MACS and More 15 2 2013

Vol 152/2013
Translational
research
Dendritic cell
vaccination
pDC- and Mo-DCbased
vaccines for clinical research
on melanoma or glioblastoma
p. 11 and p. 16
NK cells for cancer

immunotherapy
Manufacture of NK cells for
clinical trials on solid tumors
and leukemia
p. 25
Treg cells in solid

organ transplantation
Isolation and expansion of Treg
cells for clinical studies
p. 30
Supporting the quest

for a cure
MACS&more (ISSN 1610-4994) is published by

Miltenyi Biotec GmbH.
Contents

Page
News
MACSQuant Tyto the microchip revolution
in cell sorting
Seamless translation with MACS GMP Products
Reports
Clinically applicable CD304 (BDCA-4)+ human plasmacytoid
dendritic cells a new era in dendritic cellbased immunotherapy
Jurjen Tel, Erik Aarntzen, Gregor Winkels, Carl G. Figdor,
and I. Jolanda M. de Vries
11
An optimized method for the manufacture of a dendritic cellbased

vaccine for translational glioblastoma research
Sara Nava, Marta Dossena, Simona Pogliani, Daniela Lisini,
Serena Pellegatta, Eugenio A. Parati, Gaetano Finocchiaro,
and Simona Frigerio
16
Enrichment, stimulation, and viral transduction of naive and

central memory CD8+ T cells under GMP conditions for
translational research towards the development of
adoptive cell therapy of cancer patients
Anna Casati, Azam Varghaei-Nahvi, Steven A. Feldman,
Mario Assenmacher, Steven A. Rosenberg, Mark E. Dudley,
and Alexander Scheffold
20
Clinical-grade purification and expansion of CD56+CD3 NK cells

for adoptive immunotherapy of solid tumors and leukemia
Ulrike Koehl, Ruth Esser, Lutz Uharek, Martin Stern, and Jakob Passweg
25
Clinical-grade separation and expansion of regulatory T cells

for clinical studies on cell therapy in solid organ transplantation
Henrieta Fazekasova, Sarah Thirkell, Katie Lowe, Andrew Bushell,
and Giovanna Lombardi
30
Editorial board: Anthony Allen, Adrian Arechiga, Volker Arps,

Dirk Balshsemann, Caroline Blumer Toti, Hermann Bohnenkamp,
Oliver Burlon, Vincent Chu, Steven Dublin, Guy Hewlett, Christoph
Hintzen, Manuela Konradt, Joumana Masri, Shane Oram, Katja Petry,
Kai Pinkernell, Martina Simon, Gerd Steffens, Ilka Wege
Editor: Ralph Schaloske
Graphics & Layout: Miltenyi Biotec GmbH, Graphics team
Miltenyi Biotec GmbH
Friedrich-Ebert-Strae 68,
51429 Bergisch Gladbach, Germany
Phone +49 2204 8306-0
macs@miltenyibiotec.de
The CliniMACS System components: Reagents, Tubing Sets,

Instruments and PBS/EDTA Buffer, are manufactured and controlled
under an ISO 13485 certified quality system. In Europe, the
CliniMACS System components are available as CE-marked medical
devices. In the USA, the CliniMACS System components including
the CliniMACS Reagents are available for use only under an approved
Investigational New Drug (IND) application or Investigational Device
Exemption (IDE). In the US, the components of the CliniMACS
Prodigy line are for research use only and not for human therapeutic
or diagnostic use. CliniMACS MicroBeads are for research use only
and not for human therapeutic or diagnostic use.
CryoMACS Freezing Bags are manufactured by Miltenyi Biotec
GmbH and controlled under an ISO13485 certified quality system.
These products are available in Europe as CE-marked medical devices
and are marketed in the USA under FDA 510(k) clearance.
MACS GMP Products are for research use and ex vivo cell
culture processing only, and are not intended for human in vivo
applications. For regulatory status in the USA, please contact your
local representative. MACS GMP Products are manufactured and
tested under a certified ISO 9001 quality system and in compliance
with relevant GMP guidelines. They are designed following the
recommendations of USP <1043> on ancillary materials. No animalor human-derived materials were used for manufacture of these
products.
Unless otherwise specifically indicated, Miltenyi Biotec products
and services are for research use only and not for therapeutic or
diagnostic use.
autoMACS, CliniMACS, CliniMACS Prodigy, CryoMACS,
DendriMACS, gentleMACS, MACS, the MACS logo, MACSiBead,
MACSiMAG, MACSQuant, MultiMACS, TexMACS, and TransAct
are registered trademarks or trademarks of Miltenyi Biotec GmbH
or its affiliates in Germany, the United States, and/or other countries.
All other trademarks mentioned in this document are the property of
their respective owners and are used for identification purposes only.
Copyright 2013 Miltenyi Biotec GmbH. All rights reserved.
Cover image: The picture shows mature plasmacytoid dendritic cells

(PDCs). Human PDCs were isolated from PBMCs using the CD304
(BDCA-4/Neuropilin-1) MicroBead Kit. Isolated PDCs were cultured
for 48 hours in medium with IL-3 and CpG ODN at 37C. Cells
were then labeled with Anti-HLA-DR (red) and CD107a (green)
antibodies for confocal laser scanning microscopy.
2 MACS&more
Vol 15 2/2013
www.miltenyibiotec.com
MACS&more
MACS&more
Vol.Vol.
14 15
2/2012
2/2013
Dear Researcher,
Every day, researchers around the world
are devoting their energy to discovering
new ways to treat or cure diseases,
contributing to future advances in
medicine. Translational research propels
the discoveries that make a real difference
in peoples lives. Miltenyi Biotecs integrated
product portfolio provides the tools for
advancing research from bench to bedside,
at every level.
Translational research is commonly defined
as the practical application of scientific
discoveries to improve human health. At
Miltenyi Biotec, we believe that translational
research means much more. Developing a
research idea with a specific disease in mind
usually begins with basic studies in cell or
tissue culture models, deciphering molecular
pathways, or making other observations to
establish causalities. All of this has just one
goal: finding a new therapeutic solution for a
clinical problem. Any new discovery will then
need to go through preclinical studies before
a new approach can be tested for safety and
efficacy in registered clinical trials.
One of the keys for the successful translation
of scientific discovery into clinical application
is the necessity for high-quality data right
from the beginning. We understand how
important it is for researchers to be able to
reach this level consistently and reproducibly.
To achieve this, Miltenyi Biotec has developed
a spectrum of products for all aspects of
translational research that allows researchers
to go from bench to bedside without
unnecessary, time-consuming, and expensive
extra studies for a truly smooth and
seamless translation.
Miltenyi Biotecs rich product portfolio
addresses many critical aspects of cellular
therapy development. Potential applications
of cell therapies include graft engineering,
tissue regeneration, and immunotherapy,
for example.
There are many different cell types that
hold great promise, for example, for cancer
immunotherapy: 1) In their function
of professional antigen-presenting cells,
dendritic cells (DCs) loaded with tumor
antigens can induce T cells to exert antitumor responses. 2) Cytotoxic CD8+ T cells
genetically engineered to express tumor
antigenspecific T cell receptors have the
capacity to mediate tumor regression. 3) As
part of the innate immune system, NK cells
have the potential to kill tumor cells without
prior exposure to tumor antigens.
The key challenge in solid organ
transplantation is graft rejection caused
by immune responses to the transplanted
organ. Current therapies for the prevention
of graft rejection involve broad-spectrum
immunosuppressive drugs, which have
severe side effects. Regulatory T (Treg) cells
play a prominent role in the maintenance of
immunological self tolerance. As they also
can mitigate responses to foreign antigens,
Treg cells could be an invaluable tool for the
prevention of graft rejection, avoiding lifelong immunosuppression.
In this MACS&more issue you will find
reports on all these topics by scientists who
are on their way to advancing their research
to cellular therapy. They rely on Miltenyi
Biotecs integrated technologies for sample
preparation, cell separation, cell stimulation
and expansion, and cell analysis.
naive and central memory CD8+ T cells

which will be used for future clinical studies
on adoptive cell therapy of cancer. A number
of CliniMACS Reagents helped them to
separate the T cell subsets (see p. 20). This
fruitful collaboration between scientists
from the Charit in Berlin, the NIH, and
Miltenyi Biotec propelled the development
of new easy-to-handle, GMP-compliant
tools for Tcell stimulation, which will soon
be available as part of the Miltenyi Biotec
product portfolio.
In a further report, Ulrike Koehl et al. used
the CliniMACS System to enrich NK cells to
high purities by depletion of CD3+ cells and
subsequent enrichment of CD56+ cells. The
NK cells could be activated and expanded
efficiently to obtain clinically relevant
cell numbers for studies on the adoptive
immunotherapy of solid tumors and leukemia
(see p. 25).
Henrieta Fazekasova et al. are developing a
protocol for the manufacture of CD4+CD25+
Treg cells for use in clinical studies on the
prevention of graft rejection in solid organ
transplantation. Depletion of CD8+ cells
and subsequent enrichment of CD25+
cells with the CliniMACS System yielded
a cell population of high purity. Following
expansion, the cells fulfilled all the predefined release criteria for a Treg cell therapy
product (see p. 30). Fazekasova et al. are
therefore well on their way to a clinical trial,
taking another important step forward in the
translation of their basic findings into clinical
application.
Jurjen Tel et al. report on the first clinical

study of therapeutic vaccination against
cancer using naturally occurring plasmacytoid
DCs. The authors enriched these cells using
the CliniMACS System. First results of their
study are promising: median overall survival
of melanoma patients treated with the pDCs
We hope you will enjoy reading this
was improved compared to matched control
MACS&more issue and find inspiration for
patients (see p. 11).
your own research.
In their translational glioblastoma research,
Sara Nava et al. developed an optimized
method for the GMP-compatible preparation
of a Mo-DC-based vaccine. They used a
variety of tools from Miltenyi Biotec for the
Your MACS&more team
enrichment of monocytes and extraction
of autologous tumor lysates, as they moved
towards the manufacture of a DC vaccine that
will be tailored to the individual glioblastoma
patients (see p. 16).
Anna Casati et al. developed a GMPcompliant process for the enrichment,
stimulation, and
viral www.miltenyibiotec.com/macs&more
transduction of both
MACS&more
online:
Vol 15 2/2013
MACS&more 3
NEWS
MACSQuant Tyto the microchip revolution

in cell sorting
Microchip technology expands into cell sorting: Based on cutting-edge microchip technology, the MACSQuant Tyto ensures high-speed,
high-purity, fluorescence-based cell sorting in a fully enclosed, sterile cartridge system. The instrument ties in perfectly with Miltenyi
Biotecs magnetic cell separation and flow cytometry tools.
Microchip-based flow sorting

revolutionizing cell separation
Microchips have become an integral part of
our everyday lives in the form of a plethora
of electronic devices computers, telephones,
and kitchen appliances, to name just a few.
Microchip technology is now about to change
the possibilities of cell sorting fundamentally.
Utilizing cutting-edge microchip technology,
Owl biomedical Inc., in Santa Barbara,
California, developed a high-speed flow
sorting technology involving a fully enclosed
cartridge system. Miltenyi Biotec and Owl
biomedical joined forces earlier this year to
bring the MACSQuant Tyto to the scientific
community a benchtop cell sorter, which
is a perfect complement to Miltenyi Biotecs
extensive portfolio of instruments and reagents.
Cell sorting with the MACSQuant Tyto relies
on fluorescence detection with up to eight
colors detected, enabling flexible isolation
strategies based on multiple parameters. The
key component of the MACSQuant Tyto,

however, is a sterile, closed cartridge (fig. 1).
These disposable cartridges are easy to use,
keep the samples free from contamination,
and also minimize the exposure of the user to
potentially harmful samples.
In contrast to common flow sorting devices,
the cells are directed through microchannels
within the cartridge by exposure to low,
Microchip-based cell sorting coping
with the challenges of fluorescence-based
flow sorting
High cell viability, no sheath fluids, no
droplet formation.
High-speed valve allows sorting of high
numbers of cells in a short period of time.
Enclosed system keeps samples free from
contamination and prevents exposure
of the operator to potentially harmful
sample materials.
positive pressures, ensuring minimal stress to

