tetramer tagged T cells are analyzed using a flow
cytometer. The majority of reagents currently available
are for class I peptide complexes although class II com-
plexes are becoming more available with time. Since
tetramers are specific for a unique MHC-peptide complex,
this assay is most useful for the study of HLA-restricted
peptide vaccines, although tetramers are currently not
available for all antigens and all HLA types.
The coupling of immune assessment assays and clinical
responses is critical for an overall understanding of the
benefit of CI. While examples exist showing that certain
immune assessment assays correlate with tumor response,
this has not been a consistent finding. The difficulty at the
moment is identifying the best in vitro assay which shows
the greatest correlation across a range of studies done at
different clinical centers. There is much focus in this area
and what are needed are standardization guidelines and
consistent controls to allow comparison across both study
time points and across different geographic sites. When
this is accomplished it is likely that immune assays such
as ELISPOT will gain favor as a primary endpoint analy-
sis in clinical immunotherapy studies.
Summary
As stated in the beginning of this Review, there has been
tremendous progress made in the field of CI since the
initial reports using LAK and TIL. Therapies have
progressed from using nonspecific killer cells to highly
specific T cells over the course of almost 20 years. The
techniques used to generate these cells have been
improved to the point where it is possible for any labo-
ratory with immunologic expertise to participate in
cellular immunotherapy clinical trials. This is true of
course as long as the participating lab follows GLP
procedures for the generation of therapeutic cells. The
techniques exist for safe production and efficient moni-
toring of cellular products for bacterial and viral contami-
nation. The field has progressed even further with the
discovery and molecular characterization of a variety of
LDTAs. The knowledge of these antigens has allowed
their use in generating more specific populations of T
cells for infusion into patients. In addition, these LDTAs
have been used as targets for vaccines. Finally, the
elucidation of the differentiation steps of DCs and the
knowledge of large scale culture requirements has intro-
duced DCs as another major component of CI. Most
importantly, these cells have allowed the application of
immunotherapy to tumors other than melanoma.
518 Cellular immunotherapy (CI), where have we been and where are we going?
In closing, however, no matter what is accomplished
over the next 10 years we need additional insight into
the mechanisms where-by tumors evade immune
responses. While tumor and host induced suppression
has been a major area of study over the years, it must
receive continued and more focused attention in order
for these therapies to work either alone or in the adju-
vant setting. It is especially important to determine why
the immune system is failing to recognize developing
tumors in the micro-metastatic stage, that is, before the
tumor vasculature is developed. Thus, there needs to be
a better understanding of what the exact mechanisms
are where-by tumor cells are evading the immune sys-
tem and its recognition properties. If we understood this
in the first place, strategies could be implemented to
strengthen the early immune response in patients at risk
and correct the deficits so the tumor never takes hold nor
has the opportunity to metastasize. It is hoped that at the
next revision of this Chapter, a complete understanding
of tumor and host induced suppression is obtained and it
is coupled with accepted and proven CI approaches to
cancer treatment.
Acknowledgement This Chapter is dedicated to the
many patients who have given themselves for these
studies. Additionally, on a local level, this Chapter is
dedicated to Dr. Bonnie Sigafus whose tireless dedica-
tion to patients with lung cancer has increased funding
to scientists in need. Dr. Sigafus recently passed on but
her memory will always remain.
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16 Growth and differentiation factors as cancer
therapeutics
KAPIL MEHTA, ROBERT K. OLDHAM, AND BULENT OZPOLAT
Abbreviations used ADCC, antibody-dependent cyto-
toxicity; ANLL, acute non-lymphoblastic leukemias;
APL, acute promyelocytic leukemia; AML, acute myel-
oid leukemia; ATRA, all-trans-retinoic acid; CML,
chronic myelogenous leukemia; CSF, colony-stimulating
factor; DBD, DNA-binding domain; DMSO, dimethyl-
sulfoxide; HMBA, hexamethylenebisacetamide; IL-1, -2,
etc., interleukin-1, -2, etc.; LBD, ligand-binding domain;
MDS, myelodysplastic syndromes; PKC, protein kinase
C; RAR, retinoic recepotr; RXR, retinoid X receptor;
TNF, tumor necrosis factor
Differentiation therapy for the treatment of malignant dis-
orders offers an attractive alternative to the conventional
cytotoxic chemotherapy. The concept of differentiation
therapy is based on the principal of ‘reform’ rather than
‘retaliation’. The perception that malignant cell results
from a block in the differentiation pathway has led to a
conceptual strategy to remove this block and to re-estab-
lish normal homeostasis. Differentiation therapy, in gen-
eral, is associated with a growth arrest and long-term
commitment of the cell to die via apoptosis or senescence.
The conventional cytotoxic therapy contrasts from the
differentiation therapy in that there is no attempt to restore
normal homeostasis in the former case and it is accompa-
nied by immediate cell death. The treatment of acute pro-
myelocytic leukemia (APL) by all-trans-retinoic acid
(ATRA) revolutionized the treatment of the cancer and
provided the first example and proof of concept of dif-
ferentiation therapy. Therefore, the differentiation therapy
may offer the opportunity for the use of the new, rela-
tively non-toxic agents as well as the use of current che-
motherapeutic agents at doses significantly lower than
those maximally tolerated. Combining low dose of che-
motherapy with one or more differentiation agents may
also be of particular interest in the management of disor-
ders resistant to conventional drug therapy.
Interest in the therapeutic use of differentiation agents
has resurged following the discovery of the dramatic
effects of retinoic acid in the treatment of acute promyelo-
cytic leukemia (APL). Treatment with all-trans retinoic
acid (ATRA) induces differentiation of APL cells into
mature phenotype such that they are no longer capable of
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
further division and are destined to undergo programmed
cell death. Another line of evidence suggesting that growth
and maturation factors may be operative in some form of
cancers is the observation that patients with neuroblas-
toma and germ cell tumors can show maturational effects
in the tumor biopsies post treatment. It is not known
whether this relates to the treatment since occasional
spontaneous maturation had been seen prior to effective
therapy for these cancers. These lines of evidence have led
some researchers and clinicians to speculate that cancer-
ous growth remains under some degree of control by dif-
ferentiation and growth factors and that these processes
might be augmented or altered therapeutically.
A number of observations suggest that differentiation of
human myeloid leukemic cells occurs in vivo. The most
obvious example is chronic myeloid leukemia (CML),
where glucose-6-phosphate dehydrogenase (G6PD) poly-
morphism revealed the derivation of mature granulocytes,
red cells, and platelets from the leukemic clone. Similar
analysis revealed differentiation of acute myeloid leukemia
(AML) clones to red blood cells and platelets in some
patients [95], and the presence of Auer rods in mature neu-
trophils of AML patients in remission and persistence of
cytogenetic abnormalities in the absence of normal meta-
phases in patients with AML in clinical remission [26, 89].
DNA recombinant technology has also demonstrated
leukemic cell differentiation in AML and preleukemia/
myelodysplastic syndrome (MDS). DNA restriction frag-
ment-length polymorphism in heterozygous individuals
has provided strong evidence for in vivo differentiation
[95, 417]. HPRT gene analysis, quantitative analysis of
chromosome 8 trisomy and chromosome 7 monosomy,
and immunoglobin (Ig) gene rearrangement analysis
showed that five of six patients with AML had maturation
to mature granulocytes in the active phase of the disease
even in aneuploid leukemia. Another approach has been
to label myeloblasts in vivo with bromodeoxyuridine
(BUdR), which allowed the demonstration of BUdR in
mature granulocytes of AML patients [326].
Premature chromosome condensation has been used
to detect the presence of abnormal cytogenetic clones in
mature cells. Fusion of mitotic tissue culture cells with
the mature cell in question (e.g., granulocytes) results in
immediate chromosome condensation of the chromatin
527
528
of interphase nuclei into discrete chromosomes. This
permitted karyotype analysis of nondividing cells of 13
patients with CML, and with MDS or AML, after low-
and high-dose chemotherapy [159]. Twelve of these
cases showed evidence of maturation of cytogenetically
abnormal leukemic clones in vivo.
Cell lines such as the human myelomonocytic leuke-
mia HL-60 cells are promiscuous in undergoing drug-
induced differentiation. Indeed, over 80 distinct
compounds (not including an even greater number of
analogues) have been shown to induce differentiation of
the HL-60 cell line (vide infra). Many of these agents
may operate via common pathways; nevertheless they
belong to distinct categories of compounds and can be
divided into the following groups:
1. Vitamins, including retinoids and vitamin D3
[1,(25-(OH)2 D3), and vitamin E.
2. Polar/planar compounds such as dimethylsulfoxide
(DMSO), hexamethylene bisacetamide (HMBA),
N-methylformamide (NMF), cotylenin A
3. Cytokines and hematopoietic growth factors (e.g.,
Erythropoietin (Epo), G-CSF, GM-CSF, IL-1, IL-3,
IL-6, LIF, TNF, TGFβ, IFNα, IFNβ, IFNγ)
4. Phorbol esters (e.g., phorbol myristate acetate)
5. Chemotherapeutic agents that interfere with DNA
synthesis (e.g., cytosine arabinoside, tiazofurin,
6-thioguanine)
6. Chemotherapeutic agents that influence topoi-
somerase II
7. Chemotherapeutic agents that inhibit DNA methyltrans-
ferase (e.g., 5-azacytidine, 5-aza-2′-deoxycytidine)
8. Hormones (steroids-dexamethasone and predniso-
lone) [429].
9. Chromatine remodeling: Histone deaceltilase inhib-
itors (HDAC) (butyrates, valproic acid)
10. Others: adenosine analogs (neplanocin A, deoxyco-
formycin), arsenic trioxide, peroxisosome prolifer-
ator-activated recptor gamma (PPARγ)
Human Leukemic Cell Lines
as Models for Differentiation
Therapy
Much of the history of the development of an effective
chemotherapy for leukemia was based on the availability
of cell lines, or transplantable leukemias, with sensitivity
to cell cycle-specific agents. The development of biologi-
cal response modification therapy and the recognition of
leukemic cell differentiation as an obtainable goal led to
Growth and differentiation factors as cancer therapeutics
the use of leukemic cell lines of myeloid lineage capable
of terminal maturation.
The human myeloid leukemic cell lines HL-60, NB4
and U937 that can undergo myeloid differentiation by
all-trans-retinoic acid (ATRA) or other differentiation
inducing agents have been studied most extensively.
HL-60 was isolated from a patient with acute promyelo-
cytic leukemia (APL), and it retains a promyelocytic
morphology but does not express translocation t(15;17)
represent M2 type of AML (FAB classification) [110].
NB4 cells was isolated from long-term cultures of leu-
kemia blast cells on bone-marrow stromal fibroblasts of
a patient with APL [430]. NB4 cells (M3-AML) are true
APL cells harboring t(15;17); thus expressing PML-
RARα fusion protein, leading to a differentiation block
at the promyelocytic stage. U937 (M4/M5-AML) was
established from the pleural fluid of a patient with true
macrophage-type diffuse histiocytic lymphoma, and has
an immature monoblast-macrophage morphology [365].
These lines are cloned, readily maintained in culture
and, while resembling immature cells of their lineage,
can be induced to terminal neutrophil, eosinophil, and
macrophage (HL-60) or monocyte-macrophage (U937)
differentiation. NB4 cells have bileneage potential and
undergo phenotypic change along monocytoid/mac-
rophage lines with phorbol ester induction and granulo-
cytic differentiation by retinoic acid [431].
The histochemistry and morphology of these cell lines
typify them as immature cells of myelomonocytic lin-
eage. The HL-60 cell line exists in many variants with
differing doubling times and extents of spontaneous dif-
ferentiation [123]. The cells weakly phagocytize latex
particles or yeast in the uninduced state and possess low
numbers of Fc C3b, insulin, and chemotactic fMLP recep-
tors, whose expression can be greatly enhanced following
exposure to differentiation-inducing agents [4, 286].
HL-60 cells are positive to myeloperoxidase, ASD
chloracetate esterase, and Sudan black B, but unlike nor-
mal promyelocytes they do not stain for alkaline phos-
phatase, and unlike macrophages they are essentially
acid phosphatase-negative [110, 410]. Activated forms
of beta-glucuronidase, lysozyme, and G6PD, found at
high levels in the granules of normal granulocytes, are
present at low concentrations in HL-60 and are mini-
mally involved in degranulation. It has been proposed
that these enzymes are already synthesized in HL-60 and
stored within the granules as zymogens, but are only
converted to active enzymes upon stimulation [204,
422]. Uninduced HL-60 cells have a marginal capacity
to produce H2O2 and O2−, have a low level of HMPS
activity [284], and have a greatly impaired ability to kill
staphylococcus and other microorganisms [110].
Kapil Mehta et al.
The U937 cell line as originally reported grew slowly;
however, later passages of the line have an accelerated
population doubling of 20–48 h. The cells have a mono-
blastic morphology and histochemical profile (strong
ANAE, NASDAE, and beta-glucuronidase positivity
and weak acid and alkaline phosphatase) [365, 410].
U937 cells also secrete lysozyme, and neutral protease
elastase is present within the cells; these are monocyte-
specific characteristics [323, 365]. The cells bear few
Fc, C3, and chemotactic peptide receptors when com-
pared with normal monocytes, but histamine and insulin
receptors are expressed [5, 365]. Only a small percentage
of U937 cells are phagocytic, and weakly produce H2O2
and O2− and are incapable of killing microorganisms or
tumor cells [204, 324, 355].
Upon induction of differentiation, morphological
changes involve significant increases in substratum
adherence. The HL-60 cell line changes morphology
from promyelocytic to later stages of granulocyte
degranule formation when treated with 2% DMSO and
retinoic acid [37, 41, 110], whereas phorbol esters trans-
form the cells to monocyte-macrophages with the disap-
pearance of azurophilic granules and appearance of
pseudopodia and a cerebriform nucleus [337]. There is
evidence that these cells can differentiate to eosinophils
spontaneously, and in response to stimulatory activities
in human placenta-conditioned media [222]. U937 cells,
in response to differentiation stimuli, increase in size
and acquire lobated nuclei, and cytoplasmic granules
are replaced by vacuoles, duplicating monoblast to
monocyte transformation. Various granulocyte lysozyme,
acid phosphatase, and beta-glucuronidase activities
have been measured in induced cells; when HL-60 is
induced with phorbol ester (TPA), the activity of these
enzymes is increased up to 20-fold [337]. Similar
increases in activity are usually seen after induction
with all agents used for differentiation induction. The
distinctive monocyte lysosomal enzyme, acid phos-
phatase, can be detected after exposure of HL-60 cells
to phorbol ester for several days. The elaboration of this
enzyme into surrounding media is also a monocyte-
associated characteristic and accompanies the phorbol-
induced maturation of these cells. Myeloperoxidase, an
enzyme specific to the myelomonocytic lineage, is con-
stitutively expressed in HL-60. However, activity is lost
after induction with phorbol esters, lymphokines, or
retinoic acid, but is unaffected when DMSO or cAMP-
inducing agents are used [338].
Lactoferrin, a metal-binding glycoprotein, is present in
normal granulocytes in the secondary (specific) granules
and is one of the most specific markers for the neutrophil
lineage. It has not been detected in HL-60 cells [284],
529
or in the segmented neutrophils of the majority of patients
with leukemia [38]. Under normal conditions, lactofer-
rin is active as a suppressor molecule, and inhibits the
production and/or release of granulocyte-macrophage
colony-stimulating factors from monocytes and mac-
rophages at concentrations as low as 10−15 M. In vivo
lactoferrin inhibits myelopoiesis in mice at concentra-
tions as low as 10−4 mg/mouse [34]. Therefore, detection
of the synthesis of lactoferrin in HL-60 cells, even at
low concentrations, would be of importance, especially
if the synthesis could be increased by differentiation-
inducing agents and if the lactoferrin so induced was
functionally active. We have detected lactoferrin in
HL-60 cells at very low levels, using radioimmunoas-
say; in biosynthesis studies, using autoradiographic gel
analysis (3H) leucine incorporated into material immu-
noprecipitated with lactoferrin antibody. Following
induction of differentiation with DMSO or retinoic acid,
a two- to fourfold increase in lactoferrin has been noted.
This is still much lower than the 10−12 g per segmented
neutrophil reported in normal peripheral blood, indicat-
ing a persisting abnormality in the differentiated HL-60
neutrophil, comparable to that noted in neutrophils of
patients with leukemia [38].
The isozyme pattern of lactate dehydrogenase in
HL-60 cells is reported to change after induction with
DMSO; however, the resultant pattern is not character-
istic of mature granulocytes or macrophages but rather
of more immature stages [303]. Simultaneous expres-
sion of other granulocytic and monocytic specific mark-
ers may also be the result of incomplete or asynchronous
maturation. Of particular interest is the observation that
cell division is not required for the induction of many of
these characteristics and may account for their uncoor-
dinated expression [37].
Messenger RNA species have been isolated from
HL-60 cells translated in vitro and analyzed by two-
dimensional SDS-PAGE after phorbol ester or DMSO
induction. The phorbol-induced protein pattern differed
significantly from the DMSO pattern and was character-
ized as monocytoid rather than granulocytic [61]. Poly
ADP-ribose metabolism has been linked to the maturation
of granulocyte-macrophage progenitors. ADP-ribosyl
transferase is a chromatin-bound enzyme catalyzing the
transfer of ADP-ribose to chromatin proteins. The activity
of this enzyme increases during the CSF-stimulated dif-
ferentiation of marrow precursors to monocytes [104].
Using DNA array, suppression-subtractive hybridization,
and differential-display PCR techniques, the expression
of about 169 genes was shown to be modulated in
ATRA-treated human APL (NB4) cells [230]. Of these
32 genes appeared to be the direct targets of the ATRA
530
signaling pathway since their transcriptional regulation
was not blocked by cyclohexamide. A similar detailed
analysis of genes regulated by ATRA-treatment in mouse
APL cells (MPRO) was reported [223]. Both the studies
suggested the involvement of several transcription regu-
latory factors during the process of ATRA-induced
granulocytic differentiation.
One of the most frequently employed and convenient
qualitative measures of reactive oxygen intermediates as
important microbicidal products or granulocytes of mac-
rophages is the reduction of NBT to a black formizan
precipitate. A variety of factors, both physiologic and
pharmacologic, can induce HL-60 production of H2O2
[141, 117, 294]. The microbicidal capacity of granulo-
cytes and HL-60 cells is metabolically linked to an
increase in the hexose monophosphate shunt activity and
consequent production of superoxide anion activity.
Arachidonic acid metabolism has been analyzed after
induction of maturation with several agents, and recent
results suggest that there is enhanced synthesis and release
of prostoglandins of the E and F2-alpha type [142].
Maturation of U937 cells is usually characterized by
quantitative rather than qualitative changes, since the
cells constitutively produce a variety of enzymes and
growth factors. Because U937 cells are of monocytic
origin, agents that effect a maturational change may also
be potent immunoadjuvants. Blood monocytes and
U937 cells are characterized by their content of fluoride-
inhabitable esterase, alkaline and acid phosphatase, and
β-glucuronidase. These enzyme levels are elevated by
exposure of normal or neoplastic cells to agents known
to influence monocytic activation, such as vitamin D
metabolites, phorbol esters, gamma-interferon, and
lymphokines [141, 142, 147]. Other enzymatic activi-
ties that have been studied in U937 cells include elastase,
collagenase, and plasminogen activator. U937 elastase
is not released constitutively and is not readily modu-
lated [287, 353].
The production of reactive oxygen species and the
percentage of NBT-positive cells are increased in U937
cells by a variety of agents, such as phorbol esters,
DMSO, lymphokines, α- and β-interferons, retinoic
acid, 1,25-dihydroxy vitamin D3, prostaglandin (PG) E
and other cAMP-inducing agents, cytosine arabinoside
(ara-C), and protein DIFs [2, 34, 141, 292, 293]. On a
more quantitative basis, the activity of the hexose mono-
phosphate shunt has been examined. Notably, db-cAMP
and PGE are able to increase activity of the shunt, but
cannot induce other characteristics of maturity in U937
[291]. Under certain conditions, U937 cells did not appear
to produce prostaglandin E2 in response to lipopolysac-
charide (LPS), endotoxin, or concanavalin A (Con A)
Growth and differentiation factors as cancer therapeutics
stimulation [200], and differed from peripheral blood
monocytes and macrophages in this respect. However,
if supplied with exogenous arachidonic acid, the cells
can release PGE2 [60]. The failure to release esterified
arachidonic acid to the cyclooxygenase pathway was
believed to be a property shared by U937 cells and certain
macrophage subsets.
The production of certain biologically active macro-
molecules such as, interleukin 1 (IL-1) (endogenous
pyrogen, LAF) can be induced by treatment of U937
cells with agents like endotoxin or phorbol esters [302].
IL-2 is probably the factor released by U937 that
increases the production of collagenase and PGE2 in
cultured synovial cells [9]. U937 cells also secrete
hematopoietic growth-stimulating factors that may act
as autostimulatory growth factors [14]. The release of
such factors can be suppressed, as in normal myelopoi-
esis, by agents such as lactoferrin [38]. U937 also pro-
duces the second component of complement, and the
release of C2 can be augmented sevenfold by inducing
maturation of U937 with phorbol ester or a lymphokine
[302, 313]. Similarly, treatment of U937 cells with Con
A has been shown to induce the synthesis of a 65 kDa
heat resistant lymphocyte proliferation inhibitor that is
distinct from lymphotoxin or interferons [405].
The qualitative and quantitative changes in the
expression of certain plasma membrane constituents in
HL-60 and U937 have been associated with differentia-
tion. Induction of HL-60 with DMSO or phorbol ester,
for example, modifies the expression of “fast-eluting”
glycopeptides [387]. Comparison of the elution profiles
of fucosyl-labeled glycopeptides with normal granulo-
cytes and macrophages revealed that HL-60 glycopep-
tides were larger and more complex than those on a
normal band of polymorphonuclear neutrophils. DMSO
or phorbol treatment of HL-60 did not induce a normal-
ization of the elution profile to a mature conformation
[385]. Induction of cell-surface glycoproteins on HL-60,
particularly as seen in the time of appearance of gp130
following DMSO treatment, was concomitant with
development of chemotactic ability [109]. In this con-
text, patients with impaired granulocyte chemotaxis
have greatly reduced amounts of GP130 in their granu-
locytes [342].
Cytoskeletal proteins of HL-60 have also been studied.
During induced maturation, there is increased synthesis of
vimentin and other structural proteins. As may be expected,
the profiles of cytoskeletal proteins induced by DMSO
resemble those of granulocytes and, following phorbol
ester induction, resemble those of macrophages [27].
Modulation of expression of several cell surface anti-
gens has been characterized by the use of monoclonal
Kapil Mehta et al.
antibodies. In one study, two monoclonal antibodies,
B9.8.1 (specific for monocytes and metamyelocytes),
were used to characterize maturational changes [310].
Treatment of HL-60 with granulocyte- or macrophage-
inducing agents resulted in enhanced antigen expres-
sion. In a similar study, reactivity with the monoclonal
antibodies Mo1 and Mo2, recognizing determinants
specific to the myelomonocytic lineage, could be
induced with phorbol esters, as well as protein inducers
of differentiation [69, 379].
HL-60 constitutively expresses HLA-A and -B anti-
gens, and induction of HLA-DR determinants has been
reported [69]. Normal myelomonocytic precursors
express the DR antigens twice: transiently during early
stages (CFU-GM) [98], and later as monocytes.
HLA-DR determinants of HL-60 have been detected
following treatment with inducers of monocyte mac-
rophage differentiation, but not with agents that induce
granulocyte maturation.
The expression of Fc and C3 receptors has been studied
extensively in U937. The receptor for IgG1 can be induced
by a variety of agents, both physiologic and pharmaco-
logic [141, 200]. The receptor for IgE is also present,
but its modulation has not been studied. The C3B receptor
(as recognized by EAC or Mac-1 monoclonal antibody)
is inducible in a similar manner, as are Mac-1 and mac-
rophage-restricted Mac-3 cell-surface antigens [325].
The chemotactic receptor for the potent chemotactic
peptide fMLP is weakly expressed in U937 (7,505 sites/
cell). Within 3 days of exposure to phorbol esters, dex-
amethasone, or protein inducers of differentiation
(excluding native or recombinant γ-interferon), up to
fivefold increase in fMLP receptors is seen [142, 313].
U937 cells are generally considered devoid of any Ia
antigen on their surface; however, small numbers of
cells react with antibodies to HLA-DR and HLA-DS/
CD molecules. It has proved possible to clone such
Ia-positive cells and develop constitutively Ia antigen-
positive variant lines of U937 [121]. Induction of
HLA-DR determinants in response to treatment with
gamma-interferon and PG has been reported [311].
Beta2-microglobulin expression has also been shown to
increase in response to protein inducers of differentia-
tion [287]. Insulin may play an important role in the
growth regulation of U937 cells, since the number of
insulin receptors is reduced after incubation with differ-
entiation-inducing agents, and this may have a causal
role in the observed growth inhibition. The association
between differentiation induction and growth inhibition
of U937 and HL-60 cells may also be linked to the ability
of potent inducers of macrophage differentiation, such
as phorbol ester, to induce cellular production of the
531
protein of molecular weight 17,000, termed tumor
necrosis factor (TNF) [6, 309]. This activity is inhibi-
tory to neoplastic cell proliferation, including that of
leukemic cells and normal hematopoietic stem cells.
The intriguing possibility of autoregulation of leukemic
cells by production of an endogenous growth-inhibitory
molecule (TNF) is suggested.
More than 80% of cells from a human promyelocytic
leukemic cell line (HL-60) possess the capacity for self-
renewal as evidenced by their ability to form large pri-
mary colonies in semisolid medium and the presence
within these colonies of cells capable of subsequent
colony formation. Colony development is independent
of colony-stimulating factor. The observed autostimula-
tion suggests the production of specific growth promot-
ers by the cells. Differentiation, either to mature
granulocytes or to macrophages, induced by various
agents is associated with reduced cloning potential.
Nevertheless, colonies containing differentiated cells
can be developed either by cloning cells in the presence
of suboptimal concentrations of inducer or by adding
inducers to colonies developed in its absence. The loss
of self-renewal was found to be one of the early proper-
ties that changed following the initiation of differentia-
tion. The loss preceded not only the overt expression of
maturation-specific functions but also cellular commit-
ment to terminal differentiation; shorter contact with the
inducer is required to cause loss of self-renewal than to
induce an irreversible transition to differentiation. This
results in cells that lose their self-renewal potential
without being able to complete their program of differ-
entiation [96]. Increased migration of tumor cells upon
differentiation to form diffuse colonies may be the result
of increased mobility and chemotactic responsiveness.
In a modified chemotaxis assay, HL-60 cells increased
their migration in response to fMLP after a 5-day of
induction with db-cAMP or DMF [99, 388].
Phagocytosis of latex beads or opsonized yeast and
the capacity to kill microorganisms are readily induced
by a wide variety of differentiation-inducing agents.
Phagocytosis is one of the earliest acquired effector
functions of both granulocytes and monocytes. Phorbol
ester or DMSO-induced HL-60 cells effectively kill
staphylococcus [190]. It has been demonstrated that dif-
ferentiation-induced HL-60 cells can mediate monocyte
ADCC-like reaction against antibody-coated chicken
erythrocytes [69].
The U937 cell line is capable of being induced to
mediate antibody-dependent cellular cytotoxicity
(ADCC) effector function. First reports of ADCC capacity
against tumor markers used lymphokine, interferon, and
phorbol ester preparations to activate or “induce” the
532
cells [147, 324, 365]. ADCC activity of U937 cells
against erythrocytes can be induced by as little as ten
units of γ-interferon and 300 units of α- and β-interferon.
U937 cells have been studied for their ability to support
the intracellular multiplication of Toxoplasma gondii.
A small portion of U937 cells can spontaneously phago-
cytize these protozoans, and after 24 h those cells have
either lysed or contain numerous trophozoites within their
vacuoles. After a 3-day incubation with lymphokine
preparations there is a significant decrease in the num-
ber of organisms found within the vacuoles, in spite of a
generalized increase in phagocytosis [408].
In an effort to analyze the pathways involved in dif-
ferentiation of cell lines such as HL-60, many attempts
have been made to select sublines capable of sustaining
exponential growth in the presence of a single inducer of
differentiation but not in the presence of different, struc-
turally unrelated inducers of differentiation. Neutrophilic
granulocytic and monocyte/macrophage programs of
HL-60 are mechanistically different and separable, and
both agent-specific and common quantitative alterations
contribute to the mechanism for resistance to granulo-
cyte differentiation.
Numerous studies have demonstrated that the oncogene
c-myc is amplified 20- to 40-fold in HL-60 cells compared
with normal human DNA and is associated with an ele-
vated level of cellular myc-mRNA, and a decrease in this
mRNA follows chemically induced differentiation [37,
132]. Within 5 days of addition of DMSO or phorbol ester
to HL-60 cultures, transcription of the c-myc gene was
markedly reduced when compared with control cultures.
This decrease was not accompanied by alteration in either
the bulk rate of transcription or the c-myc copy number,
suggesting that decreased cellular myc RNA levels are
due to decreased transcription of the myc protooncogene
[132]. The expression of c-myc in HL-60 does not corre-
late with the proportion of proliferating cells, and the
kinetics of decrease upon DMSO induction is paralleled
closely by an increasing proportion of histochemically
detected, differentiated myeloid cells and by a decrease in
clonogenic potential, but not by changes in the proportion
of proliferating cells [97]. Changes in c-myc expression
subsequent to differentiation of HL-60 can therefore be
directly related to the differentiation process rather than to
a cell cycle-related phenomenon. Gallagher et al. [112]
observed a little change or decrease in the amplification
level of the known amplified c-myc gene in various drug-
resistant sublines in comparison with wild-type HL-60
cells and despite the existence of numerous double, min-
ute chromosomes (indicators of amplified genes) in some
drug-resistant sublines. Differential response to differen-
tiation agents could still be related to amplification of
Growth and differentiation factors as cancer therapeutics
genes other than c-myc; however, tests with several other
oncogene probes, including N-ras, Ha-ras, Ki-ras, myb,
and abl, showed no evidence of amplification or gross
rearrangement of these genes in DNA in any HL-60 line
or subline [65]. High levels of c-fos expression have been
detected in macrophages, but not in uninduced HL-60
cells. However, when HL-60 cells were induced to mac-
rophages, c-fos expression was readily detectable in the
differentiated cells [272].
Vitamin A Analogues
as Leukemia Differentiation-
Inducing Agents
Retinoids (vitamin A and its analogues) and retinoic acid,
in particular, are a family of fat soluble compounds that
exert a potent effect on the cell growth and differentiation
of various cell types including promyelocytic leukemia,
neuroblastoma, teratocarcinoma, breast cancer, prostate
cancer, melanoma, bladder cancer, squamous cell carcino-
mas of cervix, skin cancer, head and neck cancer and rhab-
domyosarcoma [68, 342, 361, 434]. Their antiproliferative
and differentiation-inducing properties have brought them
under the close scrutiny of oncologists. A major develop-
ment in the clinical application of retinoids was the dis-
covery that ATRA could induce the mature phenotype in
myeloid leukemia cells thus resulting in complete remis-
sion in patients with acute promyelocytic leukemia. [46,
72, 80, 90, 91, 170, 399] In light of high complete remis-
sion rates achieved (80–90%), ATRA is currently being
used as a frontline therapy for treatment of APL patients.
Retinoids exert their biological effects by interacting
with two families of intracellular nuclear receptors: the
retinoic acid receptors (RARs), which are activated by
ATRA and 9-cis retinoic acid, and the retinoid X receptors
(RXRs), which are activated by 9-cis retinoic acid only
[32, 227]. Each of these receptor families consists of three
subtypes (- α-β and -γ), and each subtype exists in multiple
isoforms that arise due to alternative splicing and differen-
tial use of two promoters (Table 1).
Table 1. Retinoic acid (RAR) and retinoid X (RXR) receptors
Receptor Isoforms Chromosomal location Ligand
RARα α1, α2 17θ21.1
RARβ β1, β2, β3, β4 3p24 ATRA &
RARγ γ1, γ2 12q13 9-cis RA
RXRα α1, α2 9q34.3
RXRβ β1, β2 6q21.3 9-cis RA
RXRγ γ1, γ2 1q22–q23
Kapil Mehta et al. 533

RARs and RXRs share – along with other members
of the nuclear hormone receptor superfamily – a modular
structure having a highly conserved DNA binding
domain (DBD), a ligand-binding domain, and a less
well conserved amino-terminal domain and a hinge
region present between the DBD and the LBD. The
DBD has two zinc fingers and is highly conserved
between the members of the superfamily. The LBD is
also involved in dimerization and has a carboxyl-termi-
nal activation domain, called AF-2. The AF-2 domain
has been shown to function as an autonomous transcrip-
tional activation domain when fused to a heterologous
DBD. The hinge region of the retinoid receptors has
been shown to constitute the domain responsible for
interaction with some co-repressors [51, 52].
The presence of intermediary factors that link nuclear
receptors with basal transcriptional factors had been
proposed based on transcriptional interference/squelch-
ing observed between transcriptional activation domains
of different receptors. Two kinds of factors have been
identified: (i) the corepressors, which bind to unliganded
receptors but dissociate upon ligand binding (Fig. 1a),
and (ii) the co-activators that bind only to the liganded
receptors (Fig. 1b). Two corepressors, SMRT and
N-CoR, have been identified that associate with unli-
ganded retinoid receptors and suppress the basal tran-
scriptional activity. Another co-repressor, mSin3A,
which is a homologue of the yeast global-transcriptional
repressor Sin3p, has been shown to interact directly
with SMRT and N-CoR1. Sin3A and SMRT, in turn, can
interact with histone deacytylase 1 (HDAC1) to form a
multisubunit repressor complex [277].
Similarly, a growing number of coactivator proteins
have been identified, though their mechanism of action
is not well understood except for it has been known that
they bind to the liganded receptor at the AF-1 domain
[208]. One factor that is known to potentiate the tran-
scriptional activation of the retinoid receptors is the steroid
receptor coactivator SRC-1 [412]. The formation of a
RXR/RAR heterodimer is required for the high- affinity
binding to specific DNA sequences known as retinoic acid
response elements (RAREs) that is critical for subsequent

a +0.1-1 µM ATRA +9-cis RA

RARα RARα
RXRα RXRα
HL - 60 Cell/WT Differentiation Apoptosis

b
1 µM ATRA
Differentiation blocked due to mutation in the RARα gene

HL-60R

c RARα-transfected HL-60 R cells regain

0,1-1 µM ATRA differentiation potential in response to ATRA
treatment

HL-60R/RARα
d
0.1 µM 9-cis-RA RXR -transfected HL-60R cells undergo
apoptosis in response to RXR ligation

HL-60R/RXRa

Figure 1. A model showing the basal repression and retinoid-induced transcriptional regulation of the target gene(s). (a) The
RAR/RXR heterodimer binds to a specific direct-repeat regulatory element that is separated by 5 bp (DR5-RARE) in the regula-
tory site of the target gene. In the absence of ligand (ATRA), the RAR/RXR heterodimer recruits a transcriptional repression
complex that comprises N-CoR, mSin3a, SMRT, and a histone deacetylase (HDAC-1). The HDAC-1 activity helps to suppress
transcriptional activity. (b) Addition of ATRA results in a conformational change in the RAR/RXR heterodimer that leads to the
release of the repressor complex and recruitment of a transcriptional activator complex that has histone acetyltransferase (HAT)
activity. This activity destabilizes nucleosomes and creates a permissive state for promoter activation
534
retinoid-induced transcription of target genes. Most
RAREs have been identified in the regulatory regions of
genes whose transcription is induced by retinoids. These
cognate response elements consist of direct repeats (DR)
of two core motifs in the form of PUG(G/T)TCA(X)
nPUG(G/T)TCA or similar but degenerate motifs. The
most common spacing observed in RAREs is 5 bp(DR5);
however, RAREs containing 1 (DR1) and 2 bp (DR2) are
also common.
HL-60 and NB4 cells have been extensively used to
investigate retinoid-mediated signaling pathways dur-
ing normal myelopoesis. Like normal granulocytes,
they undergo terminal differentiation in response to
retinoid treatment. Moreover, like normal granulocytes,
ATRA-induced HL-60 and NB4 cells have a limited
in vitro life span and undergo apoptosis in response to
certain stimuli [233, 431]. Thus, Both of these cells
can also serve as a model to investigate the molecular
mechanisms of apoptosis in terminally differentiated
hematopoietic cells. However, the interpretation of
retinoids’ action on cell growth, differentiation, or apop-
tosis becomes complicated in view of the fact that retin-
oids can mediate these effects by binding and activating
two different types of nuclear receptors: the RARs and
the RXRs, which as described above, differ in their
sequences and exhibit distinct ligand-binding proper-
ties. Since most of the blood cells, including HL-60
and NB4, express both types of receptors [144, 327],
retinoid-induced effects in these cells may be a result of
activation of either RARs, RXRs or both types of receptors.
We addressed this problem by using a mutant subclone
of the HL-60 cell line (HL-60R) in which retinoid receptor
function has been abrogated as a result of a -trans dominant
negative regulatory point mutation in the ligand-binding
domain of the receptor [333]. HL-60R subclones express-
ing specific receptors were generated by retrovirus-
mediated transduction of RARα or RXRα specific
coding sequences [334]. Our results suggested that the
introduction of RARα into HL-60R cells completely
restored their sensitivity to ATRA-induced granulocytic
differentiation. In contrast, the introduction of RXRα
cDNA rendered these cells remarkably sensitive to
apoptosis in response to the RXR-specific legends [239].
These observations provided a direct evidence for
RARα involvement in retinoid-induced differentiation
and of RXRα in programmed cell death (Fig. 2). Using
the receptor-selective retinoids, other investigators
arrived at similar conclusion that specific retinoid recep-
tors are involved in the regulation of differentiation and
apoptotic events in HL-60 cells [278, 386].
More recently, it was demonstrated that ATRA induces
post-maturation apoptosis of APL cells by regulating
Growth and differentiation factors as cancer therapeutics
TRAIL (tumor necrosis factor related apoptosis induc-
ing ligand) expression. Thus, induction of TRAIL-
mediated death signaling may contribute to the thera-
peutic value of retinoids [8].
A two-step model has been proposed for induction
of differentiation where early events anteceding
precommitment regulate growth arrest and late events,
and subsequent to precommitment regulate the choice
of a specific differentiation lineage [414]. Thus the
lineage specificity of cells treated sequentially with
two discrete exposures to alternative inducers depends
on the order of exposure. Retinoic acid exposure of
HL-60 for 24–72 h followed by phorbol ester treatment
produces monocyte-macrophage differentiation, whereas
reversing the order of treatment favors granulocyte
differentiation [425]. The precommitment phase has a
characteristic duration following retinoic acid exposure,
persisting for several cell cycles despite removal of
retinoic acid [134]. This precommitment is paralleled
by elevations in c-myc. Other events associated with
retinoid induction of differentiation involve elevation of
tyrosine kinase activity [74] and a protein kinase C cascade
system. Sphinganine, a potent inhibitor of PKC, enhances
differentiation of HL-60 induced by retinoids, and the
granulocytes produced are more fully differentiated as
indicated by enhanced superoxide production in response
to fMLP [363]. A role for topoisomerase II in retinoid-
induced granulocytic differentiation has been suggested
[102]. In HL-60 differentiation, retinoic acid stimulates
transient relocation of DNA supercoiling, and this is
associated with the formation of small numbers of
protein-linked DNA breaks (a characteristic of topoi-
somerase reactions). Both events are perturbed by VP16,
which inhibits differentiation [102].
Combinations of differentiation-inducing agents have
been explored in vitro with retinoic acid. Twenty-two of
24 patients with ANLL showed differentiation of bone
marrow in cultures with a combination of retinoic acid
(10−6 M), aclacinomycin A (80 nM), and dimethylforma-
mide (100 mM) [145]. Enhancement of differentiation
was seen with retinoid combined with interferons-α and
-β [183, 193, 196], and -&gammabdot; [153, 383].
Tumor necrosis factor (TNF) at 2.5 U/ml inhibited
growth and, synergistically with retinoic acid, induced
differentiation in HL-60 and KG-1 cultures and in mar-
row cultures from four to nine patients with ANLL
[378]. Retinoic acid reversed TNF inhibition of normal
marrow myeloid colonies and leukemic growth marrow
cultures from three to nine patients with ANLL.
In this context, leukemia differentiation-inducing factor
(GM-DF) produced by mitogen-stimulated human leu-
kocytes acted synergistically with retinoic acid in inducing
Kapil Mehta et al. 535

mSin3a HDAC-1
C Repression
a complex
N-CoR SMRT

LBD LBD
RARα TRANSCRIPTIONAL
Histones REPRESSION
DBD DBD

Closed chromatin
(repressed)

5 bp
AGGTCA AGGTCA
DR-5 RARE

ATRA
b SRCs
CPB/p300
(HATs)
LBD LBD

Ligated RARα TRANSCRIPTIONAL

ACTIVATION
DBD DBD

Ac Ac Ac Ac Open chromatin
Ac (activated)
Ac

5 bp
AGGTCA AGGTCA
DR-5 RARE

Figure 2. Retinoid-mediated differentiation and apoptosis of myeloid leukemia HL-60 cells

(a) The RARα nuclear receptors in HL-60 cells when bind to and are activated by appropriate ligand (e.g. ATRA) results in
granulocytic differentiation of the cells. The differentiation process is associated with induction of several new genes, including
RXRα receptors. It is conceivable that ATRA is isomerized to 9-cis RA in situ, which can then bind and activate RXRα receptors,
leading to the onset of apoptosis in differentiated HL-60 cells. (b) HL-60R cells harbor a functional mutation in the RARα gene
that results in non-functional RARα protein and renders the cells resistant to ATRA. (c) Retrovirally-transduced expression of
functional RARα in HL-60R cells restores the ability of these cells to differentiate in response to ATRA treatment. (d) However,
transduction of RXRα cDNA, renders the HL-60R cells highly sensitive to RXR-selective ligand(s) (such as, 9-cis RA) and
induce massive apoptosis in these cells without morphological differentiation.

maturation of the human leukemic lines U937 and
HL-60 [292]. T-cell-derived GM-DF was subsequently
shown to be due to the synergistic action of IFNγ, lym-
photoxin, and GM-DSF [154]. Compounds elevating
intracellular levels of cAMP, such as dibutryl cAMP,
PGE, and choleratoxin, acted synergistically with retinoic
acid to induce maturation of both cell lines. In contrast
to the requirement for continuous presence of retinoic
acid for up to 5 days in order to achieve terminal
differentiation of HL-60 cells, differentiation proteins
or cAMP-elevating compounds are active on leukemic
cells primed with retinoic acid within 8–16 h [292].
Retinoid-mediated differentiation of myeloid leuke-
mia cells is not a universal phenomenon. While the
murine myelomonocytic leukemic cell line WEHI-3 can
be induced to mature neutrophil differentiation [74] and
retinoic acid induces the human malignant monoblast
line U937 to monocyte-like cells with the capacity to
reduce nitroblue tetrazolium [292], the human myeloid
cell lines KG-1 and K562 cannot be induced to differen-
tiate [77]. The mouse myeloid leukemia M1 can be
induced to increased levels of lysomal enzyme produc-
tion without induction of phagocytosis, locomotive
activity, or morphological maturation. Fresh leukemic
cells from patients with various myeloid leukemias have
also been exposed to retinoic acid in short-term primary
suspension cultures, and morphological and functional
maturation was observed only in cases of acute promy-
elocytic leukemia [113, 193]. The differential sensitivity
of various leukemias to retinoic acid induction of terminal
536
differentiation is not dependent on the presence or
absence of cellular retinoic acid binding protein [77, 371].
High-affinity retinoic acid receptors (RARs), predom-
inantly alpha-type, were found in 12 leukemic cell lines
and in marrow blasts from 32 patients with ANLL [203,
283, 396]. While some correlation was found between
RAR expression and retinoid response with four leuke-
mic cell lines [203], extensive analysis of marrow cul-
tures from a large group of primary AML patients showed
no correlation between receptor expression and ability of
retinoic acid to inhibit colony formation or induce dif-
ferentiation [396]. The potential for terminal differentia-
tion may be irreversibly lost in many cases of acute
myeloid leukemia, but this need not negate the therapeu-
tic value of retinoic acid treatment in a wide range of
leukemias, since considerable evidence has accumulated
to suggest that retinoids can selectively inhibit leukemic
cell self-renewal independently of activation of a differ-
entiation program in the leukemic stem cell. Retinoic
acid is a potent inhibitor of the clonal growth in vitro of
myeloid leukemic cells, and a 50% growth inhibition of
HL-60 was achieved by 2.4 nM retinoic acid. The human
myeloid leukemic line KG-1, which is not inducible to
differentiation, was nevertheless extremely sensitive to
retinoic acid, with 50% of the colonies inhibited by
2.4 nM concentrations of the drug [77].
Thus, antiproliferative action of retinoids upon leu-
kemic cells is more general than the incidence of
induction of terminal differentiation, and is seen with
retinoid concentrations readily attainable in vivo. The
potential efficacy of retinoic acid in the treatment of
human leukemia is further suggested by the observa-
tion that it enhances colony-stimulating factor (CSF)
induced clonal growth of normal human myeloid and
erythroid cells in vitro [78]. In long-term bone marrow
culture it also enhanced progenitor cell production.
Maximal stimulation occurred at a retinoic acid con-
centration of 3 × 10−7 M acid, which increased the
mean number of colonies by 213 ± 8% over plates con-
taining CSF alone [31]. Retinoic acid has no direct
CSF activity, nor does it stimulate CSF production by
the cultured bone marrow cells or marrow stroma. This
stimulation may be mediated by increased responsive-
ness of the progenitor cells, possibly by increasing the
number of growth factor receptors per cell. Retinoids
are reported to enhance the binding of epidermal
growth factor (EGF) to fibroblasts and epidermal cells
by increasing the number of EGF receptors per cell
[176, 188, 319]. Enhancement of normal myelopoiesis
[31] and inhibition of myeloid leukemic cell prolifera-
tion by retinoic acid suggest that 13-cis-retinoic acid,
which is significantly less toxic in vivo than retinoic
Growth and differentiation factors as cancer therapeutics
acid, might be effective in the therapy of patients with
myelodysplastic syndrome because of the possibility
of prevention of progression to overt leukemia of these
preleukemic patients, for whom no other effective
treatment is currently available.
Table 2 lists some of the clinical studies conducted in
patients with myelodysplastic syndromes (MDS). These
studies employed oral doses of retinoids ranging from
10–100 mg/m2/d for 4 weeks to 5 years of duration
[171]. The percentages of response described in these
studies have been encouraging, but toxicity often lim-
ited the duration of the therapy. Hepatotoxicity was
dose-limiting but was completely reversible upon cessa-
tion of the therapy. In addition, cheilosis, hyperkerato-
sis, stomatitits, and increase in serum triglyceride levels
were reported. For example, Besa et al. [29] in their
study observed a 47% response including complete
remissions in 17 patients with MDS receiving 100 mg/m2
13-cis-retinoic acid with an improved survival in
responders of 33 months versus 10 months in the nonre-
sponders. However, no beneficial effects were reported
with either retinoic acid or isoretinoin in two studies of
14 MDS patients [146, 164]. N-4-Hydroxyphenyl-
retinamide (Fenretinide) lacked clinical effect in 15
MDS patients and in some may have enhanced leukemic
progression [115]. In a double-blind, placebo-controlled
trial of 13-cis-retinoic acid in 68 MDS patients with
100 mg/m2 for 6 months, no significant differences were
observed between treatment groups [195]. Approximately
30% of patients in both groups had progression of the
disease and survival was virtually identical. Ninety per-
cent of the treated patients developed mild to moderate
skin toxicity.
In view of these generally negative results, MDS
therapy with retinoic acid has been extended to combi-
nations with other agents with potential differentiation-
inducing capacity (Table 2). In a comparative study of
such combination therapy in 62 MDS patients, 50%
response was seen with a combination of retinoic acid,
1,25(OH)2 D3, and interferon, a response rate compara-
ble to that obtained with low-dose cytosine arabinoside
[151]. Combining all four agents proved too toxic.
Combining retinoic acid with low-dose ara-C produced
favorable results in one study [103], but in another study
of 14 MDS patients the response was no better than
either agent alone [160]. Combining a tocopherol
(800 mg/m2) with retinoic acid (100 mg/m2) produced a
62% response in 13 MDS patients and reduced the
toxicity involving liver damage, hyperkeratosis, and
mucositis [29].
In poor-prognosis acute nonlymphoblastic leukemia,
13-cis-retinoic acid has some efficacy on its own in one
Kapil Mehta et al. 537

Table 2. Clinical studies with retinoids in MDS patients

Retinod No. of Pts. Dose (mg/m2/day) Duration (weeks) Response (%) Ref.
13-cis RA
15 20–125 7–30 33 [125]
18 50–100 >8 16 [128]
15 100 8 20 [368]
10 100 8 30 [312]
8 20–100 6 50 [183]
33 20 8 9 [58]
30 50 >4 56 [205]
35 100 6 3 [195]
66 100 6 23 [28]
34 10–60 >12 (5 years) 12 [35]
ATRA
2 45 8–10 100 [392]
14 30–90 12 0 [15]
29 10–250 8 3 [15]
23 45 >4 13 [289]
10 45 6 50 [393]
Retinoid + others
13-cis RA + LD-Ara-C 2 [160]
13-cis RA + LD-Ara-C 3 [59]
13-cis RA + VCR 77 [103]
13-cis RA + Vit.D3 + IFNα 44 [151]
13-cis RA + Vit.D3 + LD-Ara-C 26 [152]
ATRA + G-CSF 40 [114]
ATRA + G-CSF + EPO + tocopherol 90 [235]
IFNα2 + thymopentin + LD-Ara-C 75 [391]
13-cis RA + Vit.D3 + 6-TH 52–61 [93]
ATRA + IFNα + G-CSF 23 [165]
6-TH, 6-thyoguanine; LD-Ara-C, Low dose Ara-C