the cells and high viability. No sheath fluids
are involved, and no aerosols or droplets are
formed. Instead, separation of the desired cells
occurs through a microchip-based high-speed
valve. The valve opens upon detection of a
fluorescently labeled target cell, redirecting
it into a collection chamber (fig. 2). Open/
close cycles are in the range of microseconds.
After sorting, both positive and negative cell
populations can be removed from the cartridge
for all downstream applications.
This technology is a huge leap forward in cell
sorting comparable to the invention of the
transistor radio after decades with the tube
radio, Stefan Miltenyi, founder and president
of Miltenyi Biotec pointed out.
Limitless applications
Microchip-based cell sorting is being used in
basic and clinical research to isolate a variety of
cell types for investigative purposes: stem cells
Figure 1 Microchip and the "plug-and-play" cell sorting cartridge. The fully closed, sterile cartridge keeps cells and operator safe.
4 MACS&more
Vol 15 2/2013
NEWS
from bone marrow and immune or cancer cells

from blood, for example.
In our lab we require multiparameter isolation
of cells from infected animal tissues with high
viability and yield. Some of the cells of interest
express low levels of cell surface markers and
thus are challenging to isolate using other
cell separation approaches. In addition to
T cells and monocytes, we are particularly
interested in specific CD34+ subsets of stem
and progenitor cells, explained Professor
David DiGiusto, Professor of Virology and
Director of the Laboratory for Cellular
Medicine at the Beckman Research Institute
of the City of Hope. With its closed system,
high sensitivity and sorting speeds of up to
50,000,000 cells per hour, the microchipbased cell sorting technology addresses these
needs. Our experience has been that cells are
sorted reproducibly from run to run, with high
viabilities, Professor DiGiusto pointed out.
As cells are sorted in a closed system and at
high numbers, the technology opens up a
vast potential for use in clinical settings in the
future.
Figure 3 shows two examples of microchipbased sorting of T cells. In one experiment
CD4+ cells were enriched from diluted whole
blood to a purity of about 92% (A). In the
second experiment, a MART-1specific T cell
clone was spiked into PBMCs and then labeled
with a PE-conjugated tetramer loaded with
a MART-1 peptide. The MART-1specific
T cells were isolated to a purity of about 98%
(B). Importantly, the purified antigen-specific
T cells were capable of killing MART-1+ tumor
cells and could be expanded in vitro.
The perfect complement to
MACS Technology, MACSQuant Flow
Cytometers, and MACS Antibodies
Microchip-based cell sorting is a natural
complement to our proven expertise in
magnetic separation and to our MACSQuant
Flow Cytometry family, said Stefan Miltenyi.
It ensures that the scientific community will
have access to a spectrum of cell sorting
technologies and allow them to carry out
cellular research and to translate their
discoveries into important new cellular
therapies. This technology will revolutionize
cell sorting for new diagnostic and therapeutic
applications, he concluded.
laser
laser
laser
sample
positive cells
negative cells
selected cells
laser
Figure 2 Mechanism of microchip-based sorting. As the labeled cell sample approaches the sort area, each
cell is analyzed. When a fluorescently labeled target cell is identified, a magnetic pulse opens the valve and the
cell is redirected to a collection chamber. An integrated single-crystal silicon spring returns the valve to its
original position, and undesired cells are allowed to flow through.
Vol 15 2/2013
MACS&more 5
NEWS
Before sorting
After sorting
10
10
10
10
10
CD4-PE+
0.0027
SSC
SSC
10
10
10
10
CD4-PE+
91.9
10
10
10
10
10
10
10
10
10
10
10
10
CD4-PE
10
PE+
0.87
10
10
10
10
10
10
Tetramer-PE
Fast
Magnetic cell isolation directly
from whole blood in 20 minutes
Safe
No centrifugation, less pipetting,
minimized aerosol formation
Scalable
Sample sizes from 2 to 30 mL
Excellent performance
High purity and recovery
Convenient
No density gradient
centrifugation
macsxpress.com
10
PE+
97.8
0
10
MACSxpress
Technology
Why wait?
10
SSC
10
SSC
10
CD4-PE
10
10
10
10
10
Tetramer-PE
Figure 3Isolation of T cells using microchip-based cell sorting. (A) Whole blood was diluted 1:5
with PBS and labeled with a CD4-PE antibody prior to enrichment of CD4+ cells. (B) MART-1specific
T cells were spiked into human PBMCs and labeled with a PE-conjugated tetramer loaded with a MART-1
peptide. The antigen-specific T cells were isolated by microchip-based flow sorting to high purity. Data from
Owl biomedical.
Magnetic cell separation by MACS Technology

is the best option to pre-enrich a certain cell
population or deplete unwanted cells prior to
multiparameter microchip-based flow sorting.
The combination of both technologies enables
sorting of an unparalleled large numbers of
cells at high speed and to high purities. Both
technologies ensure gentle cell processing,
making for viable, functional cells.
Integration of magnetic and microchip-based
cell sorting in a single device will be the next
step in the natural evolution of these high-end
technologies.
The new technology also fully integrates
with Miltenyi Biotecs proven and tested flow
cytometry platform including the multilaser
MACSQuant Family of flow cytometers
and thousands of flow-validated antibodies.
GMP-compliant antibodies will form a solid
foundation for high-performance flow sorting
with the MACSQuant Tyto and complex
cell analysis with the MACSQuant Flow
Cytometers.
For more information visit

www.macsquant.com/tyto
The MACSQuant Flow Cytometers and the MACSQuant Tyto
are for research use only and not for therapeutic or diagnostic use.
Available for research use only

in the United States.
6 MACS&more
Vol 15 2/2013
Report
by Miltenyi Biotec
Coming soon!
MACSQuant Tyto
Revolutionizing cell sorting with microchip-based technology
A new era has arrived. The MACSQuant Tyto
ensures high-speed, high-purity cell sorting in
a fully closed cartridge system. This first-ever
plug-and-play format makes cell sorting easier
than ever and keeps your samples safe and
free from contamination. Discover how the
MACSQuant Tyto can revolutionize your research
today.
K
eep cells and operator safe with the
sterile, closed cartridge system
Ensure minimal stress and high cell viability
no sheath fluids, aerosols, or droplets
G
ently isolate any cell population using
11-parameter fluorescence-based sorting
No cleaning or carryover between sorts
macsquant.com/tyto
Vol 15 2/2013
MACS&more 7
NEWS
Seamless translation with MACS GMP Products

GMP guidelines for the manufacture of cellular products for therapies specify the use of GMP-compliant materials. Miltenyi Biotec
provides a vast range of MACS GMP Products, covering entire workflows from cell enrichment to expansion, making the translation of
scientific discovery into clinical application as straightforward as possible.
Conducting research with

an eye to the future
Translation of scientific discovery into
clinical application involves several critical
phases. In particular, clinical trial phases are
highly regulated, requiring quality materials
throughout the pre-clinical research stage. For
the development and manufacture of cellular
therapies, strict procedures compliant with
GMP guidelines are required. All materials
used, such as antibodies, antigens, cytokines,
cell culture bags, and cell culture media, must
comply with GMP regulations. In order to avoid
time-consuming and expensive revalidation
during implementation of a new manufacturing
process for clinical studies, it is crucial to use
high-quality, GMP-compliant products as
early in the course of the development process
as possible. Miltenyi Biotec has developed a
comprehensive portfolio of GMP products,
which allow a straightforward translation.
Long-standing expertise
the basis for trusted solutions
With over 14 years of experience in GMPcompliant manufacturing, Miltenyi Biotec
offers trusted, integrated solutions for
cell manufacture according to the highly
demanding GMP requirements. The
CliniMACS Prodigy (fig. 2) enables fully
automated cell processing in an enclosed,
GMP-compliant system. MACS GMP Products,
including various specialized media, cytokines,
tools for cell activation and expansion, and cell
culture bags, provide the firm foundation for
excellent cell growth or differentiation. MACS
GMP Products are optimally adapted to each
other and therefore ensure a seamless workflow.
Complete workflows for GMP-compliant
cell manufacture
Cell therapy approaches are multifaceted and
involve various different cell types. Dendritic
cell (DC)-based vaccines, for example,
modulate the immune system by stimulating
T cells to attack tumor cells.
8 MACS&more
Vol 15 2/2013
Figure 1 The Miltenyi Biotec GMP facility in Teterow, Germany.
MACS GMP Products are manufactured

in our ISO 9001, 13485certified GMP
facility, equipped with state-of-the-art
production areas of GMP/ISO clean room
classes. All products are manufactured
and tested in compliance with relevant
For the first clinical study of plasmacytoid

DC (pDC)-based vaccination against cancer,
Jurjen Tel et al. used cells isolated with the
CliniMACS CD304 (BDCA-4) Reagent (see
p. 11). The most widely used DC type for
clinical trials however are monocyte-derived
DCs (Mo-DCs), which are differentiated ex
vivo from CD14+ peripheral blood monocytes
in a culture supplemented with GM-CSF and
IL-4. Sara Nava et al. developed an optimized
method for the manufacture of a Mo-DC-based
vaccine for their translational glioblastoma
research (see p. 16).
Miltenyi Biotec offers a sophisticated platform
enabling a complete, automated workflow for
the GMP-compliant manufacture of Mo-DCs,
including isolation of monocytes and efficient
differentiation into Mo-DCs. The heart of
the platform is the CliniMACS Prodigy.
A variety of MACS GMP Products reagents
and consumables perfectly complement the
instrument: Monocytes are enriched using
the CliniMACS CD14 Reagent. DendriMACS
GMP guidelines and designed following

the recommendations of USP <1043> on
ancillary material. Every processing step,
from development to production and
quality control (QC) testing according to
EP is extensively documented.
Figure 2 The CliniMACS Prodigy.

The instrument allows cell manufacture from sample
to final product including cell washing procedures,
density gradient centrifugation, cell enrichment, and
cell culture in an enclosed, GMP-compliant system.
The CliniMACS Prodigy is a CE-marked medical
device in Europe and available for research use only
NEWS
Leukapheresis
Treg cell
separation
Treg cell
expansion
Analysis tools
Cryopreservation
CliniMACS
Instruments
TexMACS GMP
Medium
MACSQuant
Instruments (RUO)
CryoMACS
Freezing Bags
CliniMACS
CD25 Reagent
MACS GMP
ExpAct Treg Kit
Treg Detection
Kit (RUO)
CryoMACS
DMSO (EP, USP)
CliniMACS
CD8 Reagent
MACS GMP
Rapamycin
Treg Suppression
Inspector (RUO)
CliniMACS
CD19 Reagent
MACS GMP
IL-2
Ready to use
Treg cells
MACS GMP
Cell Culture Bags
Figure 3 Miltenyi Biotec products supporting the workflow for GMP-compliant manufacture of Treg cells. MACS GMP Products and CryoMACS DMSO are for
research use and ex vivo cell culture processing only, and are not intended for human in vivo applications.
GMP Medium, MACS GMP GM-CSF, and

MACS GMP IL-4 facilitate the differentiation
of monocytes into DCs, and the MACS GMP
Cell Differentiation Bags make for simple cell
culturing. Purity of CD14+ monocytes and ex
vivo generation of Mo-DCs are monitored on
the MACSQuant Flow Cytometer using the
MC CD14 Monocyte Cocktail and the Mo-DC
Differentiation Inspector, respectively.
Regulatory T (Treg) cells play a critical role
in the maintenance of peripheral tolerance
and have the potential to curtail immune
responses towards foreign antigens. Therefore,
Treg cells hold great potential for clinical
applications, in which a down-regulation of
immune responses is beneficial, for example,
in the context of organ transplantation. Figure
3 depicts a workflow for the efficient, clinicalscale manufacture of Treg cells. Henrieta
Fazekasova et al. took advantage of a variety
of Miltenyi Biotecs GMP-compliant tools for
the enrichment and expansion of Treg cells,
suitable for use in a phase I clinical trial (see
p. 30).
The various efforts such as SOP development
and protocol establishment to obtain a
manufacturing permission are fewer when

all the tools for cell manufacture are from
the same provider, pointed out Dr. Caroline
Blumer Toti, Global Marketing Manager Cell
Culture. To make the crucial steps from preclinical research towards clinical application for
our customers as easy as possible, we develop
harmonized solutions for complete GMPcompliant workflows. DCs and Treg cells are
just two examples among many cell types
that can be manufactured with our MACS
GMP products, Dr. Hermann Bohnenkamp,
For more information on MACS GMP
Products, please contact:
Dr. Caroline Blumer Toti

CarolineB@miltenyibiotec.de
Dr. Hermann Bohnenkamp

hermannb@miltenyibiotec.de
Business Area Manager, Clinical Business

added to this.
Find out more about the complete portfolio of
MACS GMP Products at
www.miltenyibiotec.com/gmp
Instruments and PBS/EDTA Buffer are manufactured and
controlled under an ISO 13485 certified quality system. In
Europe, the CliniMACS System components are available as
CE-marked medical devices. In the USA, the CliniMACS System
components including the CliniMACS Reagents are available for
use only under an approved Investigational New Drug (IND)
application or Investigational Device Exemption (IDE). In the
US, the components of the CliniMACS Prodigy line are for
research use only and not for human therapeutic or diagnostic
use. CliniMACS MicroBeads are for research use only and not for
human therapeutic or diagnostic use.
MACS GMP Products and CryoMACS DMSO are for research
use and ex vivo cell culture processing only, and are not intended
for human in vivo applications. For regulatory status in the USA,
please contact your local representative.
Quality statement: MACS GMP Products are manufactured
and tested under a certified ISO 9001 quality system and in
compliance with relevant GMP guidelines. They are designed
following the recommendations of USP <1043> on ancillary
materials. No animal- or human-derived materials were used for
manufacture of these products.
In the USA, the MACS GMP ExpAct Treg Kit is available for
research use only.
CryoMACS Freezing Bags are manufactured by Miltenyi Biotec
GmbH and controlled under an ISO13485 certified quality
system. These products are available in Europe as CE-marked
medical devices and are marketed in the USA under FDA 510(k)
clearance.
and services are for research only and not for therapeutic or
diagnostic use.
Vol 15 2/2013
MACS&more 9
Report
MACS GMP Cytokines

Fulfill stringent demands for GMP-compliant cell culture
Designed and manufactured to
the highest quality standards
Extensive quality control

and documentation
Completely free of animal- and

human-derived materials
High and standardized

biological activity (IU/mg)
Aseptically filled and

tested for sterility
Lot-specific certificates of analysis
macscytokines.com
MACS GMP Products are for research use and ex vivo cell culture processing only, and are not intended for human in vivo applications. For regulatory status in the USA, please
contact your local representative. MACS GMP Products are manufactured and tested under a certified ISO 9001 quality system and in compliance with relevant GMP guidelines.
They are designed following the recommendations of USP <1043> on ancillary materials. No animal- or human-derived materials were used for manufacture of these products.
10 MACS&more
Vol 15 2/2013
Report
Clinically applicable CD304 (BDCA-4)+

human plasmacytoid dendritic cells
a new era in dendritic cellbased
immunotherapy
Jurjen Tel1, Erik Aarntzen1,2, Gregor Winkels3, Carl G. Figdor1, and I. Jolanda M. de Vries1,2
Department of Tumor Immunology, 2 Medical Oncology Radboud University Nijmegen Medical Centre and
Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands. 3 Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Introduction
Dendritic cells (DCs) constitute a family
of antigen-presenting cells defined by their
morphology, phenotype, and unique capacity
to process exogenously encountered antigens
and to present them to naive T cells. Following
infection or inflammation, DCs undergo a
complex process of maturation and migrate to
lymph nodes to present antigens and activate
Tcells. This decisive role in inducing immunity
was the rationale for DC-based immunotherapy,
in which DCs loaded with tumor antigens were
injected into cancer patients to stimulate T cells
to eradicate tumors,. Now, after more than a
decade, we know that monocyte-derived DCs
(Mo-DCs) can induce immune responses in a
significant number of patients, in particular
when both CD4+ and CD8+ T cell responses
are obtained, as also recently shown by our DC
vaccination studies. Nevertheless, the number
of objective clinical responses has been limited,
hampering its implementation as a novel form
of standard treatment. Although the evidence
of clinical responses is still scarce, expectations
are high because the clinical responses that
are induced are often long lasting. It remains
unclear whether DCs differentiated ex vivo
from monocytes or CD34+ progenitors are
the optimal source of DCs for the induction approved by the local Institutional Review
of potent immune responses. Because of the Board (Committee on Research Involving
limited number of naturally circulating DCs, Human Subjects Arnhem-Nijmegen) and in
virtually all vaccination studies for more than a accordance with the declaration of Helsinki.
decade have been based on DCs differentiated Written informed consent was obtained from
ex vivo from monocytes or CD34+ progenitors. all patients. The clinical trial registration
Recently, it was proposed that these artificial number is NCT01690377.
DCs may be less effective than their natural
counterparts that circulate in the blood because Isolation of pDCs with the
of the extensive culture period. Moreover, CliniMACS System
compounds required to differentiate precursors The pDCs were directly isolated from apheresis
into DCs might negatively affect DC function. products using the fully enclosed CliniMACS
Especially their migratory capacity towards Plus Instrument and GMP-grade magnetic
Tcell areas in lymph nodes might decrease due beadcoupled Anti-BDCA-4 antibodies
to exhaustion of the DCs. We reported on the (CliniMACS CD304 (BDCA-4) Reagent).
first clinical study of therapeutic vaccination The purity of pDCs after immunomagnetic
against cancer exploiting naturally occurring isolation was determined by flow cytometry.
plasmacytoid DCs (pDCs) purified by using The following primary monoclonal antibodies
MACS Technology.
(mAbs) and the appropriate isotype controls
were used: CD45-FITC, CD303 (BDCA-2)-PE
Materials and methods
and CD123-APC (all Miltenyi Biotec). The
Patient characteristics and clinical protocol
procedure resulted in clinically applicable
Sixteen distant metastatic melanoma patients purified pDCs, which had an average purity
(according to the 2001 American Joint of 75% and a yield between 13106 and
Committee on Cancer staging system) were 33106 cells (fig. 1). Following apheresis
enrolled in this feasibility study. The primary and CliniMACS Cell Separation, pDCs
endpoint was toxicity related to vaccination were cultured overnight at a concentration
and immunological response. The trial was of 106 cells/mL in X-VIVO 15 medium
Vol 15 2/2013
MACS&more 11
Report
510
410
75
310
50
210
25
Cell count
Purity or recovery (%)
100
(Applied Biosystems). Analysis was done using

the Bio-Rad CFX Manager v1.6 software, and
expression levels were determined relative to
PBGD expression.
110
0
Purity
Purity Recovery Cell

count
pDC vaccine
CliniMACS Cell
Separation
Figure 1 pDC isolation based on the CliniMACS

System is feasible. The right panel in the graph
shows the purity (%), recovery (%), and number
of cells obtained in 18 isolations from patient
samples. The left panel shows the purity (%) of the
administered pDC vaccines. The pDC vaccines were
checked for contaminating cells by determining the
presence of T cells (CD3) and B cells (CD19). T and
B cells were routinely less than 3% and 10% of all cells,
respectively (data not shown). Data were adapted
from reference 6.
(Lonza) containing 2% pooled human serum,

supplemented with 10 ng/mL recombinant
human interleukin-3. The pDCs were
subsequently activated for 6 hours by addition
of FSME-IMMUN (1:10 v/v) (Baxter AG).
During the last 3 hours of activation, pDCs
were loaded with the melanoma-associated
peptides gp100154, gp100280, and tyrosinase.
Purity of the vaccine was determined by
flow cytometry (fig. 1). The peptide-loaded
pDCs were administered intranodally in a
clinically tumor-free lymph node region under
ultrasound guidance.
In-oxinate labeling and

scintigraphic imaging
pDCs activated for 6 hours with FSME
were labeled with 5 MBq 111In-oxinate (GE
Healthcare) in 0.1 M Tris-HCl (pH 7.0) for
15 minutes at room temperature as described
previously. In vivo planar scintigraphic
images (256256 matrix, 174 and 247 keV 111In
photopeaks with 15% energy window) of the
injection depot and corresponding lymph node
basin were acquired with a gamma camera
(Siemens e.cam) equipped with medium
energy collimators, 15 minutes, 24 hours,
and 48 hours after injection. Migration was
quantified by region-of-interest analysis of the
individual nodes visualized on the images and
expressed as the fraction of 111In-labeled DCs
that had migrated from the injection depot
to following lymph nodes after 15 minutes,
24hours, and 48 hours.
111
Immunomonitoring of patients
Four days after the third vaccination, a DTH
skin test was performed and frequencies of
anti-vaccine CD8+ T cells in the blood were
estimated using mixed lymphocyte peptide
cultures (MLPCs) as described previously6.
From positive DTH sites, half of the biopsy was
manually cut and cultured in Gibco RPMI
1640 (Life Technologies) containing 7% HS and
IL-2 (100 U/mL, Proleukin, Chiron). Every 7
days, half of the medium was replaced by fresh
medium. After 2 to 5 weeks of culturing, Tcells
were tested for specificity against gp100 and
tyrosinase. DTH-derived cells were stained
RNA isolation and quantitative PCR
with tetrameric MHC complexes containing
To determine mRNA expression of IFN- and the gp100154-167, gp100280-288, or tyrosinase369-376
RIG-I, blood was drawn before vaccination peptides (Sanquin) combined with CD8
and at 4 and 24 hours after vaccination. RNA staining.
isolation from patient samples was performed For the MLPCs, PBMCs isolated before and after
using the PAXgene Blood RNA Kit (Qiagen) one cycle of three pDC injections were thawed,
according to the manufacturers instructions. divided into three groups, and incubated for
RNA isolations from PBMCs from healthy 1 hour at room temperature in Gibco IMDM
volunteers were done using the ZR RNA (Life Technologies) with 1% HS and 2 M of
isolation kit (Zymo Research) according to the peptides tyrosinase369 (YMDGTMSQV),
the manufacturers instructions. mRNA levels wild type gp100 154 (KTWGQYWQV), or
for the genes of interest were determined by wild type gp100280 (YLEPGPVVTA). These
quantitative PCR (qPCR) with a Bio-Rad pulsed cells were then washed, pooled, and
CFX apparatus (Bio-Rad) with SYBR Green distributed at 2105 cells/0.2 mL in round-
12 MACS&more
Vol 15 2/2013
bottom microwells in IMDM with 10% HS,