small study [164], and clinical improvements were seen
using combinations with low-dose ara-C [349] or
vincristine or 6-thioguanine [103]. Evidence of differ-
entiation-induction was obtained in one case of a patient
who achieved a complete remission with cytogenetic
evidence of persistence of an abnormal clone in the
marrow [349].
Acute promyelocytic leukemias (APL) almost uni-
formly respond to retinoic acid treatment. APL consti-
tutes approximately 5–10% of all cases of acute
myeloid leukemia (AML) and is characterized by M3
morphology of FAB classification and chromosomal
translocations fusing retinoic acid receptor alpha (RARα)
gene on chromosome 17 and one of four different genes,
including promyelocytic leukemia (PML), promyelo-
cytic zinc finger (PLZF), nucleophosmin (NPM) or
nuclear matrix associated (NuMA) gene [300]. The
most common forms of translocations are t(15,17)
(q22,q21) encoding PML-RARα and t(11,17)(q23,q21)
encoding PLZF-RARα fusion receptor proteins, found
in 99% and >1% of APL patients, respectively. The trans-
locations are usually reciprocal chromosomal transloca-
tions, leading to creation of reciprocal hybrid receptor
proteins (X-RARα and RARα-X). APL expressing
PML-RARα, NPM-RARα or NuMA-RARα are respon-
sive to ATRA-induced differentiation effects with
the exception of PLZF-RARα type APL that is resis-
tant to ATRA.
ATRA induces differentiation of APL blasts into ter-
minally differentiated granulocytic cells that is associated
with clinical remissions. ATRA-induced differentiation
of APL blasts requires expression of PML-RARα recep-
tor protein [360]. PML-RARα can heterodimerize with
RXR or form homodimers and subsequently bind to
retinoic acid response element (RARE), located in the
promoters of the ATRA-responsive target genes. ATRA
can bind to PML-RARα with an affinity comparable to
RARα. In the absence of ligand, RAR-RXR in normal
blasts and PML-RARα-RXR heterodimers in APL cells,
recruit nuclear co-repressor proteins, N-CoR or SMRT,
538
and Sin3A or Sin3B which in turn form complex with
histone deacetylase enzymes (HDAC1 or HDAC2),
resulting in transcriptional repression or silencing [300].
The transcriptional suppression occurs because deacyla-
tion of histone protein creates conformational changes,
limiting access and binding of transcription factors and
RNA polymerase to related genes [198]. At physiologic
concentrations of ATRA (10−9−10−8 M), the nuclear co-
repressors protein and HDAC complex are dissociated
from RARα in normal blasts, which in turn results in
recruitment of co-activators with histone acetyltrans-
ferase (HAT) activity, such as steroid receptor coactiva-
tor-1 (SRC-1), PCAF, p300/CBP, ACTR, TIF2 or P/CIP
(Fig. 1b). Acetylation of lysine residues in the N-terminal
of histones by HAT activity results in transactivation of
responsive genes leading to differentiation. However,
the physiologic concentration of ATRA does not cause
dissociation of nuclear co-repressors protein and histone
deacetylase complex from the PML-RARα fusion
receptors in APL blasts, leading to differentiation block.
The co-repressor complex is dissociated from PML-RARα
at only pharmacological concentrations (10−7−10−6 M)
of ATRA, resulting in removal of transcriptional repression
and transcription of genes related to differentiation.
In addition to release of transcriptional repression, the
other possible mechanism involved in ATRA effective-
ness in myeloid cell differentiation include expression
of different class of genes [207, 212] including induc-
tion of expression of p21WAF1/Cip1 cyclin-dependent kinase
inhibitor [45], upregulation of C/EBP- [270], interferon
regulatory factor-1 (IRF-1) [308], PDCD4 [418] and
regulation of the localization of PML oncogenic domains
(PODs) [403]. More interestingly, the treatment of APL
cells with ATRA inhibited the synthesis of vascular
endothelial growth factor that in turn resulted in decrease
in bone marrow microvessel density [185].
More recent results with acute promyelocytic leukemia
have clarified the role of ATRA in this disorder. In multi-
ple studies [54, 91, 364, 400], it is now clear that a dose
of 45 mg/m2/day given by mouth in one or two divided
doses until complete remission followed by anthrocycline-
based combination chemotherapy for consolidation,
will induce a high response rate and improve survival in
patients with APL. On the basis of these and other studies,
ATRA has recently been approved by the FDA as standard
treatment for APL.
Although trans-retinoic acid is effective in the treat-
ment of APL, resistance is common with the return of
leukemia cells shortly after treatment in the absence of
effective programs of consolidation. The role of chemo-
therapy during induction treatment of APL is still
unclear. European approaches have utilized high doses
Growth and differentiation factors as cancer therapeutics
of chemotherapy including an anthracycline plus cyto-
sine arabinoside in patients with white counts exceeding
10,000/m2 at initiation of therapy. The clinical studies
conducted over the past years have determined that the
combination of ATRA and chemotherapy gives better
survival than chemotherapy alone in newly diagnosed
APL; the relapses were less and complete remissions
are slightly higher. These studies also revealed that
maintenance treatment with ATRA, and possibly with
low dose of chemotherapy, can further reduce the inci-
dence of relapse [92, 171].
Although ATRA therapy is usually well tolerated, two
problems are frequently encountered. The first is leuko-
cytosis, an increase in peripheral leukocytes to ≥20,000
cells/μL, which occurs in about half of the APL patients
treated with tRA [106]. The second, more serious prob-
lem, retinoic acid syndrome (RAS), develops in about
20–30% of APL patients treated with tRA. RAS is char-
acterized by high fever, respiratory distress, weight gain,
pulmonary infiltrates, hypotension, pleural effusion, and
sometimes renal failure [70, 126]; findings at autopsy
show extensive infiltration of mature myeloid cells into
lungs, skin, kidney, liver, and lymph nodes. Although leu-
kocytosis is often present in RAS, one third of APL
patients who have normal leukocyte counts also develop
this syndrome. Steroids such as dexamethasone have
ameliorated RAS [106]; possibly by affecting leukocyte
activation, cytokine production, or endothelial reactions.
Since RAS occurs only in patients with APL or AML
who have been treated with ATRA, this syndrome is
considered to reflect an aberrant interaction between
maturing granulocytes and host tissues. Because the
clinical signs of RAS resemble those of endotoxin shock
and adult respiratory distress syndrome it has been sug-
gested that ATRA treatment may affect the expression
of cytokines by myeloid cells. Induction of IL-1 and
G-CSF secretion by APL cells under the influence of
ATRA may contribute to hyperleukocytosis in vivo. On
the other hand, secretion of IL-1, IL-6, TNF-α, and IL-8
could contribute to the pathogenesis of RAS, since they
are involved in leukocyte activation and adherence and
have been implicated in the development of the adult
respiratory distress syndrome.
No evidence of synergism of 13-cis-retinoic acid with
chemotherapeutic agents was found in the treatment of
patients with chronic myeloid leukemia. Based upon
in vitro observations that retinoic acid significantly
reduced the recloning capacity of bone marrow myeloid
progenitors in vitro in certain cases of this disease, Arlin
et al. [12] added retinoic acid to an intensive chemo-
therapy protocol including daunorubicin, cytosine
arabinoside, and thioguanine, and in 17 evaluable patients
Kapil Mehta et al.
did not observe any increase in the incidence and dura-
tion of true Ph chromosome-negative remission in the
chronic phase of the disease.
The future of retinoid therapy resides in (a) develop-
ment of analogues with enhanced differentiation inducing
action with reduced toxicity, (b) developing combinations
with either conventional chemotherapeutic drugs or other
differentiation-inducing agents, applying retinoids more
extensively as a maintenance therapy, and (c) developing
approaches to reduce toxicity such as use of liposome-
encapsulated retinoic acid. In this context, a recent study
suggested that tyrosine kinase inhibitor ST1571 could
augment the cyto-differentiating, antiproliferative, and
apoptogenic activity of ATRA [120]. Moreover, in APL
cell lines made resistant to ATRA, the STI1571 could
relieve the resistance. Similarly, the use of liposomal-
ATRA was shown to be superior to the oral ATRA in
terms of maintaining higher plasma levels in animal
models and in humans [86, 88, 238, 305]. These results
demonstrated that encapsulation of ATRA in liposomes
and I.V. administration generate better pharmacokinetic
profile than oral ATRA by circumventing hepatic metab-
olism of ATRA.
Evaluation of liposomal ATRA in phase I trial in
patients with refractory hematological malignancies
showed that in contrast to the decline in plasma AUC
(area under the concentration time curve) of ATRA seen
3 to 4 days of initiation of oral ATRA, there were no dif-
ference between the AUC on day 1 and day 15 following
liposomal ATRA treatment [34]. In the same study, lipo-
somal ATRA was shown to be safe and toxicity profiles
were similar to oral ATRA, although liposomal ATRA
produced much higher AUC. I.V. administration of lipo-
somal ATRA (90 mg/m2) monotherapy was shown to be
effective in newly diagnosed APL patients, inducing
PCR-negative molecular CRs in a high proportion of
patients [76, 86] These studies supported the hypothesis
that I.V. liposomal administration may improve activity
of ATRA by altering its pharmacological profile and
remain elevated following extended treatment, providing
a basis for long-term remissions in APL patients.
New synthetic retinoid compounds: These include
Am-80, Am580 and 4-HPR, CD437, MX3350-1 and
LGD1069 (Targretin, Bexarotene), a RXR selective retin-
oid. Am-80, a synthetic retinoic acid receptor (RAR)-
specific agonist, has been successful in patients with
relapsed APL previously treated with ATRA, inducing
CR in about 60% of patients in a limited study [435, 436].
Am80 is approximately ten times more potent than ATRA
in terms of induction of differentiation in vitro, and more
stable to light, heat, and oxidation than ATRA. Am80 has
a low affinity for cellular retinoic acid binding protein,
539
and does not bind to retinoic acid receptor-gamma. Am80
has been shown to be effective in patient with APL that
had relapsed from CR induced by ATRA. Of 24 evaluable
patients, who received Am80, 6 mg/m2, orally alone daily
until CR, 14 (58%) achieved CR [435]. The interval from
the last ATRA therapy was not different between CR and
failure cases. The outcome was well correlated with the
in vitro response to Am80 in patients examined. Adverse
events observed in patient treated with Am80 included
retinoic acid syndrome, hyperleukocytosis, xerosis, cheil-
itis, hypertriglyceridemia, and hypercholesterolemia, but
generally milder than those of ATRA, which all patients
had received previously. Am80 is effective in APL
relapsed from ATRA-induced CR and deserves further
clinical trials, especially in combination with chemother-
apy. Am580, a stable retinobenzoic derivative and a
RAR-alpha agonist, is a powerful inducer of granulocytic
maturation in NB4 and in freshly isolated APL blasts
[437]. After treatment of APL cells with AM580 either alone
or in combination with granulocyte colony-stimulating
factor (G-CSF) at concentrations that are 10- to 100-fold
lower than those of ATRA necessary to produce similar
effects. AM580 is more powerful than ATRA in modulat-
ing the expression of differentiation antigens only in cells
in which PML-RAR is present.
In addition, Fenretinide (N-(4-hydroxyphenyl) reti-
namide, 4-HPR), a synthetic derivative of ATRA [438],
and nonretinoid compounds such as, 1,25-dihydroxyvi-
tamin D3 [138, 438, 440] and vitamin K2 in combina-
tion with ATRA [441] have been shown to be effective
in inducing differentiation in ATRA-resistant APL cell
lines and in cases to whom ATRA cannot be used [442–
444]. 4-HPR inhibits cell growth through the induction
of apoptosis in hematopoietic malignancies and variety
of solid tumor cells including head and neck, cervical
carcinomas, neuroblastoma, lung carcinoma and breast
cancer cells [445–450]. Recently, 4-HPR received a sig-
nificant amount of attention because of its promise as
chemopreventive and therapeutic activity in breast,
prostate and ovarian cancers in preclinical and clinical
models with minimal toxicity [451, 453]. The ability of
4-HPR to induce apoptosis in ATRA-resistant tumor
cell lines, such as acute myeloid leukemia (HL-60R)
and promyelocytic leukemia (NB306) [446], neuroblas-
toma [449], head and neck carcinoma [455] and breast
cancer cells [456] suggested that 4-HPR acts indepen-
dently of retinoid receptors to mediate its biological
actions. However, a potential role for RARs in 4-HPR
induced growth inhibition or apoptosis has been sup-
ported by other studies [450, 457–459]. 4-HPR inhibits
cell growth through the induction of apoptosis rather
than differentiation in HL-60 and NB4 cells [103, 111].
540
Vitamin D Metabolites
and Analogs as Leukemia
Differentiation-Inducing Agents
The term vitamin D is generally used to describe a num-
ber of chemically related compounds having common
antirachitic properties, but differing in the rapidity of
their action and the conditions under which biologic
activity is observed. In humans, cholecalciferol (D3)
produced in the skin and the fraction obtained from the
diet undergo sequential hydroxylation reactions, first in
the liver microsomes and then in the kidney mitochon-
dria, resulting in the formation of 25-(OH) D3 and 1,25-
(OH)2 D3, respectively. The latter is thought to be the
active form of D3 in enhancing bone resorption medi-
ated by osteoclasts and in enhancing absorption of cal-
cium and phosphorus by the intestine. In addition, it
appears to act on the kidney in concert with parathyroid
hormone to promote calcium resorption. It was formerly
believed that 1,25-(OH)2 D3 was synthesized in placenta
and by calvarial cell suspension containing osteoclasts,
osteoblasts, fibroblasts, and endothelial cells [366]. The
local production of active metabolites of vitamin D by
target organs such as bone raises interesting questions as
to the role of these tissues in mediating vitamin D action,
and may provide indications of a new dimension to local
actions of vitamin D metabolites on normal or leukemic
marrow cell function, as well as more conventional
aspects of mineral metabolism. Interest in vitamin D
action in hematopoiesis was prompted by the observa-
tions that osteoclasts originate by fusion of circulating
mononuclear precursor cells and almost certainly repre-
sent one of the end-stage cells of mononuclear phago-
cyte differentiation [43, 177, 178, 376]. Like osteoclasts,
other mature, nonproliferating phagocytic mononuclear
cells, such as monocytes and macrophages, possess the
capacity to attach to and degrade bone matrix [178].
Monocytes and macrophages possess receptors for 1,25-
(OH)2 D3, and the culture of human monocytes with 10−8
M of this metabolite results in macrophage maturation
[320]. In cultures of normal human bone marrow, 1,25-
(OH)2 D3 induces extensive macrophage differentiation
[236]. Long-term cultures of human cord blood myeloid
cells also terminally differentiate to monocytes and
macrophages with vitamin D [344]. Direct evidence of
1,25-(OH)2 D3 induction of osteoclast development has
been reported by Abe et al. [72], who demonstrated that
a 1.2 nM concentration of the metabolite induced exten-
sive fusion of mouse alveolar macrophages, and that in
the presence of the lymphokine macrophage fusion fac-
tor, as little as 0.012 nM was active in producing multi-
nucleated giant cells. Multinucleated giant cell formation
Growth and differentiation factors as cancer therapeutics
has also been observed when the HL-60 cell line and the
mouse macrophage-like J774.2 cell line were exposed to
10−7−10 M 1,25-(OH)2 D3 for 24–72 h [22]. In this context,
in vivo injection of 1,25-(OH)2 D3 into patients with
malignant osteopetrosis corrected bone binding and
resorptive deficiencies in the patients’ monocytes [22].
Koeffler et al. [191] have shown that 10−7 to 10−9 M
concentrations of either the active metabolite of vitamin D
(1,25-(OH)2 D3) or certain fluorinated analogs of vitamin
D can induce normal human myeloid progenitors
(GM-CFC) in the presence of CSF to differentiate prefer-
entially to macrophages in vitro. Marrow cultures exhib-
ited an absolute, not just a proportional, increase in
macrophage colonies, indicating that the action was not
simply inhibition of granulopoiesis. The plasma concen-
tration of 1,25-(OH)2 D3 in humans is approximately 7.7 ×
10−11 M [148], and the concentration of 1,25-(OH)2 D3
inducing macrophage differentiation of progenitors
in vitro is >10−9 M, raising the question of physiologic
relevance of this observation. Certainly, patients receiving
superphysiologic doses of 1,25-(OH)2 D3 have not been
reported to have monocytosis; likewise, patients with vita-
min D-resistant rickets have not been reported to have low
monocyte or macrophage levels. Nevertheless, the possi-
bility of local production of the active form of vitamin D
by cellular components of the marrow microenvironment
[385], possibly even by tissue macrophages, raises the
possibility of much higher local concentrations of 1,25-
(OH)2 D3 in the environment of the progenitor cells than
plasma levels may suggest. Furthermore, recurrent infec-
tions, impaired phagocytic function, and decreased mobil-
ity of leukocytes have been shown in vitamin D-deficient
states. This defect in phagocytic function has been reversed
by in vitro culture of macrophages with 1,25-(OH)2 D3
[22, 332]. The immediate biological precursor of 1,25-
(OH)2 D3, 25-(OH) D3, is without biologic effect unless
used at 10- to 100-fold higher concentrations than its
hydroxylated metabolite. Thus, evidence that macrophages
themselves have 1-hydroxylase activity and can synthe-
size 1,25-(OH)2 D3 from its precursor suggests that this
molecule may also be a monokine with important local
actions in recruitment and activation of macrophages
[332] and of osteoclasts, with which they share a common
derivation from a marrow hematopoietic progenitor.
Action of Vitamin D Metabolites
on Cancer Cells
Besides playing a crucial role in maintaining the calcium
homeostasis in the body, 1,25-(OH)2 D3 can also affect
cell growth and differentiation in several cell types,
Kapil Mehta et al.
including cancer cells. Based on these properties of
1,25-(OH)2 D3, it has been studied for its ability to
inhibit and prevent the cancer growth. The results from
various clinical studies were though encouraging but
due to the calcemic adverse effects the therapeutic win-
dow of this compound was extremely narrow [36]. Abe
et al. [46] were among the first ones to demonstrate that
1,25-(OH)2 D3 could induce macrophage differentiation
of myeloid leukemic cells. Induction of phagocytes,
lysozyme production, and locomotive activity were seen
with concentrations as low as 10−10 M of the vitamin.
The potency of this differentiation inducer was such that
a 1,000-fold higher concentration of the next most
potent inducer of M1 differentiation, dexamethasone,
was required to achieve a comparable level of matura-
tion. Simultaneous treatment of M1 cells with low,
physiologic concentrations of 1,25-(OH)2 D3 (0.12 nM)
and dexamethasone (10 nM) induced a degree of differ-
entiation equivalent to the response obtained with higher
concentrations of either agent alone [249]. The isolation
of two variant clones of M1, one resistant to dexametha-
sone and the other to 1,25-(OH)2 D3, strongly suggests
that these differentiation-inducing agents act in different
ways and that combination therapy with both steroids
may be useful in reducing leukemogenicity [249].
The mouse myelomonocytic leukemic cell line
WEHI-3 is also inducible to macrophage differentiation
when exposed to 1,25-(OH)2 D3 in a clonal assay sys-
tem. The differentiation-susceptible D+ line was both
growth-inhibited and macrophage-differentiated to the
50% level with 10−8−10−10 M 1,25-(OH)2 D3. Of particu-
lar interest was the observation that a subline of WEHI-3
refractory to other differentiation-inducing agents was
exquisitely sensitive to 1,25-(OH)2 D3, with 50% growth
inhibition and macrophage differentiation seen with
10−13 M vitamin.
The first reports of the capacity of 1,25-(OH)2 D3 to
induce differentiation of human leukemic cells indicated
that HL-60 was growth-suppressed, and phagocytosis
and C3-rosette formation were markedly induced in a
dose-dependent manner over a range of 10−8 to 10−10
[248]. Unfortunately, these investigators concluded
that HL-60 was induced to form granulocytes, as had
previously been observed for retinoids and DMSO, rather
than the uniform pattern of monocyte-macrophage
differentiation reported in subsequent studies [189, 228,
234, 236, 275, 352, 356]. HL-60 cells following their
treatment with 1,25-(OH)2 D3 acquire several phenotypic
changes that were similar to the mature monoyctes/mac-
rophages. These maturation features were induced in a
dose-dependent (10−11 to 10−7 M) and time-dependent
(1–6 days) manner, and resulted in a functional phenotype
541
and two-dimensional gel pattern of proteins close to, but
not identical with, those of peripheral blood monocytes
[251]. Cell division apparently is not required for expres-
sion of these differentiation features [94, 191].
The ability of low concentrations of 1,25-(OH)2 D3 to
induce macrophage differentiation of HL-60 is similar to
that reported for phorbol esters; however, this is not due
to similar binding sites, since 1,25-(OH)2 D3 did not
compete for phorbol diester binding sites as measured by
(3H)phorbol dibutyrate binding on HL-60 [275]. Variants
of HL-60 have been developed which are resistant to dif-
ferentiation induction by DMSO, retinoic acid, TPA, and
1,25-(OH)2 D3. One variant resistant to phorbol esters
was also resistant to 1,25-(OH)2 D3 [275], suggesting
that mutants that cannot be induced to differentiate can
involve common events following the receptor binding
stage. The possibility of synergism between 1,25-(OH)2
D3 and phorbol esters or other differentiation inducers is
definitely indicated in the case of HL-60, and has been
reported with retinoic acid and with DMSO since low
concentrations of 1,25-(OH)2 D3 in combination with
DMSO produced cessation of proliferation of HL-60
cells within 2 days, as well as a greater expression of
differentiation [352]. Simultaneous treatment of HL-60
with suboptimal concentrations of 1,25-(OH)2 D3 (0.12–
1.2 nM) showed additive effects in reducing nitroblue
tetrazolium, a common marker for monocyte-macrophage
and granulocyte differentiation [250].
The human monocytoid cell line U937 is also induced
to macrophage differentiation with loss of plating effi-
ciency in the presence of 10−10 M 1,25-(OH)2 D3 [245,
293]. Differentiation involves development of adher-
ence, macrophage morphology, lysozyme production,
capacity to reduce NBT, expression of β-glucuronidase
and alkaline phosphatase, Fc receptor expression, phago-
cytosis, and reactivity with antimonocyte-specific mono-
clonal antibodies [293]. As in the case of HL-60, the
differentiation of U937 is not blocked by inhibitors of
DNA synthesis, but is by the calcium ionophore A23187
[175]. The existence of synergism between 1,25-(OH)2
D3 and other biological response modifiers has been
established with retinoic acid. U937 cells can be primed
by short incubation with 1,25-(OH)2 D3 to respond by
maturation to agents such as cAMP, PGE, and cholera
toxin, which alone do not induce differentiation [293].
1,25-(OH)2 D3 or retinoic acid plus dibutyryl cAMP
is effective in inducing a variety of differentiation mark-
ers in U937. Their actions on insulin receptors were the
opposite, however. 1,25-(OH)2 D3 increased the bind-
ing, while retinoid decreased the binding. This effect
was specific for insulin, since the transferrin receptors
were reduced by both methods of differentiation [335].
542
Thus, changes in insulin receptors during maturation
in vitro depend on the inducing agent and are not caus-
ally related to the differentiation process.
Other differentiation-inducing agents have been com-
pared with 1,25-(OH)2 D3, which was the most effec-
tive. The polar/planar compound HMBA was most
effective in reducing cell recovery, but did not induce
cell maturation. Retinoic acid-reduced cell and total
blast cell recovery with an increase in neutrophil dif-
ferentiation. Protein inducers of differentiation, α- and
γ-interferon, showed slight activity in reducing cell and
blast recovery, whereas a murine serum source of dif-
ferentiation factor (GM-DF) was highly effective in
inducing macrophage differentiation and reduction in
recovery of immature cells. 1,25(OH)2 D3 or IFN-γ
decreased blast cells and increased macrophage differ-
entiation in suspension cultures of marrow from patients
with myelodysplastic syndrome [380, 417].
As an alternative to the suspension culture technique
for monitoring differentiation induction of fresh leukemic
marrow, an agar cloning assay has been used, in which
colony or cluster incidence was measured in 7-day cul-
tures of 105 leukemic marrow cells. Unlike the clonal
assay for differentiation-induction of HL-60 or WEHI-3
cells, primary human leukemia cultures formed small
clusters of 10–20 cells (microclusters), 20–40 cells (mac-
roclusters), small colonies with an excess of clusters
(microcolonies), or colonies of normal size with a normal
cluster-to-colony ratios [148, 191, 265]. In most cases,
the clonal growth was diffuse and colonies or clusters
failed to differentiate (with the exception of most patients
with chronic myeloid leukemia and some patients with
preleukemia) [250, 332, 336, 382]. In view of this growth
pattern, clonal differentiation can only be measured by
isolation and staining of individual clones, a laborious
and inexact procedure at best. As an alternative, 1,25-
(OH)2 D3-induced reduction of leukemic cloning capacity
was used as an index of differentiation. The legitimacy of
this approach has been validated by studies showing that
50% growth inhibition of proliferation generally approxi-
mated to 50% growth inhibition when 1,25-(OH)2 D3 or
its derivatives were used to induce differentiation. It
should be stressed that this linkage between proliferation
inhibition in clonal assay and differentiation induction
generally does not hold true for other types of differentia-
tion agents. For example, inhibition of the colony growth
by most chemotherapeutic agents is not associated with
differentiation, and protein sources of differentiation
activity may not influence primary leukemic cloning
capacity, but only recloning capacity.
In a more extensive analysis of heterogeneity of
responsiveness to the growth-inhibitory/differentiation
Growth and differentiation factors as cancer therapeutics
capacity of 1,25-(OH)2 D3, it was observed that preleu-
kemic marrows exhibiting colony formation analogous
to normal marrow were least responsive to 1,25-(OH)2
D3, whereas preleukemic marrows with cluster-forming
acute myeloid leukemia-type clonal growth were most
responsive, being comparable to the clinical observa-
tions. These observations suggest the interesting, per-
haps surprising, conclusion that the more acute the
leukemia the more it is responsive to 1,25-(OH)2 D3.
Koeffler et al. [191] have also shown that 1,25-(OH)2
D3 and two fluorinated analogs, 24, 24-F2-1,25-(OH)2
D3, inhibited colony formation of marrow from patients
with ANLL (four cases) and chronic myeloid leukemia
(four cases) at concentrations of 10−8 M, with 50–80%
of leukemic colony-forming cells assuming a mac-
rophage-like morphology. This result is comparable to
the report of Moore et al. [265], with the exception that
in the latter study more patients were investigated and
the greater sensitivity seen in acute myeloid leukemia
patients (50% inhibition at 4 × 10−12 M) may be explained
by the use of total clonogenic units measured (i.e., colo-
nies of >40 cells and clusters of 3–40 cells) rather than
restricting inhibition analysis to colonies of >40 cells.
The cells of the majority of patients with acute myeloid
leukemia do not form colonies of 40 cells, and the leu-
kemic cells in general form small clusters.
A variety of metabolites and analogs of vitamin D3
have been developed and tested for biological activity
and in vivo toxicity. Recent studies have extended bio-
logical screening to leukemia differentiation systems.
HL-60 is induced to differentiate upon exposure to
10−7−10−10 M 1,24-(OH)2 D3 or 1,24R-(OH)2 D3 in a
fashion comparable to that with 1,25-(OH)2 D3. A differ-
ent analog of 1,25-(OH)2 D3 has been found highly
active in stimulating intestinal calcium transport and
bone calcium mobilization in vitamin D-deficient rats
[374]. This 24, 24-F2-1,25-(OH)2 is highly active in
induction of WEHI-3 and HL-60 differentiation and
growth inhibition, with 50% activity at 10−14 M, thus
making it the most active D3 analog tested in the leuke-
mic assay system [258]. Unfortunately, this analog is
considerably more toxic in vivo than is 1,25-(OH)2 D3.
These compounds showed the same relative activities,
in that normal marrow was always least sensitive to
growth inhibition and acute myeloid leukemia marrow
most sensitive, with preleukemia and chronic myeloid
leukemia occupying intermediate positions.
24-Homo-, and 26-homo-1,25(OH)2 D3 and delta [42]
analogues were ten-fold more potent than 1,25(OH)2 D3
in inducing differentiation of HL-60 [298]. The
24-homo-analogue was significantly less active in mobi-
lizing calcium from bone.
Kapil Mehta et al.
It is obvious that more extensive screening must be
undertaken to determine if the calcium-mobilizing
activity and consequent toxicity as a feature of D3
metabolites are invariably related to efficacy in induc-
tion of leukemic growth inhibition and differentiation.
Obviously, as compared with a long in vivo half-life,
low toxicity and retention of selective leukemia-cell
differentiating activity would be highly desirable for
clinical studies. Clinical trials with vitamin D have been
limited because of intolerable hypercalcemia which
often develops with this compound. Some responses
have been seen in patients with myelodysplastic
syndromes when treated at 2 mg/day [192]. With new
analogues of vitamin D3 now available, a separation of
the effects of calcium metabolism and those on cell
differentiation is now possible and clinical investiga-
tions are underway [301].
Receptors for Vitamin D and its
Metabolites
The role of 1,25-(OH)2 D3 as an agent responsible for
mineral homeostasis has been extensively studied. Its
mechanisms of action within target cells is retinoid and
steroid hormone-like in that the binding of 1,25-(OH)2
D3 to vitamin D receptor (VDR) induces conformational
changes in the VDR which promote heterdimerization
with retinoid X receptor (RXR) and recruitment of a
number of nuclear receptor coactivator proteins including
the SCR family members (Fig. 2) [224]. All known 1,25-
(OH)2 D3-responsive tissues, such as the intestine, bone,
and kidney, contain VDRs. In addition, many other
tissues and cultured cells have been shown to possess
1,25-(OH)2 D3 receptors, including some tumor cells.
Breast cancer, melanoma, and osteogenic sarcoma cells
possess a low density (8,000–15,000 per cell) of recep-
tors with high affinity (Kd 10−10−10−11 M) for 1,25-(OH)2
D3 [84, 100, 105, 229, 186]. Receptors with similar affinity
and density have been reported on human monocytes,
malignant B and T leukemic cell lines, activated T-cells,
Epstein-Barr virus-transformed B-lymphocytes, and
cell lines such as K562, HL-60 and U937 [228, 229,
320, 352]. Identification of 1,25-(OH)2 D3 receptors by
specific immunochemical reactivity and selective chem-
ical dissociation has shown that nuclear binding of the
vitamin receptor is a rapid event (minutes) in HL-60,
whereas the cellular differentiation response is delayed
(6–7 days) [228]. This may be reconciled if the receptor
must be maintained within the nucleus over the long
term. A differentiation-noninducible variant of HL-60
has been reported to have only 8% of receptor copy
numbers of the parent line, suggesting that assay of
543
1,25-(OH)2 D3 receptors in leukemic patients may be
predictive of the ultimate response of the patient to
adjunct therapy with 1,25-(OH)2 D3 [228]. This possi-
bility is supported by the observation that the equilib-
rium dissociation constant of the 1,25-(OH)2 D3 receptor
on HL-60 is close to the vitamin concentration, causing
50% of HL-60 cells to reduce NBT [189]. Against this
view, there is the fact that the human myeloblastic leu-
kemic cell line KG-1 has approximately the same num-
ber of 1,25-(OH)2 D3 receptors as HL-60, yet cannot be
induced to differentiate by the vitamin [189].
There is extensive evidence for a classic steroid
receptor-DNA interaction for the 1,25-(OH)2 D3 recep-
tor, with selective binding of the receptor to A + T-rich
segments of double-stranded DNA. Franceschi [101]
demonstrated that the receptor can also interact with
RNA as well as DNA. The physiologic significance of
this observation remains obscure. However, in other ste-
roid hormone systems, steroids can influence certain
nontranscriptional processes, such as the stability of
hormone-dependent mRNA, as well as posttranscrip-
tional processing of secretory proteins, the regulation of
5S RNA synthesis, and the processing of heterogeneous
nuclear RNAs.
Interaction of 1,25-(OH)2 D3 with receptors on normal
or malignant target cells results in variable and complex
changes in proliferation. A biphasic effect on the growth
of breast tumor [116, 175] and osteosarcoma cells [229]
has been reported, with physiologic concentrations of
1,25-(OH)2 D3 (10−10–10−11 M) stimulating growth, and
higher concentration (10−7–10−8 M) inhibiting it. In con-
trast, a uniform pattern of growth inhibition is seen with
the leukemic cell lines HL-60, U937 [294], M-1 and
WEHI-3, and primary myeloid leukemia at concentra-
tions as low as 10−12 M. No inhibition of normal marrow
CFU-GM colony formation is seen with 1,25-(OH)2 D3
except at very high concentrations of 0.1–1 μg/ml; growth
stimulation of normal mouse and human CFU-GM
colony formation can be observed.
Kuribayashi et al. [199] have reported on two variant
clones of HL-60, resistant to differentiation and growth
inhibition in the presence of 1,25-(OH)2 D3. One clone
was also unresponsive to phorbol ester, actinomycin-D,
and DMSO. The variant clones were found to possess
reduced amounts of cytosol receptor protein, to which
1,25-(OH)2 D3 was specifically bound, but the hormone-
receptor complex could be transferred to the chromatin
acceptor site in both the wild-type and variant clones.
This would indicate that 1,25-(OH)2 D3 resistance is due
to a reduction in specific cytosol receptors.
Freake et al. [105] reported that whole chronic myeloid
leukemic cells specifically took up 1,25-(OH)2 D3 with
544
high affinity (Kd = 3.6 × 10−11 M) and low capacity.
Subcellular fractionation of labeled cells showed that
binding was restricted to cytosols and nuclei; however,
chronic myeloid leukemic cells appeared to contain
both the receptor for 1,25-(OH)2 D3 and an unknown
substance that prevents its detection following the prep-
aration of cytosol. Cells from patients in chronic phase
specifically bound more vitamin (18 fmol/107 cells) than
did those in the blastic phase (7 fmol/107 cells), or cells
from patients with acute myeloid leukemia (2.6 fmol/107
cells). From observing that only cells from patients with
chronic myeloid leukemia responded to 1,25-(OH)2 D3
by differentiation along the monocyte-macrophage
pathway, it was concluded that differentiation induction
was most likely dependent upon adequate levels of
receptors, and that intact cells rather than cytosol prepa-
rations should be studied before cells of a particular
tissue are designated as receptor negative. In view of
the heterogeneity of the cellular composition of chronic
myeloid leukemia and the heterogeneity of morpho-
logical type in blastic chronic myeloid leukemia
(30% terminal transferase positive) and ANLL, general
conclusions on receptor display with such small groups
of patients should be treated with caution.
The HL-60 genome contains several different onco-
genes, but only one, c-myc, is significantly amplified
and transcribed at high levels (×20). Westin et al. [404]
reported that myc on mRNA is no longer present in
HL-60 cells induced to granulocytic differentiation by
DMSO or retinoic acid. Reitsma et al. [330] have shown
that 1,25-(OH)2 D3 reduced myc nRNA levels in HL-60
within 4 h of exposure to the vitamin/hormone, and this
change preceded the onset of other measurable pheno-
typic changes by at least 8 h. It remains to be determined
whether the altered transcription is the consequence of
the hormone interaction with the c-myc promoter or a
spectrum of promoters, each associated with a gene or
gene cluster involved in determining cell phenotype.
Action of 1,25-(OH)2D3 on other Aspects
of Hematopoiesis
Myelofibrosis with myeloid metaplasia is considered a
neoplastic disorder in which fibroblast proliferation and
collagen synthesis in the marrow are increased by plate-
let-derived growth factor, or related substances, released
by neoplastic megakaryocytes [48]. It has been postu-
lated that 1,25-(OH)2 D3 may inhibit the formation of
fibrous tissue (mainly collagen) in bone marrow and
also may increase its degradation [237]. The hormone
also inhibits the proliferation of megakaryocytes that
normally promote collagen synthesis. Degradation of
Growth and differentiation factors as cancer therapeutics
fibrous tissue is also mediated by monocytes and mac-
rophages, and the number and activity of these cells are
increased by 1,25-(OH)2 D3. Thus, the various actions of
this vitamin contribute to a reduction in the collagen
content; conversely, a deficiency of it may allow abnor-
mal accumulation of collagen in the marrow. In this
context, myelofibrosis in a rachitic infant regressed fol-
lowing vitamin D therapy [66], and a group of rachitic
children with anemia and a blood picture typical of
myelofibrosis also responded to vitamin D [415].
A novel immunoregulatory role has been proposed for
1,25-(OH)2 D3. Intracellular receptors that bind the vita-
min and sediment at 3.35 were not detected in “resting”
T or B lymphocytes, but T cells activated by Epstein-
Barr virus produced the receptor, and the amount of the
macro-molecule induced was the same as in normal
monocytes. Since the D3-binding macro-molecule is
seen in actively mitotic cells, it may exert an antiprolif-
erative-differentiative influence in the immune system.
1,25-(OH)2 D3 at picomolar concentrations has also
been shown to inhibit production of the T-lymphocyte
growth-promoting lymphokine interleukin 2 (IL-2).
Other metabolites of vitamin D3 were less effective, and
their order of potency corresponded to their respective
affinity for the 1,25-(OH)2 D3 receptor, suggesting that
suppression of T-cell production of IL-2 was mediated
by this specific receptor [384]. 1,25-(OH)2 D3 may
selectively inhibit the action of IL-1 in stimulation of
thymocyte proliferation [273]. It also modulates GM-CSF
production by T cells by posttranscriptional reduction in
the half-life of GM-CSF, nRNA in mitogen-activated T
cells and T-cell lines [377].
In view of the well-established calcium-mobilizing
effect of 1,25-(OH)2 D3 in its classic target tissues [226],
it is possible that the suppressive effect of this hormone
on IL-2 is mediated by an influence on calcium translo-
cation and again indicates a physiologic role of this
hormone in immunoregulation.
In vivo Effects of Vitamin D Metabolites
Sato et al. [347] have evaluated the effect of 1-α-(OH)
D3 on the growth of two solid tumors transplanted sub-
cutaneously in mice. 1-α-(OH) D3 administered orally
by stomach tube at daily doses of 0.1 and 0.2 μg/kg body
weight for 114 days suppressed sarcoma 180 tumor
growth by 37 and 64% respectively, without signifi-
cantly affecting serum calcium levels. Similar oral treat-
ment with 0.1 and 0.2 μg/kg of 1-α-(OH) D3 for 21 days
resulted in a 75–80% decrease in pulmonary metastases
in mice injected subcutaneously with Lewis lung carci-
noma fragments. While the data illustrate a potential
Kapil Mehta et al.
therapeutic anticarcinogenic effect of this metabolite,
they should be considered as preliminary, since small
numbers of mice (three to six) were tested in each treat-
ment group.
1-α-(OH) D3 and 1,25-(OH)2 D3 have marked effects
on the growth and differentiation of cultured murine
myeloid leukemia cells (M1 cells), established from an
SL mouse with myeloid leukemia. These results, and the
fact that syngeneic SL mice inoculated with M1 cells all
die of leukemia, prompted a recent evaluation of the
in vivo effects of 1-α-(OH) D3 and 1,25-(OH)2 D3 by
Honna et al. [166]. Thrice-weekly intraperitoneal injections
of picomole amounts of 1-α-(OH) D3 and 1,25-(OH)2 D3
considerably prolonged the survival time of syngeneic SL
mice inoculated with M1 cells. A similar, marked prolon-
gation of survival time was observed in athymic nude
mice with M1 leukemia and treated intraperitoneally with
1-α-(OH) D3. The results with the athymic mice sug-
gested to the authors that T-lymphocyte-mediated immune
responses were not directly involved in the effects of the
vitamin D3 metabolite. Serum levels of calcium and phos-
phorus were not significantly affected in the nude mice
given M1 cells and 1-α-(OH) D3 for 30 days, compared
with mice given M1 cells alone.
Attempts to duplicate these observations in BALB/c
mice inoculated with syngeneic WEHI-3 myelomono-
cytic leukemia cells proved unsuccessful [143]. Neither
dexamethasone nor a combination of it with 1,25-(OH)2
D3 prolonged the survival of WEHI-3 tumor-bearing
mice [143, 166]. The disparity between the in vivo
observations with M1 and WEHI-3 leukemias could
have a number of explanations, which require further
testing before dismissing the therapeutic potential of
vitamin D derivatives in leukemia. The WEHI-3B grows
more rapidly than M1, and 105 cells generally result in
100% mortality within 21 days. However, treatment
with conventional cytotoxic agents, such as Cytoxan,
produces an increase in life span, even producing cures
in a dose-dependent fashion [143]. In retrospect, however,
the explanation may reside in the relative resistance of
the WEHI-3D+ line to 1,25-(OH)2 D3-induced growth
inhibition (50% inhibition in vitro with 4 × 10−4 M), in
contrast to the much greater sensitivity of the WEHI-3D−,
the HL-60, M1, and fresh acute myeloid leukemic cells
(10−9–10−12 M).
Potter and Moore [317] have extended in vivo studies
with successful growth and maintenance of the human
myeloid leukemia lines U937, KG-1, and K562, as subcu-
taneously induced granulocytic sarcomas in nude mice.
Studies involving routine and special histochemistry,
enzyme histochemistry, immunocytochemistry, surface
markers, cytogenetics, and electron microscopy have
545
demonstrated virtual identity of cells from the induced
granulocytic sarcomas in nude mice with respective
cells from the cultured lines. The validity and reliability
of the living model have thus been established. Using
this in vivo model, preliminary experiments were under-
taken to evaluate the effect(s) of 1,25-(OH)2 D3 on the
development of these subcutaneous leukemic tumors
induced on nude mice from cultured cells. Based on the
work of Hartmann and Moore [143], a dosage of 1 μg/
ml of 1,25-(OH)2 D3 was chosen as potentially the most
effective, but least toxic. The method of administration
involved subcutaneous implantation of an osmotic
minipump (OMP) at a site remote from the concurrently
inoculated tumorigenic cells. One group of mice
received only washed cultured leukemic cells, another
received cells and an OMP containing only solvent, and
a third group of mice received leukemic cells and an
OMP delivering 1 μg/ml of 1,25-(OH)2 D3. All mice
were weighed on alternate days and observed for tumor
development.
Of the mice receiving cultured K562 or HL-60 cells
only, almost all developed granulocytic sarcomas within
an average of 4–5 weeks after inoculation. A similar
result was seen in mice inoculated with cultured cells
from either of these lines that also bore implants of an
OMP containing only solvent. Granulocytic sarcomas
either failed to develop, or developed infrequently and
(on the average) later in the group similarly inoculated
with cultured cells from these lines, and bearing OMPs
containing 1,25-(OH)2 D3.
Since each mouse underwent the same priming and
received the same number of cultured human leukemic
cells, it appears that the absence of, or delay in the
development of, induced granulocytic sarcomas may be
attributable to the influence of the administered 1,25-
(OH)2 D3 in the system. These results suggest that 1,25-
(OH)2 D3 might have an inhibitory effect on the
proliferative capacity of human leukemic cells in vivo.
Whether the mechanism involves induction of terminal
differentiation on the human tumor cell inoculum
requires investigation. The involved mechanism(s) of
inhibition are also of interest. In those few treated mice
demonstrating proliferation of xenogenic (human) leu-
kemic cells, the possibility of emergence of 1,25-(OH)2
D3-resistant clones should not be overlooked, and the
availability of receptors for 1,25-(OH)2 D3 should be
determined on cells derived from these tumors. Recently
developed analogues of 1,25-(OH)2 D3 notably 1,25
(OH)2-16-ene-23-yne-D3, were remarkably effective in
“curing” mice bearing the WEHI-3B + leukemia [32].
Differentiation was induced at dosages that did not
produce hypercalcemia.
546
Translating these observations into a clinical applica-
tion, it is clear that in vitro screening for response to
1,25-(OH)2 D3 can be used to identify resistant and
susceptible patients with leukemia and preleukemia.
While it is unlikely that the vitamin could prove effec-
tive on its own, it could be combined with conventional
chemotherapy or used as maintenance therapy in patients
achieving remission by conventional protocols. The
toxicity of 1,25-(OH)2 D3 in mice at dosages unable to
produce leukemic regression in vivo would require pre-
cise dose-response analysis in humans. In this regard,
1,25-(OH)2 D3 has been used effectively in patients with
post-menopausal osteoporosis [64]. Short-term treat-
ment (6–8 months) with 0.5 mg/day restored calcium
absorption to normal, and calcium balance improved
and the bone resorption rate decreased. With long-term
therapy (2 years) both bone resorption and formation
rates increased. The lack of side effects in long-term
treatment with 1,25-(OH)2 D3 provides a dosage guide-
line for studies in leukemia [111].
Oral administration of 1-α-(OH) D3 in two patients
with AML and one with MDS was reported to reduce
the number of leukemic cells in the marrow. In a study
of 18 patients with MDS, 1,25-(OH)2 D3 produced a par-
tial or minor response in blood in eight cases but with no
significant improvement in blood or marrow blasts
[192]. Seven of the patients developed leukemia before
or by 12 weeks of treatment and half the patients devel-
oped hypercalcemia. It was not possible to sustain serum
levels of 1,25-(OH)2 D3 at levels necessary to induce
differentiation or growth inhibition without producing
unacceptable toxicity. Future clinical trials await the use
of recently developed analogues with reduced calcium
mobilizing action and enhanced differentiating activity
[32]. A further rationale for therapy with 1,25-(OH)2 D3
and its analogues is provided by the observation that the
metabolic pathway of 1,25-(OH)2 D3 is defective in
patients with MDS or AML [30]. Bone marrow plasma
levels of 1,25-(OH)2 D3, but not its immediate precursor
25-(OH) D3, was decreased significantly in 50% of
MDS and 30% of AML patients.
Polar-Planar Compounds
as Differentiation Inducers
Differentiation-inducing activity has been reported
for various polar-planar compounds, most particu-
larly DMSO, hexamethylene bisacetamide (HMBA),
N-methylformamide and, cotylenin A [62, 63, 169, 427,
Growth and differentiation factors as cancer therapeutics
428]. The differentiation-inducing action has been most
extensively analyzed in murine erythroleukemic (MEL)
cells. Inducer-mediated differentiation is a multistep
process characterized by a latent period when a number
of changes occur [232, 348]. These include alterations
in ion flux, increase in membrane bound PKC, appearance
of CA2+ and phospholipid-independent PKC activity in
the cytosol and modulation of the expression of genes
such as c-myc, c-myb, c-fos and p53. Commitment to
differentiation is seen within 12 h and increases stochas-
tically over 48 h and is associated with suppression of c-myb
expression and a 10- to 30-fold increase in globin gene
expression [232, 331]. The levels of ornithine decarbox-
ylase are also regulated by HMBA. HMBA induces a
transient, genome-wide hypomethylation of DNA achieved
by replacement of 5-methylcytosine with cytosine resi-
dues [327]. This may be a necessary but not sufficient
step in triggering the whole program of differentiation.
Superoxide dismutase activity is induced by HMBA in
parallel with differentiation and enzyme levels are
directly related to the degree of cytosolic hemoglobin-
ization [24, 304]. Introduction of superoxide dismutase
into MEL cells with liposomes induces differentiation
as do other oxidative treatments (liposome amino acid
oxidase, xanthine oxidase, potassium superoxide). In
contrast, antioxidants inhibit HMBA-induced differen-
tiation [24]. The induction of superoxide dismutase in
MEL cells may also be a cellular response to oxidative
stress from hemoglobin autooxidation.
Potential improvements in efficacy of HMBA may be
accomplished by changes in the chemical structure of
the inducing agents and by increasing the sensitivity of
tumor cells to inducers of differentiation. MEL cell lines
that have acquired low levels of resistance to vincristine
display a markedly increased sensitivity to HMBA
[231]. A series of hybrid increased polar/apolar com-
pounds have been produced that in certain instances are
more active than HMBA in vitro and whose chemical
structure makes it likely that they have different phar-
macokinetics [231].
The first Phase I trials of HMBA involved continu-
ous infusion of escalating doses of HMBA for 5 or 10
days in patients with refractory solid tumors [11, 83,
213, 340]. Dose limiting toxicity, specifically thrombo-
cytopenia, was observed at 20–40 g/m2. The MTD of
continuously infused HMBA was 28 g/m2/day and aci-
dosis and CNS dysfunction were toxicities, as well as
hemorrhage related to thrombocytopenia. The plasma
half-life was 2.5 h and plasma levels of 1.42 nM or
higher could be achieved [419]. In Phase I trials nasogas-
tric or oral administration of HMBA at 30–36 g/m2/day
Kapil Mehta et al.
was also associated with thrombocytopenia and neutro-
toxicity. Attempts have been made to individualize
patient dosage based on plasma levels and clearance
rates of HMBA [64]. Plasma levels of 1.5–2.0 mM
could be sustained but toxicity was seen. The cause of
the thrombocytopenia is obscure but appears to be a
production defect rather than a peripheral destruction
or pooling of platelets. At the dosages of HMBA
required to produce differentiation, significant inhibi-
tion of cloning of normal myeloid, erythroid, and mega-
karyocyte progenitors is seen in vitro in murine bone
marrow culture. Fifty percent inhibition of cloning, and
suppression myelopoiesis in long-term bone marrow
cultures, are seen with doses of HMBA of 1.2 mM with
an unusually steep dose response. This observation
does not fully explain the observed clinical toxicity,
since the thrombocytopenia was not usually associated
with a neutropenia. The development of analogs of
HMBA with differentiation-inducing capacity and
reduced suppressive activity against myeloid progeni-
tors is a like direction in development of an effective
clinical differentiation protocol [231] that could be
applied in leukemia and MDS as well as in a variety of
solid tumor systems (colon, bladder, breast).
Chemotherapeutic Agents
as Differentiation Inducers
It is becoming apparent that of the variety of agents uti-
lized in cancer chemotherapy, some may be effective
because of their capacity to induce, selectively, tumor
cell differentiation, and this property may be more
important than cytotoxic potential. After extensive
screening (for reviews, see [169, 189, 343]) a number of
agents have been found to have differentiation capacity
[63, 65, 169, 214, 246]: mitomycin, doxorubicin, bleo-
mycin, daunomycin [169, 189, 214, 246], cytosine arabi-
noside [130, 167, 169, 189, 214, 274, 369], hypoxanthine
[63], 3-deazauridine, 5-azacytidine, methotrexate [33,
351], 5-aza-2-deoxycytodine [51, 57, 254, 314], aphidi-
colin [125], and adenine arabinoside [274].
In all the preceding cases the differentiation action
was observed at concentrations either below cytotoxic
levels or well below maximum growth inhibition in sus-
pension or clonal assays of leukemic cells. Most com-
pounds have been tested against murine (M1) and human
(HL-60) myeloid leukemic cell lines, in which differen-
tiation into macrophages and granulocytes is reported.
When tested in vitro against leukemic blast cells from
547
patients with acute myeloid leukemia, actinomycin D
was reported effective in differentiation induction in all 14
cases studied [169]. In all three cases in another study
cytosine arabinoside [246] and 5-aza-2-deoxycytidine
[314] were also effective in inducing macrophage
differentiation of leukemic blast cells. In the absence of
an in vivo measure of leukemogenicity, the ability of
these agents to eliminate the proliferative potential of the
leukemic clone must be measured indirectly. In reclon-
ing studies of primary human leukemias cultured in
methylcellulose, a decrease self-renewal potential of clo-
nogenic cells (plating efficiency 2, PE2) can be observed
with various chemotherapeutic agents, independent of
their chemosuppressive action on primary leukemic
cloning efficiency [41]. This action on PE2 is a likely
differentiation index, since more primitive leukemic
stem cells with extensive proliferative potential are
“differentiated” into a non-self-renewing compartment.
Furthermore, this PE2 parameter correlated with clinical
response [42]. The combination of low doses of the
above-mentioned chemotherapeutic agents with other
differentiation-inducing agents may prove effective
when neither type of agent can induce differentiation
directly. In this regard, differentiation-resistant clones of
M1 (D−) that failed to respond to protein differentiation
factor or chemotherapeutic agents could be ‘sensitized’
by as little as 0.25 μg/ml of actinomycin D, daunomycin,
mitomycin C, hydroxyurea, bleomycin, 5-fluorouracil,
prednisone, or dexamethasone to terminal differentiation
when combined with protein factor [149, 169].
Lotem et al. [219] report that in vitro screening for
differentiation-inducing compounds and compounds that
show toxicity to blast cells may be useful in selecting
appropriate treatments. However, their results were
ambiguous, in that with some patients studied both before
and after in vivo chemotherapy there was a similar dif-
ferentiation response, or an apparent loss, or a gain of
response in vitro of the remaining leukemic cells tested.
In further studies, using five compounds known to induce
HL-60 differentiation (DMSO, hexamethylene bisacet-
amide (HMBA), hypoxanthine, actinomycin D, and
6-thioguanine), differentiation of fresh myelogenous leu-
kemic cells was tested in vitro [192]. Of 12 patients studied,
the blast cells in most cases showed little morphological,
histological, or functional maturation after exposure to
the various compounds, as compared with the blast cells
cultured without the compound. Actinomycin D was the
only agent capable of causing significant maturation. This
study suggests that many compounds shown to differenti-
ate HL-60 may not trigger differentiation of less mature
myeloid leukemic cells.
548
Molecular Mechanism Implicated
in Leukemia Cell Differentiation
Cytosine arabinoside is one of the most effective single
agents for the treatment of myeloid leukemia and is con-
ventionally considered to act by incorporation into DNA
and by inhibiting DNA replication through production
poor primer termini. Cytosine arabinoside induces non-
specific esterase activity in HL-60 cells [107] and increases
surface expression of the monocyte surface antigen MY-4.
Aphidicolin, an analogue of deoxycytodine, also induces
HL-60 differentiation and slows DNA synthesis, but,
unlike cytosine arabinoside, it is not incorporated into
DNA and acts as an inhibitor of DNA polymerase [130].
Using a purine rather than a pyrimidine antimetabolite-
adenosinearabinoside, Monroe et al. [274] also observed
differentiation of HL-60 that correlated with slowing of
DNA synthesis by an agent known to act at the level
of the DNA polymerase template complex. The relation-
ship of differentiation to DNA synthesis is complicated
by observations that terminal differentiation of HL-60
to macrophages induced by the tumor promoter TPA can
occur in the absence of DNA synthesis [339] and that
terminal differentiation to granulocytes without cell
division is observed following treatment with actinomy-
cin D, DMSO, and butyric acid [236].
Agents can, however, induce HL-60 differentiation
without inhibiting cell proliferation [33], or inhibit pro-
liferation without inducing differentiation [351]. Thus,
it remains to be determined whether inhibition of DNA
synthesis is causally or indirectly related to differentia-
tion. Other mechanisms of action of chemotherapeutic
agents directed toward differentiation induction may
involve cell membrane effects. Anthracyclines may play
a role in alteration of leukemic cell glycoproteins, and
there is evidence that these drugs bind extensively to
membrane-lipid domains, altering membrane fluidity
and directly modifying the synthesis or expression of
glycoproteins at the cell surface [350].
A more specific action has been suggested involving
DNA methylation changes: for example, cytosine arabi-
noside may have a direct influence on methylation of
the c-myc oncogene [130], the expression of which is
considerably amplified in myeloid leukemic cells and
rapidly reduced once the cells are exposed to differenti-
ation-inducing agents [330, 404].
5-Azacytidine and the less toxic 2-deoxy derivatives are
also interesting candidates for in vivo use in leukemia
differentiation therapy. These compounds have been
shown to trigger gene expression in several systems,
including globin gene expression, when given in vivo
to thalassemic and sickle-cell anemia patients [206].
Growth and differentiation factors as cancer therapeutics
The mechanism by which 5-aza-deoxycytidine induces
leukemic cell differentiation most likely involves DNA
hypomethylating ability [254, 314]. Synthesis of hypom-
ethylated DNA takes place after incorporation of the
drug into DNA and is due partially to the chemical
structure of the compound and mainly to the trapping of
DNA methyltransferases, thus blocking their action.
The known differences regarding the molecular targets
of the two drugs could account for the greater differen-
tiation-inducing ability and lower toxicity of the 2-deoxy
derivative. It is well known that 5-azacytidine is actively
incorporated into mRNA and tRNA, thus producing its
major toxic effects.
A cautionary note should be introduced in considering
the potential therapeutic role of 5-azacytodine. Motojo
et al. [271] exposed blast cells from patients with acute
myeloid leukemia to 5-azacytidine, 6-azacytidine, and
the 2-deoxy derivative. Simple negative exponential
colony survival curves were obtained for the three drugs,
with the 5-aza-2-deoxy compound being most toxic and
the 6-aza least toxic. Although confirming other reports
of increased expression of certain antigenically defined
phenotypic markers of leukemic blast cell differentiation,
colonies surviving drug exposure to 5-aza and 5-aza-2-
deoxy compounds had increased secondary replating
efficiency. This suggests that hypomethylation of DNA
may promote leukemic cell self-renewal.
In vivo Induction of Differentiation with
low-dose Cytosine Arabinoside or
Azocytidine in Patients with acute
Myeloid Leukemia and Myelodysplastic
Syndrome (MDS)
The preceding in vitro observations provide a strong
case for the efficacy of low doses of ara-C in induction
of terminal differentiation of myeloid leukemic cell lines
and fresh leukemic blast cells, this action being either
direct or by synergy with endogenous differentiation-
inducing factors. It was thus of interest to investigate the
actions of low-dose ara-C in patients with acute myeloid
leukemia and myelodysplastic refractory anemia with
excess of blasts. Baccarani and Tura [17] reported the
first remission in a patient with RAB and subsequently
extended the study to 20 patients with myelodysplastic
syndrome (MDS), observing one complete and two par-
tial remissions. Complete or partial remissions in MDS
have been reported by other groups [167, 253, 318, 402,
409], but no response to low-dose-ara-C was observed in
a large cooperative study of refractory anemia with
excess of blasts [280]. In acute myeloid leukemia,
Kapil Mehta et al.
Housset et al. [168] reported remission in all three
patients, and Weh et al. [402] had seven complete and
two partial remissions in 12 cases. Encouraging results
have been obtained by others [253], but no response was
reported by Hagenbeek et al. [136] in four acute myel-
oid leukemia patients treated with the same protocol that
produced responses in other studies [168, 402]. The rea-
sons for variable results are unclear, but the patient pop-
ulation, particularly the MDS cases, was heterogeneous,
and the dosage and timing of drug administration in dif-
ferent studies varied between 10 and 30 mg/m2, every 12 or
24 h, for 7–28 days. Indeed, the more encouraging results
obtained by Housset et al. [168], compared with those
of Baccarani et al. [16], may be due to the more frequent
and longer duration of ara-C administration in the former
study, thereby probably achieving a rather constant in
vivo concentration of the drug.
In one series of 21 patient (five with refractory anemias
with an excess of blasts and 16 with acute leukemias)
treated with small doses of ara-C (10 mg/m2/12 h for
15–21 days), improvement was noted in 15 cases (71%),
and complete remission was observed in 12 (57%) [47].
Complete remission was obtained after one course of
treatment in eight cases. The fact that these patients
entered remission relatively slowly and did not suffer
marrow aplasia suggests that low-dose ara-C was func-
tioning by inducing differentiation. Generally, when clin-
ical response has been obtained, the evidence points to a
differentiating role for the drug rather than an antitumor
effect, with progressive evolution of recovery, absence of
aplasia, and the simultaneous presence of normal islets of
promyelocytes and leukemic myeloblasts [168].
The availability of the in vitro assay for detection of
leukemic cell responses to differentiation agents should
mandate prescreening of patients for in vitro sensitivity
before enrollment in a differentiation protocol such as
low-dose ara-C. Toward this goal, leukemic marrow
cultures were exposed to low doses of ara-C, and a
reduction in blast cells and an increase in more mature
granulocyte and macrophage elements were noted in
two or three patients. Subsequent low-dose ara-C treat-
ment in vivo resulted in complete remission in the two
patients that showed a strong in vitro response [246].
In a large study of low-dose (10 mg/m2) ara-C in
poor-risk ANLL, overall survival was comparable to
that observed following conventional therapy with high-
dose (200 mg/m2) ara-C and anthrocyclin [71]. While the
high-dose regimen produced more complete remissions
(55%) than the low dose (33%), there were more early
deaths in the intensive therapy group. Very-low-dose
(3 mg/m2) ara-C produced hematologic improvement in
the majority of patients in a study trial of 73 MDS
549
patients [71]. In other recent studies of low-dose ara-C
in 73 patients with ANLL and MDS [10] and 40 patients
with ANLL and MDS, complete remission rates of 24–31%
were reported with 35–45% responders. In these studies
evidence for leukemic cell differentiation was obtained.
Cytogenetic and morphologic studies suggested that
cytotoxicity rather than differentiation was responsible
for remissions observed in two other studies with low-dose
ara-C in poor-risk ANLL [19, 49].
Low-dose 5-azacytodine has also proved effective in
MDS, and in one study of 44 patients, 48% responded
with 11% complete and 25% partial remissions [357].
The median duration of remission was 53 weeks. 5-Aza-
2′-deoxycytidine in 134 patients with AML, CML in
blastic crisis, and MDS produced two complete and four
partial remissions. The drug appeared to modify the leu-
kemic phenotype in vivo as well as producing a direct
cytolytic effect [423].
Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carbox-
amide) is an inhibitor of inosine5-monophosphate (IMP)
dehydrogenase and is the enzyme in the rate-limiting
synthesis of guanylate [401]. Tiazofurin has both cyto-
toxic and cytodifferentiation activities and has been
used to induce responses in patients with blast crisis in
CML [381]. Toxicity has been severe with nausea, rash,
myalgia and serositis. Given the high toxicity, further
clinical trials to this approach must be conducted to
determine optimal dose and schedule. Combinations of
tiazofurin with other differentiations may be of interest
to investigate in the near future.
Cytokines and Hematopoietic
Growth Factors active in Regulating
Proliferation and Differentiation
of Leukemic Hematopoietic Cells
Recognition that physiologic inducers of differentiation of
normal hematopoietic cells could also influence prolifera-
tion and differentiation of leukemic cell lines led to a series
of studies over the last 2 decades involving characterization
of hematopoietic growth factors, in many cases using leu-
kemic cell lines as sources of growth factor and/or as target
for growth factors in various bioassays. As discussed in
other chapters, the genes for these factors have been cloned
and recombinant factors have been tested in vitro and in
vivo for biological activity. This large family of cytokines
and polypeptides includes factors with pleotropic and over-
lapping activities, and additive or synergistic interactions
are frequently observed [139, 209, 321, 426].
550
CSF-Dependence of Myeloid Leukemic
Progenitors
The cloning of normal or leukemic human CFU-GM in
either agar or methylcellulose has permitted analysis of
both quantitative and qualitative changes in this cell
compartment in leukemia and other myeloproliferative
disorders. Changes observed include abnormalities in
the maturation of leukemic cells in vitro, defective pro-
liferation as measured by colon size or cluster-to-colony
ratio, abnormalities in biophysical characteristics of leu-
kemic GM-CFC, the existence of cytogenetic abnor-
malities in vitro, and regulatory defects in responsiveness
to positive and negative feedback control mechanisms
(for reviews, see refs. [255–258, 261, 263–265]).
Detection of this spectrum of abnormalities has proved
to be of clinical use in diagnosis of leukemia and preleu-
kemic states, in classification of leukemias, and in pre-
dicting remission in acute myeloid leukemia. Variation
has been reported among different groups investigating
the characteristics of human acute myeloid leukemia
cells in culture. These differences reflect, in part, the
heterogeneity of the disease as well as variation in
the culture criteria, the source and activity of CSF, and the
timing of the culture. The preceding studies indicated
that leukemic cells from patients with acute or chronic
myeloid leukemia or preleukemic states were absolutely
dependent upon a source of stimulatory factors for their
clonal proliferation in culture at low plating densities.
Analysis of the dose response of myeloid leukemia
CFU-GM indicates that in the majority of cases they do
not differ markedly from normal in their responsiveness
to various sources of CSF, although occasionally, hyper-
responsiveness of leukemic cells is seen [255, 257, 258,
261, 263, 265].
In an analysis of growth-factor responsiveness in sus-
pension cultures of leukemic marrow from 25 patients,
Lowenberg et al. [220] showed that 17/25 cases exhib-
ited spontaneous “autocrine” proliferation, [362] and
this was enhanced in 21 cases by IL-3, in 17 cases by
GM-CSF of G-CSF, and in five cases by M-CSF. In four
cases the cells responded to all factors and the remain-
der responded to three, two, or only one source of stimu-
lus. As with normal progenitor assays, “spontaneous”
leukemic cloning is observed as the marrow cell or
peripheral blood leukocyte plating density increases,
owing to endogenous production of CSFs by accessory
populations, which may be residual normal cells of
T-cell lineage or leukemic cell subpopulations. The
autocrine concept of malignant transformation proposes
that cells become malignant by the endogenous produc-
tion of polypeptide growth factors that act upon their
Growth and differentiation factors as cancer therapeutics
producer cells through functional external receptors.
Support for the concept has been obtained by studies on
oncogene action, since oncogenes may confer growth-
factor autonomy on cancer cells by coding directly for
autocrine polypeptide growth factors or their receptors,
or by amplifying the mitogenic signals generated as a
consequence of growth factor-receptor interaction.
Strong evidence for an autocrine role for GM-CSF
has been provided in a study of 22 cases of primary
human acute myeloid leukemia in which Northern blot
analysis revealed expression of the GM-CSF gene in 11
cases [420]. GM-CSF expression was not found in nor-
mal hematopoietic tissue, with the exception of acti-
vated T-cells, strongly indicating that the GM-CSF gene
activation was intimately associated with the transfor-
mation event. Furthermore, in six cases, GM-CSF was
secreted by leukemic cells of both early and late stages
of differentiation, and activity was specifically neutral-
ized by antiserum to GM-CSF [129].
The paradox of CSF dependence of primary human
myeloid leukemias in clonal assay, and the autocrine
production of GM-CSF, can be answered in part by the
concentration at which the cells are plated: at high con-
centrations, ‘spontaneous’ leukemic colony or cluster
formation is the norm. In the study of Young et al. [420],
9 of 22 cases showed autonomous growth of leukemic
clusters when cells were plated at 5 × 104/ml, yet exog-
enous GM-CSF increased cloning number and clone
size in 15 of 22 cases. It is possible that in some cases
GM-CSF is not actually secreted by the leukemic cells,
but is present in active form as a membrane-bound moi-
ety. In this regard, Nara and McCulloch [281] showed
that purified cell membranes from cells of some acute
myeloid leukemia patients, but not normal bone marrow
of ALL cells, could promote proliferation of other acute
myeloid leukemic cells in short-term suspension cul-
ture, enhancing self-renewal.
A direct link between constitutive GM-CSF expres-
sion and leukemic transformation was provided by Lang
et al. [202], who transfected the murine GM-CSF gene
into CSF-dependent, nonleukemic, myeloid cell lines
and produced leukemic cell lines that constitutively
secreted GM-CSF. To date, no example of human myel-
oid leukemia constitutively producing and responding
to IL-3 has been reported, but in the mouse system, the
well-characterized spontaneous murine myelomono-
cytic leukemia WEHI-3 constitutively secretes IL-3,
apparently because of insertion of retroviral sequences
adjacent to the IL-3 gene [416].
Retroviral insertion of the IL-3 gene with a viral long
terminal repeat promoter produces leukemic transfor-
mation [140]. The fact that insertion of G-CSF, GM-CSF,
Kapil Mehta et al.
or IL-3 genes into normal hematopoietic stem cells or in
transgenic mice did not lead to leukemia [240] suggests
that autocrine induction of factor-independence is nec-
essary, but not sufficient, for converting normal cells to
a transformed state. Genetic alterations may predispose
them to undergo leukemic transformation by an auto-
crine mechanism. In this context the murine factor-
dependent cell line FDC-P1 develops into fully leukemic
cells when transplanted into whole-body-irradiated mice
associated with development of autocrine GM-CSF or
IL-3 production [80, 81].
Expression of mRNA for GM-CSF, G-CSF and
M-CSF is ubiquitous in the blast cells of AML but not
CML [55, 285, 397, 420, 421]. In a minority of these
cases significant quantities of bioactive CSF were pro-
duced, sufficient to stimulate autocrine blast cell prolif-
eration [329, 420]. The significance of these observations
was questioned by studies showing that enhanced expres-
sion of the GM-CSF gene in ABL blast cells was a con-
sequence of manipulations used to enrich blast cell
populations [180, 367].
Constitutive expression of IL-1 on mRNA and bioac-
tive protein production is a characteristic of the majority
of AMLs and is not a consequence of manipulation of
the cells. IN 10/17 cases of AML, IL-1β mRNA was
identified [131], and this autocrine IL-1 can induce
AML proliferation [247]. The proliferative effect of
IL-1 on leukemic blast cells, like its action on normal
cells, is mediated in synergy with growth factors such as
IL-3, GM-CSF, and G-CSF [161, 162]. Since IL-1
induces G-CSF, GM-CSF, and M-CSF production by
bone marrow stromal cells (endothelium, fibroblasts,
macrophages; see Moore [260] for review), and these
factors stimulate leukemic cell proliferation, a paracrine
mode of stimulation of leukemic cell proliferation has
been proposed for AML [131], and juvenile CML [18].
An IL-1-mediated autocrine growth stimulation was
proposed in a study of 13 cases of AML [67]. In all
cases, immunofluorescence showed that up to 80% of
all fresh leukemic blast cells in all patients contained
either the 33-kDa IL-1β propeptide or both the 33- and
17-dDa mature form. The bioactive IL-1α propeptide
was also detected in all cases but was less frequently
released. In six cases studied, anti-IL-1β and to a lesser
extent anti-IL-1α inhibited spontaneous proliferation,
and in 10/12 cases sufficient exogenous IL-1 was pro-
duced to stimulate significant proliferation. AML cells
constitutively released as much IL-1 as did endotoxin-
stimulated normal monocytes, and 2/12 patients that did
not respond to exogenous IL-2 were both high endoge-
nous producers and presumably were maximally stimu-
lated. In most instances, exogenous IL-1 supported the
551
establishment of continuous lines of AML cells that
grew for >2 months [67].
A more complex autocrine loop is suggested by the
studies of Bradbury et al. [36]. AML cells at low cell density
were stimulated independently by exogenous GM-CSF
or IL-1, and the response to both factors was inhibited by
antibody to GM-CSF. Antibody to IL-1 inhibited sponta-
neous proliferation of these cells, and endogenous
GM-CSF could be detected. Thus, IL-1 is produced by
the leukemic cells, particularly from the more differenti-
ated cells, and this in turn induces GM-CSF production,
which directly mediates autocrine proliferation. Autocrine
IL-1 induction of GM-CSF may account for the appear-
ance of GM-CSF mRNA in cultured rather than fresh
AML blasts [180]. Therapeutic strategy in the face of
IL-1, GM-CSF-, or IL-3-dependent leukemogenesis,
either autocrine or paracrine, would require intervention
to block CSF or IL-1 production or action. High-affinity
antibodies to the factor or its receptor, in the latter case
coupled to some form of toxin, may be one possible strat-
egy which is under active consideration as a therapy in
the case of IL-2-dependent T-cell lymphoma (anti-IL-2
receptor antibody and IL-2 toxin conjugates). A second
strategy envisages the use of GM-CSF to recruit resting
leukemic stem cells into cell cycle and synchronize the
population in S phase in conjunction with cycle-specific
chemotherapy [87, 217, 322].
Colony-Stimulating Factors as Leukemia
Differentiating Agents
The G-CSF and GM-CSF induce granulocytic differen-
tiation of HL-60 and WEHI-3B leukemic cell lines
[24, 25, 108, 315], but leukemic blast-cell proliferation
is the more general response in primary cultures of AML
bone marrow. GM-CSF is a universal proliferative stim-
ulus for AML cells, generally without differentiation,
whereas G-CSF stimulates proliferation and in a
proportion of cases granulocytic differentiation [73].
This area is reviewed elsewhere in this volume and the
actions of G-CSF and GM-CSF in clinical trials in
myelodysplastic syndrome and AML are discussed.
M-CSF is also a proliferative stimulus for some AML
cells, but in many instances it induces differentiation to
adherent macrophages [397]. Most leukemic blast pop-
ulations express the M-CSF receptor, fms, and M-CSF
mRNA is found in about half the blast population.
Marked heterogeneity of M-CSF response is seen with
different patients, ranging from cases where blast popu-
lations were unresponsive to M-CSF, to examples where
blast-cell self-renewal was stimulated, to instances
where the major effect of M-CSF was the generation of
552
terminally differentiated cells with monocyte-mac-
rophage characteristics. This heterogeneity in prolifera-
tive versus differentiation responses of leukemic blasts
to various CSF species in different patients indicates the
necessity of prescreening for factor response on an indi-
vidual patient basis.
Interleukin 1 and Cell Proliferation and
Differentiation
Interleukin 1 has been implicated in an autocrine or para-
crine mode of stimulation of myeloid leukemic cells in
synergy with GM-CSF [36, 67, 131, 161, 162, 247].
It has also been reported to inhibit proliferation of a human
myelomonocytic leukemic cell in the presence of low to
intermediate, but not high, levels of GM-CSF [346].
The IL-1 inhibition of tritiated thymidine incorpora-
tion into murine M1 cells was first reported by Onozaki
et al. [295]. On its own, IL-1 did not induce differentia-
tion, but both growth inhibition and macrophage differ-
entiation were induced synergistically by IL-1 and LPS
coadministration. In further studies these investigators
reported that while IL-1α, TNFα, and IFNβ1 all had
antiproliferative, but not differentiation-inducing, action
on M1 cells, as little as 1 unit of IL-1, in conjunction
with TNF or IFNβ1, induced FcR, phagocytic activity,
and morphological charge [297]. The differentiation
induced by IL-1 plus TNF was inhibited by antibodies
to IFNβ1, as was the antiproliferative effect of TNF (but
not IL-1), indicating autocrine IFNβ1 is induced and
mediates direct differentiation and antiproliferative
effects. Thus IFNβ1 is one of two signals required for
M1 differentiation, the other being IL-1, and the anti-
proliferative effect of IL-1 appeared to be a direct one.
The differentiation-inducing factor for M1 cells in LPS-
stimulated peritoneal macrophage conditioned medium
was identified as TNF, synergistically active with IL-1
[372]. Interleukin 1 induction of another cytokine,
MG1-2 or IL-6, has also been implicated in IL-1 induc-
tion of M1 differentiation. Lotem and Sachs [215, 216],
in contrast to the results of Onozaki et al. [297], showed
that IL-1 on its own induced M1 differentiation as mea-
sured by FcR and C3R induction, lysozyme secretion,
and morphology. In vitro, and in vivo in diffusion cham-
bers implanted in mice, IL-1 induced granulocytic dif-
ferentiation of M1 cells. IL-1 also acted synergistically
with GM-CSF to induce differentiation of a GM-CSF-
responsive, IL-1-unresponsive clone of leukemic cells.
The in vivo action of IL-1 was associated with rapid
induction of elevated serum levels of MGI-2/IL-6, but
serum levels were not sufficiently elevated to account
for the differentiation observed within the diffusion
Growth and differentiation factors as cancer therapeutics
chamber. A more likely mechanism implicates IL-1
induction of autocrine production of IL-6 by leukemic
cells, and this was observed within 6 h in IL-1-treated
M1 cells, with production increasing by 2–3 days [215,
216]. The indirect differentiation induction mechanism
was blocked using antibodies to IL-6. As with the auto-
crine IFNβ route for differentiation, the endogenous
IL-6 may require a synergistic interaction with IL-1 to
produce optimal differentiation. These results point to
the importance of considering combinations of cytokines,
both exogenous and endogenous, in designing optimal
differentiation strategies.
The ability of IL-1 to induce certain leukemic cells to
differentiate in association with growth inhibition
involves synergistic interactions with other cytokines,
some of which are produced by leukemic cells in response
to IL-1. In normal hematopoiesis, IL-1 also induces a
spectrum of hematopoietic growth factors (G-CSF,
GM-CSF, M-CSF) by an action on endothelial cells,
fibroblasts, and macrophages and acts synergistically
with these and other (e.g., IL-3) hematopoietic growth
factors to stimulate proliferation of early hematopoietic
stem cells [65, 79, 262, 266]. In vivo IL-1 alone or in
combination with G-CSF was particularly effective in
accelerating myeloid regeneration following high-dose
chemotherapy or radiation [259, 262].
Interleukin-6 and Leukemic Cell
Proliferation and Differentiation
Interleukin 6 promotes the terminal maturation of B
cells to antibody-producing cells, augmenting IgM, IgG,
and IgA production in stimulated B cells [184]. It was
originally identified as novel fibroblast-type interferon
(IFNβ2) and was found to be identical to a B-cell dif-
ferentiation factor, BSF2 [158], a hybridoma/plasmacy-
toma growth factor [390], and a macrophage-granulocyte
inducer of leukemic cell differentiation (MGI-2) [354].
Its action on normal hematopoiesis includes stimulation
of multilineage blast cell colonies in synergy with IL-6
[172]. It may act to induce the entry of Go stem cells
into cell cycle [197]. Interleukin 6 potentiates thrombo-
cytosis [155, 174]. Chronic stimulation of B cells was
achieved in transgenic mice carrying the human IL-6
gene conjugated with the Ig enhancer [184]. These mice
developed polyclonal nontransplantable plasmocy-
tomas. Freshly isolated human myeloma cells also pro-
duce and respond to IL-6, indicating an autocrine role of
this molecule in the proliferation, but not differentia-
tion, of certain malignant B cells [13, 424]. Dependence
of myelomas on IL-6 may also involve a paracrine
mechanism involving marrow stromal cell production
Kapil Mehta et al.
of IL-6 in response to myeloma cells [187]. This could
involve malignant B-cell production of IL-1, which is a
potent inducer of IL-6 gene transcription and translation
[260]. In contrast to its action on normal B cells, IL-6
does not augment Ig secretion in myeloma cells [373].
The IL-6-induced proliferation and autocrine IL-6
action have also been reported for certain human B-cell
lymphomas, and elevated serum levels of IL-6 were
found in 50% of patients with active lymphoma [413].
Interleukin 6 was found to be identical to a mac-
rophage-granulocyte inducer (MGI-2A) that caused dif-
ferentiation of myeloid leukemic cells but lacked the
ability to stimulate colony formation [218, 354]. It was
also identical to a differentiation factor (DIF or D
Factor) produced by Krebs ascites tumor cells, although
it should be noted that this source contains a second
D-factor, which is leukemia inhibitory factor (LIF, see
following section). IL-6 produces complete growth
arrest of the murine myeloid leukemia M1 within 48 h,
and this is associated with morphologic maturation of
the blast cells to macrophages with upregulation of
cfms, FcR, C3R, lysozyme secretion, and development
of phagocytic capacity [241, 251, 252]. In clonogenic
assay of M1 cells diffuse differentiated colonies develop
within 48 h of addition of IL-6 and the number of colo-
nies is reduced [241]. The murine myelomonocytic leu-
kemic WEHI-3 (D+ variant but not D−) was also induced
by IL-6 to macrophage differentiation with up-regula-
tion of FcR and cfms [50, 240]. Interleukin 6 did not
reduce the W3 leukemic clonogenic capacity.
Synergistic or additive interactions between IL-6 and
other cytokines influence both proliferation and differ-
entiation. The IL-6 and LIF or G-CSF have additive or
supraadditive effects on M1 differentiation [240, 241,
251, 252]. The IL-1 and IL-6 were additive or synergis-
tic in growth inhibition of U937, HL-60, and M1 [296,
316]. Interleukin 1 alone induced lysozyme production
by M1 cells but had no direct effect on the expression of
FcR or on morphology. Interleukin 6 independently
induced FcR and lysozyme, but the combination trig-
gered the entire sequence of differentiation markers
[316]. The M-CSF also synergized with IL-6 to enhance
differentiation of M1 cells but it counteracted the growth
inhibition, resulting in increased size and number of
macrophage colonies [240, 241]. The possibility existed
that these interacting cytokines were in fact members of
a cascade involving autocrine production of factors by
leukemic cells. Interleukin 6, IL-1, LIF, and G-CSF are
all differentiation-inducing factors, they are all endo-
toxin-inducible macrophage products, and they can be
produced by myelomonocytic leukemic cells. The M1
cells produce IL-6, and levels are increased following
553
LIF treatment [240, 241]; however, antibody to IL-6
does not block LIF induction of M1 cells. In U937 cells,
IL-6 did not induce IL-1, nor did IL-1 induce IL-6, and
both factors appear to provide distinct signals for differ-
entiation of this neoplastic macrophage cell line [296].
The action of IL-6 on human acute myeloid leukemic
blast cells is observed only when it is combined with
GM-CSF or IL-3. A heterogeneous response is seen
involving synergistic blast cell proliferation [131, 161,
162]. Like IL-1, IL-6 is secreted by most leukemic cells
where there is evidence of monocytic differentiation
(12/15 patients with ANLL: of FAB type M4 or M5)
[389]. The significance of this potential autocrine look
is uncertain.
Leukemia Inhibitory Factor
A spectrum of factors have been described that induce
differentiation of the murine M1 myeloblastic leukemic
line. This includes G-CSF, IL-1, IL-6, and TNF as well
as a unique factor termed leukemia inhibitory factor
(LIF) on the basis of its potent and selective inhibition
of M1 cell proliferation in association with induction of
macrophage differentiation [127, 118]. This factor had
earlier been characterized in the conditioned media of
L929 cells and termed D-factor, inducing morphologi-
cal maturation in M1 cells with the development of
locomotor activity, phagocytic capacity, and upregula-
tion of FcR, C3R, and prostaglandin E [381]. The factor
was purified from Krebs II ascites conditioned medium
as a 58-kDa glycoprotein [118] and from mouse Ehrlich
ascites cells as a 40-kDa activity [221]. The native mol-
ecule of 179 amino acids [118] or 180 amino acids [221]
has a molecular weight of 20 kDa with seven N-linked
glycosylation sites. The human gene is located on human
chromosome 22q11–q 12.2 [366]. The murine and
human molecules are active across species and are 78%
homologous at the amino acid level.
LIF was found to be identical to a variety of other
cytokines that had been reported to possess diverse
functions. Alloreactive human T cell clones obtained
from rejected kidney, when stimulated with a specific
antigen and IL-2, produced a factor that triggered the
proliferation of a subline of the IL-3-sensitive murine
factor-dependent cell line DA-1 [268, 269]. This factor,
called HILDA (human interleukin DA), was a 38- to
41-kDa glycoprotein that was also reported to have
eosinophil chemotactic and activating properties and
erythroid burst-promoting activity [122]. In retrospect
these latter activities were probably due to a minor con-
tamination of the protein with GM-CSF and/or other
lymphokines.
554
HILDA cDNA cloned from human lectin-stimulated
T cells was shown to be identical to LIF [269]. A human
macrophage differentiation-inducing factor (DIF) for
M1 cells isolated from a human monocytic leukemia
line, THP-1, was also shown by amino acid sequencing
to be identical to LIF [3]. The growth of totipotential
embryonic stem cells in vitro is sustained by a differen-
tiation inhibitory activity (DIA) produced by a number
of sources, including the 5,637 human bladder carci-
noma cell line. Purified DIA and LIF were very similar
in biochemical features, and purified recombinant LIF
can substitute for DIA in the maintenance of totipotent
embryonic stem cells that retain the potential to form
chimeric mice [406]. A factor constitutively produced
by human squamous carcinoma cells stimulated hepato-
cytes to produce the same spectrum of acute phase pro-
teins as IL-6 [23]. This hepatocyte stimulating factor III
was distinct from IL-6 but was identical to LIF. Rat
heart tissue produces a cholinergic differentiation factor
that controls the phenotypic choice in neurons without
affecting their survival or growth [411]. This factor
was also identical to LIF and acted on postmitotic rat
sympathetic neurons to specifically induce expression
of acetylcholine synthesis and cholinergic function and
suppress adrenergic function.
Receptor analysis with 125LIF indicated that M1 cells
possess a single class of high-affinity (Kd 100–200 pM)
receptors of relatively low frequency (300–500 per cell)
[156]. In bone marrow, spleen, and peritoneum, mono-
cytes, macrophages, and their precursors were the most
obvious cells binding LIF.
The in vivo action of LIF was revealed by studies in
which mice were engrafted with cells of the murine
hematopoietic cell line FDC-P1 multiply infected with a
retroviral construct containing cDNA encoding LIF
[242, 243]. The mice developed within 12–70 days a
fatal syndrome characterized by cachexia, excess osteo-
blasts with new bone formation, calcification in heart
and skeletal muscle, pancreatitis, thymus atrophy, and
abnormalities in the adrenal cortex and ovarian corpora
lutea. The development of this osteosclerotic syndrome,
with marked increases in osteoblasts, indicates a role for
LIF in osteoblast production and function and in regulation
of bone formation and calcium metabolism. The presence
of LIF receptors on osteoblasts further indicates the direct
nature of the stimulation [242, 243].
The action of LIF on leukemic cells appears to be
highly restricted, inducing M1 cell differentiation
alone, or synergistically with G-CSF, IL-6, or M-CSF
[244]. The growth inhibition and differentiation effect
was rapid, being evident at 24 h and marked at 48 h.
LIF was without effect on normal CFU-GM or WEHI-3
Growth and differentiation factors as cancer therapeutics
B myelomonocytic leukemic cells. Abe et al. [3] reported
a direct differentiation-inducing, and proliferation-
inhibiting, action on HL-60 and U937 cells. Maekawa
and Metcalf [225] did not observe a direct action of LIF
on HL-60 or U937 cells but reported clonal suppres-
sion of these leukemic cells by LIF in combination with
GM-CSF or C-CSF. Sequential recloning of these cells
was also suppressed by LIF-CSF combinations.
Despite the relatively weak action of LIF on human
leukemic cells, LIF clearly is able to exhibit suppressive
actions. This, coupled with its lack of suppressive effects
on normal hematopoietic cells, suggests a role in sup-
pressing human leukemias, possibly in combination
with other hematopoietic factors.
Tumor Necrosis Factor (TNFa)
and Lymphotoxin (TNFb)
Tumor necrosis factor was identified as a protein with
antitumor activity in serum of mice infected with Bacillus
Calmette-Guerin and treated with endotoxin [335].
TNFα has significant sequence homology to human
lymphotoxin β and binds to the same receptor. The bio-
logical actions of TNF are multifaceted, involving both
stimulatory and inhibitory actions depending upon the
target cell population.
In normal human hematopoiesis TNFα was reported
to inhibit CFU-GM day 7 (ED50 = 10 U) and CFU-GM
on day 14, BFU-E, CFU-E, and CFU-GEMM (ED50 =
50 U) [39, 223, 306]. Even greater inhibition of BFU-E,
CFU-GEMM and CFU-E was reported with both TNF
alpha or β in other studies [276]. TNF inhibition was
enhanced in a synergistic manner by interferon γ [39,
276, 306, 307]. TNF inhibition was reported to be nonre-
versible within 60 min, suggesting a cytotoxic mecha-
nism [39], but others have reported either full reversibility
following a 24 h preincubation [276] or partial revers-
ibility [279]. The variable results can be attributed to the
use of different sources of CSF and the variable presence
of accessory cells in the preparation. With G-CSF as a
stimulus, the ED50 with TNFα or -β was 10 U whereas
with a GM-CSF stimulus only a 20% inhibition was seen
with 1,000 U TNF [20]. Thus in normal marrow one cell
in 500 will form a colony with G-CSF or GM-CSF, but
with TNF only one cell in 100,000 will respond to
G-CSF, yet the frequency of cells responding to GM-CSF
remains essentially unchanged [21, 194]. With highly
purified marrow progenitors, the ED50 for TNF was 5 U
with G-CSF-stimulated colonies, and 500 U with
GM-CSF stimulation [267]. Using CD34-positive selec-
tion for CFU-GM, G-CSF-stimulated colonies were
strongly inhibited by TNF, but an actual enhancement of
Kapil Mehta et al.
GM-CSF or IL-3-stimulated colonies was recently
reported [345]. In murine serum-free bone marrow cul-
tures the ED50 for CFU-GM was 20–200 U or murine
TNF and for BFU-E and CFU-GEMM it was 2,000 U
[85]. Using human TNF, a lower degree of inhibition is
generally found. In most studies, human TNF does not
inhibit at doses below 10,000 U/ml, and indeed some
slight potentiation of M-CSF stimulated colonies was
reported [407].
In long-term murine or human bone marrow culture,
TNF exhibits a dose-dependent (10–1,000 U/ml) inhibi-
tion of CFU-GM production and neutrophil generation
[85, 267]. The inhibition induced by TNF appears to be
selective for neutrophil differentiation and is not manifest
in reduction of eosinophils or monocyte/macrophages.
The interaction of TNF with leukemic cells is a com-
plex one, variously resulting in differentiation, and
growth inhibition or growth potentiation. A differentiat-
ing factor for HL-60 and ML-1 leukemic cells, produced
by PHA-stimulated lymphocytes or the HUT 102 T cell
line was shown to be TNF [370]. As little as 1, 10 U of
TNF alone or in synergy with IFNγ promoted mono-
cytic differentiation of HL-60, U937 and ML-1 [135,
194, 306, 346].
The action of TNF on fresh AML marrow cells has
been investigated in clonogenic (PE1) and recloning (PE2)
assays, and suspension culture. Early reports uniformly
reported inhibition. In one study of ten patients 50% inhi-
bition was seen with 15 pM TNF and there was synergy
with IFNγ [306]. Inhibition to a degree comparable to
normal was reported in seven patients [39], and in nine of
ten patients 75% inhibition of leukemic cell cloning was
seen with 100 U TNF compared to 44–48% inhibition in
remission marrow. Enhanced differentiation to monocyte
macrophages with enhanced NBT-reducing activity was
seen with TNF or TNF plus IFNγ in suspension cultures
of marrow from 1 AML and 5 CML in blastic crisis [119].
Differentiated macrophages were observed with Auer
rods, indicating their leukemic origin.
In other, more recent studies, a more complex picture
emerges. Clonogenic assays stimulated with G-CSF or
GM-CSF and exposed to high (1,000 U) or low (100 U)
doses of TNFβ showed four types of response in AML
and preleukemic myelodysplastic syndrome: inhibition
greater than normal or remission inhibition equivalent
to normal, no response, and significant enhancement.
This latter pattern, seen in 4/13 cases, involved a syner-
gism with either G-CSF or GM-CSF and clonogeneic
capacity increased up to 60-fold. Assessment of plating
efficiency (PE1, PE2) using 5637 CM as a source of
stimulus revealed three patterns of response with TNF
[279]; (a) PE1, PE2, and suspension cell were inhibited
555
90–100% with 100–1,000 U TNF; (b) PE1 and PE2 were
not inhibited and possibly potentiated at low doses of
TNF but suspension cells were markedly inhibited; and
(c) marked inhibition of PE1 and PE2 but not of suspen-
sion cells. In no case was there evidence of TNF induc-
tion of differentiation. The nature of the stimulus may
determine whether the TNF response is inhibitory or
stimulatory. Proliferation of AML blast cells (ten
patients), induced by G-CSF, was further stimulated by
TNF, and clonogenic assays were potentiated 50–250%
of maximum [163]. In contrast, IL-3- or G-CSF-
stimulated cultures were inhibited 100% and 5,637
CM-stimulated cultures by 30% with 600 pM of TNF.
Autocrine stimulation of AML cell proliferation seen
at high cell densities was also enhanced by TNF, sug-
gesting synergism with leukemia cell-derived GM-CSF.
The mechanism of TNF potentiation is probably indi-
rect and was inhibited by antibodies to IL-1. Furthermore,
IL-1 synergises with GM-CSF in stimulating leukemic
blast cells [162], and TNF synergism was not seen in
cultures in the presence of exogenous IL-1. IL-1 and
TNF have been shown to be constitutive products of
AML cells and production of bioactive IL-1 can be
increased by TNF treatment [131, 163, 398]. The prolif-
erative response of leukemic blast progenitors to TNF
under conditions that favor autocrine stimulation may
represent one property that allows leukemic cells to
escape from negative regulation.
In the chronic phase of CML, TNF substantially
inhibits DNA synthesis in cultures of purified progeni-
tors and inhibits day 7 and day 14 CFU-GM at doses of
10–500 U [82, 194]. Some patient-to-patient variation is
seen with inhibition in the normal to less-than-normal
range. Antibodies to TNFα blocked the TNF inhibition
and enhanced GM-CSF-stimulated colony formation
twofold in cultures of accessory cell depleted progeni-
tors [82]. Autocrine production of TNF by immature
CML cells was confirmed by Northern analysis and
ELISA assays. The quantities of TNF produced by leu-
kemic cells were sufficient to inhibit normal hematopoi-
esis and may provide a selective advantage for leukemic
clones if CML progenitors are less sensitive to TNF
inhibition than normal. In this context TNF production
by hairy-cell leukemic cells has been identified as the
possible cause of myelosuppression and neutropenia
seen in this cancer [211].
Transforming Growth Factor b
Transforming growth factor β (TGFβ) is a member of a
group of polypeptide growth factors that regulate cell
growth and differentiation. TGFβ exists as a 25-dK
556
disulfide-linked dimer, and subtypes include TGFβ1,
TGFβ2, TGFβ1, 2, existing in homodimer and heterodi-
mer forms, and TGFβ3. The factor is produced by most
normal cells and its pleiotropic actions include growth
stimulation of fibroblasts and growth inhibition of epi-
thelial cells, endothelial cells, and various malignant
cells. It also affects differentiation of cells as varied as
adipocytes, myoblasts, chondrocytes, and epithelium
and is involved in production of extracellular matrix,
bone remodeling, and repair. TGFβ acts as a modifier of
the immune response and may play an important role in
hematopoiesis. It is a potent monocyte chemoattractant
and induces these cells to produce IL-1 and TNF [394].
In vitro, TGFβ1 and -β2 (2–4 pM) inhibit megakaryo-
cytopoiesis and CFU-MK [40, 173]. In both murine and
human systems, pluripotential progenitors (high prolif-
erative potential CFU, CFU-GEMM) and BFU-E are
strongly inhibited [7, 137, 182, 299, 340, 358].
Erythropoietin-stimulated CFU-E are not inhibited
[181, 182, 210, 341]. The action of TGFβ on in vitro
myelopoiesis is determined by the stage of differentia-
tion of the progenitor the type of CSF used, and the spe-
cies. In the murine and human systems, G-CSF and
M-CSF stimulated colonies are not inhibited [137, 181,
182, 358]. The GM-CSF-stimulated murine colonies are
variously reported to be resistant to TGFβ [181, 182] or
inhibited, but to a lesser extent than earlier progenitors,
at doses of 0.2–0.5 ng/ml [75, 137, 157, 288, 341].
Human day 7 CFU-GM (predominantly G-CSF respon-
sive granulocyte-committed progenitors) were enhanced
150–175% by TGFβ1 or -β2 even in the presence of
plateau concentrations of G-CSF, GM-CSF, or IL-3 [56,
299]. Day 14 CFU-GM were variously reported to be
unaffected by TGFβ [7, 157], or inhibited to a lesser
degree than earlier progenitors [182, 358]. Synergistic
or additive interactions occur between TGFβ and other
cytokines. BFU-E were synergistically inhibited by
TGFβ plus TNFα, and these cytokines additively inhib-
ited CFU-GEMM and CFU-GM [359]. Interferon
gamma and TGFβ synergistically inhibited CFU-GM
and additively inhibited BFU-E and CFU-GEMM.
In long-term bone marrow culture, TGFβ serves as a
potent inhibitor of myelopoiesis, probably by inhibiting
proliferation of early stem cells in the adherent stromal
layer [44, 150]. Indeed, TGFβ production by marrow
stromal cells may be an important negative regulator of
steady-state hematopoiesis. Further evidence for a phys-
iological role for TGFβ as negative regulator was provided
in studies in which TGFβ1 (1–5 μg/mouse) was injected
via the femoral artery into normal mice [123, 124]. The
CFU-GEMM stimulated by IL-3 were completely inhib-
ited in the femur, and CFU-GM were inhibited 50% by
Growth and differentiation factors as cancer therapeutics
24 h with reversal of inhibition at later times. This obser-
vation suggests that TGFβ, by reversibly inhibiting the
cycling of early stem cells, may be effective in protecting
such cells from damage inflicted by cell cycle-specific
chemotherapy.
The action of TGFβ on myeloid leukemic cells gener-
ally involves potent growth inhibition; however, an
influence on differentiation is seen in some systems.
TGFβ alone induced monocyte-macrophage differentia-
tion of the U937 and THP-1 cell lines but was only a weak
inducer of HL-60 [179]. Synergism between TGFβ and
TNFα, IFNγ, dexamethasone and phorbol esters is seen
in differentiation induction of U937, THP-1, and ML-1 in
human monocytic or myeloblastic leukemias [133, 179].
Low doses (0.5 ng) of TGFβ induced hemoglobinization
of K562 with growth inhibition [53]. Inhibition of prolif-
eration of M1 murine myeloid leukemic cells is produced
by low doses of TGFβ with enhanced adherence, but
differentiation-associated properties such as phagocytic
activity, lysozyme secretion, and morphologic change
were not induced [290]. Indeed, TGFβ inhibited the
dexamethasone-induced differentiation of this cell line.
Potent inhibition of proliferation was seen when TGFβ
was added to a variety of factor-dependent cell lines
(NFS-60, 32D, FDCP-1, B6 S4t A, DA-3) and to the
IL-3-producing WEHI-3 myelomonocytic leukemic cell
line [181]. Inhibition was also seen with the human leu-
kemic cell lines U937, THP-1, and KG-1 but not with
various other lines (e.g., HL-60, various T and B leukemias
and lymphomas) [182]. The response of the cell lines to
TGFβ appeared to correlate with the extent of display of
TGFβ receptors.
In primary cultures of bone marrow from 15 patients
with chronic myeloid leukemia, all showed TGFβ inhi-
bition of D14 CFU = GM but only 4/15 showed the
stimulation of D7 CFU-GM, which is the response seen
with normal marrow, and in 11/15 TGFβ inhibited D7
CFU-GM [7]. Both TGFβ1 and -β2 inhibited 45% of
CFU-GM stimulated by GM-CSF in marrow cultures
from five CML patients but, in contrast to normal,
G-CSF-stimulated colonies were also inhibited [358]. In
studies of acute myeloid leukemia (18 patients), TGFβ
suppressed both primary and secondary leukemic clo-
nogenic capacity [282, 375]. These results showed that
1–10 ng/ml of TGFβ delayed progression of leukemic
blast-cell progenitors from G1 to S phase in a cytostatic
manner with no induction of differentiation.
It is now apparent that a large number of factors
responsible for growth and differentiation have been
identified. In addition, their receptors on normal and
neoplastic cells are also being identified and investi-
gated. It is clear that some of these factors are produced
Kapil Mehta et al.
by tumor cells and represent autocrine growth factors.
Obviously, the blockage of these growth factors by
reducing their production or by blocking their attach-
ment to receptors could be an effective strategy for cancer
treatment. Likewise, the production of stimulatory factors
for angiogenesis by tumor cells has now been well char-
acterized and the blockage of their paracrine effect on
adjacent blood vessels in normal tissue is another poten-
tial method of therapy. Some tumors produce a consider-
able quantity of growth factor proteins and peptides and
others produced smaller amounts or a wider diversity of
these factors. In some, factors which promote the growth
of tumor cells clearly play a role in cancer growth and
metastasis and the autonomy that cancer seems to enjoy.
An interruption of this process will clearly lead to further
approaches in cancer treatment.
A good example of the clinical significance of growth
factors has been demonstrated in studies showing that
approximately 50% of tumor tissue from breast cancer
patients expresses epidermal growth factor receptor.
These receptors appear to be inversely related to steroid
receptors and therefore bring a poor prognosis to patients
who are steroid-receptor negative and epidermal growth
factor receptor positive. Since thousands of tumors have
been studied with regard to this particular characteristic,
it is abundantly clear that epidermal growth factor recep-
tor should be explored as a target for cancer treatment.
Monoclonal antibodies have been produced by various
investigators which are being administered to patients
with breast cancer in an attempt to block the epidermal
growth factor receptor. Many of these studies are being
done in association with chemotherapy to try to get an
effective one–two punch in inhibiting tumor growth.
Oncogenes such as erb and v-cis are known to encode
for growth factor receptors. Soluble receptors with high
affinity binding might be useful in therapy to bind with
growth factors and reduce their activities on membrane-
bound receptors present in the tumor cell or adjacent
blood vessel. Factors which are structurally similar to
growth factors but are inactive in inducing receptor
function (blockers) are also being explored as treatment
strategies. Growth factor interaction can also be blocked
by certain drugs such as Suramin. Suramin and its ana-
logues are currently in clinical trials for these effects.
Anti-angiogenesis factors have also been identified and
these are also in clinical trials. In summary, the applica-
tion of techniques to block growth factors and/or the
transduction of biochemical effects from growth factor
receptor stimulation represent advent technologies for
the millennium. As more is known about autocrine and
paracrine effects of growth factors, more can be done
with respect to altering their effects on tumor cells and
557
adjacent tissues. This approach, when employed in con-
cert with the use of differentiation agents, may allow for
the regulation and control of tumor growth in a manner
very different than chemotherapy and radiation therapy.
Perhaps one can regulate cancer in a manner similar to
the use of insulin to regulate diabetes rather than
attempting to destroy every last cancer cell with all the
toxicity inherent in these historical approaches.
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myelopoiesis. Leuk Res 2006; 30(1):60–68.
430. Lanotte M, Martin-Thouvenin V, Najman S, Ballerini P, Valensi
F, Berger R. NB4, a maturation inducible cell line with t(15;17)
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show bilineage potential and an aberrant pattern of neutrophil sec-
ondary granule protein gene expression. Blood 1994; 84:294–302
17 Granulocyte colony-stimulating factor:
biology and clinical potential
MARYANN FOOTE and GEORGE MORSTYN
Introduction
The study of hematopoiesis was greatly facilitated in the
mid-1960s when techniques for studying hematopoietic
cells in clonal culture were developed. Initially, serum
or conditioned medium was added to cultures as a source
of growth factors, i.e., the colony-stimulating factors
(CSFs) [56, 57, 85]. One of the factors that was isolated,
purified, cloned, and produced in commercial quantities
was granulocyte colony-stimulating factor (G-CSF), a
protein that acts on the neutrophil lineage to selectively
stimulate the proliferation and differentiation of com-
mitted progenitor cells and activation of mature neutro-
phils (Fig. 1). A property that distinguished G-CSF from
other CSF and facilitated its purification, molecular
cloning, and large-scale production in prokaryotic cells
was its ability to induce terminal differentiation of a
murine leukemic cell line (WFHI-3B). After observing
that serum from endotoxin-treated mice was capable of
causing the differentiation of a WFHI-3B myelomono-
cytic leukemic cell line, Metcalf [55] named the activity
GM-DF (granulocyte-macrophage differentiating fac-
tor). Further analysis showed that this serum contained
G-CSF as well as granulocyte-macrophage colony-
stimulating factor (GM-CSF). Nicola et al. [66, 68] fur-
ther purified G-CSF from medium conditioned by lung
tissue of endotoxin-treated mice. This G-CSF could
stimulate WFHI-3B cells as well as normal cells, sup-
porting the formation of numerous small, neutrophil-
containing colonies at a concentration similar to that
needed for WFHI-3B differentiation [67]. Subsequently,
murine G-CSF was identified as a protein and was
shown to have both differentiation-inducing activity for
WFHI-3B and granulocyte colony-stimulating activity
in bone marrow cells [68]. Other researchers, notably
Asano et al. [2] and Welte et al. [102], found several
human carcinoma cells that constitutively produce colony-
stimulating factors. One of these factors was purified to
apparent homogeneity from the conditioned medium of
bladder carcinoma 5637 cells [102] or a squamous car-
cinoma cell line [71]. The purified CSF selectively stim-
ulated neutrophilic granulocyte colony formation from
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
bone marrow cells, so it was concluded that this factor
was the human counterpart to mouse G-CSF. The protein
initially identified as G-CSF also was called CSF-13
and pluripoietin (pCSF).
The study of G-CSF progressed to the purification
and molecular cloning of both murine and human forms
and then to the first clinical trials of recombinant human
(rHu) G-CSF in cancer patients [7–9, 26, 27, 60–62].
Because of its unique biologic activities, rHuG-CSF is
used for reversing or ameliorating neutropenias of vari-
ous causes, for allowing increased cancer chemotherapy
dose intensity, and for mobilizing hematopoietic stem
cells for transplantation.
Filgrastim, the nonglycosylated rHuG-CSF, was the
first hematopoietic growth factor approved for commer-
cial use. A second form of rHuG-CSF, a glycosylated
version, lenograstim, was developed and approved for
commercial use in Europe. Nartograstim (also known as
marograstim, KW-2228, or NTG), a mutein rHuG-CSF,
was developed, but it had limited clinical development
[89]. A polyethylene glycol-modified rHuG-CSF (peg-
filgrastim) has been approved for commercial use [28].
Another pegylated rHuG-CSF is RO 258315; it is nar-
tograstim with polyethylene glycol molecules at the
amino terminus and the four lysine residues. The final
product of pegylation of RO 258315 is a mixture of one,
two, three, or four polyethylene glycol molecules
attached to each molecule of KW-2228, with the dipe-
gylated protein most common (60%) [94]. Table 1 sum-
marizes what is known about endogenous G-CSF and
Table 2 summarizes some characteristics of the rHuG-
CSFs. The molecule will be specified in this review
when the term ‘rHuG-CSF’ is insufficient. G-CSF’ will
be used to discuss the endogenous protein.
Biochemistry and Structure
Native human G-CSF appears to exist as a mixture of
two forms, one having 177 amino acids and the other
having 174 amino acids [1, 76, 86]. The gene for human
G-CSF is positioned at 17q11–22 and is approximately
569
570 Granulocyte colony-stimulating factor: biology and clinical potential

Pluripotential Stem Cell

CFU-Blast

flk-2/flt-3 ligand flk-2/flt-3 ligand flk-2/flt-3 ligand flk-2/flt-3 ligand

SCF SCF SCF SCF

Lymphoid NK
CFU-GEMM
Stem Cell Precursor

SCF MGDF/TPO SCF SCF SCF SCF SCF SCF

IL-3 SCF IL-3 IL-3 IL-3 IL-3 IL-7 IL-7
IL-3 flk-2/flt-3 ligand flk-2/flt-3 ligand flk-2/flt-3 ligand

CFU-GM

BFU-E CFU-Meg IL-3 IL-3 CFU-Eo CFU-Ba CFU-Mast Pre-B Cell Pre-T Cell
GM-CSF G-CSF
SCF SCF GM-CSF
IL-3 IL-3
GM-CSF GM-CSF
EPO IL-11
IL-6
MGDF/TPO IL-3 IL-3 IL-3 IL-7
CFU-M CFU-G GM-CSF SCF

IL-3 IL-3
CFU-E GM-CSF GM-CSF
IL-3 M-CSF G-CSF
GM-CSF
EPO
Megakaryocyte
IL-6

Monocyte SCF
IL-2
GM-CSF IL-7 IL-2
Reticulocyte M-CSF B Lymphocyte

Proplatelet

Red Blood Cell Platelet Macrophage Neutrophil Eosinophil Basophil Tissue Mast Cell Plasma Cell T Lymphocyte NK Cell

Figure 1. Hematopoietic tree (Figure courtesy of Amgen Inc., Thousand Oaks, CA)

Table 1. Summary of characteristics of human G-CSF

Characteristic
Location of gene Chromosome 17q11-22 (human)
Gene size Approximately 2500 nucleotides (approximately 2.5 kb)
Number of exons 5
Protein produced Proform
Number of amino acids in mature protein 174 (18.6 kDa)
Glycosylation site Thr 133
Disulfide bonds 2 (between 36 and 42, and 64 and 74)
Basic structure 4 helices arranged in antiparallel fashion; and sheet pleating
Receptor structure Single 150-kDa chain-forming homodimer
M. Foote and G. Morstyn
Table 2. Characteristics of the forms of recombinant human G-CSF
Generic name No. of amino acids Cell source
Filgrastim 175 E. coli
Lenograstim 174 Chinese hamster ovary
Nartograstim 174a E. coli
Pegfilgrastim 175 E. coli
RO 25–8315 174 E. coli
a
Changes to amino acids 1, 3, 4, 5, and 17
2.5 kbp [84]. It is encoded by the CSF3 gene. One chro-
mosomal gene exists per haploid genome.
The core protein of native human G-CSF has a molec-
ular weight of 18.8 kDa and the glycosylated protein
20–23.5 kDa [76]. Filgrastim is a highly purified protein
consisting of a single 175-amino acid polypeptide;
lenograsfim, lacking an N-terminal methiorryl residue,
has 174 amino acids. Lenograstim exists as two formu-
lations, one using gelatin and one using human serum
albumin as a stabilizer. The gelatin lenograstim formu-
lation reportedly allows faster neutrophil recovery after
chemotherapy than lenograstim [90]. Although nar-
tograstim also has 174 amino acids, the molecule has
been modified at the first, third, fourth, fifth, and 17th
amino acids [70]. Nartograstim is conjugated with an
average of two polyethylene glycol chains per protein
molecule [23]. Pegfilgrastim has a 20-kDa polyethylene
glycol molecule added to filgrastim, giving the protein
an extended half-life in the serum [58].
Native human G-CSF and lenograstim are O-glycosylated
[64, 86] whereas filgrastim and pegfilgrastim are not glyco-
sylated. The sugar chains are responsible for the differences
in molecular mass. The presence and type of glycosylation of
the recombinant protein depends on the cellular source (i.e.,
yeast, mammalian cell, or bacterial cell) [62]. Lenograstim
has heterogenous glycosylation: not all the sugar chains are
identical. Not all structural components of native human
G-CSF have been identified, but it is known that its sugar
composition is not identical to that of lenograstim [72, 73].
Native human G-CSF does not bind to concanavalin, sug-
gesting that it does not have mannose-containing carbohy-
drates [69]. When G-CSF was treated with neuraminidase, it
showed reduced charge heterogeneity in isoelectric focusing,
suggesting sialic acid-containing O-glycosylation [68].
Crystallography studies of filgrastim have shown that
the glycosylation site (threonine 134) is attached to the
C-D loop at a distance from the active biologic sites [43,
74]. Although the glycosylation does not seem to have a
role in the biologic function of the molecule, it may par-
tially protect the molecule from proteolytic degradation.
The observation that a limited proteolytic degradation
571
Other information Marketing company
Non-glycosylated Amgen, Kirin, Roche
Glycosylated Chugai, Rhône-Poulenc
Mutein form Kyowa-Hakko
Non-glycosylated, pegylated Amgen
Mutein form, pegylated Roche
of filgrastim results in the cleavage of the molecule near
threonine 134 points to a role of glycosylation for prote-
olytic protection and indicates that the residues along
this portion of the protein structure may serve as ‘handles’
for proteolytic degradation [76]. Although it has been
suggested that glycosylation of lenograstim increases
biologic activity in vitro compared with filgrastim [103],
this advantage has not been verified in randomized double-
blind comparative studies [35]. Glycosylation possibly
influences the antigenicity of recombinant proteins [48].
Several cell types in the human body produce G-CSF,
including stromal cells, endothelial cells, fibroblasts,
macrophages, and monocytes [19, 24, 61, 76, 79, 96,
105]. These cell types are widely distributed in the body,
suggesting that G-CSF participates in the production
and functional enhancement of neutrophils that occur in
response to local infection or other causes.
In a variety of pathologic conditions, including expo-
sure to endotoxin, response to an infection or to neutro-
penia, the amount of circulating endogenous G-CSF in
the blood increases [10, 40, 56, 98]. In humans, G-CSF
is the primary factor for the upregulation of neutrophils
in infection and in various pathologic conditions with
decreased neutrophil counts [10]. It has been suggested
that the Toll-like receptors on macrophages and den-
dritic cells detect gram-negative bacteria and induce
IL-23 production that, in turn, induces IL-17 production
by T cells, which then stimulates G-CSF production.
Mechanistically, as G-CSF concentrations increase during
bacterial infections and circulate in the blood, neutro-
poiesis is stimulated in the marrow. Hence, in cases of
gram-negative and fungal infections, serum G-CSF con-
centrations are increased [40]. Indeed, the highest
G-CSF concentrations have been found in patients with
neutropenia and febrile neutropenia [10].
G-CSF selectively stimulates the proliferation and
differentiation of neutrophil precursors by binding to a
specific cell-surface receptor [18]. The G-CSF receptor
is expressed on cells of the neutrophil lineage from
myeloblast to the mature neutrophil and on a subset of
cells of the monocyte lineage [56]. It has been reported
572 Granulocyte colony-stimulating factor: biology and clinical potential
that G-CSF receptor expression on cell types is not usu-
ally associated with its activity including endothelial
cells, activated T lymphocytes, and nonhemopoietic
tumor cell lines. The role of these receptors is unclear.
Both filgrastim and lenograstim, as well as native HuG-
CSF derived from a tumor cell line, have identical dose-
dependent affinity for G-CSF receptors (Fig. 2). The
binding of G-CSF to its receptor decreases the number
of available surface receptors as the surface receptor
complex is internalized and degraded.
Both endogenous and recombinant G-CSFs work on
neutrophil precursor cells and mature neutrophils.
rHuG-CSF stimulates the proliferation, differentiation,
and activation of committed progenitor cells of the neu-
trophil lineage and reduces neutrophil maturation time
from 5 days to 1 day, leading to a rapid release of mature
neutrophils from the bone marrow into the circulation
[53, 54]. Neutrophils previously treated with rHuG-CSF
have normal intravascular half-life [8, 54]. In the pres-
ence of rHuG-CSF, neutrophils have enhanced superox-
ide production in response to chemoattractants [1, 100].
G-CSF also enhances chemotaxis by increasing the
binding of fMLP (formyl-methionyl-leucyl-phenylala-
nine) [14] as well as enhancing anti-Candida activity.
rHuG-CSF does not stimulate release of other cytokines
by neutrophils and mononuclear cells [78].
Figure 2. Inhibition of labeled filgrastim, lenograstim, and tumor cell-derived human G-CSF binding to neutrophils. Those data
demonstrate that the three forms have the same affinity for G-CSF receptors in vitro. (Figure courtesy of Amgen Inc., Thousand
Oaks, CA)
Lenograstim also increases superoxide anion produc-
tion in neutrophils in response to fMLP [93]. Neutrophils
from healthy volunteers treated with lenograstim showed
evidence of enhanced stimulation by agonists, including
adhesion to nylon fibers and physiologic substrates
[33, 36].
In vivo studies in animals and some studies in humans
have shown that rHuG-CSF causes a dose-dependent
increase in the number of neutrophils in the peripheral
blood. This increase is thought to be a result of decreased
maturation time, increased number of cell divisions, and
accelerated release into the peripheral blood. The action
of rHuG-CSF causes rapid dose-dependent increases in
neutrophils and small or no effects on monocytes and
eosinophils, respectively [50, 52, 63].
Physiology
G-CSF is an indispensable cytokine for normal murine
myelopoiesis, as has been shown by knock-out-mouse
experiments [47]. Circulating neutrophil concentrations
were reduced by 70–80% with less of a reduction in mar-
row stores of progenitors (50%) compared with those of
normal mice. Despite appearing superficially healthy, these
mice have a diminished ability to mount neutrophilia
M. Foote and G. Morstyn
and monocytosis in response to infection and have a
marked impairment in ability to control Listeria infec-
tions. The observations from this study indicate that
G-CSF is required for maintaining the normal quantita-
tive balance of neutrophil production during steady-state
granulopoiesis in vivo and implicate rHuG-CSF in emer-
gency granulopoiesis during infectious episodes.
Pharmacokinetics
Pharmacokinetic/pharmacodynamic data from the differ-
ent rHuG-CSF products are difficult to compare directly,
as different study designs, doses, regimens, routes of
administration, and populations were evaluated. Table 3
summarizes these data.
Healthy Volunteer Studies
Table 3 summarizes pharmacokinetic and pharmacody-
namic data from healthy volunteer studies. Healthy
men given single doses of 3.45 μg/kg filgrastim by
30-min intravenous (IV) infusion had a mean serum
concentration of 20.8 ng/ml 5 min after the end of infu-
sion [3]. The mean (SD) elimination half-life was 163
(±7.4) min, and G-CSF concentrations returned to nor-
mal values within 14–18 h.
After single subcutaneous (SC) injections of
lenograstim 10, 20, or 40 μg in healthy volunteers, Cmax
values were 0.09, 0.18, and 0.48 μg/L, respectively,
within 3.5–4.5 h, peak serum concentrations were main-
tained for almost 4 h, and lenograstim was almost elimi-
nated from the serum by 48 h [81]. Lenograstim exhibits
dose-dependent pharmacokinetic characteristics with
peak serum concentrations after repeated doses (SC or
IV) that are proportional to the administered dose [13].
Lenograstim does not appear to accumulate after repeated
Table 3. Summary of pharmacokinetic/pharmacodynamic data in normal volunteers. Pharmacokinetic
data are difficult to compare directly because of differences in study designs, doses, and populations
Generic Name Cmax
Filgrastim (75 μg/kg dose)
a
1.65±0.80 ng/ml
Lenograstimb (5 μg/kg dose) 10.3–11.9 mg/L
Nartograstimc (2 μg/kg dose) 1.52±0.58*
Pegfilgrastima (30 μg/kg dose) 43.6±20.0 ng/ml
Ro 25–8315d 2.5±1.6 ng/ml
Cmax, peak serum concentration; tmax, time to Cmax; AUC, area under the serum drug concentration curve versus
time curve
*
Units not provided
a
Amgen data on file; bDunn and Goa [24]; cSuzuki et al. [92]; dVan der Auwera et al. [97]
573
administration, and no dose sequence effects were observed
in a crossover study [38].
Administration of single 10, 30, 60,100, or 150 μg/kg
doses of RO 258315 to healthy men produced dose-
dependent increases in peak neutrophil count [94]. The
time to reach peak concentration and the area under the
serum concentration time curve increased 100-fold over
the range of doses studied.
A healthy volunteer study was reported with pegfil-
grastim [58]. Pegfilgrastim was administered as a single
injection of 30, 60, 100, or 300 μg/kg, and blood samples
were taken at specific time points for 48 h. Neutrophil
counts increased in a dose-dependent manner, and the peak
neutrophil count attained and the length of the response
were dose dependent.
Patients with Disease
Patients with cancer receiving filgrastim 11.5 μg/kg as a
30-min IV infusion had a peak serum concentration of
384 ng/ml [26, 27]. When patients with cancer received
SC bolus or SC infusion doses, serum concentrations of
filgrastim reflected rapid absorption [60]. The serum half-
life of filgrastim has a t1/2 of 3.5 h [48]. With filgrastim,
the maximum increase in neutrophil count can be achieved
by all routes of administration tested [60, 80].
Patients with nonmyelogenous malignancies receiving
lenograstim at 5, 10, or 20 μg/kg/day as a SC injection
had peak plasma concentrations of 10.1, 35.0, and
49.7 mg/l, respectively [25]. Cmax was reached within
4–8 h. The serum half-life of lenograstim was 3.0, 4.8,
and 6.0 h, respectively, for the 5-, 10-, and 20-μg/kg per
day cohorts.
Results of a randomized, dose-escalation study with
pegfilgrastim in patients with nonsmall cell lung cancer
receiving chemotherapy were published [39]. Thirteen
patients were randomly assigned to receive daily injections
tmax (h) AUC
5.5±1.8 14.3±4.3 (0–24 h, ng/L×h)
6.0 89.8–102.3 (0–24 h, mg/L×h)
4.0±2.0 12.7±4.78 ng×hr/ml
9.50±3.51 887±336 (0–infinity, ng/L×h)
13±5.9 184±77 ng × h/ml
574 Granulocyte colony-stimulating factor: biology and clinical potential
of filgrastim 5 μg/kg per day or a single injection of peg-
filgrastim at 30, 100, or 300 μg, 2 weeks before chemo-
therapy and 24 h after completion of chemotherapy.
Peak serum concentrations of filgrastim and the duration
of increased serum concentrations were dose dependent.
The concentration of serum filgrastim remained high for
a longer duration in the setting of chemotherapy-induced
neutropenia until neutrophil recovery occurred. Patients
treated with pegfilgrastim had a higher prechemother-
apy neutrophil count than patients treated with filgrastim
(who did not receive the study drug until after chemo-
therapy was completed). After chemotherapy, the neu-
trophil count nadirs were similar among patients
receiving filgrastim 5 μg/kg per day or pegfilgrastim
30 μg; higher neutrophil count nadirs were seen in
patients receiving the higher doses of pegfilgrastim.
Pharmacodynamics
The exposure of filgrastim may be influenced by the
increased neutrophils formed after administration of
the cytokine [80]. Increased numbers of neutrophils
were shown to be associated with increased clearance
of filgrastim in patients with cancer, which suggests
that a negative feedback mechanism is involved in
maintaining neutrophil counts at optimal values [45].
The bioavailability of filgrastim was an average of 53%
when administered at very low doses (1 μg/kg) to
healthy volunteers [91]; when administered at thera-
peutic doses, its bioavailability has been reported as
high as 80% [80].
Lenograstim has an absolute bioavailability of 30%
after SC doses of 2–5 μg/kg, with an apparent volume of
distribution of 1 L/kg [13]. The serum elimination half-
life after SC administration is approximately 3 h, but it
is shorter after repeated IV infusion (1–1.5 h) [38].
The absolute bioavailability of pegfilgrastim after SC
administration is estimated to be approximately 15% [31].
The exposure of pegfilgrastim is affected by the neutrophil
status; pegfilgrastim concentrations for patients who expe-
rienced severe neutropenia were sustained longer and
higher than those who did not experience severe neutrope-
nia. Based on pharmacokinetic/pharmacodynamic model-
ing, clearance of pegfilgrastim is almost completely
(>99%) regulated by neutrophil-mediated clearance.
While renal clearance plays a significant role in the elimi-
nation of filgrastim, it is absent for pegfilgrastim, based on
results from a bilateral nephrectomy rat study [58].The
predominant dependency of pegfilgrastim clearance on
the neutrophil-mediated pathway allows pegfilgrastim to
be under highly efficient ‘self-regulation’.
Clinical Implications
Much clinical data have been published about the various
forms of rHuG-CSF and their use in numerous thera-
peutic areas. To cite all the important references in this
review paper is impossible, and we have concentrated
on the pivotal phase 3 trials used in obtaining marketing
authorization whenever possible. The interested reader
is directed to the cited reviews [21, 49, 59, 88, 101].
Chemotherapy-induced Neutropenia
Neutropenia and infection are common dose-limiting
effects of cancer chemotherapy. It has long been known
that the risk of infection is directly related to the depth
and duration of neutropenia [6]. The severity of chemo-
therapy-induced neutropenia depends on the dose inten-
sity of the therapy and other factors. Often, oncologists
withhold or delay the next cycle of chemotherapy if a
patient has low neutrophil counts. Such practice,
although done to prevent infectious complications, can
compromise otherwise effective cancer chemotherapy.
Two randomized, placebo-controlled, double-blind
studies involving more than 300 patients with small-cell
lung cancer receiving cyclophosphamide, doxorubicin,
and etoposide (CAE) chemotherapy showed that fil-
grastim decreased the incidence, severity, and duration
of severe neutropenia [15, 92]. These two studies
showed that, in the placebo control group, most of the
febrile neutropenic events occurred in the first cycle.
Two randomized placebo-controlled phase 3 trials with
lenograstim showed significant reductions in the median
duration of neutropenia in patients with inflammatory
breast cancer [12] or non-Hodgkin’s lymphoma [29]. The
median neutrophil nadir was higher in lenograstim-treated
patients who had inflammatory breast cancer than in
patients administered placebo. Treatment with lenograstim
was associated with reductions in culture-confirmed
infections during periods of neutropenia, shorter durations
of hospitalization for infections, and reduced need for
antibacterial drugs.
In two double-blind randomized studies, use of peg-
filgrastim reduced neutropenia and its complications in
patients receiving chemotherapy [31, 37]. In one study,
patients with high-risk stage II or stage III/IV breast
cancer received a single 6-mg injection of pegfilgrastim
or daily 5 μg/kg SC injections of filgrastim for up to four
cycles of chemotherapy [31]. The single injection of
pegfilgrastim was as effective as a mean of 11 daily
injections of filgrastim with respect to the observed mean
duration of grade 4 neutropenia. The incidence of febrile
neutropenia was lower, but not statistically significantly,
M. Foote and G. Morstyn
in the pegfilgrastim group compared with the filgrastim
group. In the second study, patients received either
100 μg/kg pegfilgrastim once per cycle of chemotherapy
or daily injections of 5 μg/kg filgrastim [37]. Again,
single doses of pegfilgrastim were comparable to multiple
doses of filgrastim in terms of duration of grade 4 neu-
tropenia and depth of neutrophil nadir counts in all
cycles. In both studies, pegfilgrastim and filgrastim were
well tolerated.
Since these pivotal randomized clinical trials were
completed, additional studies were conducted. This has
led to major organizations such as ASCO, EORTC,
NCCN, and ESMO evaluating these data and producing
guidelines. These data have been reviewed by an expert
panel in Europe [32]. The ASCO evaluation was pub-
lished in 2006. There is agreement in all studies that
rhuG-CSF reduces the rate of febrile neutropenia by
50% what ever the background febrile neutropenia rate.
Much discussion, however, ensued about the high back-
ground febrile neutropenia rate in the initial registration
trials and whether the use of rHuG-CSF was appropriate
in settings with a lower febrile neutropenia rate. One
issue in the early studies was the frequent measures of
neutrophil counts and temperature led to the identifica-
tion of neutropenia that might have been missed if blood
counts were performed less frequently. Nevertheless,
recent studies were performed in settings in which the
febrile neutropenia rate was approximately 20% and so
current guidelines recommend the use of CSFs in set-
tings in which the risk of febrile neutropenia is greater
than 20%. The existence of risk factors such as age,
treatment intent such as dose intensity, and prior toxicity
from chemotherapy may increase risk.
Bone Marrow or Stem Cell
Transplantation
Bone marrow or stem cell transplantation allows the use
of very high doses of chemotherapy, with or without
radiotherapy, to eliminate cancer cells from patients with
refractory tumors. After ablative therapy and before the
patient’s bone marrow recovers full function, the patient
often experiences profound pancytopenia that requires
multiple transfusions of blood and blood products.
In the pivotal phase 3 trial for filgrastim in the high-
dose chemotherapy and autologous bone marrow trans-
plantation setting, patients were administered filgrastim
10 or 30 μg by continuous IV infusion or placebo for 5
days, starting 1 day after bone marrow infusion [87].
The dose of filgrastim was adjusted as needed during
neutrophil recovery for a scheduled maximum of 28
days of administration. The median number of days of
575
neutropenia (neutrophil count <0.5 × 109/L) was 23 days
for the control group, 11 days for the 10-μg/kg per day
group, and 14 days for the 30-μg/kg per day group.
For the control group, compared with both treatment
groups combined, the difference in the number of days
of neutropenia was statistically significant. Moreover,
filgrastim reduced the duration of fever and febrile
neutropenia.
A phase 3 trial of lenograstim was performed in 298
patients receiving either autologous or allogeneic bone
marrow transplantation [30]. The addition of lenograstim
5 μg/kg per day produced 30% reduction relative to
placebo in the median time to reach neutrophil counts of
0.5 × 109/L. Other significant reductions compared with
placebo were seen in lenograstim-treated patients for
median time in hospital, duration of antibiotics, and dura-
tion of parenteral nutrition. The incidence of infections,
febrile neutropenia, and septicemia were similar between
the two groups, but the median duration of infection and
febrile neutropenia were reduced with lenograstim.
In another study with lenograstim [51], reduction of
neutropenia was associated with a reduction in the number
of days of fever and a shorter duration of parenteral anti-
biotic use after transplantation.
Recombinant HuG-CSF alone or in combination with
chemotherapy is an effective agent for recruiting periph-
eral blood progenitor cells (PBPC) with long-term
reconstituting ability [11, 22, 41, 82, 83]. In a histori-
cally controlled study, filgrastim-generated PBPC, in
conjunction with autologous bone marrow transplanta-
tion and daily filgrastim, accelerated recovery of neutro-
phil and platelet count [82]. In a study of 85 patients
with relapsed Hodgkin’s disease [11], use of filgrastim
for mobilization resulted in an accelerated time to recov-
ery of granulocytes compared with nonmobilized PBPC
recipients. The use of mobilized PBPC resulted in an
accelerated time to platelet engraftment compared with
nonmobilized PBPC recipients. Costs were lower in
patients who received filgrastim-mobilized PBPC com-
pared with patients who did not.
Use of lenograstim for PBPC mobilization has pri-
marily been reported in abstract form. Patients with
lymphoma previously treated with lenograstim had a
median time to neutrophil recovery of 11–12 days after
chemotherapy and infusion of lenograstim-assisted
autologous PBPC transplantation [99].
A comparison of lenograstim and filgrastim administered
after chemotherapy to increase PBPC yield was reported
as a 126-patient retrospective study [46]. No differences
were observed between groups for median CD34 cells
harvested or number of leukophereses needed to obtain
the 3 × 106 CD34 cells/kg required for transplantation.
576 Granulocyte colony-stimulating factor: biology and clinical potential
Severe Chronic Neutropenia
A phase 3 trial of filgrastim in patients with severe chronic
neutropenia has shown long-term efficacy in boosting
neutrophil counts, with hematologic and clinical benefits
sustained during prolonged maintenance treatment [16,
17]. Of the 120 patients who received filgrastim, 108 had
a median neutrophil count of >1.5 × 109/L during the
study. Their bone marrow had increased proportions of
maturing neutrophils. The incidence and duration of infec-
tion-related events was reduced by approximately 50%
and the duration of antibiotic use was reduced by 70%.
No phase 3 studies of lenograstim in patients with
severe chronic neutropenia have been reported, but sev-
eral small groups of patients reported have been treated
with lenograstim [21]. One European phase 2 study
reported that all 19 children treated with lenograstim had
neutrophil recovery to >1.0 × 109/L [20]. Normal bone
marrow cytology was attained by ten of the 19 children.
Bone Marrow Failure States
Both filgrastim and lenograstim are licensed in Japan
and China for the treatment of aplastic anemia [77].
Studies suggest that rHuG-CSF increases neutrophil
counts in some patients with moderate aplastic anemia;
however, patients with very severe hypoplasia generally
do not respond to growth factors [5, 42]. The European
Group for Bone Marrow Transplantation (EBMT)
Working Party discourages the use of rHuG-CSF as a
single agent in patients with newly diagnosed aplastic
anemia [4]. Although published data do not fully sup-
port the use of growth factor as first-line treatment for
aplastic anemia, rHuG-CSF in combination with other
approaches for this disorder (e.g., with immunosuppres-
sive therapy [76]) may have a role.
The myelodysplastic syndromes are a group of neoplas-
tic hematopoietic disorders. In a phase 3 randomized study
involving 102 patients with RAEB or RAEB-t subtypes
of myelodysplastic syndromes, filgrastim was efficacious
in increasing neutrophil counts [65], although imbalances
in patient characteristics made the overall benefit, if any,
of the use of rHuG-CSF in this setting difficult to define.
In a phase 2 study to demonstrate the efficacy of
lenograstim, most patients responded to IV doses of 2 or
5 μg/kg per day [104]. Administration of lenograstim
increased neutrophil counts in 18 Japanese patients and
no patient progressed to acute myeloid leukemia.
AIDS/HIV Infection
Filgrastim has been approved in Australia, Canada, the
European Union, and Japan for use in patients with HIV
infection, for reversal of clinically significant neutropenia,
and for maintenance of adequate neutrophil counts during
treatment with antiretroviral therapy. A phase 3 study
reported the effect of filgrastim on the incidence of severe
neutropenia in patients with advanced HIV infection and
its effect on the prevention of infectious morbidity [44].
In this 24-week study of 258 patients, filgrastim was
administered daily at 1 μg/kg and adjusted to as much as
10 μg/kg, or was administered intermittently at 300 μg
daily 1–3 days per week. Patients in a control group
received filgrastim only if they developed severe neutro-
penia. Both daily and intermittent administration of
filgrastim lowered the incidence of bacterial infection
rates compared with patients in the control group. The
filgrastim-treated patients had 31% fewer bacterial infec-
tions than did the control patients, suggesting that the use
of filgrastim can reduce the risk of several bacterial infec-
tions and subsequent number of days of hospitalization.
No phase 3 studies with lenograstim in the setting of
AIDS/HIV infection appear to have been published as
full reports. One study using lenograstim in the setting of
AIDS showed that lenograstim could safely ameliorate
the neutropenia associated with zidovudine treatment in
patients with AIDS or AIDS-related complex [95].
Current Issues
Although both filgrastim and lenograstim have been
marketed for more than a decade and pegfilgrastim was
approved in 2002, and are licensed for many uses in
countries around the world, work continues to fully
understand the mechanisms of action and long-term
results of chronic administration and to discover other
conditions and disease states where rHuG-CSF may be
useful. One issue that may not be fully resolved in the
perceived relationship between use of hematopoietic
growth factors in several patient populations and the
risk of developing leukemia [34]. It is not clear if the
slightly increased risk of developing leukemia is due to
the underlying disease concurrent with chemotherapy or
if the use of a growth factor increases the risk.
Surveillance is on going and the benefits appear to out-
weigh any potential risks.
Immunomodulation
Immune reconstitution depends upon hematopoietic
reconstitution as the first step, and hematopoietic recon-
stitution, of course, depends on the fine actions and inter-
actions of hematopoietic growth factors. Hematopoietic
growth factors, including G-CSF, may be important
modulators of immune reconstitution because of their
ability to increase the number of leukocytes, recruit
M. Foote and G. Morstyn
immature leukocytes, cause maturation and augmenta-
tion of leukocytes, and suppress leukocytes [97].
Use in Patients with Sickle-cell Anemia
Increases in leukocyte counts may be associated with
worsened prognosis in patients with sickle-cell anemia.
A study of 3,764 patients with sickle-cell disease, rang-
ing in age from birth to 66 years of age, was done to
determine the life expectancy, median age at death, and
circumstances of death [75]. Patients with sickle-cell
anemia who had hemoglobin values <7.1 g/dL (below
the tenth percentile) had a slightly higher risk of death
than those patients with increased white blood cell
counts (>1.5 × 109/L).
Conclusions
The discovery, isolation, and cloning of G-CSF and the
subsequent production of rHuG-CSF have had profound
effects on the practice of medicine, particularly oncol-
ogy. The use of rHuG-CSF has allowed increased
administration of cytodoxic drugs, thereby allowing
increased efficacy of chemotherapy and eliminating the
dose-limiting toxicity of myelosuppression; however,
the treatment of many diseases, including nonmalignant
disease and non-neutropenic infections, also has been
altered by the use of rHuG-CSF. Although rHuG-CSFs
have been commercially available for more than 2
decades, research continues to find new formulations
that improve patient compliance or that have enhanced
biologic activity.
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18 Granulocyte-macrophage colony-stimulating
factor
MARYANN FOOTE AND GEORGE MORSTYN
Introduction
Granulocyte-macrophage colony-stimulating factor
(GM-CSF) is one of the colony-stimulating factors
whose name is derived from its major target cell lin-
eages. Since the original studies, which characterized
its ability to stimulate the clonal proliferation of myel-
oid precursors, however, a broader range of biologic
effects on mature, effector cells, and the immune sys-
tem, have been identified. These expanded areas of
activity have carried its more recent clinical application
beyond that of ‘colony-stimulating factor’ into the
realm of immunomodulation.
GM-CSF was initially purified from media used to
culture an HTLV-II-infected, T lymphoblastoid cell line
(Mo) [5], but was subsequently found to be produced by
a variety of cell types including macrophages, endothe-
lial cells, and certain mesenchymal cells. Although basal
serum concentrations of GM-CSF are often not measur-
able in normal adults, its elaboration is readily inducible
in ‘producer’ cells by immune stimuli and many of the
mediators of inflammation.
In 1985, GM-CSF was the first myeloid hematopoi-
etic growth factor, to have its gene sequenced and cloned
[20, 92]. GM-CSF is used for the native form of the
molecule or when it is not necessary to distinguish a
specific form of the recombinant molecule, which is
referred to as rHuGM-CSF.
Biochemistry and Structure
The gene for GM-CSF has been mapped to the long
arm of chromosome 5 (5q21–32) [51] in the cluster
region of genes for other growth factor proteins and their
cell-surface receptors, such as interleukin-3 (IL-3) [56],
IL-4 [93], IL-5 [83], macrophage colony-stimulating
factor and its receptor (the c-fms proto-oncogene
product) [70], and the receptor for platelet-derived
growth factor [93].
The gene for GM-CSF encodes a protein of 127
amino acids whose molecular weight varies from l4 to
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
35,000 daltons depending upon the degree of glycosyla-
tion. The gene has been expressed using recombinant
DNA technology in bacteria (Escherischia coli), Chinese
hamster ovary (CHO) cells, and yeast (Saccharomyces
cerevisiae) resulting in the availability of large amounts
of glycosylated and nonglycosylated material for clinical
trials. Small differences in the amino acid sequence, as
well as major differences in the degree of glycosylation,
can be found among the preparations. The growth factor
produced by expression of the cDNA in bacteria is not
glycosylated [16], but that expressed in mammalian
cells and yeast demonstrates variable degrees of glyco-
sylation [20]. Both the glycosylated and nonglycosy-
lated forms are active in vitro or in vivo, and have
similar activity to the natural form of the protein, but the
degree of glycosylation plays a role in inducing antibody
formation, as well as altering some specific biologic
activities and toxicities.
Physiology
Both endogenous and recombinant GM-CSF generate
predominantly granulocyte and monocyte colonies in
semisolid culture systems. It acts as a potent stimulus for
the growth of uncommitted and committed bone marrow
progenitors including colony-forming unit–granulocyte,
erythrocyte, monocyte, and megakaryocyte (CFU–
GEMM) [61, 77]. It is a potent inducer of granulocyte-
monocyte differentiation, and potentiates the growth of
human blast-forming units–erythroid (BFU-E) in the
presence of erythropoietin in vitro.
At the level of mature cells, GM-CSF enhances the
function of neutrophilic and eosinophilic granulocytes,
and of monocyte/macrophages. Exposure of neutrophils
to GM-CSF produces increased expression of cellular
adhesion molecules [5, 23, 41] such as CDllb, increased
expression of class II MHC molecules [38, 63], increased
number of FMLP receptors [88], and increased fMLP-
induced superoxide production [87], chemotaxis, anti-
body dependent cellular cytotoxicity (ADCC), and
phagocytosis. In vitro, it was reported to inhibit random
581
582
neutrophil granulocyte migration (8,334) and in vivo,
when administered by continuous infusion, it prevented
neutrophils from migrating into areas of inflammation [69].
GM-CSF prolongs the survival of neutrophilic and
eosinophilic granulocytes in vitro [60] by inhibiting
programmed cell death [11], and enhances microbicidal
activity and their leukotriene synthesis [79]. Exposure
of macrophages to GM-CSF increases expression of
cell-surface adhesion molecules such as CD11a, b, and
c; and Fc RII (CDw32) receptors involved in phagocy-
tosis [72, 91]. In vitro, it enhances ADCC, phagocyto-
sis, microbicidal and tumoricidal activity [39, 44, 89],
and the synthesis and secretion of other cytokines
including those related to inflammation such as tumor
necrosis factor (TNF) and IL-1.
GM-CSF exerts its biologic activity by binding to spe-
cific transmembrane surface receptors, which are subse-
quently internalized, on the target cells [24, 33]. These
receptors have been detected on mature neutrophils,
monocytes/macrophages, some lymphocytes, normal
bone marrow progenitors, dendritic cells (DC), fibroblasts,
endometrial and endothelial cells, fresh leukemic cells,
and leukemic cell lines [13, 14,18, 66]. GM-CSF itself can
downregulate these receptors on cells such as neutrophils,
monocytes, and normal bone marrow myeloid cells
in vitro [18].
Crosslinking experiments have revealed molecular
weights of 75–156 kD for the receptors. At least two
types of GM-CSF receptors have been identified: one
with high affinity (20–100 pmol/L) and one with low
affinity (1 nmol/L), but as many as four types may exist
on AML cells [13]. Two subunits (alpha and beta)
comprise the heterodimer receptor and both have been
cloned [46]. The alpha subunit is specific to the GM-CSF
receptor. At least two distinct regions within the
cytoplasmic domain of the common beta subunit have
been shown to be responsible for different signals [74].
A membrane proximal region of approximately 60
amino acids has been shown to be essential for induction
of c-myc and activation of DNA replication, which
involves the tyrosine phosphorylation of a Janus kinase
(JAK2) and activation of its in vitro kinase activity [71].
A second, distal region of about 140 amino acids is
required for activation of Ras, Raf-1, MAP kinase and
p70 S6 kinase, and induction of c-fos and c-jun [74].
Pharmacology
Both glycosylated and nonglycosylated forms of
rHuGM-CSF have been studied clinically. Glycosylated
rHuGM-CSF (sargramostim), produced in yeast, is
available in the United States. Molgramostim, which is
Granulocyte-macrophage colony-stimulating factor
not glycosylated, is available in Europe and other
countries.
Pharmacodynamics and
Pharmacokinetics
Several early pharmacodynamic studies defined the
pharmacology of rHuGM-CSF [58]. Radiolabeling of
leukocytes suggested that a transient leukopenia is due
to sequestration of the cells within the lung. In vitro and
in vivo studies have suggested that this effect may be
due to a change in cell surface adhesion molecules
on the leukocytes such as those identified by CD11b
[5, 80], which is upregulated on neutrophils, as well as
LAM-1, which is downregulated, and adherence to
endothelial cells of the blood vessels [41].
The major effect of rhGM-CSF on hematopoiesis is
the leukocytosis that was reported to be dose dependent
in most of the early studies [58] when either glycosy-
lated or nonglycosylated rHuGM-CSF was administered
daily subcutaneously (SC) or by prolonged intravenous
(IV) or SC infusions. The pattern of the leukocytosis in
many of these trials appeared as a biphasic response: an
initial increase that frequently plateaued during the first
few days of treatment, followed by a decline. With con-
tinued dosing, however, another increase in leukocytes,
which often contained more immature myeloid cells
such as myelocytes, promyelocytes, and myeloblasts,
was observed. Appearance of monocytes and eosino-
phils generally occurred later in the course of treatment
and at higher doses of rHuGM-CSF. Morphologic
changes in neutrophils included toxic granulation with
an increase in leukocyte alkaline phosphatase, promi-
nent Döhle bodies, cytoplasmic vacuolization and, at
higher doses, increasing numbers of hypersegmented
neutrophils. At the higher doses of rHuGM-CSF in
patients with normal marrows, the cellularity of the
bone marrow was increased with a left-shift and an
increase in the myeloid-to-erythroid ratio [50, 58].
An important observation made during these early
trials, which later found significant clinical application,
was the dose-dependent increase in peripheral blood
erythroid and myeloid progenitors [59]. Bone marrow
progenitors were found to be unchanged or decreased,
probably somewhat dependent upon the degree of
expansion of the non-colony forming myeloid mononu-
clear cells [62].
Pharmacokinetic studies were conducted using both
glycosylated and nonglycosylated rHuGM-CSF by
radioimmunoassay and by bioassay. Route of adminis-
tration affected the peak serum concentration, area
under the concentration-time curve, and the time during
which GM-CSF was detectable in the serum.
Maryann Foote and George Morstyn
Clinical Implications
Much clinical data have been published about the various
forms of rHuGM-CSF and their uses in many therapeutic
areas. It is impossible to cite all the important papers, and
we have concentrated on reports of pivotal studies.
Chemotherapy-induced Neutropenia
Many clinical trials have studied the ability of rHuGM-
CSF to abrogate the hematologic toxicity of chemother-
apy (both standard and dose-intensified) and bone
marrow/stem cell transplantation. Most of the early
studies were not randomized studies, included relatively
small numbers of patients, compared the results of
cycles of chemotherapy given without growth factor to
cycles given with growth factor, and cycles of chemo-
therapy given with growth factor compared with data of
historical controls. In most studies, rHuGM-CSF did
not completely eliminate profound leukopenia. In gen-
eral, the neutrophil nadir occurred earlier in the courses
of chemotherapy in which rHuGM-CSF was adminis-
tered than in the courses without. Higher doses of the
growth factor in some studies decreased the depth of the
absolute neutrophil count (ANC) nadir, and frequently
the duration of neutropenia and time to an ANC > 1.0 ×
109/L was shortened at during the first cycle of chemo-
therapy administered with rHuGM-CSF. The results
with respect to the ability to reduce the morbidity (hema-
tologic, and in some cases the incidence of infection and
mucositis) of dose intensification was variable.
Furthermore, even in the larger randomized trials [6, 15,
35, 53], efficacy was variable. In most cases, duration of
neutropenia was reduced at least during the first cycle;
however, persistence of this effect on subsequent cycles,
and impact on infections was much more variable.
Bone Marrow and Stem Cell
Transplantation
One of the areas of more intense research has been derived
from the ability to mobilize peripheral blood stem cells
(PBPC). Although it is difficult to collect PBPC from a
patient who is in a hematologic steady-state, the number
of circulating stem cells increases during blood cell count
recovery after treatment with chemotherapy, especially
after cyclophosphamide. The early trials showed that
CSF alone could increase the number of circulating PBPC
[80], and when administered after chemotherapy the
number was enhanced even further [78].
The PBPC harvested by leukopheresis during rHuGM-
CSF priming without chemotherapy or while a patient is
receiving growth factor to abrogate the myelosuppression
583
of chemotherapy have been used to supplement bone
marrow after autologous bone marrow transplantation
[36]; to support dose-intensified chemotherapy when
growth factor alone is not adequate [76]; and when autol-
ogous bone marrow alone was used for hematopoietic
support [27, 43]. Although the early trials were conducted
primarily in patients undergoing autologous transplant,
the technique has been applied to normal donors and for
allogeneic stem cell transplant harvesting.
PBPC were initially used in combination with autolo-
gous bone marrow, and were found to reduce the time to
hematologic reconstitution [12, 36, 68]. Durable reconsti-
tution was demonstrated using PBPC alone [37, 68, 76].
CSF-mobilized PBPC are preferred because of the ability
to harvest great numbers of PBPC, the simplicity of har-
vesting, the ability to harvest from patients with a history
of pelvic irradiation and/or other causes of poor bone
marrow reserve, and the rapidity of engraftment [9].
For autologous, allogeneic, and syngeneic transplan-
tation, rHuGM-CSF has been administered after the con-
ditioning regimen and reinfusion of bone marrow and/or
PBPC to shorten the duration and reduce the severity of
neutropenia, and thus decrease the requirement for sup-
portive care, and the duration of hospitalization [10, 55,
64, 65].
Bone Marrow Failure States
GM-CSF has multilineage activity, in vitro and some-
what more variably in vivo. It can also enhance effector
cell function, and induces differentiation of some leuke-
mic cell lines. Based on these activities, it was thought
to be a natural candidate to study as therapy for patients
with myelodysplastic syndromes (MDS), who frequently
have bi- and tri-lineage cytopenias, as well as dysfunc-
tional granulocytes. The focus of most of these studies
was improvement in blood counts, and determination if
myeloblasts were stimulated [3, 26, 32, 40, 49, 84, 85].
A wide range of doses were administered by a variety of
dosing schedules for all of the preparations of rHuGM-
CSF. The reported studies, for the most part, have
included relatively small numbers of patients. Their dis-
ease status covered the spectrum from refractory anemia
(RA) to the poorer prognosis refractory anemia with
excess of blasts (RAEB), refractory anemia with excess
of blasts in transformation (RAEB-t), and chronic myel-
omonocytic leukemia (CMMoL). In general, these studies
reported comparable increases in neutrophil counts but
no consistent improvement in the other hematopoietic
cell lines [29]. An increase in bone marrow and periph-
eral blood myeloblasts and conversion to acute myelog-
enous leukemia (AML) occurred more often in patients
with RAEB and RAEB-t, but in many cases, the number
584
of myeloblasts decreased and/or returned to baseline
upon discontinuation of the growth factor. In a number of
patients, the drug was discontinued due to progression
of disease or to side effects. Several investigators tried to
circumvent the issues of toxicity and myeloblast stimu-
lation by manipulating the dosing of the GM-CSF [54].
Acute Leukemia
Use of hematopoietic growth factors has taken a number
of approaches in patients with acute leukemia: for facili-
tating recovery from the myelosuppressive effects of
induction and/or consolidation chemotherapy to reduce
morbidity; as a means of sensitizing myeloid leukemic
cells to chemotherapy by recruitment of these cells into
S-phase followed by treatment with a cycle-specific
drug, for harvesting PBPC (after intensive chemotherapy)
that may be used for stem cell support after bone marrow
ablation; and as differentiation/maturation therapy in
myeloid leukemia. There has been appropriate concern
about possibly stimulating the leukemic population,
especially in AML, with rHuGM-CSF since such activity
has been reported in vitro [42, 62, 86]. Sargramostim
showed reduction in early deaths due to fungal infections
and reduction in neutropenia in a small randomized trial
that led to its registration in this setting.
The second approach taken in the use of myeloid
growth factors in patients with AML has been an attempt
to cycle activate leukemic cells, thus sensitizing them to
cycle-specific chemotherapy, such as cytosine arabino-
side, and potentially improving remission rates [4, 8, 17,
25, 30, 47]. None of the studies showed an absolute
change >15% in the S-phase fraction of cells during
treatment with rHuGM-CSF; in general the percent
change was small.
The use of rHuGM-CSF in acute lymphoblastic leu-
kemia (ALL) has been less controversial, but even fewer
studies have been reported. The complex nature of most
ALL chemotherapy regimens, which include frequent
dosing, and the difficulty in determining appropriate
growth factor dosing, has undoubtedly contributed to
the limited number of clinical trials.
Current Issues
Use in Children
Although G- and GM-CSF were placed into clinical trials
in the late 1980s and approved by the FDA in 1991,
most clinical trials have been conducted in adult patients.
As is commonly the case with new agents, the evaluation
Granulocyte-macrophage colony-stimulating factor
of these agents in the pediatric population was extremely
limited by concerns about their long-term effects. One
of the earliest areas of investigation for myeloid growth
factors in pediatric patients was chronic severe neutro-
penia [31, 90]. Because of the limited numbers of studies
using these agents, that permitted enrollment of children,
the safety and efficacy was not established until consid-
erably later than when the same was defined for adults.
In pediatric oncology, these agents were used as adjuncts
to chemotherapy and radiation to facilitate engraftment
after transplantation, and to mobilize PBPC. Guidelines
for their use in children were not available until a
European expert panel convened to define them and
finally published their recommendations in 1998 [75].
Until that time, guidelines published by ASCO were
extended to children, despite the lack of clinical trials to
support those recommendations [1, 2]. The ASCO
guidelines for clinical use of CSFs: 2000 update made
no significant changes to its previous recommendations
for their use in the pediatric population [67].
Clinical trials using G- and GM-CSF as mobilizing
agents for PBPC have demonstrated efficacy for collect-
ing autologous stem cells that can reduce the period of
neutropenia compared with bone marrow. Based on
such studies, the European guidelines recommended the
routine use of CSF in children for this purpose. On the
other hand, despite the now common use of CSF to
mobilize PBPC from normal donors in the adult popula-
tion, there has been significant hesitation in extending
that practice to the pediatric population. The limited
information available regarding the long-term effects of
CSF in normal pediatric subjects resulted in the state-
ment by the European panel that such use was
contraindicated.
Immunomodulation
GM-CSF has a broad spectrum of activities that can con-
tribute to the immune response in humans. Early in the
studies characterizing this protein, it was recognized that
GM-CSF could induce production of other cytokines
and growth factors such as IL-1, IL-6, and tumor necro-
sis factor (TNF) that also contribute to the expansion of
B and T cells, as well as induce or coinduce TNF-α gene
expression with interferon (IFN)-γ in monocytes [19, 45]
and in conjunction with IL-2, costimulates T-cell prolif-
eration [73].
As the biology of DC has been better defined, it has
become evident that in vitro and possibly in vivo,
GM-CSF is a cofactor in the differentiation and func-
tional activities of these cells. DC are antigen-presenting
cells that have a critical role in T-cell immune responses
Maryann Foote and George Morstyn
[7, 11, 15, 82]. DC can be differentiated from CD34+
hematopoietic progenitor cells in human bone marrow,
peripheral blood, and cord blood [21, 22] when cultured
with GM-CSF plus TNF. They can be cultured from
monocytes using GM-CSF plus IL-4. Antigens are pre-
sented on their surface in association with class II MHC
molecules, to be recognized by T4 helper cells. The
CD4+ cells participate in the development of B cells,
antigen-specific cytotoxic T8 cells; and macrophages,
eosinophils, and natural killer (NK) cells, which are
antigen nonspecific [7]. All these cells may be involved
in an immune response. In addition to its activity on the
function of DC, GM-CSF increases expression of class
II MHC molecules on the DC, which are essential to
recognition. It is an enhancer of costimulatory molecule
expression – such as B7 and adhesion molecules like
ICAM that are necessary for the interaction of antigen
presenting cells and T cells.
Vaccine Therapy
Since the mid 1990s, many nonclinical and clinical studies
have used genetic engineering techniques to harness the
immunomodulatory activity of GM-CSF to augment
antitumor immunity. Using tumor cell lines or autologous
tumor cells genetically modified to produce GM-CSF,
investigators have shown that this immunostimulatory
protein can enhance tumor immunogenicity in a variety
of tumors. Replicating tumor cells are transduced with
viral supernatants harvested from CRIP packaging cell
lines transfected with MFG-S-human GM-CSF. They
are irradiated and administered as vaccine to tumor-
containing animals or patients. Delayed-type hypersen-
sitivity skin responses were demonstrated against
irradiated autologous cancer cells after vaccination [52].
Biopsies of vaccine sites showed recruitment of DC, T
cells, and eosinophils [48, 81], regression of metastatic
lesions was observed in some studies [28, 52, 57] and
prolongation of survival [25, 48].
Conclusions
rHuGM-CSF has found limited adoption for the pre-
vention of neutropenia and infections in the induction
therapy of acute myeloid leukemia. It has been used
for the mobilization of progenitor cells for PBPC
transplantation. Its effects on other cells of the immune
system acting as a vaccine adjuvant and in DC therapy
are not approved by the FDA for clinical use. In the
future, rHuGM-CSF in these settings may have great
utility.
585
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19 Cancer gene therapy
DONALD J. BUCHSBAUM, C. RYAN MILLER, LACEY R. MCNALLY,
AND SERGEY A. KALIBEROV
Introduction
Cancer is the product of a multi-step process involving
an accumulation of genetic alterations in somatic cells
[203]. Advances in the understanding of the molecular
basis of neoplasia and host–tumor relationships have
resulted in new cancer therapy strategies with transla-
tion into clinical trials. Gene therapy has emerged as a
treatment strategy which relies on the introduction of
genetic material to cure, slow the progression, or possi-
bly even prevent a variety of diseases including genetic,
infectious (HIV in particular), cardiovascular, and
arthritic diseases [11, 66]. Gene therapy has shown
potential for the treatment of cancer. A number of can-
cer gene therapy approaches have been developed based
on direct correction of genetic lesions. These “mutation
compensation” approaches, designed to correct the
molecular lesions underlying neoplastic transformation
and progression, have demonstrated efficacy in the con-
text of in vitro and preclinical model systems. Although
many therapeutic genes exist an efficient, non-toxic
gene delivery system is not currently available. The effi-
ciency and accuracy of gene delivery remain the most
significant barriers to the success of cancer gene therapy
[200]. However, preclinical studies have demonstrated
approaches that may have promise in the clinical set-
ting. The approaches that have been explored include
genetic immunopotentiation with cytokine gene transfer
and tumor cell vaccination, molecular chemotherapy
with prodrug activation by suicide genes or an increase
in tumor cell sensitivity to chemotherapy or radiation
therapy, protection of bone marrow cells from the toxic
effects of chemotherapeutic drugs, inhibition of acti-
vated oncogenes by antisense treatment, transfer of
tumor suppressor genes, and pro-apoptotic gene therapy.
Each of these approaches have been translated into clin-
ical cancer therapy trials [32, 66].
Gene therapy is likely to play a prominent role in the
treatment of cancer in the future. The purpose of this
article is to provide an update on research in gene ther-
apy for the treatment of cancer. Several excellent recent
reviews of cancer gene therapy have been published
[32, 48, 64, 66, 105, 168]. In this paper, current cancer
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
gene therapy strategies for the treatment of cancer are
summarized in Table 1. However, insufficient informa-
tion is available to evaluate the therapeutic efficacy of
these strategies in most clinical trials. The first clinical
gene transfer trial involved the transfer of gene-marked
tumor-infiltrating lymphocytes into patients with
advanced cancer. There are now approximately 190
active gene therapy protocols which involve thousands
of cancer patients [39]. About 50% have been designed
to augment an immune response against cancers by vac-
cine or direct cytokine gene transduction and about 20
protocols involve a drug-sensitization strategy. Approved
protocols exist for overcoming multi-drug resistance in
breast and ovarian cancer patients receiving myelosup-
pressive chemotherapy.
Gene Transfer Vectors
Vectors are vehicles to carry the genetic material. The
vectors for modification of tumor cells or normal tissues
should allow definitive therapeutic or preventive inter-
ventions. To achieve this end, there is a need for gene
delivery vectors capable of efficient and selective gene
transfer to tumor cells in vivo. One of the major prob-
lems has been a low level of gene transfer in human gene
therapy clinical trials. Recent advances in the develop-
ment of vectors for gene transfer make it likely that gene
therapy will have an increasing role in the clinical treat-
ment of cancer. Vectors are being engineered that will
target specific tumor cell types. Vector requirements
depend on the specific approach and disease. Gene transfer
vectors are either nonviral or viral [96].
Viruses are currently the most effective means of
gene delivery and can be manipulated to express thera-
peutic genes or to replicate specifically in certain cells.
Viral vectors used in in vivo pre-clinical studies have
included retrovirus, adenovirus, adeno-associated virus,
and lentivirus. Each has limitations, and studies leading
to a better understanding of virus–cell interactions are
needed to improve vector technology. Some viruses,
such as adenovirus, only result in transient production
of the therapeutic protein as they do not incorporate the
589
590 Cancer gene therapy

Table 1. Current strategies for cancer gene therapya

Strategy Specific agent Reference
Mutation Compensation
Induction of tumor suppressor genes P53, Rb, BRCA-1 [32, 33, 66, 141, 170]
Pro-apoptotic and cell cycle gene therapy bax, bak, Fas ligand, caspase-9, p21 [88, 101, 148, 149, 211]
Inactivation of oncogenes
Intracellular knockout of growth factor receptors erbB-2 [8, 43, 66]
Antisense k-ras, c-myc, TGFß [66]
Genetic immunopotentiation
Genetic modification of tumor cells cytokines, B7, MHC [41, 50, 66, 201, 219]
Genetic modification of immune effector cells Cytokines [66, 167]
Molecular chemotherapy
Suicide gene therapy HSV-tk, CD [5, 9, 66, 72, 108, 146, 168, 169,
183, 216]
Chemoprotection of bone marrow MDR [36, 126]
Inhibition of angiogenesis flt-1, flk-1, endostatin, angiostatin [13, 52, 104, 174]
Replicative vector oncolysis Adp53, herpes virus [66, 102, 123, 187, 193]
Chemosensitization and radiosensitization bax, bcl-2, erbB-2, p53, cytokines, [14, 30, 31, 42, 47,51, 66, 70, 89, 110,
angiostatin, HSV-tk, CD 117, 143, 151, 154, 169, 180, 181, 216]

a
Reprinted with permission from Mary Ann Liebert, Inc.

Table 2. Characteristics of most commonly used vectorsa

Cell division Duration of
Type Insert size Genome required expression Advantages Disadvantages
Retrovirus 5–7 kb RNA Yes Long-term Potential to integrate into Insertional mutagenesis
genome of target cell
Adenovirus 7–35 kb DNA No Transient Relatively high Local tissue inflammation
transduction efficiency and immune response
into normal and tumor
cells; easy production
and at high titers;
tropism can be modified
Adeno- 2–4 kb DNA No Long-term High transduction Insertional mutagenesis;
associated- efficiency into muscle difficulties with production;
virus and brain do not work in all organs
Herpes simplex Up to 30 kb DNA Yes Transient High transduction Difficult to obtain long-term
virus efficiency gene expression; difficult
to target
Nonviral vectors No limitation RNA or DNA No Transient Repetitive and safe Low efficiency gene transfer
administration feasible

a
Adapted from [66, 105]; Reprinted with permission from Mary Ann Liebert, Inc.

genes into the chromosomes of the target cells [125].
Other vectors, such as adeno-associated viruses, are
limited in the size of therapeutic genes that can be incor-
porated or produce low levels of protein [208]. The
mechanisms by which adeno-associated virus integrates
into the human genome are unclear, and HIV lentiviral
vectors may have biosafety issues. Major limitations of
different vector systems include biosafety risk, random
integration into the genome, need for cell proliferation,
potential for immune responses to the vector, lack of a
tissue-specific response, and unknown duration of effect.
The characteristics of the most commonly used vectors
used in cancer gene therapy are summarized in Table 2.
Retroviral Vectors
Retroviral vectors have been used for many gene transfer
protocols because of their ability to produce stable, high
level gene expression in many cell types as a consequence
of their integration into the target cell genome. There
are several limitations of retroviral vectors, including
the size of the inserted gene, low titers, difficulty in
Donald J. Buchsbaum et al.
large-scale production, and they can only insert their
genes into the genome of actively dividing cells. Vectors
derived from lentiviral retroviruses can transduce gene
expression in both dividing and nondividing cells [96],
and have been used for cancer gene therapy in animal
models [45]. The issues and assays needed to ensure
patient safety with this new vector system are being
defined [22, 49, 156]. Efforts are ongoing to increase the
infectivity of retroviruses through the development of
retroviral vectors which display ligands on their surface
for which receptors are present on target cells. Serious
adverse events in some clinical trials have highlighted
safety concerns when retroviral viral vectors are used for
gene transfer [142].
Adenoviral Vectors
Adenoviral vectors for gene transfer are adenoviruses
(Ad) that have been genetically modified through dele-
tions of the viral genome to create space for insertion of
a foreign transgene [96]. Both replication-incompetent
and replicative Ad vectors have been used in clinical
cancer gene therapy trials. There is no risk for insertion
mutagenesis since they do not incorporate into the
genome. An entire issue of Human Gene Therapy was
devoted to Ad vector safety and toxicity [134]. Ad vec-
tors produce higher levels of gene expression and can be
produced in greater quantities than retroviral vectors
[96]. Another advantage of Ad vectors is their ability to
infect both dividing and nondividing cancer cells. A dis-
advantage is that Ad vectors tend to be recognized as
foreign and therefore elicit host cellular and neutralizing
humoral immune responses directed against the viral
capsid. These inflammatory reactions limit the efficacy
of repetitive administrations of Ad vectors and compro-
mise the persistence of transduced cells in vivo [48].
A solution to overcoming the immune response has been
to produce Ad vectors deleted of all Ad gene products.
Although gene expression is transient following Ad
infection, since the transferred genes do not integrate
into the genome, this produces a biosafety advantage.
To sustain transgene expression levels, repeat adminis-
trations of the Ad vectors are required. Analysis of a
variety of primary human tumors has demonstrated
deficiencies in the primary receptor for Ad, coxsackie
and Ad receptor (CAR), which explains the lack of gene
transfer in many human clinical trials.
Modification of Ad vectors may reduce expression of
endogenous Ad proteins and diminish immune responses
to Ad vectors. Second generation Ad vectors have been
developed to reduce the host immune response against
viral proteins and to increase the duration of therapeutic
591
gene expression [66, 105]. The recognition of CAR
deficiency in most human carcinomas has resulted in the
generation of Ad vectors capable of CAR independent
gene transfer [36, 66]. Strategies that have been devel-
oped to achieve this utilize retargeting ligands as well as
genetic capsid modifications. This has allowed infection
and gene transfer to otherwise refractory tumor cells,
and resulted in therapeutic gain in preclinical models.
These new vectors are currently being evaluated in
human clinical trials [107].
Replication-competent adenovirus vectors, which
cause cytolysis as part of their natural life cycle, repre-
sent an emerging technology that shows considerable
promise as a novel treatment option, particularly for
armed vectors which carry therapeutic genes [135]. The
use of replication competent viruses is an attractive
strategy for tumor therapy because the virus can repli-
cate and spread in situ, exhibiting oncolytic activity
through a direct cytopathic effect, and produce higher
levels of therapeutic gene expression achieved as a
result of adenovirus replication.
Adeno-associated Viruses
Adeno-associated viruses (AAV) have received consid-
erable attention for gene transfer studies and cancer
gene therapy [95, 197]. AAV are capable of infecting
both dividing and nondividing cells, producing stable
and efficient integration of viral DNA into the host
genome, and achieving long-term gene expression. Only
minimal viral gene products are retained in current vec-
tors so that they are not very immunogenic and do not
markedly elicit host cytotoxic T-cell immune responses
against vector-transduced cells. AAV vectors may be
useful for antiangiogenic gene therapy and cytokine
gene transfer and in vivo immunization approaches
[140, 157]. However, major obstacles in the develop-
ment of effective recombinant AAV mediated gene
therapy are infection specificity and gene targeting.
Herpes Simplex Virus
The herpes simplex virus (HSV) has been used to deliver
therapeutic genes to some forms of brain cancer and
prostate adenocarcinoma [96, 202]. HSV displays a
broad host cell range and its cellular receptors, heparan
sulfate (HS), herpes virus entry mediator (HVEM), and
nectin-1 and 2, are widely expressed on the cell surface
of numerous cell types. Herpes viruses have been engi-
neered to replicate selectively in tumors but poorly or
not at all in normal tissues. The problems with HSV-
mediated gene transfer include the cytopathic nature of
592
HSV, the difficulty in maintaining long-term expression
of inserted genes, and low infection efficiency [105].
This suboptimal result may reflect viral gene deletions,
which can reduce the replicative potential of viruses
in tumor cells. For example, deletion of the γ34.5
gene significantly reduced viral growth even in rapidly
dividing cells [194]. Biosafety concerns of HSV relate
to the fact that wild-type virus is highly pathogenic and
cerebral injection causes fatal encephalitis. Toxic and/or
pathogenic properties of the virus must, therefore, be
disabled prior to its use as a gene delivery vector.
Other Viral Vectors
Vaccinia viruses, poxviruses, baculoviruses, and RNA
replicons derived from poliovirus [12] have also been
investigated for use in the delivery of genes for thera-
peutic purposes. A variety of replicative viruses have
been employed for gene transfer including parvovirus,
herpes virus, reovirus, and Ad [66]. The antitumoral
effect is achieved by virtue of the replicative cycle of
the virus to produce oncolysis selectively in tumor cells.
An attractive aspect of replicative viruses is their ability
to achieve an amplified effect as a result of their capac-
ity to spread and infect tumor cells within solid tumors
[7, 37, 65]. Replicative viral vectors have been tested in
human clinical gene therapy trials. Vector modifications
to overcome viral receptor deficiency and to achieve
tumor replicative specificity are being investigated.
Nonviral Vectors
Nonviral vectors have been another promising area of
vector development. Several lipid-, peptide-, and poly-
mer-based systems are being investigated for gene deliv-
ery [34, 73, 144]. Liposomes have been used for gene
transfer of foreign MHC genes in a clinical trial. The
liposomes can be modified to provide for selectivity in
cell targeting. However, the levels of gene expression
are limited by the liposomal degradation of the internal-
ized particles. Systemic administration of cationic lipid/
plasmid complexes yields predominantly transfection of
the lungs, with the major transfected cell type being
endothelial cells [13]. Improved cationic liposomes
delivered the p53 tumor suppressor gene to primary and
metastatic murine and human lung tumors in mice and
this was associated with suppressed tumor growth and
prolonged animal survival with minimal toxicity [160].
Direct injection of naked DNA into tumors using
mechanical methods has been shown to result in gene
transfer and expression [105]. A problem is the inability
to transduce a large number of cells and the fact that the
Cancer gene therapy
DNA is only transiently maintained. This approach has
also been used for the generation of cancer vaccines.
Nonviral vectors are attractive with respect to ease of
large-scale production and lack of specific immune
response [111].
Targeting of Viral Vectors
Transductional Targeting
Several studies have shown that viral vectors can be tar-
geted to specific cell types after attachment of ligands
(e.g. transferrin, folate, epidermal growth factor, fibro-
blast growth factor, peptides, and single chain antibod-
ies (sFv) that bind to tumor cell surface receptors) to the
viral capsid, either by chemical conjugation employing
the Fab fragment of an anti-knob monoclonal antibody,
or by genetic engineering for transductional regulation
of vector infection (Fig. 1) [16, 36, 68, 86]. Tropism-
modified Ad vectors can infect cells that are refractory
to transduction by the native Ad, resulting in enhanced
gene transfer and therapeutic benefit in vivo [162].
Efforts have been made to genetically engineer the viral
capsid proteins to contain cell-targeting ligands. Our
group and others have modified the carboxy terminus of
the Ad fiber protein to incorporate peptides or growth
factors with specificity for tumor cellular receptors [36,
66]. Ad vectors which have been engineered to incorpo-
rate a RGD motif at the carboxy terminus or in the HI
loop for binding to tumor cell surface integrins have
shown markedly enhanced transduction of human cancer
cell lines and primary tumor cells, which express low
levels of the CAR receptor [36, 65, 94]. Combined target-
ing of Ad to glioma cell surface integrins and epidermal
growth factor receptors increased gene transfer into
primary glioma cells [66]. It has been shown that Ad
vectors can be targeted to vascular receptors by using
peptide-based molecular adaptors [196]. Alternatively,
the therapeutic gene can be placed under the control of
a tissue- or tumor-specific promoter which is activated
in tumor cells but not normal cells, and therefore restrict
expression to the tumor cell.
Transcriptional Targeting
Targeted expression of therapeutic genes has been
obtained using transcriptional regulatory sequences from
tumor-specific genes that are ectopically expressed in
cancers, viral genes expressed in virus-associated can-
cers, and tissue-specific genes expressed in cancers
and their tissues of origin [125]. Examples include the
Donald J. Buchsbaum et al. 593

a b c

Anti-fiber
antibody Vector with TSP
(Fab) gene construct

Ligand Peptide Peptide

Primary Cell surface Receptors

Receptors
receptor receptor
(CAR) (CAR)
TSP Gene
Specific Transcriptional
Regulatory Sequence
Tumor Cell Tumor Cell
Gene Expression

Figure 1. Strategies to target Ad vector gene transfer include (a) transductional targeting to modify Ad binding tropism to the
CAR receptor in which a ligand that binds to tumor cell surface receptors is conjugated to a Fab of an anti-knob monoclonal
antibody, (b) transductional targeting by genetic engineering of a peptide with specificity for tumor cellular receptors into the
knob of the Ad fiber protein, and (c) transcriptional targeting using a tumor-specific promoter (TSP) to produce targeted
expression of a therapeutic gene in a tumor cell with specific transcriptional regulatory sequences in combination with genetically
modified Ad. Reprinted with permission from Mary Ann Liebert, Inc.

alpha-fetoprotein promoter for hepatocellular carcinoma,

the hTERT promoter in telemorase positive tumors, the
PSA, PSMA, and probasin promoters for prostate can-
cer, and the MUC1 and erbB-2 promoters for breast can-
cer [61, 125]. The incorporation of these promoters into
gene therapy vectors allows selective expression of the
transduced therapeutic gene. Other examples of induc-
ible promoters that have been investigated are hypoxia-
responsive elements that are induced by hypoxia which
is present in many solid tumors [82], and radiation
responsive promoters that provide a three- to four-fold
induction in gene expression in the radiation field [19].
Regulatory sequences from genes expressed in tumor
endothelial cells and from cell cycle-regulated genes are
also candidates for transcriptional targeting [125].
For example, we have developed Ad vectors encoding
apoptosis-inducing (bax or TRAIL) genes under control
of the VEGF or flt-1 promoter, respectively, and
angiogenesis-suppressing soluble VEGF receptor 2 gene
under control of the VEGF promoter element. Using VEGF
and VEGFR1 promoter for targeting of gene expression
in VEGF/VEGFR1 positive tumor cells as well as tumor-
associated blood vessel endothelial cells significantly
increased therapeutic efficacy of gene therapy in human
prostate tumor xenograft models [90–92].
Mutation Compensation
Induction of Tumor Suppressor Genes
The p53 protein is a multifunctional regulator of cell
growth and plays a critical role in regulating the cell
cycle. When the p53 gene is mutant or absent, the lack
of control of p21 results in lack of inhibition of cyclin
dependent kinases and unregulated cell growth. The p53
protein also plays a central role in the apoptosis path-
way. A large proportion of human cancers carry impaired
p53 gene function. In certain tumor types, dysfunctional
p53 has been associated with more aggressive tumor
growth, enhanced tendency for tumor metastases, and
poor survival rates. The approach of p53 gene transfer
has been used by a number of investigators [141]. When
p53 was transfected into tumor cells and overexpressed,
apoptosis and growth arrest has been observed. It was
shown that transfecting normal p53 to cells with deleted
or mutated p53 decreased anchorage independent
growth in vitro and tumorigenicity in animals [32].
The approach taken by Clayman et al. [32] to p53
gene replacement therapy has been to inject head and
neck squamous cell cancer with an Ad vector encoding
wild-type p53. Patients with head and neck cancer have
594
accessible lesions allowing for direct delivery of vectors.
Intratumoral injection of Ad containing wild-type p53
produced growth inhibition of established head and
neck squamous cell tumors in animal models [32]. The Ad
vector was not toxic to normal squamous oral epithelial
cells or normal fibroblasts in culture, and minimal toxicity
was produced in animal models. Studies performed in
mouse models of non-small cell lung cancer also have
shown a significant therapeutic effect of Adp53 gene
therapy [32]. Studies have shown that vascular endothelial
growth factor (VEGF) expression is reduced in cancer
cells following Adp53 infection, resulting in inhibition
of angiogenesis in vivo [32].
Phase I clinical trials of Adp53 gene transfer have
been completed [32, 33]. The studies demonstrated that
Adp53 gene transfer is safe and well tolerated. Transient
p53 expression occurred in solid tumors despite high
levels of antibodies to Ad. The most frequently observed
toxicities were fever, chills, and injection site pain [32].
A Phase II study of 170 patients with recurrent head and
neck squamous cell cancer further established the safety
and lack of toxicity of direct intratumoral Adp53 injections
[32]. A small percentage of patients had stable disease.
Randomized trials of Adp53 gene therapy in head and
neck cancer patients are currently ongoing.
Other clinical trials have confirmed the safety and effi-
cacy of retroviral or Ad p53 gene replacement therapy in
non-small cell lung cancer, prostate cancer, recurrent
ovarian carcinoma, and esophageal cancer [32, 170,
177]. The results of a recent Phase II study indicated that
intratumoral Ad p53 provided no additional benefits in
patients receiving first-line chemotherapy for advanced
non-small cell lung cancer [176]. In addition, other
replacement strategies that are being investigated clini-
cally include induction of Rb and BRCA-1 gene expres-
sion [65]. Preclinical studies showed expression of the
wild-type Rb and BRCA-1 proteins after gene transfer
and reversion of the malignant phenotype, often associ-
ated with the induction of apoptosis in tumor cells [141].
Another tumor suppressor gene that has been investi-
gated is PTEN [129]. Multiple gene replacements have
been examined as a potential treatment of cancer and
have generally resulted in additive or synergistic effects.
Combination delivery of both p16 and p53 into cancer
cells by adenovirus resulted in an additive or synergistic
apoptotic effect for treatment of cancer [100, 173].
Pro-apoptotic and Cell Cycle Gene
Therapy
Another approach that is receiving attention is pro-apop-
totic cancer gene therapy in which the ratio of pro-apop-
Cancer gene therapy
totic and anti-apoptotic proteins is modified in order
to increase apoptosis. Human tumor cells have been
transduced with bax, bak, and Fas ligand genes in Ad vec-
tors and shown to undergo apoptosis [88, 101, 148, 149].
In addition, the gene for caspase-9 has been used in an Ad
vector to induce apoptosis in human prostate cancer cells
and suppress tumor growth in nude mice [211]. A tumor-
specific apoptotic effect of TRAIL (tumour necrosis fac-
tor-related apoptosis-inducing ligand) has been shown, in
which the TRAIL gene was delivered using Ad or AAV
and shown to have significant anti-tumour efficacy in
animal models of aggressive primary and metastatic
cancer [119]. A limitation of these approaches is that
every cell of the primary tumor and metastases be affected
by the treatment, unless these approaches are used in
combination with other therapeutic modalities.
Induction of apoptosis following p21 gene transfer
which was originally identified as a molecule that regu-
lates transition from the G1 phase to the S phase of the
cell cycle has been reported [87].
Inactivation of Oncogenes
Although most oncogenes are silenced after fetal devel-
opment to prevent abnormal tissue growth, cancer cells
may propagate by activating or amplifying oncogenes.
One form of cancer gene therapy is the targeted disrup-
tion of tumor oncogenes by: (1) inhibition of oncogene
transcription; (2) reduction of mRNA translation into
protein; and (3) interference with protein function.
Inhibition of erbB-2, and blockade of k-ras, c-myc,
c-fos, TGFß and insulin-like growth factor 1 are
approaches that are being investigated clinically [66].
Transcription of dominant oncogenes has been inhibited
using triplex-forming oligonucleotides. Ad gene E1A
that inhibits transcription of the human c-erbB-2 pro-
moter suppressed tumorigenicity and metastatic poten-
tial induced by the erbB-2 oncogene. Translation of
oncogene messenger RNA has been blocked using spe-
cific antisense sequences [66]. These include antisense
treatment against k-ras in lung cancer, c-myc in breast
and prostate cancer, and TGFß in glioma. The erbB-2
oncoprotein has been inhibited with the use of intracel-
lular antibodies. In this regard, Deshane et al. [43] from
our group showed that intracellular expression of an anti
erbB-2 sFv following Ad gene transfer resulted in down-
regulation of cell surface erbB-2 expression, and cyto-
toxicity in human ovarian cancer cells both in vitro and
in vivo in an animal model. This approach was translated
into a clinical Phase I trial in patients with ovarian cancer
[8]. Of the 13 patients evaluable for response, 5 (38%)
had stable disease and 8 (62%) had progressive disease.
Donald J. Buchsbaum et al.
Genetic Immunopotentiation
Genetic immunopotentiation strategies are designed to
achieve active immunization against tumor associated
antigens by gene modification of tumor cells to enhance
their immunogenicity, or enhance the anti-tumor activity
of immune system cells [53]. Genetic immunopotenta-
tion is most utilized for the treatment of melanoma.
Tumor vaccines are used to initiate an immune response
against an unrecognized or poorly antigenic tumor.
Treatment with unmethylated cytosine–phosphate–gua-
nine (CpG) activates plasmacytoid dendritic cells, thus
releasing IFN alpha and boosting T-cell and natural killer
cell responses as well as activating conventional myeloid
dendritic cells to treat early stage melanoma [131].
Alternatively, metastatic melanoma treatment with retro-
viral vectors armed with IFN-gamma is currently in
phase I clinical trials as a tumor cell vaccine [60].
Genetic Modification of Tumor Cells
It has been hypothesized that vaccination against the
tumor by exposing tumor antigens to the immune sys-
tem in a more favorable context will result in tumor
rejection [66]. A major focus of research is the identifi-
cation of new tumor antigens and the development of
cancer vaccines and new anticancer drugs. The first
clinical trials with cDNA vaccination with carcinoem-
bryonic antigen demonstrated limited benefit. Cytokine
or co-stimulatory molecule B7 gene modified tumor
cells and defined tumor antigens have also been used.
Numerous studies have shown that tumor cells can be
transfected with various cytokine genes and become tar-
gets for specific immune rejection [201]. Transduction
of a cytokine gene into tumor cells elicits an inflamma-
tory host reaction that impairs tumor growth [124, 207].
This approach has been used in clinical trials [63, 201].
Several investigators have shown that exposure to tumor
cells induced to express B7 can be highly effective at
enabling normal animals to reject subsequent challenge
by tumor cells, but this approach has been much less
effective at enabling tumor-bearing animals to kill their
pre-existing tumor [50]. In a melanoma trial with autol-
ogous irradiated tumor cells transduced with GM-CSF,
20% of patients who received three vaccinations were
alive after a follow-up of 3–5 years [41]. Live attenuated
viruses have been used in vaccines. Cationic liposomes
that encapsulate DNA expression plasmids which
encode allogeneic MHC molecules have been used to
generate tumor vaccines in situ [219]. In these approaches
the genetic modification of tumor cells and effector
immune cells can be performed ex vivo, thus enhancing
595
the level of gene transfer and avoiding toxicity com-
pared to in vivo gene transfer. However, the level of gene
transfer into tumor and immune effector cells in clinical
trials has so far been limited [66].
Genetic Modification of Immune
Effector Cells
Cells of the immune system including lymphocytes, NK
cells, and dendritic cells have been genetically modified
to augment their capacity to kill tumors [66]. Tumor
infiltrating lymphocytes were the first immune cells to
be genetically transduced and used in a human gene
therapy trial against melanoma [167]. Although attempts
have been made to modify their binding tropism [66],
the modest localization and toxicity of these lympho-
cytes remains a limitation for this form of therapy. Using
dendritic cell vaccines requires the cells to be loaded
with antigen for presentation to elicit an immune
response to tumor [175]. The first reported dendritic cell
vaccination study was for indolent lymphoma with other
trials for lymphoma [78]. In the case of glioma, tumors
have been targeted by bone marrow-derived dendritic
cells loaded with glioma cell mRNA resulting in a spe-
cific T cell response to protect against intracerebrally
implanted glioma tumor cells [114]. Numerous studies
continue to evaluate the effectiveness of dendritic cell
vaccines in preventing tumor relapses and extending
patients’ survival [218].
Molecular Chemotherapy
Evolution of Molecular Chemotherapy
Paradigm
The narrow therapeutic index of drug toxicity to tumor
versus normal tissues, has significantly limited conven-
tional systemic chemotherapy and necessitated further
drug development research aimed at designing more
selective chemotherapeutic agents.
Because systemic toxicity is the main limitation to
cytotoxic chemotherapy, one approach towards increasing
its therapeutic index is through research on alternative
forms of drug delivery. One promising alternative
employs gene transfer to engineer either (1) increased
tumor cell sensitivity or (2) decreased normal tissue
sensitivity to systemically administered cytotoxic agents.
This approach, which has been termed molecular chemo-
therapy, gene-directed enzyme-prodrug therapy (GDEPT),
or suicide gene therapy involves delivery of a specific
enzyme that can produce cell death through the conversion
596
of an inactive non-toxic prodrug into a cytotoxic drug
metabolite. Specifically targeted expression of the prod-
rug-activating enzyme avoids systemic toxicity, and
results in high drug concentration in the tumor mass and
an improved therapeutic index compared to systemic
drug administration. The key element of a GDEPT therapy
system is a gene that encodes an enzyme, which con-
verts a prodrug to an active cytotoxic drug. Importantly,
prodrug-activating enzymes are normally absent or
poorly expressed in mammalian cells. This means tumor-
targeting of gene therapy, using specific delivery vehicles,
restricts enzyme expression to the transduced tumor cells
and adjacent surrounding tumor cells through diffusion
of the drug metabolite to generate a bystander effect.
From Concept to Clinical Trials
A number of enzyme-prodrug (EP) systems have been
investigated for cancer gene therapy over the last 20
years. Of the nine classes of traditional chemotherapeutic
agents, alkylating agents, anthracyclines, antibiotics,
antimetabolites, camptothecins, platinum agents, podo-
phyllotoxins, taxanes, and vinca alkaloids, EP systems
have been described for five. A comprehensive overview
of these systems and their development in cell culture and
animal model systems was outlined in the previous edition
of this text [21]. Here, we will focus on the clinical devel-
opment of these approaches over the past 5 years.
One of the most widely studied suicide gene-prodrug
systems for cancer gene therapy utilizes the herpes
simplex virus-thymidine kinase (HSV-tk) in combina-
tion with anti-herpetic drugs such as a 9-[2-hydroxy-1-
(hydroxymethyl)-ethoxy]-methylguanine (ganciclovir;
GCV) or its analogues (acyclovir and valacyclovir). The
HSV-tk/GCV system is characterized by the effective
phosphorylation of GCV into a toxic compound that is
incorporated into DNA during its replication. This incorpo-
ration into guanine sites in newly synthesized DNA chains
causes termination of synthesis and the selective killing
of dividing cells by activation of apoptosis pathways.
The first report of GDEPT for cancer appeared in the
literature in 1986 and involved HSV-tk and the prodrug
GCV [132]. HSV-tk GDEPT was tested in the first clini-
cal gene therapy trial for cancer in humans, which began
patient accrual in 1993 [145] and paved the way for a
series of similar clinical trials involving retrovirus-
mediated delivery of HSV-tk. Initial studies in animal
models suggested that the main limitation to this strategy
was the limited HSV-tk gene transfer efficiency to tumor
cells mediated by murine fibroblast-based retroviral vector
producer cells. These findings were later confirmed in a
large, multinational phase III prospective, open-label,
Cancer gene therapy
randomized controlled trial, the most comprehensive
cancer gene therapy clinical trial to date, published by
the GLI-328 International Study Group in November
2000 [159]. This study assessed the efficacy of standard
therapy, consisting of surgery plus fractionated external
beam radiotherapy, versus standard therapy plus adju-
vant, intratumoral HSV-tk retroviral gene therapy at the
time of surgery in 248 patients with previously untreated
(grade IV) glioblastoma multiforme (GBM). No signifi-
cant differences were found between the two treatment
arms in either overall median survival (354 versus 365
days, respectively, p > 0.05) or progression-free survival
(183 versus 180 days, respectively, p > 0.05). At autopsy
or post-therapy biopsy, 7 of 17 (41%) tumors and 1 of
13 (8%) normal brains tested positive for retroviral vec-
tor DNA by PCR. Thus, anecdotes and case reports of
response notwithstanding [198], 20 years of preclinical
and clinical testing of retroviral vector-mediated, HSV-
tk-based cancer gene therapy failed to achieve signifi-
cant clinical benefit in patients with GBM.
As of April 2008, 121 clinical trials of enzyme-prod-
rug cancer gene therapy had been initiated in 14 differ-
ent countries for over 20 different tumor types [1].
Despite the major setbacks to the field in the early 2000s,
the number of new enzyme-prodrug clinical trials
worldwide continues to grow at approximately the same
rate (six new trials per year). The majority (77 of 90,
86%) have utilized either retroviruses or adenoviruses
to effect gene delivery. Although retroviruses remain the
single most common gene transfer vector employed
worldwide (Fig. 2a), its use has plateaued in the United
States since 1996 (Fig. 2b) and no new enzyme-prodrug
trials for cancer have been presented at the NIH
Recombinant DNA Advisory Committee (RAC) since
2000 [2]. Problems with retrovirus vectors are well-
established [190] and alternative vectors, including rep-
lication-competent adenovirus and herpes simplex
viruses (HSV) are becoming increasingly employed.
HSV-tk/GCV remains the most actively investigated
enzyme-prodrug combination, but alternative systems,
including cytosine deaminase (CD) and the combina-
tion of HSV-tk and CD either as separate or fused trans-
genes, are becoming more frequent, both in the US and
worldwide (Fig. 3). CD/5-fluorocytosine (CD/5-FC)
was proposed as an alternative enzyme-prodrug system
in 1992, shortly after the initiation of phase I clinical
trials with HSV-tk/GCV [133]. In contrast to GCV,
5-FC, a non-toxic antifungal agent utilized clinically for
CNS mycoses, produces a well-characterized, highly
effective chemotherapeutic agent, 5-FU, upon intratumoral
conversion by CD. Moreover, 5-FU is freely diffusible
and, unlike GCV, does not require gap junction-mediated
Donald J. Buchsbaum et al. 597

Figure 2. Enzyme-prodrug cancer gene therapy clinical trials worldwide (a) and in the United States (b). The cumulative
number of clinical trials were determined from the gene medicine [1] and NIH Recombinant DNA Advisory Committee (RAC)
databases [2] and stratified by gene transfer vector employed. Abbreviations: Ad, adenovirus; HSV, herpes simplex virus
598 Cancer gene therapy