L-arginine (116 mg/L), L-asparagine (36
mg/L), L-glutamine (216 mg/L), 1-methyl-Ltryptophan (100 M), IL2 (20 U/mL), and
IL7 (10 ng/mL). On day 7, 50% of the medium
was replaced by fresh medium containing IL-2
and peptides at 4 M. Tetramer labeling was
performed on day 14 as described previously.6
Anti-gp100154 T cell clones were derived that
represented either the spontaneous anti-gp100
T cells present prior to vaccination in patients 2,
5, 6, 10, and 11, or the pDC-induced anti-gp100
T cells present after vaccination in patients 1, 4,
8, and 12. Tetramer-positive CD8+ T cells were
sorted at 1 cell/well and restimulated weekly
with irradiated HLA-A2+ EBV-transformed B
cells pulsed with the gp100154 peptide at 2 M,
and irradiated allogeneic PBMCs as feeder cells,
in medium supplemented with IL-2 and IL-7.
Matched historical controls
Matched historical controls were identified
from records of metastatic melanoma patients
from the Radboud University Nijmegen
Medical Centre (Nijmegen, The Netherlands),
The Netherlands Cancer Institute Antoni
van Leeuwenhoek Hospital (Amsterdam, The
Netherlands), and University Hospital Essen
(Essen, Germany) who had received first-line
dacarbazine (DTIC) chemotherapy at 850
1000 mg/m2 i.v. at 3 weekly intervals, between
March 2000 and March 20106.
Statistical analysis
Significant differences from controls were
determined according to paired Students t-test
or by one-way ANOVA analysis followed by
the Tukeys post-hoc test. Differences between
pre- and post-vaccination were evaluated with
a Wilcoxon signed-rank test. Kaplan-Meier
probability estimates of overall survival were
calculated, and statistical differences between
the survival of the groups were determined
with a log-rank test. Statistical significance
was defined as p<0.05. SPSS 19.0 was used for
survival analyses.
Report
Results and discussion
B
RIG-I
RIG-I
RIG-I
IFN-
IFN-
IFN-
depot
IFN-
40
40
40
50
0.15
0.10
0.10
0.10
30
30
30
40
0.10
10- --
10
10
**
Healthy
Healthy
Healthy
Healthy
1
1
1hhh
1 h
444hhh
4h
24
24
24hhh
24 h
gp100
gp100
gp100
+
+
10-
100
1000 0
10
101
10
1000
10
104
1044
10
101
1011
10
102
104
1044
10
0.2%
0.2%
0.2%
3
10
100
1000 0
10
101
10
1000
10
104
1044
10
101
1011
10
100
104 103 100
1033
10
104
101
102
1022
10
103
1033
10
102
104
1044
10
0.1%
0.1%
0.1%
3
10
104
0.1%
102
1022
10
103
101
1011
10
102
100
1000 0
10
101
10
1000
10
1044
104
10
Tetramer-APC
Tetramer-APC
Tetramer-APC
Tetramer-APC
After
After
After333
After 3 vaccinations
vaccinations
vaccinations
vaccinations
Tyrosinase
Before
Before
Before
Before vaccination
vaccination
vaccination
vaccination
10-
101
103
1033
10
0.2%
101
1011
10
100
103 100
1033
10
104
10- --
10
10
102
1022
10
101
1011
10
102
102
1022
10
101
102
103
1033
10
104
1044
10
0.3%
0.3%
0.3%
103
100
1000 0
10
101
10
1000
10
1044
104
10
103
102
1022
10
103
101
1011
10
102
101
1011
10
102
100
1000 0
10
101
10
100
10
101
1011
10
CD8-FITC
CD8-FITC
CD8-FITC
102
1022
10
103
1033
10
101
1011
10
100
104 103 100
1033
10
104
0.3%
102
1022
10
0.2%
102
1022
10
103
101
1011
10
102
102
1022
10
103
gp100280
0.2%
0.2%
0.2%
Tyrosinase
103
1033
10
104
0.4%
100
103 100
1033
10
104
gp100280
gp100
gp100
280
280
Tyrosinase
Tyrosinase
Tyrosinase
104
1044
10
0.4%
0.4%
0.4%
gp100
101
1011
10
102
gp100154
Tyrosinase
Tyrosinase
Tyrosinase
102
1022
10
103
gp100154
gp100
gp100
154
154
10-
10- --
10
10
0
0010
n.d.
103
1033
10
104
gp100154
gp100
gp100
154
154
**
**
**
gp100154
n.d.
104
1044
10
**
1020
10
10
0.05
0.00
**
**
**
20
20
20
30
0.05
0.05
0.05
n.d. n.d.
n.d.
n.d.
n.d.
0.00 n.d.
0.00
0.00
***
Healthy
Healthy
Healthy
Healthy
1
1
1hhh
1 h
444hhh
4h
24
24
24hhh
24 h
1.9%
1.0%
**
**
**
***
***
***
RIG-I
**
50
50
50
Expression
Expression
relative
totoPBGD
PBGD
Expressionrelative
relativeto
PBGD
Expression relative to PBGD
7.0%
1.2%
Expression
Expression
relative
totoPBGD
PBGD
Expressionrelative
relativeto
PBGD
Expression relative to PBGD
0.15
0.15
0.15
++
TTTcells
Frequency
+CD8
CD8
cells
Frequency
ofofantigen-specific
antigen-specific
CD3
cells
Frequencyof
antigen-specificCD3
CD3+CD8
Frequency of antigen-specific CD3+CD8+ T cells
Clinical study with natural pDCs

We have embarked on exploiting pDCs that
naturally circulate in the peripheral blood
for cancer immunotherapy. Although it is
well known that intratumoral pDCs, when
inactive, are associated with tumor progression,
activated interferon alpha (IFN-)-producing
pDCs can activate NK cells and induce antigenspecific T and B cell responses. Our finding
that commonly used prophylactic vaccines
against infectious diseases could simply be
used as adjuvants to activate pDCs to secrete
significant amounts of IFN- has certainly
expedited the use of naturally circulating DCs
in the clinic10.
We initiated the first study exploiting pDCs
to treat advanced stage IV melanoma patients.
Designed as a safety study, we treated 15
patients in three cohorts with dose-escalating
pDC vaccines ranging from 0.3 to 3 million
cells per injection. The vaccines were well
tolerated and no signs of severe toxicity
(common toxicity criteria grade 34) were
observed. Six vaccinated patients developed
grade 1 flu-like symptoms and one patient
reported grade 2 non-treatment related pain
resulting from progressive subcutaneous
metastasis. In none of the vaccinated patients
did we detect antibodies to the murine
antibody used during the isolation procedure
(data not shown). We conclude that it is
feasible and safe to administer activated and
tumor-peptide loaded pDCs to patients. As the
number of cells was low and no data existed
on their migratory capacity in vivo, we decided
to inject the cells intranodally to maximize
the possibility to raise an immune response.
Forty-eight hours after intranodal injection, a
significant proportion of 111In-labeled pDCs
remained at the injection site, and distinct
amounts were detected in distant lymph nodes
(fig. 2A). To verify whether activated pDCs
secreted significant quantities of type I IFNs in
vivo, we investigated gene expression of IFN-
and the IFN-induced gene RIG-I in blood
mononuclear cells before and after vaccination.
We compared these expression levels to those of
cells from healthy individuals. In line with their
ability to secrete large amounts of type I IFNs,
transcription of IFN- was clearly induced
four hours after vaccination and decreased
20 hours later (fig. 2B), indicating a temporal
systemic induction of type I IFNs. As expected,
104
1044
10
100
100
1000 0
10
101
10
100
10
0
101
102
1022
10
103
1033
10
102
104
1044
10
0.4%
0.4%
0.4%
103
104
0.4%
101
1011
10
102
1022
10
103
1033
10
104
1044
10
100
100
101
102
103
104
100
101
102
103
104
CD8-FITC
Figure 2Activated pDCs are mature and migrate to distinct lymph nodes in vivo. (A) Migration and
biodistribution of 111In-labeled pDCs visualized by scintigraphical imaging. Forty-eight hours after
administration, 11% of injected pDCs were distributed over up to 4 distant lymph nodes away from the
injection depot. (B) The graphs show the IFN- and RIG-I gene expression levels in the blood relative to PBGD
expression at the indicated times after each vaccination. Healthy indicates steady-state expression levels of
indicated genes in 8 healthy individuals (n.d.: not detected; ** p<0.01; *** p<0.001). (C) pDC vaccinerelated
CD8+ T cell responses were detected in the blood after MLPCs. The graph shows the frequencies of gp100154
tetramer+CD8+ T cells in the blood before and after one cycle of vaccination. (D) pDC vaccine-related CD8+
Tcell responses were detected in biopsies taken from DTH skin tests. Two weeks after the third pDC injection,
a DTH skin test was performed by intradermally injecting pDCs loaded with either the gp100 or the tyrosinase
peptides. Biopsies taken 2 days later were cultured for 34 weeks in low-dose IL-2, and proliferating T cells were
stained with specific tetramers. Dot plots show DTH-infiltrating lymphocytes stained with gp100154 tetramers
(** p<0.01). Data were adapted from reference 6.
Vol 15 2/2013
MACS&more 13
Report
Percent survival
Although the initial endpoint of this study

was safety and feasibility, we obtained some
100
surprising clinical results: the median overall
survival showed a remarkable improvement
80
compared to matched control patients: 22.0
60
months (95% CI: 1.842.2) versus 7.6 months
P=0.001
(95% CI: 5.89.4) (fig. 3). We observed highly
40
improved clinical responses with these freshly
isolated naturally circulating DCs when
20
compared to conventional Mo-DCs: 7 out of
15 stage IV melanoma patients are still alive
0
0
10
20
30
40
50
two years after start of treatment. Interestingly,
Overall survival (months)
we obtained these results even with a tenfold
DTIC
pDCs
lower dose, demonstrating the potency of these
natural pDCs. Interestingly, Celli et al. recently
Figure 3pDC vaccination improves overall
reported that as little as one hundred DCs are
survival. Clinical outcome of pDC vaccination was
needed to elicit a T cell response, indicating
compared to a group of carefully matched historical
that indeed perhaps only small amounts of
control patients who received dacarbazine as firstDCs
are needed for the induction of anti-tumor
line treatment. Median overall survival data showed
11
.
responses
a remarkable improvement compared to matched
control patients: 22.0 months versus 7.6 months.

Furthermore, the overall survival of pDC-vaccinated
patients was significantly improved compared to
matched controls. Statistical significance between the
survival of the groups was determined by a log-rank
test, p=0.001. Data were adapted from reference 6.
we observed increases in the expression of

the IFN-induced gene RIG-I after four hours,
which further increased after 24 hours (fig. 2B).
We used tetramers to detect the presence of
tumor antigenspecific CD8+ T cells in blood,
and in biopsies taken from skin delayed-type
hypersensitivity (DTH) reactions. Ex vivo
tetramer staining was negative both before and
after vaccination (data not shown). Therefore,
we resorted to an in vitro restimulation of
blood mononuclear cells under limiting
dilution conditions over two weeks with the
three antigenic peptides, before screening
all microcultures for the presence of CD8+
tetramer+ cells. This procedure allowed us to
estimate the frequencies of blood CD8+ T cells
that recognize a given antigen and proliferate
in vitro in response to this antigen. As shown
in figure 2C, 7 out of 15 patients showed
a significant increase in the frequency of
gp100154-specific CD8+ T cells. Furthermore,
we detected anti-gp100154 CD8+ T cells in DTH
biopsies in two patients after vaccination (fig.
2D). We conclude that vaccination with small
numbers of peptide-loaded and activated
pDCs can induce tumor-specific CD8+ T cell
responses in metastatic melanoma patients6.
14 MACS&more
Vol 15 2/2013
Conclusion
Taken together, our results demonstrate that
1) the pDCs injected into a single lymph
node distributed into downstream nodes, 2)
after each vaccination even small numbers
of injected pDCs induced a systemic type I
IFN signature, and 3) injected pDCs primed
tumor-specific CD8+ T cells. These findings
indicate that it is worthwhile to further explore
the potential of pDCs and other naturally
circulating DCs for cancer immunotherapy.
It will also be interesting to see if other DC
subsets, such as the myeloid DCs, are equally
or even more effective.
7. de Vries, I.J. et al. (2003) Clin. Cancer Res. 9:

50915100.
8. de Vries, I.J. et al. (2005) Nat. Biotech. 23: 1407
1413.
9. de Vries, I.J. et al. (2002) J. Immunother. 25: 429
438.
10. de Vries, I.J. et al. (2011) Mol. Immunol. 48: 810
817.
11. Celli, S. et al. (2012) Blood 120: 39453948.
MACS Product
Order no.
CliniMACS Plus Instrument
151-01
CliniMACS CD304 (BDCA-4)

Reagent
278-01
CliniMACS Tubing Set
161-01
CliniMACS PBS/EDTA Buffer
700-25
CD45-FITC, human; CD303