Figure 3. Enzyme-prodrug cancer gene therapy clinical trials worldwide (a) and in the United States (b). The cumulative number
of clinical trials were determined from the gene medicine [1] and NIH Recombinant DNA Advisory Committee (RAC) databases
[2] and stratified by transgene delivered. Abbreviations: CD, cytosine deaminase; CYP1B1, cytochrome P450 1B1; HSV, herpes
simplex virus; HSV-tk*, genetically modified HSV-thymidine kinase transgene or co-delivery of HSV-tk and other transgenes;
TK, thymidine kinase
Donald J. Buchsbaum et al.
intracellular communication to elicit a potent bystander
effect [67, 179].
5-FU is also a well-established, clinically useful radi-
osensitizing agent commonly employed for chemoradi-
ation of gastrointestinal (GI) malignancies [189]. We
have previously demonstrated that adenovirus (Ad)-
mediated expression of E. coli CD (AdCMVCD),
together with 5-FC, a combination termed virus-directed
EP therapy (VDEPT), and external beam radiation
(XRT) significantly enhanced in vitro cytotoxicity and
in vivo tumor growth control in both GI [151, 152] and
non-GI [128] tumors relative to those receiving
AdCMVCD/5-FC alone. Moreover, incorporation of
AdCMVCD/5-FC into clinically appropriate fraction-
ated XRT schemes significantly enhanced local tumor
growth control relative to single high-dose XRT [181].
One promising approach towards generating more
potent enzymes for EP gene therapy involves utilizing
either homologous enzymes from different microbial
species or mutated endogenous enzymes that possess
improved kinetics, facilitating enhanced prodrug con-
version. For example, CD/5-FC EP systems have been
described using CD from at least three different micro-
organisms: the bacterium E. coli [133], the yeast
Saccharomyces cerevisiae [98] and Candida albicans [10].
Lawrence and colleagues demonstrated the catalytic
superiority of recombinant CD derived from the yeast
Saccharomyces cerevisiae (yCD) relative to E. coli CD
(bCD) [98] and that the favorable enzyme kinetics of
yCD translate into (1) enhanced tumor regression after
systemic 5-FC [98], (2) 19F-NMR-based detection of
CD activity [182], and (3) radiosensitization [99].
We have recently described benefits of the second
approach, specifically mutation of E. coli CD [93].
Substitution of alanine (A) for aspartic acid (D) at posi-
tion 314 of CD increased the relative specificity of the
mutant CD-D314A enzyme to 5-FC in comparison with
wild-type CD [121] and increased prodrug conversion
and cytotoxicity to human glioma cells in vitro [93]. We
constructed the AdbCD-D314A vector, encoding mutant
bCD-D314A gene and investigated mutant bCD gene
transfer in an Ad-directed molecular chemotherapy
approach for treatment of human glioma xenografts.
This study demonstrated that AdbCD-D314A infection
resulted in increased 5-FC-mediated cell killing, compared
with AdbCDwt. Therapy with a replication-incompetent
adenovirus encoding CD-D314A with 5-FC and XRT
showed significant reduction in clonogenic glioma
cell outgrowth in vitro and subcutaneous xenograft
growth in vivo compared to wild-type CD/5-FC/XRT.
Finally, increased 5-FC conversion efficiency of this
CD-D314A system led to improved survival of mice
599
bearing orthotopic, intracranial xenografts of human
glioblastomas after 5-FC therapy and efficacy was further
potentiated by concomitant fractionated external-beam
XRT [93].
Co-delivery of cDNAs encoding multiple prodrug
metabolizing enzymes, either as separate or fused coding
sequences, is another promising approach first described
for the two most common EP systems, HSV-tk/GCV and
CD/5-FC. The CD-TK fusion gene proved to be synergis-
tic when co-expressed in tumor cells from two separate
coding sequences [6]. Freytag and colleagues extended
this approach by combining enzyme-prodrug and onco-
lytic viral therapy through use of a CD-TK fusion con-
struct delivered by a replication-competent adenovirus
(Ad5-CD/TKrep) [166]. Freytag, Kim, and colleagues at
Henry Ford Hospital have initiated three phase I clinical
trials with Ad5-CD/TKrep in patients with prostate carci-
noma, either alone [54], with concomitant conformal
XRT [59], or with intensity-modulated radiation therapy
(IMRT) [54]. CD-TK gene therapy proved to be safe in
all three trials. Although the first trial was a phase I dose-
escalation trial and was not powered to determine effi-
cacy, preliminary analyses of 5-year follow-up data from
16 patients treated with direct in situ injection of 1010–
1012 Ad5-CD/TKrep particles and a 1 week course of
GCV/5-FC showed an increase in the prognostically
meaningful surrogate endpoint of mean prostate-specific
antigen (PSA) doubling time (PSADT) [59]. The authors
demonstrated a significant increase in PSADT from 16 to
31 months. Androgen suppression therapy (AST) is a pal-
liative approach for patients with treatment-refractory
prostate carcinoma and is associated with significant
patient morbidity. The authors demonstrated that the pro-
jected onset of AST in the treated cohort of patients was
delayed by 2 years [59]. The exact mechanisms of the
apparent therapeutic effect will require additional clinical
trials and follow-up correlative molecular studies.
However, at a time of decreased pharmaceutical company
interest in enzyme-prodrug gene therapy and tight fund-
ing of biomedical research, these results have been hailed
in the gene therapy and urological communities [191] as
evidence that this line of research merits continued finan-
cial support and multi-institutional testing of this approach
in phase II clinical trials.
Inhibition of Angiogenesis
Extensive preclinical and clinical data support the con-
cept that tumor growth is dependent on angiogenesis
and that VEGF plays a central role in this process.
Inhibition of angiogenesis is one of the major targets for
600
anti-cancer gene therapy strategies [52]. An advantage
of the anti-angiogenic approach is the highly amplified
death of a large number of tumor cells when deprived of
their vasculature, which can partially overcome current
limitations in the number of tumor cells modified by
gene transfer in vivo, as well, it offers an alternative
means of tackling multidrug-resistant tumors that have
proved intractable to conventional chemotherapies
because unlike cancer cells, endothelial cells are stable
and do not mutate [20]. An additional advantage is the
direct access of vectors delivered systemically to vascu-
lar endothelial cells. Both suppression of cellular angio-
genic signals and augmentation of inhibitors of
angiogenesis have proven to be feasible strategies in
preclinical tumor models [104]. It has been shown that
Ad-mediated anti-VEGF therapy using a gene encoding
a soluble VEGFR1/flt-1, a naturally encoded, alterna-
tively spliced form of flt-1 VEGF receptor, can be used
to control tumor growth [52, 122]. Treatment with
AdVEGF-sKDR, encoding a soluble VEGF receptor 2
under control of the VEGF promoter, significantly
inhibited the proliferation and migration of human vas-
cular endothelial cells and prostate cancer cells.
AdVEGF-sKDR also sensitized cancer cells to ionizing
radiation. In vivo tumor therapy studies demonstrated
significant inhibition of tumor growth in mice that
received combined AdVEGF-sKDR infection and radi-
ation versus AdVEGF-sKDR alone or radiation therapy
alone [92]. Similarly, suppression of other angiogenic
factors such as basic fibroblast growth factor (bFGF) by
Ad mediated expression of antisense basic fibroblast
growth factor (bFGF-AS) resulted in significant inhibi-
tion of transitional cell carcinoma growth [83]. RNasin,
the placental ribonuclease inhibitor, is known to have
anti-angiogenic activity through the inhibition of angio-
genin and bFGF. A plasmid-mediated gene therapy
approach employing Rnasin in B16 murine melanoma
cell lines significantly inhibited angiogenesis and tumor
metastatic progression [18].
Angiopoietins regulate blood vessel assembly and
mediate their activity through the receptor tyrosine
kinases Tie-1 and Tie-2. Administration of an Ad vector
expressing a soluble Tie-2 receptor (AdExTek) an
endothelium-specific receptor tyrosine kinase, which is
capable of blocking Tie-2 activation, significantly inhib-
ited the growth rate of tumors in mice with two different
well established primary tumors, a murine mammary
carcinoma (4T1) or a murine melanoma (B16F10.9),
64% and 47%, respectively. It has also been shown that
administration of AdExTek therapy inhibited tumor
metastasis when delivered at the time of surgical exci-
sion of primary tumors [116].
Cancer gene therapy
Several immuno-cytokines and or chemokines have
been employed in anti-angiogenic gene therapy strategies.
Interferon alpha (IFN-alpha) gene delivery by electropo-
ration produced regression of squamous cell carcinoma
(SCVII) tumors in 50% of the mice and increased sur-
vival time more than two fold [112]. Similarly, inhibition
of tumorigenicity and metastasis of human bladder car-
cinoma in athymic mice by Ad-mediated IFN-beta gene
therapy is also attributed to the inhibition of angiogenesis
[85]. IFN-gamma gene therapy employing retrovirus
completely eradicated intracranial C6 glioma tumors in
an immunocompetent mouse model [171]. The IFN-
gamma mediated tumoricidal activity is due to an appar-
ent interplay of B and T cell components of the immune
system, as well as the inhibition of tumor angiogenesis.
Further, Ad-mediated gene therapy of proliferin-related
protein (PRP), a strong inhibitor of endothelial cell
migration or interferon inducible protein (IP10), a sup-
pressor of capillary tube formation, significantly sup-
pressed murine melanoma tumor growth [163]. A recent
study also reported that gene transfer of human IP10
using replication-competent retroviral (RCR) vectors
markedly reduced growth in xenograft and syngeneic
mouse models associated with a marked reduction in
microvessel density [185].
Interleukin-8 (IL-8) is a mediator of angiogenesis.
Based on this mechanism, an Ad-mediated antisense
IL-8 gene therapy significantly inhibited human bladder
carcinoma in athymic nude mice [84]. Similarly, treat-
ment of subcutaneous and intracranially established rat
C6 cell glioma by retroviral delivery of interleukin-4
(IL-4) in situ, resulted in tumor growth arrest and was
associated with eosinophil infiltration and inhibition of
angiogenesis [172].
Matrix metalloproteinases (MMPs) play a critical role
in degradation of endothelial basement membrane which
are required to initiate angiogenesis, thus promoting
angiogenesis and cancer progression. TIMP-2 is a natural
MMP inhibitor. Ad-mediated TIMP-2 gene therapy
significantly reduced tumor growth rates by 60–80%,
angiogenesis by 25–75%, and increased survival by 90%
in murine lung, colon, and human breast cancer models
in mice [108]. TIMP-1 gene transfer delivered by an AAV
vector inhibited angiogenesis via reduced endothelial
cell migration and invasion in vitro and inhibited tumor
growth in a murine xenotransplant model [222].
Treatment with this low dose of AdhTIMP-2 produced a
synergistic effect in combination with TNF/IFN-gamma
using the murine B16BL6 melanoma model [199].
Angiogenesis is regulated by several angiogenic
agents and at multiple levels [103]. The anti-angiogenic
molecules function by different mechanisms, including
Donald J. Buchsbaum et al.
endothelial cell proliferation, migration, protease activity,
and tubule formation. Therefore, it can be inhibited by
different methods either by a combination of angiogenic
inhibitors or combination of tumor suppressor agents
and angiogenic inhibitors, and/or combination of immu-
notherapy and angiogenesis inhibitors. For example, ret-
roviral mediated angiostatin and endostatin combination
gene therapy showed synergistic anti-tumor activity and
survival in murine leukemia and melanoma models with
complete loss of tumorigenicity in 40% of animals in the
leukemia model [174]. Thrombospondin I has been
shown to inhibit angiogenesis. However, a synergistic
inhibition of MDA-MB-435 breast cancer tumor growth
has been shown with a combination gene therapy of lipo-
somes complexed with p53 and thrombospondin I encod-
ing plasmids BAP-TSPf and BAP-p53 when compared
to either BAP-p53 or BAP-TSPf alone [214].
Antigen specific cancer immunotherapy and anti-
angiogenesis have emerged as two attractive strategies
for cancer treatment. An innovative approach that com-
bines both mechanisms which has potent anti-tumor
activity has been reported using Calreticulin (CRT)
which has the ability to enhance MHC class I presenta-
tion and exhibit an antiangiogenic effect [28]. Another
study reported that treatment of murine breast carcinoma
with combined Ad-mediated murine angiostatin and
Ad-mediated murine IL-12 resulted in 96% of the ani-
mals developing initial regression with 54% undergoing
total regression of the tumor. Further, mice were resistant
to rechallenge and developed strong CTL response [71].
One novel form of gene therapy has been reported
wherein a cell based anti-angiogenic gene therapy
approach has been employed. Fibroblasts were retrovi-
rally transduced to overexpress thrombospondin-2 to
inhibit human squamous cell carcinoma, malignant mel-
anoma, and Lewis lung carcinoma growth [184].
However, a similar study using hematopoietic stem cells
transduced with retrovirus encoding a secretable form
of endostatin did not show inhibition of neoangiogene-
sis or anti-tumor activity [150]. Another study employed
a gene therapy approach with murine bone marrow-
derived cells encoding soluble flk-1 to inhibit tumor
growth. Significant reduction in tumor size was reported
when challenged with tumor within 3 months after
transplantation with bone marrow-derived cells encod-
ing truncated soluble flk-1 [40].
The induction of several inhibitors of angiogenesis
has been carried out by transfection of cells with the
Thrombospondin-1 gene, or by using viral vectors that
encode the genes for soluble platelet factor-4 and
angiostatin [52]. Cationic lipid based delivery of endostatin
gene sequences and its expression in muscle suppressed
601
primary tumor growth and development of metastases in
the lungs of mice [13]. Further, retroviral-mediated
delivery of angiostatin and endostatin in both murine
leukemia and melanoma models produced enhanced
antitumor efficacy [174]. Also, Ad-mediated murine
endostatin gene therapy prolonged survival and in 25%
of the mice completely prevented tumor growth in a pro-
phylactic human colon/liver metastasis xenograft murine
model [25]. However, high dose intravenous delivery of
Ad-mediated human endostatin was associated with
severe acute toxicity in mice that included loss of weight,
bleeding, death of animals [204].
Although there is convincing proof of concept in ani-
mal models that an anti-angiogenesis gene therapy
approach can be used to treat cancer, realization of its
full potential for the treatment of a broad array of dis-
eases will require several challenging technical hurdles
such as duration of expression, induction of immune
response, cytotoxicity of the vectors and tissue specific-
ity to be overcome and safety concerns to be alleviated
[24, 94, 113, 168, 206].
Replicative Vector Oncolysis
One approach to overcome suboptimal tumor transduc-
tion of non-replicative viral vectors is the use of condi-
tionally replicative viral vectors, in which replication
competent virus selectively replicates within tumor cells
but not in normal tissues. Release of progeny virions
from the initially infected tumor cells would infect
neighboring tumor cells and thereby spread throughout
the tumor volume. In addition, the use of viruses that
have a lytic life cycle would result in virus-mediated
oncolysis [66].
The currently available replicating vectors for cancer
oncolytic virotherapy include Ad or HSV-1 and can be
divided into two large groups: genetically engineered
viruses with enhanced selectivity and/or decreased tox-
icity (including a wide variety of vectors with specific
deletions in viral genes or that encode essential genes
under control of tumor specific promoters) and vectors
engineered to function as therapeutic gene delivery
vehicles by incorporating an expression cassette con-
taining a transgene to improve the antitumor efficacy of
oncolytic virotherapy (including GM-CSF or CD/TK
genes). Importantly, some of these vectors have already
been explored in extensive preclinical studies with clinical
evaluation in patients showing significant antitumor
effects. As of April 2008, 28 clinical trials had been initi-
ated employing Ad, HSV-1, Vaccinia Virus and Poliovirus
for different tumor types [1].
602
Since the E1B 55kD gene product in Ad vectors is
responsible for p53 binding and inactivation, it was
hypothesized that an E1B 55kD deletion mutant Ad
would be unable to inactivate p53 in normal cells and
thus would be unable to replicate efficiently. Because
p53 is absent in many tumors, the replication of a mutant
Ad would be selective in tumors. A selectively replica-
tion-competent E1B 55kD gene-deleted Ad, dl1520
(ONYX-015) has been injected into solid tumors of
patients whose tumors carry mutant p53. Clinical trials
using ONYX-015 alone produced minimal tumor
responses in patients with head and neck cancer while
combination treatment with chemotherapy produced a
63% response rate [102].
In recent years, two major strategies have been devel-
oped for specific retargeting of conditionally replicating
Ad vectors. In the first one, transcription of essential
viral genes has been controlled by replacing the native
viral promoters with tumor or tissue-specific promoters.
A number of oncolytic Ads have been generated in
which the gene essential for viral replication is under
the control of exogenous promoters that are preferen-
tially active in tumor cells [77, 158, 197]. One group has
produced a conditionally replicative Ad in which the
expression of E1A is controlled by the midkine pro-
moter that induced tumor cell killing of neuroblastoma
and Ewing’s sarcoma cells [3].
CN706 is an oncolytic conditionally replicating Ad
vector, encoding the E1A gene under the control of an
exogenous minimal enhancer/promoter construct
derived from the 5′ flank of the human PSA gene pro-
moter [165]. CN706 was tested in a phase I clinical trial,
in which virus was intratumorally injected into patients
with locally recurrent prostate cancer without dose-lim-
iting toxicity [44]. Another example of a specifically
targeted replication-competent Ad vector, Ad-hOC-E1,
which contains a single bidirectional human osteocalcin
(hOC) promoter to drive both the early viral E1A and
E1B genes was constructed. This vector selectively rep-
licated in OC-expressing prostate cancer cells and viral
replication was enhanced at least ten-fold with vitamin
D3 exposure. Unlike Ad-sPSA-E1, an Ad vector with
viral replication controlled by a strong super prostate-
specific antigen (sPSA) promoter which only replicates
in PSA-expressing cells with androgen receptor (AR),
Ad-hOC-E1 retarded the growth of both androgen-
dependent and androgen-independent prostate cancer
cells irrespective of their basal level of AR and PSA
expression [60]. CG7870, a prostate selective replica-
tion-competent Ad with improved efficacy, contains the
prostate-specific rat probasin promoter, driving the E1A
gene, and the human prostate-specific enhancer/promoter,
Cancer gene therapy
driving the E1B gene. In nu/nu mice carrying LNCaP
xenografts, a single tail vein injection of CG7870 elimi-
nated tumors within 4 weeks [221]. Also, this agent was
evaluated in a phase I trial of patients with hormone-
refractory metastatic prostate cancer. Systemic injection
of CG7870 produced a transient minor decrease of pros-
tate-specific antigen levels [178]. Studies using an Ad
vector encoding the E1A gene under transcriptional
control of the human telomerase reverse transcriptase
promoter have shown that viral genome replication
and productive infection is primarily restricted to telom-
erase-positive tumor cells. Administration of the virus
into nude mice bearing human prostate xenografts pro-
duced significant tumor reduction [81].
The second strategy is to genetically modify the fiber
protein to present a tumor-specific binding ligand.
Progress in this direction has included the successful
insertion of small peptides such as an RGD motif in the
Ad fiber, resulting in Ad retargeting [15]. It was shown
that the efficacy of a replicating Ad can be improved by
incorporating a RGD peptide motif into the fiber protein
[186]. Also, an Ad that secretes a fusion molecule con-
sisting of the extracellular domain of CAR (sCAR) and
epidermal growth factor (EGF) was constructed.
Infection of tumor cells with a sCAR-EGF-retargeted
replication-competent virus system resulted in increased
oncolysis in vitro and a therapeutic benefit against tumor
xenografts [76].
In an attempt to improve both the efficacy and safety of
oncolytic virotherapy, a replication-competent Ad vector
encoding a CD and HSV-tk fusion gene was constructed.
A single injection of a replicative HSV-tk vector in
established s.c. human glioma xenografts resulted in a
significant reduction of tumor growth. The addition of
ganciclovir produced an additional slowing of tumor
growth and prolonged survival [138]. Phase I studies
demonstrated the safety of intraprostatic administration
of this Ad in combination with conventional-dose three-
dimensional conformal radiation therapy in patients with
intermediate to high risk prostate cancer [54, 57, 59].
Herpes simplex virus 1 (HSV-1) vectors have also
been developed that replicate conditionally in dividing
tumor cells based on mutations engineered in the viral
genome [193]. So far, four oncolytic HSV vectors have
been tested in several clinical trials with encouraging
results. G207 is an attenuated/replication-competent
HSV-1 mutant that lacks both copies of ICP34.5 (RL1)
gene and contains an insertion of lacZ in the ICP6
(UL39) gene [130]. These gene modifications offer a
new modality in cancer therapy through the ability of
G207 to selectively replicate within and kill malignant
cells with minimal toxicity to normal tissues [106].
Donald J. Buchsbaum et al.
G207 induced a significant inhibition of the tumor
growth alone and in combination with radiation in
human prostate tumor xenograft models [202]. Based
on positive results in preclinical brain tumor models,
G207 has been tested in clinical trials in patients with
glioblastoma, with some evidence of disease stabiliza-
tion [123]. A gamma-34.5 gene deleted HSV1716 vector
was also evaluated in patients with recurrent malignant
glioma following surgical resection in phase I/II trials
[74, 147, 161] and in patients with metastatic melanoma
[120]. NV1020 HSV-1 mutant vector has been tested
followed by chemotherapy in 12 patients with colorectal
cancer hepatic metastases with some minor responses
[114]. All the patients in these clinical trials tolerated
the treatment well. The safety of ICP34.5-deleted HSV
has been shown in multiple clinical studies.
Recently, a phase I clinical trial with a second-gen-
eration oncolytic HSV expressing granulocyte mac-
rophage colony-stimulating factor (Onco VEXGM-CSF)
has been conducted. This virus was administered intra-
tumorally in patients with cutaneous or s.c. deposits of
breast, head and neck and gastrointestinal cancers, and
malignant melanoma [79]. Improved oncolysis was
achieved through the use of a more potent clinical isolate
of HSV for construction of the virus, and, in addition
to the deletion of ICP34.5, the expression of the US11
gene as an immediate early rather than late gene was
used since this has previously been shown to boost
tumor-selective virus replication [23]. Also, co-
expression of GM-CSF has shown promising preclinical
and clinical results [192, 205]. Incorporation of a gene
encoding a cell membrane fusion glycoprotein of the
gibbon ape leukemia virus into the HSV genome sig-
nificantly increased the antitumor potency in mouse
models of primary and metastatic human prostate
cancer [137].
Chemosensitization
and Radiosensitization
The limitations of surgery and radiation therapy con-
cern treatment of cancer metastases. The problem with
chemotherapy is its low therapeutic ratio for many
tumors and the development of multi-drug resistance.
Gene therapy offers the potential to enhance radiobio-
logical effects through various mechanisms, and com-
bination treatment with radiation therapy might amplify
tumor cell killing with selected gene transfer events.
Chemosensitization can be achieved by genetic induc-
tion of apoptosis, by inhibition of molecules involved
603
in tumor cell resistance, or by enhancing intratumoral
production of cytotoxic drugs [154, 223].
Ionizing radiation and many chemotherapies depend
on wild-type p53 function for their cytotoxic effect.
Thus, restoration of wild-type p53 function in tumor
cells can be used to potentiate the effects of radiation
therapy and chemotherapy [47, 213]. Animal studies
have shown that the effects of cisplatin can be synergistic
in combination with Adp53 gene transfer in human lung
cancer cells in vivo. A trial was carried out in non-small
cell lung cancer patients receiving Adp53 alone, or pre-
ceded by cisplatin. Clinical responses were observed,
and progression-free survival was prolonged with the
combined treatment [169]. Colon and nasopharyngeal
cancer cells transfected with Adp53 showed increased
sensitivity to radiation [110, 169]. Studies of Adp53
gene therapy combined with surgery, chemotherapy, or
radiation therapy have been initiated in patients with
head and neck cancer and non-small cell lung cancer
[17, 51, 139, 188]. Results show that Adp53 is well tol-
erated and produces efficacious treatment in combination
with radiation and/or chemotherapy agents [62].
Restoring or enhancing the capacity of tumor cells to
undergo apoptosis through genetic modification of Bax
or Bcl-2 expression has resulted in tumors being more
sensitive to chemotherapeutic drugs and radiation ther-
apy [14, 64, 210]. The cellular transcription factor E2F1
promotes apoptosis. Intratumoral injection of E2F1-
expressing Ad vector in combination with gemcitabine
produced a significant reduction in pancreatic tumor
xenograft size [164]. Transduction of the caspase-3 gene
in rat hepatoma cells in the liver with an Ad vector
induced extensive apoptosis and reduced tumor volume
when combined with etoposide administration [215].
A HSV type 1 mutant in combination with mitomycin C
exhibited a synergistic cytotoxic effect against non-
small cell lung cancer cells in vitro and an additive effect
against tumor xenografts [195].
Transcriptional targeting via radiation inducible pro-
moters in a vector can selectively produce gene transfer
in a tumor in combination with radiation therapy [31].
Promoters triggered by radiation confine the cytotoxic
effect to the high dose target volume. Examples of radi-
ation inducible promoters include various early response
genes (c-jun, c-fos, Egr-1, and NFκB), which can be
coupled to genes which produce proteins that enhance
radiosensitivity. Similarly, the Egr-1 promoter in an Ad
vector carrying the TNF-α gene has been activated by
several chemotherapy drugs [118].
Tumor injection of an Ad expressing IL-12 and B7.1
following fractionated radiation therapy resulted in a
greater therapeutic effect in murine tumor models than
604
with either treatment alone [117]. Expression of IL-3 in
transfected murine tumors increased their response to
radiation, and systemic administration of IL-3 gene
transduced tumor cells in combination with local radia-
tion therapy resulted in enhanced efficacy [30]. Rat
glioma cells transduced to express HSV-tk and treated
with prodrug acyclovir or bromovinyldeoxyuridine had
enhanced sensitivity to radiation in vitro and in vivo
[31]. Weichselbaum et al. showed that irradiation medi-
ates enhancement of HSV replication and produces
enhanced antitumor activity [4, 127]. It was shown that
HSV-tk/ganciclovir suicide gene therapy, vector-based
TNF-α expression, and radiosurgery was more effective
in controlling human glioblastoma xenografts than single
or dual-component protocols [143]. Triple therapy with
Ad.Egr-TNF, radiation, and temozolomide produced
increased survival in mice bearing glioma xenografts
compared with dual treatment [217]. A phase I trial of a
replication-deficient Ad vector expressing TNF-α under
control of the Egr-1 promoter in combination with radi-
ation therapy in patients with soft tissue sarcomas of the
extremity was well tolerated and produced a high num-
ber of objective responses [136]. The combination of
radiation therapy and angiostatin gene therapy produced
enhanced antitumor effects in a rat glioma model [70].
The combination treatment with anti angiogenic agents
and chemotherapy or radiation therapy has been shown
to produce an enhanced anti-tumor effect in preclinical
models [89, 212]. Based on this observation, genetic
modification of tumor vascular endothelial cells would
be expected to produce an enhanced therapeutic effect
in combined modality therapy.
Several chemotherapy drugs are proven radiosensi-
tizers. One such drug, 5-FU, when produced by the
CD/5-FC suicide gene therapy approach following
intratumoral injection of an Ad encoding the CD gene,
has been shown by our group to enhance the effects of
radiation therapy in preclinical models of cholangiocar-
cinoma, glioma, pancreatic cancer, and colon cancer
[93, 151, 181]. Thus, strategies to increase chemosensi-
tivity and radiosensitivity by gene transfer have poten-
tially wide applicability for the treatment of cancer
clinically. Freytag et al. evaluated the efficacy and toxic-
ity of replicative Ad-mediated suicide gene therapy in
combination with radiation therapy in a preclinical
model of prostate cancer, and in prostate cancer clinical
trials [54–56, 58, 59].
Replication-selective oncolytic Ad have been con-
structed to target tumor tissue for selective replication
and amplification at the tumor site with limited replica-
tion in normal cells, minimizing toxic side effects.
Although low efficacy was documented with oncolytic
Cancer gene therapy
Ad as a single agent therapy, favorable interactions with
the cytotoxic drugs cisplatin and 5-FU were reported in
phase II and III trials with recurrent squamous cell car-
cinoma of the head and neck [29, 58, 59, 97, 209].
Synergistic effects between oncolytic Ad vectors and
chemotherapeutic drugs (e.g. paclitaxel, docetaxel, dox-
orubicin, cisplatin, and 5-FU) have been reported in ani-
mal models and in clinical trials [75, 102, 220].
Combination with radiation therapy allowed a 50-fold
decrease in the dose of the oncolytic Ad in a prostate
xenograft model [26, 44]. Combination of oncolytic Ad
with radiotherapy significantly increased antitumor effi-
cacy against prostate cancer xenografts compared to
either agent alone [46].
Current Limitations and Future
Directions of Cancer Gene
Therapy
The development of sophisticated molecular biological
techniques over the past 25 years and their application
to the study of the molecular mechanisms of cancer
have made gene therapy both a logical and practical
extension of classic treatment paradigms that is poised
to make major advances in new treatments for cancer in
the near future.
The major limitation of gene therapy for the treat-
ment of cancer arises from the relative inefficiency of
current vectors in transducing target cells, the inability
to transfer therapeutic genes into sufficient numbers of
target cells in situ to elicit the desired biological effect,
the development of both vector- and transgene-induced
humoral and cellular immunity, and the inability of vec-
tors to selectively localize and transduce tumor cells fol-
lowing systemic administration. Both viral and non-viral
vectors are rapidly cleared from the circulation, primar-
ily by hepatic sequestration, following intravenous
injection. These four limitations pose significant prob-
lems for the development of cancer gene therapy
employing systemic vector administration. Future
research is likely to be focused on generating modified
vectors with reduced toxicity and immunogenicity,
increased transduction efficiency, increased duration
and regulation of gene expression, and enhanced vector
specificity and targeting [66, 105]. Refinements directed
towards these goals will be made with replicative viral
vectors capable of both therapeutic gene delivery and
direct oncolysis [102]. A universal gene delivery system
has yet to be identified, so that further optimization of
existing vector delivery systems is likely to occur.
Donald J. Buchsbaum et al.
Small interfering RNA (siRNA) has been used to
knock down or silence gene expression. This has been
accomplished using chemically synthesized oligonuco-
leotides delivered in nanoparticles and liposomes, or
vector-based siRNA [80, 155]. Targets have included
oncogenes, and genes involved in angiogenesis, metas-
tasis, anti-apoptosis, and resistance to chemotherapy.
Further research into this promising gene therapy tech-
nique is required.
Gene-modified stem cells have been used for cancer
therapy [27, 153]. Their ability to traffic to tumor sites
makes them an attractive gene delivery vehicle. They
may be useful in conjunction with immunotherapy and
gene therapy for treating a variety of cancers.
The targeting of tumor blood vessels should be a
useful approach for the treatment of a variety of cancers.
Issues that need to be addressed are highly efficient
gene delivery and long-term expression of the anti-
angiogenic genes in metastatic cancer. The development
of targeted vectors should aid in the development
of this approach. Gene-directed enzyme prodrug
therapy uses transcriptional differences between normal
and cancer cells to drive the selective expression of a
metabolic “suicide gene” that is able to convert
a nontoxic prodrug into its toxic metabolite. A variety
of enzyme prodrug systems have been evaluated in
phase I/II clinical trials using either retrovirus or
adenovirus to deliver the enzyme to tumor cells [38].
The evaluation of gene therapy in combination with
surgery, radiation therapy, and chemotherapy is likely to
undergo further development.
Gene therapy will continue to be applied to many
fields of medicine, and particularly to the treatment of
cancer. Whereas human gene therapy trials for cancer
have not yet yielded clear benefit, these studies highlight
the deficiencies of current approaches. Gene therapy will
likely become an integral component of a multimodality
strategy for the treatment of cancer. Improvements in
vectors that increase the specificity and duration of gene
expression, the reduction of immunogenicity and toxic-
ity, and targeted vector delivery are required for advance-
ment of the clinical practice of gene therapy with an
increase in response rate. As knowledge of the molecular
basis of cancer accumulates, and as genetic screening
programs are introduced to identify high-risk patients, it
might be possible to use gene therapy for early treat-
ment, including vaccination against cancer.
Acknowledgements We acknowledge research support from
the National Institutes of Health (P20 CA101955). We are
grateful to Sally Lagan for preparation of the manuscript.
605
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20 Regulatory process for approval of biologicals
for cancer therapy
ANTONIO J. GRILLO-LÓPEZ
Abstract The tenet ‘Above all do no harm’ is necessary
and important but not sufficient [1]. Today’s physicians
have a responsibility to actively participate in making
new therapies available to the cancer patient. We know
that cancer can be cured. However, biologicals and
all new anticancer agents must be efficiently and
rapidly evaluated to find those that are most promising.
They must then be incorporated into combination
regimens that provide the greatest opportunity for cures.
Our regulatory agencies and processes were originally
established with the goal of protecting the patient
from unscrupulous investigators. Now it appears as if
they exist to protect the cancer and not the patient.
Where did we go wrong? What is the track record of the
regulatory agencies? How can regulatory obstacles be
overcome? How can regulatory challenges be turned
into opportunities? What is the optimal process for the
development of new anti-cancer therapies?
Keywords Biological therapy • targeted therapy
• antibodies • rituximab • FDA • EMEA • regulatory
• IND • NDA • BLA • ODAC • accelerated approval
• regular approval • clinical trials • endpoints
The Doctor–Patient Relationship:
Duty and Responsibility
As to diseases, make a habit of two things – to help, or at least to
do no harm.
Hippocrates
Primum non nocere (above all do no harm) is an impor-
tant consideration for the physician caring for the cancer
patient [1]. The extreme, inaction for fear of doing harm,
is simply not acceptable particularly when challenged
by a potentially curable illness. In cancer therapy all
new agents present benefits and risks that must be
weighed with the patient’s best interest in mind. In the
face of a serious illness like cancer, patients are generally
willing to take greater risks and accept more toxicity in
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
the expectation of a therapeutic benefit that will make
their efforts worthwhile. Likewise, Oncologists have
traditionally been more willing to accept a higher level
of adverse events in treating their patients than physi-
cians in other specialties. In cancer therapy primum non
nocere really implies a very delicate weighing of risk
versus benefit where the balance weighs somewhat
more heavily on the benefits side while accepting more
risk than one would accept for drugs in other therapeutic
areas. A patient may be denied the possibility of a cure
because of therapeutic nihilism or therapeutic conservatism.
One extreme is the nihilistic approach that says – we
have nothing that can cure you and therefore we will
‘watch and wait’. This approach, originally devised by
Portlock and Rosenberg [2, 3] for patients with early
stage Follicular Non-Hodgkin’s Lymphoma (NHL) is
today obsolete as we develop potentially curative com-
bination regimens for this disease [4]. At the other
extreme is the urge to persist in administering one che-
motherapy regimen after the next to a patient who is
refractory and at the latter stages of the disease.
Hippocrates said, first – to help, and then (when you can
no longer help) – to do no harm.
Physicians are bound by a sacred oath to care for their
patients, to offer them the best that modern science
provides for their disease, complications, and general
wellbeing [5]. This duty, this responsibility, is critical to
the patient with an incurable illness such as cancer. It is
even more important when the potential for cure exists
for a cancer that was previously held to be incurable.
When that potential exists for a new agent still under
investigation, time is of the essence. Developing that
agent then becomes a race where the losers (patients)
will perish because that curative therapy took too long
to be evaluated and approved. The physician directly
caring for the patient must seek the most effective
therapies even when investigational and provide his
patient with access to those via participation in clinical
trials or referral to centers where those therapies are
available. This duty, this responsibility extends beyond
the physician directly caring for the patient. Physicians in
613
614 Regulatory process for approval of biologicals for cancer therapy
the pharmaceutical industry must do everything within
their reach to ensure that new agents are developed and
submitted for approval in the most expeditious fashion.
Physicians in our regulatory agencies [Food and Drug
Administration (FDA), European Medicines Evaluation
Agency (EMEA) and others] must likewise understand
that they have similar duties and responsibilities to the
patient. Being employed by a bureaucracy does not
mean that your sacred oath is no longer valid. In fact,
physicians in regulatory agencies could be an important
catalyst in making new promising therapies available to
patients in a timely fashion. However, the opposite has
occurred and as a result, regulatory agencies and their
bureaucratic processes are today the most important
impediment to the timely development and availability
of new (some of them potentially curative) anticancer
therapies. The patient has great expectations of his
personal physician but may not understand the role that
physicians in the regulatory agencies are playing in defining
their destiny. Regulatory agencies need to recognize that
new anti-cancer agents are not truly available to all that
can benefit from them until approved and marketed.
Many types of expanded access and ‘compassionate
use’ programs have been tried but these will never
substitute for approval and marketing. The fact is that,
prior to marketing, anti-cancer therapeutics are available
only to a select minority of patients that qualify for
these programs while the majority are deprived of this
opportunity. Thus, regulatory agencies have a very
serious responsibility to expedite the approval of new
anti-cancer agents and make them broadly available. It
must be made clear that the majority of physicians in the
regulatory agencies are good professionals and well
meaning individuals who work very hard for less than
adequate salaries. They deserve respect and support. It
is a minority that, strategically placed, has created and
enforced the obstacles that impede progress.
However, single agent approval although necessary
and important is not sufficient as the majority of effec-
tive therapies (and specifically, curative therapies) are
not based on single agents but on combination regimens
[6–9]. The regulatory agency’s responsibility, as estab-
lished by law, is the review and approval of new anti-
cancer agents (single agents). Pharmaceutical industry
is responsible for the discovery and development of new
single agents and this is what the agency is required to
regulate. Once the single agent is approved, it is the
oncology community’s responsibility to find its optimal
use in combination with other agents. The oncology
community is then responsible for defining the role of
the new agent within a combination regimen and its
place in the therapeutic armamentarium for the target
disease. Regulatory agencies have no important role in
this. Pharmaceutical industry can be a partner but should
play a secondary role. Thus, investigators in academia
and in the community (including academic institutions,
cancer centers, cooperative groups, consortia, and others)
have very important duties and responsibilities to the
patient in the development and optimization of combi-
nation regimens.
Development of Cancer
Therapeutics
The mistakes are all there, waiting to be made.
Chessmaster S.G Tartakower
Progress towards more effective and less toxic therapies
and progress towards curative therapies depends on the
very complex process of drug development and approval.
It is only after approval that a new agent will be widely
available to the patients who will benefit from its use.
However, even after a new agent is approved it is still
necessary to continue the clinical research process that
will determine its optimal use within a combination
regimen. On the average, it takes about 12 years for a new
agent to proceed from discovery to approval (Fig. 1).
Preclinical development accounts for the first 4 or 5
years; and clinical development, regulatory review and
approval take over 7 years. It is thought that regulatory
time accounts for 4 to 5 of these 12 years (FDA imposed
delays, clinical holds, excessive and unreasonable
requirements, waiting for meetings to be scheduled,
review time, waiting for FDA responses, etc.).
The clinical development process begins prior to the
first patient being treated. The Phase I protocols must be
written, a clinical plan defined, and all preclinical data
submitted to the regulatory agencies before one can pro-
ceed. All of these documents are reviewed by the regu-
latory agency and a determination is made as to whether
or not the Phase I (PI) clinical trials can be initiated.
Once a recommended dose for PII has been established
in PI, the PII trials can commence. Ideally this occurs
with some overlap with the PI studies. Both the PI and
the PII trials should include, as early as possible, combi-
nation regimens. These pilot combination studies are
not necessary for the evaluation of the safety profile or
the activity of the single agent; however, they are impor-
tant for two reasons: (a) getting an early start towards
treatment optimization within a combination regimen,
and (b) evaluating possible combinations suitable for
Antonio J. Grillo-López
Figure 1. Timeline: New anticancer agents
PIII studies. It is unusual to find the optimal combina-
tion regimen this early in development. Following PII,
the PIII trials are then carried out with a view to regula-
tory approvals.
The clinical development plan incorporates all activi-
ties from PI to approval and beyond. It is usually written
by a physician (project clinician) with support from other
professionals. The extent and overall scope of this criti-
cal road map depends heavily on projections of the
resources that will be available over the 6 to 10 years it
will take to complete clinical development. Most new
agents are developed by pharmaceutical companies.
A large and well established company is more likely to
dedicate substantial resources than a small start-up bio-
technology company that depends, at least initially, on
venture capital. Regardless of available resources, the
clinical development plan must clearly define the path to
a regulatory approval. Given a new agent with reasonable
efficacy and tolerable toxicity, this is the main objective.
It is the objective not only for the pharmaceutical company
and its investors but also for the patient and all who have
a role in the care of the patient. The road to therapeutic
success in cancer is paved with many compounds that
failed to show the desired efficacy or were too toxic. One
must, however, wonder how many active compounds
were trampled under and became part of that pavement
due to simple mistakes, inappropriate development
plans, technical problems in the conduct of clinical trials,
companies going bankrupt, inadequate resources, misin-
terpretation of clinical data, and/or any combination of
the myriad problems and challenges that plague the clin-
ical phases of development. One important problem is
615
the shortage of well qualified and experienced physi-
cians to assume the role of project clinician (or of Chief
Medical Officer, CMO) in a pharmaceutical company. It
used to be that any physician considering such a role
would unequivocally be given the message, by his peers
in academia, that this would be a black mark on his cur-
riculum vitae and that his career might not survive that
experience. This has changed over time and today there are
many more qualified and experienced physicians, many
with an academic background, in the pharmaceutical
industry. However, over time, the need has also increased.
As an example, in San Diego, California there are over
400 bio-medical companies. They all need a CMO and
most also need one or more project clinicians depending
on how many new agents they have in clinical trials.
There are simply not enough physicians with clinical trials
and regulatory expertise to satisfy the need.
Simple mistakes can have major impact in the lengthy
and complex process of cancer drug development. As an
example, one small company contracted with a contract
research organization (CRO) to have them create a data-
base, perform data entry, and carry out the statistical anal-
ysis for one of their clinical trials. Despite the company’s
extraordinary efforts to make sure that the CRO’s database
was identical to the company’s database for their other
studies, one simple mistake was made. Due to this, the
summary data tables for safety and efficacy cutting across
all studies were very obviously incorrect. After extensive
investigation (double checking, and painstaking review of
source documents, data entry, programming, queries, dic-
tionaries, coding conventions, quality control, and of all
the data), it was determined that the CRO had reversed the
616 Regulatory process for approval of biologicals for cancer therapy
coding conventions followed by the company for gender.
This one simple mistake cost the company a 2 months
delay (or $3.0 million for this particular company) in filing
the regulatory dossier. At the other extreme (more serious
mistakes) some new agents have been turned down by the
FDA for incomplete or inadequate data collection. When
this happens at the end of PIII after many years of devel-
opment, the losses are measured in the hundreds of mil-
lions and more importantly in thousands of lives when an
effective product (with some curative potential) is lost to
the cancer patient.
Some may marvel at how well we have performed in
terms of cancer drug development over the past few
decades given all the challenges, mistakes and prob-
lems. Such complaisance is unacceptable. I would prefer
to tackle the challenges and problems, avoid/preempt
the mistakes and find solutions that will enable us to
perform at peak efficiency as we head towards better
and hopefully curative therapies. We owe that and more
to the patients that we serve.
Biologicals and the Treatment
of Cancer
Rationale for Developing Biological
Therapies
According to the theory of aerodynamics, as may be readily dem-
onstrated through wind tunnel experiments, the bumblebee is
unable to fly. But the bumblebee, being ignorant of these scientific
truths, goes ahead and flies anyway – and makes a little honey
every day!
Ross E. Hutchins
Back in the late 1980s and early 1990s, antibodies
(MAbs) were the bumblebees of cancer therapeutics.
Practically no one, after years of frustrating clinical
experiences, believed that MAbs would ever fly. The
1994 review by Dillman listed the many MAbs taken
to clinical trials with disappointing results [10].
Frustration and disappointment were understandable.
After all, it had been nearly 100 years since Paul
Ehrlich first spoke about MAbs and ‘magic bullets’
[11]. And yet some of us knew, even then, that har-
nessing the power of MAbs for therapeutic applica-
tions was a question of time [7]. Anticancer therapies,
such as radiotherapy and chemotherapy, had been the
standard for too many years, as we sought additional
and better alternatives. They had served us well; how-
ever, their toxicity was tolerated, only because we
lacked other options. MAbs had arrived; it was just a
question of time!
The rationale is relatively simple. The intent is to
make the patient’s immune system an ally. Not to impair
it but to find ways of working with and enhancing the
immune system. This is readily understood in the case
of MAbs. They work either by utilizing the patient’s
immune system (complement, effector cells, etc.) to kill
the tumor cell or by binding to the target antigen and
transmitting an apoptotic signal. MAbs are also used
as per Ehrlich, as ‘magic bullets’. They are used to
deliver to the tumor cell a payload consisting of a
toxin, chemotherapeutic agent, or a radioisotope. Other
biological therapies, in the broad definition of the term
(treatments that are or resemble natural body substances
or that are made from natural body substances), have
very specific molecular targets (targeted therapies).
The end of the last millennium ushered in a new era in
cancer therapeutics. MAbs, led by rituximab, had arrived.
Some other MAbs, as well as a variety of other targeted
therapies, are now approved (Herceptin, Mylotarg,
Campath, Zevalin, Bexxar, Erbitux, Avastin) and many
are under investigation. We witnessed the development
and approval of the first MAbs for the treatment of can-
cer, rituximab [12, 13]. Early in this new millennium, we
witnessed the approval of the first radio immunotherapy
[14, 15]. Other firsts will soon follow: the first vaccine for
cancer, the first antisense product, the first genomics-
derived therapeutic, and others. This will be the era of
MAbs, of biologicals, of targeted therapies. Gone are the
days when chemotherapy was the only and, unfortunately,
usually the last resource. These new therapeutics open the
door for new combination regimens and new multimo-
dality therapies with curative potential. We now have much
more selective and specific therapeutics. We now have
agents with very different mechanisms of action that are
ideal for combination therapies. Importantly, never before,
in the history of cancer therapy, have we been able to har-
ness, control, and direct the immune system as we can
today. The immune system can now become an important
and effective partner in our efforts to cure cancer. It is
only the beginning. There is a bright future ahead for the
many cancer patients who will benefit from all the prom-
ising advances that we envision today and that will
become reality in this new era.
Rituximab as an Example
The human mind treats a new idea the way the body treats a strange
protein, it rejects it.
P. B. Medawar
The development of rituximab began at IDEC Pharma-
ceutical Corporation’s (IDEC) laboratories in San Diego,
California in 1991. Mice were injected with human
Antonio J. Grillo-López
CD20 antigen and the anti-CD20 MAbs they produced
were isolated and characterized. One of those, the
murine IDEC-2B8, was chosen for chimerization. The
chimeric MAb (IDEC-C2B8) was favored in order to
reduce the immunogenicity of the murine parent MAb,
to utilize a human framework which allowed for com-
plement and effector cell binding, and to selectively
effect B-cell depletion. The technology necessary for
humanization was not yet fully developed at that time
and thus was not pursued. A vector was engineered that
enabled high MAb yields when inserted in CHO cells
and used in a high volume fermentation process. The
appropriate preclinical pharmacology and toxicology
studies were carried out including B-cell depletion studies
in monkeys. Mechanistic studies confirmed the desired
complement binding, complement dependent cytolysis
(CDC), effector cell binding and antibody dependent
cellular cytotoxicity (ADCC). The MAb was clearly
shown to induce apoptosis. All of this pre-clinical work
was carried out within a year at IDEC’s laboratories [16,
17] (Table 1). Synergism studies were completed in col-
laboration with scientists at UCLA [18]. All the pre-
clinical development work was performed at IDEC’s
laboratories in San Diego, California with no govern-
ment or NIH support. This MAb was ‘made in the USA’,
at a small biotechnology company, a start up funded by
venture capital, and with no taxpayer’s monies. Rituximab
is an example of American entrepreneurship at its best.
IDEC-C2B8 was ready for clinical trials by late 1992.
The Investigational New Drug application (IND) was
submitted to the FDA who placed the project on a clinical
hold due to their concerns about the effect of B-cell deple-
tion on immunoglobulin levels and immunocompetence.
Finally, after months of scientific discussions, the FDA
allowed the PI clinical trials to proceed (February 1993).
The desired B-cell depletion was observed. The MAb
Table 1. IDEC’s pre-clinical scientists responsible for the
development of rituximab (IDEC-C2B8)a
Name Responsibilities
Nabil Hanna, Ph.D. VP, Pre-Clinical R&D – in charge of
all preclinical development
Darrel Anderson, Ph.D. Isolation and characterization of the
murine parent antibody, IDEC-2B8
Roland Newman, Ph.D. Gene identification and cloning
experiments; antibody engineering
Mitchell Reff, Ph.D. Engineering of the chimeric antibody
IDEC-C2B8 and vector engineering
Chris Burman, Ph.D. VP, Manufacturing – in charge of
antibody production
a
Key scientists. Many others at IDEC contributed and are not
listed for space reasons.
617
was doing what it was intended to do. Significant reac-
tions (primarily fever and chills) were seen even in the
first patient treated. These were biologic manifestations
of MAb induced B-cell depletion. The total absence of
such reactions might indicate the MAb was not effec-
tive. Two patients had objective responses, to a single
infusion of MAb, which lasted for months [19]. The PI/
II study was designed to define the pharmacokinetic
profile [20], confirm the dose to be used in a four infu-
sion schedule [21] and to establish a response rate in
patients with relapsed or refractory Non-Hodgkin’s
Lymphoma (NHL) [22, 23]. The PIII (pivotal trial) com-
pleted enrollment in 1 year, the data was collected and
analyzed, and the complete filing (including all preclini-
cal and clinical studies) was submitted simultaneously
to the US FDA (electronic and paper copy) and in
Europe to the EMEA in February 1997 [24–27]. The
clinical work including the design of the overall clinical
plan; writing and implementing all of the protocol for
the initial eight studies; establishing collaborations with
numerous academic institutions in North America and
Europe; collecting, analyzing and interpreting the data;
writing all reports, abstracts and publications; generating
the electronic dossier; filing, presenting, and defending
the data with regulatory agencies in the US and Europe;
and many other activities critical to the ultimate success
of this project were carried out by the Medical and
Regulatory Affairs Division at IDEC [7] (Table 2). The
clinical phase of development was completed in 3 years
(first patient enrolled in Phase I to last patient enrolled
in PIII), a new record in NHL as even the NCI’s coop-
erative groups will take at least 3 years to complete one
PIII study. It took 1 year (total time from last patient
enrolled in PIII to BLA filing) to complete the necessary
observation time on the Phase III patients, and prepare
and file the regulatory dossier (same day filing of a US
BLA and an European dossier). Regulatory review and
approval, in the US, took 6 months plus a 3 months delay
imposed by the FDA while resolving manufacturing
issues. Clinical/regulatory development time was a record
setting 4 years and 9 months as compared to the average
of over 7 years for cancer drugs in general (Fig. 2).
The five things that made this clinical development
program successful were: an active agent, the right people,
good leadership, ironclad data and peer level relationship
with the FDA. Clearly rituximab was bound to be suc-
cessful. It worked exactly as it had been designed. IDEC
had the right people empowered by good leaders. The
company had a core group of professionals who had years
of experience in clinical development. They were not
inventing the wheel. IDEC was not a ‘virtual company’
outsourcing a lot of work. It was a small company and yet
618 Regulatory process for approval of biologicals for cancer therapy

Table 2. IDEC’s clinical scientists responsible for the development of rituximab (IDEC-C2B8)a
Name
Antonio J. Grillo-López, M.D.
Brian K. Dallaire, Pharm. D.
John Leonard, Ph.D.
Anne McClure, M.S.
Jay Rosenberg, Ph.D.
David Shen, Ph.D.
Chester Varns
Alice Wei
Christine White, M.D.
Responsibilities
Chief Medical Officer and Senior VP Medical and Regulatory Affairs – in charge of all clinical
development and regulatory affairs
Senior Director, Clinical Operations – conduct of clinical trials including strategy, timelines, budgets
and resources
Senior Director, Project Planning and Regulatory Affairs – planning and timelines for all clinical
trials; communications with the regulatory agencies; filings and dossiers
Director, Medical Writing – documentation (protocols, clinical trial reports) for each study,
regulatory filings (INDs, periodic reports, regulatory dossiers); publications
Head, Clinical Immunology Laboratory – analysis of all patient samples for bcl-2 (pcr), pharmacoki-
netics; and other specialized tests
Senior Director, Biometrics – data entry, programming, queries, tracking, statistical planning and
strategy, data analysis, interaction with regulatory agency’s statisticians
Director, Clinical Trials Monitoring – protocol design, study implementation, clinical trial monitoring
and GCP, data collection and clean-up, interaction with clinical sites
Senior Director, Regulatory Affairs - planning and strategy for regulatory evens, communications
with regulatory agency’s staff; filings and dossiers
Senior Director, Hematology and Oncology – safety officer; interactions with clinicians and their
staff at study sites; publications and presentations

a
Many others at IDEC Pharmaceuticals, as well as investigators and staff at investigational sites, made important contributions and
are not listed due to space constraints.

Figure 2. Rituxan: Clinical development

timeline

able to handle the work load for this program mostly inter-
nally. Virtual companies generate virtual data. That was not
the case at IDEC. The clinical program was subject to
careful prospective planning, meticulous study imple-
mentation and conduct, rigorous adherence to GCP and
GLP requirements, strict auditing procedures, precise sta-
tistical methodology, and painstaking attention to correct
response and duration adjudication. The clinical data, the
cornerstone critical to the approval of any new agent, was
ironclad. Not a single patient’s response or duration was
ever contested by reviewers, auditors, peers, or editors.
The FDA contested only one patient, a patient in the
Phase III study that had been classified as inevaluable. The
result of the joint review by he FDA and the clinical group
at IDEC was that the patient’s classification was revised to
evaluable, response was a CR, and duration was long.
The FDA had actually added a CR and increased the
overall response rate (not their original intent). Lastly, a
peer level relationship with the FDA is of the utmost
importance. Establishing a relationship that is amicable,
Antonio J. Grillo-López
collegial, professional and based on mutual respect is the
key to success. It requires openness, timeliness, and the
most professional and scientific approach. In such a situ-
ation the FDA staff can be your allies and partners. Ideally
you end up with what amounts to a joint development
team as happened with the rituximab CALA (Table 3).
On a personal note, there was one new idea in the
development of rituximab that was rejected by several
prominent lymphoma experts. I knew that rituximab’s
ultimate contribution to the care of the lymphoma
patient would be determined by its contribution within a
curative combination regimen such as CHOP in the
treatment of Diffuse Large Cell B-Cell Lymphoma
(DLBCL). Thus, one of the first clinical trials I designed
was the Phase II pilot study of rituximab plus CHOP
(R + CHOP) [28]. The definitive goal was to treat patients
in a disease where CHOP was known to be curative
(about 40% cure rate). Synergism between rituximab
and doxorubicin had already been shown [29]. However,
the toxicity of the combination and its impact on the
cure rate of CHOP were unknown. Therefore, we chose
to perform the first study of R + CHOP in patients with
Low Grade NHL. Several prominent lymphoma experts
rejected this new idea of the simultaneous administra-
tion of rituximab and CHOP as they might reject a foreign
protein. Their objection was that ‘antibodies have lim-
ited efficacy and should be used only after chemother-
apy, to treat minimal residual disease’. They argued for
sequential rather than simultaneous administration. My
new idea, that the synergism shown in cell lines would
lead to enhanced efficacy in clinical trials and that we
would miss that opportunity if we gave the antibody in
sequence rather than simultaneously with chemotherapy,
was not considered valid. One investigator did accept
my new idea as valid (did not reject it as a foreign protein)
and enthusiastically participated in this study.
The study revealed a 100% response rate (7+ years
median TTP). Side effects were similar to those of CHOP
and rituximab with no additive toxicity. We conducted a
similar study in DLBCL and found a 94% ORR [82%
Table 3. Rituximab’s CALA Team (1996–97)
Responsibility FDA IDEC
Medical Bernard Parker, M.D. Antonio
Grillo-López, M.D.
Susan Jerian, M.D. – interpretations are often issued in the form of guidelines
Patricia Keegan, M.D. –
Regulatory – Alice Wei
– Augusta Cerny
Product Reviewer Mark Brunswick, Ph.D. –
Statistical Jawahar Tiwari, Ph.D. David Shen, Ph.D.
Technical Support Robin Jones Ken Fite
CALA Coordinator Michael Fauntleroy – major reconstructive surgery. A few within the agency
619
progression free survival (PFS) at 5 years] [30, 31].
These studies led to the GELA study, led by Bertrand
Coiffier, which showed that the R + CHOP combination
almost quadrupled the event free survival of CHOP [32].
This study established R + CHOP as the gold standard
for the treatment of DLBCL. Rituximab had reached its
curative potential in combination with CHOP. No other
agent, alone or in combination, has ever achieved a sig-
nificant improvement over CHOP alone.
The USA’s Food and Drug
Administration
Want of foresight, unwillingness to act, lack of clear thinking, con-
fusion of counsel – these are the features which constitute the endless
repetition of history.
Sir Winston Churchill
The FDA was created in 1938 by an act of congress
(FDAC Act) and charged originally with protecting the
consumer by evaluating the safety of new products [33].
The role of the FDA has evolved over time. The act was
revised in 1962 and thereafter the FDA required that,
in addition to safety, efficacy be established in two
adequate and well-controlled clinical trials. In 1997,
the FDA’s Modernization Act (FDAMA) was approved
and it allowed the FDA to accept one clinical trial and
other supportive studies as evidence of efficacy [34].
However, in practice the FDA generally continues to
operate as if efficacy should be shown in ‘two adequate
and well controlled clinical trials’ and as if the only
acceptable endpoint is overall survival. One unwritten
rule is that short of the ‘two adequate and well con-
trolled clinical trials’ the one Phase III study would have
to reach high statistical significance for its primary end-
point at the p = 0.01 level (rather than 0.05 in the case of
two studies). The presence of good supportive trials is
of no consequence in this situation. Any application
needs to contain ‘two adequate and well controlled clinical
trials’ that show significance at the p = 0.05 level or one
that that shows significance at the p = 0.01 level.
The acts of Congress that created and, over the years,
have modified the FDA and its mandates have been
interpreted by the agency in their own manner. These
which are not always followed by the FDA itself. All of
this has evolved into a bureaucratic process that is com-
plex, costly and time consuming. Each subsequent law,
each subsequent guideline is one more band aid on a
gaping wound that in reality can only be addressed by
620
contribute to this bureaucracy. The majority try to do
their best to work within this flawed system. Leadership
is a major problem. The agency has for long periods of
time operated under interim leaders. The commissioner
of the FDA is appointed by the President and must be
sanctioned by Congress. This is a highly political, drawn
out process for a job that few want. In the absence of true
leadership, the agency falls back on the more primitive
instincts of self protection. Any publicity is bound to be
negative and any misstep can affect their Congress
approved budget. Any action taken by the FDA will be
subject to intense scrutiny and criticism. Adverse events
occurring post approval will be assumed to have hap-
pened because of the FDA’s negligence in scrutinizing
safety data. In this environment it is easier to say no or
not to act at all. Nevertheless, FDA reform is a buzzword
that one hears mostly from politicians prior to elections.
After that, it seems like no one dares tackle this subject.
The Oncologic Drugs Advisory
Committee of the FDA
Life is short, Art is long, Opportunity is fleeting,
Experience is deceitful, Judgment is difficult.
Hippocrates
The US Congress has enacted legislation authorizing
the establishment of committees of experts to advise
government agencies in discharging responsibilities.
The US Food and Drug Administration (FDA), in turn,
created the Oncologic Drugs Advisory Committee
(ODAC) whose structure and function is defined in the
Code of Federal Regulations (CFR), in the charter pub-
lished by the FDA, and other documents [35–37].
According to the charter, the committee is expected to
“review and evaluate data concerning the safety and
effectiveness of marketed and investigational human
drug products for use in the treatment of cancer and
make appropriate recommendations to the Commissioner
of Food and Drugs”.
The FDA requests advice from the committee members
(members) on a variety of matters, including various
aspects of clinical investigations and applications for
marketing approval of drug products. They receive sum-
mary information about the applications and copies of
the FDA’s review of the application documents. Based
on this information, the committee may recommend
approval or disapproval of a drug’s marketing applica-
tion. The FDA generally follows the committee’s recom-
mendation, but is not bound to do so. The FDA must
notify affected persons of their decision within 90 calen-
Regulatory process for approval of biologicals for cancer therapy
dar days of the committee’s recommendation. However,
the FDA requests the committee’s advice via a series of
questions that are delivered to committee members usu-
ally the day before the meeting [38, 39]. The list does not
usually include the key question – should this product be
approved; or, more appropriately, will this new agent
benefit the patients who may receive it. The questions
usually bring up technical issues regarding study con-
duct, data collection, choice of endpoints, or statistical
analysis. The FDA’s desire for the committee to provide
a certain answer or recommendation is many times trans-
parent, equivalent to a jury being asked – do you find this
guilty person guilty. Sometimes a question is asked in a
negative way that could serve to lead the committee to
respond negatively. Many times questions are asked that
require an in-depth understanding of regulatory law at
which committee members are not experts.
One could argue that the committee is set up to fail.
The members are academicians with a wealth of aca-
demic knowledge. There is not a single community
Oncologist on the committee. In the US, the majority of
cancer patients are treated in the community, not at aca-
demic centers. Members have experience mostly with
Phase II carried out in their own institution and Phase III
trials conducted by the cooperative groups. Not one com-
mittee member has ever been responsible for taking a
new agent all the way through clinical development
(Phase I to III) and to an approval. Not one committee
member has ever had to produce, negotiate, file and
defend a regulatory dossier submitted to the FDA and had
that product approved. The one member of the committee
with this kind of experience is the Industry Representative
who has a voice but not a vote. Committee members are
not trained on their role at ODAC or on regulatory law.
There is information on an FDA website but no active
training is provided. Members are not allowed to meet in
the absence of an FDA representative. Why not? This
would allow for an exchange of experiences and ideas
that could only have positive results. ODAC’s recom-
mendations are just that, the agency can accept them or
not. They should probably be mandatory. After all, the
agency brought the agent in question to the committee
because they felt that en expert recommendation was
required. Why should they then decide not to follow that
recommendation? Members do not see all of the applica-
tions that are filed with the FDA. The agency selectively
presents those where there are issues that require the
committee’s expertise. How do we know that there were
no similar issues with the other applications? Why can’t
the committee learn from the discussion of those other
applications? What precedents are set in the approval or
denial of applications never seen by the committee?
Antonio J. Grillo-López
Wouldn’t it be better for the committee to review all
applications? For these and many other reasons judgment
is difficult. And yet, the FDA makes it look like their
questions are reasonably addressed by the committee as if
judgment were simple. Each committee member should
ask – am I being manipulated by the FDA? Actually,
judgment could be simple as will be shown below.
Regulatory Review and
Approval Process
There are risks and costs to a program of action. But they are far
less than the long-range risks and costs of comfortable inaction.
John F. Kennedy
The current process for preclinical development entails
meeting all regulatory requirements (for pharmacology,
toxicology, and manufacturing), filing reports on all the
data on an ongoing basis and having periodic meetings
with the agency. At the end of this process following
another meeting with the FDA, the IND is filed. The
first clinical trial can begin after a mandatory 30 days
wait provided that the agency does not impose a clinical
hold. During clinical development periodic meetings
are held with the FDA, data on all studies is filed (clinical
study reports), study conduct is subject to GCP and end
of Phase II or pre-licensing application (BLA or NDA)
meeting is scheduled. At this meeting all plans for the
content of the application (BLA) are presented and
discussed with particular emphasis on the protocol,
endpoints, and statistical analysis. The company may
also submit a Special Protocol Assessment (SPA) where
the FDA reviews the Phase III protocol and an agree-
ment is reached as to its validity for approval purposes
(given the appropriate results). Phase III is then imple-
mented and conducted meeting all GCP requirements
and with the appropriate audits during and at the end of
the study. Nowadays, most Phase III studies will include
provisions for a Data Safety Monitoring Board (DSMB)
or a Data Safety and Efficacy Monitoring Board
(DSEMB), a mechanism for histopathological review
of biopsies and a mechanism for third party clinical
review and classification of responders or of radiological
exams. Procedures for blinding and for reporting are
carefully defined. Interim analyses require additional
rigorously documented processes.
The cost of all of this has risen exponentially over the
past few years. It is said that the cost of bringing one
new drug to market exceeds a billion dollars and in a
Phase III solid tumor study the per-patient, fully allo-
cated, costs can exceed $100,000.00.
621
The BLA is filed with the FDA upon the completion of
Phase III after the necessary patient observation period.
Completing all of the documentation necessary for the
BLA will take an average of 6 to 8 months from last
patient observation date (last patient last visit) or, depend-
ing on the primary endpoint, from the median patient’s
last visit. The FDA then has a certain period of time to
review and approve the application. In the case of an
application for accelerated approval that might be 6
months. Nevertheless, the FDA can ‘stop the clock’ at any
time or occasionally even ‘restart the clock’. The agency
can also request that the new agent be presented to ODAC.
Again, a long and drawn out process that favors inaction
over action. There are numerous possibilities for improve-
ment. An action oriented process is sorely needed.
A different way of looking at the regulatory review
and approval process is by considering its overall place
in the larger scheme leading to treatment optimization.
It is through combination therapies that patients get the
most benefit. The optimal role of any new agent within
a combination regimen takes many years to define.
Nitrogen mustard was available and used effectively as
a single agent (despite its toxicity) for over 20 years
before its role in the MOPP regimen was defined and
found to constitute a curative therapy for Hodgkin’s
Disease (Fig. 3). Many patients benefited from Nitrogen
Mustard during those 20 years. Thus, one could envi-
sion a process that begins with discovery, followed by
preclinical development, clinical development, regula-
tory review and approval, and reaching completion with
treatment optimization. The FDA’s role is modest when
viewed from this perspective. Their responsibility should
only be that of ensuring the safety of new anticancer
agents and thus limited to the review and approval of
data generated during clinical development. Everything
else they regulate today is really the responsibility of the
company developing the new agent (discovery to approval)
or of the oncology community (approval to completion
of treatment optimization) (Fig. 4).
Accelerated Versus Regular Approvals
Take calculated risks. That is quite different from being rash.
George S. Patton
The two mechanisms for the approval of new anticancer
agents of approval are (1) Regular Approval and (2)
Accelerated Approval. Requirements for Regular
Approval include evidence of clinical benefit as mea-
sured by endpoints reflecting improved quality or quan-
tity of life or the use of established surrogate endpoints
for clinical benefit such as survival, disease free survival,
622 Regulatory process for approval of biologicals for cancer therapy
Figure 3. Nitrogen mustard
Figure 4. New anticancer agents:
Optimization
and improvement in tumor-related symptoms. In con-
trast, Accelerated Approval requirements allow the use
of surrogate endpoints reasonably likely to predict clini-
cal benefit (for example, durable response) provided
there is “substantial evidence” of efficacy from well-
controlled clinical trials [40, 41]. This mechanism is
specifically for situations involving “serious and life-
threatening” illness where patients’ diseases are unre-
sponsive or patients are intolerant of available therapies
(i.e. medical need). The new therapy must provide an
advantage over existing therapies. Importantly, an addi-
tional requirement is the implementation of post-
approval, confirmatory studies. The FDA’s approval is
contingent on subsequent confirmation of efficacy pro-
vided by these studies, and approval can be withdrawn
depending on their results.
Accelerated approval was instituted because Regular
Approvals took too long and there was a desire to bring
new agents earlier to the patients who needed them.
Clearly there is an element of risk as Accelerated
approval occurs earlier when data is not as mature as for
a Regular approval. Regular Approval, however, signi-
fies that large numbers of patients may have to go
untreated because an effective new agent is not avail-
able. Accelerated Approval implies a calculated risk.
This risk is minimized when safety is acceptable and
when mature data will be available in a reasonable
period of time.
Antonio J. Grillo-López
The FDA, as will be seen in 8.0 below, has a less than
desirable track record with Accelerated Approvals. They
are bound by their own regulations to accept this mech-
anism and yet they oppose it and have impeded its use.
A recent example is the case of satraplatin. This is an
oral platinum compound that was presented at the
ODAC meeting of July 2007. Satraplatin has been eval-
uated in a large Phase III clinical trial (randomized,
double blind) for the indication of androgen indepen-
dent Prostate Cancer in patients who have failed at least
one prior chemotherapy regimen [42]. The company
applied for and obtained an SPA (reviewed and approved
by the FDA). They then applied for an Accelerated
Approval based on the protocol and SPA defined
endpoint, progression free survival (PFS), for an interim
analysis. The median PFS was 9.7 weeks for the control
arm (prednisone) and 11.1 weeks for the satraplatin +
prednisone arm. This 15% increase in PFS is a modest
but statistically significant improvement in PFS and
valuable to the patient that has already failed at least one
chemotherapy regimen. The FDA asked ODAC several
questions, some were endpoint related technical issues
and the last was whether or not they should wait until
completion of the study and availability of OS data for
consideration of the application. At the end of the day,
the committee voted only on this last question agreeing
unanimously that the FDA should postpone any deci-
sion on the application pending completion of the Phase
III study and availability of OS data [43]. I believe that
the consequences of this decision are not well under-
stood by the committee, the media or the public. The
situation is as follows:
1. The question was totally inappropriate. The commit-
tee is not there to rule on whether an Accelerated or
a Regular Approval should be considered. This is a
regulatory issue to be decided by the FDA. The
application was for an Accelerated Approval and that
is what the FDA should have asked the committee –
is there a medical benefit to patients and should this
drug be granted Accelerated Approval. This question
needed to be addressed independently of the avail-
ability, at some later point, of OS data.
2. Why was the question asked? The FDA had several
options regarding the application: (a) They could
have denied it and yet leave the door open for
re-submission when OS data was available. This option
presented relatively little risk for the FDA. (b) They
could have approved; but then, they would have to
face the uncertainty as to whether the OS data would
support approval. This option is feared by the FDA
because of the negative publicity it generates if the
data does not support approval. And yet, this has
623
happened only exceptionally. There is some risk but
this is the risk that the Accelerated Approval mandate
recognized and intended be taken. Most Accelerated
Approvals will make useful new agents available
earlier. A minority will later fail to have a confirmed
advantage and will need to be withdrawn. The FDA
is fully authorized to withdraw approvals in such cases.
(c) The FDA could ask the question of ODAC and
thus shift the responsibility and the possible negative
fallout from the agency to the committee, and this is
what they chose to do.
3. What response was sought? The FDA must have
thought that there was some merit to this new
agent; otherwise they would have denied the applica-
tion outright. However, it was much less risky for
them to wait until there was confirmatory OS data.
They wanted the committee’s support and wanted
the committee to be responsible for their decision to
wait. The ODAC was manipulated by the FDA to
achieve their desired end result.
Unfortunately, all of this means that satraplatin will not
be available for approximately another 2 years even if
all goes well with the re-submitted application (based
on OS data). This does not hurt the FDA, the agency
doesn’t seem to care and doesn’t want to take the risk.
This is not understood by committee members, they
have been manipulated before. Most of the media mis-
interprets the situation. The public is biased against
pharmaceutical industry because of high drug prices and
any setback for a company is not viewed negatively. It is
only the patients who could benefit from satraplatin who
will have to wait and suffer. It is difficult to understand
how the availability of an oral platinum, the first of its
kind, could be anything other than a positive outcome.
The oncology community should have the opportunity
to evaluate satraplatin in different combinations and
find the optimal treatment regimen.
Clinical Trial Endpoints
We will never have all the facts to make a perfect judgment, but
with the aid of basic experience we must leap bravely into the
future.
Russell R. McIntyre
The agency has developed guidelines for the conduct of
clinical trials and the endpoints to be measured in deter-
mining efficacy. They have reviewed their performance
regarding endpoints for approval of new anti-cancer
agents in 2003 [44] and more recently they have issued
new guidelines [45]. The bottom-line is that the FDA con-
tinues to favor the most conservative approach. They favor
624 Regulatory process for approval of biologicals for cancer therapy
OS as the ‘most reliable…, precise and easy to measure
…, preferred endpoint’ and state that it should be ‘evalu-
ated in randomized controlled studies’ [45]. It is obvious
that they do not favor Accelerated Approval because it
allows for single arm trials with endpoints other than OS.
Nevertheless, one may still be able to negotiate and come
to an agreement with the FDA to utilize TTP or PFS as
primary endpoints. In fact, some new agents have even
been approved with RR (response rate) as the primary
endpoint. What the FDA does not realize is that OS is
plagued by multiple factors that introduce bias when this
endpoint is utilized. They claim that (in the case of OS)
‘bias is not a factor in endpoint measurement’.
Obviously, prolongation of survival when cure is not
possible is a most desirable objective for the individual
patient as well as for those around him. Even so, this is
not a strong argument for OS as an endpoint because
survival as a goal for the patient is an entirely different
issue than survival as an endpoint for clinical trials.
Using OS as an endpoint has major drawbacks. These
studies require more patients, more time, more resources,
cost much more, and delay the availability of useful
anti-cancer agents. Using OS as an endpoint prolongs
clinical trials, delays cancer drug approval and is not in
the better interests of all patients, current and future.
Shouldn’t we be trying “to help or at least to do no harm?”
That is not achieved by using OS as an endpoint.
OS feels comfortable to the FDA as it appears to
decrease the risks that they take whenever they approve
a new agent. It only requires two time-points: date on
study and date of death. Death is easily confirmed via a
death certificate. OS is tantalizingly deceptive as it gives
the impression of being so firm, simple, well defined
and definitive that little judgment might be required in
its interpretation whereas other endpoints might appear
to be more problematic [34]. Actually, TTP and PFS are
better endpoints even though they do require the perfor-
mance of CTs, bone scans, or MRIs. These endpoints
are not subject to the multiple factors affecting OS, par-
ticularly subsequent therapies, which are unrelated to
the experimental therapy under evaluation and make OS
a poor endpoint even for randomized and controlled
clinical trials. Studies utilizing OS as the primary end-
point require more patients, more time, more resources,
cost much more, and delay the availability of useful
anti-cancer agents. Using OS as an endpoint prolongs
clinical trials, delays cancer drug approval and is not in
the better interests of the cancer patient. Instead of OS
we should use endpoints with clear cause and effect. In
the case of TTP, the cause is the biologic activity of the
anti-cancer agent being studied, and the effect is lack of
progression, an effect that ends when progression occurs.
Both the onset and the end of the effect are easily
measured (on-study date to date when progression is
identified) and occur during the study before any other
therapy is administered. In the case of OS, the effect is
survival duration. The cause is the biologic activity of
the anti-cancer agent being studied as influenced by
subsequent therapies, supportive care, etc. The effect, as
modified by all of these factors, ends when death occurs.
Death occurs after the study has ended and patients may
have gone on to other studies with experimental drugs
and/or received additional conventional chemotherapy.
Endpoints such as TTP and PFS are much better end-
points for clinical trials than OS. In fact, the FDA has
approved more new anti-cancer agents in the past 15
years based on these than based on OS. Choosing the
perfect endpoint is not easy but ‘with the aid of basic
experience we must leap bravely into the future’ and the
future lies with the shorter timeframes of TTP and PFS.
The FDA’s Track Record
Statistics is a tool, it is meant to serve, not to enslave [9].
In the past 15 years, since Accelerated Approval regula-
tions have existed, the FDA has granted approval for
around 30 anticancer indications [46–48]. If one counts
only the first indication for each new agent, there have
been a total of 23 new agents that have been granted
Accelerated Approval. However, only 12 of these 23
agents were approved based on response as the sole
endpoint and based on a single arm trial. This is the sub-
set that truly represents the intent of “Accelerated
Approval”. In the case of the other 11 agents there were
multiple indications, and randomized trials were avail-
able for some, or the endpoints included some measure
of response duration. Twelve agents in 15 years, less
than one agent per year, is hardly a record of which to be
proud and clearly indicative of the FDA’s bias against
Accelerated Approval. Accelerated approval has been
made more and more difficult since it became available
in 1992. It appears as if the intent is to dissuade appli-
cants from utilizing this mechanism. Pharmaceutical
companies have become increasingly reluctant to use
this mechanism. The FDA’s track record to date repre-
sents a failure to capitalize on an opportunity to bring
many more promising new agents early-on to the patient.
They do use their statistics to make it look otherwise.
But then, the FDA does use statistics in different ways
such as a tool in their decision making. However, they
do go to the extreme of expecting that statistics will sub-
stitute for common sense and good judgment [9]. Some
believe that Statistics is precise, an exact, science. They
would have you believe that an analysis is correct if the
p-value is less than 0.05 or better yet, less than 0.01 and
that truth lies in a favorable hazard ratio, a significant
Antonio J. Grillo-López
log-rank test, a pair of Kaplan Meier (KM) curves that
do not meet, or a median PFS projection from such
curves. The truth is that statistics are fallible and often
inaccurate. A p-value of 0.0298 (or any p-value with
four decimal places) is a joke in spite of the apparent
accuracy conferred by the four decimal places. A p-value
is worth only as much as the data from which it is
derived. There is truth in the saying that Statistics is the
science of producing reliable fact from unreliable figures.
Clinical data is never 100% accurate, relevant, or spe-
cific. Patients and patient populations are remarkably
heterogeneous biological entities that change and evolve
constantly. It is impossible to reliably quantify an end-
point that depends on multiple and variable factors.
Patient populations even in the most carefully stratified,
randomized and balanced clinical trials will be hetero-
geneous. You may think that prognostic factors are bal-
anced, but, as an example, two arms balanced on the
number of prior therapies can be quite different depending
on the nature of the therapies and the patient’s response.
How can you stratify or balance for immune system
responsiveness or for bone marrow reserves when we
can’t even measure those very accurately? Some are
concerned when two curves on a Kaplan Meier graph
for PFS come together at the tail end. The only way that
two KM curves will not come together at some point [in
a TTP, PFS or OS graph] is when one of the two thera-
pies confers immortality. A median PFS, from a Kaplan
Meier graph, will be just a “projected or estimated PFS”
until all events prior to that median have occurred or
there are no remaining temporarily censored patients
before that median. Only then will that median be fixed,
before that there is every probability that it will change.
The shape of the curve also changes over time. And,
likewise, it will be fixed only when all events affecting
the curve have occurred. Few clinicians truly under-
stand Kaplan Meier graphs, their intricacies or short-
comings. The FDA errs in the opposite direction. They
expect too much of statistics.
Is there a Simpler Approach to
the Development of New
Therapies? A Proposal
I don’t know the key to success, but the key to failure is trying to
please everybody.
Bill Cosby
A simpler approach would be to approve new anti-cancer
agents based on reasonable safety and efficacy in Phase
II clinical trials [6]. This would reduce average clinical
development time from 7 to 3 years. Clinical cancer
625
drug development and clinical cancer treatment devel-
opment are two related but distinct objectives. It is criti-
cally important to understand this distinction. The FDA
is intended to regulate drug development and not treat-
ment development.
Drug Development Versus Treatment
Development
Clinical cancer drug development may be defined as the
process required for the development of a new antican-
cer agent (single agent). It is usually carried out by a
pharmaceutical company, is tightly regulated and influ-
enced by the FDA, and has drug approval and marketing
as its goal. Clinical cancer drug development consists of
a lengthy series of activities that require expert profes-
sional staff and complex and costly equipment, systems
and procedures.
Clinical cancer treatment development may be
defined as the process required for the development of a
new anticancer treatment (combination or multimodal-
ity). It is usually carried out by academic institutions,
consortia, or cooperative groups, is loosely regulated
and not significantly influenced by the FDA, and has the
development of new combinations or multimodality
treatments as its goal (Fig. 5).
The FDA’s responsibility, as established by law, is
the review and approval of new anti-cancer agents.
Pharmaceutical industry is responsible for cancer drug
development and this is what the agency is required to
regulate. Clinical cancer drug development should con-
sist of the appropriate Phase I and Phase II clinical trials
(sponsored by a pharmaceutical company) to determine
the safety and clinical activity of a new anti-cancer
agent as monotherapy. Development should be col-
lapsed to the shortest possible timeframe and allow for
early termination of ineffective agents. This would also
allow for the expeditious development of the truly
worthwhile drugs and decrease overall development
time and cost (Fig. 6).
The responsibility for cancer treatment development
should be squarely on the shoulders of the oncology
community. It is the oncology community (including
academic institutions, cancer centers, cooperative groups,
consortia, and others) that, in collaboration with pharma-
ceutical industry, should take a new anti-cancer agent
through the appropriate Phase II and Phase III clinical
trials to find its optimal use in combination with other
agents. It is the oncology community that should be
responsible for defining the role of a new agent within a
treatment regimen and its place in the therapeutic arma-
mentarium. This task is complex and requires multiple
clinical trials of different combinations. The Phase III
626 Regulatory process for approval of biologicals for cancer therapy
Figure 5. Cancer treatment development
Figure 6. Cancer drug approval: FDA
studies require large numbers of patients and take a
long time to complete. It can take decades before the
ultimate use of a new anti-cancer agent, within combi-
nation regimens, can be elucidated (Fig. 4). Nevertheless,
during this time, patients can benefit from treatment
with the new agent even though its optimal use in
combination is still in the process of being defined.
This process, cancer treatment development, should
not be regulated by the FDA.
Kinds of Useful Anticancer Agents
It is practical to classify clinically useful anti-cancer
agents in three categories from the development as well
as from the regulatory viewpoint. These are: (a) agents
with significant activity as single agents, (b) agents with
some activity and/or synergy with other agents, and (c)
agents with no significant clinical activity as single
agents but with significant synergism with other agents.
Agents with significant activity as single agents
have a response rate that compares favorably with that
of other available agents (when used as monotherapy).
They should be approved on the basis of Phase II trials
with data from historical controls. The entire applica-
tion need not include more than 300 to 350 patients.
Some measurement of response duration, such as TTP
or PFS, would be useful but should not be an absolute
requirement. After all, these new anti-cancer agents
Antonio J. Grillo-López
are most frequently combined with other agents rather
than used as monotherapy. It is the response duration
of the combination that is of interest. However, the
optimal combination may take years to define. In the
meantime, these agents could be approved and made
available to patients who may benefit from them. What
is the downside of such an approach? At the extreme,
the FDA may approve a drug that later shows toxic
effects not seen in the original experience included in
the application, and/or the response rate may turn
out to be lower than originally determined, and/or
no additive effect is seen when used in combination
regimens. It is unlikely that all of these will be
observed. If this extreme situation does occur, then
approval can be withdrawn or, alternatively, the drug
will die on its own as it will not be prescribed (a sort of
market induced apoptosis). There are risks but they are
small relative to the potential benefit of having an
active agent approved earlier rather than much later.
Agents with some activity and/or synergy with other
agents have a response rate lower than that of other avail-
able agents (when used as monotherapy). They may or
may not have synergism with other drugs or combina-
tions. Their approval requires a risk benefit determina-
tion. The FDA will not approve such agents unless they
show superiority or non-inferiority to a standard regimen
in a Phase III trial. Thus, in most instances, such an agent
will live or die based on its activity within a combination
that will probably not be the optimal combination. These
agents should be approved based on Phase II trials and
the oncology community allowed to conduct the multiple
studies that will eventually show how they may best be
utilized in combination regimens. As above, there are
risks, but not much to lose in approving such an agent.
Agents with no significant clinical activity as single
agents but with significant synergism with other agents
are inactive as monotherapy but exhibit significant syn-
ergy with other drugs or combinations. In this case,
there is no choice but to carry out controlled, random-
ized Phase III trials to show the synergistic effect.
Two of these three kinds of useful anti-cancer agents
can be approved based on reasonable safety and efficacy
in Phase II clinical trials. They would be available to
patients earlier, development costs and drug prices should
decrease and the oncology community could proceed with
combination studies earlier so that new curative regimens
might be identified. A criticism might be that Pharmaceutical
Industry will benefit from these earlier approvals. This
should be tempered by the additional risk and cost that
early approvals entail including the occasional approval
withdrawal or failure in the marketplace. All in all, it
seems like a win–win situation for the patient.
627
It is hard to please everybody but this does seem like
a reasonable and simple approach.
Conclusions
We cannot avoid meeting great issues. All that we can determine
for ourselves is whether we shall meet them well or ill.
Theodore Roosevelt
There is a regulatory process in place for the approval of
biologicals for cancer therapy and, whether we like it or
not, it must be followed if new anticancer agents are to
be developed and approved. The process is unduly com-
plex and the requirements many times unnecessary or
overly rigorous. The FDA’s interpretation of their man-
date has gone above and beyond what was originally
envisioned; but then, they themselves interpret the laws
by which they must abide. Additionally the current pro-
cess significantly delays the availability of new agents
to the patients who can benefit from them. This must
change. The proposal discussed in section 9.0 above
(approval based on reasonable safety and efficacy in
Phase II clinical trials) is a possibility that should be
seriously considered. It would serve to reduce average
clinical development time from 7 to 3 years (Fig. 6)
making the new agent available to patients earlier. Also,
it would make the new agent available to the oncology
community earlier for the broad combination studies
that would eventually lead to the identification of the
agent’s role within the optimal treatment regimen (Fig. 5).
Every person interested in the wellbeing of the cancer
patient must help effect this change. These are great
issues and no one can avoid meeting them.
Curing Cancer
Destiny is not a matter of chance, it is a matter of choice; it is not a
thing to be waited for, it is a thing to be achieved.
William Jennings Bryan
We know that we can cure cancer. Rituximab is an example
of a biological that has significantly increased the
cure rate in an important cancer (DLBCL). Every year
there are in excess of 15,000 NHL patients in the US,
another 15,000 in Europe, and a total of over 50,000
worldwide who are potentially curable if treated with R
+ CHOP. This also means that every year delay in getting
rituximab to the market cost over 50,000 lives. We need
more efficient and faster processes for the approval of
cancer therapies. In 2004 there were over 150 new anti-
cancer agents in clinical trials, including 59 biologicals.
Of those, 13 were in Phase III. Of the 59 biologicals only
628 Regulatory process for approval of biologicals for cancer therapy
5 have been approved. We need to do better than this.
The destiny of the cancer patient is a matter of choice
and the choice must be to act faster, decrease bureau-
cracy and delays, approve earlier and make promising
new agents available to those who need them. Just like
rituximab there may be new agents in development that
can extend lives or be curative. They must be evaluated
earlier with a view to their optimal role in combination
regimens. Regulatory review and approval processes
should help and not impede this ultimate objective.
The Patient’s Plight
Were we directed from Washington when to sow, and when to reap,
we should soon want bread.
Thomas Jefferson
The patient is at the receiving end of drug development.
The system is such that it takes a long time for effective
drugs to go from discovery to being broadly available to
patients. It is the government that imposes the delays,
hurdles and restrictions. The patient has to wait and yet,
the patient does not have the luxury of time. FDA reform
must stop being a buzz word heard only prior to elec-
tions. It must become reality.
What does the Future Hold?
I believe that the essence of government lies with unceasing con-
cern for the welfare and dignity and decency and innate integrity of
life for every individual.
Lyndon B. Johnson
Today’s physicians have a responsibility to actively par-
ticipate in making new therapies available to the cancer
patient. We know that cancer can be cured. However,
biologicals and all new anticancer agents must be effi-
ciently and rapidly evaluated to find those that are most
promising. They must then be incorporated into combi-
nation regimens that provide the greatest opportunity
for cures. Physicians in the pharmaceutical industry
must do everything within their reach to ensure that new
agents are developed and submitted for approval in the
most expeditious fashion. Physicians in our regulatory
agencies (FDA, EMEA, and others) must likewise
understand that they have similar duties and responsi-
bilities to the patient. Being employed by a bureaucracy
does not mean that your sacred oath is no longer valid.
In fact, physicians in regulatory agencies could be an
important catalyst in making new promising therapies
available to patients in a timely fashion. Government
must come to understand that they can correct the issues
that we face given the current regulatory process for the
approval of biologicals and other cancer therapies. They
need to act and show they are truly ‘concerned for the
welfare and dignity and decency and innate integrity of
life for every individual’.
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Association of serum rituximab (IDEC-C2B8) concentration and
anti-tumor response in the treatment of recurrent low-grade or fol-
licular non-Hodgkin’s lymphoma. Ann Oncol 9:995–1001.
21. Grillo-López AJ (2000). Rituximab: An insider’s historical per-
spective. Semin Oncol 27(6, 12 Suppl):9–16.
22. Maloney DG, Grillo-López AJ, Bodkin DJ, et al. (1997). IDEC-
C2B8: Results of a Phase I multiple-dose trial in patients with relapsed
non-Hodgkin’s lymphoma. J Clin Oncol 15(10):3266–3274.
23. Maloney DG, Grillo-López AJ, Bodkin D, et al. (1997). IDEC-
C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in
patients with relapsed low-grade non-Hodgkin’s lymphoma. Blood
90(6):2188–2195.
24. McLaughlin P, Grillo-López AJ, Link BK, et al. (1998). Chimeric
anti-CD20 monoclonal antibody therapy for relapsed indolent
lymphoma: Half of patients respond to a 4-dose treatment pro-
gram. J Clin Oncol 16:2825–2833.
25. McLaughlin P, Hagemeister FB, and Grillo-López AJ (1999).
Rituximab in indolent lymphoma: The single-agent pivotal trial.
Semin Oncol 26(5, 14 Suppl):79–87.
26. Grillo-López AJ, Varns C, Shen D, et al. (1999). Overview of the clini-
cal development of rituximab: First monoclonal antibody approved for
the treatment of lymphoma. Semin Oncol 26(5, 14 Suppl):66–73.
27. Wei A, Shen D, Kim D, et al. (2003). The Electronic Solution to
the FDA Submission – Notes From the Field. Appl Clin Trials
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28. Czuczman M, Grillo-López AJ, Saleh M, et al. (1995). Phase II
clinical trial of IDEC-C2B8/CHOP combination therapy in low
grade lymphoma: Preliminary results. Proc Am Soc Clin Oncol
14:401 (#1261).
29. Demidem A, Lam T, Alas S, et al. (1997). Chimeric anti-CD20
(IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell
line to cell killing by cytotoxic drugs. Cancer Biother Radiopharm
12(3):177–185.
30. Vose JM, Link BK, Grossboard ML, et al. (2001). Phase II Study
of Rituximab in combination with CHOP chemotherapy in patients
with previously untreated, aggressive non-Hodgkin’s lymphoma.
J Clin Oncol 19:389–397.
31. Vose JM, Link BK, Grossbard ML, et al. (2005). Long-term update
of a phase II study of rituximab in combination with CHOP che-
motherapy in patients with previously untreated, aggressive non-
Hodgkin’s lymphoma. Leuk Lymphoma 46(11):1569–1573.
32. Coiffier B, Lepage E, Briere J, et al. (2002). CHOP chemother-
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structure and function (Editorial). Expert Rev Anticancer Ther
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structure and function (Editorial). Expert Rev Anticancer Ther
5(5):753–756.
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21CFR601.40).
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States Food and Drug Administration approval of oncology drugs.
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of cancer drugs and biologics (2007). http://www.fda.gov/cder/
guidance/7478fnl.pdf
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and Research Last Updated: 08/20/2007 07:47:37. Originator:
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scripts/cder/onctools/statistics.cfm
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of oncology products: A decade of experience. J Natl Cancer Inst
96:1500–1509.
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pre- and post-accelerated approval (Editorial). Expert Rev Anticancer
Ther 5(2):197–200.
21.1 Cancer biotherapy: 2009 disease-related
activity
ROBERT K. OLDHAM AND ROBERT O. DILLMAN
In the last decade, we have witnessed a burgeoning number
of biologicals under clinical investigation, either singly
or in combination with other biologicals or chemothera-
peutic agents. Biotherapies have demonstrated efficacy
against certain malignancies and are approved for gen-
eral use in medicine. It is expected that their assimilation
into our standard anticancer armamentarium will con-
tinue to broaden in the next decade, leading to the domi-
nance of biotherapy in cancer treatment soon after the
year 2010. This chapter will summarize disease-related
activity for selected biotherapies as we move into the
second decade of the new millennium.
Historically, cancers have been classified by histo-
pathological features, based on the pathologist’s ability
to discern similarities and differences among cancers by
scrutinizing tissue sections under the microscope. Over
100 types of cancers have been so categorized. This
pathology-based classification scheme, upon which the
previous standard modalities of cancer treatment – surgery,
radiation therapy, and chemotherapy – were structured,
has contributed to major advances in the treatment of
selected malignancies.
As cancer biology and cellular genetics, including
cancer susceptibility genes become better understood,
we are learning that the unregulated growth of tissues,
the activation of oncogenes, mutations, the persistence
of cells that normally become senescent, and matura-
tional aberrations may segregate in patterns not amena-
ble to simplistic histopathological classification. Early
evidence of such conflicts in categorization is provided
by the presence of similar antigens on cancer cells aris-
ing from very different tissues. For example, antibodies
have been described that react very clearly with breast
cancer, colon cancer, and certain other adenocarcino-
mas. As such, these antibodies cross histopathological
and anatomic borders. Similarly, there may be common
reactivities among tumors of lymphoid origin, of
squamous differentiation, and of mesenchymal deriva-
tion. A better understanding of oncogene activation,
genomics and of normal and aberrant regulation of
growth and differentiation may provide for a dynamic
classification scheme rather than a static descriptive one
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
we now use. As new approaches in biotherapy are tested,
scientists and clinicians must be constantly aware that
our current histological anatomic classification system
is largely artificial and that clues to the clinical activity
of biotherapy may allow us to reclassify neoplasms
according to biological features and behavior patterns.
Indeed, recent advances in the molecular analysis of
tumors by gene expression profiling and proteomics
have begun to lead to a new paradigm for the classifica-
tion of malignancies based on the molecular signature
of an individual’s tumor derived from the global assess-
ment of thousands of genes. By examining the clustering
of gene expression, new categories of malignancy have
begun to emerge that go beyond traditional distinctions
based on histology alone [6].
Gene profiling and microarray analysis have recently
been shown, by retrospective studies, to correlate with
prognosis. Current efforts are focused on using this kind
of genetic information to design clinical trials in breast,
colon and lung cancer. The hypothesis in breast cancer
is that a certain profile might predict for a better prognosis
using hormone therapy alone vs. adding chemotherapy
for those individuals having a gene profile consistent
with more aggressive disease. Once such a hypothesis is
validated through current prospective clinical trials, the
adjuvant therapy of breast cancer will be much improved,
given that we can focus chemotherapy on the most likely
subgroup to benefit [2, 3, 4, 7, 13].
Such considerations transcend all aspects of classical
developmental therapeutics, in which surgery, radiation
therapy, and chemotherapy evolved along disease-specific
and anatomic lines. Thus, although phase I toxicity trials
are conducted across histological boundaries, phase II and
III studies progress within a fairly rigid disease-specific
format. As a consequence, antitumor activities in lung
cancer, breast cancer, colon cancer, etc. are discussed
and debated. While such a testing paradigm may have
served oncologists well for protocol construction and for
regulatory oversight, this mindset of classical pathology
oriented phase II–III studies may actually inhibit the
development of biotherapy [8]. Furthermore, the devel-
opment of biological therapies and molecularly targeted
631
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anticancer agents poses special challenges to the design of
clinical trials to include models that assess the effect of the
agent on its putative target and the validation of new sur-
rogate endpoints [12]. The traditional objectives of phase
I and II studies (maximally tolerated dose and response
rate, respectively) developed for cytotoxic agents are less
relevant to biological and targeted therapies where a
“biologically effective dose” may prevent tumor growth
and induce stabilization of disease that is of important
clinical benefit but is not reflected by a radiographically
measurable objective response. With these new modali-
ties, we must be open to the possibility that the biologi-
cal activity of these agents and approaches may sort
unpredictably and may require a more individualistic
orientation. It is abundantly clear that monoclonal anti-
bodies, proteomics, and gene expression profiling tech-
nology can allow for the precise description of an
individual patient’s cancer using panels of antibodies,
mass spectroscopy signatures, and microarrays that rep-
resent all of the expressed genes in the human genome
[1, 5, 10, 11]. The use of the laboratory to develop
patient-specific therapies may allow scientists and clini-
cians to approach the cancer problem from a completely
different and more individualized perspective [9].
Many biotherapies are supportive or ancillary to the
anti-tumor activity of standard approaches such as che-
motherapy. Use of colony-stimulating factors, erythro-
poietin, blood transfusions, marrow transplantation, etc.
has broad application in oncology. These techniques
were described in earlier chapters.
While recognizing the limitations of organ-specific
categorization and the likely reclassification of tumors
based on either gene expression clusters or the expres-
sion of common molecular therapeutic targets, it remains
a standard and useful exercise to describe the clinical
activities in biotherapy within such a historical frame-
work. Therefore, in this chapter, a brief summary of the
activity of various forms of biotherapy will be provided
by cancer type. This chapter is designed to describe
Cancer biotherapy: 2009 disease-related activity
these activities briefly with references into 2009 and is
for quick reference only. The reader is encouraged to
refer to specific, subject-oriented chapters and refer-
ences for detailed information on specific methods of
cancer biotherapy.
References
1. Avner BP, Liao SK, Avner B, DeCell K, Oldham RK. Therapeutic
murine monoclonal antibodies developed for individual cancer
patients. J Biol Response Mod 1989; 8:25–36.
2. Brenton JD, Carey, LA, Ahmed AA, et al. Molecular classification
and molecular forecasting of breast cancer: Ready for clinical
application? J Clin Oncol 2005; 23(29):7350–7360.
3. Cheang MCU, Voduc D, Bajdik C, et al. Basal-like breast cancer
defined by five biomarkers has superior prognostic value than triple-
negative phenotype. Clin Cancer Res 2008; 14(5):1368–1376.
4. Cronin M, Sangli C, Liu M-L, et al. Analytical validation of the
oncotype DX genomic diagnostic test for recurrence prognosis and
therapeutic response prediction in node-negative, estrogen receptor-
positive breast cancer. Clin Chem 2007; 53(6):1084–1091.
5. Lakhani SR, Ashworth A. Microarray and histopathological analysis
of tumours: the future and the past? Nat Rev Cancer 2001;
1:151–157.
6. Liotta L, Petricoin E. Molecular profiling of human cancer. Nat
Rev Genet 2000; 1:48–56.
7. Loi S, Sotiriou C, Buyse M, et al. Molecular forecasting of breast
cancer: Time to move forward with clinical testing. J Clin Oncol
2006; 24(4):721–722.
8. Oldham RK. Biologicals and biological response modifiers: design
of clinical trials. J Biol Response Mod 1985; 4:117–128.
9. Oldham RK. Custom-tailored drug immunoconjugates in cancer
therapy. Mol Biother 1991; 3:148–162.
10. Oldham RK, Lewis M, Orr DW et al. Adriamycin custom-tailored
immunoconjugates in the treatment of human malignancies. Mol
Biother 1988; 1:103–113.
11. Orr D, Oldham R, Lewis M et al. Phase I trial of mitomycin C
immunoconjugates cocktails in human malignancies. Mol Biother
1989; 1:229–240.
12. Tan AR, Swain SM. Novel agents: clinical trial design. Semin
Oncol 2001; 28:148–153.
13. van ‘t Veer LJ, Mongyue D, van de Vijver MJ, et al. Expression
profiling predicts outcome in breast cancer. Breast Cancer Res
2003; 5:57–58.
21.2 Biological therapy of melanoma
ROBERT K. OLDHAM
Melanoma is among the human solid tumors that best
exemplify the application of biological therapy to the
treatment of cancer. In the first decade of the twenty-first
century, more than 4 million people will be diagnosed
with malignant melanoma, and the incidence continues to
increase in developed countries worldwide [9]. Although
primary cutaneous melanoma is highly curable, with 85%
of patients enjoying long-term survival after surgical
excision, disseminated melanoma was amenable only to
palliation with radiation therapy and chemotherapy [41].
Although short term responses to chemotherapy are not
uncommon, particularly in skin, lung and subcutaneous
lesions, significant long-term benefit in visceral disease is
infrequent, with 5-year survival remaining less than 5%
for disseminated disease. Because melanoma frequently
affects young individuals, half of the patients who die of
melanoma had a life expectancy of greater than 25 years,
and the productive years of life lost exceed those of any
other solid tumor [121].
Melanoma and the Immune
System
Melanoma has long been known to be a tumor with
unusual behavior in certain patients. Spontaneous, com-
plete regression occurs in one patient in 1,000–10,000
with lesser degrees of temporary spontaneous partial
regression in as high as 0.5% of patients. Such observa-
tions indicate that the body has some inherent capacity
to induce regression of this disease in selected instances.
Despite considerable investigation, the mechanisms of
such spontaneous regressions remain unclear, and formal
proof of the involvement of an innate or adaptive anti-
melanoma immune response is still lacking.
Several features of malignant melanoma suggest that the
in vivo biological responses that play a role in the progres-
sion and regression of this form of cancer involve the
immune system. For this reason, melanoma is an attractive
and well-examined model for the therapeutic use of bio-
logical agents, particularly those designed to enhance or
manipulate the host immune response. First, the infrequent
spontaneous regression of cutaneous and disseminated
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
melanoma has been carefully documented [92]. Indeed,
approximately 5% of patients with disseminated melanoma
do not have an identifiable primary lesion, suggesting that
the cutaneous disease may have spontaneously regressed
before the growth of metastases is detectable [94].
Paradoxically, partial regression of primary melanoma
lesions has been reported by some observers to be associated
with a worse prognosis; however, this is likely due to the
difficulty that this feature adds to the assessment of risk in
the pathological specimen. This phenomenon has recently
been shown to involve cytolytic T-lymphocytes [37, 71].
Second, paraneoplastic depigmentation events, such as the
development of vitiligo and “halo” formation around
primary skin melanomas and nevi are thought to indicate
an immune mediated reaction against the pigmented cells
in these lesions, and the development of vitiligo in
malignant melanoma during biotherapy carries an improved
prognosis [24]. The corollary of this clinical observation
may have therapeutic implications: if the immune
recognition of melanoma can lead to the destruction of
normal melanocytes, immunization with melanocyte
antigens may lead to the rejection of melanomas [7]. Third,
the presence of tumor infiltrating lymphocytes in primary
melanoma lesions indicates a host immune response,
and the degree of lymphocyte invasion is of prognostic
significance [15, 25]. Fourth, there are patients whose
melanoma may remain dormant for many years, suggesting
innate immune surveillance and control of micrometastatic
disease, who subsequently relapse and die from dissemi-
nated melanoma 10 or more years after the diagnosis of the
primary lesion [108, 110]. Finally, the incidence of mela-
noma is higher and the prognosis worse among immuno-
suppressed kidney transplant recipients, providing
additional support for the role of immune surveillance in
the evolution of malignant melanoma [23, 34].
Biotherapy of Malignant
Melanoma
Early observations indicated that the immune system
could be harnessed therapeutically to treat malignant mel-
anoma. Intratumoral injection of bacterial extracts such as
633
634
Bacille Calmette-Guerin (BCG) or Corynebacterium
parvum frequently induced the regression of the injected
melanoma and occasionally lead to tumor regression at
sites of lymphatic drainage and rarely distant metastases
[90]. The natural history of malignant melanoma and these
early therapeutic observations provide a compelling ratio-
nale for the systematic evaluation of biological therapy in
malignant melanoma. In this light, many agents have now
been evaluated to enhance or mimic the host immune
response against melanoma. These agents have included
effector cells such as lymphocytes derived from resected
tumors or the blood of melanoma patients; antibodies
directed at various epitopes on the melanoma cell; vac-
cines composed of natural or synthetic components of the
melanoma cell or derived from mimic “anti-idiotype”
antibodies; non-specific stimulators of the immune system
such as BCG and the cytokines. Until recently, only the
cytokines have consistently shown clinical, anti-tumor
activity in melanoma, and two of these cytokines, inter-
feron alpha and interleukin-2, have emerged as useful in
clinical practice for the treatment of patients with malig-
nant melanoma [10, 53, 62].
Although durable responses are rare, cytotoxic therapy
for melanoma leads to tumor regression in a substantial
number of patients, and the activity of single agents and
chemotherapy combinations is well established. As a
result, treatment regimens that combine chemotherapy
and biological therapy agents, usually cytokines, are
referred to as biochemotherapy [46, 96, 109, 119].
Monoclonal antibodies that influence the immune
response are currently in clinical trials. Chief among
these are anti-CTLA-4 antibodies, which have been
promising in early-phase clinical trials with the induc-
tion of both partial and complete responses in patients
with advanced melanoma. Toxicities have been substantial
with bowel inflammation and severe colitis as well as
iritis being seen in many of these patients. Vitiligo also
is a side effect of this form of biological therapy and
may predict a good response to the monoclonal antibody
therapy [6, 27, 33, 42, 91, 107].
The gene therapies for malignant melanoma are new
topics of interest; in these strategies, a therapeutic gene is
introduced into an effector cell or a gene encoding a strong
antigen is introduced directly into the melanoma cell.
Clinical trials with gene therapies are now underway.
Immunostimulation
and Vaccines
Attempts to augment native immunologic defenses using
various non-specific immunostimulators, particularly
Bacillus Calmette-Guerin (BCG), have been intensively
Biological therapy of melanoma
studied in melanoma. Up to 90% of melanoma nodules
injected intralesionally with BCG will demonstrate
regression in immunocompetent patients. In 15–20% of
such patients, associated distant shrinkage of nodules
may be seen [77]. Tumor regression by the local applica-
tion of BCG has also been successfully applied to the
treatment of superficial bladder cancer, and the mecha-
nism of tumor regression has been investigated in this
model. The local application of BCG sensitizes host
T-cells to peptides in the extracts, and the presentation of
these peptides by host antigen presenting cells within the
injected tumor bed triggers local T-cell cytokine produc-
tion that can activate host cells that can directly recog-
nize and kill tumor cells [98, 99, 108]. Preparations of
cellular constituents, such as cell-wall skeleton, may also
serve as effective adjuvants [123]. Systemic effects have
been demonstrated by the emergence of antimelanoma
antibodies and by lymphocytic infiltration of regressing
noninjected lesions [77].
Vaccines derived from allogeneic melanoma cells have
long been of interest. In one early report, an enriched
tumor cell vaccine was infused intra-lymphatically, pro-
viding proof of principle for the concept of inducing
host antitumor immunity with antigens from allogeneic
melanoma cells by demonstrating that the effects of
such intra-lymphatic immunotherapy were not limited
to regional lymph nodes and that systemic response
were seen [1]. These initially encouraging results have
lead to intense study of this strategy of immunomodula-
tion for at least the last quarter century, yet these efforts
have not significantly improved the survival of mela-
noma patients [78]. However, immunizing melanoma
patients with a pure Gm2 ganglioside, known to be on
melanoma cell membranes, has induced circulating IgG
and IgM antibodies. The induction of such antibodies
has been reported to be accompanied by prolonged
survival [69, 75, 80].
While definitive proof of the success of vaccines
derived from allogeneic melanoma cells is lacking, at
least one allogeneic cell lysate vaccine (Melacine) has
undergone rigorous analysis and was approved in 1999
for the treatment of disseminated melanoma in Canada
based on phase III results demonstrating superior quality
of life during active therapy for disseminated melanoma
compared to a four-drug chemotherapy combination
[39]. The two therapies achieved similar efficacy results.
Survival data was also compared from the Phase III
study with a comprehensive meta-analysis of therapies
for Stage IV melanoma showing that the median sur-
vival of 11 months among evaluable patients receiving
this allogeneic vaccine was better than that achieved by
other available therapies [59]. Further, a significantly
longer median survival of 18.2 months was observed in
Robert K. Oldham 635

patients who were clinical responders to Melacine therapy.
Melacine has not been approved by the FDA for use in
the U.S. as of 2008.
More recent studies using genetically engineered
vaccines have been performed. Details can be found in
the chapter on vaccines and the chapter on gene therapy.
This work was prompted by previous studies using par-
tially purified tumor vaccines (Table 1).
More recent studies have used highly purified or
genetically engineered vaccines with a variety of anti-
gens and adjuvants which are immunogenic in humans
(Tables 2 and 3).
Clinical trials with genetically engineered vaccines
are ongoing and while there is evidence of stimulation
of cytotoxic T cells, enhancement of antibody titers and
augmentation of DTH responses, the anti-tumor activi-
ties of these vaccines remain to be confirmed.
Interferon
In phase I trials with alpha-interferon, an occasional
melanoma patient experienced objective tumor regression.
Subsequent phase II studies using partially purified
human leukocyte or lymphoblastoid alpha-interferon
showed response rates as high as 12% [43, 95]. The com-
posite experience using recombinant alpha-interferon
has yielded a response rate of approximately 20% [51,
81]. On occasion, these responses have been complete,
but activity in visceral disease has been infrequent.
Perhaps as many as 30% of patients achieving a com-
plete response enjoy an enduring remission. Studies
using alpha-interferon in the adjuvant surgical setting
demonstrated a reduced recurrence rate with substantial
doses of interferon. This study lead to FDA approval of
interferon for the treatment of melanoma. Beta and gamma
interferon were also evaluated in melanoma with both
now approved for non-cancer indications. A suggested
potentiation by cimetidine of human leukocyte inter-
feron activity in malignant melanoma [11, 47] has not
been confirmed by later studies using natural or recom-
binant alpha-interferon [51, 68].
There has been great interest in exploring the benefits
of adjuvant IFN for high risk, surgically excised mela-
noma. Based on the findings in metastatic disease, it
was hoped that IFN would be most active when the
tumor burden was low and would increase the overall
survival of patients at high risk of relapse after surgery
for melanoma. Multi-institutional, randomized con-
trolled trials (RCTs) have been conducted in the United
States and Europe in an attempt to demonstrate a benefit
to adjuvant IFN. The inter-group United States trials,
lead by the Eastern Cooperative Oncology Group
(ECOG), E1684, E1690, and E1694, have received the

Table 1. Tumor antigen vaccine trials

Stage of disease No. of patients Vaccine preparation Adjuvant Controls Results Ref.
Resected II 25 Autologous soluble BCG Historical Improved survival over 162
membrane extract historical growth
Disseminated 56 Specific TAA Freund’s complete None Regression seen in 137
approximately 25%
of patients
Disseminated 13 Polyvalent TAA None None CR 1 of 13 PR 1 of 13 36
Resected stage II 94 Polyvalent melanomatumor 40 pts – Alum Historical Improved survival over 38
antigen vaccine 17 pts – Cytoxan historical controls

Table 2. Adjuvants: effects on immunogenicity

Mechanisms of action
Approach Depot effect Macrophage CD4+ T cell and B cell CTL
Conjugate vaccines + − ++ −
Recombinant vector vaccines
BCG + + + ++
Vaccinia + − + ++
Immunological adjuvants
Alum or oil (squalene) + − − −
BCG or BCG CWS + + + −
Endotoxin (lipid A) − ++ + −
Liposomes + − − +
MDP derivatives − + + +
636
Table 3. Clinical trials with immunogenic antigens
Antigen Vaccine No. of patients responding Ref.
GM2 BCG 50/58 37
GM2 KLH 30/30 131, 217
GD2 KLH 4/6 131, 217
Mage 1 – Ongoing trial 361
Mage 3 – Ongoing trial 106
Tyrosinase – Ongoing trial 29
most attention and scrutiny in North America. E1684
enrolled 287 patients and randomly assigned patients to
observation versus 1 year of high dose IFN for 1 year
(“high dose” defined here as at least 10 million units per
square meter) versus observation. Disease free and
overall survival was both improved by IFN treatment
[55], and long-term follow-up data from this study was
confirmatory [55, 56]. In follow-up, E1690 accrued 642
patients to observation or treatment with high dose IFN
or low dose IFN. Although median survival for high
dose IFN was essentially identical to the median sur-
vival for high dose IFN on E1684, overall survival was
not improved by treatment with high dose IFN (or low
dose IFN) [54]. The lack of benefit for IFN in E1690
appears to derive from an unexplained improvement in
the median survival of the observation group in E1690
compared to E1684. A significant number of patients on
observation on E1690 received IFN at the time of recur-
rence, which may confound interpretation of the results
of IFN treatment. Finally, E1694, enrolled 774 patients
to treatment with high dose IFN or a vaccine, and dem-
onstrated that the high dose IFN significantly prolonged
both disease free and overall survival [54]. The vaccine
treatment arm of E1694 had a similar survival compared
to the control arm of E1690, indicating that the improve-
ment in disease free and overall survival resulted from
the treatment with IFN rather than any detrimental effect
of the vaccine treatment.
Other large studies have explored the role of adjuvant
IFN in melanoma. For example, among the more
recently reported, the AIM High Study randomized 654
patients to treatment with low dose IFN for 2 years or
observation. No significant effect was observed on dis-
ease free or overall survival [12]. Several other impor-
tant studies, also using low dose IFN, have also been
performed and failed to convincingly demonstrate an
overall survival benefit for adjuvant therapy for mela-
noma [18]. Definition of the high risk population has
differed among randomized studies, primarily depend-
ing on the inclusion of patients with intermediate or
deep primary lesions or involved regional lymph nodes.
In the E1684 study that initially demonstrated a benefit to
adjuvant IFN, the benefits of adjuvant IFN were limited
Biological therapy of melanoma
to patients with stage III disease that had involved
regional lymph nodes [55]. In addition, the dose and
duration of therapy differ and among studies and this
issue remains contentious. Data from the United States
intergroup studies suggest that higher doses of IFN in the
adjuvant setting result in greater effectiveness. A meta-
analysis of ten randomized controlled trials was recently
conducted and concluded that there was a clear benefit
to adjuvant IFN for prolonging disease free survival but
the benefits were less clear in terms of overall survival
[125]. In this meta-analysis, high dose versus low dose
IFN was compared, and there was no statistically signifi-
cant difference between high dose and low dose IFN treat-
ment. However, a systematic review of eight randomized
controlled trials of systemic adjuvant interferon as
monotherapy versus no treatment (and thus excluded
E1694 from analysis) for high risk melanoma concluded
that there was no clear benefit of IFN therapy on overall
survival and that heterogeneity between the trials made
meta-analysis inappropriate [64]. Thus, despite fairly
rigorous and intensive study, controversy remains regard-
ing the benefits of IFN in the adjuvant setting [56].
Studies of interferon alpha in metastatic melanoma
patients have shown response rates ranging from 0–30%
with an overall average of 16% [15, 19, 20, 32, 35, 52,
63, 112, 117]. As such, about one in six patients with
metastatic melanoma will benefit from interferon alpha
therapy. However, it is notable that a small percentage of
the total group responding to this treatment will achieve
a complete response, and occasionally, long remissions
have been observed. A review of 11 phase II studies of
single agent IFN conducted in the 1980s and comprising
315 patients described an overall response rate of 15%
and a median survival of 8 months [61]. The dose, route
and schedule of IFN administration differed among the
studies, but intramuscular injections given three times
weekly were usually used, and doses of 10 million units
per square meter or higher (up to 50 million units per
square meter) were used in all cases. Complete remis-
sions were observed in 0–4% of patients. It is difficult to
extrapolate the optimal dose and frequency of adminis-
tration of interferon alpha from the available data; a very
wide range of doses and frequencies have been evaluated
in the reported clinical trials (Tables 4 and 5). It is also
not clear that a strong dose–response relationship exists
for interferon alpha in malignant melanoma.
It is important to compare these responses to those
observed for single agent chemotherapy (also in the
15–20% range). Higher response rates were seen with
drug combinations and the best three drug combinations
report responses in the 30–40% range and generally
include cisplatin and dacarbazine. However, even the
Robert K. Oldham 637

Table 4. Clinical trials with recombinant interferon activity for the two drugs [3, 36] while a third showed
Study Dose (MU) route/ Evaluable CR/PR no improvement over dacarbazine alone [120].
schedule patients (%) A very encouraging randomized phase II trial has dem-
Creagen [56] 12/M2 i.m. 3 times 30 20 onstrated that bevacizumab (Avastin) has shown activity
per. week in metastatic melanoma when compared in a randomized
Creagen [58] 50/M2 i.m. 3 times 31 23 phase II trial with interferon. A large randomized phase
per week III study is in progress to compare bevacizumab plus
Coates [49] 20/M2 i.v. daily × 5 15 0
interferon with interferon alone.
Hersey [134] 50/M2 i.m. 3 times 18 11
per week
Legha [178] 3–36 i.m. daily or 3 62 8
times per week
Interleukin 2/lymphokine-
activated Killer (LAK) Cells
Table 5. Biochemotherapy treatment programs
Interleukin-2/lymphokine activated killer (LAK) cells
Study Evaluable patients CR/PR (%)
have been reported to provide a response rate of 50% for
Interferon + DTIC patients with advanced metastatic melanoma [100, 124]
Kirkwood [159] 23 4
(Table 6). Most of these responses have been partial and
Gundersen [124] 15 20
Mulder [220] 31 35 of only several months duration, although occasional
Thompson [354] 86 21 patients do experience complete responses. Subsequent
Sertoli [322] 72 26 studies with interleukin 2/LAK cells and studies using
Interferon + platinum/combinations interleukin 2 in combination with interferon continue to
Hamblin [130] 12 83
Richards [286] 74 57
demonstrate that melanoma is a neoplasm sensitive to
Legha [179] 30 56 these kinds of biological approaches [11, 28, 31, 58,
Pyrhonen [271] 45 53 106, 114]. Overall, response rates have been in the
Richner [288] 20 35 20–30% range. Responses continue to be most frequent
in patients with skin, lymph node, and lung metastasis
and less frequent with abdominal and bony metastasis.
best chemotherapy combinations have failed to demon- The propensity of melanoma to metastasize to the brain
strate a survival advantage, and the duration of response is has been a limiting factor, since many of these patients
usually less than 6 months [60]. A recent meta-analysis have responded peripherally only to relapse and die
explored the net benefit of IFN therapy in metastatic with central nervous metastases. The major challenge
malignant melanoma by compiling all randomized trials from these studies using interleukin-2-based biothera-
that compared a treatment regimen with IFN to a non- pies is to determine why one patient will respond with a
IFN containing treatment regimen. Eleven studies were complete remission and another patient will not respond
identified (five unpublished) that collectively comprised at all. Is this related to the state of immunological acti-
1,164 patients. The meta-analysis showed that the regi- vation inherent to the patient or the state achieved by
mens including IFN-alpha improved response rates and these biotherapies or does it somehow relate to the sen-
overall survival compared with regimens without IFN- sitivity of the melanoma cells to biotherapy?
alpha with odds ratios of 0.65 (for response) and 0.69 If it is possible to summarize all the available data
(for survival). In melanoma, the response rate for the into a single response rate by combining a very heterog-
IFN containing regimens was 24% compared with 17% enous group of clinical trials, IL-2 would appear to ben-
(range, 5–30%) for those without IFN [45]. Taken together, efit about one melanoma patient out of every five treated
these data indicate that IFN is an active agent and an with an overall response rate of 18% (Table 7). Complete
important component of treatment regimens for meta- responses with long-term remissions, rare in chemother-
static melanoma. The combination of interferon alpha apy treated patients, are occasionally seen with IL-2.
with chemotherapy met with early encouragement. Many studies have shown a higher percentage of com-
Several phase II studies showed the combination of dac- plete responders with high dose IV bolus IL-2, but no
arbazine and alpha interferon to induce responses in difference in survival compared to lower dose regimens.
25–30% of patients with metastatic melanoma [15, 36, Similar to the situation with alpha interferon, the opti-
93, 113]. In randomized studies comparing the combi- mal dose and schedule of administration have not been
nation to single agent dacarbazine, two showed superior determined, and a clear dose–response relationship has
638 Biological therapy of melanoma

not been demonstrated. IL-2 has been used alone or in
combination with lymphokine-activated killer (“LAK”)
cells. The addition of LAK cells, while of historical
importance in the original description of IL-2 therapy
for advanced malignancies, provides, at best, marginal
improvement over IL-2 alone [104, 105]. Good preclinical
models support the combination of IL-2 and interferon
alpha, but studies, both phase II and randomized trials,
showed no benefit for this combination in metastatic
melanoma [115].
Of historical interest is the combination of chemotherapy
and IL-2. Although the clinical trials combining single
agent dacarbazine with IL-2 did not demonstrate a ben-
efit for combining these modalities (Table 8) [29, 38, 40,
118], cisplatin based regimens were more promising
[2, 8, 26, 50, 63, 97]. Four trials with cisplatin and IL-2
have shown overall response rates in excess of 50%, and
all of these also included alpha interferon. Randomized
studies have not confirmed these data [48].
Interleukin 2/T-cells
As further evidence for the interleukin-2 mediated effect
on melanoma, Rosenberg et al. reported a 50% response
rate using T-cells isolated from melanoma nodules and
grown to large numbers in vitro, another example of
adoptive immunotherapy of cancer [101, 105]. These
tumor-infiltrating lymphocytes (TIL) appear to have a
higher level of specific activity and, in large part,
are MHC-restricted T cells specifically reactive with
the individual’s melanoma from whom the T cells were
derived. Concomitant studies by Dillman and Oldham
[30, 65] have confirmed the feasibility of this approach
using tumor-derived activated cells (TDAC) although
with much lower response rates. This approach of growing
T cells from the tumor and/or draining lymph nodes is
technologically demanding and very expensive [65, 66,
83]. To date, the results are not clearly superior to the
use of interleukin 2 alone or interleukin with peripheral
blood cells (LAK) activated in vitro. On the other hand,
these cells are exquisitely more specific for the mela-
noma cells. Clearly, this approach validates the belief of
many investigators that specific T-cell reactivity occurs
in advanced cancer and demonstrates the feasibility of
applying such approaches as a clinical biotherapy [29,
66, 82, 107].
Rosenberg has pioneered the use of TIL cells plus
interleukin-2 in the treatment of cancer utilizing IL-2/
TIL with and without cyclophosphamide and in various
schedules and doses in several clinical trials. In sum-
mary, approximately 86 patients were treated; 28 of
whom had had prior IL-2 and 58 had no prior IL-2.
A 34% response rate was seen for the total group of
patients with the same response rates in the both the IL-2
pretreated patients and the IL-2 naive patients. Both the
response rate and duration of response were equivalent
in the two groups and some of the remissions were of
very long duration.
111
In labeled TIL cells were given to selected patients
and with nuclear imaging the presence of these labeled
cells in tumor tissue was verified. Thus, both based on
the in vitro studies showing enhanced killing by T cells
compared to LAK cells and in studies using TIL
cells clinically, where a higher response rate was seen,
the evidence is accumulating that these more specific T
cells are more powerful when used with interleukin-2 in
the treatment of advanced melanoma. It’s of particular
interest to note that patients resistant to interleukin-2
respond to interleukin-2 plus TIL cells, indicating a role
for the cells alone as a therapeutic approach in treating
advanced melanoma. Curiously, no one has performed the
kind of T cell dose response studies normally done with
drugs in patients with advanced cancer. The major question

Table 8. The activity of combined IL-2 + chemotherapy in malignant melanoma

First author Year Chemotherapy Other therapy No. of patients CR + PR
Papadopoulos [244] 1990 DTIC 30 33% (10)
Flaherty [138] 1990 DTIC 32 22% (7)
Dillman [102] 1990 DTIC 27 26% (7)
Stoter [435] 1991 DTIC 25 24% (6)
Fiedler [131] 1992 DTIC 16 13% (2)
Blair 1991 CDDP, DTIC 28 43% (12)
Demchak [93] 1991 CDDP 27 37% (10)
Hamblin 1991 CDDP, DTIC IFN 12 83% (10)
Richards [372] 1992 CDDP, BCNU IFN, TAM 74 57% (40)
Legha [244] 1992 CDDP, DTIC, VLB IFN 30 56% (17)
Khayat [212] 1993 CDDP IFN 39 54% (21)
Atkins [13] 1994 CDDP, DTIC TAM 38 42% (16)
Robert K. Oldham
that remains is what would be the effect of giving
repeated massive doses of activated T cells to patients
with advanced melanoma? What would be the influence
of dose, schedule, type of T cell, etc.? These studies
need to be done before conclusions on the efficacy of T cell
therapy are drawn.
Studies conducted by Oldham and co-workers reported
a series of patients with various types of cancer treated
with a combination of interleukin-2 and tumor-derived
activated cells (TDAC) [65–67, 72, 73, 82, 84, 85, 87,
88]. These tumor-derived T cells are similar to the TIL
cells described by Rosenberg and in these studies some
366 tumor biopsies were tested with the T cells grown
successfully in 75% of the cases. Sixty-three patients
were treated with a minimum of at least 1010 TDAC by
intravenous infusion and partial responses were seen in
eight patients, including the dramatic complete regres-
sion of scalp nodules in a patient with renal cancer. The
cells were well tolerated, responses were seen in patients
with renal cancer and melanoma, most of whom were
previously resistant to interleukin-2 and all of whom had
bulky, advanced cancer [66]. Taken together, the results
of T-cell therapy indicate that these cells are active in
advanced cancer. The observation that they are active in
patients previously resistant to Interleukin-2 demon-
strates that the cells alone have consistent activity in a
minority of patients with advanced cancer.
Over the last few years, Rosenberg and a few other
investigators in Europe have begun to look at ways to
manipulate the immune system prior to infusing the
activated T-cells. Very aggressive therapy such as com-
binations of high-dose chemotherapy in the setting of an
autologous transplant plus whole body radiation therapy
followed by the infusion of autologous T-cells and
Interleukin-2, has resulted in a greater than 75%
response rate in the first few patients treated with this
approach at the NCI. Many of these patients had bulky,
Interleukin-2 resistant disease, and the induction of
complete responses in such patients is very encouraging
[70, 103].
Gene therapy
A new category of biological therapy has become recog-
nized, utilizing gene engineered cells. By definition,
gene therapy is the introduction of genetic material into
cells for therapeutic purposes. The gene therapy of cancer
has focused on advanced melanoma and renal cancer
models for the development of this new therapeutic
approach. The first patient treated on an approved gene
therapy protocol was a patient with malignant melanoma
639
who on May 22, 1989, received an infusion of tumor
infiltrating lymphocytes (TILs) marked with the neomy-
cin phosphotransferase gene (a gene which carries neo-
mycin resistance in bacteria). While this gene transfer
was not designed for the therapeutic enhancement of
TIL activity, the ten melanoma patients who consented
to this gene “therapy” demonstrated that foreign genes
could be effectively and safely transferred into human
cells and administered to patients without apparent
adverse effects on the patient nor risk to the caregivers
or public [102]. This study also confirmed that the engi-
neered T cells trafficked to the melanoma nodules.
These results allowed Rosenberg and colleagues to pro-
ceed to alter the TILs with therapeutic intent. On January
29, 1991, the first patient treated on a true gene therapy
protocol was an advanced melanoma patient who
received an infusion of TILs that contained the gene for
tumor necrosis factor (TNF). Several metastatic mela-
noma and other advanced cancer patients were treated
in this early study, and responses have been observed
[101]. The rationale for this strategy is to provide high
local concentrations of TNF at tumor sites. TNF is quite
toxic when systemically administered, but presumably
the TILs provide sufficient local concentrations of TNF
for an antitumor effect while sparing systemic toxicity.
Another gene therapy approach initially described by
Nabel and colleagues is to introduce the HLA B-7 gene
directly into cutaneous melanomas of non-HLA B-7
patients with metastatic disease [79]. The concept was
to induce a cytotoxic immune response against a foreign
major histocompatibility complex (MHC) class I antigen,
leading to the recognition of the injected cutaneous
melanoma cell as foreign and ultimately generating a
systemic anti-melanoma immune response. In the initial
study, the five patients treated locally injected with the
HLA B7 gene all showed evidence of safe and success-
ful gene transfer, cytotoxic T-lymphocytes were shown
to invade the lesions, and one patient showed tumor
regression not only of the lesion that had the HLA B7
gene delivery but also of distant cutaneous and pulmo-
nary metastases [79]. The initial success of this strategy
has lead to a number studies with the HLA B7 gene
(commercialized as “Allovectin 7”), now most com-
monly administered intramuscularly as naked DNA.
Further details of this line of investigation may be found
in the chapters on gene therapy and vaccines elsewhere
in this book. Other genes under evaluation include gran-
ulocyte macrophage colony stimulating factor (GM-CSF)
and B2-microglobin, whose gene products will induce an
antitumor immune response when inactivated autologous
tumor cells are reinfused into the patient. The cytokine
gene therapy concept has been expanded in other clinical
640
trials that will evaluate the feasibility of genes for IL-2,
gamma interferon, TNF, and IL-4 transduced into autol-
ogous tumor cells or fibroblasts to produce active cytok-
ines effective in the treatment of melanoma and other
cancers [22]. To date, the clinical benefit and activity of
gene therapy for melanoma remains to be fully delin-
eated [5, 17, 49, 74, 76].
Antibodies
Relatively specific visualization of tumor nodules with
radiolabeled antibody has been reported [13] and anti-
body given intravenously can subsequently be demon-
strated on the melanoma cell surface by histopathologic
techniques [111]. However, only minor clinical activity
has been observed with either unconjugated antibody or
with preparations of radiolabeled, drug-conjugated, or
toxin-conjugated anti-melanoma monoclonal antibodies.
The 9.2.27 antimelanoma monoclonal antibody rec-
ognizes a melanoma-associated antigen (a 250-kD
chondroitin sulfate proteoglycan core glycoprotein) that
is found on 90% of melanoma cells and relatively few
non-melanoma cells [86]. Oldham et al. reported the
selective targeting of this antibody to biopsied mela-
noma nodules in eight patients [86]. The 9.2.27 antibody
does not activate complement, poorly activates ADCC,
but may have application because its target antigen
modulates very little. Their studies suggest that quanti-
ties on the order of 0.5 to several grams of antibody are
necessary for saturation of target antigens in vivo.
Schroff et al. have observed that an interrupted infu-
sional schedule appears to compromise the in vivo local-
ization of antibody to tumor, presumably by the
development of human anti-murine antibodies (HAMA),
which impede antibody localization and accelerate
clearance [111]. Goodman et al. later administered
MAb96.5 and MAb48.7 to four patients and MAb96.5
alone to a fifth patient. MAb96.5 is an IgG2a immuno-
globulin that recognizes p97, a transferrin-like cell sur-
face glycoprotein of 97 kD [44, 45]. MAb48.7 is an
IgG1 immunoglobulin recognizing a melanoma-associ-
ated cell surface proteoglycan. Although the infusion of
antibody was well tolerated, there were no objective
remissions nor histopathologic changes found in biop-
sied tissue, although impressive antibody binding to
melanoma cells was observed. Perhaps the lack of activity
reflected the lack of in vitro activation of human com-
plement and very modest ADCC.
Studies by Vadhan-Raj et al. [122] are of interest in
that the IgG3 anti-GD3 mouse monoclonal antibody
R24, directed against the sialoganglioside membrane
Biological therapy of melanoma
antigen GD3, appears to have induced clinical responses
(four partial and two minor responses among 21 patients)
using unconjugated antibody [122]. These studies are in
contrast to previous studies using other anti-melanoma
antibodies. MAb R24 provoked a clear inflammatory
reaction with increased number of mast cells with evi-
dence of their degranulation, an influx of polymorpho-
nuclear cells, complement deposition, particularly C3,
C5, and C9, and infiltration with T3+/T8+/Ia+ lympho-
cytes. MAb R24 has been studied in conjunction with
other biological modalities such as IL-2 [4], interferon-
alfa-2a, with total lymphoid irradiation (which did not
interdict the human anti-murine antibody response as
had been hoped) and by novel routes of administration
(e.g., isolated limb perfusion) [16]. Cheung et al. have
studied another IgG3 anti-ganglioside MAb, 3F8, in a
phase I trial [14]. Two of nine patients with metastatic
malignant melanoma achieved partial responses, one
lasting 22 weeks and the other continuing at 56 weeks.
The most prominent toxicities consisted of severe pain,
hypertension, and focal urticaria; a decrease in serum
complement activity was observed when dosages
equaled or exceeded 20 mg/m2. Houghton reported that
the combination of MAb R24 and MAb 3F8 yielded one
complete remission among 13 patients with melanoma.
Goodman et al. observed no anti-tumor activity among
eight patients given MAb MG-21, another antibody rec-
ognizing a GD3 surface antigen commonly displayed
by human melanoma cells [44, 45]. In an intriguing
report, Livingston and associates reported one complete
response among 13 patients with metastatic malignant
melanoma who received murine IgG2a MAb ME-36.1,
which recognizes the melanoma-associated ganglio-
sides GD2 and GD3. This patient’s posttreatment B
lymphocytes, when stimulated with polyclonal goat
anti-idiotypic ME-36.1, were found to synthesize human
antibodies preferentially and specifically recognizing
GD2, suggesting to the researchers that the induction of
human anti-melanoma ganglioside antibodies provoked
by ME-36.1 may have mediated the observed clinical
response. These observations that murine monoclonal
antibodies can induce complement activation, active
antibody-dependent cellular cytotoxicity, and generate
human anti-melanoma antibodies may support a further
role for the investigation of unconjugated antibody in
malignant melanoma.
A murine monoclonal anti-melanoma antibody-ricin
A chain immunotoxin (XOMAZYME-MEL) was stud-
ied in several clinical trials, since the initial report of its
use in 22 patients in early 1987 [113]. The monoclonal
antibody moiety of this immunotoxin is an IgG2A anti-
body recognizing melanoma-associated proteoglycan
Robert K. Oldham
membrane antigens of 220 kD and greater than 500 kD.
One patient demonstrated complete disappearance of
detectable melanoma (a retrocardiac pulmonary metasta-
sis) over an 8-month period following a single infusion
of immunotoxin, with maintenance of a complete remis-
sion for greater than 2 years until relapse occurred in the
brain. However, no major responses were seen in the
remaining 21 patients. The treatment was accompanied
by a transient reduction of serum albumin with associated
fluid retention and weight gain, alopecia, fever, fatigue,
and malaise, with mild allergic reactions occurring in
three patients. In a phase II trial with this immunotoxin,
three partial responses were observed among 43 patients,
these responses lasting for 10+, 13+, and 15+ months
[87, 116]. Cyclophosphamide at a dose of 1 g/m2 admin-
istered following the infusion of immunotoxin has not
inhibited the generation of host immune responses
directed against the immunotoxin preparation.
Given the studies by various investigators using unla-
beled antibodies, which have been marginally useful in
the treatment of melanoma, several investigators have
pursued studies using antibody in combination with
cytokines [4, 21] with no evidence of activity over and
above using the cytokines alone. Similarly, early trials
with radiolabeled antibodies for therapy of melanoma
have not proven useful [57].
Since antibodies can induce inflammatory infiltrates
and activities around melanoma nodules, further work is
ongoing with regard to using these antibodies to produce
inflammatory effects and combining these effects with
cytokines and chemotherapy. Engineered subfragments
of antibody as well as humanized antibodies are currently
in clinical trials and it is felt some of these improved mol-
ecules may improve the delivery of immunotoxins and/or
radionuclides to melanoma deposits [89].
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21.3 Biological therapy of genitourinary cancer
ROBERT K. OLDHAM

Kidney Cancer kidney cancer, these statistics illustrate the large potential
patient population that can benefit from an active biologi-
Overview cal and targeted therapy of cancer.
Recent progress in targeted therapy for renal cancer
Kidney cancer remains one of the best examples of a has lead to the identification of several agents, which
disease that responds to the immune system and is ame- target either vascular endothelial growth factor or a
nable to intervention with biological therapy. Cancer of tyrosine kinase enzyme. These molecular findings have
the kidney and renal pelvis was estimated to account for lead to the development of what is now being called
51,200 new cancer cases and cause 12,900 cancer deaths “targeted therapy” of kidney cancer and many other
in the United States in 2007. In the United States, kidney types of cancer [15].
cancer is the 12th leading cause of cancer death, com-
prising 3% of all malignancies in men and 2% in women.
The 5-year relative cancer free survival has improved Cytotoxic Chemotherapy is Ineffective
over the last 2 decades, from 52% in the years 1974–
Kidney cancer remains one of few modern examples of a
1976 to the current rate of 61% in the years 1989–2007,
malignant disease for which currently available cytotoxic
causing 2% of all cancer deaths in this country [32].
chemotherapy agents are essentially inactive. An exhaus-
Internationally, kidney cancer has the highest incidence
tive review of 143 published or nationally presented sin-
in North America and northern Europe, the lowest inci-
gle agent chemotherapy trials concluded that no agent
dence in India, China, Japan, and South America, and
achieved significant activity in the treatment of renal cell
accounts for 2% of all cancers worldwide [43, 56, 79].
carcinoma. This review included studies reported from
The incidence of kidney cancer has increased by
1975–1995 involving almost 4,000 patients treated with
approximately 2% per year over the past 2 decades. The
80 different single agents, showing a 4% overall response
greatest rise in incidence is in patients with localized dis-
rate [2]. Newer cytotoxic agents have had similarly disap-
ease (3.7% per year] and may be ascribed in large part to
pointing results in renal cell carcinoma. A review of che-
the increase in abdominal imaging in the population at
motherapy trials reported from 1990–1998 included 33
large. However, the incidence of regionally advanced
chemotherapy agents given to 1,347 patients on 51 phase
and distantly metastatic kidney cancer has also increased
II clinical trials lead to the conclusion that no chemother-
over this time period, indicating that factors in addition
apy has produced response rates that justify use as a sin-
to the increase in abdominal imaging may be contribut-
gle agent, although synergy may exist for certain
ing to the rising incidence of this disease [38].
combinations that warrant further study [63]. Responses
Currently, approximately 25% of patients with kidney
to chemotherapy in renal cell carcinoma must be evalu-
cancer present with disseminated disease while an addi-
ated in light of the low but finite level of background
tional one third to one half of patients without dissemina-
noise created by spontaneous regression of metastases.
tion at initial diagnosis develop metastases [22, 38]. Thus,
The lack of an active cytotoxic agent for this disease pro-
more than half of all patients with kidney cancer will
vides further rationale for developing and optimizing bio-
present with or develop disease for which systemic anti-
logical and targeted therapy for kidney cancer.
cancer therapy is needed. Five year survivals of over 90%
have recently been reported for patients with small, inci-
dentally detected kidney cancers [100]. However, relapse Kidney Cancer is Responsive
rates of 50% or higher in patients with larger or more
to the Immune System
extensive tumors provide a strong rationale for the devel-
opment of a systemic adjuvant post-surgical therapy for Several clinical observations similar to those described
patients with resectable kidney cancer. Since traditional for malignant melanoma indicate a role for the immune
forms of cytotoxic chemotherapy remain ineffective for system to influence the natural history and treatment of

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, 645
© Springer Science + Business Media B.V. 2009
646
kidney cancer. First, patients with established metastatic
kidney cancer may have long disease or treatment free
intervals, irrespective of the treatment received, sug-
gesting a degree of innate immune control. In a large,
retrospective, single institution analysis, 5-year survival
was observed in 4.5% of 670 patients with advanced
kidney cancer treated on one of several cytokine, che-
motherapy, or hormonal therapy trials from 1975–1996,
including 9 of 294 patients (3%) treated with chemo-
therapy or hormonal therapy agents subsequently dem-
onstrated to lack activity in this disease [61]. Similarly,
patients may relapse many years, even decades after a
nephrectomy with curative intent for renal cell carci-
noma, indicating a potential role for immune surveillance
to control micrometastatic kidney cancer effectively for
many years [99]. Second, immunosuppression increases
the risk of the development of kidney cancer, as seen in
the increased risk of the development of renal cell carci-
noma in kidney allograft recipients [46]. Third, immune
stimulation with cytokine therapy can induce tumor
regression and prolong survival in patients with meta-
static kidney cancer and will be described in detail in
this chapter. Fourth, spontaneous regression of metastatic
kidney cancer has been carefully documented [102].
Indeed, the response rates for observation, placebo, and
“placebo-equivalent” therapy in phase II and III trials of
metastatic renal cell carcinoma have been as high as 7%.
The most notable example of a clinical study showing
a high rate of apparently spontaneous regression of meta-
static kidney cancer was a phase II trial was performed on
73 patients with metastatic renal cell carcinoma who were
identified prospectively and treated with observation until
evidence of progression. Five patients (7%) achieved a
partial or complete remission on observation and 12%
were progression free for at least 1 year [68]. Recently
reported phase III trials in metastatic renal cell cancer
also demonstrated a spontaneous remission rate of greater
than 5%. A randomized trial comparing interferon gamma
to placebo in 197 patients with advanced renal cell carci-
noma showed a 7% response rate in the placebo group
that was higher than the response rate achieved in the
treatment group [35]. Another recent phase III trial has
demonstrated modest rates of regression with what is
likely to be “placebo-equivalent” treatment in compari-
son to interferon alpha. Medroxyprogesterone acetate has
been evaluated for single agent activity in renal cell car-
cinoma in a number of trials, is generally considered to be
inactive, and may be thought of as a “placebo-equivalent”
in this setting [29]. A trial comparing interferon-alpha to
medroxyprogesterone demonstrated the superiority of
interferon alpha, but a 3% overall objective response rate
was observed in the medroxyprogesterone arm, and the
Biological therapy of genitourinary cancer
authors reported a 7% response rate to medroxyproges-
terone in the subset of patients who remained on treatment
for at least 6 months [57]. Taken together, the results of
these studies demonstrate the potential role of the innate
immune system in mediating the spontaneous regression
of established metastases from renal cancer.
Based on these clinical observations, renal cell carci-
noma, like melanoma, appeared to constitute an ideal
human tumor system for biotherapy. Early attempts to
harness the immune system in the treatment of kidney
cancer are of historical interest and employed a variety
of active specific immunization techniques. Tykka, in
1974, reported a 17% complete response rate of pulmo-
nary metastatic lesions in patients receiving a monthly
vaccine composed of homogenized tumor plus either
tuberculin or Candida every 4 weeks [101]. Subsequently,
three separate trials using either tumor homogenate plus
tuberculin, enzymatically digested tumor tissue plus
Corynebacterium parvum, or whole tumor cells plus
attached dimethyldioctadecyl ammonium bromide
(DDA) as vaccine have all reported responses in 15–25%
of patients [54, 66, 70]. Furthermore, immune RNA has
been extracted from lymphocytes of guinea pigs specifi-
cally immunized with renal tumor cells and coincubated
with autologous lymphocytes; these reinfused lympho-
cytes induced a response in three of six patients [96].
Thymosin fraction V, a partially purified extract of calf
thymus glands, has induced objective responses in
approximately 15% of advanced renal cancer patients.
These early studies provided the impetus for developing
biological therapies for renal cell carcinoma.
Important laboratory observations provide evidence
of an immunologic response to RCC and have been
reviewed elsewhere in this book. Several lines of evi-
dence indicate that cytotoxic T-lymphocytes (CTLs)
play a central role in mediating the regression of RCC.
First, lymphocytic infiltrates within renal tumors are
comprised primarily of CD3+ T-lymphocytes, and
molecular analysis of these lymphocytic infiltrates has
demonstrated clonally expanded populations of T-cell
receptor alpha and beta restricted T-cells that are gener-
ally not found in peripheral blood, normal kidney or
lymph nodes [47, 72]. Second, tumor-specific cytotoxic
T-cell lines from tumor infiltrating lymphocytes have
been developed from patients with RCC that are capable
of recognizing and lysing autologous tumor cells [4].
Third, specific T-cell defined tumor associated antigens
have been identified from patients with RCC, including
HER-2/neu, PRAME, and RAGE [65]. Taken together,
these correlative laboratory observations demonstrate
the central role of the CTL in mediating the immune
response to RCC.
Robert K. Oldham
Cytokine Therapy
The treatment of MRCC with the cytokines interferon-
alpha and interleukin-2 results in objective responses in
about 15% of patients. A recent retrospective analysis of a
large, single institution patient database provides an exam-
ple of the benefits that derive from treatment of MRCC
[60]. This review of 670 patients with advanced RCC ana-
lyzed the outcomes of patients treated with cytokine
therapy versus chemotherapy or hormonal therapy in clin-
ical trials from 1976–1996. About 60% of patients were
treated with cytokine therapy with interferon-alpha, inter-
leukin-2, or interferon-alpha plus interleukin-2 versus and
about 40% were treated with chemotherapy or hormonal
therapy. A highly significant and clinically meaningful
survival advantage was demonstrated for cytokine therapy
versus chemotherapy or hormonal therapy, with median
survival times of 13 versus 6 months (p < 0.001, 95%
confidence intervals of 12–15 months versus 5–8 months).
The survival advantage for cytokine therapy extended to
all prognostic groups, with median survivals of 27 versus
15 months for the favorable subset of patients, although
patients with the poorest prognosis survived less than 6
months even with treatment. This important analysis dem-
onstrated that in each risk group, there was an approxi-
mate doubling of the median overall survival when
patients were treated with cytokine therapy compared
with chemotherapy or hormonal therapy.
Trials of cytokine therapy in metastatic renal cell carci-
noma, have demonstrated wide variations in response
rate, including some trials that failed to demonstrate
responses to high dose interleukin-2 [1] while others have
demonstrated responses as high as 33% [86]. From these
observations, several conclusions can be drawn. First,
advanced kidney cancer has a variable natural history
determined at least in part by the innate immune response
to renal cell carcinoma. Second, pre-treatment patient and
tumor characteristics have a large impact on the outcome
of a given treatment or observation strategy. Third, a mea-
sure of caution must be exercised when evaluating the
results of clinical trials in renal cell carcinoma, particu-
larly when the results indicate marginal activity of a par-
ticular treatment. Several recent reports have identified
some of the important prognostic variables that help to
predict clinical outcomes in kidney cancer, including
TNM stage, tumor grade, performance status, and certain
indicators of disease burden (lactate dehydrogenase level,
presence of anemia, and presence of hypercalcemia). It is
possible to identify a favorable subset of patients with
advanced kidney cancer whose median survival exceeds
2 years with cytokine therapy and an unfavorable subset
of patients with a median survival of approximately 6
647
months or less for whom cytokine therapy may be less
beneficial [60, 109]. Another important variable in the
outcome of patients with metastatic kidney cancer treated
with cytokine therapy is the presence of the primary kid-
ney tumor [26, 27, 58].
It is also becoming increasingly evident that not all his-
tological types of kidney cancer are equally capable of
response to immune modulation. It is important to recog-
nize that conventional (clear cell) renal cell carcinoma and
its histological variants (conventional renal cell carcinomas
may show a mixture of cytoplasmic features, including
variants that have formerly been classified as “granular
cell” and “sarcomatoid” renal cell carcinomas) represent a
specific clinico-pathological disease entity characterized
by typical findings on light microscopy and loss of the von
Hippel-Lindau tumor suppressor gene. This disease entity,
now classified as conventional renal cell carcinoma, com-
prises approximately 60% of all epithelial tumors arising
from the kidney [78]. It is clear that the other histological
types of tumors of renal epithelial origin (e.g. papillary,
chromophobe, oncocytoma, and collecting duct tumors)
represent distinct clinico-pathological diseases with dif-
ferent clinical behavior, but it is not clear whether other
histological types of kidney cancer are capable of respond-
ing to immune manipulation [69].
Recognition of known prognostic indicators as deter-
minants of eligibility and stratification is a critical com-
ponent in the design of clinical trials and the evaluation
of their results. Furthermore, because of the major con-
tribution of prognostic factors on the outcome of patients
with metastatic kidney cancer (MRCC) irrespective of
treatment, randomized trials are required to demonstrate
the relative merits of a particular therapy. The overall
response rates observed with cytokine therapy for kid-
ney cancer have been relatively low, and the natural his-
tory of patients with MRCC varies widely independent
of treatment. As a result, the impact of treatment with
these cytokines on survival has been controversial and
can only be directly assessed in phase III clinical trials.
Until recently, phase III trial data have been lacking, but
several important phase III studies have recently been
completed and reported that shed light on the impact of
cytokine therapy on the survival of patients with MRCC,
the role of “cytoreductive” nephrectomy prior to cytokine
therapy in with metastases at initial presentation, and the
incremental benefits that may be achieved with intensive
therapy with high dose interleukin-2 [32, 45, 92].
Interferon-alpha
Interferon-alpha has produced objective responses in
10–20% of patients with MRCC. An earlier review of
648 Biological therapy of genitourinary cancer

the data from multiple phase II studies showed a response
rate of 12% in 1,042 patients treated with a variety of
doses and schedules of interferon, and concluded that
longer survival could not be demonstrated in patients
with MRCC treated with interferon-alpha [9, 105]. The
mean time to response may be as long as 3–4 months. It
can be argued that an optimal dose and schedule for
alpha-interferon has never been defined for renal cell
carcinoma. The anti-tumor activity of interferon can be
attributed to its ability to modulate cellular immunity, to
its direct (non-immunologically mediated) anti-prolifer-
ative or pro-apoptotic activity, or to an effect on tumor
angiogenesis [52] It is not clear that all of these proper-
ties of interferon can be evoked optimally and simulta-
neously from a single dose and schedule of interferon in
vivo. Some data appear to demonstrate a dose–response
relationship for interferon, suggesting that the clinical
responses induced by interferon are likely to be due to
its known anti-proliferative activity rather than to immu-
nomodulation. However, evidence for a dose–response
relationship for interferon has not been consistently
observed among investigators. While some toxicities
from interferon do appear to be dose-related, not all
antitumor responses appear to be dose-related. From his
extensive review of the literature relating to the treat-
ment of renal cell carcinoma with interferon, Muss [64]
concluded that toxicity from interferon was dose-related
and that the highest therapeutic index for interferon
occurred at doses ranging from 5–10 million units/m2.
However, it is interesting to note that some studies have
demonstrated responses using very low doses of inter-
feron. For example, a single-arm phase II trial conducted
by the Southwest Oncology Group [63] accrued 40 fully
evaluable, previously untreated patients with advanced
renal cell carcinoma treated with alpha-interferon 1 million
units subcutaneously daily continually until there was
evidence of disease progression, resulting in 6 responses
(response rate, 15%; 95% confidence interval, 5.7–30%).
The ability of interferon-alpha to prolong survival has
now been evaluated in several randomized trials in com-
parison to a non-cytokine containing control arm [32, 48,
57, 73, 97]. The results are presented in Table 1. The two
smaller studies showed no survival benefit for treatment
with interferon-alpha, but included few patients and
allowed crossover in one case. The two larger studies
both demonstrated a statistically significant improve-
ment in survival for the interferon-containing arm at
doses of 10–18 MU three times weekly. Because the
addition of vinblastine has been shown not to improve
survival over interferon alone, the improvement in sur-
vival of interferon plus vinblastine over vinblastine alone
can be attributed to the interferon. Taken together, the
results of the four randomized, prospective trials com-
paring cytokine therapy versus non-cytokine therapy in
several hundred patients, demonstrated an objective
response rate of 16% versus 2% and average median sur-
vival of approximately 12 versus 8 months. These results
also closely resemble the results of the large, retrospec-
tive single-institution analysis of cytokine versus non-
cytokine therapy described above in which median
survivals were 13 versus 6 months [32].
A randomized trial involving interferon alpha was
conducted through the Southwestern Oncology Group
and was designed to answer the important question of
whether nephrectomy in the setting of known metastatic
disease is of clinical benefit [27]. Almost 350 patients
with metastatic kidney cancer were randomly assigned
to receive interferon alpha immediately upon enroll-
ment into the trial or following a nephrectomy.
Interestingly, the objective response rate to interferon
alpha was unexpectedly low and not different between
the two arms of the study (less than 4%), yet the median

Table 1. Randomized trials comparing interferon alpha with non-cytokine treatment

Treatment N Response (%) Median survival (months) P value Publication
IFN 30 6 7 NA Steineck 1990(97)
MPA 30 3 7
IFN + VBL 44 20 16 0.19 Kriegmair 1995(48)
MPA 45 0 10
IFN + VBL 79 16 17 0.0049 Pyrhonen 1999(73)
VBL 81 2 10
IFN 167 16 8.5 0.011 MRC 1999(57)
MPA 168 2 6
Total
IFN +/− 320 16 11.5
VBL or MPA 324 2 7.7

IFN interferon-alpha, MPA medroxyprogesterone acetate, VBL vinblastine

Robert K. Oldham
survival was significantly improved (by 3 months) in
the patients who underwent nephrectomy before immu-
notherapy, independent of other prognostic factors.
More rigorous assessment of objective response was
proposed to explain the low response rate observed in
this trial; dose and schedule were comparable to studies
reporting much higher responses. A similar improve-
ment in survival for nephrectomy in the setting of meta-
static disease was also observed in a somewhat smaller
study of similar design, and responses to interferon were
in the usual range (12–19%) [58]. A similar European
trial demonstrated an improvement in the time to dis-
ease progression as well as the median survival for
patients who had a nephrectomy compared to those who
did not prior to initiating interferon-alpha therapy. In
addition, there are some studies that indicate resection
of solitary or a small number of metastasis may lead to
long-term survival in approximately 30% of patients
with metastatic renal cancer. Thus, surgery plays an
important role in MRCC, especially in conjunction with
effective systemic therapy [22].
PEG-interferon
The treatment of kidney cancer with inferon alpha ther-
apy requires daily or thrice weekly subcutaneous admin-
istration. The preparation of the interferon with
polyethylene glycol (PEG) sustains the absorption of
interferon from a subcutaneous injection, slows its clear-
ance, and improves its pharmacokinetic parameters. This
improvement in pharmacokinetics allows for less fre-
quent administration, for example once weekly, and may
also improve the effectiveness and side effect profile by
avoiding the frequent peaks and troughs associated with
the shorter half-life of conventional interferon. PEG-
interferon alpha has become commercially available for
the treatment of hepatitis C, and studies were done to
evaluate the effectiveness and tolerability of this agent in
renal cell carcinoma. Preliminary studies suggest that
PEG-interferon retains anti-tumor activity in renal cell
carcinoma and has a spectrum of side effects similar to
conventional interferon alpha, but improves the conve-
nience of administration to a once weekly dose [62].
Other Interferons
The role of beta-interferon in the treatment of renal-cell
carcinoma remains to be established. A trial of combina-
tion interleukin-2 and beta-interferon evoked objective
responses in 6 of 22 evaluable patients, with one com-
plete remission and five partial remissions. Two of these
responses lasted for almost 2 years [32]. Although this
649
27% overall response rate is similar to that achieved
with interleukin-2 and lymphokine-activated killer
(LAK) cell therapy and to higher doses of single agent
interleukin-2, the contribution of beta-interferon is
uncertain.
Early experience with gamma-interferon suggested
little if any activity [75, 80]. Gamma-interferon is a
product of activated T cells and is unique in its ability to
stimulate macrophages. Although approved for chronic
granulomatous disease, it has demonstrated some activ-
ity in metastatic renal cancer and in one study using a
low-dose weekly subcutaneous injection of 100 mg, a
30% response rate was observed in patients with meta-
static renal cancer [5]. A second study reported a 15%
response rate [19]. Combinations of alpha and gamma-
interferon have shown additive toxicities without added
therapeutic benefit [74]. An effort to improve response
by combining alpha-interferon and gamma-interferon
failed to achieve this intent, perhaps attributable to addi-
tive toxicities and inability to suitably escalate dosages
[28]. Some of the same principles regarding dose and
schedule of alpha-interferon, discussed above, pertain
to gamma-interferon. A dose–response relationship for
gamma-interferon may not be straightforward [6]. Foon
et al. conducted a phase I trial of gamma-interferon in
which anti-tumor responses were observed. However,
the responses occurred at the lowest doses tested [28].
Follow-up trials with both beta and gamma interferon
have yielded equivocal results and only interferon-alpha
is approved for use in MRCC.
Interleukin-2
In the United States, high-dose interleukin-2 was
approved by the Food and Drug Administration for the
treatment of advanced kidney cancer, whereas in Europe,
lower doses and infusional IL-2 were approved and are
more commonly used in MRCC. Kidney cancer cells in
culture can grow in spite of the presence of high concen-
trations of interleukin-2, demonstrating that interleukin-2
has no directly cytotoxic activity. The action of interleu-
kin-2 appears to be the activation of lymphocytes and
natural killer cells that mediate the antitumor activity of
this agent. About 10–20% of patients treated with inter-
leukin-2 will achieve an objective response, and a small,
but important fraction of these patients will achieve a
complete remission, which may be quite durable. A
review of over 1,900 patients treated with single agent
interleukin-2 demonstrated an overall response rate of
15% that included complete remissions in 4% of the total
patient population (Table 2) [7, 8]. This review included
patients treated on phase I and II studies using various
dose levels, schedules, and routes of administration.
650
Table 2. Results of therapy with interleukin-2
Route of Administration N % % complete
responding responders
Intravenous bolus 733 16 5 monitoring and the administration of vasopressors to
Continuous intravenous 922 13.3 2.5
infusion
Subcutaneous 290 15.0 3 is nonetheless associated with significant toxicity and
Total 1945 15.0 3.6
Table 2 adapted from(7,8).
It is likely that the dose intensity of interleukin-2 admin-
istration influences the proportion of patients that
respond to treatment as well as the quality and durability
of the responses, but a linear dose-response relationship
has not been demonstrated for interleukin-2, and ques-
tions of dose and schedule have been the subject of
several important clinical studies in the late 1990s.
Interleukin-2 was approved by the United States Food
and Drug Administration in 1992 based on a data set of
over 450 patients treated on a series of phase II studies
with high dose, intravenous bolus interleukin-2 with
and without lymphokine-activated killer (LAK) cells,
demonstrating an overall response rate of 21% with 6%
complete and 15% partial responses [18, 25, 53]).
Subsequently, studies have demonstrated that the bene-
fits of high dose interleukin-2 plus LAK cells are derived
from the interleukin-2 (as discussed below and reviewed
in [48]), and of the over 450 patients analyzed, 255 were
treated with high dose interleukin-2 without LAK cells.
The results of treatment in these 255 patients have been
rigorously scrutinized and followed long-term, updated
reports have been published periodically [24, 25, 30,
31]. These reports are instructive in an analysis of the
outcome of treatment of metastatic kidney cancer with
interleukin-2. Objective responses following high dose
interleukin-2 have been confirmed in 15% (7% com-
plete and 8% partial), with a median response duration
for the entire group of 15 months. Considering this
group as a whole, the results of treatment with high dose
interleukin-2 appear to be fairly similar to the results
presented above for treatment with interferon alpha in
which the overall response rate of 16% and median sur-
vivals of 7–16 months have been observed in rigorously
conducted trials. However, the importance of aggressive
immunotherapy with high dose interleukin-2 treatment
may be the duration and quality of the responses
observed. In this 255 patient data set treated with high
dose interleukin-2, the median survival for responding
patients is over 84 months, and the median response
duration for completely responding patients has not
been reached, which is tantamount to cure [24].
Biological therapy of genitourinary cancer
The administration of interleukin-2 at high doses
requires hospital admission to an intensive care unit or a
dedicated, specialized nursing unit capable of close
approximately half of patients, a skilled treatment team,
and in spite of the careful attention of this skilled team
occasional mortality. A 4% treatment related death rate
was noted in the original 255 patient dataset, although
more recent series have found the treatment related
death rate to be 1% or less [34, 44, 107]. Because of the
toxicity associated with the capillary leak syndrome and
the potential cardiac toxicity associated with high dose
interleukin-2, patient selection is very important for its
safe administration [44]. The toxicity of high dose inter-
leukin-2 has limited its application to patients that are
relatively young, have limited co-morbidities, an excel-
lent performance status, and access to one of the rela-
tively few treatment centers that offer this intensive
therapy. Because lower intensity and outpatient regi-
mens of interleukin-2 lead to responses in a fairly simi-
lar proportion of patients in phase II studies, it has been
tempting to replace high dose interleukin-2 with low
dose, intravenous or subcutaneous, outpatient regime ns
of interleukin-2. While considerably easier to adminis-
ter and applicable to a much broader patient population,
it is not clear that the response rate and quality of
responses are preserved with this approach.
In order to directly compare the results of therapy with
high dose interleukin-2 compared to lower intensity and
less toxic regimens, two large, prospective, randomized
trials have completed patient accrual at the National
Cancer Institute (NCI) and at multiple institutions through
the Cytokine Working Group (CWG) comparing high
dose interleukin-2 with lower dose regimens [55, 107].
In the former study from the NCI, patients were random-
ized to high dose intravenous bolus interleukin-2, low
dose intravenous bolus interleukin-2, or subcutaneous
interleukin-2. In the latter study through the CWG,
patients were randomized to receive high dose intrave-
nous bolus interleukin-2 or subcutaneous interleukin-2
plus interferon alpha. The response rates with high-dose
interleukin-2 are approximately twice that observed with
lower doses of interleukin-2 with or without interferon
alpha in groups of patients that have been comparably
screened and selected on the basis of their ability to
undergo the rigors of treatment with high dose interleu-
kin-2 [55, 107]. The survival and the response duration
from these trials await further maturation of the data;
however, the trend from both studies appears to indicate
that the durability of the responses in patients treated
with high dose interleukin-2 is superior to that achieved
Robert K. Oldham 651

with low dose interleukin-2 [55, 107]. Overall survival was
not significantly different in the two groups. [55, 107].
In summary, the consistent results of the numerous
studies of the treatment of metastatic kidney cancer with
cytokine therapy are that a relatively small and consis-
tent fraction of patients, 10–20%, will achieve evidence
of tumor regression with occasional complete remissions
and long survival noted for a few patients. For the subset
of patients who are able to receive aggressive immuno-
therapy with high dose interleukin-2, this intensive therapy
may result in a higher percentage of complete and partial
remissions and more durable responses(Table 3). Even
enthusiasts of high dose interleukin-2 hoped to replace
this labor intensive and toxic treatment with a more
widely applicable but therapeutically equivalent regimen
(in designing the randomized trial described above, the
Cytokine Working Group had expected equivalent thera-
peutic results from the low dose regimen and performed
this trial “before accepting the low dose regimens as the
standard”). However, at the time of this writing, high
dose interleukin-2 cannot be dismissed and remains one
of the standard treatment approaches for appropriately
selected patients treated at centers with expertise in the
administration of this therapy.
Cytokine Combinations
Pre-clinical studies of interleukin-2 and interferon alpha
suggested additive or synergistic activity and led to a
number of phase I and II studies to investigate the clinical
activity of this combination. A review of over 1,400
patients has been published demonstrating response rates
of approximately 20% and complete remissions in 5% of
patients irrespective of the route of interleukin-2 adminis-
tration [8]. Because of this apparent slight but potentially
significant improvement in the response rate, several ran-
domized trials have been performed comparing combina-
tion versus single agent cytokine therapy. The largest of
these, reported by Negrier et al., comprised over 400
patients and demonstrated an improved response rate for
interleukin-2 plus interferon alpha (18.6%) compared to
interleukin-2 (6.5%) or interferon alpha (7.5%) alone
[65]. Unfortunately, the improvement in the response rate
did not lead to a significant improvement in median sur-
vival, although the crossover design for patients failing to
respond to interleukin-2 or interferon alpha monotherapy
makes comparison of the survival times difficult. It is
important to note that the overall response rate to cytokine
monotherapy in this study was lower than what has gen-
erally been observed, again underscoring the importance
of patient selection and the importance of randomized trials
for comparing the outcomes of different treatments. An
important observation resulted from the crossover design
of this study: responses to interleukin-2 after interferon
failure or interferon alpha after interleukin-2 failure are
rare [20]. Thus, at present, monotherapy with either inter-
leukin-2 or interferon alpha at modest doses remains the
standard of cytokine care for most patients with kidney
cancer and can be expected to result in tumor regression in
10–20% and overall median survivals of slightly greater
than a year [32].
A cytokine therapy must be reinterpreted in light of the
newer molecularly targeted agents described in a subse-
quent section. Perspective randomized trials of cytokine
therapy versus targeted agents, and/or combinations
thereof, are currently underway [32].

Table 3. Randomized trials comparing high dose versus low dose interleukin-2
Study/treatment N Complete responses (%) Partial responses (%) Objective response rate (%)
a
NCI Study(107)
HD-IL-2 115 9 (8) 13 (11) 22 (19)
LD-IL-2 (IV) 112 5 (4) 6 (5) 11 (10)
LD-IL-2 (SC) 53 3 (6) 3 (6) 6 (11)
CWG Study(55)
HD-IL-2 99 8 (8) 17 (17) 25 (25)
LD-IL-2 (SC) + IFN (SC) 94 2 (2) 10 (11) 12 (13)
Totals
HD-IL-2 214 17 (8) 30 (14) 47 (22)
LD-IL-2 (IV or SC) +/− IFN 259 10 (4) 19 (7) 29 (11)

HD-IL-2 high dose interleukin-2 by intravenous bolus injection; LD-IL-2 low dose interleukin-2 by intravenous bolus injection (IV) or
subcutaneous injection (SC); IFN interferon alpha by subcutaneous injection
a
The NCI study was initially designed to evaluate high dose versus low dose intravenous bolus injections of interleukin-2. After the first
117 patients were randomized (56–57 per arm), the third arm of the study was added to compare subcutaneous interleukin-2 admin-
istration. For a statistically valid comparison, these data have been reported comparing only concurrently randomized patients [104].
For informational comparison in this table, the data have been presented as if all patients had been randomized concurrently.
652
Adoptive Cellular Therapy
Lymphocyte Activated Killer Cells and Tumor
Infiltrating Lymphocytes
Historically and for the purposes of review, cancer immu-
notherapy has been classified as “active,” including agents
such as cytokines that activate the host anti-tumor immune
response, and “passive,” including agents such as mono-
clonal antibodies or immune effector cells that directly
(or sometimes indirectly) mediate the anti-tumor response
themselves. The transfer of immune effector cells with
direct anti-tumor reactivity has also been termed “adop-
tive cellular therapy.” In kidney cancer, the use of lym-
phokine-activated killer (LAK) cells is of great historical
significance, since the development of modern cytokine
therapy using interleukin-2 for renal cell carcinoma was
originally based on the observation, in the early 1980s,
that LAK cells are lymphoid cells that circulate in the
peripheral blood, are activated in vitro by exposure to
high concentrations of interleukin-2, and have the ability
to selectively lyse neoplastic cells [44]. Early clinical trials
of interleukin-2 were therefore done in combination with
LAK cells, or more accurately stated, early trials of LAK
cells required the systemic administration of interleukin-2
to demonstrate efficacy of the LAK cells [36, 85, 98,
104]. An initial response rate in excess of 75% was
reported [85], but a series of phase II studies including
over 500 patients with renal cell carcinoma demonstrated
that this treatment approach lead to anti-tumor responses
in 22% of patients (range 9–35%) [40]. Moreover, most
of these responses have been partial and transient, typi-
cally lasting no more than several months. Subsequently,
randomized trials at the National Cancer Institute [86]
and elsewhere [40]) comparing interleukin-2 plus or
minus LAK cells failed to demonstrate a contribution to
response rate or survival by the LAK cells.
In the hope of reducing the number of cells needed for
passive transfer and decreasing the requirement for the
co-administration of high dose interleukin-2, tumor infil-
trating lymphocytes (TIL) were discovered in the search
for a more potent lymphoid effector cell [108]. Whereas
LAK cells are nonspecific natural killer cells, TIL are
activated cytotoxic T cells, which show a greater propen-
sity for target-specific killing. TIL are isolated from a
primary or metastatic tumor, and, similar to LAK cells,
are expanded ex vivo in the presence of interleukin-2,
then reinfused in combination with systemic cytokine
therapy. In murine models, TIL were found to be 50–100
times more potent on a per cell basis in the treatment of
established lung micrometastases than LAK cells [87].
Sufficient studies utilizing acceptable doses of interleukin-2
along with escalating doses of activated cells have not
Biological therapy of genitourinary cancer
been done. Perhaps the most extensive dose-related studies
have been accomplished by Oldham and co-workers
[51] in utilizing as many as ten doses of activated T cells
in treatment of patients with advanced malignancy. With
regard to patients with renal cancer, only four doses have
been used, giving total cell doses up to 2 × 1011 cells.
A series of phase I–II trials were performed, including
114 patients with renal cell carcinoma, treated with TIL
plus IL-2, and demonstrating an objective response rate
of 23% (range 0–35%) [40]. Again, in a randomized trial
comparing interleukin-2 therapy alone to the combina-
tion of interleukin-2 plus TIL in 178 patients with renal
cell carcinoma, there was no advantage in terms of
response rate, duration of response, or survival to the
addition of the TIL compared to interleukin-2 alone [23].
The addition of an expanded population of autologous
tumor killing cells in the form of TIL or LAK cells to
interleukin-2 has been carefully studied and in its present
form offers no advantage to the treatment of kidney can-
cer over cytokine therapy alone. Nonetheless, early
observations demonstrated that melanoma patients
treated with TIL plus interleukin-2 were capable of
responding even when a prior response to interleukin-2
alone was not observed [88]. The administration of an
expanded population of highly selected T cells derived
from autologous TILs in combination with high dose
IL-2 and following a non-myeloablative chemotherapy
conditioning regimen mediated the regression of meta-
static melanoma and MRC patients refractory to stan-
dard therapy, including high dose IL-2. This study
demonstrated that prior lymphodepletion improves the
persistence and function of adoptively transferred cells
[17]. Ongoing studies at the National Cancer Institute
and in Europe indicate that TIL cells can be genetically
engineered to enhance their activity against both mela-
noma and kidney cancer. Such genetically retargeted
cells sometimes, accompanied by lympho-depletion
strategies, have shown high response rates in a small
series of patients over the last 5 years [17, 50].
Allogeneic Stem Cell Transplantion
A novel approach to the biological treatment of meta-
static renal cell carcinoma has employed allogeneic
hematopoietic stem cell transplantation in an attempt to
mediate anti-tumor activity from donor T-cells from an
HLA matched sibling donor. This treatment strategy rep-
resents a form of adoptive cellular immunotherapy.
Based on data that the transfer of allogeneic stem cells in
the treatment of hematalogic malignancies can induce a
graft-versus-leukemia reaction that may improve the
cure of these malignancies, a similar graft-versus-tumor
Robert K. Oldham
effect has been sought in the treatment of metastatic kid-
ney cancer. Childs et al. established proof-of-principle
for the existence of the GVT effect in metastatic kidney
cancer [10, 11] and have recently reviewed their experi-
ence at the National Institutes of Health using this treat-
ment modality [12]. Over 50 patients have been treated,
and 47% have achieved evidence of tumor regression,
including 18 (38%) partial responses and 4 (9%) com-
plete responses [12]. Three of the four complete respond-
ers remain disease free, including the first patient treated
whose remission exceeds 5 years. Interestingly, the
majority of patients that eventually respond to this treat-
ment experience initial tumor progression in the first few
months following the procedure, and tumor regression
occurs late in the course of the transplant at a median of
5 months. Disease regression typically occurred after the
withdrawal of immunosuppression and after the conver-
sion of the host to full donor T-cell chimerism, implying
that these responses resulted from the GVT activity of
donor T-cells. These results demonstrate the proof-of-
principle that a clinically meaningful graft-versus-tumor
response can be achieved in the setting of metastatic
renal cell carcinoma. The wide applicability of this
approach may be limited by the long interval of time
required from the initiation of the evaluation of the
patient for treatment with stem cell therapy until the
withdrawal of immunosuppression when a graft-versus-
tumor effect may be expected. This time interval has so
far required many months, during which time patients
with even a moderate rate of tumor progression may
worsen clinically and become ineligible for aggressive
intervention or succumb to their disease, particularly in
light of the fact that most patients will have been treated
with a traditional treatment approach before consider-
ation of this novel form of immunotherapy. Indeed, as
expected, of the first 19 patients reported by Childs et al.,
eight patients died of disease progression within approx-
imately 1 year while awaiting the GVT effect during
follow-up after transplant [10]. As a result, treatment of
patients with stem cell transplantation requires careful
selection of a subset of kidney cancer patients who are fit
enough to undergo an aggressive treatment and who
have a slowly progressive form of the disease. The role
of stem cell therapy for the treatment of metastatic renal
cell carcinoma will require further study with large num-
bers of patients and confirmation of these intriguing
results by other investigators at other sites [84].
Vaccines and Gene Therapy
Vaccines and gene therapy approaches to the treatment
of kidney cancer have generally attempted to improve
653
tumor antigen presentation and patient T-lymphocyte
activation by enhancing tumor antigenic peptide presen-
tation in the context of major histocompatibility complex
(MHC) molecules, by restoring co-stimulatory signals
that are frequently deficient in tumor cells, and by
amplifying recruitment of the patient’s immune effector
cells. Gene therapy strategies for kidney cancer are based
on the premise that genetic modification of immune
cells can enhance function or that the introduction of a
particular gene into tumor cells can increase their immu-
nogenicity and induce an effective immune response not
only against the gene-modified tumor, but also against
the unmodified tumor cells that are present elsewhere in
the individual [39, 67]. Tumor cells may be modified in
vitro or in vivo to express cytokines, co-stimulatory
molecules, or foreign MHC molecules.
Vaccine therapy
Dendritic cells (DC) are the most potent, professional
antigen presenting cells (APC) of the immune system and
may be uniquely able to stimulate T-cells because they
possess all of the required co-stimulatory molecules.
Dendritic cells can be isolated from peripheral blood
mononuclear cells and expanded and modified ex vivo,
making vaccination with DC feasible. Strategies to induce
the expression of tumor antigens on dendritic cells include
gene transfer into DC using viral or non-viral vectors,
tumor cell lysates, apoptotic bodies, tumor-derived RNA,
and DC-tumor cell hybrids. Alternately, dendritic cells
may be expanded in vivo by the systemic administration
of combinations of cytokines such as GM-CSF and IL-4
[90]. These approaches indicate there is a need to reach a
balance between immunity and tolerance [16].
A study in patients with metastatic renal cell carci-
noma demonstrated the potential effectiveness of DC
vaccines using electrofusion to generate autologous
tumor cell-allogeneic DC hybrids [49]. Seventeen
patients were vaccinated subcutaneously, and after vac-
cination, four patients achieved complete responses and
two patients had partial responses. Fever and injection
site pain were the only important toxicities reported.
Controversy exists regarding the effectiveness and repro-
ducibility of the electrofusion technique used in this
study, but other human clinical trials employing dendritic
cell vaccines in patients with metastatic renal cell cancer
have also shown anti-tumor responses [33, 41].
The field of cancer vaccination has been advanced by
evidence that both cytotoxic and helper T cells recog-
nize intracellularly degraded peptides that are processed
by specialized antigen presenting cells via the protea-
some apparatus, inserted into the endoplasmic reticu-
lum, and transported to the cell surface for association
654
with major histocompatibility (MHC) molecules [103].
Taking advantage of this finding, a specific approach to
improving effective antigen presentation for the treat-
ment of renal cell carcinoma involves the use of peptide
vaccines derived from heat shock protein–peptide com-
plexes from kidney tumors. Heat shock proteins (HSP)
are molecular chaperones implicated in “loading”
immunogenic peptides onto MHC molecules for pre-
sentation by antigen presenting cells to T-cells. In this
vaccination strategy, autologous tumor derived peptides
associated with HSP administered to patients are taken
up by antigen presenting cells such as DC and processed
by the APC so that tumor antigen is “re-presented” to
naïve T-cells, leading to an antigen specific T-cell
response [94]. Amato et al. have treated patients with
metastatic renal cell carcinoma with autologous gp96
heat shock protein–peptide complex vaccination in a
phase I–II trial [3]. The vaccine, at one of three doses,
was administered once with a week for 4 weeks with
follow-up doses at weeks 12 and 20 depending on
response. Responses were observed in 4 of 16 patients
(25%) at the 25 ug dose level, including one patient with a
complete response; three additional patients experienced
prolonged stabilization of disease in excess of 1 year.
Follow on phase III randomized studies have not con-
firmed these earlier trials, and there has not been a sig-
nificant difference seen between the HSP arm and the
placebo arm in these adjuvant studies [95].
Gene Therapy
Human gene therapy generally involves the insertion of
functional DNA into a human cell either in vivo or ex
vivo to replace a defective gene or to provide a new
function to the cell. Traditionally, the therapeutic DNA
has been packaged in a viral or lipid vector for delivery
of a functional gene into human cells. Alternately, naked
DNA, generally in the form of a plasmid, may be directly
injected into skin or muscle or adsorbed onto micropar-
ticles and forcibly bombarded into the skin in vivo or
other cells ex vivo. Naked DNA injections have been
alternately termed “polynucleotide vaccines.” Human
gene therapy approaches to the treatment of kidney can-
cer that have advanced into clinical trials have included
altering immune effector cells with a gene designed to
improve their function or altering tumor cells with a
gene designed to improve their immunogenicity [33].
The “polynucleotide vaccine” strategy of transferring a
gene encoding a tumor antigen into endogenous antigen
presenting cells has yet to be exploited in the clinical
model of renal cell carcinoma, although this approach
has been attempted clinically in other solid tumors [89].
In an example of the former approach, investigators
Biological therapy of genitourinary cancer
used natural killer cells transfected with the interleu-
kin-2 gene for the adoptive immunotherapy of renal cell
carcinoma and other malignancies [91]. This trial dem-
onstrated the feasibility of this approach, the relative
lack of toxicity, a significant increases in other immuno-
stimulatory cytokines (interferon gamma, transforming
growth factor beta, and granulocyte-macrophage colony
stimulating factor), and clinical response in one patient
with lymphoma. There are several examples of the latter
gene therapy approach of genetically modifying tumor
cells to increase their immunogenicity in the treatment
of kidney cancer. Autologous renal cell carcinoma cells
have been retrovirally transduced with the gene for
granulocyte-macrophage colony stimulating factor and
re-administered as a vaccine to 16 patients with meta-
static renal cell carcinoma [93]. No significant toxicities
were observed, and one patient experienced a partial
remission. Another strategy to make tumor cells more
immunogenic has involved the transfer of the gene encod-
ing human leukocyte antigen (HLA) molecule B7 into
autologous renal cell carcinoma cells. In one study, lipid-
DNA complexes (commercially called “Allovectin-7”)
were transferred into accessible metastatic lesions in 15
HLA B7 negative renal cell carcinoma patients [83].
Conceptually, the intent of these intratumoral injection
studies is to develop an in vivo autologous tumor vac-
cine that will lead to a systemic disease response. In this
example, neither toxicity nor responses were observed.
A similar example of this approach includes the direct
transfer of the lipid complexed interleukin-2 gene into
accessible metastatic lesions in patients with renal cell
carcinoma. Further details of gene therapy are presented
in Chapter 19.
Targeted therapy
It has been known for many years that renal cancer over-
expresses vascular endothelial growth factor (VEGF).
Because of this finding, recent efforts have targeted
VEGF in an attempt to block vascular supply and blood
supply to MRCC.
Bevacizumab (Avastin) is a recombinant human
monoclonal antibody against VEGF [32, 71]. In a phase
II trial or more than 100 patients with MRCC, random-
ized placebo versus a low-dose (3 mg/kg) or high-dose
(10 mg/kg) of bevacizumab given intravenously in treat-
ment-resistant patients every 2 weeks (over 90% had
received prior IL-2) demonstrated an improved time to
progression for the bevacizumab group [106].
As a result of this interesting phase II trial, there are
ongoing studies with bevacizumab plus other agents
active in MRCC [81]. Bevacizumab plus interferon is
Robert K. Oldham
more active than interferon alone [106], and bevaci-
zumab combined with erlotinib (Tarceva), an EGRF
inhibitor, has shown objective responses in patients with
resistant, advanced renal cancer [37].
Many clinical trials are underway using various com-
binations of cytokines and targeted therapies, but no
specific combination has proven superior to an alternative
combination to-date.
In addition to a large molecule inhibitor of VEGF,
such as the monoclonal antibody bevacizumab, there
are small molecule inhibitors, which work through the
super family of tyrosine kinases. Three of the agents are
currently approved for the treatment of MRCC and are
being used alone and in various combinations with the
other active agents.
Sorafenib (Nexavar) is an oral agent that inhibits Raf
kinase. Sorafenib also is a direct inhibitor of the VEGF
receptor family. In a phase II randomized study with over
200 patients with MRCC, patients received 400 mg twice
a day of sorafenib versus placebo. The median progres-
sion free for survival was longer in the sorafenib group..
This study was followed by a phase III randomized trial
or sorafenib versus placebo in cytokine-refractory MRCC
[21]. Although only a small percentage of patients had a
complete or partial response, and many of the patients
exhibit stable disease while receiving the drug. The
median progression free survival for the sorafenib group
was 24 weeks, double that of placebo. Sorafenib was well
tolerated, but does have significant side effects which are
generally manageable, including dermatological, gastro-
intestinal and neurologic symptoms, along with fatigue
and elevates blood pressures [76].
Another oral tyrosine kinase inhibitor of VEGF recep-
tor is sunitinib (Sutent). This drug was effective in phase
II trials and was subsequently approved based on random-
ized phase III trial in patients with MRCC [59]. Again, the
median progression free for survival was approximately
double in the treated group versus the placebo group.
A third small molecule, temsirolimus (Torisel), has
recently been approved for poor risk MRCC patients. This
drug is given intravenously and is an inhibitor or rapamy-
cin (mTOR), a molecule important in tumor-promoting
intracellular signaling pathways. Studies comparing this
agent to interferon-alpha demonstrated an improvement in
survival amounting to 3–6 months [42]
Other agents, such as lenalidomide [13], tykerb lapa-
tinib [77] and axitinib [82], are underway.
While it is clear that these small molecule and large
molecule agents targeting various receptors and mole-
cules that support neovascularization and/or promote
tumor growth are important individually, it is unknown
how best to combine them with other agents active in
655
MRCC. The daunting task of working through this will
require many randomized trials for patients with MRCC
[14]. While the best combination is not known, the
future looks much brighter for these patients than it did
a mere 5 years ago.
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21.4 Biological therapy of colon cancer
ROBERT O. DILLMAN
Colon Cancer
Non-specific Immune Stimulants
Levamisole
Levamisole is a synthetic phenylimidazothiazole oral
antihelminth that exhibited non-specific immunostimu-
latory properties in animal models [82]. Interest in this
agent as a cancer therapy dates back to 1971. Small
exploratory clinical studies were inconclusive, but con-
sidered encouraging enough to lead to randomized
phase III trials in colon cancer. Two randomized trials
failed to demonstrate a benefit for levamisole alone
compared to placebo in the adjuvant setting. In one
small double-blind U.S. trial 78 patients were random-
ized to receive 18 months of levamisole 2.5 mg/kg/day
given on days 1 and 2 of each week, or placebo follow-
ing resection of Dukes B or C colorectal cancer [8]. No
benefit for levamisole was observed. In a double-blind
randomized European trial comparing levamisole at 100
to 250 mg twice a week to placebo as adjuvant therapy
for 297 patients with node-positive colon cancer, there
again was no demonstrable benefit for levamisole [1].
Other trials combined levamisole with 5-fluorouracil
(5FU). In a large adjuvant trial 1,296 patients, who had
resected colon cancer that was either locally invasive
(n = 318) or metastatic to regional lymph nodes (n = 929),
were randomized to observation, or to 5FU plus levami-
sole for 1 year, or to levamisole alone [58]. Patients
were assigned to observation, or to levamisole alone
(50 mg p.o. three times a day for 3 days, repeated every
2 weeks for 1 year), or to this regimen of levamisole
plus 5-FU (450 mg/m2 i.v. daily for 5 days and then,
beginning at 28 days, weekly for 48 weeks). Once again
levamisole alone provided no benefit compared to
observation, but after a median follow up of 3 years, the
combination of 5-FU plus levamisole was associated
with a significantly reduced risk of recurrence and death
for patients who had lymph node metastases (p = 0.006).
After a median follow up of 6.5 years the advantages of
levamisole + 5FU persisted in stage III disease, with a
40% reduced recurrence rate (p < 0.0001) and 33%
reduced death rate (p = 0.0007) [59]. No benefit was
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
demonstrated in the smaller subset of patients with stage
II disease [60]. On the basis of this trial, 5FU plus
levamisole became standard therapy for the adjuvant
treatment of stage III colon cancer during 1990–1997.
A major limitation of the early levamisole trial was
that it did not have a control arm of 5FU plus folinic
acid (leucovorin), which was shown to be more active
than 5FU alone while the trial was being conducted.
Two large U.S. trials subsequently showed that levami-
sole did not add benefit to such a regimen. During
1989–1990 2,151 patients who had stage II or III colon
cancer were randomized to receive weekly 5FU plus
leucovorin, 5FU plus levamisole, or 5FU plus leuco-
vorin plus levamisole [98]. After more than 7 years of
follow up, the 5-year survival rates were 74% for 5FU
plus leucovorin, 73% for all three drugs, and 70% for
5FU plus levamisole. During 1988–1992 3,794 patients
were randomized in a four-arm trial to receive 5FU plus
levamisole for 1 year or 6 months of low-dose leuco-
vorin plus 5FU (“Mayo Clinic regimen”), or high-dose
leucovorin plus 5FU (“Roswell Park regimen”) or the
Mayo clinic regimen plus levamisole, plus FU (LDLV
plus LEV) regimen [33]. After 10 years of follow up
there were no differences in survival among the arms,
and the 5FU plus leucovorin regimens replaced the
levamisole regimens as standard therapy when the
results were first reported in 1997.
Other randomized trials of adjuvant therapy in colon
cancer confirmed beyond a doubt that the addition of
levamisole provided no benefit in the adjuvant therapy
of colon cancer. In a trial in which 379 patients received
5FU plus folinic acid, and 374 received 5FU plus
levamisole, after a median follow up of 6.8 years, there
was a 72% survival rate in both arms [63]. In a four-arm
trial in which 598 patients were randomized to receive 1
year of weekly 5FU, or 5-FU plus levamisole, or 5-FU
plus IFN-α or all three agents, recurrence rates and sur-
vival were worse in the combined arms that contained
levamisole (p = 0.003) [77]. In another randomized
trial there was no difference in survival for 92 patients
who received 5FU plus levamisole compared to 92 who
received 5FU alone, but leucopenia and hepatic toxicity
were more frequent in the levamisole arm [5]. In a two-arm
659
660 Biological therapy of colon cancer

trial in which 1,703 colon cancer patients were random-
ized to 5FU plus levamisole or 5FU plus leucovorin
plus levamisole, there was no difference in survival, but
the three drug regimen was more toxic [13].
Thymosin Fraction 5
The importance of the thymus gland and thymic proteins
for normal T-lymphoycte cellular immunity has been
recognized for many decades. One of the preparations of
interest was a crude lysate called thymosin fraction 5. As
summarized in Table 1, subsequent dose-exploring and
phase II trials failed to confirm significant activity in
lung cancer or immune enhancing activity for thymosin
fraction 5 [14, 15].
Interferons
As shown in Table 1, single agent alpha-interferon (IFN-
α) rarely produces objective tumor responses in patients
with metastatic colorectal cancer [4, 62]. In three phase II
trials there were no objective responses observed with either
lymphoblastoid interferon or alpha interferon [6, 9, 54].
However, there is laboratory evidence that IFN-α can
enhance the antitumor activity of 5-FU by a mechanism
which is different from that of leucovorin [61, 89].
An early clinical trial which combined 5-FU with IFN-α
for the treatment of advanced colorectal carcinoma
claimed a 75% objective response rate among patients
who had not been treated previously with chemotherapy,
but no responses in previously treated patients [89–91].
Unfortunately, other investigators typically reported much
lower response rates for this combination in colorectal
cancer, with a range of 20% to 50% [12, 49, 68, 92, 93].
Randomized trials were unable to confirm response
rates any higher than 20% to 25%, which is no different
that 5FU alone. In a randomized trial for patients with
previously untreated advanced colon cancer, the response
rate was 21% for 104 patients who received 5FU alone
(750 mg/m2 as a 4-h i.v. infusion on days 1–5 and then
i.v. bolus weekly) compared to 25% for 101 patients who
received 5FU plus IFN (3 to 9 MIU thrice weekly s.c.)
[67]. In a similar trial that utilized the same dose and
schedule of 5FU, but IFN at a dose of 10 MIU s.c. thrice
weekly, the response rate was 30% for 54 patients who
received 5FU alone and only 19% for 52 patients
who received 5FU plus IFN [39]. The lower response rate
was probably due to patients withdrawing from the trial
because of toxicity. Patients who received IFN experienced
more toxicity, including four toxic deaths, leucopenia,
lymphopenia, depression, and alopecia.
Subsequent trials combined 5FU, folinic acid, and
IFN-α, but the response rates were no higher than the
doublets [32, 44, 69]. Randomized trials confirmed that
IFN-α adds toxicity, but not benefit, to 5FU and folinic
acid. In a trial that included 204 untreated patients, there
was no difference in response rate was (23% to 24%) or
survival (medians 11 to 12 months) [10]. The addition
of IFN was associated with more toxicity, especially
fever, diarrhea, and mucositis. In another trial 102