(BDCA-2)-PE, human;
CD123-APC, human
Visit www.macsantibodies.com
Instruments and PBS/EDTA Buffer, are manufactured and
controlled under an ISO 13485 certified quality system. In Europe,
the CliniMACS System components are available as CE-marked
medical devices. In the USA, the CliniMACS CD304 (BDCA-4)
Reagent is available for research use only, other CliniMACS
System components are available for use only under an approved
Investigational New Drug (IND) application or Investigational
Device Exemption (IDE). CliniMACS MicroBeads are for
research use only and not for human therapeutic or diagnostic use.
The CD45-FITC, human, CD303 (BDCA-2)-PE, human, and
CD123-APC, human antibodies are for research use only.
diagnostic use.
Acknowledgments
This research was supported by NWO
Vidi grant 917.76.363 to IJMdV, by EU
grants Cancerimmunotherapy (LSHCCT-2006- 518234) and DC-THERA (LSHBCT-2004-512074), by the NOTK foundation,
the TIL foundation, and by the Cancer Plan of
the Belgian Federal Public Service. CGF was
awarded with a NWO Spinoza prize.
References
1. Banchereau, J. and Palucka, A.K. (2005) Nat. Rev.
Immunol. 5: 296306.
2. den Brok, M.H. et al. (2005) Expert Rev. Vaccines
4: 699710.
3. Aarntzen, E.H. et al. (2013) Cancer Res. 73: 1929.
4. Figdor, C.G. et al. (2004) Nat. Med. 10: 475480.
5. Schreibelt, G. et al. (2010) Blood 116: 564574.
6. Tel, J. et al. (2013) Cancer Res. 73: 10631075.
b
va i l a
a
w
o
N
le as
ed
k
r
a
CE-m
vice
a
medic
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p
o
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in E
The CliniMACS Prodigy

Mastering the complexity of cell processing
Automated cell processing from cell sample to final product

Flexible programming options
Fully automated, closed system
MACS Cell Separation
Density gradient fractionation
Cell washing procedures
Optimized cell culture
clinimacs-prodigy.com
The CliniMACS System components: Reagents, Tubing Sets, Instruments and PBS/EDTA Buffer are manufactured and controlled under an ISO 13485 certified quality
system. In Europe, the CliniMACS System components are available as CE-marked medical devices. In the USA, the CliniMACS System components including the
CliniMACS Reagents are available for use only under an approved Investigational New Drug (IND) application or Investigational Device Exemption (IDE). In the US, the
components of the CliniMACS Prodigy line are for research use only and not for human therapeutic or diagnostic use. CliniMACS MicroBeads are for research use only
and not for human therapeutic or diagnostic use.
Report
An optimized method for the manufacture of

a dendritic cellbased vaccine for translational
glioblastoma research
Sara Nava1, Marta Dossena1, Simona Pogliani1, Daniela Lisini1, Serena Pellegatta2, Eugenio A. Parati1, Gaetano Finocchiaro2, and Simona Frigerio1
1
2
Cell Therapy Production Unit UPTC, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
Unit of Molecular Neuro-Oncology, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
Introduction
mg protein/gram tissue
pharmaceutical products, as assessed by

Glioblastoma (GB) is the most prevalent and microbiological safety, viability, phenotype,
aggressive malignancy in the central nervous and functionality of mDCs produced.
system (CNS). Immunotherapy represents a
promising approach to support the immune Materials and methods
system in the elimination of tumors, and The study was approved by the local
dendritic cells (DCs) hold great potential institutional review board of the Fondazione
for the induction of anti-tumor responses as IRCCS Istituto Neurologico Carlo Besta (Milan,
shown in animal models- as well as various Italy), and informed written consent was
clinical trials,. DCs are professional antigen- obtained from all patients. The whole process
presenting cells playing a central role in the was performed in the clean-room facility of the
regulation of the adaptive immune response via Cell Therapy Production Unit in the Istituto
T cells and B cells. Remarkably, DCs incubated Neurologico Carlo Besta.
with tumor lysates or peptides generate specific
anti-tumor responses in the CNS-.
For the manufacture of DC products that are
p < 0.05
appropriate for translational research towards
25
immunotherapy, it is mandatory to follow good
manufacturing practice (GMP) guidelines.
20
Moreover, a high level of standardization is
required to ensure maximum reproducibility.
15
We developed an optimized method for the
production of a DC-based vaccine, involving i)
10
the automated isolation of CD14+ monocytes
from peripheral blood mononuclear cells
5
(PBMCs) using the closed CliniMACS System,
ii) differentiation of monocytes into immature
0
DCs (iDCs), iii) standardized and reproducible
Classical
New
preparation of autologous tumor lysates using
method
method
the gentleMACS Dissociator, and iv) efficient
maturation of iDCs into mature DCs (mDCs)
Figure 1Comparison of the new tumor lysis
using the tumor lysates.
The resulting mDCs were functional as protocol vs. a traditional standard method. Lysates
were prepared as described in the materials and
evaluated using a mixed lymphocyte methods section. Data indicate meanssd from
reaction (MLR). This method is reproducible 12 and 19 preparations using the classical and new
and conforms to GMP guidelines for protocol, respectively. Data were adapted from ref. 11.
16 MACS&more
Vol 15 2/2013
DC culture
CD14+ cells were isolated from leukapheresis
products from GB patients using the
CliniMACS System (Miltenyi Biotec)
according to the manufacturers guidelines.
The positive fraction was cultured at 35106
cells/mL in a VueLife Closed Culture Systems
(Afc) in CellGRO Medium (CellGenix),
supplemented with 20 ng/mL IL-4 and 50
ng/mL GM-CSF. All reagents were of clinical
grade. On day 5 of culture, iDCs were pulsed
with autologous tumor lysate (see below) at the
concentration of 50 g protein/106 living cells
plus 50 g/mL keyhole limpet hemocyanin
(KLH, Calbiochem), and 10 ng/mL IL-4 and
25 ng/mL GM-CSF for 24 hours. On day 6,
antigen-loaded DCs (aDCs) were cultured
with a pro-inflammatory cocktail including
10 ng/mL of TNF-, IL-1, IL-6, and 1 g/mL
PGE2. After 24 hours, mDCs were collected
and frozen. All samples were stored in a GMPdedicated area and managed with a Good
Automated Manufacturing Practices-4 (GAMP
4) software.
Preparation of tumor lysates
GB specimens removed during surgery were
used for the preparation of tumor lysates.
Samples were washed in a sterile 0.9% NaCl
solution, weighed, and snap-frozen in nitrogen
gas until use. For tumor lysate preparation two
alternative methods were applied.
The classical method was previously
described by Ashley and colleagues. In brief,
tumor samples were minced and mechanically
dispersed using syringes with decreasing
Report
Flow cytometry
PBMCs from healthy donors and DCs
harvested at different culture steps (iDCs day
5, aDCs day 6, mDCs day 7) were analyzed by
flow cytometry using CD14-PE, CD80-FITC,
CD83-PE, CD86-PE, and HLA-DR-FITC
antibodies. Specific staining was determined
with appropriate isotype controls.
Mixed lymphocyte reaction (MLR)
PBMCs were isolated from patient or unrelated
healthy donor blood by centrifugation over
a Ficoll-Paque gradient and resuspended
in CellGRO medium (CellGenix). For
unidirectional MLRs 2105 responder cells
(PBMCs) were co-cultured with stimulating
cells in a 96-well plate (Corning). Stimulating
cells were 1104 DCs, 2105 autologous
PBMCs (for auto-MLR, i.e., negative control),
or 2105 allogeneic PBMCs (for allo-MLR,
i.e., positive control). Stimulating cells were
pre-treated with mitomycin C (50 g/mL,
Sigma Aldrich) for 20 minutes at 37 C and
used after extensive wash. After 5 days, 1 Ci
CD14
80
% of Max.
Positive cells (%)
100
p < 0.01
40
30
20
60
40
10
20
iDCs
aDCs
mDCs
100
100
75
80
% of Max.
p < 0.001
50
HLA-DR
100
Positive cells (%)
60
50
25
aDCs
iDCs
mDCs
100
25
aDCs
mDCs
monocytes
40
aDCs
iDCs
mDCs
CD83 (MFI)
100
60
40
20
20
iDCs
100
10
10
10
10
CD86-PE
aDCs
80
50
40
p < 0.05
80
MFI
50
101 102 103

CD83-PE
60
20
100
80
iDCs
% of Max.
20
CD86
p < 0.001
p < 0.001
100
80
40
101 102 103

CD80-FITC
100
60
% of Max.
aDCs
75
Positive cells (%)
% of Max.
40
20
% of Max.
Positive cells (%)
60
20
100
Positive cells (%)
80
iDCs
p < 0.001
100
80
40
101 102 103

HLA-DR-FITC
p < 0.001
80
60
100
CD83
p < 0.001
p < 0.001
mDCs
CD80
100
40
20
101 102 103

CD14-PE
60
mDCs
60
40
20
Classical
method
New
method
Classical
method
100
101 102 103

CD83-PE
New method
Figure 2 Phenotypic analysis of DCs. Cells were cultured as described in the materials and methods section.
iDCs (day 5), aDCs (day 6), and mDCs (day 7) were stained with fluorochrome-conjugated antibodies and
analyzed by flow cytometry. (A) The bar charts show percentages of the cell types that were positive for the
respective markers. Data represent meanssd from 31 independent experiments. The histograms show the
results from one representative experiment. (B) CD83 expression in mDCs that were prepared by using tumor
lysates from either the classical or the new method. The bar chart indicates the MFI normalized to isotype
controls (left bar: 12 experiments, right bar: 19 experiments). The histogram shows the results from one
representative experiment. Data were adapted from reference 11.
100
p = ns
p < 0.001
80
60
SI
needle sizes (18G, 20G, and 21G). The

single-cell suspension was diluted in PBS and
centrifuged at 300g for 5 minutes. The pellet
was resuspended in PBS and filtered through
70 m and 30 m filters. The suspension
was centrifuged at 300g for 5 minutes and
sonicated in a bath for at least 1 hour.
In our new method tumor samples were
minced, transferred to a gentleMACS
C Tube (Miltenyi Biotec) and mechanically
dissociated with the gentleMACS Dissociator
(Miltenyi Biotec) using the installed software
program m_spleen04. The resulting singlecell suspension was centrifuged at 1000g for
10 minutes. The pellet was resuspended in PBS,
transferred to a gentleMACS M Tube (Miltenyi
Biotec) and homogenized with the gentleMACS
Dissociator using program protein_01. The
homogenate was filtered through 70 m and 30
m filters and then sonicated for 30 minutes.
For both methods the presence of latent live
tumor cells was determined by trypan blue
exclusion. If viable cells were present, further
sonication steps were performed until 0%
viability was obtained. Protein content was
determined by reaction with bicinchoninic
acid (BCA, Pierce Biotechnology/Thermo
Fisher Scientific) following the manufacturers
instructions.
40
20
0
iDCs
mDCs
both
methods
mDCs
classical
method
mDCs
new
method
autologous
MLR
Figure 3 Functional analysis of DCs. The capacity of iDCs and mDCs to induce MLR was assessed as
described in the materials and methods section. The results indicate meanssd from the following numbers of
experiments: 18 (iDCs), 31 (mDCs both methods), 12 (mDCs classical method), 19 (mDCs new method), and
18 (autologous MLR). Data are expressed as stimulation index (SI) and represent meanssd. ns: not significant.
Data were adapted from reference 11.
Vol 15 2/2013
MACS&more 17
Report
[3H]-thymidine (Amersham Biosciences) was

added for further 18 hours. The radioactivity
incorporated into DNA was measured in a
-scintillation counter. Results were expressed
as stimulation index (SI) allowing the
comparison of results from different donors.
SI was calculated as follows: mean counts per
minute (cpm) from stimulated cells divided
by mean cpm from non-stimulated cells. MLR
responses were considered positive when SI
was 3 for PBMC-induced stimulation and
SI6 for DC-induced stimulation.
One-way ANOVA and two-tailed test were
utilized for all statistical analyses.
Results
Isolation of monocytes and preparation of
mature DCs
DCs can be obtained through differentiation
of CD14+ monocytes into iDCs in the presence
of GM-CSF and IL-4, and subsequent
maturation to mDCs with a pro-inflammatory
cocktail (TNF-, IL-1, IL-6, and PGE2). As
the first step, we isolated CD14+ monocytes
from leukapheresis products using the
closed, automated CliniMACS System. In
31 preparations we achieved CD14+ cell
purities of 98.72.3% (meansd). A starting
population of at least 5109 WBCs was used
for the isolation of CD14+ monocytes to end
up with a reasonable amount of DC vaccine, a
final number of at least 55106 mDCs. In 31
preparations, we used (11.524.0)109 WBCs
(meansd), which resulted in (1.30.5)109
CD14+ monocytes. Following differentiation
and maturation, we obtained (117.147.0)106
mDCs, which corresponds to a yield of
1.20.7% and 9.13.1%, in relation to the
numbers of WBCs and CD14+ monocytes,
respectively. Cell viability after one freeze-thaw
cycle amounted to 94.33.9% (meansd).
Novel method for the preparation of
tumor lysates
DC-based vaccines can be generated by pulsing
DCs with synthetic peptides or recombinant
proteins representing certain tumor-associated
antigens (TAA). However, expression of TAA
in GB is heterogeneous, and selected peptides
or proteins might not reflect the actual
expression profile of relevant (and possibly
unknown) TAA in a particular tumor. To tailor
18 MACS&more
Vol 15 2/2013
a DC-based vaccine to a specific glioblastoma, regardless of whether mDCs were prepared