Table 1. Single-agent activity of various biologicals in patients with metastatic colon cancer
Modality class Biological agent Reference Patients Response rate (%)
Non specific Thymosin fraction 5 Dillman 12 0
Cytokine Lymphoblastoid Interferon [6] 19 0
Cytokine Interferon-α [54] 18 0
Cytokine Interferon-α [9] 36 0
Cytokine Interleukin-2 (IL-2) [73, 74] 22 0
Cell therapy IL-2 + LAK [73, 74] 38 18
Cell therapy IL-2 + LAK [17] 35 0
Cell therapy IL-2 + TIL [64] 8 0
Cell therapy IL-2 + TIL [20] 8 0
Cell therapy CD4+ T cells [19] 15 7
Mab Anti-CEA [16] 30 0
Mab Edrocolomab 40 0
Mab Edrocolomab [24] 20 5
Mab Edrocolomab [52] 25 4
Mab Edrocolomab [57] 52 2
Mab Bevacizumab [28] 234 3

Mab = monoclonal antibody

LAK = lymphokine activated killer cells
TIL = tumor infiltrating lymphocytes
CEA = carcinoembryonic antigen
Robert O. Dillman
patients were randomized to receive 5FU and leuco-
vorin with or without IFN; there was no difference in
response rates (8% and 10%) but time to progression
and overall survival both favored the group that did not
received IFN (p = 0.002) [50]. Thus, clinical trials
showed that 5FU plus IFN-α was no better than 5FU
plus leucovorin, but more toxic, and that there was no
advantage for the addition of IFN-α to 5FU plus leuco-
vorin in the treatment of metastatic colorectal cancer.
Several randomized trials have established that there
is no role for IFN-α in the adjuvant treatment of high-
risk colon cancer. In a Greek trial that included stage II
and III colon cancer 139 patients received 5FU plus foli-
nic acid and 141 received the same therapy plus IFN (3
MU s.c. thrice weekly) [23]. After a median follow up of
4 years, there was no difference in disease free survival
or overall survival. In an Italian trial in which 322
patients with stage II or III colon cancer were random-
ized to infusional 5FU plus leucovorin with or without
IFN (5 MU s.c. thrice weekly), there was no difference in
either disease-free survival or overall survival but toxic-
ity was greater in the IFN-arm because of flu-like syn-
drome [26]. In a three-arm German trial, 813 patients
with stage II and 750 with stage III colon cancer were
randomized to 5FU plus levamisole alone or with leuco-
vorin, or with IFN [81]. The 4-year survival rate was
highest in the arm that included leucovorin (78%), but
was no better in the IFN-arm than 5FU plus levamisole
alone (both 66%). In a four-arm trial in which 598
patients were randomized to receive 1 year of weekly
5FU, or 5-FU plus levamisole, or 5-FU plus IFN-α or all
three agents, there was increased toxicity in the IFN-
arms but no effect on survival [77]. The National Surgical
Adjuvant Breast and Bowel Project (NSABP) group ran-
domized 2,176 patients with stage II or III colon cancer
to receive 5FU plus leucovorin with or without IFN [97].
After 4.5 years of follow up there was no difference
in disease free survival (69–70%) or overall survival
(80–81%), but there was more toxicity associated with IFN.
In one small randomized trial 99 patients with stage
II, III, or IV colon cancer were randomized to receive
IFN-gamma 0.2 mg s.c. daily for 6 months or observa-
tion after surgical resection of their disease [96]. Despite
evidence that IFN-γ induced immune response, after a
median follow-up of almost 5 years, disease free sur-
vival was worse for patients who received IFN-γ (p =
0.03) and they had a trend to worse survival (p = 0.12).
Interleukin-2
There is only limited clinical trial data with interleukin-2
(IL-2) in colorectal cancer, and most of the experience
661
has been in combination with adoptive cell therapy or in
combination with other biologicals. High-dose bolus IL-2
yielded no objective responses in 22 patients [73, 74].
The response rate for high-dose continuous infusion
IL-2 and tumor necrosis factor (TNF-α) was 0/16 [18].
The response rate for high-dose continuous infusion
IL-2 and IFN-α was 1/10 [65].
Adoptive Cell Therapy
There has been only limited exploration of cell-based
therapies for colon cancer. As described in detail else-
where in this book, lymphokine activated killer [LAK]
cells are derived by taking peripheral blood lymphocytes
(PBL) and stimulating them in vitro with IL-2 while
tumor infiltrating lymphocytes (TIL) are produced by
incubating tumor samples with IL-2. For patients with
metstatic colon cancer, LAK administered with high dose
bolus IL-2 produced responses in 7/38 (18%) patients
[73, 74] AK cells infused with high-dose continuous
infusion IL-2 (civ IL-2) produced no responses in 35
patients [18]. There were no responses among 16 patients
treated with TIL, eight of whom received high doses of
TIL plus civ IL-2, and eight who received variable doses
of TIL with other schedules of IL-2 [20, 64].
Autolymphocyte therapy and autologous activated lym-
phocytes were used to describe autologous lymphocyte
preparations that were derived from peripheral blood and
stimulated with anti-CD3 monoclonal antibodies in vitro to
produce an auto-cytokine preparation that was then used
for incubation with additional peripheral blood lympho-
cytes [19, 66]. The autolymphokine preparation contained
significant amounts of TNF-α, IL-1β, interferon-γ, and
IL-6, but no IL-2. Culturing peripheral blood lymphocytes
in this media resulted in enrichment of CD3+ cells, but
decreased CD3+/CD56+ cells (NK), decreased CD4+/
CD45RA(2H4) suppressor T cells, and increased CD8+/
HLA-Dr+, and CD4+/CD29(4B4)/HLA-DR+ helper T cells.
There was a slight decrease in NK cytolytic activity in Cr51
assays compared to the mononuclear cells obtained prior to
incubation in ALK. These cells, which lacked cytolytic
activity, were infused in doses of about 109 cells in patients
who were receiving high doses of cimetidine to inhibit
suppressor or negative regulatory T cells. A response rate
of 1/15 was observed with such a product in the setting of
metastatic colon cancer [19].
Monoclonal Antibodies
Early trials with murine anti-CEA monoclonal antibodies
(Mabs) were not associated with clinical responses [16].
However as described in detail elsewhere in this book,
662
there are now three monoclonal antibody products that
are commercially available with marketing indications for
colon cancer. These include the anti-VEGF Mab bevaci-
zumab, the chimeric anti-EGFR Mab cetuximab, and
the human anti-EGFR Mab panitumumab. The murine
anti-Epcam Mab edrocolomab was approved in Germany
for the treatment of colon cancer several years earlier.
Edrocolomab (17-1A, PANOREX®)
Edrecolomab is a murine IgG2a now known to react with
the 37–40 kd glycoprotein epithelial cell adhesion mole-
cule (epcam) that is expressed on various adenocarcino-
mas and on normal epithelial tissues [31, 38]. Single
injections of edrecolomab at doses of 15–1,000 mg were
well tolerated, but 50% of patients developed HAMA after
a single injection [22, 78, 79] summarized in Table 1, as a
single agent, edrocolomab exhibited minimal anti-tumor
effects against colon cancer. There were no objective
responses among 40 patients who received single infu-
sions of 15–1,000 mg of 17-1A during phase I trials in
patients with various gastrointestinal adenocarcinomas
[78, 79]. In a phase II trial of 20 patients who received
200–850 mg of 17-1A, there was one response in a patient
with recurrent rectal cancer [24]. In a trial of 25 patients
who received one to four doses of 400 mg of 17-1A, one
patient had a complete response [52]. Subsequently, this
group treated eight patients with a 17-1A chimeric anti-
body with a human IgG4 subclass heavy chain but there
were no responders [53]. Mellstedt and colleagues reported
one objective response among 52 patients who received
17-1A by a variety of doses and schedules [24, 57].
An apparent benefit for adjuvant edrocolomab was
demonstrated in a multicenter randomized German trial in
patients with resected stage III colorectal cancer [71].
There were 90 patients randomized to observation, and 99
to a post-operative regimen of edrecolomab 500 mg i.v.,
then 100 mg i.v. once a month for 4 months. After a median
follow up of 5 years, there was superior disease-free and
overall survival for the Mab-treated group. This included
a 30% reduction in death rate and 27% reduction in tumor
recurrence, which was similar to results reported in U.S.
trials of 5-fluououracil and levamisole that had led to
acceptance of 5-FU based chemotherapy in the adjuvant
treatment of colon cancer. After a median follow up of 7
years, the apparent benefits of edrecolomab persisted with
a 23% reduction in recurrence and 32% reduction of death
in the antibody group [72]. On the basis of this trial,
edrecolomab was approved for the treatment of Dukes C
colorectal cancer in Germany in 1995.
Unfortunately these promising results were not con-
firmed by four additional phase III trials. North American
Biological therapy of colon cancer
trials compared 5-FU + levamisole, or 5-FU + leuco-
vorin to the same agents plus edrecolomab in Dukes C
colon cancer using the same dose and schedule used
in the German trial. European trials compared 17-1A
antibody alone, to 5-FU + eucovorin, and to 5-FU +
leucovorin + 17-1A. Edrecolomab was also studied in
combination with chemotherapy and radiotherapy
in Dukes B and C rectal cancer. Results of the large ran-
domized European adjuvant trial did not demonstrate a
benefit for the use of edrecolomab as adjuvant therapy
[70]. There were 2,761 patients randomized in the three-
arm trial. The major toxicities observed in the edreco-
lomab-alone arm were diarrhea in 32%, although severe
or life-threatening diarrhea was noted in only 2%.
Results in the edrecolomab-alone arm were inferior to
the 5FU-containing arms (p < 0.05), and there was no
evidence of better results when edrocolomab was com-
bined with 5FU (p = 0.53). Three-year survival rates
were 76% for 5FU plus leuovorin, 75% for the antibody
plus chemotherapy combination, and 70% for the anti-
body alone. The differences in 3-year event free survival
also revealed inferior results for antibody alone, and no
advantage for the chemotherapy plus antibody combi-
nation. In another trial 377 patients with stage 2 colon
cancer, stratified by whether the primary tumor was T3
or T4, were randomized post-operatively to treatment
with 900 mg edrecolomab or observation [35]. The
study was stopped because of discontinuation of drug
supply in Germany after negative results from other tri-
als became public. After a median follow-up of 3.5 years
there was no difference in overall survival or disease-
free survival.
Several trials have explored edrecolomab in combina-
tion with other biologicals with or without chemother-
apy. Weiner et al. gave 150 mg of 17-1A on days 2 to 4 in
combination with 1.0 MIU/m2 of IFN-γ on days 1 to 4, to
19 colorectal cancer patients, but no antitumor responses
were noted [95]. In a second trial 27 patients with colon
or pancreatic cancer were given IFN-γ at doses up to 40
MIU/day for 4 days followed by 400 mg 17-1A on day 5
[94]. No objective tumor responses were seen and the
authors concluded that the low dose of IFN-γ was as
effective as higher doses. Saleh et al. treated 15 colorec-
tal cancer patients with 0.1 mg/m2 IFN-γ on days 1 to 15
and 400 mg of 17-1A at a dose of 400 mg on days 5, 7, 9
and 22 [75]. No significant objective tumor responses
were described.
Bevacizumab (Avastin®)
Bevacizumab is a humanized Mab that binds to the
VEGF ligand. As a single agent bevacizumab rarely
Robert O. Dillman
produced objective responses in a three-arm random-
ized trial in patients with metastatic colorectal cancer
[28]. However, a three-arm randomized phase II trial in
patients who had untreated metastatic colorectal cancer
suggested that bevacizumab enhanced 5-FU based che-
motherapy [46]. The 144 patients were randomized to
fluorouracil and leucovorin (both at 500 mg/m2) as the
control arm, or to the same therapy plus bevacizumab q
2 weeks at either 5 mg/kg or 10 mg/kg. 5FU and leuco-
vorin were given weekly for the first 6 weeks of each
8-week cycle. Better results were seen with the addition
of bevacizumab, and there appeared to be an advantage
for the lower dose.
In the pivotal trial for regulatory approval, 923 patients
with metastatic colorectal cancer were randomized to
receive irinotecan, 5-FU and leucovorin (IFL) and beva-
cizumab (IFL-BV), IFL and placebo (control), or FL
with bevacizumab (FL-BV) at a dose of 5 mg/kg every 2
weeks [42, 43] the first phase of the trial, 313 patients
were randomly assigned to these three arms, then; after a
planned initial analysis, the FL-BV arm was discontin-
ued after enrollment of 110 patients, not because of infe-
rior results, but because IFL had become the standard
control arm based on other trials in metastatic colorectal
cancer. IFL-BV was superior to IFL-placebo for the 813
patients randomized to treatment with one of these arms,
in terms of response rate (p = 0.004), PFS (HR 0.54, p <
0.001), and OS (HR 0.66, p < 0.001) [42]. The IFL-BV
arm was associated with more grade 3 or 4 hypertension
(11% versus 2%). The FL-BV arm also was superior to
the IFL-placebo arm [43].
In a trial of initial therapy for patients with metastatic
colorectal cancer who were not considered optimal can-
didates for first-line irinotecan treatment, 209 patients
were randomized to receive FL + becacizumab at 5 mg/kg
or FL + placebo [48]. The addition of bevacizumab
resulted in a better PFS (HR = 0.50, p < 0.001), higher
response rate (p = 0.055), and a trend toward better sur-
vival (HR = 0.79, p = 0.16). Hypertension was more
frequent with bevacizumab.
In a three-arm trial 829 patients whose cancers had
recurred after IFL therapy were randomized to receive
oxaliplatin, fluorouracil, and leucovorin (FOLFOX4)
with bevacizumab or to FOLFOX4 alone, or to bevaci-
zumab alone [28]. In this trial bevacizumab was given at
10 mg/kg every 2 weeks. As noted earlier, bevacizumab
showed little activity as a single agent, but FOLFOX4
plus bevacizumab was superior to FOLFOX alone for all
key endpoints, including OS (HR 0.75, p = 0.001), PFS
(HR 0.61, p < 0.0001) and response rate (p < 0.0001).
A metanalysis was performed using the raw data
from three randomized trials that included 5-FU plus
663
leucovorin and bevacizumab (FL-BV) as initial treat-
ment for patients with metastatic colorectal cancer [47].
For the combined data, FL-BV (n = 249) was superior to
FL (n = 241) in terms of response rate (34% versus 24%,
p = 0.019), PFS (9 versus 6 months, HR = 0.63, p < 0.001),
and OS (18 versus 15 months, HR 0.74, p = 0.008). In
contrast to these excellent results in previously untreated
patients, in an expanded access trial 350 patients with
metastatic colorectal cancer, who had relapsed after or
progressed during both irinotecan and oxaloplatin based
therapy, the response rate was only 4% in the first 100
patients enrolled by investigator analysis, and only 1%
based on blinded central review [7].
A response rate of 49% was observed among 81 eval-
uable patients with untreated advanced colorectal cancer
who were treated with IFL and bevacizumab [27]. The
doses of irinotecan and 5FU both had to be reduced by
20% to 25% because of vomiting, diarrhea and neutro-
penia. Bleeding occurred in 37 patients (46%) and nine
patients (11%) had grade 3 or 4 thromboembolic events.
In another trial FOLFOX4 and bevacizumab was used
to treat 53 patients with previously untreated metastatic
colorectal cancer [21]. The response rate was 68%.
Hemorrhage was not a problem in this trial, and only
one patient had a severe thromboembolic event.
Cetuximab (Erbitux®)
Cetuximab is a chimeric Mab that binds to EGFR.
Clinical trials with cetuximab used an initial loading
dose of 400 mg/m2 i.v. over 2 h, then 250 mg/m2 i.v. over
1 h weekly. Saltz et al. treated 57 patients whose met-
static cancer had not responded to prior irinotecan alone
or as part of combination chemotherapy [76]. Patients
were required to have EGFR demonstrated by immuno-
histochemistry on formalin-fixed paraffin-embedded
tumor tissue, but it is now known that quantitative
expression of EGFR is not predictive of response to
cetuximab treatment. The most common adverse events
were an acne-like skin rash, predominantly on the face
and upper chest (86%) and the constellation of asthenia,
fatigue, malaise, or lethargy (56%). Three patients were
reported to have had a grade 3 allergic reaction; two
were withdrawn.
Lenz et al. treated reported a response rate of 12% for
346 patients whose metastatic cancer was considered
refractory to fluoropyrimidines, irinotecan and oxalipla-
tin [51]. EGFR positivity by IHC was an eligibility
requirement, but degree of positivity did not correlate
with clinical benefit. The most prevalent toxicity was
the acneiform rash which was observed in 83% of
patients, and which was predictive of clinical benefit.
664
In a randomized trial, 572 patients who had pro-
gressed despite 5FU, irinotecan, and oxaloplatin, were
either observed or received standard dose cetuximab
[45]. The objective response rate was only 8%, but PFS
and OS were better in the cetuximab arm. Skin rash,
infection and hypomagnesemia were more common in
the cetuximab arm. Eleven patients discontinued cetux-
imab because of infusion reactions.
In the pivotal trial Cunningham et al. randomized 329
patients with colorectal cancer that had progressed
during or within 3 months after discontinuation of
irinotecan, to receive either, irinotecan plus cetuximab,
or cetuximab alone, using a 2:1 randomization [11].
Protocol prescribed irinotecan was to be the same as
used prestudy. The response rate was higher for the
combination therapy (23% versus 11%, p = 0.007), as
was PFS (4.0 months versus 1.5 months, p < 0.001).
Gebbia et al. reported a response rate of 20% for
cetuximab plus irinotecan in 60 patients who had
received at least two prior therapies, and whose meta-
static cancer was considered refractory to oxaliplatin
and irinotecan [25]. Standard cetuximab dosing was
combined with irinotecan 120 mg/m2 weekly for 4 out
of 6 weeks. Responses were not predicted by EGFR
expression. Other than the acneiform skin rash that was
severe in 13% of patients, the main grade 3 or 4 toxici-
ties were attributable to the chemotherapy.
Vincenzi et al. reported a 25% response rate for cetux-
imab and irinotecan in 55 patients whose metastatic can-
cer was considered refractory to oxaliplatin and irinotecan
[88]. Standard cetuximab dosing was combined with iri-
notecan 90 mg/m2 weekly. Skin toxicity was observed in
89% of patients. The most common grade 3 to 4 adverse
events were dermatologic (33%), diarrhea (16%), fatigue
(13%) and stomatitis (7%). Fever was noted in 25%,
typically in association with the first infusion of cetux-
imab, but no allergic reactions were recorded.
More recently cetuximab has been combined with
modern combination therapies such as FOLFOX and
FOLFIRI as the initial treatment of patients with metastatic
colorectal cancer. Tabernero et al. used FOLFOX and
cetuximab as the initial treatment for 43 patients with
metastatic colorectal cancer [83]. Cetuximab was given
day 1 at a dose of 400 mg/m2 during week 1, and then
250 mg/m2 weekly thereafter. Treatment was well-tolerated
and the response rate was 72%. In a randomized trial of
337 patients, a preliminary report showed that cetuximab
plus FOLFOX was superior to FOLFOX alone in terms of
response rate (46% versus 36% p = 0.064) [3]. In a ran-
domized trial of 1,198 patients, an initial report showed
that cetuximab plus FOLFIRI was superior to FOLFIRI
alone in terms of response rate (47% versus 39%, p = 0.004)
and PFS (8.9 versus 8.0 months p = 0.048) [85].
Biological therapy of colon cancer
Panitumumab (Vectibix®)
Panitumumab is a human Mab that reacts with EGFR.
A pivotal trial for regulatory approval compared panitu-
mumab at 6 mg/kg i.v. every 2 weeks to best supportive
care in patients with metastatic colorectal cancer whose
disease had progressed during or after standard therapy
with fluoropyrimidine-, oxaliplatin-, and irinotecan-
containing chemotherapy regimens [29, 30, 84, 86]. All
patients had epidermal growth factor receptor (EGFR)-
expressing tumors. Panitumumab produced a 10%
objective response rate and a longer progression free
survival compared to supportive care alone. There was
no difference in survival, but approximately 75% of
patients in the supportive care alone arm crossed over to
receive panitumumab after disease progression. Quality
of life was also better in the patients who received pani-
tumumab [80]. For 176 patients who progressed on the
supportive care arm, and then subsequently did receive
panitumumab, 12% had an objective response and two
patients had complete responses [86].
Hecht et al. treated 148 patients whose metastatic col-
orectal cancer had progressed on chemotherapy that
included a fluoropyrimidine and irinotecan or oxaliplatin,
or both [37]. Panitumumab was given i.v. at a dose of
2.5 mg/kg weekly for 8 of each 9 weeks until disease pro-
gression or excessive toxicity. Skin toxicity occurred in
95% and 5% were grade 3 or 4. Four patients discontinued
therapy because of toxicity and one patient had an infu-
sion reaction but was able to resume treatment. EGFR of
at least 1+ by IHC was an eligibility requirement. There
was no difference in response for 105 patients who were
judged as having high EGFR by IHC compared to 43
patients who were characterized as having a low EGFR.
Berlin et al. tested the combination of irinotecan, 5-FU
and leucovorin, and panitumumab as initial therapy in
patients with metastatic colorectal cancer [2]. This was a
two-part, multicenter, phase II study of panitumumab
2.5 mg/kg weekly with bolus 5-FU (IFL) in the first part
of the trial, and infusional 5-FU (FOLFIRI) in the second
part. Grade 3 to 4 diarrhea occurred in 11 patients (58%)
in part 1 and six patients (25%) in part 2. All patients had
dermatologic toxicity, but none was grade 4. The authors
concluded that panitumumab + FOLFIRI was better tol-
erated than panitumumab + IFL.
Vaccines
A number of trials were carried out with a vaccine consist-
ing of about 10 million irradiated autologous cells obtained
directly from fresh tumor, admixed with about 10 million
bacillus Calmette-Guérin (BCG) organisms and injected
i.d. weekly for 2 weeks, and then an additional injection
Robert O. Dillman
of irradiated tumor cells during the third week. Initial
trials had shown that injections of such a product were
well-tolerated, associated with local delayed type hyper-
sensitivity reactions to autologous tumor, and isolation of
human monoclonal antibodies from circulating lympho-
cytes [36, 40]. In a European phase III trial 254 patients
with stage II or stage III colon cancer were randomized to
observation or active specific immunotherapy with this
preparation beginning 4 weeks after surgery [87]. At a
median follow-up of more than 5 years, there was a 44%
reduction in the risk of recurrence in the treatment arm
(p = 0.023). In the subset analysis there was no benefit for
patients with stage III disease, but there was a great benefit
in terms of decrease risk of recurrence and death in the
vaccine arm. In a U.S. phase III trial 297 patients with
stage II colon cancer, and 115 with stage III colon cancer,
were randomized to observation or to the autologous
active specific immunotherapy starting 1 month after
surgery [34]. After a median follow up of more than 7.5
years, there was no difference in outcomes between the
two treatments based on an intent-to-treat analysis,
although there were substantial problems in compliance
with the injection schedule. This product was not granted
regulatory approval. In addition to the inconclusive trials,
there were issues regarding sterility of such a product
derived from fresh colon tumor, and the variability of the
product in terms of tumor cells versus stromal and immune
cells in fresh tumor.
Another vaccine that has been explored in colon
cancer consists of a non-replicating canarypoxvirus
(ALVAC) engineered to express carcinoembryonic anti-
gen (CEA) and the B7.1 costimulatory molecule. In a
phase I trial eight patients were treated with the vaccine
[55]. The investigators were able to isolate cytotoxic T
cells that could lyse allogeneic and autologous tumor
cells in a MHC-restricted manner [99]. In a second phase
I trial involving patients with CEA-expressing adenocar-
cinomas, three cohorts of six patients were treated with
increasing doses of an ALVAC-CEA-B7.1 vaccine at
doses of 45 million, 450 million, and 0.45 billion plaque-
forming units delivered i.m. monthly for 3 months [41,
56]. In this trial there again was evidence of induction of
specific T cell immune reactivity to CEA.
Summary
The history of immunotherapy in colon cancer is note-
worthy because of the brief periods of success enjoyed
in the adjuvant setting by the BCG-tumor cell vaccine,
the non-specific immune stimulator levamisole in com-
bination with 5FU, and the anti-epcam murine Mab
665
edrocolomab. All three of these products had no or little
activity in the advanced disease setting, and all eventu-
ally fell out of favor in the adjuvant setting because of
results from randomized trials. Combinations of inter-
feron and 5FU appeared encouraging in patients with
advanced disease, but eventually were found to be want-
ing in both the metastatic and adjuvant settings based on
results of large randomized trials. Recently, despite lim-
ited activity as single agents in the advanced setting,
two monoclonal antibodies have gained approval based
on their impressive activity in combination with chemo-
therapy in patients with metastatic disease, including
enhanced survival. Bevacizumab and cetuximab are
now standard components of systemic treatment regi-
mens, but only in combination with chemotherapy.
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and effector cell activation in a phase II trial of recombinant
gamma-interferon and the murine monoclonal antibody CO17–
1A in advanced colorectal carcinoma. Cancer Res 1988;48:
2568–73.
96. Wiesenfeld M, O’Connell MJ, Wieand HS, et al. Controlled
clinical trial of interferon-gamma as postoperative surgical
adjuvant therapy for colon cancer. J Clin Oncol 1995;13:
2324–9.
97. Wolmark N, Bryant J, Smith R, et al. Adjuvant 5-fluorouracil and
leucovorin with or without interferon alfa-2a in colon carcinoma:
National Surgical Adjuvant Breast and Bowel Project protocol
C-05. J Natl Cancer Inst 1998;90:1810–6.
98. Wolmark N, Rockette H, Mamounas E, et al. Clinical trial to assess
the relative efficacy of fluorouracil and leucovorin, fluorouracil
and levamisole, and fluorouracil, leucovorin, and levamisole in
patients with Dukes’ B and C carcinoma of the colon: results from
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Oncol 1999;17:3553–9.
99. Zhu MZ, Marshall J, Cole D, et al. Specific cytolytic T-cell
responses to human CEA from patients immunized with recombi-
nant avipox-CEA vaccine. Clin Cancer Res 2000;6:24–33.
21.5 Biological therapy of breast cancer
ROBERT O. DILLMAN
Breast Cancer
Several studies indicate that patients with carcinoma
of the breast remain reasonably immune competent
throughout the natural history of their disease [45, 91].
Anti-tumor immunity is demonstrable in patients by a
number of techniques. An in vitro immune reaction to
tumor-associated antigens was demonstrated using the
leukocyte migration inhibition (LMI) and the leukocyte
adherence inhibition (LAI) assays, using as stimulants
tumor cell extracts, soluble membrane extracts of MCF-7
cells (a breast cancer cell line), and soluble membrane
extracts of biopsy-derived tumor tissue [15, 32, 75].
Several of these studies demonstrated blocking factors,
presumably immune complexes, in the serum [83]. The
T-antigen, a precursor of the M and N blood group anti-
gens whose expression is masked on normal tissue, is
expressed on nearly all breast carcinoma tissue. Patients
also mount a strong cellular immune reaction to this
antigen as demonstrated by both delayed hypersensitivity
and in vitro LMI. The demonstration of delayed hyper-
sensitivity to tumor-associated antigens has not been
particularly useful clinically. However 85% of breast
cancer patients had such a reaction to a crude membrane
extract prepared from cultured MCF-7 breast tumor cells
previously infected with vesicular stomatitis virus [3].
Non-specific Immune Stimulation
Because of the prevalence of breast cancer, in the 1970s
many studies were carried out with various nonspecific
immune stimulating agents including BCG, levamisole,
Corynbacterium parvum, poly A:U, and cis-retinoic
acid. As a generalization, these agents failed to demon-
strate significant benefit.
Bacillus Calmette Guerre (BCG)
In a European trial 254 patients with surgically resected
breast cancer (stages T1–3a/N0–1/M0) were random-
ized to either surgery alone or surgery plus adjuvant
chemotherapy with chlorambucil, methotrexate and 5FU
with BCG [78]. After 10 years of follow up, the treat-
ment arm was associated with better relapse and overall
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
survival. However, because of its design, this trial did
not address whether BCG made any contribution to
these results. U.S. trials of adjuvant cyclophosphamide,
doxorubicin, and 5-fluorouracil, with or without BCG,
in patients with stage II or III breast cancer [12], or stage
IV breast cancer [52]. A randomized trial of BCG after
radiation therapy for locally advanced breast cancer also
found no advantage for BCG [79].
A methanol-extracted residue (MER) of BCG was
tested in a trial in which 395 evaluable patients with
metastatic breast cancer were randomized to one of
three different chemotherapy combinations with or
without MER [1]. MER was injected at five sites at a
dose of 100 μg or at the lowest tenfold dilution that pro-
duced a 1-cm indurated lesion. After adjustments for
chemotherapy regimen, collectively response rates were
inferior in the combined chemoimmunotherapy arms
(38% vs. 52%, p = 0.02). MER was associated with sig-
nificant toxicity including painful ulcers and fevers, but
without response or survival benefit.
Corynbacterium Parvum
NSABP conducted a trial in which breast cancer
patients were randomized to receive l-phenylalanine
mustard (L-PAM) and 5FU (PF) with or without C par-
vum [30]. After 8 years of follow-up there was no
improvement in disease free survival (DFS) or overall
survival (OS), and the trend was actually for inferiority
in the immunomodulating arm. There was also more
toxicity, especially fever and chills associated with
C parvum administration.
Pseudomonas Antigens
In a trial for metastatic breast cancer patients, 133 previ-
ously untreated women were treated with combination
chemotherapy and then randomized to no further treat-
ment or nonspecific immunotherapy with a heptavalent
pseudomonas vaccine [38]. Women with estrogen receptor
positive tumors also received tamoxifen after completion
of the chemotherapy. The addition of the pseudomonas
vaccine failed to increase the response rate, duration of
response, or survival.
669
670
Levamisole
The antihelminth levamisole exhibited limited benefit
as a single-agent in patients with metastatic breast cancer.
The agent was generally well-tolerated although granu-
locytopenia and agranulocytosis were observed in some
patients. Most trials focused on utilizing the agent as an
adjunctive therapy. In an early small randomized trial in
patients with inoperable stage III breast cancer who had
been treated with radiation therapy, there was a better
median DFS (25 vs. 9 months) and OS at 30 months
(90% vs. 35%) for 20 patients who received adjuvant
levamisole compared to 23 patient who did not [72].
In an adjuvant study conducted before adjuvant che-
motherapy and/or hormonal therapy had been estab-
lished as standard therapy, 72 post-surgery stage II
breast cancer patients were randomized to receive
levamisole or placebo [43]. After 5 years of follow up,
there was a trend for survival advantage in the levami-
sole arm, primarily because of an apparent benefit in
post-menopausal patients.
During 1976 to 1978 101 patients with advanced breast
cancer were randomized to receive doxorubicin, vincristine
and cyclophosphamide with either placebo or levamisole
2.5 mg/kg given on 2 consecutive days every week except
on the days chemotherapy was given [44]. There was a
higher response rate in patients treated with levamisole
(63% vs. 47%) and after 10 years of follow up, they had
experienced longer survival.
In another trial conducted in metastatic breast cancer, 97
women were randomized to receive cyclophosphamide,
doxorubicin, and 5-fluorouracil chemotherapy with pla-
cebo or levamisole, 2.5 mg/kg, 2 days of each week [16].
The patient population was limited to those with only bone
metastases or local chest wall recurrence. There was no
significant difference in response rate or survival.
Poly A:U
Poly A:U, an interferon inducer and stimulator of natural
killer cytotoxicity, has been evaluated in a randomized
adjuvant study (not incorporating chemotherapy) in
patients with stage II carcinoma of the breast, all of whom
had previously undergone mastectomy [42]. Patients
receiving poly A:U had a better overall survival than
those not receiving this polynucleotide. At the time this
benefit was comparable to other studies using adjuvant
chemotherapy in breast cancer. Benefit was essentially
limited to patients with one to three involved lymph
nodes and could be correlated with natural killer cyto-
toxicity augmentation and 2′,5′-A synthetase production
(an indicator of interferon induction).
Biological therapy of breast cancer
Immunoactivation/Absorption/
Ultrafiltration
Staphylococcal protein A is a polypeptide with a mole-
cular weight of 42,000 that has a high affinity for the Fc
portion of mammalian immunoglobulin (IgG). Preclincal
studies demonstrated therapeutic efficacy in a spontane-
ous canine mammary carcinoma 2and a chemically
induced mammary carcinoma of rats) following the
exposure of plasma to staphylococcal protein A [37, 70, 86].
It was postulated that the removal of immunosuppres-
sive immune complexes was enabling an endogenous
anti-tumor response. In a highly publicized pilot study,
four of five patients with advanced breast cancer exhib-
ited significant anti-tumor effects following repeated
exposure of plasma to protein A imbedded in a colloidal
charcoal mixture [87]. Unfortunately several attempts to
confirm the anti-tumor effects results were unsuccessful
[29, 60]. It was eventually determined that the anti-
tumor effects were the result of elution of the Staph A
protein and possibly other enterotoxins off the column
[59, 85]. In the blood stream this protein acted as a
“super antigen”, and induced a cytokine cascade associ-
ated with fever, chills, hypotension, and other systemic
symptoms in patients.
The Prosorba® immunoadsorbent column consisting of
Staphylococcal aureus protein A bound covalently to an
inert silica matrix to remove immune complexes, received
regulatory approval for the treatment of immune-mediated
thrombocytopenia (IMRE Corporation, Seattle, WA). In
one trial that enrolled 114 patients with various tumor
types, objective partial responses were reported for 5/22
patients with measurable metastatic breast cancer [51].
The final commercial product appeared to overcome the
problems with elution of the protein A off the column, but
this also appeared to remove clinical activity since there
was no evidence of anti-tumor effects in another clinical
trial conducted in 16 patients with metastatic breast
cancer [27].
A related approach, utilized dialysis filters to ultrafil-
trate plasma to remove suppressive factors or induce
immune activation, which appeared to induce tumor
regression [47]. As was the case with the Staph A col-
umns, the patients who had tumor responses were the
same ones who developed symptoms consistent with a
cytokine-release syndrome. An effort to confirm the ben-
efits of this approach by other investigators was aborted
by the proponents of the procedure after no responses
were seen in seven patients (R.O. Dillman, personal
observation 1990). The clinical reactions experienced by
these patients suggested that impurities eluted off the
dialysis filters were responsible for the severe immune
Robert O. Dillman 671

reactions that were producing anti-tumor effects in some
patients.
Cytokines
Interferon
When clinical trials with interferon began, there was
great optimism that these natural proteins would be clin-
ically useful in most malignancies [7]. As summarized
in Table 1, several early trials suggested interferon-
alpha (IFN-α) had significant anti-tumor activity against
breast cancer. Early studies investigating leukocyte-
derived IFN-α reported response rates of 20% to 40%
[8, 35]. However, a subsequent study using human lym-
phoblastoid interferon documented responses in only
1/29 evaluable patients [46]. A trial involving 32 women
with recurrent loco-regional breast cancer randomized
patients to observation or recombinant human IFN-α at
a dose of 3 MU s.c. daily for 1 year after local treatment
[28]. There were no differences in the rates of new or
recurrent breast cancer, but there was a high incidence
of side effects. Homogenates of breast cancer cells
exposed to interferon express increased levels of estro-
gen receptor protein. In a trial for postmenopausal
patients with advanced breast cancer that was not known
to be hormone receptor negative, 99 women were ran-
domized to receive tamoxifen alone, or with IFN-α2a 3
MU i.m. thrice weekly, or daily oral 13-cis-retinoic acid
[19]. Response rates did not differ significantly, and
ranged from 38% to 44%. After 8 years median follow-
up, there was no significant difference in overall sur-
vival. In terms of trends, the interferon arm had the
lowest response rate and lowest median survival, perhaps
because more patients discontinued treatment because
of the various toxicities that accompanied administration
of interferon.
Since there were over 12 IFN-α molecules in the
human-derived leukocyte interferon preparation, and at
least eight in the lymphoblastoid product, but only one in
each recombinant IFN-α product, it is possible that other
molecules present in the natural products are important
for anti-tumor effect. However, an analysis of many
reported studies suggests that that IFN-α has negligible
activity in breast cancer [61].
Interleukin-2
Because of the recognized efficacy of chemotherapy and
hormonal therapy, and the substantial toxicity of IL-2, few
patients with breast cancer were included in early clinical
trials of IL-2 [24, 74]. As shown in Table 1, no anti-tumor
responses were seen with these high dose regimens. Lower
doses of IL-2 have been explored in combination with
other therapy. Following high-dose chemotherapy with
autologous peripheral blood stem cell rescue, 72 women
with high-risk stage II or III breast cancer were random-
ized to receive either a low dose of IL-2 (1 MU/m2) s.c.
daily for 28 days or combined cyclosporine A at 1.25 mg/
kg i.v. daily from day 0 to +28 and interferon gamma
0.025 mg/m2 s.c. every 2 days from day +7 to +28 [88].
Both treatments were well tolerated. At a median follow-
up of 67 months, there were no significant differences in
progression free or overall survival. The combination of
high dose continuous infusion IL-2+ IFN-α yielded an
objective response in 4/23 patients [62].

Table 1. Single-agent activity of various biologicals in patients with metastatic breast cancer
Modality class Biological agent Reference Patients Response rate
Non specific Staph A protein column Terman 1981 5 80%
Non-specific Staph A protein column Messerschmidt 1988 22 23%
Non specific Staph A protein column Fennelly 1995 16 0%
Cytokine Interferon-α Gutterman 1980 16 44%
Cytokine Interferon-α Borden 1982 23 22%
Cytokine Lymphoblastoid Interferon Laszlo 1986 29 3%
Cytokine Interleukin-2 ± LAK Dillman 1993 7 0%
Cytokine Interleukin-2 ± LAK Rosenberg 1989 6 0%
Mab Trastuzumab Burris 2004 52 33%
Mab Trastuzumab Vogel 2002 114 26%
Mab Trastuzumab Baselga 2005 105 19%
Mab Trastuzumab Cobleigh 1999 222 15%
Mab Trastuzumab Baselga 1996 43 12%
Mab Bevacizumab Cobleigh 75 9%
Retinoid Bexarotene Esteva-2003 95 6%

Mab = monoclonal antibody.

LAK = lymphokine activated killer cells.
672
Retinoids
Cis-retinoic Acid
In a trial for postmenopausal women with advanced
breast cancer that was not known to be hormone receptor
negative, 99 patients were randomized to receive tamox-
ifen alone, or with s.c. IFN-α with 13-cis-retinoic acid
1 mg/kg p.o. daily [19]. After 8 years median follow-up,
response rates and overall survival were similar for the
tamoxifen control arm and the retinoid containing arm.
Bexarotene
Bexarotene is a retinoid X receptor-selective retinoid
that exhibited anti-tumor activity against breast cancer in
preclinical testing. A multicenter phase II trial random-
ized 145 women with metastatic breast cancer to oral
bexarotene at a doses of 200 or 500 mg/m2 per day or
placebo [26]. A response rate of 6% was observed in 48
tamoxifen-refractory patients and in 47 chemotherapy-
refractory patients, and 3% in 51 patients with tamox-
ifen-resistant disease. Hypertriglyceridemia is noted in
84% of patients. Other common drug-related adverse
events were dry skin (34%), asthenia (30%), and head-
ache (27%).
Monoclonal Antibodies
Trastuzumab
As summarized in Table 1, an extensive clinical experi-
ence has confirmed the anti-tumor activity of the anti-
Her2 humanized Mab trastuzumab in patients whose
breast cancers over express Her2. Trastuzumab is now
standard therapy for the treatment of patients with high-
risk or advanced breast cancer. Various clinical trials
have confirmed the efficacy of trastuzumab, especially
in combination with chemotherapy, for the treatment of
Her-2 positive breast cancer in patients with recurrent
metastatic breast cancer, as the initial treatment for met-
astatic breast cancer, and as adjuvant therapy. At this
time most physicians continue trastuzumab maintenance
indefinitely or until disease progression because the
Her2 receptor is continually being produced by some
malignant cells.
As shown in Table 1, as a single agent, trastuzumab
alone produced objective response rates of 12–33% in
patients with Her2-positive metastatic breast cancer that
had relapsed after chemotherapy eHer2 [5, 6, 9, 20, 90].
In a phase II trial in 46 patients with metastatic breast
cancer in whom at least 25% of tumor cells expressed
Her2 by immunohistochemistry (IHC), a schedule of
Biological therapy of breast cancer
250 mg i.v. followed by weekly doses of 100 mg for 9
additional weeks yielded five responses among 43 eval-
uable patients [5]. The largest trial of trastuzumab as a
single-agent was conducted in 222 patients with Her2-
positive metastatic breast cancer that had recurred after
chemotherapy [20]. Trastuzumab alone was given at an
initial dose of 4mg/kg followed by weekly doses of
2 mg/kg. There were eight complete and 26 partial
responses for a response rate of 15% with a median
duration of response of 13 months. Burris et al. gave
trastuzumab at twice the standard doses by the weekly
schedule as initial treatment for 8 weeks for patients
with Her2-positive metastatic breast cancer who had
never received chemotherapy [9]. They reported a
response rate of 33% among 52 evaluable patients in a
trial that enrolled 61 patients. This response rate is the
highest reported for single-agent trastuzumab. However,
in a randomized phase II trial conducted by Vogel et al.,
there was no suggestion that this higher dose produced
a higher response rate [90]. In this trial there was no
significant difference in results for an 8 mg/kg loading
dose and 4 mg/kg per week maintenance versus the stan-
dard 4 mg/kg loading dose and 2 mg/kg per week main-
tenance. The failure to demonstrate any difference
between these doses is not surprising in view of the sus-
tained serum levels of trastuzumab that were achieved
at the lower dose. In this trial trastuzumab alone as ini-
tial treatment for metastatic breast cancer resulted in
seven complete and 23 partial responses among 114
patients (26%), but the response rates were 35% for
patients whose tumors were IHC3+ compared to 0% for
tumors that were IHC2+, and 34% among patients
whose tumors were FISH+ compared to 7% for tumors
that were FISH-. Baselga et al. tested an every 3-week
schedule of trastuzumab delivery as initial therapy for
105 patients with Her2-positive metastatic breast cancer
[6]. They observed a response rate was 19%, but it was
34% for patients whose tumors were IHC3+ and/or
FISH+ for Her2.
Numerous phase II trials have been reported in which
trastuzumab was combined with single agent chemo-
therapy for treatment of patients with Her2-positive
breast cancer. These doublet combinations were associ-
ated with response rates between 25% and 75% with
median PFS of 6 to 12 months. Response rates of 50%
or greater were reported in 17 of the 20 trials, and
appeared to be much higher than observed with chemo-
therapy alone. Many of these reports found higher
response rates in the subsets of patients who had 3+ IHC
Her2 expression by IHC and/or overexpression by
FISH, as opposed to 2+ IHC Her2 or negative FISH.
The addition of trastuzumab was not associated with
Robert O. Dillman
any additive toxicities, other than cardiac disease in
association with anthracyclines.
Most of the single agents used in these trials were the
taxanes paclitaxel or docetaxel, or the vinca vinorelbine.
In phase II trials with 23 to 40 patients, various sched-
ules of docetaxel were associated with response rates
ranging from 50% to 70% depending on prior treatment
[2, 26, 57, 76, 84]. In phase II trials with 25 to 88
patients, various schedules of paclitaxel and trastu-
zumab produced response rates ranging from 36% to
62% depending on prior treatment [31, 34, 48, 71, 77].
In phase II trials with 30 to 62 patients, various sched-
ules of vinorelbine and trastuzumab produced response
rates ranging from 50% to 78% depending on prior
treatment [4, 10, 11, 17, 24, 40, 64]. There was a 38%
response rate for gemcitabine and trastuzumab among
61 patients who had previously been treated with a tax-
ane and anthracycline [63]. Liposomal doxubicin and
trastuzumab produced a 52% rate in 29 patients who
had received little or no prior chemotherapy [18]. A
response rate of 24% was observed for cisplatin and
trastuzumab in 37 patients who had received multiple
prior chemotherapy regimens [66].
Many phase II clinical trials have used combination
chemotherapy plus trastuzumab for treatment of patients
with Her2-positive breast cancer. Most of these trials uti-
lized chemotherapy combinations of taxanes and plati-
num agents. Combination chemotherapy has consistently
produced higher response rates and longer progression
free survival in clinical trials in patients with metastatic
breast cancer, although survival advantages have been
harder to establish. Phase II trials of combination chemo-
therapy plus trastuzumab appear to produce slightly
higher response rates (40% to 92%) than were seen with
single agents plus trastuzumab, and the PFS was some-
what longer (range 6–16 months).
Docetaxel, carboplatin and trastuzumab yielded a
response rate of 58% in 59 patients with metastatic
breast cancer, 15% of whom had received prior taxane
therapy in the adjuvant setting [67]. Docetaxel, cisplatin
and trastuzumab produced response rate of 79% in 62
previously untreated patients [67]. Docetaxel, epirubicin
and trastuzumab produced a response rate of 67% as ini-
tial therapy for 45 patients, but the use of an anthracy-
cline was associated with an unacceptable rate of cardiac
toxicity [89].
In a randomized trial, trastuzumab and paclitaxel with
or without carboplatin were compared as first-line therapy
for 196 women with HER-2-overexpressing (2+ or 3+ by
IHC) metastatic breast cancer [71]. The addition of
carboplatin produced a higher response rate 52% versus
36% (p = 0.04), and longer median PFS (11 vs. 7 months,
673
hazard ratio 0.66, p = 0.03). In consecutive phase II trials,
weekly paclitaxel and carboplatin plus trastuzumab
appeared superior to an every 3-week schedule of the
same agents with response rates of 81% versus 65%, median
PFS 14 versus 10 months, and median OS 3.2 versus 2.3
years, and was associated with less hematologic toxicity
[68]. In 40 patients who had received no prior chemo-
therapy the combination of paclitaxel, gemcitabine and
trastuzumab produced a response rate of 52% [31]. The
same combination had a response rate of 92% in 13 previ-
ously untreated patients [54]. Gemcitabine, cisplatin and
trasutuzumab produced a response rate of 40% in 20
patients, but most had previously received both a taxane
and anthracycline [82]. Gemcitabine, vinorelbine and
trastuzumab as second-line therapy produced a 50%
response rate in 30 patients [58].
Randomized trials have established the superiority of
combining trastuzumab with chemotherapy compared
to chemotherapy alone. One of the pivotal trials that led
to regulatory approval of trastuzumab compared trastu-
zumab plus chemotherapy to chemotherapy alone in
469 patients with metastatic disease who had not
received prior chemotherapy for metastatic disease [80].
All patients had tumors that overexpressed Her2, which
was defined as 2+ or 3+ by IHC on a scale of 0–3. An
initial 4 mg/kg trastuzumab dose was followed by 2 mg/kg
weekly. The first dose of Mab was infused i.v. over
90 min, but in the absence of significant infusion related
toxicity, subsequent doses were infused i.v. over 30 min.
Patients who had not received an anthracycline previ-
ously were randomized to receive trastuzumab alone or
with cyclophosphamide 600 mg/m2 i.v. and doxorubicin
60 mg/m2 or epirubicin (AC) i.v. every 3 weeks for six
cycles. Patients who had received adjuvant chemother-
apy that included an anthracycline were randomized to
receive paclitaxel 175 mg/m2 i.v. over 3 h alone every 3
weeks, or with trastuzumab. The combination of che-
motherapy plus trastuzumab was superior for key end
points including: response rate (50% vs. 32%, p <
0.0001), duration of response (9.1 vs. 6.1 months), PFS
(median 7.4 vs. 4.6 months, p < 0.001), and OS (death at
1 year 22% vs. 33%, p = 0.008; median survival 25 vs.
20 months, p = 0.046) with a 20% risk reduction of
death. The differences were most striking for paclitaxel
plus trastuzumab versus paclitaxel alone. The median
OS was 22 months for paclitaxel plus trastuzumab ver-
sus 18 months for paclitaxel alone (p = 0.17) and 27
months for AC plus trastuzumab versus 21 months for
AC alone (p = 0.16).
In another randomized trial, docetaxel plus trastu-
zumab was superior to docetaxel alone in patients with
metastatic breast cancer who had received no prior
674
chemotherapy [50]. Docetaxel was given every 3 weeks
and trastuzumab was given weekly. The addition of tras-
tuzumab roughly doubled the response rate and PFS, and
yielded a better OS 31 versus 23 months (p = 0.032).
Three large randomized trials have confirmed the
expected advantage of combining trastuzumab with che-
motherapy in the adjuvant treatment of patients with
Her2 positive breast cancer [41, 69, 73, 81]. Two U.S.
cooperative group trials were combined for one prelimi-
nary analysis [73]. The National Surgical Adjuvant
Breast and Bowel Project (NSABP) trial B-31 compared
doxorubicin and cyclophosphamide followed by pacli-
taxel every 3 weeks with the same regimen plus 52 weeks
of trastuzumab beginning with the first dose of pacli-
taxel. The North Central Cancer Treatment Group
(NCCTG) trial N9831 compared three regimens: doxo-
rubicin and cyclophosphamide followed by weekly
paclitaxel, the same regimen plus 52 weeks of trastu-
zumab initiated concomitantly with paclitaxel, and the
same chemotherapy regimen followed by 52 weeks of
trastuzumab after paclitaxel. For an early analysis at a
median follow up of 2 years, the two similar arms from
each trial were combined and compared: doxorubicin
plus cyclophosphamide followed by paclitaxel with or
without trastuzumab concurrent with paclitaxel [73]. At
a median follow up of 3 years, DFS was superior in the
there trastuzumab arm (87% vs. 75%, p < 0.0001) with a
52% reduction in the risk of disease recurrence. The
3-year OS also favored the use of trastuzumab (94% vs.
92%, p = 0.015). Because of the use of anthracyclines in
this trial, patients have been closely monitored for car-
diac toxicity, which has been higher in the trastuzumab
arms. In the NSABP B-31 trial 16% of women discontin-
ued trastuzumab therapy due to clinical evidence of
myocardial dysfunction or significant decline in left ven-
tricular function.
An international, multicenter trial (HERA) random-
ized more than 5,000 women with HER2-positive node
positive or high-risk node negative breast cancer, com-
pared 1 or 2 years of trastuzumab given every 3 weeks
with observation after completion of locoregional ther-
apy and at least four cycles of neoadjuvant or adjuvant
chemotherapy (Piccart-Gebhart et al. 2007) [81]. Initial
reports after 2 years of follow up indicated that 1-year of
trastuzumab conveys an advantage over observation
with a 46% reduction in disease recurrence (p < 0.0001)
(Piccart-Gebhart et al. 2007), and a 34% reduction in
death (p < 0.0001) [81].
In a European trial 1,010 women with node-positive
or high risk node-negative breast cancer were random-
ized to adjuvant therapy that consisted of three cycles of
either docetaxel or vinorelbine, followed by three cycles
Biological therapy of breast cancer
of fluorouracil, epirubicin, and cyclophosphamide in both
groups, with a secondary post-chemotherapy random-
ization for the 232 patients whose tumors were Her2-
positive to either observation or trastuzumab for 9 weeks
[41]. The 3-year DFS was better for patients who received
trastuzumab (91% vs. 86%, p = 0.005) with a 42%
reduction in the risk of recurrence.
Based on Her2 biology, it is probable that there is
more of an advantage to give trastuzumab during, rather
than only after chemotherapy, but this has not yet been
established in trials. It is also probable that there is an
advantage for giving trastuzumab indefinitely, even if it
has already been given with chemotherapy, but this also
has not been proven. Finally, completed trials have
attempted to address whether it is better to give adjuvant
trastuzumab for 2 years or 1 year, but it is probable that
it is better to give trastuzumab indefinitely if there is
reason to believe that the breast cancer has not been
completely eliminated.
Trastuzumab is also being combined with chemother-
apy as part of neo-adjuvant therapies with pathologic
complete response rates ranging from 13% to 65% [11,
13, 14, 22, 39, 56]. After 3 weeks of neoadjuvant treat-
ment with trastuzumab alone, 23% of patients had a par-
tial response before going on to receive 12 more weeks
of trastuzumab combined with paclitaxel before surgery
[56]. One randomized trial, which was designed to
accrue 164 patients, was closed early because of the
marked superiority of the trastuzumab-containing arm
[13]. Chemotherapy consisted of four cycles of pacli-
taxel followed by four cycles of Z5FU, epirubicin, and
cyclophosphamide, with weekly trastuzumab for all 24
weeks. After 3 years of follow up, there had been no
recurrences in the patients who received neoadjuvant
therapy, and they have a better PFS (p = 0.041) [14].
About half of Her2-positive patients are hormone
receptor positive which has led to interest in the interac-
tion “cross-talk” between Her2 and hormone receptors
which appears to increase resistance to hormonal thera-
pies. There is a recent report of a 26% response rate for 31
evaluable patients who were treated with the aromatase
inhibitor letrozole in combination with weekly trastu-
zumab [49]. Her2 positivity was confirmed for 82%
(IHC3+ and/or FISH+) and 82% had previously been
treated with tamoxifen. The median PFS was 6 months.
The anti-vascular endothelial growth factor Mab bev-
acizumab has been approved for the treatment of breast
cancer in combination with chemotherapy. As shown in
Table 1, bevacizumab had demonstrable, but limited
activity as a single agent in breast cancer [21]. In that
phase I/II trial, cohorts of patients received 3, 10 or
20 mg/kg of bevacizumab i.v. every other week. However,
Robert O. Dillman
as a third-line treatment for patients with metastatic
breast cancer, the addition of bevacizumab to capecit-
abine produced a higher response rate compared to
capecitabine alone (19% vs. 9%, p = 0.001), although it
did not increase DFS or OS [55]. In subsequent trial the
addition of bevacizumab to paclitaxel as initial therapy
for metastatic disease, produced higher response rates
(37% vs. 21%) and doubled the PFS from 6 to 12 months
compared to paclitaxel alone [53].
Many other antibody products are in development,
but none have been approved by regulatory authorities
for standard treatment of breast cancer. These are
described elsewhere in this text in other chapters that
cover monoclonal antibodies, radioimmunotherapy and
immunotoxins.
Vaccines
Because of the importance of HER-2 as a target, a vaccine
was formulated with peptides 15–18 amino acids in
length derived from the HER-2/neu domains and
admixed with GM-CSF as an adjuvant [25]. Patients
with breast or ovarian cancer underwent i.d. immunization
once a month for a total of two to six immunizations. All
eight patients immunized with HER-2/neu peptides
developed HER-2/neu peptide-specific T-cell responses
and six also developed HER-2/neu protein-specific
responses.
BA7072 is a recombinant fusion protein consisting of
sequences from intracellular and extracellular domains
of HER-2 linked to GM-CSF. Lapuleucel-T (APC8024)
is product produced by Dendreon Inc. that consists of
patient-specific peripheral-blood mononuclear cells
obtained by leukapheresis and then activated in vitro
with BA7072. In an exploratory trial, 18 patients with
HER-2 positive breast cancer underwent leukapheresis
and subsequent lapuleucel-T infusion 2 days later every
2 weeks for at least three doses [65]. Laboratory assays
demonstrated cellular immune responses specific for
BA7072 and HER-2 sequences. One patient had a par-
tial response that persisted for 6 months.
MUC1 is a tumor-associated antigen that is expressed
on many adenocarcinomas including breast cancer.
Sialyl-Tn is a carbohydrate associated with MUC1.
Theratope® is a synthetic Sialyl-Tn antigen produced by
Biomira, Inc. Most of the clinical trials with this product
have been conducted in breast cancer patients [36]. A
related formulation combined clustered sTn conjugated
to keyhole limpit hemocyanin (KLH) (sTn(c)-KLH)
combined with the adjuvant QS-21. sTn(c)-KLH was
tested in 27 patients with high risk breast cancer who
received 1, 3, 10, or 30 μg. Immunizations consisted of
675
sTn(c)-KLH conjugate containing 30, 10, 3, or 1 μg of
Tn(c) plus 100 micrograms of QS-21 [33]. All patients
developed significant IgM and IgG antibody titers
against sTn(c).
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21.6 Biological therapy of lung cancer
ROBERT O. DILLMAN
Non-specific Immunostimulants
Bacillus Calmette Guerin (BCG)
Bacillus Calmette Guerin (BCG) was the first biotherapy
tested in lung cancer in the modern era. Early randomized
trials suggested that various forms or extracts of BCG
could induce immune responses in patients with lung
cancer [88]. However, after the reporting of numerous
randomized trials that explored intrapleural, cutaneous,
and intralesional BCG, this approach was abandoned.
Intrapleural BCG: Following an initial encouraging
report based on 40 patients, after 4 years of follow up
of 169 patients with stage I lung cancer, there appeared
to be a survival advantage for intrapleural BCG follow-
ing surgery [68, 69]. However, other small randomized
trials of intrapleural BCG involving 52, 92 and 118
patients respectively, found no difference in survival
and documented significant toxicity [56, 61, 89]. A
large U.S. randomized trial of 425 patients also failed
to confirm a survival advantage for intrapleural BCG
in patients with stage I lung cancer [72]. A large
European randomized trial of 407 patients also found no
advantage for intrapleural BCG, and noted a shortened
survival in patients who received BCG and underwent
pneumonectomy [58].
Intratumoral BCG: A randomized trial in 88 patients
with lung cancer examined the effects of pre-operative
intratumoral treatment with BCG, and found no survival
advantage [64].
Cutaneous BCG: A report of 48 patients suggested an
increased progression free survival (PFS) for s.c. BCG
in lung cancer patients treated with radiotherapy [80].
After 2 years of follow up, a randomized trial of 500
patients suggested a slight, but statistically insignificant,
survival advantage for patients who received i.d. BCG
post-operatively [23]. In a randomized trial of 103
patients with advanced or metastatic cancer, a methanol
extracted residue (MER) of BCG provided no response
or survival benefit beyond chemotherapy with CCNU,
methotrexate, and doxorubicin [85]. In a three-arm trial
in 92 surgically resected patients, investigators observed
the same 37% 5-year survival rate in all three-arms that
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
included two different types of cutaneous BCG therapy
or no immuotherapy [70].
SRL172, a preparation of heat-killed Mycobacterium
vaccae, was combined with chemotherapy to treat
patients with inoperable non-small cell lung cancer
(NSCLC) or mesothelioma [75]. In a small study, 28
patients were randomized to receive chemotherapy alone
or with monthly i.d. injections of SRL172. The response
rate was higher for the SRL172 arm (54% versus 33%),
as was median survival (9.7 months versus 7.5 months)
and 1-year survival (42% versus 18%). SRL172 pro-
duced mild inflammation at the injection site.
In 298 patients with small cell lung cancer (SCLC) a
randomized trial of induction chemotherapy and radiation
therapy with or without concurrent BCG by scarification
or found no beneficial effect on response rate, duration of
response, or survival [67]. A second study in 102 patients
SCLC found no advantage for the MER form of BCG
[34]. A third randomized trial in limited stage SCLC com-
pared MER to observation after four cycles of chemother-
apy and again there was no difference in outcome in terms
of maximum response or survival [66].
Corynebacterium Parvum
Another non-specific immunostimulant form of biother-
apy that was studied in lung cancer is Corynebacterium
parvum (C parvum). There was no difference in response
rate in a small randomized trial of 49 NSCLC patients
who received C parvum in combination with ifos-
phomide and doxorubicin chemotherapy compared to
those who received chemotherapy alone [33]. A 49
patient randomized trial of i.v. C parvum as an adjuvant
to surgery also found no advantage for this approach
[111]. A randomized trial in 79 patients failed to confirm
a positive effect on response rate or PFS in patients who
received chemotherapy with doxorubicin, cyclophosph-
amide and vincristine, and methotrexate with or without
C-parvum [96]. A large European trial randomized 286
evaluable patients with stage I or II non-small cell lung
cancer to no further treatment or adjuvant therapy with
a combination of intrapleural and intravenous C parvum
[57]. There was no difference in outcome.
679
680
Levamisole
Levamisole alone: The antihelminth levamisole has also
been studied in lung cancer. A randomized trial in which
211 post-operative patients were either observed or
treated with levamisole found no difference in survival
[1]. In a randomized trial of 318 patients, there was a
15% increase risk of death associated with a regimen of
pre and post operative levamisole compared to a placebo
group, primarily because of post surgical respiratory
failure [2].
Levamisole and chemotherapy: A Veterans Adminis-
tration trial randomized 381 patients with metastatic
lung cancer to cyclophosphamide and CCNU alone or
with levamisole [20]. After statistical adjustments,
levamisole had a statistically significant negative effect
on survival.
Levamisole and radiation therapy: In a four-arm
trial in 107 patients with squamous cell NSCLC, there
was no advantage for levamisole or levamisole plus
doxorubicin over radiation therapy alone [110]. The
Southeastern Cancer Study Group randomized 251
patients with inoperable NSCLC to radiation therapy
alone or with levamisole [46]. The trends for response
rate and local control favored radiation alone; survival
was the same in both arms. The Radiation Therapy
Oncology Group (RTOG) conducted a similar trial in
285 patients with inoperable stage I or II or unresectable
T3 N0 or N1 NSCLC [79]. Patients received radiation
therapy and were randomized to either placebo or
twice weekly levamisole for 2 years. Levamisole failed
improve PFS or OS. RTOG also found no difference in
a study of 74 patients with resected stage II–III non-
small cell lung cancer with positive nodes randomized
to post-operative thoracic irradiation plus either placebo
or levamisole [31].
BCG and/or C parvum and/or levamisole: Some ran-
domized trials explored C parvum and/or levamisole
and/or BCG simultaneously. In one trial, 76 patients
with stage III lung cancer were randomized to chemo-
therapy alone (cisplatin, doxorubicin, cyclophosph-
amide and vincristine) chemotherapy plus BCG, or
chemotherapy plus C-parvum. Survival favored the
group that received C-parvum (p = 0.05) [12]. A ran-
domized trial in 100 patients with resectable lung cancer
showed no difference in post-operative survival among
intrapleural BCG, BCG plus C-Parvum, or placebo
[112]. A total of 109 patients with advanced lung cancer
of various cell types, were randomized to MACC che-
motherapy alone (methotrexate, doxorubicin cyclophos-
phamide, and CCNU), or with oral levamisole or with
s.c. C parvum. There were no differences among the
Biological therapy of lung cancer
three treatment groups in terms of response rate or sur-
vival [16]. Another trial randomized 141 patients with
resected stage II and III adenocarcinoma and large-cell
undifferentiated carcinoma to receive postoperative
chemotherapy (cyclophosphamide, cisplatin and doxo-
rubicin) or immunotherapy (intrapleural BCG and oral
levamisole) [32]. Survival was better for the patients
who received chemotherapy.
OK-432
OK-432 (Picibanil) is a lyophilized preparation of peni-
cillin-treated and heat killed Streptococcus pyogenes that
has been studied for many years as an immunotherapy in
Japan. OK-432 has also been explored as treatment for
malignant ascites and malignant pleural effusions, and
also as inhalational therapy for bronchoalveolar cell car-
cinoma [74, 113]. A prospective randomized study in 93
patients with primary lung cancer failed to demonstrate
an immunological or survival benefit from adjuvant intra-
pleural OK-432 immunotherapy after complete resection
of the primary lung tumor [49]. A recent three-arm ran-
domized trial compared intrapleural OK-432, intrapleural
bleomycin, or systemic cisplatin plus etoposide in 102
patients with NSCLC who had malignant pleural effu-
sions [114]. Pleural PFS and OS both favored the OK-432
arm, but the differences were not statistically different.
Intrapleural OK-432 as also been given in combina-
tion with chemotherapy as adjuvant treatment for
patients with resected non-small-cell lung cancer. A
recent meta-analysis was conducted based on data from
1,520 such patients enrolled in 11 randomized clinical
trials, all of which were started before 1991 in which
standard chemotherapy was compared with the chemo-
therapy plus OK-432 [92]. The 5-year survival rate for
all eligible patients in the 11 trials was 51% in the immu-
nochemotherapy group versus 44% in the chemotherapy
group (hazard ratio 0.70, p = 0.001). Because, only four
of these trials actually utilized a centralized randomiza-
tion, an analysis of that subset of trials was analyzed and
also showed longer survival time for the OK-432 plus
chemotherapy group (odds ratio = 0.66, p = 0.049).
Confirmatory trials have not been performed in the
United States or Europe.
Thymic Hormones
A number of different thymic hormones, including crude
preparations such as Thymosin fraction V, and purified
agents such as thymosin alpha-1, have significant effects
on T cells and cell mediated immunity that led to inves-
tigation of their immunopotientiation in lung cancer [55].
Robert O. Dillman 681

In a small randomized trial in SCCL, thymosin fraction

V plus chemotherapy did not increase the response rate
compared to chemotherapy alone, but was associated with
a significant prolongation of survival [19]. Unfortunately,
a larger study of 91 patients found no advantage for the
addition of thymosin fraction 5 over chemotherapy alone
in terms of response rate or survival [97]. A randomized
trial involving 105 NSCLC patients found a higher
response rate for patients who received VAP (vindesine,
doxorubicin, cisplatin) alone compared to patients who
received VAP plus thymosin fraction V, and there was a
statistically insignificant trend for better survival for
chemotherapy alone [8].
As summarized in Table 1, dose-exploring and phase
II trials failed to confirm significant anti-tumor effects
or enhancing activity for thymosin alpha-1 in NSCLC
[21, 22]n a phase II trial in patients with advanced
NSCLC, the combination of cisplatin and etoposide
chemotherapy combined with thymosin-alpha 1 and
low-dose IFN-α2a produced a response rate of 43%
(24/56) and a median survival of 12.6 months [25]. In a
small randomized trial involving 22 patients with
advanced NSCLC, following ifosfamide chemotherapy
half of patients received the combination of thymosin
alpha 1 with interferon alpha (IFN-α) [93]. The chemo-
immunotherapy cohort had a higher response rate (33%
versus 10%) and longer PFS as well as less suppression
of T cell counts and less hematologic toxicity.
Interferon alpha
Non Small Cell Lung Cancer: In a phase II trial in 38
patients with measurable NSCLC, human leukocyte
interferon, which is predominantly interferon-alpha,
produced no objective responses [95] Preclinical and
preliminary clinical data suggested IFN-α potentiates
the cytotoxic activity of several chemotherapy agents
against NSCLC which provided the rationale for several
trials of combination therapy in NSCLC. In one trial
182 patients were randomized to receive either cisplatin-
epidoxorubicin-cyclophosphamide (CEP) alone or with
i.m. IFN-α [3]. The response rate and toxicity were
statistically higher in the combination therapy arm,
but there was no difference in OS.. In a phase II trial
the 3/41 (7%) response rate and median survival of only
6 months for patients treated with carboplatin and IFN-α
was considered disappointing [62]. In a randomized
phase II trial a combination of ifosfamide, platinum
and IFN-α produced a response rate that similar to
the same chemotherapy alone, but was associated
with much greater neutropenia [82]. In a small pilot
study the combination of 5FU, leucovorin, and IFN-α
resulted in responses in 7/18 (39%) of patients with
metastatic NSCLC, but this same response rate has been
reported for 5FU chemotherapy alone in patients
with adenocarcinoma for the lung [82]. A phase II study
in 100 previously untreated patients with unresectable,

Table 1. Single-agent activity of various biologicals in patients with metastatic lung cancer
Modality class Biological agent Lead author Patients Response rate (%)
Non-specific Thymosin alpha 1 [22] 10 0
Non-specific All-trans retinoic acid [104b] 28 7
Cytokine Interferon-α (IFN) [95] 38 0
Cytokine Interferon-α + isotretinoin [5] 17 0
Cytokine Interleukin-2 (IL-2) [4] 11 0
Cyokines IL-2 ± IFN-β [103] 76 3
Cytokines IL-2 + IFN-α [76] 7 0
Cytokines IL-2 + IFN-α [35] 11 0
Cytokine IL-4 [109] 55 2
Cell therapy IL-2 + LAK [9] 15 6
Immunotoxin Ricin A-Anti-CD56 [59] 21 4
Mab Anti-Epcam [24] 6 0
Mab Anti-GRP [40] 13 8
Mab Cetuximab [29] 66 5
Mab Trastuzumab [18] 24 1
Mab Trastuzumab [48] 13 0
Vaccine Belagenpumatucel-L [73] 61 15