it is therefore crucial to pulse the DCs with with lysates from the new method or the
autologous whole tumor lysates containing all classical method. As a negative control, we also
potential TAA. Both soluble and membrane- tested antigen-loaded mDCs in combination
bound proteins are extracted in our new with PBMCs from the same patient prior to
method for the preparation of tumor lysates. vaccination. We found that the mDCs did not
The procedure is highly standardized through induce autologous MLR.
automation, and takes place in a closed system.
The process is highly efficient as reflected by a Conclusions
130% increase in the protein yield compared to The CliniMACS System enables isolation of
CD14+ monocytes with high purity.
a traditional standard method (fig. 1). The high
amount of protein in the tumor lysate allowed We developed a new procedure for the
efficient preparation of tumor lysates in
us to load all the iDCs obtained after monocyte
an automated, closed system using the
differentiation.
gentleMACS Dissociator.
mDCs pulsed with tumor lysates from the
Phenotypic analysis of iDCs, aDCs,
new method showed a higher and more
and mDCs
consistent CD83 expression than mDCs
To evaluate the differentiation and maturation
pulsed with the classical method, indicating
status of the DCs, we used the monocyte
a more effective and uniform maturation of
marker CD14 as well as the maturation markers
DCs by lysates from the new method.
CD80, CD83, and CD86. HLA-DR was used
as a marker for antigen-presenting cells. Flow The mDCS are functional antigen-presenting
cells as assessed by MLR.
cytometric analysis showed that the maturation
markers were significantly up-regulated in The high yields of isolated monocytes and
tumor lysates enable the manufacture of
mDCs compared to iDCs and aDCs. CD14
mDCs in numbers that are sufficient for a
was down-regulated accordingly. Expression
clinical trial.
of HLA-DR persisted indicating that the
mDCs were functional antigen-presenting cells
References
(fig. 2A).
1. Pellegatta, S. et al. (2006) Cancer Res. 66: 10247
CD83 is currently the most specific marker
10252.
for mature DCs and plays a key role in the 2. Pellegatta, S. et al. (2006) Neurol. Res. 28: 527
531.
initiation of anti-tumor immune responses.
Therefore, we compared expression of 3. Mayordomo, J.I. et al. (1995) Nat. Med. 1: 1297
1302.
CD83 in mDCs prepared with tumor lysates 4. Pellegatta, S. and Finocchiaro, G. (2009) Methods
from either the classical method or the new
Mol. Biol. 568: 233247.
method. The mean fluorescence index (MFI) 5. Thurner, B. et al. (1999) J. Immunol. Methods
223: 115.
of CD83+ cells prepared with the new method
6. Liau, L.M. et al. (2005) Clin. Cancer Res. 11:
was significantly higher (fig. 2B; 75.38.1 vs.
55155525.
38.913.4; p<0.05). The histogram in figure 7. Liau, L.M. et al. (2000) Neurosurg. Focus. 9: e8.
2B shows that CD83 fluorescence was higher 8. Daga, A. et al. (2011) Curr. Pharm. Des. 17: 2439
2467.
and more uniform in cells prepared with the
new method, indicating that CD83 expression 9. Wheeler, C.J. et al. (2004) Clin. Neurosurg. 51:
132139.
in these cells occurred at a higher level and 10. Ardon, H. et al. (2012) Cancer Immunol.
more consistently than in cells prepared with
Immunother. 61: 20332044.
the classical method. Thus, the lysate prepared 11. Nava, S. et al. (2012) PLoS One 7: e52301.
with the new method resulted in a more 12. Ashley, D.M. et al. (1997) J. Exp. Med. 186: 1177
1182.
efficient maturation of DCs.
Functional analysis of DCs
To study DC functionality we used a one-way
MLR with iDCs and mDCs as stimulating cells
and PBMCs from unrelated healthy volunteers
as responder cells. Overall, the mDCs induced
a higher allogeneic MLR than iDCs (fig. 3),
13. Sallusto, F. and Lanzavecchia, A. (1994) J. Exp.

Med. 179: 11091118.
14. Iwamoto, M. et al. (2003) Int. J. Cancer 104: 9297.
Report
MACS Product
Order no.
151-01
CliniMACS CD14 Reagent
272-01
700-25
MACS GMP Recombinant

Human GM-CSF
170-076-112

Human IL-1
170-076-102

Human IL-4
170-076-101

Human IL-6
170-076-104

Human TNF-
170-076-103
gentleMACS Dissociator
130-093-235
gentleMACS C Tubes
130-096-334
gentleMACS M Tubes
130-096-335

Europe, the CliniMACS System components are available as
components including the CliniMACS Reagents are available
for use only under an approved Investigational New Drug
(IND) application or Investigational Device Exemption (IDE).
CliniMACS MicroBeads are for research use only and not for
local representative. MACS GMP Products are manufactured
The gentleMACS Dissociator and gentleMACS Tubes are
for research use only. Unless otherwise specifically indicated,
Miltenyi Biotec products and services are for research use only
and not for therapeutic or diagnostic use.
Regulatory T cells for clinical

research exclusive solutions
iscover our solutions for GMPD
compliant enrichment and
expansion of regulatory T cells
(Tregcells).
CliniMACS CD25 System
Robust large-scale enrichment
CE-marked components
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Efficient expansion of Treg cells
TexMACS GMP Medium
Free of serum and animal
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erfect for the demands of Treg
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ISO 13485 certified

manufacturing of reagents
miltenyibiotec.com
Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use
only and not for therapeutic or diagnostic use. The CliniMACS System components: Instruments,
Reagents, Tubing Sets, and PBS/EDTA Buffer are manufactured and controlled under an
ISO 13485 certified quality system. In Europe, the CliniMACS System components are available
as CE-marked medical devices. In the USA, the CliniMACS System components including the
CliniMACS Reagents are available for use only under an approved Investigational New Drug (IND)
application or Investigational Device Exemption (IDE). MACS GMP Products are for research use
and ex vivo cell culture processing only, and are not intended for human in vivo applications.
The products are manufactured and tested under a certified ISO 9001:2000 quality system and
in compliance with relevant GMP guidelines and the recommendations of USP chapter 1043 on
ancillary materials. In the USA, the MACS GMP ExpAct Treg Kit is available for research use only.
Vol 15 2/2013
MACS&more 19
Report
Enrichment, stimulation, and viral transduction

of naive and central memory CD8+ T cells under
GMP conditions for translational research
towards the development of adoptive cell
therapy of cancer patients
Anna Casati1, Azam Varghaei-Nahvi2, Steven A. Feldman2, Mario Assenmacher1, Steven A. Rosenberg2, Mark E. Dudley2, and Alexander Scheffold3
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Surgery Branch, National Cancer Institute, National Institute of Health, Bethesda, MD, USA
3
Department of Rheumatology and Clinical Immunology, Charit, Universittsmedizin Berlin, Berlin, Germany
Introduction
for cell activation and transduction.
Adoptive immunotherapy involving the transfer One of the current standard procedures for
of genetically engineered T cells can mediate cell activation involves soluble CD3 antibodies
tumor regression in patients with metastatic in combination with, for example, autologous
melanoma or other forms of cancer.- However, antigen-presenting cells (APCs) for crossthe efficacy of T cell immunotherapy depends linking. This protocol is highly dependent
on a number of critical steps. Autologous on the quality of the cross-linking cells and
T cells collected from the patients are activated, requires large amounts of viral supernatants,
genetically modified, expanded, and finally which is a major cost factor in this process. The
infused back into the patient to unfold their other standard T cell activation process involves
anti-tumor activity.
CD3 and CD28 antibodies immobilized on
Peripheral blood T cells are heterogeneous with solid surfaces, such as cell culture vessels or
regard to their function and differentiation large cell-sized particles., These methods
states, i.e., their self-renewal capacity have major drawbacks, as they are timeand persistence. In particular terminally consuming and difficult to standardize for
differentiated effector memory T (Tem) GMP compliance. Moreover, large particles
cells show poor persistence and only weak need to be removed prior to infusion of T cells
capacity to mediate tumor regression-. It has into the patient.
been shown that engineering of T cells can We developed a reliable method for the
improve their anti-tumor reactivity and in vivo enrichment of CD8+ Tn and Tcm cells under
persistence., Moreover, naive T cells (Tn) GMP conditions. We also introduced a new
show a higher efficacy in adoptive cell transfer flexible nanomatrix conjugated with CD3
in a cancer setting than memory T cells.- and CD28 antibodies (TransAct Reagent),
Among memory T cells, central memory Tcells which allows easier handling for the activation
(Tcm) were shown to exert a higher anti-tumor of T cells in a closed system under GMP
activity than Tem cells in mice,, non-human conditions. The activated Tn and Tcm cells
primate, and humanized mouse models. isolated from blood of melanoma patients
However, the translation of this principle into were expanded and transduced efficiently.
therapeutic strategies is hampered by the lack Tn and Tcm cells activated with TransAct
of appropriate protocols for the clinical-scale Reagent or CD3/CD28-conjugated cell-sized
isolation of CD8+ Tn and Tcm cells, as well as MACSiBead Particles showed only minimal
GMP-compliant reagents and reliable processes effector differentiation.
20 MACS&more
Vol 15 2/2013

Enrichment of human CD8+ Tn and
Tcm cells
For all experiments fresh leukapheresis samples
from patients with metastatic melanoma were
used. Patients were enrolled in clinical trials
approved by the National Cancer Institute
Institutional Review Board. PBMCs were
prepared by density gradient centrifugation
(Ficoll Hypaque) and washed twice in PBS.
For each enrichment, 24109 PBMCs were
used.
Tn cells were enriched with a three-step
procedure. Firstly, PBMCs were incubated with
CliniMACS CD4 MicroBeads on a rotator for
30 min at room temperature (RT). Cells were
washed by centrifuging and resuspending
in 100 mL of buffer A, i.e., CliniMACS PBS/
EDTA Buffer supplemented with 0.5% human
albumin (Plasbumin-25 25%, USP; Talecris
Biotherapeutics), and transferred to a collection
bag. Magnetically labeled CD4+ cells were
depleted using the CliniMACS Plus Instrument
(program Depletion 2.1) and the CliniMACS
Tubing Set LS. Secondly, the unlabeled target
cells were incubated with CliniMACS CD62L
MicroBeads for 15 min in cold buffer A and
washed. Target cells were enriched using
the CliniMACS Plus Instrument (program
Enrichment 3.2) and the CliniMACS Tubing
Set. Finally, the target fraction was labeled with
a CD45RA-Biotin, human antibody and Anti-
Report
Biotin MACSiBead Particles. Cells were further