Mab = monoclonal antibody

LAK = lymphokine activated killer cells
Epcam = epithelial cell adhesion molecule
GRP = gastrin releasing peptide
682
measurable or evaluable stage III or IV NSCLC examined
the combination of IFN-α2a and cisplatin in NSCLC
[39]. After an initial planned 6 months of therapy,
responding patients could receive maintenance IFN-α
for up to 6 more months. The overall response rate was
33% among 84 evaluable patients with a median survival
of 6.4 months. As would be expected, better results
were achieved in stage III than IV patients. In another
phase II trial 35 patients received MVP chemotherapy
(mitomycin C, vindesine or vinblastine, cisplatin) plus
IFN-α2a-IFN, 3 MIU during the first week of each 28
day cycle of therapy [99]. There was a tumor response
rate of 51% (18/35), median PFS of 6 months, and
median OS of 9.5 months.
Small Cell Lung Cancer: Following induction che-
motherapy with cisplatin, doxorubicin, cyclophosph-
amide (CAP) 237 SCLC patients were randomized to
receive no further treatment or IFN-α as maintenance
therapy [65]. Although there were more long term survi-
vors among the subset of patients with limited stage dis-
ease who received IFN-α, there was no difference in
OS. In a trial involving 215 patients with limited stage
SCLC, 171 responded to induction chemoradiotherapy
and 140 were randomized to receive maintenance IFN-α
or no further treatment [41]. The IFN-α afforded no
improvement in duration of response or survival, and
was tolerated poorly. In a trial that included patients
with both limited and extensive stage SCLC, 90 previ-
ously untreated patients were randomly assigned to
receive Ifosfamide, Carboplatin, and Etoposide (ICE)
alone or in combination with twice weekly low dose s.c.
IFN-α [115]. There was no significant difference in
overall response rates, but toxicity, complete responses
and survival were higher in the IFN-α arm (p < 0.05).
Most of the impact was in patients who had limited
stage disease. In a similar trial with different induction
chemotherapy 85 patients were randomized to chemo-
therapy alone or concurrent with IFN [81]. The IFN-α
arm was associated with higher rates of complete (30%
versus 15%) and partial remission (42% versus 29%),
and longer survival (p < 0.02). In patients with exten-
sive stage SCLC, a single arm phase II trial that included
IFN-α in both the induction and maintenance therapy
did not suggest results that were better than the histori-
cal experience with the etoposide-cisplatin chemother-
apy alone [42]. A three-arm trial in 219 patients with
any stage of SCLC randomized patients to cisplatin plus
etoposide alone, or to low doses of natural or recombi-
nant IFN [90]. Hematologic toxicity was greater in the
interferon arms and there was no difference in survival.
Radiation Therapy and Interferon Alpha: In vitro
studies suggest that IFN-α may be a radiosensitizer for
Biological therapy of lung cancer
both NSCLC and SCLC, but this has been explored in
only a limited manner clinically. In one study 20 patients
with inoperable NSCLC, were randomly assigned to
receive either hyperfractionation radiotherapy alone or
concurrently with IFN-α 3MU i.m. and 1.5 MU inhaled
via a dosimeter-equipped jet nebulizer 30 min before
each radiotherapy session [60]. Combined treatment
with radiotherapy was feasible but impractical. There
was no difference in response rate and some early deaths
occurred in the combination arm that could have been
treatment related. In a phase I study, escalating doses of
IFN-α2a were combined with three different schedules
of cisplatin and twice-daily radiotherapy [108]. IFN-α was
injected s.c. 2 h before the first daily fraction of radia-
tion. The objective response rate was 46% (11/24).
The Radiation Therapy Oncology Group (RTOG)
conducted a trial in 123 patients with stage III NSCLC
in which patients were randomized to 50 Gy RT over 6
weeks alone or with IFN-α 16 MIU by i.v. bolus every
other day thrice weekly on weeks 1, 3, and 5 during
radiation therapy [14]. Because of toxicity, only 76%
completed IFN-α, and only 82% completed radiotherapy
compared to 94% in the control arm. Median survival
and 1-year survival rates were similar, respectively 9.5
to 10.3 months and 42% to 44%.
Retinoids
Prevention Trials
It had been proposed that analogs of vitamin A such as
arytenoids and retinoids might help prevent lung cancer
and heart disease. The effects of a combination of 30 mg
of beta carotene per day and 25,000 IU of retinol (vita-
min A) in the form of retinyl palmitate were explored in
a multicenter, randomized, double-blind, placebo-con-
trolled primary prevention trial involving 18,314 smok-
ers, former smokers, and workers exposed to asbestos
[77]. There were 388 new cases of lung cancer diag-
nosed during a mean follow-up of 4 years. Surprisingly,
the vitamin-treated group had a higher relative risk 1.28
(p = 0.02) of developing lung cancer compared with the
placebo group. There were no differences in the risks of
other types of cancer, but there was also a higher risk of
cardiac related mortality in the retinoid treatment group.
The investigators concluded that the combination of
beta carotene and vitamin A had no benefit and may
have had an adverse effect on the incidence of lung
cancer and on the risk of death from lung cancer, cardio-
vascular disease, and for these reasons the study was
stopped early.
Robert O. Dillman
Beta carotene (50 mg on alternate days) alone was
tested in a similar randomized, double-blind, placebo-
controlled trial involving some 22,071 male physicians,
about half of whom were current or former smokers
[30]. After 12 years of follow up 170 cases of lung can-
cer had been diagnosed. There were no differences in
the overall incidence of lung cancer, overall malignancy
or cardiovascular disease, or in overall mortality.
National Cancer Institute (NCI) Intergroup phase III
trial (NCI #I91-0001) tested the hypothesis that retinoid
chemoprevention would decrease recurrences or second
primary tumors in the setting of stage I NSCLC [52]. The
1,166 patients had pathologic stage I NSCLC and were 6
weeks to 3 years post potentially curative surgery without
other adjuvant therapy. They were randomized to received
placebo or 13-cisretinoic acid (isotretinoin, CRA,
Accutane®) at a dose of 30 mg/day for 3 years. After a
median follow-up of 3.5 years, there were no differences
between the placebo and isotretinoin arms, but there was
more mucocutaneous toxicity and noncompliance in the
isotretinoin arm. Secondary multivariate and subset anal-
yses suggested that isotretinoin was harmful in current
smokers, but beneficial in those who had never smoked.
Squamous metaplasia is frequently observed in bron-
chial biopsy samples from chronic smokers. In a small
randomized trial the synthetic retinoid etretinate was
evaluated to see if could reverse such squamous meta-
plasia [50]. Following bronchoscopy 86 individuals
with significant dysplasia and/or metaplasia were ran-
domized to receive either 1 mg/kg isotretinoin or pla-
cebo daily for 6 months, 69 were reevaluated at the
completion of treatment. A reduction in the metaplasia
was noted in more than 50% of subjects in each group,
but no significant change in metaplasia was found
among those who continued to smoke.
In an exploratory trial, the frequency of abnormalities
in the expression of retinoic acid receptor-beta (RARbeta)
in bronchial cells and the ability of CRA to correct such
abnormalities, were examined in 188 smokers who under-
went bronchoscopy [7]. Bronchial brushing samples were
obtained for cytology analysis and for molecular analy-
sis. Forty-four individuals with diminished RARbeta
expression were randomized to receive a placebo or CRA
30 mg p.o. daily for 6 months. Only 27 patients completed
treatment and 18 underwent repeat bronchoscopy. There
was no difference between the results of RARbeta expres-
sion before and after treatment in the placebo group (p =
0.43), but there was upregulation of RARbeta expression
in the CRA group (p = 0.001). The authors felt that these
results supported undertaking a phase III chemopreven-
tion trial of CRA treatment for lung cancer. In another
trial 307 post operative patients with stage I NSCLC were
683
randomized to no treatment or retinol palmitate (300,000
IU p.o. daily for 12 months) [78]. After 46 months there
were fewer new tumors in the retinol group (p = 0.045)
but few recurrences had taken place.
Cis-retinoic Acid and Interferon
The combination of IFN-α plus CRA, which had pro-
duced high objective response rates in squamous cell
cancers of the skin and cervix, produced objective
responses in only 1/21 patients with squamous cell cancer
of the lung, and that response was in a patient who only
had regionally advanced disease [86]. A three-arm ran-
domized phase II multi-center study of 85 patients with
SCLC revealed no differences in survival among the
three arms, one of which included IFN-α during induc-
tion, and IFN plus CRA as maintenance therapy [91].
Cis-retinoic Acid and Chemotherapy
CRA has also been combined with chemotherapy.
Carboplatin, vindesine and 5-fluorouracil/leucovorin were
combined CRA 1 mg/kg orally in 28 patients with
advanced, measurable NSCLC [84]. Toxicity was man-
ageable. The response rate was 39% (11/28) with a median
survival of 9.7 months for the whole group.
Trans-retinoic Acid and Interferon
The combination of all trans-retinoic acid (ATRA) and
IFN-α was tested in patients with various types of non-
small cell lung cancer that were unresectable, locally
advanced, or metastatic [6]. The authors reported an
objective response rate of 21% (6/29) with several
responses lasting more than a year in duration. ATRA
alone produced two objective response in 28 patients
with NSCLC [104b].
Transretinoic Acid and Chemotherapy
In a phase II study 38 patients with advanced squamous
cell NSCL were treated with a combination of low-dose
ATRA (40 mg/m2/day), IFN-α (6 MU/day) and monthly
cisplatin (40 mg/m2) for 12 weeks [27]. There was a
response rate of 21% but only 16% of patients were able
to complete the planned treatment.
Two large randomized trials were conducted in which
patients with pleural effusions or distant metastatic NSCLC
were randomized to receive standard combination chemo-
therapy with placebo or bexarotene capsules (Targretin®) at
a dose of 400 mg/m2 p.o. daily [106]. In both trials the
2-year survival rate was 16% for chemotherapy alone, and
684
12% to 13% in the chemotherapy plus bexarotene arms; so
the studies were conclusively negative in terms of any ben-
efit for this retinoid [13, 36].
Interferon Gamma
Randomized trials have failed to support a benefit for
IFN-gamma in lung cancer, even in the setting of mini-
mal residual disease.
Non Small Cell Lung Cancer
In a small randomized phase II trial, 37 patients with
inoperable NSCLC were randomized to receive either
two cycles of etoposide and cisplatin chemotherapy or 6
weeks of thrice weekly IFN-β (30 MU) plus IFN-γ
(200 μg) followed by two cycles of chemotherapy [98].
There was more hematologic toxicity during chemo-
therapy on the combined modality arm (p = 0.02), but
no difference in response rates (11–17%) or survival.
Small Cell Lung Cancer
Of 71 patients with extensive stage SCLC, 41 had a com-
plete or partial response after four cycles of PACE (cis-
platin, doxorubicin, cyclophosphamide, and etoposide)
and then were treated with INF-γ 0.2 mg s.c. daily until
grade IV toxicities or disease progression occurred [11].
There was an increased tumor regression in only 2/30
(7%) patients who had a partial response at the end of
chemotherapy, and no suggestion of a survival benefit.
Based on this the authors concluded that INF-γ was not
active against SCLC. In a phase III trial, 100 patients in
complete remission following treatment with six cycles
of combination chemotherapy, thoracic radiotherapy,
and prophylactic cranial irradiation (PCI), were random-
ized to observation or IFN-γ at a dose of 4 MU s.c. per
day for 6 months [37]. Significant toxicity including
chills, myalgia, lethargy, and alteration of mood-person-
ality were observed in the IFN- γ group. There was no
difference in progression free survival or overall sur-
vival, although the trends favored the group who did not
receive IFN-γ. The study had sufficient power to exclude
a 33% improvement in survival (P = 0.04) for the IFN-γ.
In another phase III trial, 127 of 177 patients with SCLC
who were in complete or nearly-complete response fol-
lowing chemotherapy with or without thoracic radiother-
apy, were randomized to observation or to receive either
IFN-γ 4 MU (0.2 mg) sc every other day for 4 months or
observation [107]. There was no difference in progres-
sion free or overall survival.
Biological therapy of lung cancer
Interleukin-2 Alone,
in Combination, or with
Adoptive Cellular Therapy
IL-2 in NSCLC
Eleven subjects with Stage III–IV non small cell lung
cancer were treated with continuous i.v. IL-2 at a dose of
18 MIU/m2/day from day 1 to day 13 with a rest on day
7 [4]. There were no tumor responses, but immune
effects were noted. In an Italian study, 20 patients with
NSCLC received the pineal hormone melatonin orally at
a dose of 10 mg day-1 each evening starting 1 week
before starting s.c. IL-2 at a dose of 3 MIU/m2 every 12 h
for 5 days/week for 4 weeks [53]. The authors reported
partial responses in 4/20 (20%), with little toxicity. In a
subsequent trial, the same group conducted a random-
ized trial in 60 patients with locally advanced n = 8) or
metastatic NSCLC (n = 52) who received either low-
dose s.c. IL-2 plus melatonin or cisplatin plus etoposide
chemotherapy [54]. Similar response rates (all partial)
were observed (7/29 patients treated with biotherapy and
6/31 in patients receiving biotherapy, but the survival
rate at 1 year actually favored the biotherapy group (45%
versus 19%, p = 0.02). This study is of interest because it
suggests that a relative non toxic immunotherapy regimen
may be as efficacious as standard chemotherapy in the
treatment of advanced NSCLC.
In a phase II trial, 76 patients with stage IV NSCLC
were randomized to receive either IL-2 alone 18 MIU/
m2 i.v. 3 days weekly or IL-2 plus i.v. IFN-β (6 MU/m2),
both given thrice weekly [103]. Objective responses
were observed in only 3/76 (4%) patients; grade 4 toxicity
was about 10% in each arm.
Continuous infusion IL-2 (18 MIU/m2/day for 5 days)
combined with IFN-α (5 MU s.c. every other day during
IL-2) produced no responses in seven patients with
NSCLC [76]. In another trial of continuous i.v. IL-2 (18
MIU/m2/day for 3 days) and IFNα (5 MU/m2/day i.m.
for 3 days) produced no responses in 11 patients [35].
IL-2 at a dose of 3 MU/m2 s.c. twice daily, and IFN-α
at a dose of 3 MU once daily, 5 days a week was given
as a consolidation treatment to patients with NSCLC
cancer who had responded to chemotherapy, but no ben-
efit was observed in a study of 52 patients in which
many patients dropped out because of toxicity [105].
IL-2 in SCLC
In a phase II trial, 24 of 50 patients with extensive
SCLC who had not achieved complete remission with
Robert O. Dillman
PACE (cisplatin, doxorubicin, cyclophosphamide, and
etoposide) chemotherapy, were treated with IL-2 by
4-day continuous i.v. infusion of 4.5 MIU/m2 per day
for up to 8 weeks [17]. Four patients improved to a
complete response and one improved from stable dis-
ease to a partial response for an improvement rate of
21% (5/21), but the treatment was quite toxic, and only
five patients completed the planned 8 weeks of treat-
ment. These radiographic improvements in response
may have been because of faster resolution of dead
tumor cells by activated macrophages rather than a
cytotoxic anti-tumor effect.
IL-2 and LAK
A phase II study of interleukin-2 (IL-2) at a fixed dose of
6 MU/m2 per day as a 24 h continuous intravenous infu-
sion (CIV) with lymphokine-activated killer (LAK) cells
yielded one near complete response that lasted 18 months
[9]. Two Japanese trials have suggested that IL-2 plus
LAK may be of value in NSCLC. A randomized trial of
immunotherapy with IL-2 plus LAK cell was conducted
in 105 patients after noncurative resection of primary
lung cancer [43]. All patients received standard chemo-
therapy and/or radiotherapy with or without the addition
of the immunotherapy. The 7-year survival rate was
greater in the immunotherapy group than in the control
group (39% versus 13%, p < 0.01). The same investiga-
tors conducted a randomized prospective study of post
surgical adjuvant immunotherapy using IL-2 and LAK
cells in 82 patients who had undergone a curative resec-
tion of primary lung cancer [44]. The three arms included
observation alone, adjuvant chemotherapy (cisplatin,
vindesine, and mitomycin C) and adjuvant chemotherapy
for two cycles followed by IL-2 plus LAK. Chemotherapy
plus immunotherapy produced a 5-year survival rate
of 50% compared to 33% for observation and 30% for
chemotherapy alone (p = 0.03).
IL-2 and TIL
In a Phase I clinical-trial retroviral-mediated IL-2 genes
were transferred to tumor infiltrating lymphocytes (TIL)
that were infused into ten lung cancer patients with
refractory pleural effusions [101]. Autologous TIL were
exposed to the retroviral plasmid pL(IL-2)SN contain-
ing the human IL-2 gene with about 10 to 16 billion
IL-2-transfected TIL cells infused into the chest cavity
of each patient. Pleural effusions did not re-accumulate
for at least 4 weeks in six of ten patients.
In a randomized study, TIL cultures were successfully
established for 113 of 131 patients, 13 stage II, 42 IIIA,
685
and 32 IIIB NSCLC who were randomized to receive
s.c. IL-2 with TIL with either chemotherapy or radiation
for stage III A disease, or to observation alone in patients
with stage II disease [83]. The radiation therapy con-
sisted of 60 Gy and the chemotherapy of cisplatin and
vinblastine followed by radiation therapy. The radia-
tion/chemotherapy was not given until 2–3 months after
the administration of IL-2 and TIL. Better survival was
seen for the patients who received IL-2 plus TIL in
stage IIIA (median survival 22 months versus 9 months,
p = 0.06) and stage III B (median survival 24 months
versus 7 months, p < 0.01). There was no difference in
survival for patients with stage II disease. There has
been no effort to replicate this study in the United States.
One criticism of this study is that the median survival
rates for this chemotherapy followed by radiation therapy
has been 13–14 months in U.S. trials, and the analysis
was based on treatment rather than intent to treat.
Interleukin-4
In a randomized phase II trial, 63 patients with advanced
NSCLC, 44 of whom had received prior combination
chemotherapy were randomized to receive one of two
different dose of Interleukin-4 (IL-4) (0.25 μg/kg and
1.0 μg/kg) given s.c. thrice weekly [109]. Common side
effects were fatigue and fever. Only one of 55 evaluable
patients had an objective tumor response. A subsequent
randomized three-arm placebo-controlled phase III trial
of chemotherapy alone or with one of two different dose of
IL-4 showed no difference in response rate or survival
(unpublished results).
Amifostine (Ethyol™)
Amifostine is an analog of cysteamine that selectively
protects normal tissues of various organ systems against
the toxic effects of various cytotoxic drugs and radiation.
Because of this it has been suggested that amifostine be
given concurrently with chemotherapy and radiation
therapy in the treatment of lung cancer in an effort to
decrease toxicity [102, 15]. It was originally named
Walter Reed 2721, a reflection of its origin from a clas-
sified military research program that was trying to
develop agents that would protect personnel from the
lethal effects of radiation. Amifostine is actually a prod-
rug that is converted by tissue alkaline phosphatase to
WR-1065, a free thiol with a sulfhydryl group that binds
to oxygen-free radicals generated by radiation and chemo-
therapy. It may also upregulate p53 as an additional
686
mechanism of chemoresistance. It acts a broad-spectrum
cytoprotective agent that seems to protect virtually all
normal tissues except perhaps the brain. The alkaline
phosphatase enzyme that converts WR-2721 to WR-1065
is expressed in much greater amounts in normal blood
vessels and normal tissues than in neoplastic vasculature
or membranes of malignant cells. Numerous clinical
trials have shown that amifostine decreases the toxic
effects of chemotherapy and radiation on normal tissues
without any diminution in clinical efficacy as manifest
by equivalent response rates and survival. Cytoprotective
effects without decreased anti-tumor efficacy were dem-
onstrated for patients with NSCLC who were receiving
carboplatin [10, 51]. A small randomized trial in 62
patients suggested a better survival for patients with
stage II and III NSCLC who received chemoradiotherapy
with amifostine compared to chemoradiotherapy alone
[45]. However, a larger national trial in 242 patients
stage II to IIIA/B non-small-cell lung cancer failed to
confirm any benefit for amifostine to carboplatin/paclitaxel
chemotherapy [71].
Monoclonal Antibodies
The murine anti-Epcam monoclonal antibody (Mab)
KS1/4 and KS1/4-methotrexate immunoconjugate were
administered to patients with Stage IIIB or IV NSCLC.
Six patients received KS1/4 alone and five patients
received KS1/4-methotrexate conjugate [24]. Mild to
moderate side effects including fever, chills, anorexia,
nausea, vomiting, diarrhea, anemia, and brief transami-
nasemia were seen in both groups. There was one possible
clinical response in a patient treated with unconjugated
antibody. Results were not better with a KS1/4 metho-
trexate immunoconjugate, but the latter was associated
with greater GI toxicity because of reactivity with antigen
on normal small bowel.
An immunotoxin consisting of murine Mab N901, that
binds to the CD56 (neural cell adhesion molecule
[NCAM]) antigen found on cells of neuroendocrine ori-
gin, and blocked ricin was tested in a phase I trial in 21
patients with SCLC [59]. Successive cohorts of at least
three patients were treated at doses from 5 to 40 μg/kg/
day for 7 days. The dose-limiting toxicity was a capillary
leak syndrome that occurred in two of three patients at the
highest dose. No patient developed clinically significant
neuropathy. One patient achieved a partial response.
The murine Mab 2A11 binds to gastrin-releasing peptide
(GRP), which binds to receptors and stimulates growth
of SCLC cells [40]. 2A11 at a dose of 250 mg/m2 over 1 h
thrice weekly for 4 weeks was given to 13 previously
Biological therapy of lung cancer
treated SCLC patients. One patient had complete reso-
lution of radiographically detectable tumor that lasted 4
months. Four patients (33%) had stable disease. No
toxic reactions were observed.
The anti-VEGF Mab bevacizumab (Avastin®) is now
part of standard therapy for patients with non-squamous
NSCLC. In a three-arm randomized phase II trial 99
previously untreated NSCLC patients were randomized
to treatment every 3 weeks with paclitaxel 200 mg/m2
and carboplatin (AUC = 6) (PC) alone or in combina-
tion with bevacizumab at 15 or 7.5 mg/kg [38]. The
response rate and PFS were higher for the 15 mg/kg
bevacizumab arm compared to PC alone with a trend
toward better survival. Bleeding was the most signifi-
cant complication associated with bevacizumab and
included minor mucocutaneous hemorrhage, and major
hemoptysis associated with centrally located squamous
cell cancers accompanied by tumor necrosis and cavita-
tion. In a large two-arm randomized trial, 878 patients
with previously untreated non-squamous NSCLC were
randomized to PC + bevacizumab (15 mg/kg) or PC
alone on a 3 week schedule [94]. The bevacizumab arm
was associated with a higher response rate (p < 0.001),
better OS (HR = 0.79, p = 0.003), and PFS (HR 0.66,
p < 0.001). Even though patients with squamous cell
histology were not enrolled in this trial, there were still
five hemorrhagic deaths in the bevacizumab arm (1.2%)
and significant bleeding was more frequent in the beva-
cizumab arm (4.4% versus 0.7%, p < 0.001). In a large
European trial 1,043 patients with previously untreated
non-squamous NSCLC were randomized to gemcit-
abine plus cisplatin with bevacizumab at 10 mg/kg or
5 mg/kg or placebo [63]. Results favored the bevaci-
zumab arms, although the benefit was not as impressive
as in the U.S. trial with PC.
As shown in Table 1, the anti-EGFR Mab cetuximab
(Erbitux®) also enhances the effects of chemotherapy in
NSCLC. In NSCLC Hanna et al. observed a response
rate of only 5% for cetuximab alone in 66 patients with
metastatic disease who had progressed after previous
systemic therapy [29]. Other studies have combined
cetuximab with chemotherapy. Thienelt et al. reported a
26% response rate in 31 previously untreated patients
who received paclitaxel, carboplatin, and cetuximab
[104a]. Robert et al. reported a 29% response rate in 35
patients who were treated with gemcitabine, carboplatin
and cetuximab [87].
Some patients with NSCLC have tumors that express
Her2, the target of trastuzumab (Herceptin®). In one
study only 24/209 (11%) patients had Her2-positive
tumors by immunothistochemistry (IHC), and only 1/24
(4%) of them had a response [18]. In another NSCLC
Robert O. Dillman
trial 13/69 had Her2-positive tumors by IHC, and 0/13
responded to trastuzumab [48]. Trials of chemotherapy
and trastuzumab were also compromised by the low
proportion of patients with HER2-positive tumors. For
example, only 16% to 21% of patients in three NSCLC
trials had HER2-positive tumors, and in two of the trials
less than 5% of patients had tumors that were Her2 3
+ by IHC [26, 47, 116]. Gatzemeier et al. randomized
101 patients with Her2-positive NSCLC to trastuzumab
plus gemcitabine and cisplatin or the chemotherapy
alone, and found no difference in response rate (36%
versus 41%) or PFS (6.1 versus 7.0 months), but only
six patients had tumors that were 3 + Her2 by IHC, and
five of them did have an objective response to treatment
[26]. Zinner et al. observed a 38% response rate in 21
patients treated with gemcitabine and cisplatin [116].
Krug et al. observed a 28% response rate among 64
patients including 7/30 in patients treated with docetaxel
plus trastuzumab and 11/34 in the patients treated
with paclitaxel plus trastuzumab [47]. Response rates
did not appear to be higher than anticipated response
rates for chemotherapy alone, but to determine the true
contribution of trastuzumab would require randomized
trials. Collectively these studies have been disappointing.
This does not appear to be a fruitful area for further
investigation given that so few NSCLC express Her2.
Vaccines
There is great interest in the potential role of vaccines in
the adjuvant setting of lung cancer, but no products are
commercially available. In a randomized trial in which
95 post operative NSCLC received no additional treat-
ment or an i.d. dose of an autologous irradiated suspen-
sion of fresh tumour cells combined with a small dose of
C parvum, there was no difference in outcome [100].
A vaccine consisting of an anti-idiotype antibody
BEC2, a mouse Mab that mimics the structure of the
disialoganglioside GD3, was administered with BCG to
15 SCLC patients following primary therapy with che-
motherapy with or without radiation therapy [28]. All
patients developed anti-BEC2 antibodies and five were
found to have antibodies against GD3. A Phase III trial
is being conducted to evaluate BEC2 plus BCG as adju-
vant therapy after chemotherapy and irradiation.
The most encouraging vaccine in lung cancer at the
moment is belagenpumatucel-L, a transforming growth
factor beta-2 (TGF-β) antisense gene-modified alloge-
neic tumor cell vaccine designed to inhibit secretion of
TGF-β at the site of immunization. In a randomized
phase II trial, 75 NSCLC patients with stage II, III, or
687
low tumor burden stage IV NSCLC were randomized to
receive 12.5, 25, or 50 million cells per injection on a
monthly or every other month schedule to a maximum
of 16 injections [73]. There were no significant adverse
events associated with the treatment. There was a dose-
related survival benefit in patients who received the two
higher doses who had a 2-year survival rate of 52%
compared to 20% for patients in the lowest dose cohort
(p = 0.007). There was response rate of 15% among 61
patients who had stage IIIB or IV disease, although this
interpretation was confounded by previous therapy.
Belagenpumatucel-L is being tested further in a pivotal
randomized phase III trial.
Summary
As of 2008, the anti-VEGF Mab bevacizumab is the
only biotherapeutic that has a ever received a commer-
cial marketing indication in lung cancer. None of the
cytokine or cell therapy approaches are considered
promising at this time, but other Mabs are still being
tested. Some of the results with adoptive cell therapy are
provocative, but have not been replicated. IFN-α adds
little if any benefit after chemotherapy, and adds to toxi-
cicty when administered with chemotherapy. Doses of
IL-2 that are effective in other malignancies are too
toxic to administer to patients with significant underly-
ing lung disease. The results with retinoids have been
disappointing. Randomized trials will determine whether
any of the current vaccine approaches are worthwhile.
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21.7 Biological therapy of B and T cell
lymphoproliferative disorders
ROBERT O. DILLMAN
Biotherapy has consistently exhibited significant activity
in lymphoid malignancies. The first report of a complete
remission with a monoclonal antibody in 1982 was in a
patient with follicular lymphoma. Interferon alpha (IFN-
α) became the first biological approved for the treatment
of malignancy in 1986, because of its activity in hairy
cell leukemia, and for many years was used in the treat-
ment of lymphoma. The first monoclonal antibody
approved for the treatment of a malignancy was the anti-
CD20 chimeric Mab rituximab, which has become a
block-bluster drug since its approval in 1997. The first
immunotoxin to gain regulatory approval, denileukin
diftitox was approved based on activity in T cell lym-
phoma. The anti-CD52 humanized Mab alemtuzumab
was approved based on its activity in CLL. The first two
radiolabeled antibodies approved for cancer treatment
were the anti-CD20 products Y-90 ibritumomab tiuxetan
and I-131 tositumomab which are very active in the
treatment of B cell lymphoma. Finally, it is widely antici-
pated that perhaps one or more of the patient-specific
vaccines consisting of idiotype protein may become the first
tumor specific vaccines to receive regulatory approval as
treatment for follicular lymphoma.
Hairy Cell Leukemia
This section starts with this relatively uncommon B cell
lymphoproliferative disorder because of its significance
in the validation of biotherapy. In 1986, IFN-α became
the first biological to achieve regulatory approval based
on its activity in hairy cell leukemia (HCL) [66, 75, 86,
164]. In one report 75% of 64 patients with HCL, 61 of
whom had undergone prior splenectomy, who were
treated with 2 MU/m2 IFN-α2b s.c. thrice weekly dem-
onstrated an objective response [75]. In another report
78% of 212 patients with HCL, 166 of whom had under-
gone prior splenectomy, who were treated with 2 MU/
m2 s.c. IFN-α2b s.c. thrice weekly had an objective
response [164]. These responses proved to be durable
with 28% in continuous remission beyond 6 years and
83% alive beyond 6 years [141]. A small randomized
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
trial confirmed that IFN-α was superior to splenectomy
as initial treatment of the disease [155]. However, in
1991 the purine analog deoxycoformycin (pentostatin)
gained regulatory approval for the treatment of HCL
and displaced IFN-α as the treatment of choice because
was it active in interferon-refractory disease [67, 81]
less toxic, and superior to IFN-α when compared
directly in randomized trials in previously untreated
patients [77]. In 1993 another purine analog 2-chlorode-
oxyadenosine (cladribine), which like pentostatin was
also associated with durable responses in over 90% of
previously untreated patients, was also approved for the
treatment of HCL [17, 136].
The circulating lymphocytes of HCL typically express
very high levels of CD20. The anti-CD20 chimeric Mab
rituximab has been used to treat relapsed HCL using both
the 4 and 8 week treatment schedules of 375 mg/m2 per
week. The 4-week schedule produced responses in 5/10
patients in one study [103], but only in 6/24 patients who had
all relapsed after cladbribine [126]. The 8-week schedule
produced responses in 8/15 relapsed patients [163].
B Cell Lymphoma
Interferon
In early trials single agent activity for IFN-α was dem-
onstrated in the lymphomas with response rates near
50% in indolent lymphomas, and as high as 20% in
more aggressive lymphomas [185, 190, 192]. Numerous
trials focused on the role of IFN-α in combination with
chemotherapy or as maintenance therapy following
induction chemotherapy as summarized in Table 1. In a
U.S. multicenter trial 249 patients with B cell lymphoma
were randomized to combination chemotherapy with
cyclophosphamide, doxorubicin, vincristine and predni-
sone (COPA) with or without IFN-α [156, 157]. There
was better progression-free survival (PFS) and overall
survival (OS) in the IFN-α arm, but there was also more
toxicity. In a French trial 268 patients with B cell
lymphoma were randomized to combination chemo-
therapy with cyclophosphamide, doxorubicin, vindesine,
693
694 Biological therapy of B and T cell lymphoproliferative disorders

Table 1. Randomized trials of interferon-alpha in the treatment of B-cell lymphoma.

Author Year Ref. # of Pts Induction Maintenance Results
Smalley et al. 1992, 2001 NEJM [156,157] Leukemia 249 COPA ± IFN None ↑PFS ↑OS
Peterson et al. 1993 1997 ASCO [133,191] 581 CTX ± IFN ± IFN for CR NSD
Solal-Celigny et al. 1993 NEJM [158] 268 CHVP ± IFN CHVP ± IFN ↑PFS ↑OS
Aviles et al. 1994 Leuk Lymphoma [2] 384 CEOP-Bleo ± IFN for CR ↑EFS ↑OS
Hagenbeek et al. 1998 J Clin Oncol [69] 347 CVP + RT to bulky ± IFN for OR/SD ↑PFS
Unterhalt et al. 1996 Leukemia [168] 498 PmM or CVP ± IFN for CR ↑DFS
Fisher et al. 2000 J Clin Oncol [49] 571 CHOP & ProMACE ± IFN 1 yr for OR NSD
Rohatiner et al. 2001 Br J Cancer [151] 204 CLB ± IFN ± IFN for OR NSD
Neri et al. 2001 J Hematother [125] Stem 151 MXT/Leu CEOP CVP ± IFN 1 yr ↑DFS ↑OS
Cell Res

CTX = cyclophosphamide
CLB = chlorambucil
COPA = cyclophosphamide, vincristine, prednisone, doxorubicin
CHOP = cyclophosphamide, vincristine, doxorubicin,prednisone
CHVP = cyclophosphamide, doxorubicin, vindesine, prednisone
CEOP-Bleo = cyclophosphamide, epirubicin, vincristine, prednisone, bleomycin
CVP = cyclophosphamide, vincristine, prednisone
ProMACE = procarbazine, methotrexate, doxorubicin, cyclophosphamide, etoposide
PmM = prednimustine and mitoxantrone
MXT/Leu = mitoxantrone, chlorambucil
CR = complete response
OR = objective response (partial or complete response)
PFS = progression free survival
EFS = event free survival (neither death nor disease progression)
DFS = disease free survival
OS = overall survival

prednisone (CHVP) with or without IFN, and the same
treatment was continued as maintenance treatment in
responders [158]. This trial also demonstrated improve-
ment in PFS and OS. It should be noted that these two
randomized trials were not limited to patients with indolent
lymphoma. The US trial included patients with high-risk
follicular lymphoma as well as patients with intermediate
grade non-follicular lymphoma. The French trial was
conducted in patients with high-risk follicular lym-
phoma defined by tumor mass >7 cm, B-symptoms, or
multiple extranodal sites of disease.
CALBG randomized 581 indolent lymphoma patients
to cyclophosphamide with or without IFN-α, with a
second randomization to observation or IFN-α for
patients who had complete remissions [133, 191]. There
was no difference based on the initial therapy, but there
was some trend toward better PFS for the patients who
achieved a complete response (CR) and subsequently
received maintenance IFN-α. A German trial randomized
indolent lymphoma patients who had achieved a CR
with chemotherapy alone to IFN-α or observation [168].
Maintenance IFN-α was associated with longer disease
free survival. A European cooperative group trial ran-
domized indolent lymphoma patients whose disease had
no progressed during chemotherapy, to observation or
IFN-α [69]. IFN-α maintenance therapy was associated
with longer PFS. In another 151 indolent lymphoma
patients were randomized to receive IFN-α or observation
after chemotherapy [125]. Again there was improvement
in PFS for patients who experienced a CR, and improved
OS for all patients. In a Spanish trial 384 patients with
follicular lymphoma who were in complete remission
after six cycles of cyclophosphamide, epirubicin, vincris-
tine, prednisone and bleomycin (CEOP-Bleo) chemother-
apy, were randomized to observation or IFN-α maintenance
[4]. At a median follow up of almost 10 years, the IFN-α
arm had a better event free survival (64% versus 35%,
p < .01) and OS (81% versus 57%, p < .001). In contrast
to these results, in a trial in which 204 patients were
randomized to chlorambucil with or without IFN-α, and
the responders were randomized to observation or IFN-
α, there was no difference in outcome [150]. In a U.S.
cooperative group trial there was no survival difference
for 571 patients with low-grade stage III or IV lympho-
mas who were randomized to CHOP and PROMACE
induction therapy with or without maintenance IFN-α,
and subsequently 268 responders were randomized to
observation or 1 year of IFN-α [49].
Robert O. Dillman
A meta-analysis focused on ten randomized trials in
which IFN-α was tested as part of initial therapy in
2005 patients with lymphoma [151]. In seven of these
trials IFN-α was used as part of initial therapy, and in
seven IFN-α was used as maintenance therapy. In the
trials in which IFN-α was combined with chemotherapy,
there was no increase in response rates compared to che-
motherapy alone. However, the use of IFN-α either with or
after chemotherapy was associated with better survival.
The best results were obtained in four trials in which the
intensity of IFN-α therapy was greater than 36 MU per
month (p = .0004). Better survival was also noted in the
five trials in which doxorubicin or mtioxantrone were
used in the combination chemotherapy (p = .0008).
Based on these trials, prior to the introduction of
rituximab, it appeared that IFN-α added nothing to che-
motherapy in the initial treatment of patients with low-
risk indolent lymphoma, but the use of maintenance
IFN-α in responding patients was associated with pro-
longed PFS, but not OS. The converse appeared to be
true in high-risk, low-grade lymphoma. In that disease
setting it appeared that concurrent administration of
IFN-α with “CHOP-like” regimens was associated with
a progression free and perhaps overall survival advantage,
but maintenance IFN-α did not appear to add benefit in
that setting.
Two additional trials have been completed in lym-
phoma patients who had responded to induction therapy,
and then were randomized to consolidation with either
intensive chemotherapy and stem cell rescue, or IFN-α.
In patients with mantle cell lymphoma who were in CR
after chemotherapy the median PFS was much better in
the transplant arm than the IFN-α arm (39 versus 17
months, p = .011) [40]. Similarly, in 244 patients with
follicular lymphoma who were in remission following
chemotherapy, the 5-year PFS was twice as high in the
Table 2. Interleukin-2-based treatment of B-cell lymphoma
Lead author Year Ref. Regimen
Allison 1989 J Clin Oncol [1] LD bolus
Margolin 1991 J Immunother [113] HD bolus & hybrid bolus/CIV + LAK
Bernstein 1991 J Immunother [6] ID CIV + LAK
Weber 1992 J Clin Oncol [172] HD bolus + LAK
Weber 1992 J Clin Oncol [172] HD bolus
Duggan 1992 J Immunother [41] LD bolus
Gisselbrecht 1994 Blood [65] HD CIV for low-grade
Gisselbrecht 1994 Blood [65] HD CIV for intermediate-grade
HD = high dose
ID = intermediate dose
CIV = continuous intravenous
LAK = lymphokine activated killer cells
IFN = interferon
695
transplant arm than the IFN-α maintenance arm (65%
versus 33%, p < .0001) [105]. More recently the inferi-
ority of IFN-α compared to other therapies was con-
firmed in another trial that compared rituximab plus
CHOP to CHOP as induction therapy in patients with
follicular lymphoma, and then randomized responders
to high-dose chemotherapy or maintenance IFN-α [72].
Survival was similar for RCHOP-transplant, RCHOP-
IFN-α, and CHOP-transplant, all of which were supe-
rior to the CHOP-IFN-α arm. This trial seems to have
solidified chemotherapy plus rituximab as the current
standard initial therapy for patients with follicular lym-
phoma and suggests there is no general benefit for the
addition of IFN-α or high dose chemotherapy for
patients who respond to RCHOP. Thus, controversies
regarding the role of IFN-α in B-cell lymphoma have
been muted by the introduction of rituximab, which as a
single agent had much higher response rates and a much
milder toxicity profile than interferon.
Interleukin-2
The published experience in lymphoma with interleukin-2
(IL-2) alone, or with adoptive cellular therapy, is sum-
marized in Table 2. In three small pilot studies, IL-2 +
lymphokine activated killer (LAK) cells were associ-
ated with response rates of 0%, 8%, and 50% [6, 113,
172]. Five studies of IL-2 alone using high-dose bolus,
low-dose bolus, or intermediate-dose continuous infu-
sion intravenous [CIV] IL-2 were associated with
response rates of 0%, 4%, 20% and 22% [1, 41, 65,
172]. Most of these studies had a mix of all types of
lymphoma patients. Interestingly, in one French trial the
response rate for intermediate-dose CIV IL-2 was 22%
for intermediate lymphomas compared to only 4% in
indolent lymphoma [65]. In a cooperative group clinical
# Pts Response rate
9 22%
15 0%
12 8%
8 50%
11 0%
20 20%
24 4%
23 22%
696 Biological therapy of B and T cell lymphoproliferative disorders

trial patients were randomized to a combination of low-
dose bolus IL-2 with or without IFN-β [41]. Toxicity
was substantial, and there appeared to be no advantage
of the addition of IFN-β since the response rates were
4/20 for IL-2 alone and 3/21 for IL-2 plus IFN-β.
Because of the success of rituximab in lymphoma, and
the toxicity of higher dose IL-2 regimens, outpatient s.c.
IL-2 was combined with rituximab for the treatment of
relapsed follicular lymphoma with reported response
rates of 10% in a trial of 54 patients [95], 26% in a trial
of 34 patients [186], and 55% in a trial of 20 patients
[61]. An industry sponsored randomized phase 2 trial of
8 weeks of s.c. IL-2 with 4 weeks of standard rituximab
for patients who had relapsed after prior chemotherapy
was terminated because of poor accrual.
Because of the toxicity associated with high dose sched-
ules of IL-2, the limited single agent activity, and the
emergence of other agents for the treatment of lymphoma,
there has been little interest in pursuing trials with IL-2 in
recent years in the lymphomas other than as low doses for
immune reconstitution purposes following myelosuppres-
sive therapy, such as in the transplant setting.
Monoclonal Antibodies
In November 1997 Rituximab [Rituxan®] became the
first monoclonal antibody (Mab) approved with a mar-
keting indication for a malignant disease. Rituximab has
revolutionized the treatment of B cell lymphoproliferative
disorders and is now standard therapy. The clinical trial
experience with rituximab is summarized in Table 3. In
nine trials of relapsed, previously treated follicular lym-
phomas rituximab produced response rates ranging from
46% to 76% [3, 8, 15, 28, 54, 64, 116, 137, 178].
Responses were evident within 2 months in most patients,
and persisted for over a year from the date of first treatment.
Response rates of over 40% were also noted in patients
with bulky B-cell lymphoma [31] and patients who
relapsed after a previous response to rituximab [29, 85].
In four trials in previously untreated follicular lymphomas
rituximab produced response rates ranging from 67% to
78% [21, 64, 78, 179]. In small lymphocytic lymphoma
three trials in previously treated patients yielded response
rates of only 14% [54, 116, 137], but the response rate
was 65% in such patients who had previously received
chemotherapy and who received maintenance rituximab
[78]. In marginal zone lymphoma response rates were
85% in previously untreated patients with extranodal
MALT disease compared only 45% in such patients who
had relapsed after prior chemotherapy [22], and 77% in
patients who had relapsed with gastric MALT [114]. In
lymphoplasmacytic lymphoma, four trials in previously
treated patients reported response rates ranging from
16% to 50% [37, 63, 166] while three trials with untreated
patients reported response rates ranging from 35% to
65% with the best results obtained with extended rather
than only 4 weeks of therapy [37, 63, 167]. In the limited
experience reported for single agent rituximab in mantle
cell lymphoma responses were about 30% regardless of
whether patients were previously untreated or had
relapsed after prior therapy [19, 54]. There are only two
published reports regarding single-agent rituximab in
large B cell lymphoma, both of which were conducted in
patients who had relapsed after prior therapy, and both
recorded a response rate of 37% [19, 165]. In practice
rituximab is almost always used in combination with
chemotherapy in aggressive lymphomas, but is often
used alone as initial therapy of patients with follicular
lymphoma or marginal zone lymphomas.
Rituximab has been safely combined or sequenced
with various types of chemotherapy with numerous
reports of high response rates and durable remissions.
These are summarized in Chapter 10 of this text. In large
randomized trials rituximab plus chemotherapy has con-
sistently produced superior outcomes compared to the
same chemotherapy alone. In patients with previously

Table 3. Response rates for rituximab as a single agent

Lymphoma histology Setting # trials # patients Response range Median response rate
Follicular lymphoma Untreated 4 180 67–78% 73%
Follicular lymphoma Relapsed/refractory 9 547 46–76% 58%
Small lymphocytic Untreated 1 23 65% 65%
Small lymphocytic Relapsed/refractory 3 68 13–14% 14%
Marginal zone Untreated 1 24 87% 87%
Marginal zone Relapsed/refractory 2 37 45–77% 61%
Lymphoplasmacytic Untreated 4 78 35–65% 50%
Lymphoplasmacytic Relapsed/refractory 3 92 16–50% 33%
Mantle Cell Untreated 1 34 29% 29%
Mantle Cell Relapsed/refractory 2 54 30–33% 32%
Large B cell Relapsed/refractory 2 87 37% 37%
Robert O. Dillman
untreated follicular lymphomas response rates were higher
for RCHOP versus CHOP [72], RCVP versus CVP
[112], and RMCP versus MCP [71]. In previously
untreated indolent lymphoma response rates were higher
for RFND versus FND [117]. In previously untreated
lymphoplasmacytoid lymphomas response rates were
higher for RCHOP versus CHOP [11]. In relapsed folli-
cular lymphoma response rates were higher for RCHOP
versus CHOP [169] and for RFCM versus FCM [56]. In
relapsed mantle cell there was a higher response rate for
RFCM versus FCM [55]. In the trials in which it is an
endpoint that has been assessed, PFS has also been longer
in those patients who received rituximab with their che-
motherapy [71, 112, 169]. Survival advantages have not
been established in any of these trials, and probably will
not be because of the clinical benefit associated with
second and third-line therapies in these diseases.
In patients with large B cell lymphoma the addition of
rituximab has not only typically increased response rates
and complete response rates, but more importantly, it has
also produced longer progression free and overall sur-
vival and is probably curative therapy for more than half
of all patients. All four large randomized trials in which
RCHOP was compared to CHOP as initial therapy for
patients with large B cell lymphoma, RCHOP has been
associated with a superior PFS and OS with a substantial
reduction in the risk of death [20, 47, 68, 134, 135]. In
the one relatively small randomized trial in mantle cell
that has been published, RCHOP was superior to CHOP
in terms of response rate (94% versus 75%) complete
response rate (34% versus 7%), with a 50% longer median
PFS (21 versus 14 months), but with no difference in
survival [106].
As discussed in Chapter 10, the role of maintenance
rituximab remains controversial. In indolent lympho-
mas four randomized trials utilizing a variety different
maintenance schedules, have shown that administration
of additional doses or courses of rituximab clearly pro-
longs progression free survival after initial treatment
with rituximab alone in untreated patients [64], after
rituximab alone in patients who have relapsed after prior
chemotherapy [64, 70], and after chemotherapy and
rituximab in patients who had relapsed after prior
chemotherapy [56, 169]. However, none of these trials
have established a survival benefit. The only published
randomized trial that has tried to address the issue of
duration of benefit from rituximab (or time to becoming
rituximab refractory, is of uncertain relevance because it
was conducted in patients who had relapsed after che-
motherapy, and they were then treated with rituximab
alone. However, the additional rituximab therapy was
associated with a higher response rate 52% versus 35%,
697
complete response rate (27% versus 4%), was a striking
difference in the time to disease progression, medians of
31 months versus 8 months (p = .007), but no difference
in time to become “rituximab refractory” which was
about 30 months in both arms, nor in overall survival at
a median follow up of 42 months [70]. In large B cell
lymphoma the one randomized trial that addressed this
issue found there was no advantage in terms of PFS or
OS derived by giving four weekly doses of rituximab
every 6 months for 2 years after responding to RCHOP
induction chemotherapy [68]. In patients with relapsed
mantle cell lymphoma who responded to RFCM as sec-
ond line therapy, compared to observation there was
also prolongation of PFS for patients who received two
additional 4 week courses of rituximab 3 and 9 months
after completing RFCM [56]. There is no specific data
regarding this issue in small lymphocytic lymphoma,
however, one would expect that any sort of additional
rituximab would prolong PFS based on the higher
response rates achieved with prolonged rituximab in
that disorder, and the results in follicular lymphoma.
Two radiolabeled murine Mabs have been approved
for the treatment of B cell lymphoma. These are both
anti-CD20 antibodies: Y-90 ibritumomab tiuxetan
(Zevalin®) which was approved in February 2002, and
I-131 tositumomab (Bexxar®) which was approved in
June 2003 [32]. In both products the original mouse
antibodies ibritumomab tositumomab were kept as
murine proteins in order to decrease half-life in the cir-
culation to reduce non-specific total body irradiation.
Ibritumomab is the same antibody that was modified to
create the chimeric antibody rituximab; therefore, it has
exactly the same binding to CD20 as rituximab. The
clinical trial results for these two products are summa-
rized in Table 4. As expected, in phase I trials the dose
limiting toxicities of these agents were cytopenias sec-
ondary to bone marrow suppression with nadirs 5 to 8
weeks after treatment, but both produced objective
response rates of 65–70% with durabilities of 9 to 12
months [87, 176]. Both of these products have produced
response rates of about 40% with durabilities of about
1-year in patients with transformed B-cell lymphomas
that had been heavily treated with prior chemotherapy.
Both products have produced response rates of about
80% with a median duration of response of 11 to 12
months in patients with follicular lymphoma who had
failed prior chemotherapy, but were not refractory to
Rituximab [27, 89, 178]. Both products produced higher
response rates that an unconjugated Mab in randomized
trials, as compared to tositumomab for the I-131 product,
and to rituximab for the Y-90 product [30, 178]. Both
products produced response rates in over 70% of patients
698 Biological therapy of B and T cell lymphoproliferative disorders

Table 4. Radioimmunotherapy of B cell lymphoma: radiolabeled anti-CD20 antibodies

131-I tositumomab (Bexxar) 90-Y-ibritumomab tiuxetan (Zevalin)
Dose Limiting Toxicity Bone marrow suppression Bone marrow suppression
Therapeutic Dose 75 cGy total body dose 0.4 mCi/kg (max 32 mCi)
Phase I response rate 71% 67%
Response rate in relapsed follicular 81% 86%
lymphoma
Response rate in rituximab refractory 70% 74%
lymphoma
Response rate in transformed lymphoma 39% 56%
RIT versus unlabeled Moab 67% versus 28% (tositumomab) 80% versus 56% (rituximab)

with indolent B-cell lymphomas that had become refrac-
tory to Rituximab, defined as either no response or
progression with 6 months after a response [82, 177].
Long term follow up has confirmed the remissions in
these various settings are usually quite durable [50, 76,
175, 180]. In a young cohort (median age 49 years) of
76 patients with previously untreated follicular lym-
phoma, treatment with I-131 tositumomab produced a
response rate of 95% with a 75% complete response
rate, and 60% molecular complete response rate [88]. At
a median follow-up of greater than 5 years, the 5-year
progression-free survival rate was 59%, and the median
progression-free survival was projected to be 6.1 years.
Seventy percent of the 57 complete responders were
still in remission 4 to 8 years following therapy.
Despite these tremendous results, these two radiola-
beled products have struggled in the marketplace
because of the reluctance of medical oncologists to refer
their patients for this treatment. One reason for this is
the limited numbers of sites able and willing to provide
therapy, so that often patients have to travel long dis-
tances seeking such treatment. The treatment itself
requires some complex coordinated planning. Many
hospitals have kept these products off their formularies
because of the high single cost of the radioisotope and
the financial risks associated with fixed payments. Since
these treatments have to be delivered by physicians with
radiation credentials, medical oncologists do not share
in the profit margin associated with such treatment in
the way that they do with the administration of chemo-
therapy and other intravenous products. Because there
are so many systemic agents available for lymphoma,
physicians can easily rationalize that they should try all
other therapies before considering referral for radioim-
munotherapy. Despite the high response rates, to date
there are no trials directly comparing radioimmunother-
apy plus rituximab to chemotherapy plus rituximab,
although it is likely efficacy would be similar and qual-
ity of life superior in the radioimmunotherapy arm. At
this time it appears that the most acceptable application
of these products will be as consolidation treatment after
induction therapy [107, 138, 183], or as part of marrow
ablative therapy and hematopoietic stem cell rescue
[121, 124, 170, 171].
Immunotoxins
Denileukin diftitox, an immunotoxin consisting of IL-2
fused to diphtheria toxin, is approved for treatment of
CD-25 expressing cutaneous T-cell lymphomas (CTCL).
The IL-2 receptor is also expressed on many B cell
malignancies; so denileukin diftitox has been tested in
patients with relapsed indolent lymphoma. In 45 patients
with progressive B cell lymphoma, all of whom had
been treated with rituximab in the past, there was a 24%
response rate with a median duration of 7 months [24].
A second phase II trial was aborted because of slow
accrual after 35 of a planned 77 patients had been
enrolled [99]. Objective responses were observed in
3/29 evaluable patients, 1/21 with follicular lymphoma
and 2/8 with small lymphocytic lymphoma. In both of
these trials response rates were similar regardless of
whether the lymphoma tested positive or negative for
the IL-2 receptor.
The combination of denileukin diftitox and rituximab
produced a response rate of 32% in 38 evaluable
patients, 30 (80%) were rituximab-refractory [25]. The
overall response rate (ORR) was 32% and median dura-
tion of response was 8 months. Of note 6/11 patients
with rituximab-refractory follicular lymphoma had an
objective response.
Vaccines
As of early 2008 there were no vaccines approved for
use in any B cell malignancy, although three products,
MyVaxID, FavID, and BiovaxID were being evaluated
in pivotal regulatory trials [51, 145, 184]. Initial investi-
gation established that vaccination with an individual
patient’s idiotype protein could induce an endogenous
Robert O. Dillman
idiotype response [100]. In a trial of 41 patients with
indolent B cell lymphoma, vaccination with an idiotype-
keyhole limpet hemocyanin (KLH) product produced
anti-idiotype responses in 49% of patients [83]. For the
subset of 32 patients who were in their first remission,
PFS was much longer in those patients who exhibited an
immune response compared to those who did not (7.9
versus 1.3 years, p = .0001). The vast majority of
patients in these trials had follicular lymphoma, in part
because of the inherent difficulty in isolating the idiotype
from other B cell malignancies [149]. In a trial of 33
consecutive patients with relapsed follicular lymphoma,
who had relapsed after an initial complete response and
were induced into a second remission with CHOP, an
idiotype vaccine was able to be prepared for 83% [74].
Overall 80% of the 25 treated patients exhibited some
sort of a cellular idiotype-specific response. All 20
responders had a longer disease free interval after their
second remission than their first. A cellular idiotype-
specific response was detected in 18/25 (72%) while a
humoral idiotype-specific response was demonstrated in
13 patients (52%). In another trial of an idiotype-KLH
product co-administered with GM-CSF, objective tumor
responses were noted in 4/32 previously treated indo-
lent lymphoma patients [144]. Encouraging results such
as these have attracted commercial interest, and at pres-
ent there are at least anti-idiotype vaccine products that
have entered into phase III trials, all of which are being
conducted in patients with follicular lymphoma. All
three utilize the idiotype paraprotein of the malignant B
cell clone as the immunogen to produce an endogenous
anti-idiotype response as summarized in Table 5. It has
been announced that the Genitope trial did not meet the
necessary regulatory endpoints, but those patients who
were able to mount an anti-idiotype response did have a
Table 5. Anti-idiotype vaccines in follicular lymphoma
MyVaxID (Genitope)
Vaccine type Id-KLH/GM-CSF
Production Method Recombinant DNA
Disease Setting Untreated
Induction therapy CVP
Eligibility CR or PR
Accrual Finished
Id = idiotype
KLH = keyhole limpet hemocyanin
GM-CSF = granulocyte-macrophage colony stimulating factor
CVP = cyclophosphamide, vincristine, prednisone
PACE = platinum, doxorubicin, cyclophosphamide, and etoposide
RCHOP = rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone
CR = complete response
PR = partial response
699
longer PFS. It is possible the dendritic cells pulsed with
idiotype protein may be a more powerful vaccination
approach [147, 193].
T Cell Lymphoproliferative
Malignancies
Even though T cell malignancies account for less than
10% of the malignant lymphoproliferative disorders,
they have been the object of many trials of biological
therapy. Most of the trials or biologics in T cell lym-
phoma have been in mycosis fungoides cutaneous T cell
lymphoma (CTCL) with predominantly patients who
were in the Sezary syndrome stage of disease which fea-
tures a characteristic desquamative rash and circulating
malignant T cells. The single-agent activities of different
biologicals in the T cell lymphoproliferative diseases are
summarized in Table 6.
Interferon
As a single agent interferon alpha produced response
rates ranging from 40% to 60% in patients with CTLC
with most of the responses observed in patients with
mycosis fugoides [10, 39, 131]. Many trials were con-
ducted with IFN-α in combination with other therapeutic
modalities. A response rate of 83% was reported for the
combination of IFN-α and psolaren with ultraviolet light
A (PUVA) and (IFN-α2a) in 63 symptomatic early-stage
CTCL patients, with most of the responses being CRs
[18]. In another trial of IFN-α and PUVA in 25 patients
with early-stage CTCL, 91% of patients had an objective
response with 76% achieving a CR [152]. In two trials
FavID (Favrille) BiovaxID (Biovest)
Id-KLH/GM-CSF Id-KLH/GM-CSF
Recombinant DNA Hybridoma rescue
Untreated or Relapsed Untreated
Rituximab PACE or RCHOP
CR or PR or SD CR only
Finished In progress
700 Biological therapy of B and T cell lymphoproliferative disorders

Table 6. Biotherapy of T cell lymphoma with single agent biologicals

Modality Class Biological agent Reference Disease Patients Response rate
Cytokine Interferon-α [10] CTCL 20 45%
Cytokine Interferon-α [131] CTCL 22 59%
Cytokine Interferon-α [39] CTCL 45 38%
Cytokine Interleukin-2 [65] CTCL 7 71%
Cytokine Interleukin-2 [140] CTCL 22 18%
Immunotoxin Denileukin diftitox [130] CTCL 71 30%
Immunotoxin Denileukin diftitox [104, 153] CTCL 35 37%
Immunotoxin Denileukin diftitox [25] PTCL 27 48%
Mab antiCD4 [97] CTCL 8 62%
Mab antiCD5 [35] CTCL 10 40%
Mab Y90 – antiCD5 [60] CTCL 8 38%
Mab Alemtuzumab [108] PTCL 8 50%
Mab Alemtuzumab [45] PTCL 14 36%
Mab Alemtuzumab [5] CTCL 14 86%
Mab Alemtuzumab [110] CTCL 22 55%
Mab Alemtuzumab [93] CTCL 8 38%
Retinoid Isotretinoin [94] CTCL 25 44%
Retinoid Isotretinoin [120] CTCL 39 59%
Retinoid Isotretinoin [16] PTCL 12 50%
Retinoid Etretinate [120] CTCL 29 67%
Retinoid Bexarotene [42] CTCL 94 49%

CTCL = cutaneous T cell lymphoma

PTCL = peripheral T cell lymphoma

s.c. IFN-α was combined with extracorporeal photo-
chemotherapy (photopheresis) using oral 8-methoxypso-
ralen as the photosensitizer. A response rate of 90% was
observed in 39 patients, most of whom had early-stage
CTCL, with 24 achieving a CR [98]. In the second trial,
which was limited to 14 patients with stage II CTCL, a
response rate of only 50% was observed [181]. IFN-α
has combined with a variety of retinoids to treat patients
with T cell lymphomas. In an early report all seven
patients treated with IFN-α and etretinate had a response
[161]. In another small study there was an 83% CR rate
in 12 patients with CTCL who received IFN-α and acit-
retin [2]. In a more complex trial 45 patients, 13 of whom
had stage I Sezary syndrome and the other 32 mycosis
fungoides, were treated with 3 months of daily s.c. IFN-α
alone, and responders then continued to received thrice
weekly s.c. IFN-α, while non-responders were treated
with thrice weekly s.c. IFN-α plus etretinate [39]. After
1 year 28 patients (62%) were in remission. IFN-α alone
produced a response in 17 patients (38%). Among the 28
patients who had not responded after 3 months of IFN-α,
11 (39%) eventually achieved a response after changing
to IFN-α plus etretinate. The combination of s.c. IFN-α
and the oral synthetic retinoid bexarotene produced a
response rate of 39% among 18 CTCL patients, which
appeared no better than either agent used alone [160]. A
response rate of 31% was noted for 17 patients with
anthracycline-resistant peripheral T-cell lymphoma
(PTCL) who were treated with thrice weekly s.c. IFN-
α2a and daily oral 13-cis retinoic acid (isotretinoin) [84].
A response rate of 100% was reported for 28 patients
with Sezary syndrome who were treated with extracor-
poreal photochemotherapy and two or more of various
biologicals including IFN-α, retinoids, and GM-CSF
[146]. One small randomized trial compared thrice
weekly 9 MU IFNα-2a plus PUVA to 9 MU IFNα-2a
plus acitretin in 98 patients with stage I and II CTCL,
only 82 of whom were considered evaluable for response
[159]. IFNα-2a plus PUVA was not only less toxic, but
was associated with more rapid response, and a complete
response rate of 70% compared to only 38% for IFNα-2a
plus acitretin.
IFNα has also been combined with chemotherapy in
the treatment of patients with T cell lymphoma. The
purine analogs fludarabine and pentostatin have sub-
stantial single-agent activity in CTCL. A response rate
of 41% was observed in a trial of 41 CTCL patients,
only six of whom had not received prior therapy, and 29
of whom had not responded to prior therapy, who were
treated with pentostatin 4 mg/m2 i.v. days 1 through 3
and IFNα 10 MU/m2 i.m. on day 22, and 50 MU/m2 i.m.
23 through 26 [58]. A response rate of 51% was observed
in 35 patients, 10 of whom had received pentostatin pre-
viously and 21 of whom had not responded to prior
Robert O. Dillman
therapy, who were treated with fludarabine 25 mg/m2 i.v.
days 1 to 5 every 28 days with IFNα 5 MU/m2 s.c. thrice
weekly for up to eight cycles [59].
Interleukin-2
There has been limited experience with IL-2 in the T
cell malignancies. In one trial that utilized continuous
i.v. infusion of relatively high doses of IL-2, responses
were noted in 5/7 patients [65]. In a recent trial of s.c.
IL-2 a response rate of only 18% was reported for 22
heavily pretreated CTCL patients [140].
Retinoids
The vitamin analogs collectively known as the retinoids,
have been shown to modulate proliferation and differen-
tiation in premalignant and malignant cells, and also
have immunologic effects. The retinoid response is
mediated by nuclear receptors, which have been charac-
terized as retinoic acid receptors (RARs) and retinoid
“X” receptors (RXRs). Because of the dramatic effects
of retinoids in skin disorders including acne and aging
of the skin, there has been intense investigation of retin-
oids in the treatment of CTCL, especially in early stage
mycosis fungoides and Sezary syndrome, and also in
PTCL. As summarized in Table 6, as single-agents retin-
oids have produced response rates ranging from 50% to
70% in CTCL, depending on the product and the spe-
cific CTCL patient population [42, 43, 94, 120], and
50% in PTCL [16]. Substantial anti-tumor activity was
demonstrated with cis-retinoic acid (isotretinoin) and
related retinoids, but none of these agents were ever
submitted for regulatory approval for marketing these
agents in T cell malignancies.
The retinoid X receptor-selective agent LGD1069 was
given orally by mouth to 52 patients in an dose escalation
trial in which some tumor response were observed at higher
doses [119]. This product became bexarotene (Targretin
capsules; Ligand Pharmaceuticals Incorporated, San
Diego, Calif), which received regulatory approval based
on the activity of this agent in early-stage cutaneous T-cell
lymphoma at doses of 300 mg/m2 per day [42, 43].
Hypertriglyceridemia and hypothyroidism were the major
adverse events noted. High response rates have also been
observed with bexarotene gel [9, 139]. Interestingly, a sin-
gle institution retrospective comparison of all-trans retinoic
acid (RAR-specific) and bexarotene (RXR-specific) in
patients with relapsed mycosis fungoides/Sézary syndrome
detected no substantial differences between the products in
terms of duration of disease control, although the response
rate was higher for bexarotene (21% versus 12%) [140].
The combination of bexarotene and immunotoxin denileu-
701
kin diftitox yielded a response rate of 67% among 14
patients with relapsed or refractory CTCL [57].
Immunotoxins
Denileukin diftitox (Ontak™) is a genetically engineered
fusion protein that combines the active chain of diphthe-
ria toxin to the cytokine IL-2 which binds to the CD25
receptor full-length sequence for interleukin-2 (IL-2).
This immunotoxin has therapeutic potential for any
malignancy, including B cell lymphomas and Hodgkin’s
disease, in which there is over-expression of CD25, or
more specifically, the subunit of the IL-2 receptor.
Denileukin diftitox was approved by the US FDA based
on its activity in cutaneous T cell lymphoma where
response rates 30% to 40% were achieved in trials [104,
130, 153]. Response rates in peripheral T cell lymphomas
may be even higher [26] This product may be useful in
any CD25 overexpressing malignancy, which includes
some cases of B-cell lymphoma and Hodgkin’s disease
in addition to most T cell malignancies.
Monoclonal Antibodies
The humanized anti-CD52 Mab Alemtuzumab
(Campath®) was approved in May 2001 based on data
submitted for the treatment of patients with CLL that
had recurred or been refractory to the purine analog flu-
darabine. As shown in Table 6, alemtuzumab has exhib-
ited significant activity as a single agent in both CTCL
with where response rates ranged from 38% to 86% [5,
45, 110], and in PTCL where response rates ranged from
36% to 50% in two small studies [93, 108]. Alemtuzumab
has also been combined with chemotherapy in the treat-
ment of PTCL with very encouraging results. In an
Italian trial the combination of CHOP chemotherapy
and s.c. alemtuzumab yielded a response rate of 75% in
24 patients with 17 of the 18 responses recorded as CRs
[62]. In a Korean trial in 20 PTCL patients the combina-
tion of CHOP chemotherapy and i.v. alemtuzumab was
associated with a response rate of 80% with 13 of the 16
responses being CRs [96].
Vaccines
A vaccine consisting of dendritic cells loaded with the
lysate from CTCL cells and KLH was given as weekly
intranodal injections in ten patients with CTCL [111].
Tumor-specific delayed-type hypersensitivity (DTH)
reactions to tumor lysate were observed in 3/8 patients
so tested. There was a 50% objective response rate
including one CR and four PRs with all of the response
occurring in patients who had a low tumor burden.
702 Biological therapy of B and T cell lymphoproliferative disorders

rituximab with cyclophosphamide and a purine analog,

Summary quickly attracted the interest of investigators, and enthu-
Although T cell malignancies are relatively rare, they have siasm for pursuing IFN-α in CLL waned.
been the target of many biological therapies. Interferon-
alpha, the retinoid bexarotene, the immunotoxin denileu-
kin diftitox, and the Mab alemtuzumab are all routinely
Monoclonal Antibodies
used in the treatment of these malignancies both as single A summary of clinical trials of Mab in CLL are shown
agents, and increasingly in combination therapies. in Table 7. Some of the earliest trials with Mabs were
conducted in patients with CLL. The anti-CD5 murine
Mab T101 bound to CLL cells and decreased circulating
lymphocyte numbers, but at the doses given, and with
Chronic Lymphocytic Leukemia the murine construct, was not able to induce sustained
responses [33–35, 53]. Trials were never carried out at
Interferon
higher doses or with humanized constructs.
Early U.S. trials using leukocyte-derived interferon and In May 2001 the anti-CD52 monoclonal antibody
recombinant interferon in small numbers of patients alemtuzumab (Campath) became the second Mab
with chronic lymphocytic leukemia (CLL) were associ- approved by the U.S. FDA for a hematologic malignancy
ated with non-durable response rates of 11% to 15% when it was granted regulatory approval based on its
[52]. However a Greek trial reported objective responses activity in patients with CLL whose disease had recurred
in 10/26 (38%) [7], and a British trial reported responses or been refractory to the purine analog fludarabine [91].
in 8/18 (44%), although all were transient [118]. Other Trials conducted in the fludarabine resistant or refractory
trials focused on IFN-α as maintenance therapy follow- setting were associated with response rates ranging from
ing chemotherapy. A small randomized trial of Italian 45 33% to 55% with a median of 40% [23, 91, 123, 132,
patients showed a significantly longer duration of 142, 143]. In 115 patients who had progressive disease
response and fewer infections in patients who received after multiple course of prior chemotherapy, the response
IFN-α maintenance therapy [46]. However investiga- was still 23% [48] In the untreated setting alemtuzumab
tors who conducted a single-arm U.S. trial of mainte- produced responses of 83% to 87% when given by the
nance therapy in 31 patients who had responded to i.v. or s.c. route [109, 187]. In a European registration
fludarabine, concluded that adding IFN-α did not trial 297 previously untreated CLL patients were ran-
enhance the degree of chemotherapy-induced remis- domized to oral chlorambucil 40 mg/m2 monthly versus
sion, and that the time to progression was similar to a standard i.v. alemtuzumab [80]. The Mab produced
historical control group [127]. In a German trial in superior response rate, 83% versus 55% (p < .0001) and
which 44 high-risk patients were randomized to obser-
vation or IFN-α, there was no difference in PFS or OS
Table 7. Single-agent activity of monoclonal antibodies in CLL
[102]. In the largest trial of IFN-α in CLL, 133 previ-
ously untreated patients were randomized to receive flu- Antibody Reference Setting Patients Response
rate
darabine and prednisone with or without interferon, and
then 78 responders were randomized to observation ver- Anti-CD5 [35] Relapsed 10 0%
Anti-CD5 [53] Relapsed 13 0%
sus maintenance interferon [115]. The initial response Alemtuzumab [109] Untreated 38 87%
rates were similar (84% and 86%) suggesting that IFN-α Alemtuzumab [80] Untreated 149 83%
added nothing over chemotherapy alone. There was a Alemtuzumab [123] Relapsed 91 55%
longer response duration in patients in patients who Alemtuzumab [23] Relapsed 16 50%
received maintenance therapy, however, this difference Alemtuzumab [142] Relapsed 136 40%
Alemtuzumab [132] Relapsed 29 38%
could be explained by an imbalance in the distribution Alemtuzumab [91] Relapsed 93 33%
of patients who were in CR at that the time they entered Alemtuzumab [143] Relapsed 24 33%
the maintenance phase of the study. Although these were Alemtuzumab [48] Relapsed 115 23%
all relatively small studies by the standards of modern Rituximab [162] Untreated 19 90%
clinical trial design, the failure of IFN-α to consistently Rituximab [78] Untreated 26 70%
Rituximab [79] Untreated 43 58%
achieve durable disease control, and the toxicity associ- Rituximab [12] Relapsed 33 45%
ated with IFN-α, dampened enthusiasm for larger studies. Rituximab [128] Relapsed 40 36%
Furthermore, the high durable response rates achieved Rituximab [188] Relapsed 23 35%
with combinations of rituximab with fludarabine, or Rituximab [73] Relapsed 28 25%
Robert O. Dillman
PFS which led to a marketing indication as initial ther-
apy, even though in the U.S. purine-analog based therapy
is considered the treatment of choice.
Fludarabine and alemtuzumab combination therapy as
the initial treatment of patients with CLL produced
responses in 83% of 36 patients, with 11 of the 30
responses being CR [44]. Combining GM-CSF with
alemtuzumab in 14 patients with relapsed disease did not
appear to increase the response rate (36%) or decrease
the risk of infection [189]. Because of the additive toxic-
ity and immuosuppression associated with combining
alemtuzumab with chemotherapy, most of the recent
emphasis has been on using alemtuzumab after chemo-
therapy in an effort to eradicate minimal residual disease
in responding CLL patients. Thrice weekly doses of 10
to 30 mg have been administered i.v. or s.c. for 4 to 12
weeks as a consolidative therapy [122, 129, 173]. These
studies have shown that consolidation with alemtuzumab
does increase clinical, phenotypic, and molecular com-
plete response rates, but there is an increased risk of
opportunistic infections, even if anti-viral, anti-bacterial,
and anti-Pneumocystis prophylaxis is used.
Although rituximab does not have regulatory approval
for marketing as a treatment for CLL, it is widely used
in combination with chemotherapy. As shown in Table 7,
in patients with previously treated, relapsed CLL, as a
single agent rituximab was associated with response
rates ranging from 25% to 45% with a median of 35%.
over a wide range of doses and by various schedules of
administration [12, 73, 128, 188]. Response rates were
much higher in patients who had not been previously
treated with a range from 58% to 90% with the highest
rate occurring in patients with early stage disease who
were considered to be high risk. [78, 79, 162].
The popularity of rituximab in CLL is due to the abil-
ity to combine it with chemotherapy with no apparent
increase in toxicity. Rituximab combined with purine
analogs has produced response rates of about 90% in
untreated patients treated with rituximab and fludara-
bine [12, 154], and 33% in previously treated patients
who received pentostatin plus rituximab [38]. The com-
binations of purine analogs plus cyclophosphamide plus
rituximab have been associated with very high response
rates and durable complete remissions in previously
untreated patients. In single institution trials the combi-
nation of fludarabine, cyclophosphamide, and rituximab
(FCR) has produced response rates of 77% in the
relapsed setting [174], and 95% as initial therapy [92].
The combination of pentostatin, cyclophosphamide, and
rituximab (PCR) has produced response rates of 75% to
90% in previously untreated patients [36, 90, 101], but
only 33% in previously treated elderly patients in the
703
community setting [36]. In a trial of young patients with
relapsed CLL, cladribine, cyclophosphamide, and ritux-
imab (CCR) produced a response rates of 58% [148].
In one randomized phase II trial, concurrent fludarabine
plus rituximab was compared to the sequence of fludara-
bine followed by rituximab in 104 patients [13]. The
response rate was higher for concurrent therapy (90% ver-
sus 77%) as was the complete response rate (47% vs
28%). A retrospective comparison of these patients to a
similar population of patients treated with fludarabine
alone at the same dose in an earlier randomized trial sug-
gested that patients treated with the combination of flu-
darabine plus rituximab had much better outcomes,
including a higher response rate (84% versus 63%), higher
complete response rate (38% versus 20%) better progres-
sion free survival after 2 years (67% versus 45%), and bet-
ter overall survival at 2 years (93% versus 81%) [14].
Summary
Biotherapy now plays a crucial and ever increasing role in
the management of both B-cell and T cell lymphoprolif-
erative malignancies. Because of its ease of delivery,
anti-tumor activity and relatively low toxicity rate,
rituximab has become the most important single agent
in the treatment of indolent B cell lymphoma., and com-
bined with chemotherapy, has become the standard of
treatment for more aggressive B cell lymphomas.
Rituximab alone or in combination with chemotherapy
has become the initial treatment of choice for indolent
lymphomas, and CHOP + Rituximab has become the
treatment of choice for large B cell lymphoma. Despite
evidence-based data supporting the use of interferon-α
in lymphoma, because of its toxicity and need for
chronic treatment, this biological never gained wide-
spread acceptance in the treatment of lymphoma in the
U.S. IL-2 was never an important agent in these disor-
ders because of the need for hospitalization and toxicity
associated with higher doses, and the lack of clinical
activity at lower doses. Two biological agents, the
immunotoxin denileukin diftitox and the retinoid bex-
arotene were both received regulatory approval based
on studies conducted in cutaneous T cell lymphoma.
The use of denileukin difitox is limited by its immuno-
genicity. The antiCD52 Mab alemtuzumab is being used
increasingly in combination with chemotherapy for the
T cell malignancies, but its immune inhibition and tox-
icity profile make it a less attractive option in B cell
lymphoma because of the availability of rituximab.
Vaccine approaches are interesting and promising, but
at this time there is no commercial product.
704 Biological therapy of B and T cell lymphoproliferative disorders
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21.8 Biological therapy of multiple myeloma
ROBERT K. OLDHAM
Interferon
Several studies have demonstrated response rates of
about 20% in patients with refractory multiple myeloma
treated with recombinant alpha-interferon [1, 3, 7]. An
attempt to augment this response experience by using
high dose induction schedules, often with the addition of
Prednisone, did not result in enhanced response [1].
Anecdotal observations have suggested possible syner-
gism between the interferons and cytotoxic drugs [2, 5].
Because of response rates of 50% in untreated patients
[8], studies were done to evaluate the combination of
interferon with standard chemotherapeutic regimens as
initial therapy for multiple myeloma. Preliminary inter-
pretation suggested that the duration of initial response
may be extended with the addition of alpha-interferon
[6]. Follow-up studies were not confirmatory.
The availability of very active new agents such as
Velcade has precluded further studies with Interferon com-
binations. In addition, thalidomide and reilamide, with
their immunomodulating capacities, are active in multiple
myeloma [9]. Given the high degree of activity both with
chemotherapy and immunomodulators, as well as the use
of autologous bone marrow transplants, Interferon is now
little studied in multiple myeloma [4, 9].
With the advent of Velcade and revlamide as targeted
therapy for multiple myeloma [4, 9], there has been little
subsequent activity with other forms of biotherapy in this dis-
order. However, given the activity of revlamide as an immu-
nomodular, we may soon see other drugs working through
the immune system to treat this disseminated disease.
Antibodies
Plasma cells produce myeloma protein (antibody), giving
ready availability of a secreted protein target for antibody
induction. However, this very characteristic makes it
unlikely that antibody therapy will be useful in myeloma,
at least not with the myeloma protein as target, since
the therapeutic antibody would be totally absorbed in
the vascular and extracellular pool. Anti-CEA studies
in colon cancer suggest this is not a rational approach to
follow, but targeting myeloma cell membrane proteins
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
(nonsecreted) could be attempted. No major clinical
studies of antibody against other plasma cell membrane
targets have been reported.
CD20 is rarely expressed on plasma cells, but has
been reported to be present on malignant cells from up
to 20% of patients with multiple myeloma [11].
Rituximab has rarely produced objective responses in
multiple myeloma even in patients whose plasma cells
were felt to overexpress CD20. Using the standard dos-
ing of 375 mg/m2, Treon et al saw objective responses in
only 1/19 patients [11], and Hussein et al reported
responses only 2/21 patients prior to starting chemo-
therapy five weeks later [10]. There has been a much
greater unpublished experience in the community prac-
tice of medicine with anecdotal reports of occasional
excellent responses in refractory myeloma patients
whose plasma cell overexpressed CD20. Another strat-
egy is to use rituximab as an adjuvant treatment after an
intial response to chemotherapy, in an effort to suppress
the B cell clone that leads to the malignant plasma cells.
Based on this same rationale, rituximab may be more
active against smoldering myeloma than myeloma with
a high proliferative index.
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711
712
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Biological therapy of multiple myeloma
10. Hussein MA, Karam MA, McLain DA, et al. Biologic and clinical
evaluation of rituxan in the management of newly diagnosed mul-
tiple myeloma patients. Blood 1999;94:313a [abstract 1400].
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21.9 Biological therapy of squamous cell cancers
of the head and neck
ROBERT O. DILLMAN
Introduction
This category includes squamous cell cancers that originate in
the mouth, oropharynx, nasopharynx, and larynx. Many
of the cancers are associated with smoking and other
forms of tobacco abuse that are local irritants and mutants,
and some are caused by human papilloma virus, and other
viruses. Primary therapy typically includes surgery and/or
radiation therapy, and increasingly chemotherapy as part
of organ sparing approaches that minimize the morbidity
associated with extensive surgery in this area.
Non-specific Immune Stimulants
Bacillus Calmette Guerin (BCG)
There were no trials that tested single-agent activity of
BCG, but several trials addressed the issue of whether
BCG augmented methotrexate or other chemotherapy
regimens in the treatment of advanced squamous cell
cancers of the head and neck. In a large, non-randomized
phase II trial, 100 patients with advanced, recurrent, or
metastatic squamous cell carcinoma of the head and neck
were treated with the combination of chemotherapy and
BCG [49]. The objective response rate was 35%, and the
median duration of response was 17 weeks, both of which
were considered similar to historical results obtained with
chemotherapy alone. In a small randomized phase II trial,
38 patients with advanced, inoperable squamous cell car-
cinoma of the head and neck were randomized to receive
methotrexate alone or with BCG [35]. The response rates,
3/19 in the methotrexate alone arm and 4/19 for metho-
trexate plus BCG, were similar as was the duration of
response and survival. In another small randomized phase
II trial, 23 patients with advanced recurrent head and neck
carcinoma were randomized to receive either high dose
methotrexate with calcium leucovorin rescue (HDMTX)
(n = 12) or HDMTX in combination with BCG (n = 11)
[6]. There were three objective tumor responses of simi-
lar brief duration in both groups. Although this trial was
too small to be conclusive, it cast doubt on whether BCG
added anything to HDMTX chemotherapy for such
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
patients. In another randomized prospective study of
patients with advanced squamous cell carcinoma of the
head and neck, treatment with methotrexate plus BCG
was no better than methotrexate alone in terms of response
rate or survival [62].
There were two small trials with relatively positive
results. One was another small randomized phase II trial,
in which 34 patients with recurrent squamous cell cancer
of the head and neck were randomized to receive the five-
drug chemotherapy regimen BACON (bleomycin, dox-
orbucin, CCNU, vincristine and nitrogen mustard) alone
(n = 14) or with BCG by scarification (n = 20) [41]. The
patients treated with BACON plus BCG experienced a
longer survival (P = 0.014) than those treated with
BACON alone. The other positive trial was in the neoad-
juvant setting. Following neoadjuvant methotrexate and
definitive local therapy, 52 patients with locally advanced
squamous cell cancer were randomized to adjuvant meth-
otrexate alone (n = 27) or with an adjuvant treatment with
a BCG vaccine (n = 25) consisting of 2 to 4 million Tice
strain BCG organisms given i.d. in alternating sides of the
neck every 2 weeks for six doses, then every 4 weeks for
nine doses [50]. At a median follow-up of about 3.5 years,
52% of the BCG vaccine-treated group remained disease
free compared to only 26% of the controls. Similarly,
68% of the BCG vaccine-treated group were alive at 3.5
years compared to only 41% of the controls. Based on
this trial BCG showed promise as adjuvant immunother-
apy in ear, nose, and throat cancers.
Levamisole
Several small randomized trials were conducted in head
and neck cancer with the antihelminth levamisole.
A randomized double-blind study compared levamisole
(n = 31) with placebo (n = 34) as adjuvant treatment
following surgery of patients with squamous cancer
of the head and neck [61]. There was no difference in
progression free survival (PFS) or overall survival (OS).
The largest randomized trial ever conducted with levam-
isole in head and neck cancer patients, actually sug-
gested a detrimental effect. After treatment of a primary
713
714 Biological therapy of squamous cell cancers of the head and neck
squamous cell carcinoma of the head and neck, 134
patients were randomized to receive placebo (n = 65) or
levamisole, 150 mg/day orally for 3 consecutive days
every other week [33]. Patients with stage I and II disease
who were treated with levamisole had a significantly
higher incidence of recurrence than the placebo-treated
patients (p = 0.02).
Some other small studies were more encouraging. In
a small randomized study of 24 patients with squamous
cell cancer of the larynx or hypopharynx, 12 patients
received placebo and 12 received levamisole 50 mg t.i.d.
for 3 consecutive days every 2 weeks following surgery
and/or radiation [30]. After 20 months of follow up, the
recurrence rate was only 20% in the levamisole group
compared to 50% in the control group. In another trial,
following conventional radiotherapy, 82 patients with
T1 or T2 N0 M0 squamous cell carcinoma of the oral
cavity were randomized to receive either placebo or
levamisole at a dose of 150 mg daily for 3 consecutive
days every 2 weeks [34]. After a median of 3 years of
follow up, 44% of patients who received levamisole
were still disease-free compared to only 32% in the pla-
cebo group. There also was a faster recovery from radi-
ation-induced leukopenia and lymphopenia in the
levamisole group. In another small randomized study,
34 patients without distant metastases were randomized
to observation and 31 to receive futraful and uracil
(UFT) plus levamisole as adjuvant treatment for stage
III and IV squamous cell carcinomas of oral cavity,
oropharynx, hypopharynx and larynx following primary
therapy [24]. The 5-year disease free survival rate
favored the UFT-levamisole arm (57% versus 39%), but
was not statistically significant (p = 0.21). There was
also a trend for a lower rate of distant metastasis (10%
versus 32%, p = 0.06). The contribution of levamisole
beyond UFT is unclear.
Cornybacterium Parvum (C parvum)
Three randomized trials failed to demonstrate any ben-
efit for C parvum in the treatment of epidermoid cancers
of the head and neck. In a small randomized trial,
patients with advanced squamous cancer of the head
and neck were randomized to treatment with weekly
methotrexate alone or methotrexate with C parvum
given s.c. weekly as bilateral doses in sites around the
neck [63]. The addition of C. parvum did not improve
the response rate, response duration or survival. In a
randomized trial involving 57 patients with previously
untreated squamous cell carcinoma of the head and
neck, 29 received radiation alone and 28 patients
received C. parvum administered i.v. and locally into
the tumor-bearing lymph nodes of the neck, or into the
cervical node region in patients who lacked palpable
lymph nodes, before and after radiation therapy [10].
After 2.5 years of follow up, the study was terminated
when it became evident that there was no difference dis-
ease free survival. In a large trial 209 operable patients
with squamous cell head and neck cancer were random-
ized to surgery alone or pre-operative intratumoral
immunotherapy with C parvum followed by post-operative
s.c. C parvum for 2 years [31]. Only 176 were considered
fully evaluable, but there was no difference disease free
or overall survival.
OK-432
The streptococcal preparation OK-432 has been exten-
sively studied in Japan in various malignancies including
epidermoid cancers of the head and neck. During 1984
to 1989 120 newly diagnosed patients of laryngeal
squamous cell carcinoma were registered at ten differ-
ent institutions, stratified by stages of disease, treated
with radiation therapy and 5FU chemotherapy with or
without surgery, with or without chemotherapy, and
randomized to no further therapy or immunotherapy
with OK-432 [23]. There was no apparent advantage for
those patients who received OK-432. The 5-year dis-
ease free survival rate for 109 evaluable patients was
76% in the OK-432 arm and 75% in the control arm.
The 5-year overall survival rate was 84% in the OK-432
arm and 78% in the control arm.
Thymic Hormones
It has long been recognized that the thymus gland is
important in the development of cell-mediated immu-
nity and that many thymic hormones have stimulating
affects on T lymphocytes. Clinical experiments have
been performed with crude thymus extracts, isolated
purified products, and synthesized peptides. Several
thymic preparations have been of clinical interest,
including thymosin fractions 5, thymosin-α1, prothy-
mosin, thymulin, thymopoietin, thymostimulin (TP-1),
and thymic humoral factor [46]. In vitro thymostimulin
restores a number of immunologic defects noted in the
monocytes and dendritic cells of patients with head and
neck cancers. In a small study, 18 patients with squamous
cell cancer of the head and neck were treated with thy-
mostimulin at one of three dosages (0.5, 1.0, or 2.0 mg/
kg) prior to surgery, and then their exicised tumors were
examined and results compared to those from 16 con-
temporary controls who had not received treatment with
the thymic hormone [21]. Preoperative treatment with
Robert O. Dillman
thymostimulin enhanced T-cell infiltration. Anti-tumor
effects were not determined. There have been no publi-
cations of follow up studies.
Interferons
Interferon-α
Interferon-alpha (IFN-α) was studied in several trials in
squamous cell cancer of the head and neck. In a phase II
trial, 71 patients with recurrent or metastatic squamous
cell carcinoma of the head and neck were randomized to
receive recombinant IFN-α2b at a relatively low dose (6
MU/m2 daily × three every 4 weeks) or a relatively high
dose (12 MU thrice weekly) [59]. As shown in Table 1,
the response rate was 5%, with a response rate of 1/32
for evaluable patients who received the lower dose, and
2/29 for those who received the higher dose. Median
survival was about 6 months in both arms.
Other trials combined IFN-α with radiation therapy
and/or chemotherapy. In a small study, 22 patients with
operable head and neck cancer were randomized to
receive radiotherapy alone prior to surgery, or radiother-
apy with natural leukocyte IFN-α at a dose of 6 MU i.m.
daily for 4 weeks prior to surgery, and then thrice weekly
for 2 months [54]. Objective responses were observed in
4/10 patients who received IFN-α and radiotherapy,
compared to 2/12 among those who only underwent
radiation therapy, but there was no difference in survival.
The study was discontinued because of worse toxicity in
the IFN-α cohort. IFN-α was combined with 5-FU and
cisplatin to treat patients with recurrent or metastatic
squamous cell carcinoma of the head and neck who had
not received prior chemotherapy [20]. The response rate
was 25% among 50 eligible patients and the median sur-
vival was 5 months. The authors concluded that IFN-α
added to toxicity without conveying a benefit to the che-
motherapy. In 20 patients with recurrent and/or meta-
static squamous cell carcinoma of the head and neck, the
same combination of chemotherapy and IFN-α produced
Table 1. Single-agent activity of various biologicals in
patients with head and neck cancer
Modality Biological Lead Response
class agent author Patients rate (%)
Non-specific Isotretinoin [25] 19 16
Cytokine Interferon-α [59] 61 5 was determined to be 2 MU/day. There were two objective
Cytokine Interleukin-2 [12] 31 13
(IL-2)
Mab Cetuximab [57] 109 13
715
a 30% response rate in 18 evaluable patients who had an
8.5 month median survival [3]. IFN-α2b, cisplatin and
5FU were combined to treat 14 patients with previously
treated squamous cell head and neck cancers [2].
Hematologic toxicity caused treatment delays in 9/14
patients, which led to early closure of the trial, but 8/14
(54%) of the patients had an objective response rate.
Interferon-gamma
Interferon-gamma (IFN-γ) was given at a dose of
0.25 mg/m2 as a 24-h infusion i.v. weekly for 4 weeks to
eight patients with locally advanced, but resectable head
and neck cancer [42]. There were minimal side effects,
and three patients had clinically measurable responses
prior to surgery.
Interleukins
Interleukin-2
Local injections of IL-2 might enhance an immune
response in lymph nodes that drain head and neck can-
cers. In one study, 20 patients with recurrent, inoperable
head and neck squamous cell carcinoma received peri-
lymphatic injections of natural IL-2 for 10 days [11].
Irrespective of the site of the recurrence, injections were
always given 1.5 cm below the insertion of the sterno-
cleidomastoid muscle on the mastoid. Injections were
performed on the ipsilateral side if the lymphatic chain
was still present, or on the contralateral side if the patient
had previously undergone an extensive neck dissection.
Thirteen patients had brief response to such treatment,
and there was no benefit from repeated treatment. The
same study investigators treated 31 patients with recur-
rent head and neck squamous cell carcinoma with ten
daily doses of 500 or 500,000 U of IL-2 as injections
1.5 cm from the insertion of the sternocleidomastoid
muscle on the mastoid [12]. There were no toxic effects.
Objective, but non-durable tumor responses were noted
in 4/16 patients who received the 500 U dose, but the
response rate was 0/15 at the higher dose. In a U.S. dose
escalation trial of local therapy, IL-2 was injected per-
ilesionally in divided doses in each of four quadrants
and bilaterally into the superior jugular lymph nodes in
36 patients with unresectable squamous cell carcinoma
of the head and neck [58]. The maximum tolerated dose
responses for a response rate of 6%.
In a European multicenter trial, 202 patients with
advanced, but potentially resectable cancers of the oral
716 Biological therapy of squamous cell cancers of the head and neck
cavity or oropharynx, were randomized to undergo sur-
gery and radiation therapy alone or in combination with
perilymphatic recombinant IL-2 [14]. A dose of 5,000 U
IL-2 was injected around the ipsilateral cervical lymph
node chain daily for 10 days before surgery, and after
surgery the same IL-2 dose was injected into the con-
tralateral cervical lymph node chain 5 consecutive days
monthly for 1 year. Treatment was well-tolerated and
did not interfere with the treatment plan. Those patients
who received IL-2 had a longer disease free survival
(p < 0.01) and overall survival (p < 0.03).
A preparation of polyethylene glycol-modified IL-2
(PEG-IL-2) provides a slower release of IL-2 from sites
of injection, and therefore a more continuous exposure
to the IL-2. PEG-IL-2 was given to 19 patients with
recurrent head and neck squamous cell carcinoma as
intratumoral injections of 200,000 U of (PEG-IL-2) two
or three times a week for 4 weeks [29]. Temporary
regional swelling and redness developed in ten patients,
and nine patients had systemic eosinophilia. One patient
had an objective response.
Several trials combined IL-2 and IFN-α. In a phase II
study 11 patients with recurrent head and neck cancer
were treated with continuous i.v. recombinant human
IL-2 and i.m. or s.c. IFN-α2a [53]. Two patients (18%)
achieved a partial response, but toxicity was substantial.
In another phase II trial, a response rate of 36% was
reported for 14 patients with advanced head and neck
cancer, even though toxicity was such that only three
patients were able to complete three cycles of therapy
[45]. The degree of natural killer cell activation corre-
lated with response.
Other trials combined IL-2 with chemotherapy for the
treatment of squamous cell head and neck cancer. In a
non-randomized phase II study, 23 patients with
advanced (stage III or IV) head and neck squamous cell
carcinoma received chemotherapy with cisplatin, 5FU,
and vinorelbine, or the same combination with IL-2 at a
dose of 9 MIU s.c. daily from day 9 to 13 and from day
16 to 20 of every cycle [27]. The response rate was 63%
for the chemotherapy alone, and 100% with the addition
of IL-2. In a randomized, phase III, multicenter clinical
trial, only 33 patients with advanced head and neck
squamous-cell carcinoma were randomized to 5FU and
cisplatin alone, or combined with s.c. IL-2 at a dose of
4.5 MIU/day on days 8–12 and 15–19 of each treatment
cycle [28]. Response rates were similar between the
arms, 12/17 in the chemotherapy alone arm, and 10/16
in the IL-2 containing arm.
Multikine is a combination of natural interleukins
derived from leukocytes. In a pilot study, 12 previously
untreated patients with various head and neck cancers
were treated by peritumoral injection of this cytokine
combination, in addition to oral zinc sulfate, oral indo-
methacin, i.v. cyclophosphamide. [15]. Four patients
reportedly had an objective response. In a four-center
phase I/II dose-escalation clinical trial, 54 patients with
locally advanced, node-negative primary oral squamous
cell carcinoma were treated with a leukocyte multikine
interleukin product prior to surgical resection [51].
Paraffin-embedded tumor samples obtained at surgical
resection of the residual tumor showed what the authors
interpreted as increased intraepithelial T cell infiltration.
Because of the trial design, it was not possible to tell
whether significant anti-tumor effects occurred.
Interleukin-12
Interleukin-12 (IL-12) was injected intratumorally prior
to surgery once weekly, two or three times, at either 100
or 300 ng/kg, in ten previously untreated patients with
head and neck squamous cell carcinoma [55]. The injec-
tions resulted in a greater infiltration of natural killer
cells and CD20+ B cells than were seen in samples from
20 patients who had not received IL-12. Because of the
trial design, it was not possible to tell whether signifi-
cant anti-tumor effects occurred.
Retinoids and Vitamins
There is a long history of interest in the effects of vita-
mins and especially vitamin A analogs, for the treatment
and prevention of head and neck cancers. Much of the
focus has been on 13-cis-retinoic acid (isotretinoin,
CRA). As summarized in Table 1, an objective response
rate of 16% was observed in 19 evaluable patients who
were treated with isotretinoin as part of a multi-institu-
tional, randomized phase II trial that included 40 patients
with advanced head and neck squamous cell carcinoma
who were randomized to treatment with either metho-
trexate or isotretinoin [25]. In this trial there were no
objective responses in patients treated with methotrex-
ate chemotherapy.
Because of in vitro evidence of synergy, and some
evidence of single-agent activity, several small trials
combined 13-cis-retinoic acid (isotretinoin, CRA) with
IFN-α. A response rate of 13% was reported in a trial of
16 patients with unresectable recurrent head and neck
carcinomas who were treated with CRA 40 mg p.o. daily
plus IFN-α 3 MU s.c. every other day [32]. A response
rate of only 5% was reported in another phase II trial of
this regimen in 21 patients with recurrent squamous cell
carcinoma of the head and neck [60]. In another trial, no
Robert O. Dillman
responses were observed in ten patients with advanced
squamous cell carcinoma of the lung and of the head
and neck who were treated with high dose 13-cis-retinoic
acid (2 mg/kg/day) and IFN-α [44]. Collectively, the
results of these three trials were disappointing, with a
cumulative recorded objective responses of only 3/47
(6%) of patients.
The retinoids have produced impressive results in the
prevention of squamous cell cancer of the head and
neck. Patients with squamous cell cancers of the head
and neck remain at high risk for both recurrent and sec-
ond primary tumors after initial therapy. After comple-
tion of surgery or radiotherapy (or both), 103 patients
who were disease-free after primary treatment for
squamous-cell cancers of the larynx, pharynx, or oral
cavity were randomized to receive 12 months of either
isotretinoin (13-cis-retinoic acid) (50 to 100 mg/m2/day)
or placebo [19]. There was no reduction in the rate of
recurrence of the original cancer, but after a median
follow-up of 32 months, only 4% of patients in the isot-
retinoin group had second primary tumors, as compared
with 24% in the placebo group (p = 0.005).
Previously high-dose isotretinoin therapy had been
shown to be an effective treatment for leukoplakia, a
precursor of squamous cell cancer in the mouth. In a
follow up study, 70 patients with leukoplakia underwent
induction therapy with a high dose of isotretinoin
(1.5 mg/kg/day) for 3 months, then patients with
responses or stable lesions were randomly assigned to
maintenance therapy with either beta carotene (30 mg/
day) or a low dose of isotretinoin (0.5 mg/kg/day) for 9
months [26]. Of 66 evaluable patients, 55% responded
to the high-dose CRA, and 59 went on to randomization
between beta carotene and low-dose CRA. Of the 53
patients who could be evaluated, 92% in the low-dose
isotretinoin group and 45% in the beta carotene group
had sustained disease control (p < 0.001).
Trials conducted more recently outside the United
States failed to confirm these impressive results. During
1992 to 1995, 272 Italian patients who had undergone
radical treatment for advanced (stage III and IV)
squamous cell head and neck cancer were randomized
to no further treatment (n = 126) or to receive 1 year of
CRA at a dose of 0.5 mg/kg/day p.o. (n = 126) [52].
Because of difficulties in getting IFN-α, a third arm of
CRA plus IFN-α was discontinued after enrolling only
15 patients. After 3 years of follow up, the 5-year actu-
arial OS rates were similar for both arms at 59% and
57%, as were DFS rates at 66% and 63%. Adverse
effects were mostly grade I, and occurred in 69% of
treated patients. As in other trials, adverse events
included cytopenia, mucositis, conjunctivitis, cutaneous
717
toxicity, hypertriglyceridemia and hypercholester-
olemia. These authors concluded that CRA was ineffec-
tive as chemoprevention in patients with radically
treated HNSCC. In an Australian trial, 151 patients head
and neck squamous cell carcinoma, who had undergone
curative treatment, were randomized to 3 years of treat-
ment with isotretinoin at a high dose (1.0 mg/kg/day) or
a moderate dose (0.5 mg/kg/day) or placebo [36]. There
was no difference in the rate of second primary cancers
of the head and neck, lung, or bladder, or time to recur-
rence of disease. These authors concluded that the use
of CRA as prophylaxis against a second cancer in head
and neck cancer patients is not indicated.
The largest trial conducted to date was a phase III
randomized trial of 3 years of low-dose isotretinoin
(30 mg/day) versus placebo in 1,190 patients with early-
stage (stage I or II) squamous cell cancer of the head
and neck [22]. After 4 years of follow up, it was deter-
mined that isotretinoin did not statistically significantly
reduce the rate of second primary tumors or increase
survival. This study reaffirmed that active smoking was
the greatest risk for subsequently being diagnosed with
additional smoking related cancers.
Higher doses of cis-retinoic acid and interferon are
associated with some toxicity which might be mitigated
by concomitant administration of vitamin E; so, all three
agents have combined as a chemoprevention therapy
after treatment of advanced head and neck cancer. After
definitive local treatment with surgery, radiotherapy, or
both, 45 patients with locally advanced squamous cell
head and neck cancer were treated with 13-cRA (50 mg/
m2/day orally, IFN-α 3MIU/m2 s.c. thrice weekly, and
alpha-tocopherol (1,200 IU/day orally) for 12 months
with 85% completing the planned therapy [48]. The
2-year disease free survival (DFS) rate was 81%, and
overall survival (OS) 91%. In a similar trial CRA, IFN-
α2a, and vitamin E were used to treat 45 patients who
had locally advanced (III or IV) squamous cell carci-
noma of the head and neck that had been treated with
surgical resection, radiation, or both [47]. The three-
drug bioadjuvant chemopreventive treatment was con-
tinued for 12 months. The 5-year PFS was 80% and
5-year OS 81%, which the authors felt was much better
than the 40% 5-year survival that is considered the his-
torical standard.
Other combinations of biologicals have been tested
as chemoprevention strategies for these patients. In a
large European trial, from 1988 to 1994, 2,592 patients
(60% with head and neck cancer and 40% with lung
cancer) were randomized to receive one of four treat-
ment arms: retinyl palmitate (0.3 MIU daily for 1 year
followed by 0.15 MIU for an additional year),
718 Biological therapy of squamous cell cancers of the head and neck
N-acetylcysteine (600 mg daily for 2 years), the combi-
nation of retinyl palmitate and N-acetylcysteine for 2
years, or no additional treatment [56]. In terms of risk,
94% had smoked tobacco at sometime in their lives and
25% continued to smoke after the cancer diagnosis.
After a median follow-up of 4 years, 916 (35%) patients
had experienced a recurrence, second primary tumor, or
death. There was no statistically significant difference in
event-free or overall survival. or event-free survival for
any of the 2 × 2 analyses for the impact of retinyl palmi-
tate or N-acetylcysteine. There was a lower incidence of
second primary tumors in the no intervention arm that
was not statistically significant.
Retinoids have been combined with chemotherapy
agents and biologicals other than IFN-α. Ifosfamide,
cisplatin, and CRA (0.5 mg/kg) were given in combina-
tion to treat 52 patients with squamous cell carcinoma
of the head and neck, who had either locally advanced
disease, that could not be managed by surgery or radia-
tion therapy, locoregional recurrence or distant metasta-
ses [38]. Treatment was well-tolerated. The response
rate was 72%, with a median PFS of 10 months and OS
of 13 months. Cisplatin, IFN-α (6 MIU/day i.m.), and
CRA 1 (mg/kg/day) were given in combination for 12
weeks to 23 previously treated patients who had devel-
oped advanced or metastatic head and neck squamous
cell carcinoma [16]. There were four objective responses.
In a trial that enrolled 54 patients, 42 who had exhibited
clinical benefit (complete or partial response, disease
stability) from docetaxel, ifosfamide, and cisplatin che-
motherapy as treatment for recurrent or metastatic
squamous cell carcinoma of the head and neck, low-
dose s.c. IL-2 and oral CRA were given as maintenance
immunotherapy [39]. Median PFS was 11 months and
median OS was 22 months. Progressive increases in the
numbers of lymphocytes and natural killer cells were
documented, as well as a decline in levels of vascular
endothelial growth factor (VEGF).
Vaccines
The vaccines that were recently approved that prevent
infection with human papilloma virus (HPV) will pre-
vent HPV- squamous cell head and neck cancers, which
account for a small subset of all head and neck cancers.
In terms of the more traditional vaccine approach, there
have been relatively few studies in patients with head
and neck cancer.
In a pilot study, following radiation therapy 20 patients
with squamous cell cancer of the head and neck were
preconditioned with IL-2 and subsequently vaccinated
with virus-modified autologous tumor cells prepared from
short-term tumor cultures [18]. Delayed type hypersen-
sitivity reaction to unmodified tumor cells was demon-
strated, but anti-tumor effects could not be measured
because of the trial design.
Adoptive Cell Therapy
There has been only limited investigation of adoptive
cell therapy in patients with head and neck cancer. In
one study, irradiated autologous tumor cells were
admixed with BCG and injected i.d. in six patients with
advanced head and neck cancers, then vaccine-primed
lymphocytes were isolated from regional lymph nodes,
expanded in vitro with IL-2 and anti-CD3 Mab, then
infused into patients along with i.v. IL-2 [9]. Specific
cytoxicity against autologous tumor was demonstrated
in four patients. There were no significant tumor
responses after transfer of the activated lymphocytes. In
another approach, three patients with recurrent head and
neck cancer underwent leukapheresis to collect periph-
eral blood mononuclear cells (PBMC) which were then
incubated in vitro with the bifunctional monoclonal
antibody (Mab) catumaxomab, which has one arm that
binds to the epithelial cell adhesion molecule (Epcam)
and the other arm binds to the T cell antigen CD3 [40].
Intravenous administration of catumaxomab is compli-
cated by the release of cytokines when the Mab binds to
circulating T cells; so in this trial the strategy was to
activate the T cells in vitro where the release of cytok-
ines would take place ex vivo, then infused the cells i.v.
in hopes that the catumaxomab would target the cells to
tumor. Assays showed that the PBMNC indeed did
release substantial amounts of interferon gamma and
tumor necrosis factor alpha in vitro. Patients received
escalating doses from 10,000 to 10 million cells per
kilogram, with 1 million per kilogram being the maxi-
mum tolerated dose. One patient achieved a CR that
lasted more than 2 years.
Antibody Therapy
Cetuximab has been extensively evaluated in squamous
cell cancers of the head and neck. Vermorken et al. gave
single-agent cetuximab at the standard dose and sched-
ule to 109 patients who had recurrent and/or metastatic
disease that progressed during platinum chemotherapy
[57]. Acneiform skin rash, which occurred in 49%, was
the most common toxicity There was one death due to
an infusion-related allergic reaction. The 13% response
Robert O. Dillman
rate was similar to that of single-agent cetuximab in col-
orectal cancer.
Phase I trials suggested that cetuximab could be
safely given with radiation therapy and might enhance
therapeutic benefit [43]. Bonner et al. conducted a mul-
tinational trial comparing cetuximab plus radiotherapy
to radiotherapy alone in 424 patients with locoregion-
ally advanced disease, that showed a benefit for the
addition of cetuximab, including a 26% reduction in
death, with an increase in survival from 29 to 49 months
(p = 0.03) [4]. There was no increase in toxicity other
than the acneiform rash.
Pfister et al. treated 22 patients with locoregionally
advanced disease with cisplatin, cetuximab, and 70 Gy
radiotherapy [37]. This trial was closed early because of
several adverse events from infection and cardiac events.
Grade 3 or 4 cetuximab-related toxicities included acnei-
form rash in 10% and hypersensitivity in 5%. With a
median follow-up of 52 months, the 3-year overall sur-
vival rate is 76%, the 3-year progression-free survival rate
is 56%, and the 3-year locoregional control rate is 71%.
Herbst et al. treated 130 patients who did not have an
objective response or relapsed within 90 days of com-
pleting two cycles of cisplatin/paclitaxel or cisplatin/
fluorouracil, with standard cetuximab and cisplatin (75
or 100 mg/m2 i.v.) every 3 weeks [17]. The most com-
mon toxicities were anemia, acneiform skin rash, leuko-
penia, fatigue/malaise, and nausea/vomiting. Seven
patients (5%) experienced a grade 3 or 4 hypersensitiv-
ity reaction to cetuximab. Baselga et al. treated 96
patients who were refractory to cisplatin, with standard
cetuximab and cisplatin at the same dose and schedule,
during which progressive disease had occurred [1].
Acneiform rash was the most common toxicity.
Burtness et al. randomized 117 patients who had
recurrent or metastatic disease to receive cisplatin every
4 weeks with weekly cetuximab or placebo [7]. Response
rate was higher with the addition of cetuximab, but PFS
and OS were not improved. Survival was better for
those patients who developed the skin rash.
Bourhis et al. treated 53 patients who had recurrent or
metastatic disease with a combination of cetuximab,
cisplatin or carboplatin, and escalating doses of 5FU as
initial therapy [5]. Dermatologic toxicity was the most
common adverse event, but the most common grade 3
or 4 adverse events were leucopenia (38%), asthenia
(25%), thrombocytopenia (15%), and vomiting (14%).
Chan et al. treated 59 patients who had recurrent or
metastatic nasopharyngeal cancer that had recurred after
cisplatin, with cetuximab and carboplatin [8]. Six
patients (10%) experienced serious treatment-related
adverse events during cetuximab.
719
The humanized anti-EGFR Mab h-R3 was given at
four dose levels weekly for 6 weeks in combination
with radiotherapy, to 24 patients with advanced head
and neck cancer [13]. In this trial there were some infu-
sion reactions, but no dermatologic or allergic toxicities
were noted. The overall survival of patients was consid-
ered encouraging.
Summary
The role of biotherapy in squamous cell cancer of the
head and neck is very limited at this time. Enthusiasm
for retinoid therapy alone, or in combination with inter-
feron has waned with the publication of recent random-
ized trials. The results associated with regional and
intranodal injections are interesting, but have not cre-
ated commercial interest. In the near future, monoclonal
antibody combined with chemotherapy may emerge as a
new standard of care for systemic disease.
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21.10 Biological therapy of glioblastoma
ROBERT O. DILLMAN

Current Status of Therapy External Beam Radiation therapy (EBRT) is a stan-

dard component of the initial treatment of GBM, prefer-
for Glioblastomas ably following surgical resection, but is also used as
primary therapy in patients who are not surgical candi-
Glioblastoma multiforme (GBM) or glioblastoma is a
dates [30, 46]. Improvements in the delivery of radia-
collection of morphologically and genetically diverse
tion therapy have decreased toxicity, but partial brain
poorly differentiated neoplasms which collectively are
radiation to 6,000 cGy delivered over 5–6 weeks, which
the most common and most primary adult brain tumor
remains the standard of care for newly diagnosed GBM,
[50, 52]. More than 90% of GBMs are rapidly progres-
and the use of hyperfractionation radiation therapy have
sive primary tumors without clinical or histological
had little effect on survival [82]. Stereotactic radiosur-
evidence of a less malignant precursor lesion, typically
gery and gamma knife radiosurgery have enabled more
appearing in the elderly. Primary GBM are genetically
specific delivery of even higher doses of radiation [51].
characterized by loss of heterozygosity 10q (70% of
Although a randomized trial failed to show a survival
cases), EGFR amplification (36%), p16(INK4a) dele-
advantage for the addition of a radiosurgery boost in the
tion (31%), and PTEN mutations (25%). Secondary
initial management of GBM [72], more advanced uses
GBMs progress from lower grade astrocytomas that are
of this technique to areas around the edge of the radio-
diagnosed in younger patients. These are associated
graphically defined tumor are being explored.
with TP53 mutations and a G:C–>A:T mutation at
Adding chemotherapy to surgery and radiation ther-
CpG sites (60%). When these lower grade lesions
apy does improve the survival of patients with newly
progress to GBM, they acquire additional mutations
diagnosed GBM. A meta-analysis of randomized trials
including loss of heterozygosity 10q (70%). Primary
of adjunctive chemotherapy determined that the 2-year
GBM are associated with a much worse prognosis than
survival for 884 patients treated with radiation therapy
secondary GBM.
alone was only 16% compared to 23% for 1,538 patients
Despite recent therapeutic advances, GBM remains
who received radiation and chemotherapy [29]. Recently,
highly lethal when treated using standard measures [62,
concomitant and adjuvant chemoradiotherapy with oral
66, 75]. The refractoriness of GBM probably relates, in
temozolomide has become widely accepted as the stan-
part, to microscopic local extension which is beyond the
dard treatment for newly diagnosed GBM based on a
areas treated by surgery and radiation therapy [49].
randomized trial that showed improved median survival
Also, there is a limited therapeutic index between brain
from 12 months to 15 months, and the 2-year overall
and tumor which limits radiation therapy. Tumor cell
survival from 8% to 26% [74, 75] meta-analysis of 16
heterogeneity [6], and the limitations of the blood brain
randomized trials comparing chemotherapy to no che-
barrier [76], conspire to impair the effectiveness of most
motherapy showed a survival advantage for the use of
systemically administered therapies.
chemotherapy, but the impact of temozolomide was
Surgery is still the mainstay of standard therapy,
much greater than that of local or systemic carmustine
although a substantial number of patients are not
therapy [71].
candidates for surgery either because of the anatomic
location of the tumor or because of comorbid medical
conditions. Historically, following surgery alone, the
median survival for GBM was only 35 weeks, with a Biological Therapy
1-year survival of 33%, and a 2-year survival of 0%
[41, 70]. The ability to perform a near-total resection
of Glioblastoma
has been enhanced by microsurgical techniques with The potential clinical role of immunotherapy in the
navigational guidance, but GBM is rarely completely management of glioblastomas is under investigation.
resected, which is why surgery alone seldom, if ever, is The early forays with biological therapy, were not
curative [10, 42, 84]. associated with improved outcomes [37, 81]. There has

R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy, 723
© Springer Science + Business Media B.V. 2009
724 Biological therapy of glioblastoma

recently been a rekindling of enthusiasm for vaccine Interferon-alpha

approaches, anti-angiogenesis agents, gene therapy
strategies, newer monoclonal antibodies, and intracra-
nial lymphocyte therapy, but these approaches are still
under development.
Non-specific Immune Stimulators
There was limited investigation of the early non-specific
immune stimulants such as bacillus Calmette-Guerin
(BCG), Corynbacterium parvum, or levamisole. In one
small study five patients with malignant gliomas who
had undergone BCG inoculation were injected
intratumorally with PPD in an effort to induce an
intratumoral delayed type hypersensitivity reaction [2].
There was a subjective increase in inflammation based
on biopsies obtained before and after the PPD injections,
but there was no evidence of an antitumor effect. Other
studies combined these agents with other treatment. In
one trial BCG, and in another levamisole, were combined
with nitrosourea chemotherapy following surgery and/or
radiotherapy, but neither agent appeared to enhance the
anti-tumor effect [93]. No survival benefit was obtained
in a trial in which 25 patients with malignant glioma
were randomized to receive radiation therapy alone or
with oral levamisole [94].
Interferons
As summarized in Table 1, limited anti-tumor activity
has been documented in clinical trials in malignant
gliomas exploring the activity of lymphoblastoid inter-
feron [56], interferon alpha [Chang et al. 1998, 54], or
interferon beta [78, 90, 92].
Intratumor injections of leukocyte interferon were not
associated with significant responses in 17 patients with
malignant gliomas, 12 primary and five recurrent [40].
Most trials have utilized i.v. or s.c. routes to deliver
IFN-α systemically. In a trial in which IFN-α was given
for 8 weeks, three patients with glioblastoma exhibited
a clinical response [54]. There appeared to be a possible
relationship between IFN-α gene status and tumor
response. No responses were noted in 18 patients with
recurrent glioma who received thrice weekly s.c. IFN-α
with oral tamoxifen (Chang et al. 1998). In 29 patients
with recurrent anaplastic astrocytoma and glioblastoma,
the combination of IFN-α with eflornithine, an irrevers-
ible inhibitor of ornithine decarboxylase was associated
with no objective tumor responses [15]. As in other
tumor types, IFN-α was combined with chemotherapy
for patients with malignant gliomas. In one phase II trial
the combination of BCNU and IFN-α produced objec-
tive response in 7/21 (33%) [13]. In another phase II
trial in 35 patients with recurrent malignant glioma, the
combination of IFN-α and BCNU chemotherapy pro-
duced an objective response rate of 29% [14]. This
encouraging results led to a trial in which 383 malignant
glioma patients were treated with radiation therapy and
BCNU, then 275 who had experienced disease progres-
sion at the end of therapy, were randomized to receive
more BCNU alone or in combination with IFN-α [16].
There was increased toxicity associated with the use of
IFN-α but no improvement in outcome.
Several trials have explored the use of IFN-α in com-
bination with radiation therapy. A retrospective review
of 175 patients with malignant gliomas suggested that
the highest complete remission rates were in patients

Table 1. Single-agent activity of various biologicals in patients with malignant gliomas

Modality class Biological agent Lead author Patients Response rate (%)
Retinoid 13-cis retinoic acid (CRA) [69] 82 4
Retinoid 13-CRA and celecoxib [43] 25 0
Retinoid All-trans retinoic acid [96] 34 3
Retinoid All-trans retinoic acid [97] 30 10
Cytokine Interferon-α + tamoxifen [95] 18 0
Cytokine Interferon-α + eflornithine [15] 29 0
Cytokine Interferon-α [54] 30 10
Cytokine Lymphoblastoid interferon [56] 14 0
Cytokine Interferon-β [92] 14 0
Cytokine Interferon-β [78] 13 0
Cytokine Interferon-β [3] 21 19
Cytokine Interferon-β [92] 65 28
Monoclonal antibody EMD 55,900 (Mab 425) anti-EGFR [73] 16 0

EGFR = epidermal growth factor receptor.

Robert O. Dillman
who received the combination of radiation therapy,
nitrosourea, and IFN-α [87]. In 19 patients with primary
glioblastoma, only two of whom were able to undergo a
near total resection, the combination of radiation ther-
apy and thrice weekly 3–5 MIU s.c. of IFN-α was asso-
ciated with a median survival of 7.5 months [22]. In
vitro experiments suggested that combining 13-cis
retinoic acid with IFN-α was associated with even
greater radiosensitizing effects against glioblastoma cell
lines [45]. A subsequent trial of these agents was com-
bined with radiation therapy in 40 patients, only four of
whom underwent near total resection, and resulted in a
median survival of 9.3 months with a 1-year survival of
42% [23]. Several patients experienced severe radiation
necrosis, suggesting that enhanced radiosensitization
had been achieved in vivo.
Systemic IFN-α was given concurrently with intral-
esional interleukin-2 (IL-2) in five patients with recur-
rent malignant glioma [48]. IL-2 was given by this route
because of the induction of cerebral edema by high
doses of systemic IL-2. As part of this study four patients
had received IL-2 alone and cerebral edema was
observed in two patients who received 50,000 IU intral-
esionally. In the combination study, systemic IFN-α
was given at dose of 3 MIU to 50 MIU i.v. weekly and
IL-2 was limited to 10,000 IU intralesionally. The com-
bination IL-2 + IFN-α was associated with weakness
and fatigue, and two patients exhibited increased peritu-
moral edema on radiologic scans. Because of toxicity,
this combination has not been pursued.
Interferon-beta
Results with interferon-beta (IFN-β) may be somewhat
better than what has been reported with IFN-α. No
responses were observed in 14 patients with relapsed
high grade gliomas who were treated with lymphoblas-
toid interferon [56]. IFN-β (Betaseron) was given by
i.v. infusion over 30 min thrice weekly to 29 pediatric
patients with a variety of primary malignant brain
tumors, including 12 high-grade astrocytomas and nine
brain stem gliomas [3]. Objective responses were noted
in 4/21 evaluable patients. No objective responses were
recorded in ten patients in a pilot study in which IFN-β
(Betaseron) was i.v. thrice weekly at a dose of 90 MIU
to 14 adult patients with recurrent malignant glioma
[90]. In a larger trial using higher i.v. thrice weekly
dosing, 15/65 patients with recurrent or non-responsive
malignant glioma had an objective response [92].
Histologically, 41 had glioblastoma and 24 had
anaplastic astrocytoma. A thrice weekly dose of 180
MIU was felt to be the optimal dose based on toxicity
725
and clinical activity. No activity was noted in a small
study of 13 patients treated with a different IFN-β prep-
aration (Fiblaferon) [78]. No anti-tumor effects were
observed in six patients with glioblastoma multiforme
who were treated with local injection of IFN-β through
an Ommaya reservoir [8]. In another trial, 55 of 109
patients with newly diagnosed supratentorial glioblas-
toma had stable disease following 60 Gy radiation ther-
apy, and were then treated with adjuvant IFN-β [19].
Their median survival of 13.4 months was encouraging
based on comparisons to historical controls in the
Radiation Therapy Oncology Group glioma historical
database. Japanese studies have combined IFN-β with
other agents resulting in encouraging survival results
[4, 80], but randomized trials are need to see whether
IFN-β adds anything to current regimens that utilize
temozolomide.
Interferon-gamma (IFN-γ)
In a small randomized trial, 31 patients with high-grade
gliomas were randomized to receive intralesional
recombinant IFN-γ prior to and after radiation therapy,
or standard RT after surgical debulking [31]. Intratumoral
IFN-γ was given thrice weekly for 4 weeks until radio-
therapy, with doses escalated from 5 to 50 micrograms,
and after radiotherapy, was resumed at a dose of 50
micrograms twice a week for up to 9 weeks. Median
survival was 54–55 weeks in both arms.
Retinoids
Several laboratories have shown that retinoic acid
has growth-inhibitory activity against glioma cells
in vitro in addition to immune modulating and cell
differentiation effects. A small number of trials have
examined the activity of 13-cis-retinoic acid (13-CRA)
as a single agent in the treatment of malignant gliomas.
In a phase II trial conducted in 50 patients with pro-
gressive or recurrent disease after radiation and che-
motherapy, there were three objective responses [91].
These same patients were included in a retrospective
radiographic analysis of 85 patients with recurrent
GBM who were treated with 13-CRA at MD Anderson
Cancer Center, and an objective response rate of 4%
was noted for 82 patients [69]. In a small pilot study,
13 patients with GBM and ten patients with anaplastic
astrocytoma who had achieved a complete response
after primary surgery, radiation therapy, and chemother-
apy, were given 13-CRA at a dose of 60 mg/m2 daily for
3 weeks of each 4-week cycle with a dose escalation of
up to 100 mg/m2 [85]. Treatment was well tolerated,
726
but even in this highly selected group of patients with
a high representation of non-GBM histology, the
median survival was still only 17 months from the time
of entry into the study.
Other trials have combined 13-CRA with other
agents. 13-CRA plus the COX-2 inhibitor celecoxib
yielded no responses in 25 patients with recurrent glio-
blastoma [43]. In a large phase II trial, 88 patients with
recurrent or progressive malignant glioma were treated
with a combination of temozolomide plus 13-CRA [38].
Histolologies included 40 glioblastoma multiforme, 28
astrocytoma, 14 oligodendroglioma, and six mixed
glioma. There were ten responses among 84 evaluable
patients (15%). 13-CRA was added to radiation therapy
and temozolomide in the treatment of 61 adults with
newly diagnosed supratentorial glioblastoma [17]. The
median survival was 12.2 months and 1-year survival
was estimated to be 57%. 13-CRA was added to radia-
tion therapy and IFN-α in the treatment of 40 adults
with newly diagnosed supratentorial glioblastoma, only
four of whom underwent near total resection [23]. The
median survival was 9.3 months, and 1-year survival
was 42%.
All-trans-retinoic acid (ATRA) has also been explored
as a biotherapy for malignant gliomas. In a phase II trial,
one of 34 evaluable patients with recurrent glioma had
an objective response after two cycles of 120–150 mg/m2
given daily for 3 weeks of each 4-week cycle (Kaba 1997).
The authors concluded that single agent ATRA has no
significant activity against recurrent cerebral gliomas.
In another trial, 30 patients with malignant gliomas,
including 14 with glioblastoma multiforme and 14 with
anaplastic astrocytoma, three (10%) had an objectivew
response after treatment with all trans-retinoic acid
(ATRA) [97].
Adoptive Cell Therapy
The use of LAK and other cell therapies are being
explored in the management of malignant brain tumors
[77]. Systemic IL-2 often causes edema around brain
tumors and sometimes hemorrhage. For this reason,
patients with brain tumors have been excluded from
most treatment protocols utilizing systemic IL-2.
However, many investigators have explored the feasi-
bility of direct instillation of activated cells, with or
without IL-2, directly into brain tumors or areas in
which brain tumors have been excised. The validity of
alleged objective tumor regressions in many of these
studies is in question, since they LAK were injected into
a post-surgical tumor bed making interpretation of
response quite difficult.
Biological therapy of glioblastoma
Intravenous adoptive cell therapy approaches
Although the blood brain barrier is perceived as a limi-
tation to systemic infusional approaches for adoptive
cellular therapy, some investigators have attempted this
as well. In terms of systemic adoptive cell therapy, one
group has tried i.v. approaches with two different cell
products. In the first study, following resection of recur-
rent anaplastic astrocytoma or GBM, 15 patients were
immunized with BCG and their own irradiated tumor
cells, then 2 weeks later peripheral blood mononuclear
cells were harvested by leukopheresis, cultured in vitro
with irradiated autologous tumor cells and IL-2 then
infused i.v. without additional IL-2 [33]. Feasibility and
safety were confirmed, but clinical benefit was not
apparent. In the second study, these same investigators
took nine patients with recurrent, but surgically resect-
able anaplastic astrocytoma or GBM, tapered them off
steroids after total surgical resection and immunized
them with autologous cancer cells from the resected dis-
ease that had been admixed with BCG [86]. They then
collected peripheral blood mononuclear cells which
were stimulated and expanded in vitro with anti-CD3
monoclonal antibody and IL-2, then 1010–1011 activated
cells were infused into the patients. The study design
makes it hard to interpret claims of objective tumor
response in these patients, but two patients were consid-
ered disease-free more than 4 years later.
T cells derived from lymph nodes have also been
infused i.v. Following surgery, 12 patients with newly
diagnosed gliomas (half lower grade, half GBM) were
vaccinated i.d. with GM-CSF + short-term cultured
autologous irradiated tumor cells, then lymphocytes
from resected draining lymph nodes were stimulated
with staphylococcal enterotoxin A for 48 h and then
cultured in IL-2-containing medium 6–8 days, then
109–1010 cells were administered i.v. to the patients [55].
The cell products were mostly CD4+. Four patients
were interpreted as having partial regression of residual
tumor.
Localized Adoptive Cell Therapy Approaches
Most efforts with adoptive cellular therapy for malignant
gliomas have utilized localized approaches. In a phase
I trial, nine patients received LAK and/or IL-2, and one
patient received both together [36]. Escalating doses of
LAK cells, 108–1010 or recombinant IL-2, 104–106 units
were suspended in 5 ml of Hanks balanced salt solution
and were directly injected into the tissue surrounding the
surgical cavity remaining after tumor extirpation using
multiple injections via 20 gauge needles. LAK cells were
Robert O. Dillman
developed within 2–3 days of surgery. There was no
comment regarding whether patients received steroids
before and/or immediately after surgery and immuno-
therapy. None of the ten patients were felt to have exhib-
ited toxicity beyond that normally seen post-craniotomy.
In another trial, at the time of tumor resection, a catheter
was implanted in the cavity and used to deliver 6 MIU/
day IL-2 by continuous infusion for 5 days, with or with-
out LAK [11]. In five patients, LAK cells were infused
into the cavity on days 1, 3, and 5 after surgery, and eight
patients received IL-2 alone. Fever, confusion, and cere-
bral edema were observed in all patients and there was
no evidence of clinical benefit.
Several trials utilized instillation of LAK with IL-2.
In one trial, 23 recurrent glioma patients were treated
with 107–108 LAK cells with 50–400 units of a recombi-
nant IL-2 via Ommaya reservoir [88]. LAK were gener-
ated 4–6 days prior to instillation. Many patients had
substantial neurological deficits at the time of treat-
ment.. Side effects noted were chills and fever.
Substantial improvement occurred in at least three
patients. In another small study, 13 recurrent glioma
patients were treated with LAK generated 3–5 days
prior to craniotomy and suspended in 5–10 ml saline
with 106 units of Cetus IL-2 [47]. Daily injections of 106
units of IL-2 were given for 3 additional days. A second
cycle of IL-2/LAK was infused via Ommaya reservoir
1–2 weeks later. All patients had symptoms of aseptic
meningitis, including increased intracranial pressure,
headache, fever, and malaise. The authors commented
on the difficulty of attributing some of these effects
to IL-2 as opposed to post-craniotomy. They felt that
the approach was sufficiently safe to try on newly diag-
nosed patients, although all of these patients were on
dexamethasone, which is known to inhibit IL-2 and
LAK activity. Barba et al. treated ten recurrent glioma
patients with LAK cells and IL-2 given via a catheter
stereotactically placed in the tumor bed and connected
to an Ommaya reservoir [5]. Three weeks after surgery,
therapy was begun with 1010 LAK cells in 10 cc of
saline. IL-2 was infused for 5 days via the same cath-
eter at 10,000–60,000 U/kg/dose, three times per day.
Neurologic side effects occurred in all patients because
of intracerebral edema. Glucocortocoid therapy was
limited during the immunotherapy, but patients were
then treated with high-dose dexamethasone after com-
pleting IL-2/LAK. One of nine evaluable patients was
considered to have had a beneficial response. Lillehei et al.
treated 11 patients with recurrent high-grade gliomas
[98]. Peripheral blood lymphocytes were stimulated in
one of two different culture conditions and placed into
the tumor cavity with IL-2 in a plasma clot. The survival
727
following therapy ranged from 3.5 months to >3 years,
with a median survival of 4.5 months. Three of nine
patients with recurrent GBM, who were treated with
LAK and IL-2 administered directly into the tumor cav-
ity via an Ommaya reservoir, were interpreted as having
an objective response to treatment, although long-term
survival was not achieved [9]. One of ten patients with
recurrent GBM, who were treated similarly with local
instillations of LAK and IL-2, were felt to have had a
partial response [67].
Hayes et al. gave LAK cells to 19 out of 44 possible
patients with recurrent glioma who were considered
candidates for resection [32]. Fifteen tumors were clas-
sified as gliomas (grade 4) and four as anaplastic astro-
cytomas (grade 3). Patients underwent leukapheresis
within 1–6 weeks of resection after discontinuation or
reduction of corticosteroids. LAK cells were produced
by incubating the mononuclear cells in 6 million inter-
national units/ml of IL-2 for 4 days. A total of 0.5 – 1 × 106
LAK were infused via Ommaya reservoir on day 1, with
a maximum-tolerated IL-2 dose of 1.2 MIU/ml IL-2.
The same dose of IL-2 was infused without cells on
days 3, 5, 8, 10 and 12, which constituted one treatment
cycle. The 12-day cycle of therapy was repeated about 2
weeks later. Patients who had not progressed after two
treatment cycles could be re-treated at 12-week intervals.
Since all of these patients had maximum tumor debulk-
ing at the time of surgery, tumor response could not have
been a meaningful endpoint of this trial, although the
authors reported one complete and two partial responses.
However, median survival from the date of operation
was 53 weeks for the 15 glioblastoma patients, with a
1-year survival of 53% compared to a median survival
of only 26 weeks, versus a 1-year survival rate of 5% for
18 contemporary controls who underwent debulking
and then received chemotherapy.
The largest phase II trial to date enrolled 40 patients
with pathologically confirmed GBM at the time of sur-
gery, who subsequently had placement of autologous
LAK cells into the tumor cavity [25]. LAK cells were
generated by incubating peripheral blood mononuclear
cells with interleukin-2 (IL-2) for 3–5 days in vitro.
Because of the inherent difficulties in interpreting a
tumor response in such patients who have undergone
surgical resection, survival was compared to contempo-
rary matched controls. Median survival post-LAK was
9.0 months; 1-year survival was 34%. Gender, age,
location of tumor, and number of cells implanted were
unrelated to outcome. Inclusion of IL-2 at the time of
cell instillation was associated with improved survival
(p = .097). Median survival from date of original diag-
nosis for 31 patients who had GBM at initial diagnosis
728
was 17.5 months versus 13.6 months for a control
group of 41 contemporary GBM patients (p = .012).
The median survival rates were higher than reported
in most published series of patients who had undergone
re-operation for recurrent GBM. This approach is
currently being combined with surgery, radiation
therapy and temozolomide and preliminary results are
encouraging [26].
In a slightly different approach, peripheral blood
lymphocytes were stimulated with both phytohemag-
glutin (PHA) and IL-2 to produce what were called
mitogen activated killer (MAK) cells. In a phase I/II
trial involving 55 patients with various glioma histolo-
gies, MAK were generated over 7–10 days prior to cran-
iotomy, then suspended in 15 ml of autologous
EDTA-plasma (total volume 15–20 ml) for placement in
the tumor bed following surgery [34]. Therapy was gen-
erally well tolerated, other than fever and nausea, and
most patients were discharged within 5–7 days after
craniotomy. There was no effort made to judge tumor
response, but survival for the patient who underwent
treatment was considered encouraging. MAK were also
used to treat 19 patients with high-grade gliomas [39] At
the time of treatment both PHA and IL-2 were placed in
the tumor site as well in 16 patients. Three patients were
felt to have a significant decrease in radiologic enhance-
ment of the tumor area.
Because of the paucity of lymphocytes found in
malignant gliomas, few investigators have explored the
use of tumor infiltrating lymphocytes (TIL) as adoptive
cell therapy for GBM. Following surgical resection and
placement of an Ommaya reservoir, six patients were
treated with low doses of TIL every 2 weeks concurrent
with thrice weekly low doses of IL-2 for 1 month [57].
Three patients were interpreted as having a partial
response to the treatment.
Antibody Therapy
The epidermal growth factor receptor (EGFR) is
expressed on malignant glioma cells; and therefore has
been a target for antibody-based therapy of glioblas-
toma. EMD55900 (Mab 425) is a murine IgG2A Mab
directed against EGFR. Mab 425 was given i.v. to 16
patients previously treated with surgery, radiotherapy
and chemotherapy for high grade supratentorial malig-
nant gliomas (11 GBM) [73]. There were no tumor
responses including the last six patients who received
200 mg thrice weekly for 4 weeks. Single doses of 20,
40, 100, 200, or 400 mg of the murine MAb 425 were
given i.v. before surgery to 30 patients with malignant
brain tumors [28]. Treatment was well-tolerated and in
Biological therapy of glioblastoma
vivo binding was confirmed. Eight patients with primary
or recurrent EGF-R-positive glioblastomas were treated
intratumorally twice weekly through an implantable
catheter with total doses of 4 mg and 120 mg of Mab 425
[83]. The treatment induced an intense inflammatory
reaction and a substantial necrosis. In an early pilot
study, 25 patients with primary malignant glioma were
given i.v. or intra-arterial I-131-labeled anti EGFR (425)
4–6 weeks following initial surgery and radiation ther-
apy [12]. The median survival was 16 months for the 15
patients with GBM. During 1987–1997, this group
treated 180 patients following surgery and radiation
therapy, with and without chemotherapy, with an aver-
age total dose of 140 mCi given typically as three weekly
injections [27]. The median survival for patients with
GBM was 13 months.
The humanized anti-EGFR Mab h-R3 was given to
29 patients with newly diagnosed malignant gliomas
including 16 glioblastoma, 12 anaplastic astrocytoma
and one anaplastic oligodendroglioma [58]. Following
debulking surgery, patients received six 200 mg infu-
sions of h-R3 weekly during radiotherapy. Interestingly,
no patients developed acneiform rash, and there were no
allergic reactions. Median survival was 17 months for
the glioblastoma patients. A trial of the anti-EGFR mab
cetuximab with temozolomide and radiation therapy is
being conducted in patients with previously untreated
glioblastoma [21].
For many years investigators at Duke University have
been testing monoclonal antibodies (Mab) that react
tenascin, which is a large, disulfide-bonded glycopro-
tein of the extracellular matrix that is highly expressed
in the stroma of gliomas, but not normal brain tissue.
Radiommunotherapy trials have been carried out with
murine Mab 81C6, but a chimeric version has also been
tested. In a phase I trial conducted in 34 previously irra-
diated patients with recurrent or metastatic brain tumors,
the maximum tolerated dose for 131I-labeled 81C6
administered through an Ommaya reservoir into the
surgical resection cavity was 120 mCi based on dose-
limiting delayed neurologic toxicities [7]. Patients with
recurrent GBM had an encouraging survival of about
13 months. The same MTD was determined in a phase
I trial involving 42 newly diagnosed patients who had
not received prior radiation therapy or chemotherapy
[18]. Median survival for patients with GBM was 16
months. A small number of patients had to undergo sur-
gical excision of symptomatic radionecrosis. In a subse-
quent trial, newly diagnosed patients were treated with
direct injections of 131-I-labeled anti-tenascin murine
81C6 into the surgical cavity followed by conventional
external-beam radiotherapy and chemotherapy [1].
Robert O. Dillman
Biopsies from 15 patients all showed tumor and/or radi-
onecrosis. Subsequently, a phase II trial was carried out
in 33 newly diagnosed patients (27 GBM) using 120 mCi
of 131-I-labeled murine 81C6 injected directly into the
surgical resection cavity prior to receiving conventional
external-beam radiotherapy, followed by a year of alky-
lator-based chemotherapy [59]. Median survival for the
GBM patients was 18 months. In a phase II trial in 43
patients with recurrent gliomas (33 GBM), 100 mCi of
131-I-labeled murine 81C6 was injected directly into
the surgical resection cavity. The median overall sur-
vival for patients with recurrent GBM was 15 months
[60]. In animal models, a 131-I-labeled IgG2/mouse
chimeric antitenascin 81C6 (ch81C6) construct exhib-
ited higher tumor accumulation and enhanced stability
compared to the murine antibody, but in 43 patients the
maximum tolerated dose was only 80 mCi because of
dose-limiting hematologic toxicity associated with a
longer serum half life [61].
Another group has conducted trials with murine anti-
tenascin Mabs called BC-2 and BC-4, which were radi-
olabeled with I-131. In one trial, an early analysis of 17
of 23 patients with recurrent malignant gliomas who
had received intracavitary doses ranging from 15 mCi to
57 mCi, showed a median survival of about 16 months
[63]. In an expanded group of 50 patients (24 with pri-
mary and 26 with recurrent disease) the median survival
was 18 months for 26 patients with recurrent disease
and 20 months in 24 patients with newly diagnosed
lesions [64]. BC-4 was labeled with Y-90 and adminis-
tered to another 20 patients with recurrent high grade
gliomas (18 GBM) using doses ranging from 5–30 mCi
[65]. The maximum tolerated dose to the brain was
25 mCi delivering 3,200 cGy/mCi.
In patients who have experienced recurrence of GBM,
encouraging results for the combination of irinotecan
and the anti-vascular endothelial growth factor mono-
clonal antibody bevacizumab have been reported. In a
phase II trial, adult patients with recurrent anaplastic
astrocytoma or glioblastoma (grade III or IV glioma)
received bevacizumab at 10 mg/kg and irinotecan i.v.
every 2 weeks of a 6-week cycle [79]. The dose of irino-
tecan was determined based on whether patients were
taking antiepileptic drugs that induced hepatic enzymes
that accelerate metabolism of irinotecan. Patients taking
enzyme-inducing antiepileptic drugs received irinote-
can at 340 mg/m2, while other patients received irinote-
can at 125 mg/m2. Although the significance of
radiographic changes in treated gliomas is questionable,
based on study design, the authors alleged an overall
response rate of 63% for all patients. In other trials of
bevacizumab, patients with brain metastases were
729
excluded because of concerns that there might be an
increased risk of intracerebral hemorrhage. However, in
patients with malignant gliomas, there were no instances
of central nervous system hemorrhage, but four patients
(12%) experienced thromboemboic complications.
Vaccine Therapy
A variety of vaccine approaches are being explored in
patients with glioblastomas. These include Newcastle-
Disease-Virus (NDV) infected autologous tumor cells
derived from short term cultures [68], irradiated autolo-
gous tumor cells from continuously proliferating cell
cultures [24], autologous dendritic cells pulsed with
lysates of autologous tumor [89], autologous dendritic
cells pulsed with of acid-eluted peptides from autolo-
gous tumor [44], autologous tumor vaccines prepared
from formalin-fixed and/or paraffin-embedded tumor
tissue obtained at the time of surgery [35], autologous
glioma cells and interleukin (IL)-4 gene transfected
fibroblasts [53] and intratumor injection of Herpses
Simplex retroviral vector-producing cells, followed by
intravenous ganciclovir [20]. These were mostly small
pilot studies to demonstrate safety, involving only 10–15
patients in most instances.
Summary
Currently there are no biological products that have
a regulatory approval for marketing as treatment for
malignant gliomas, although several appear to have some
activity. Whether any of these products become widely
available and paid for by payment providers will depend
on the choices made by large biotechnology companies.
Because of current treatment paradigms, a successful
biological will almost certainly have to be integrated into
existing treatment algorithms as part of treatment of newly
diagnosed glioblastoma. Unintentional patient selection
issues and difficulties in measuring a tumor response,
especially as these relate to local instillation of cell therapies
and antibodies, makes it difficult to determine the clinical
significance of apparent improvements in survival.
Randomized trials will be needed to determine if any of
these therapies are adding benefit.
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22 Speculations for 2009 and beyond
ROBERT K. OLDHAM
It is now apparent that the genomic capacity of mam-
malian cells to produce biological substances that have
medicinal value is enormous. Now that a substantial
number of lymphokines/cytokines, growth and matura-
tion factors, cellular therapies, and antibody-based
approaches have been explored, there is clear evidence
of clinical activity with respect to colony-stimulating
factors (CSF), blocking factors for epidermal growth
receptors (EGF) and vascular endothelial growth factor
(VEGF), interferon (IFN), interleukin 2 (IL-2), acti-
vated cells, vaccines, monoclonal antibodies, and their
immunoconjugates. If one analyzes the current level of
clinical activity for biotherapy in the historical context
of chemotherapy, it is obvious that much more rapid
progress is being made in biotherapy, leading to devel-
opment of improved, less toxic and more selective forms
of treatment. From the earliest days of chemotherapy,
massive searches were done among tens of thousands of
chemical compounds, searching for the one right drug
that might have antitumor activity without excessive
toxicity for the patient. The successes were few. From
analyzing more than a million chemical structures,
less than 80 anticancer drugs have come to the clinic,
and no more than 30 of these can be considered even
moderately active.
This is not to say that the drug development paradigm
has not had some success. However, the search has been
arduous, complex, expensive, and almost wholly based
on the process of random screening of large numbers of
compounds to find the rare active chemical. By contrast,
the first genetically engineered biological to be approved
as an anticancer agent, alpha-interferon, has been an
unqualified success in the clinic. It has been the drug of
choice for the treatment of hairy-cell leukemia and
chronic myelogenous leukemia (until even better tar-
geted therapies were developed) and is moderately
active in other forms of leukemia and lymphoma.
Substantial activity has been reported in renal cancer,
melanoma, and selected other tumors.
IL-2 is now approved for renal cancer and melanoma.
Monoclonal antibodies and their immunoconjugates
have proven broadly effective, and the first inhibitors of
EGF receptor and VEGF have been approved. Thus,
biotherapy and small molecule targeted therapy can
R.K. Oldham and R.O. Dillman (eds.), Principles of Cancer Biotherapy,
© Springer Science + Business Media B.V. 2009
claim a high success rate in terms of new substances that
have come through the complete testing program with
approval for clinical use.
The process of drug development was historically
based on the idea that sufficient random testing would
identify those active substances that could selectively
kill cancer cells. Ancillary was the hope that specific
processes, unique to cancer cells, would be identified
that might allow chemicals to be selectively active on
those cells. A secondary hypothesis that rational drug
development could be based on structure–function rela-
tionships, receptor information, or analog development
has proven equally difficult. These concepts have been
difficult to validate, and the level of selectivity for che-
motherapy is very low. In fact, for every chemothera-
peutic agent, there are normal tissues that are probably
more affected by the drug than the cancer in which the
drug is active. While there is still hope for the discovery
of some unique chemotherapeutic drug that is selec-
tively active against cancer, this goal seems much less
likely to be achieved than it was a decade ago.
In contrast, biotherapy and targeted therapy work
through physiologic molecules for which the body has
receptors and known mechanisms of action. These sub-
stances are often used in pharmacological amounts, but
they are message molecules known to our cellular sys-
tems. Emerging evidence in laboratory animal model
systems raises the hope that cancer cells are not com-
pletely without the ability to be controlled by appropri-
ate growth, differentiation, and antiproliferative
influences. Most of the data for interferon assumed an
antiproliferative activity on cancer cells, in that higher
doses have generally been more effective, and tumors
resistant to lower doses sometimes respond to an
increased dose. In addition, the doses active in many
cancers are well above the level where interferon is most
active as an immunomodulator.
As the process of developmental therapeutics for bio-
therapy is expanded, there is a great need for all those
involved to remain open-minded with respect to new
paradigms for drug development. Simply to presume that
biologicals must fit into the mold used for chemotherapy
drug development has been a major error, since the
process of developmental therapeutics for biologics is
733
734
very different from that for drugs. Even in the preclini-
cal screening programs now in use for biologicals, the
process is more one of rational selection than of random
screening – the need is to exploit the activities of known
molecules rather than to search among the thousands for
the singularly active substance. As the information base
in cancer biology grows, it will become increasingly
important to exploit what we know of the power of bio-
logical molecules rather than to search among unknown
chemicals for active substances.
Beyond Interferon
Alpha-interferon is an approved agent for hairy-cell
leukemia, chronic myelogenous leukemia, Kaposi’s
sarcoma, renal cancer and melanoma; it is also active
against a variety of other cancers. Beta- and gamma-
interferon are both now approved for clinical use.
Beyond these three interferons, the process of genetic
engineering is bringing forth a huge number of interferon
molecules for testing. Given any small protein of 140–
160 amino acids, the possibility now exists for making
an infinite number of molecules simply for altering
portions of the amino acid chain. This process is
underway for the interferons, and will demonstrate
whether more active interferons can be developed to
enhance the immunomodularity, antiviral, or antipro-
liferative activity of the natural molecules.
Lymphokines/cytokines
Interleukin 2 came into the clinic in the mid-1980s by
way of a process that used the growth factor to activate
lymphocytes that could subsequently be used therapeu-
tically as effector cells in patients. The cancer-killing
capacity of these activated cells was apparent from their
in-vitro activity, and the major questions related to
their in-vivo use, distribution, and anticancer activity.
Interleukin 2 is being used in amounts far in excess of
its normal physiologic role, and toxicities are the natural
result. This particular approach was very expensive; it
required hospitalization and truly enormous doses of
both IL-2 and the activated cells. The future may bring
a better strategy for using this approach in patients,
whereby repeated treatment may become less toxic and
more active than the current intensive regimens.
Outpatient regimens that are much less toxic are now
used more frequently.
Interleukin 2 is part of a broad cascade of biological
molecules that have activities in cellular activation and
Speculations for 2009 and beyond
the control of cellular proliferation. These molecules
include IL-1-32 and beyond. The future may allow the
use of these components in combination or sequence
to strengthen what are natural but ineffective bodily
responses to the problem of cancer growth and metasta-
sis. Although induction of such a cascade by one par-
ticular substance may not be feasible, the artificial
programming and the medicinal use of these lymphokines
are now both feasible and practical. As the process of
administration of biotherapy becomes better defined, so
will the programmability of therapeutic regimens using
these substances. One can envision pre-programmed
infusions of combinations of lymphokines designed for
maximal exploitation of their biological activities.
Antigen-specific Lymphokines
Over and above the use of lymphokines in a more general
sense, as just described, there is evidence that certain
lymphokines act in antigen-specific ways. As the proteins
constituting tumor-associated antigens become more
thoroughly characterized, as a result of monoclonal
antibody technology and genetic engineering, one can
envision the use of specific antigens with specific
lymphokines, thus allowing oncologists to mount antigen-
specific, and perhaps cancer-specific, immunologic
responses in individual patients. It is likely that these
antigen-specific lymphokines will have to be used in
ways that are selected specifically for individual patients.
If, as will be reasoned below, the array of tumor-associated
antigens present on the tumor cells of each individual is
slightly different from the array present on cancers of a
like type in other individuals, the use of antigen-specific
lymphokines with specific antigenic preparations will of
necessity be unique to each individual. Parallels for the
clinic are already being explored in animal models for
transplantation and other specific immune responses.
The application of this technology to human therapeutics
will occur in the not-too-distant future.
Growth and Differentiation
Factors
Cancer might be likened to a juvenile delinquent; a
derivative of one’s own self but without the controls
necessary for appropriate socialization. If cancer cells
were to have only a limited spread and growth and if
subsequent therapeutic approaches could simply limit
further growth, there would be no need to eradicate the
Robert K. Oldham
cancer cells completely. Such an approach has an obvi-
ous parallel in the use of physiologic mediators for
endocrinopathies. For example, diabetes did not disap-
pear by virtue of the discovery of insulin. The genetic
anomaly continues to exist, and diabetes has become a
more frequent clinical problem in our society. However,
insulin has been used to control the pathologic manifes-
tations of this disorder. Is it possible there are growth
and differentiation factors, coded for by the mammalian
genome, that could be used similarly for treating can-
cer? There is early evidence of such mediators in vitro
and in animal models. The control of growth factors, as
well as agonists and antibodies to those factors, provide
substance to the argument that molecular manipulation
of growth and differentiation is a major area for devel-
opmental therapeutics for cancer.
If one takes the growth and differentiation of cells in
vitro as a model, the use of epidermal growth factor,
nerve growth factor, insulin, and other hormones has a
profound effect on both the rate of replication and the
degree of deviance of replicating cells from their ‘nor-
mal’ counterparts. Similarly, cancer growth, prolifera-
tion, and spread may be controllable by manipulation of
growth factors made by normal tissues and/or by the
cancer cells themselves.
Similar approaches are now in the clinic as targeted
therapy using both monoclonal antibodies and small
molecules interacting with EGF, VEGF and their recep-
tors. Rudimentary attempts using certain chemicals that
cause in-vitro differentiation, have had interesting effects
on cell lines and experimental modes; however, the
use of retinoids, butyrates, and other factors that might
regulate growth and differentiation have had minimal
impact in the clinic. With almost daily discovery of sub-
stances that support growth of cancer cells in vitro, one
might imagine that the antithesis of such factors might
also be developed, exploited, and utilized for clinical
therapeutics. In biology, as in physics, there seems to
be a reaction for every action. For many of the mole-
cules that support growth, experimental data also indi-
cates that other molecules exist to offset that increased
growth. As these molecules are isolated and charac-
terized, and subsequently produced by genetic engineer-
ing, they will broaden our potential arsenal for cancer
biotherapy.
Monoclonal Antibodies
The current evidence to support the therapeutic use of
monoclonal antibodies and their immunoconjugates has
been presented in earlier chapters. While it is early in
735
the clinical application of this biotherapeutic approach,
it is already apparent that monoclonal antibodies offer
the probability of selective cancer treatment that has
never before been available [34].
The search for the perfect ‘magic bullet’ is probably
futile. With the exception of anti-idiotypic antibodies,
which can be quite specific for the B cell clone that has
deviated and become a malignant lymphoma or leuke-
mia, it seems unlikely that such singular and specific
antigens are represented on all cancers. Rather, the evi-
dence seems to support a bewildering array of cancer-
associated antigens present in different quantities on
cancers from different individuals.
Data are accumulating concerning the use of typing
panels of monoclonal antibodies. They indicate that
one can assess the antigenic array on each individual’s
tumor cells and decide which antibodies should com-
pose a cocktail to cover the available specificities of that
cancer. This testing mechanism reveals cancer to be
individualistic. It is different for every patient and per-
haps even among the clones of cancer present within an
individual patient. This should not be surprising, since
the genetic apparatus that governs each of us is indi-
vidualistic. With the exception of identical twins, no
two humans are exactly alike, and one would be amazed
if cancers were exactly alike. Unless a specific genetic
lesion is identified as the cause of each histological type
of cancer, each malignancy in each patient may be an
individual problem in biology. It is unlikely that a singu-
lar change leads to all forms of lung cancer, breast can-
cer, or colon cancer. It is possible that genetic changes
will result in specific markers for each of those cancers,
which can be exploited in treatment. However, it is per-
fectly obvious that the cells in which those changes
occur are genetically distinct from one person to another.
Many genetic alterations, caused by the same chemical
or physical carcinogen, result in the outgrowth of cells
that markedly differ from each other. If such is the case,
one might postulate that cancer in each patient is the
individualistic expression of a response to that multi-
step, complex carcinogenic impulse, thereby embody-
ing the genetic characteristics of both the host and the
carcinogenic influence. If such is the case, then the
actual problem in developmental therapeutics will be
individualistic and each patient will have to be analyzed
as an individual problem in cancer biology.
This concept of individuality of cancers has been diffi-
cult for many cancer biologists and clinicians to accept.
This concept was first present in the 1st Edition of this text
book in 1987 and is still being debated. Recently, it has
been “rediscovered” as “Personalized Medicine”. We have
learned and use the histological classification systems
736
embedded in the minds of pathologists and transmitted
through textbooks of medicine. These concepts classify
cancers categorically according to tissue of origin and his-
tological features. In spite of the clinical and laboratory
observation that phenotypic analysis and even ‘genotype’
in cancer biology confer great diversity within cancers of
the same histological type, we continue to develop thera-
peutics as if all breast cancer, all lung cancer, and all colon
cancer are replicates. This is the simplest to teach, the sim-
plest to learn, and the simplest to practice. However, it is
fundamentally and biologically incorrect.
Heretofore, there has never been a technology that
allows cancer biologists to understand cancer on an
individualistic basis. Monoclonal antibody technology,
proteonomics, and genomics represent what may be the
solutions to the problem. If cancer is indeed diverse
and individualistic, might not the immune system,
itself individualistic and diverse, be able to solve the
problem? Each person’s immune system has the ability
to produce several hundred thousand to several million
different antibody molecules. Taken together, the diver-
sity of the immune system from the broader perspective
of a population of individuals is gigantic. By the use of
in-vitro techniques and the cellular diversity inherent in
the immune system, it is probable that the response
capabilities in antibody technology exceed the diversity
implicit to cancer. It may well be postulated that each
cancer in each patient presents with an individual array
of antigens. If such is the case, it might be possible to
generate antibodies and/or type tumors with a multipha-
sic approach, leading to the generation of cocktails of
antibody to respond to the diversity inherent in cancer
biology. Clearly, T cells could be generated and manipu-
lated in a similar manner for specific cellular therapy.
Cancer Treatment: The Future
It has been categorically stated by various authorities
that cancer is a problem that should be largely solved or
under control within 10–20 years. The National Cancer
Institute set a national priority of reducing the cancer
death rate by at least 50% by the year 2000. These
projections were certainly optimistic, and they were not
achieved. On the horizon for surgery, radiotherapy, and
chemotherapy, there seems little to support the goal of
reducing the cancer death rate by 50% by any future
date. Although it is doubtful that the cancer death rate
can easily be reduced by this magnitude, it is likely that
the approaches available through biotherapy will play a
major role in developmental therapeutics in the next
decade. If the field is allowed to progress by using
Speculations for 2009 and beyond
the best minds of science rather than proceeding in an
overly structured and rigidified way, new paradigms for
developmental therapeutics may allow for greater strides
to be made using biotherapy.
End-point reductions in cancer death rates are difficult
to achieve, or even to detect, because of time factors. To
know that the death rate from breast cancer has been
reduced by 50%, one would have to have at least a 5-year
follow up of the patients at risk since the recurrence rate,
although highest in the first 2 years, continues to be sig-
nificant for several years after diagnosis. For lung cancer,
the problem is somewhat more straightforward, in that
end point of an effective change in therapeutics can be
discerned within 3–5 years. Breast, colon, and lung can-
cers being the most common malignancies, the reduction
of death rate by 50% in these three tumors by the year
2000 would have required that the actual change would
have to have occurred between 1990 and 1995. That sim-
ply did not occur. Although biotherapy is likely to play
an increasingly important role within the next decade, it
is doubtful that changes of such magnitude can be pro-
duced in the near term. Rather, it seems more likely that
the first 2 decades of the 2000s will be those in which the
early phases of such changes may occur, and the actual
end point for significant death-rate reduction will then
fall well after the year 2020.
It is 2009. Interferon, interleukin 1–32, growth and
maturation factors, tumor necrosis factor, colony stimulat-
ing factors, activated cells, monoclonal antibodies, immu-
noconjugates, vaccines, and gene therapy are clinical
realities – but they are all in an early phase of developmen-
tal therapeutics. If developed as drugs have been tested in
the past, the average time to take these from the laboratory
to an approved therapeutic is 10 years, at a cost of over
$800 million each. If we are to move forward in develop-
mental therapeutics for biologicals, we cannot take 10
years to develop each approach individually. Certainly, it
is impossible to imagine a series of events that will allow
an end-point reduction of 50% by the year 2020 or even
2050 if each biopharmaceutical takes 10 years to be
brought from concept to widespread clinical use.
Biotherapy is not Chemotherapy
and it not just Immunotherapy
The fourth modality of cancer treatment is constituted
more broadly to include all of the factors described in
this book [3]. To take maximum advantage of the oppor-
tunities available through biotherapy, major structural
changes are necessary in our system of translation of
Robert K. Oldham
developmental therapeutics from concept to the labora-
tory and then to the clinic [2]. We cannot afford to
develop biologicals in the protracted, expensive unidi-
mensional manner of drug development. We have a
huge number of new biological substances, and the cur-
rent system of access and opportunity for patients, the
system of funding of research, our method of govern-
ment regulation, and our reimbursement system for
developmental therapeutics must undergo major change
[4–11, 15, 18, 19, 21–27, 29–33]. We are now faced
with the reality of many more opportunities for effective
cancer therapy than mechanisms by which these oppor-
tunities can be brought to clinical reality.
For more than 2 decades, cancer research and
treatment have operated on the ‘kill and cure’ hypothesis.
Developmental therapeutic programs have functioned
under a format where a new drug is brought to the clinic
test in phase I for toxicity and phase II for activity with
the presumption that short-term effects on cancer
(response rates) will ultimately lead, if positive, to
survival benefit. While this paradigm has been useful in
developing cytotoxic drugs, there is much to suggest
that we should now broaden our concept of developmental
therapeutics, as cancer biotherapy comes to the fore, to
include the idea of cancer control. Much as one treats
diabetes with insulin, it may soon be possible, through
the use of growth and maturation factors and other forms
of cancer biotherapy, to arrest the growth and spread of
cancer and thus make these diseases more amenable to
long-term control even in the absence of tumor
eradication. The implications in terms of cost and
reimbursement for clinical services are obvious. We
cannot afford to pay for developmental therapeutics as
we have in the past. How is society going to pay for the
very long-term clinical trials, perhaps constituted over a
3–5 year period, to test the various hypotheses for cancer
control? Witness the difficulty in establishing clinical
trials for cancer prevention and the high cost intrinsic
to these programs as examples of the difficulty in
envisioning clinical research for long-term cancer
control projects. Obviously, this is going to require a
major restructuring of developmental therapeutics if
society is to take advantage of these opportunities [8–10,
12, 13, 34].
For more than 3 decades, the major factors slowing
developmental therapeutics have been the regulations
promulgated by Congress and the Food and Drug
Administration (FDA) for the development of new
drugs [14]. These regulations have resulted in a system
of drug development costing more than $800 million
per new drug taken through to commercial availability.
In the last 10 years, it has become apparent that the
737
funding for clinical research is becoming less available.
Much of the funding was derived from insurance
reimbursement for clinical trials, and with the current
pressure on insurers to reduce costs, at the behest of
employers and patients who pay for their insurance
premiums, it is becoming increasingly difficult to fund
clinical research through third-party reimbursement.
Thus, while the regulation and the intrinsic cost were
the major impediments to rapid drug development prior
to 2000, reimbursement for clinical trials and even
reimbursement for the ‘off-label’ use of new forms of
therapy will become the major limiting factor in the
future. We are now faced with the probability that cancer
cures are being developed that will not be reimbursed
by government or private insurance programs [13, 16,
17, 20].
With our current reimbursement system, autologous
bone marrow transplantation for certain forms of
cancer has reached a level of cure of 20% of patients
with selected neoplasms at a cost of $100,000–150,000
per procedure. Thus, the upper limit of the price for a
human life is becoming more clear. New technology
tends to be expensive early and becomes less expensive
later. However, we now face the probability that
certain new technologies will be available and highly
effective while still very expensive [20]. Society must
decide how much life is worth and what percentage of
the gross national product should be allocated to
healthcare. This is an area that needs public debate
and should not be simply left to ‘social planners and
thought leaders’.
Therefore, it is apparent that cancer biotherapy brings
cancer research and developmental therapeutics to a
crossroads. In this millennium, we will not be limited by
our ability to conceptualize and develop highly effective
treatment. We will be limited by broader social consid-
erations of who shall pay for such approaches and how
much each life is worth [20, 28].
Thus, it is apparently that the many opportunities bio-
therapy brings to scientists and clinicians will have to be
moved at a much faster rate from laboratory to clinic if
our patients are to derive benefit from these new
approaches. Perhaps the use of the laboratory in con-
junction with the clinical practice of medicine, as sug-
gested by the Nobel Laureate, Dr. Peter Medawar, is the
true ‘breakthrough’ in the developmental therapeutics
of cancer [1]:
“The cure of cancer is never going to be found. It is
far more likely that each tumor in each patient is going
to present a unique research problem for which labora-
tory workers and clinicians between them will have to
work out a unique solution.”
738
References
1. Medawar P, ed. The Life Sciences. New York: Harper & Row, 1977.
2. Oldham, RK. Biologicals: new horizons in pharmaceutical
development. J Biol Response Modif 1983; 2:199–206.
3. Oldham RK. Biologicals and biological response modifiers: the
fourth modality of cancer treatment. Cancer Treat Rep 1984;68:
221–232.
4. Oldham RK. The cure for cancer. J Biol Response Modif
1985;4:111–116.
5. Oldham RK. Whose rights come first? J Biol Response Modif
1985;4:211–212.
6. Oldham R`K. The government-academic ‘industrial’ complex. J Biol
Response Modif 1986;5:109–111.
7. Oldham RK. Profits: who benefits? J Biol Response Modif
1986;5:203–205.
8. Oldham RK. Patient-funded cancer research. N Engl J Med
1987;316:46–47.
9. Oldham RK. Drug development: who foots the bill? Biotechnology
1987;5:648.
10. Oldham RK. False hope vs. opportunity. Cope 1987; April:66.
11. Oldham RK. Whose life is it anyway? Wall Street J 1987;April 24.
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17. Oldham RK. Clinical research in cancer: a time for consensus.
Pharm Exec 1989;July:23–34.
Speculations for 2009 and beyond
18. Oldham RK, Avent RA. Clinical research: who pays the bill?
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19. Oldham RK. Clinical research in cancer: a time for consensus.
Mol Biother 1989;1:242–243.
20. Oldham RK. Cancer cures. By the people, for the people, at what
cost? (editorial) Mol Biother 1990;2:2–3.
21. Oldham RK. Cancer and diabetes: are there similarities? Mol
Biother 1990;2:130–131.
22. Oldham RK. Whose rights come first? Mol Biother 1991;3:
58–59.
23. Oldham RK. Cancer research: a pubic trust. BioPharm 1991;4:
8–9.
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Biother 1991:3:2–5.
25. Oldham RK The rights and wrongs of national heath care. Pharm
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zation of Cancer Research. Franklin, TN: Media American, 1992.
29. Oldham RK. Fundamentally flawed. Cancer Biother 1993;8:
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30. Oldham RK. What’s the score? Cancer Biother 1993;8:187–188.
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Cancer Biother 1994;9:99–102.
32. Oldham RK. The war on cancer: new battle plan needed. Cancer
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twenty-five years of progress. JCO 26(11):1774–1777.
 Index

1,25-(OH)2D3 544 autologous bone-marrow transplantation (ABMT) 498

4-1BB ligand 199 autologous tumor cells 507, 511, 585
azocytidine 548
activation-induced cell death (AICD) 169, 204, 276
active specific immunotherapy 5 B cells 279, 333
acute leukemia 584 cytokines 200, 208, 211, 212
acute lymphocytic leukemia (ALL) 366 interleukins 194
acute myelocytic leukemia (ALM) 366 lymphocytes 85, 88
acute myeloid leukemia 548 Bacillus Calmete Guerin (BCG) 669, 679, 713
adeno-associated viruses (AAV) 589–591 bacterial toxins 409
adenoviral vectors 591 bestatin 126, 128
adjuvant chemotherapy 128 beta particles 464
adoptive cellular therapy 652, 684 bifunctional antibodies 307
AIDS/HIV, 118, 576 biological heterogeneity 25, 23, 33
alemtuzumab 340 biological response modifiers (BRM) 1, 41, 101, 364, 484
alkylating agents 112 biological 2, 5, 41, 117, 121, 123, 125, 338, 342, 349, 613,
all-trans retinoic acid (ATRA) 527, 528 616, 660, 671, 681, 700, 715, 724
allergic reactions 321 bispecific antibodies 307
allogeneic bone-marrow transplantation (ALBMT) 497, 500 blood transfusions 109
allogeneic stem cells 652 bone cells 207
allogeneic tumor cells 512 bone marrow 497, 575, 576, 583
alpha interferon 465 breast cancer 344, 347, 348, 473, 669
alpha particles 647, 660, 681, 699, 715, 724
amifostine 685 camptothecins 596
anaphylactoid reactions 320 cancer vaccines see vaccines
angiogenesis 18, 28, 29, 179, 191, 203, 216, 599 carbohydrates 148, 150
animal models 305 CD20, 325, 366
anthracyclines 596 CD27 ligand 200
anthrax 408 CD30 ligand 200
anti-angiogenesis 292 CD40 ligand 201, 369
antibiotics 112 CD52, 340
antibodies 12, 67, 68, 71, 303, 305–312, 315, 338, 363, 377, CEA gene expression 472
640, 661, 672, 686, 696, 701, 702, 711, 735 cell cycle gene therapy 594
see also monoclonal antibodies cell functions 95
anti-idiotype 363 chemoattractants 203
current trends 640, 711, 728, 735 chemokines 203
heterogeneity 451 chemosensitization 603
radiolabelled 375 chemotactic cytokines 179
antibody-dependent cell-mediated cytotoxicity (ADCC) 312 chemotherapy 29, 113, 126
anti-CD20 antibodies 469, 497 differentiation 547
antigen-specific lymphokines 734 ex-vivo purging 498
antigenic modulation 322 immunoconjugates 455
antigens 306, 363, 364, 372, 373, 380, 381, 509, 669 immunosuppression 111, 127, 128, 130
anti-idiotype antibodies 363 interferions 285
anti-idiotype vaccines 314 monoclonal antibodies 332, 333
antimetabolites 111, 596 renal cancer 645
antiproliferative action 290 chemotherapy-induced immunosuppression 111
antisera 303, 305 chemotherapy-induced neutropenia 574, 583
antitumor cocktails 453 chimeric monoclonal antibodies 325
antiviral activity 290 chimeric proteins 71
apoptosis 200, 204, 207, 213, 216, 371 chronic lymphocytic leukemia (CLL) 122, 329, 333, 702

739
740 Index

chronic myeloid leukemia (VML) 284 gene-directed enzyme-prodrug therapy (GDEPT) 595
cimetidine 128 genetic immunopotentiation 595
clinical remission 114 genetically modified tumor cells 654
clinical trials 41, 326, 340, 344, 351, 357, 361, 457, 623 genitourinary cancer 645
antisera 305 genomics 53, 65
immunoconjugates 455, 457 glioma 474
immunotoxins 426, 427 graft-versus-host disease (GVHD) 497
monoclonal antibodies 326, 340, 344, 351, 357, 361 graft-versus-tumor (GVT) effects 500
cloning 53 granulocyte-colony stimulating factor (G-CSF) 569
colon cancer 122, 659 granulocyte-macrophage colony-stimulating factor
colony-stimulating factors (CSF) 62, 551, 569, 581 (GM-CSF) 581
colorectal cancer 351, 357, 359 growth factors 10, 527, 549, 555, 734
combination chemotherapy 113
combined-modality studies 126 hair-cell leukemia (HCL) 279, 329, 693
complement-mediated cytotoxicity (CMC) 312 head cancer 123, 358, 359, 713
conjugates 67 helper T cells see T-helper cells
copper-64, 465 hematopoiesis 165, 174, 183, 191, 544
Corynebacterium parvum 679, 714 interleukins 165, 174, 183, 191
cytokines 8, 57, 155, 549, 734 malignancies 107, 109, 114
see also individual cytkines HER-2, 68, 343, 373, 430, 453, 646, 573
current trends 647, 651, 734 herpes simplkex virus (HSV) 591
cytolytic functions 106 histamine receptor antagonists 116
cytosine arabinoside 548 historical perspective 1
cytotoxicity 95, 312 HIV/AIDS, 118, 576
Hodgkin’s disease 120, 330
daclizumab 367 hormones 113, 680
delayed allergic reactions 321 host-tumor interactions 68
delayed-type hypersensitivity (DTH) 93, 105 human anti-immunoglobulin response (HAMA/HACA/
delivery see drug delivery systems HAHA) 321
dendritic cells 165, 204, 513 human monoclonal antibodies 309
differentiation 174, 292, 527, 734 humanized monoclonal antibodies 310
dimethyl sulfoxide (DMSO) 527 humoral immunity 94
dose response 324 hybridoma technology 307
drug delivery systems 74
drug development 42, 625 idiotype network 313
immune system 1, 85, 429, 505, 633, 645
effector cells 102, 595 colony-stimulating factors 551
embryonic development 206 complexes 321
empirical clinical testing 46 cytokines 180
endothelia 213 immunoselection 323
enzyme-prodrug systems 596, 605 immunotoxins 69, 407, 426, 427, 698, 701
eosinophils 174 interferons 291
epidermal growth factor receptor 314, 356, 372, 373 potentiation 595
escalating-dose trials 46 stimulation 183, 669
ex-vivo purging 498 suppression 101
expression of genes 25, 27, 53 immunity 85, 94, 183, 194, 195, 515
immunoconjugates 451, 452, 455
Fas ligand 204 immunoglobulin 95, 321
febrile neutropenia 332, 347, 571 immunosuppression 101
FISP, 196, 276 chemotherapy-induced 111
Flt-3 ligand 171, 177, 178, 205, 206 radiation therapy-induced 110
free antigen 323 in vitro assays 94, 96
fungal toxins 415 in vivo assays 93
in vivo binding 315
gamma interferon 684, 715, 725 inflammation
gastrointestinal cancer 472 cytokines 201, 208, 213, 216
gene expression 25, 27 interleukins 165, 174, 183, 191
gene therapy 589, 594, 604, 639 infusion reactions 318
Index 741

interferons 9, 45, 57, 197, 277, 649, 660, 715 LIGHT, 207, 161
alpha 647, 660, 681 lipopolysaccharide (LPS) 163
current trends 671 lung cancer 122, 123, 679, 684
gamma 161, 191, 684, 715, 725 lutetium-177, 464, 465
interleukin-1 (IL-1) 60, 156, 165, 552 lym-1 antibody 466, 467
interleukin-2 (IL-2) 61, 156, 169, 637, 638, 649, 661, 671, lymph nodes 209
684, 695, 701, 715 lymphocytes 88, 91, 498, 652
interleukin-3 (IL-3) 61, 171 lymphoid organogenesis 207, 208
interleukin-4 (IL-4) 61, 172, 156, 685 lymphokine-activated (LAK) cells 93, 505, 652
interleukin-5 (IL-5) 157, 174 lymphokines 8, 95, 734
interleukin-6 (IL-6) 157, 174, 552 lymphoma 180, 281, 326
interleukin-7 (IL-7) 157, 177 lymphoproliferative responses (LPR) 94, 105
interleukin-8 (IL-8) 157, 179 lymphotoxins 207, 208, 554
interleukin-9 (IL-9) 157, 180
interleukin-10 (IL-10) 157, 180 macrophages 92, 177
interleukin-11 (IL-11) 158, 182 malignant melanoma 121, 123, 633
interleukin-12 (IL-12) 158,183 mast cells 174, 180, 212
interleukin-13 (IL-13) 158, 185 maturation factors 5, 10
interleukin-14 (IL-14) 158, 186 melanoma 121, 123, 380, 381, 385, 633
interleukin-15 (IL-15) 158, 186 metastasis 17, 20–22, 24, 32
interleukin-16 (IL-16) 158, 187 migration 203
interleukin-17A (IL-17A) 158, 189 ML-1 191, 555, 556
interleukin-17B (IL-17B) 159, 190 molecular chemotherapy 589, 590, 595, 599
interleukin-17C (IL-17C) 159, 191 monoclonal antibodies 12, 71, 303, 305, 307, 309–311, 315,
interleukin-17E (IL-17E) 159, 191 338, 378, 661, 686, 696, 701, 702, 735
interleukin-17F (IL-17F) 159, 191 monocytes 92
interleukin-18 (IL-18) 159, 191 multidrug resistance 32
interleukin-19 (IL-19) 159, 193 multiple myeloma 280, 329, 711
interleukin-20 (IL-20) 159, 194 mutation compensation 593
interleukin-21 (IL-21) 159, 194 myelodysplastic syndrome (MDS) 548
interleukin-22 (IL-22) 159, 195
interleukin-23 (IL-23) 159, 195 natural killer (NK) cells 92, 169, 194, 208
interleukin-24 (IL-24) 159, 196 neck cancer 123, 358
interleukin-24 (IL-25) 160, 196 neutropenia 574, 576, 583
interleukin-26(IL-26) 160, 197 neutrophils 189
interleukin-27 (IL-27) 160, 197 nitric oxide synthetase-inhibiting cytokine 210
interleukin-28A (IL-28A) 160, 197 non-hematological tumors 472
interleukin-28B (IL-28B) 160, 197 non-Hodgkin’s lymphoma (NHL) 281
interleukin-29 (IL-29) 160, 197 non-small cell lung cancer (NSCLC) 684
interleukin-31(IL-31) 160, 198 nonspecific immunomodulators 1, 5
interleukin-32 (IL-32) 160, 198 nonsteriodal anti-inflammatory and antipyretic drugs
interleukin-33 (IL-33) 160, 198 (NSAIDs) 117
interleukin-35 (IL-35) 160, 199 non-viral vectors 590, 592
interleukins 8, 60, 62, 119, 715 novel neurotophin 209
iodine-131, 464, 483 NPT 15392, 117
isolating genes 53
OKT-432, 126, 129, 680, 714
Isoprinosine 124 oncogenes 594
oncostatin M, 209
Kaposi’s sarcoma 286 osteoblasts 213
kidney see renal cancer osteoclasts 212
osteopontin 210
lentinan 128 ovarian cancer 355
leukemia 284, 329, 333, 470, 471, 487, 532, 540, 548, 551, OX40 ligand 211
584, 693, 702
leukemia inhibitory factor (LIF) 206, 553 pancreatic cancer 355
levamisole 115, 123, 126, 129, 659, 670, 680, 713 pathogenesis 17
ligand conjugation 416 patient selection 48
742 Index

PEG interferon 649 stomach cancer 310

peptide cytotoxins 407 surgical adjuvant studies 122
peptides 476, 477, 479
perioperative immunosuppression 109 T cells 638
phagocytes 95 cytokines 200, 206, 209, 211
plant toxins 407 interleukins 169, 174, 177, 180, 186, 194, 195
podophyllotoxins 113 lymphocytes 88, 147
polar-planar compounds 528, 546 lymphoma 699
preclinical models 4, 426 reconstituting factor 118, 125
pro-apoptotic gene therapy 589 T-cytotoxic factor 91
proallergic cytokines 185 T-helper cells 90, 172, 209
prostaglandin antagonists 124 T-suppressor cells 90
prostate cancer 32, 355, 380, 472 technetium-99m 478
proteomics 65 thrombocytosis 182
thrombopoiesis 174
radiation therapy-induced immunosuppression 110 thymic factors 7, 117, 125, 128, 129
radioimmunotherapy (RIT) 71, 463, 466, 469 thymic hormones 680, 714
radiolabelled antibodies 463 toll-like receptors 163
radiosensitization 603 TRAIL, 216
RANKL, 212 TRANCE, 212
recombinant DNA, 7, 53, 571 transductal targeting 592
recruitment 203 transfer factor 119, 125, 129
regulatory approaches 313 transforming growth factor β 555
remission 114 transcriptional targeting 592
renal cancer 288, 353, 380, 645 trastuzumab 343, 344–346, 348, 349, 672
replicative vector oncolysis 601 tumor cell burden 105
reticuloendothelial system 94 tumor infiltrating lymphocytes (TIL) 505
retinoids 119, 125, 672, 682, 701, 716, 725 tumor lysis syndrome 319
retroviral vectors 590 tumor necrosis factors (TNF) 63, 165, 207, 213,
rhenium-186/188, 464 371, 554
rituximab 325–333, 337, 38, 616 type I toxins 415,
type II toxins 415
sarcomas 356 type III toxins 415
screening 4 unconjugated monoclonal antibodies 303
serum sickness 321 urologic cancer 122
severe chronic neutropenia 576 vaccines. 71, 147, 374, 385, 510, 513, 634, 653, 664, 675,
sickle-cell anemia 577 687, 698, 701, 718
skin development 194 current trends 634, 653, 664, 675
small cell lung cancer (SCLC) 684 vertebrate toxins 414
solid tumors 105, 108, 114, 121 vinca alkaloids 113
soluble cytokine receptors 64 viral vectors 589
stem cell factor (SCF) 162, 212 vitamin A analogs 532
stem cells vitamin D analogs 540, 543, 544
bone-marrow transplantation 497, 500, 575
colony-stimulating factors 569 wound repair 165, 209, 468
cytokines 204
radioimmunotherapy 469 Y2B8 antibody 468
renal cancer 645 yttrium90, 464–477, 481–485, 487, 488