enriched using the MACSiMAG Separator.
Tcm cells were enriched with a two-step
procedure. PBMCs were incubated with
CliniMACS CD4 MicroBeads and CliniMACS
CD45RA MicroBeads (in cold buffer A) on
a rotator for 30 min at RT. Subsequently, the
cells were washed, resuspended in 100 mL of
buffer A, and transferred to a collection bag.
Magnetically labeled cells were depleted using
the CliniMACS Plus Instrument (program
Depletion 2.1) and the CliniMACS Tubing Set
LS. The unlabeled target cells were incubated
with CliniMACS CD62L MicroBeads for
15 min in cold buffer A and washed. Target
cells were enriched using the CliniMACS Plus
Instrument (program Enrichment 3.2) and the
CliniMACS Tubing Set.
Flexible nanomatrix for T cell stimulation
(TransAct Reagent)
The TransAct Reagent was provided by Miltenyi
Biotec. TransAct Reagent is a colloidal reagent
consisting of nanoscale iron oxide crystals
embedded into a biocompatible polysaccharide
matrix with an overall diameter of about
100nm. Antibodies against CD3 (clone OKT3)
and CD28 (clone 15E8) are covalently attached
to the matrix. The matrix can be produced
under cGMP conditions, sterilized by filtration
and unbound reagent can easily be removed
from the cell suspension by centrifugation of
the cells. In the experiments described here,
the reagent was used at 0.5 g/mL of both
antibodies conjugated in a 1:1 ratio.AntiCD3
andantiCD28 TransAct Reagentscan also be
used when conjugated on separate matrices and
mixed in variable ratios and concentrations to
achieve optimal T cell activation.
T cell stimulation and transduction
Three different methods were used for
stimulation of the enriched T cell subsets:
1)CD3 antibody coated on the cell culture dish
(OKT3; 10 g/mL) in combination with soluble
CD28 antibody (15E8; 1 g/mL), referred to as
CD3/CD28, 2) MACSiBead Particles loaded
with CD3, CD28, and CD2 antibodies, and
3) TransAct Reagent. Interleukin 2 (IL-2; 300
IU/mL) was present in the cell culture for all
three approaches. T cells were transduced
using the mF5 retrovirus encoding an antiMART-1 T cell receptor (TCR) comprising
constant regions of the mouse TCR and
chains in addition to human TCR CDR

antigen-combining regions. Transductions
were performed as described.
Flow cytometry
To analyze the phenotypes of the T cell
subsets, cells were labeled with fluorochromeconjugated antibodies against CD8, CD4,
CD45RA, CD62L, CD127, IFN-, IL-2, TNF-
(all from Miltenyi Biotec), CCR7, CD27,
CD45RO (BD Biosciences), and MART-1
tetramer (Beckman-Coulter; Allele HLA-A
0201, peptide MART-1, ELAGIGILTV).
For the analysis of intracellular cytokine
production, cells were cocultured with the
624 (MART-1+HLA-A2+) melanoma cell line
in the presence of brefeldin A. After 6 hours
cells were labeled with the CD8 antibody, fixed,
and permeabilized with the Inside Stain Kit
(Miltenyi Biotec). Subsequently, cells were
analyzed by flow cytometry.
positive selection of CD62L + cells. The

unlabeled non-target fraction from the
CD62L+ cell enrichment step was enriched in
CD8+CD45RACD62L cells and represented
the Tem cells in our in vitro experiments.
Phenotypic characterization of CD8+ Tn

and Tm cells
The CD8+ cell subsets are characterized
by differential expression of a number of
additional markers. As CD62L and CCR7 are
generally co-expressed in Tn and Tcm cells,
we analyzed the enriched cell populations for
CCR7 surface expression (fig. 1) and found
that 705.5% of the Tn cells and 547% of
the Tcm cells expressed CCR7. In contrast,
only 13.22.4% of the Tem cell fraction
expressed CCR7 (see fig. 1 for representative
dot plots). A large proportion of the Tn cell
fraction (62.55.7%) expressed CD127 (IL7R), whereas expression in Tcm and Tem cells
amounted to only 303.7% and 30.77.3%,
respectively. Expression of CD27 is known to
Groups were compared using the two- positively correlate with replicative capacity
tailed unpaired t-test. The error bars in the and tumor response,. We found that CD27
graphs represent the standard error of means. expression was significantly higher in Tn and
Tcm cells than in Tem cells (881.8% and
Results
80.55.3 vs. 625.4%). In contrast, granzyme
Clinical-scale enrichment of human
A expression was higher in Tem cells (852.2%)
CD8+ Tn and Tcm cells
compared to Tn and Tcm cells (16.73.6%
Human CD8+ T cells represent a heterogeneous and 42.56.6%). This is an agreement with
population of Tn, Tcm, and Tem cells, which the notion that lymph node homing capacity
can be distinguished by their expression of and the acquisition of effector function are
CD45RA and CD62L (table 1). We used these coordinately regulated,. Taken together,
phenotypic characteristics to isolate Tn and these data indicate that the GMP-compliant
Tcm cells. Tn cells were enriched in a three- procedures used for enrichment of Tn and Tcm
step procedure, involving CD4+ cell depletion, cells yield populations with distinct, typical
positive selection of CD62L+ cells, and a second phenotypes (fig. 1).
positive selection of CD45RA+ cells. Purity and
recovery are shown in table 1. The remaining Transduction of T cell subsets with antigenCD8 cells contained in the target fraction were specific TCR
mainly NK and B cells.
The standard protocols for in vitro activation
Tcm cells were enriched in a two-step of T cells involve autologous antigenprocedure, i.e., simultaneous depletion of presenting cells (APCs), which are required
CD4+ and CD45RA+ cells and subsequent for cross-linking of soluble CD3 antibodies
T cell subset
Phenotype
Purity
Recovery
Naive T (Tn) cells
CD8+CD45RA+CD62L+
75.215.5%
44.616.7%
Central memory T (Tcm) cells
CD8+CD45RACD62L+
849.6%
60.923.8%
Effector memory T (Tem) cells
CD8+CD45RACD62L
n.d.
n.d.
Table 1 Phenotypes of human CD8+ T cell subsets. Purities (among CD8+ cells) and recoveries after
enrichment with the CliniMACS System are shown. Numbers indicate means SEM (n=6). Data were adapted
from reference 15.
Vol 15 2/2013
MACS&more 21
Report
TN
TCM
8
16
4.5
43
15
10
CCR7
70
PBMCs
TEM
CD45RO
CD45RO
58
18
48
CD45RO
CD45RO
71
25
3.4
0.7
47
11
0.1
86
40
CD62L
14
27
92
56
CD127
CD127
CD127
60
6.4
89
27
0.3
4.5
24
2.6
13
72
20
44
CD62L
11
CD127
CD27
CD27
30
CD27
50
GranzA
23
CD27
90
GranzA
80
GranzA
GranzA
Figure 1 Enriched Tn, Tcm, and Tem cells show distinct phenotypes. Enriched T cell subsets and
PBMCs were analyzed for expression of CCR7, CD45RO, CD62L, CD127, CD27, and granzyme A (GranzA).
Representative dot plots are shown. Numbers indicate the percentages of cells in the respective gates.
*0.01 < P < 0.05; **0.005 < P < 0.01; ***P < 0.005. Data were adapted from reference 15.
100
**
**
% MART-1-tetramer+
CCR7+ cells
% MART-1-tetramer+
CD62L+ cells
and costimulatory signals. Since APCs are

removed during enrichment of the T cell
subsets, an alternative approach was required
to effectively stimulate the enriched T cells. We
tested three methods: 1) CD3 antibodies coated
onto the cell culture vessel plus soluble CD28
antibody, 2) CD3 and CD28 antibodies linked
to MACSiBead Particles, and 3) TransAct
Reagent, a novel GMP-compatible reagent,
which consists of CD3 and CD28 antibodies
immobilized on a novel flexible nanomatrix. In
contrast to MACSiBead Particles, the TransAct
Reagent can be effectively removed from the
culture by centrifugation. Thus, the reagent can
be handled similarly to soluble antibodies.
CD8+ Tn, Tcm, and Tem cells were stimulated
for 60 hours and then transduced with a
retroviral vector encoding the anti-MART-1
TCR. Ten days after transduction, the
transduction efficiency was similar for all three
T cell subsets, regardless of the stimulation
protocol, as determined by MART-1 tetramer
staining (data not shown). We analyzed the
transduced cell subsets for expression of CCR7
and CD62L after 1315 days of expansion.
CD62L was expressed at equally high levels on
Tn and Tcm cells, whereas Tem cells showed a
lower cell surface expression of this marker (fig.
2). CCR7 was highly expressed on Tn cells, in
contrast to Tcm cells, which showed reduced
surface expression, and Tem cells, which
were CCR7 negative. Tn cells stimulated with
TransAct Reagent or MACSiBead Particles
showed a higher frequency of CCR7+MART-1
tetramer +CD8 + cells (57.226.8% and
58.2610.1%) compared to cells stimulated
with CD3/CD28 (30.58.4%). This indicates
that the phenotype, in particular expression of
75
50
25
0
TN
TCM TEM
CD3/CD28
TN
TCM TEM
MACSiBead
Particles
TN
TCM
TransAct
Reagent
TEM
PBMCs
100
*
*
75
***
*
***
***
***
50
25
0
TN
TCM TEM
CD3/CD28
TN
TCM TEM
MACSiBead
Particles
TN
TCM
TEM
PBMCs
TransAct
Reagent
Figure 2 Expression of CD62L and CCR7 in MART-1 tetramer+ Tn, Tcm, and Tem cells. The three T cell subsets were enriched from PBMCs and stimulated with
CD3/CD28 antibodies, MACSiBead Particles, or TransAct Reagent in the presence of IL-2. PBMCs were stimulated with soluble CD3 and CD28 antibodies. Cells were
transduced 60 hours after stimulation to express MART-1-specific TCR. Cells were analyzed by flow cytometry 1315 days after stimulation. Data indicate frequencies
of (A) MART-1 tetramer+CD62L+ cells and (B) MART-1 tetramer+CCR7+ cells among CD8+ cells. Data were adapted from reference 15.
22 MACS&more
Vol 15 2/2013
Report
Conclusion
B
% CD8%+IL-2
CD8++cells
IL-2+ cells
20
20
1.4
1.4
6.8
6.8
TransAct
Reagent
TransAct
Reagent
CD8 CD8
CD8 CD8
CD8 CD8
CD3/CD28
3.4
3.4
IL-2
IL-2
IL-2
IL-2
IL-2
IL-2
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
TN TCM TEM
TN TCM TEM
TN TCM TEM
PBMCs
*
*
*
*
10
10
0
0
% CD8%+IFN-
CD8++IFN-
cells+ cells
Functional analysis of T cell subsets

To test whether the MART-1 TCR in transduced
cells was functional, we re-stimulated the T cell
subsets with a MART-1+HLA-A2+ cell line and
analyzed the production of IL-2, TNF-, and
IFN-. The percentage of CD8+IL-2+ Tn cells
was higher when they had been stimulated
with MACSiBead Particles (134.2%) or
TransAct Reagent (7.31.3%) compared to
CD3/CD28 (3.80.7%) (fig. 3B). Similarly, Tn
cells stimulated with MACSiBead Particles
showed a higher percentage of TNF-producing cells (42.24.2%) than CD3/CD28stimulated Tn cells (225.6%) (fig. 3D). The
percentage of IFN--producing T cells was
similar for all three methods of stimulation (fig.
3C). There was a slight trend towards increased
percentages of IFN-+ cells in Tcm and Tem
cells in comparison to Tn cells.
Compared to CD3/CD28-stimulated Tn cells,
the cells stimulated with MACSiBead Particles
or TransAct Reagent showed an increase in
IL-2, equivalent or increased TNF- levels,
and equivalent or reduced IFN- levels, which
indicates less progress towards terminal
differentiation.
MACSiBead
Particles
MACSiBead
Particles
CD3/CD28
A
+
% CD8%+TNF-
CD8+TNF-
cells+ cells
the homing receptor CCR7, can be modulated

by the method of cell stimulation.
Taken together, our results show that the
enriched T cell subsets can be efficiently
stimulated, transduced, and expanded over
two weeks. However, expression patterns in
expanded cells can differ from the initial cell
population, and marker expression varies
dependent on the stimulation method, which
may have an effect on the cells behavior in vivo.
60
60
40
40
20
20
0
0
60
60
40
40
20
20
0
0
We developed a reliable and easy-to-perform

MACSiBead Particles
TransAct Reagent
CD3/CD28
GMP-compliant method for the enrichment
MACSiBead Particles
TransAct Reagent
CD3/CD28
of CD8+ Tn and Tcm cells from leukapheresis
Figure 3 Cytokine production by Tn, Tcm, and Tem cells following re-stimulation with MART-1+
products.
We developed a novel reagent for effective melanoma cells. T cells were stimulated using the different protocols as indicated, and transduced to express a
T cell stimulation, TransAct Reagent, which MART-1-specific TCR. Transduced T cell subsets and PBMCs were cultured for a total of 1315 days+ and then+
washed to remove IL-2. Cells were cultured for two more days and re-stimulated with a MART-1 HLA-A2
can be sterile filtered and added to cell melanoma cell line for 6 hours. Cytokine production was determined by intracellular staining. (A) IL-2
cultures at defined concentrations. Excess expression in CD8+ Tn cells. Numbers indicate percentages of IL-2+ cells among CD8+ cells. (B) Frequencies
reagent can be easily removed from the cells of CD8+IL-2+, CD8+IFN-+, and CD8+TNF-+ cells among CD8+MART-1 tetramer+ cells. *0.01 < P < 0.05;
**0.005 < P < 0.01; ***P < 0.005. Data were adapted from reference 15.
by centrifugation.
The enriched T cell subsets can be stimulated
expression of CD62L and CCR7 is preserved, The enrichment and expansion procedure
and efficiently transduced to produce MARTin particular on Tn cells, which may confer
yields a high number of cells providing a
1-specific cells.
improved in vivo persistence to the cells.
solid basis for future translation into clinical
Following stimulation of T cells with
studies.
TransAct Reagent or MACSiBead Particles,
Vol 15 2/2013
MACS&more 23
Report
References
1. Restifo, N.P. et al. (2012) Nat. Rev. Immunol. 12:
269281.
2. Brenner, M.K. and Heslop, H.E. (2010) Curr.
Opin. Immunol. 22: 251257.
3. Johnson, L.A. et al. (2009) Blood 114: 535546.
4. Morgan, R.A. et al. (2006) Science 314: 126129.
5. Morgan, R.A. et al. (2010) Mol. Ther. 18: 843851.
6. Lamers, C.H. et al. (2011) Blood 117: 7282.
7. Johnson, L.A. et al. (2006) J. Immunol. 177: 6548
6559.
8. Hinrichs, C.S. et al. (2009) Proc. Natl. Acad. Sci.
USA 106: 1746917474.
9. Gattinoni, L. et al. (2005) J. Clin. Invest. 115:
16161626.
10. Hinrichs, C.S. et al. (2011) Blood 17: 808814.
11. Klebanoff, C.A. et al. (2005) Proc. Natl. Acad. Sci.
USA 102: 95719576.
12. Klebanoff, C.A. et al. (2004) Proc. Natl. Acad. Sci.
USA 101: 19691974.
13. Berger, C. et al. (2008) J. Clin. Invest. 118: 294
305.
14. Wang, X. et al. (2011) Blood 117: 18881898.
15. Casati, A. et al. (2013) Cancer Immunol.
Immunother. 62: 15631573.
16. Bialer, G. et al. (2010) J. Immunol. 184: 62326241.
17. Bunnell, B.A. et al. (1995) Proc. Natl. Acad. Sci.
USA 92: 77397743.
18. Sallusto, F. et al. (1999) Nature 401: 708712.
19. Huang, J. et al. (2006) J. Immunol. 176: 77267735.
20. Huang, J. et al. (2005) J. Immunother. 28: 258267.
21. Geginat, J. et al. (2003) Blood 101: 42604266.
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diagnostic use.
Report
Clinical-grade purification and

expansion of CD56+CD3 NK cells for
adoptive immunotherapy of solid
tumors and leukemia
Ulrike Koehl1, Ruth Esser1, Lutz Uharek2, Martin Stern3, and Jakob Passweg3
Institute of Cellular Therapeutics GMP Development Unit (OE 8885), IFB-Tx and Cellular Therapy Centre Hannover Medical School (MHH), Hannover, Germany
Internal Medicine, Hematology and Oncology, Charit Berlin, Germany.
3
Division of Hematology, University Hospital Basel, Switzerland.
Introduction
Cell therapy may represent a promising
treatment option for patients who suffer
from leukemia and tumors and have a high
risk for relapse after allogeneic, especially
haploidentical, stem cell transplantation
(SCT). While established T cell therapies,
such as donor lymphocyte infusions, are
associated with the risk of graft-versus-host
disease (GvHD), natural killer (NK) cells
may mediate graft-versus-leukemia/tumor
effects without induction of GvHD. Therefore,
immunotherapy with highly purified NK cells
in recipients of haploidentical SCT could serve
as an attractive alternative cell therapy1,2.
Human NK cells are lymphocytes of the innate
immune system involved in the early defense
against infectious pathogens and against
MHC class-Inegative or low-expressing
malignant targets without the requirement
for prior immune sensitization of the host3,4.
They reside mainly in the marrow, spleen,
and peripheral blood, where they account for
approximately 218% of the peripheral blood
lymphocytes. NK cells are usually the first
lymphoid subpopulation to recover after SCT5.
Phenotypically they can be defined by the
expression of CD56, an isoform of the neural
cell adhesion molecule and the lack of the CD3

antigen on the surface. Further characterization
allows the major CD56dim CD16+ (around
90%) to be distinguished from the minor
CD56 brightCD16 NK subpopulation. The
immunoregulatory CD56bright NK cell subsets
express the high-affinity interleukin 2 (IL-2)
receptor, which enables them to proliferate in
response to IL-2 and to produce high amounts
of cytokines, such as IFN-, TNF-, TNF-,
GM-CSF, IL-10, and IL-13. The CD56 dim NK
cells are essentially cytotoxic cells that express
low levels of the IL-2 receptor. NK cells are able
to lyse targets by releasing cytotoxic granules
containing perforin and granzymes and using
antibody-dependent cellular cytotoxicity
pathways via membrane receptor binding to
the Fc portion of IgG antibody, and by the
induction of apoptosis through molecules of
the TNF superfamily (Fas/CD95, TRAIL).
Killing activity of NK cells is regulated by a
set of surface receptors that either induce or
inhibit the cytotoxic response3,6,7. Activation
of NK cells is facilitated by the engagement
of activating surface receptors through
interaction with stimulatory ligands expressed
by malignant cells. These immune recognition
receptors include NKG2D, the natural
cytotoxicity receptors (NCR) NKp30, NKp44,

and NKp46, CD16, NKp80, DNAM-1, and
2B4 (CD244)6,8. Activation with cytokines,
such as IL-2, leads to a strong up-regulation
of the NCRs and NKG2D, and this correlates
with increased NK cell cytotoxicity against
malignant cells9. Inhibitory receptors comprise
both killer cell immunoglobulin-like receptors
(KIRs) and the heterodimeric C-type lectine
receptor CD94-NKG2A/B
10. In addition,
several activating KIR and the C-type lectin
receptor CD94-NKG2C/E/F are known3. A
number of studies have demonstrated NK
cellbased killing of many different mouse and
human tumors and leukemias and have led
to the initiation of the first clinical phase I/II
trials using allogeneic NK cells for treatment
of cancer.

Clinical-scale NK cell enrichment
Protocols for the enrichment of NK cells
from non-stimulated leukapheresis products
using good manufacturing practice (GMP)
procedures have already been established (table
1; fig. 1). The aim of these procedures is to
obtain a highly purified NK cell product with
minimal T cell contamination and conserved
Vol 15 2/2013
MACS&more 25
Report
NK cell cytotoxicity. NK cell enrichment

usually consists of one or two rounds of CD3+
cell depletion with subsequent CD56+ cell
enrichment11-.
In our procedure, after steady-state
leukapheresis of unstimulated donors, the
cells were washed twice for platelet reduction
with CliniMACS PBS/EDTA Buffer (Miltenyi
Biotec, Bergisch Gladbach, Germany)
supplemented with 0.4
% human serum
albumin (Red Cross Blood Donor Service,
Baden-Wrttemberg-Hessen, Germany).
Thereafter, 5 mL of Intraglobin (Biotest,
Dreieich, Germany) were added and incubated
for five minutes to reduce non-specific antibody
binding. Cells were labeled for 30 minutes
with CliniMACS CD3 Reagent (Miltenyi
Biotec), using one vial of reagent in case of total
nucleated cell (TNC) numbers up to 40109 or
CD3+ cell numbers up to 15109 and two vials in
case of TNC numbers up to 80109 or CD3+ cell
numbers up to 30109. After washing twice, CD3+
cells were depleted with the CliniMACS Plus
Instru
ment using the separation program
DEPLETION 2.1. If necessary, the T cell
depletion step was repeated to further remove
residual T cells. Thereafter, the Tcelldepleted
harvests were concentrated and labeled with
CliniMACS CD56 Reagent (Miltenyi Biotec)

for 30 minutes (one vial CD56 Reagent for TNC
numbers up to 40109 and CD56+ cell numbers
up to 10109). After washing, CD56+CD3
NK cells were enriched using the separation
program ENRICHMENT 1.1. All steps were
performed in a closed system observing GMP.
The study protocol was approved by the local
ethics committee in Frankfurt and Basel and
informed consent of the donors has been
obtained.
Expansion and activation of NK cells
The purified CD56 + CD3 NK cells were
suspended and seeded at a concentration
of 12106 cells/mL in X-vivo 10
media (BioWhittaker, Verviers, Belgium)
supplemented with 5
% heat-inactivated
human fresh frozen plasma and 1,000 U/
mL rhIL-2 (Proleukin, Novartis, Germany)
under GMP-compliant conditions (fig. 1).
In an early phase of the study, cells had been
expanded and activated using both 175 cm2
culture flasks (Nunc, Wiesbaden, Germany)
and VueLife cell culture bags (CellGenix,
Freiburg, Germany); for the ongoing phase I/II
trial only VueLife cell culture bags were used11.
Fresh medium was added every three days,
and samples for monitoring cell content and

viability were taken directly after leukapheresis,
after each depletion/enrichment step, and every
second day during stimulation. Phenotyping
and evaluation for cytotoxicity was performed
by flow cytometry. After 10 days, stimulated
NKcells were administered to the patients or
cryopreserved in X-vivo10 medium supple
mented with 10% DMSO.
Phenotyping and functional
characterization of the product
for quality control
The absolute number of CD56+CD3 NK
cells and the number of residual T cells were
determined by flow cytometry performed on
a four- or a five-color flow cytometer (Epics
XL or FC 500, Beckman Coulter, Krefeld,
Germany) in a single-platform technique. The
gating strategy was based on the ISHAGE
single-platform stem cell enumeration method
using low scatter, high expression of CD3 and
CD45 antigens, CD16 and CD56 expression
and 7-AAD staining, in a no-wash preparation
with counting beads. Our previously described
four-color panels1 were ex
tended to the
following five-color panels: CD45-FITC/
CD56-PE/CD3-ECD/7-AAD/CD16-PC7 and
Clinical phase I/II study with allogeneic NK cells post haploidentical SCT:
Patients with high-risk leukemia and malignant tumors
Leukapheresis
NK cell purification
NK cell expansion
CD3+ depletion followed

by CD56+cell enrichment,
according to GMP
IL-2dependent,
according to GMP
NK cell infusion
Donor/Parents
Recipient
haploidentical
SCT
NK cell
infusion
NK cell
infusion
+3
+40
NK cell
infusion
+100
(days post SCT)
Figure 1 Purification, ex vivo expansion, and infusion of donor NKcells. In an ongoing clinical phase I/II trial patients receive freshly isolated NKcells on day (+3),
+40, +100 or IL-2activated NKcells on day +40 and +100 post haploidentical SCT.
26 MACS&more
Vol 15 2/2013
Report
CD45-FITC/CD3-PE/CD14-ECD/7AAD/ Results and discussion

CD56-PC7. Samples were prepared in Technical aspects clinical-scale NK cell
triplicate and CD45-FITC/IgG1-PE/ product manufacture and NK cell activation
CD14-ECD/7AAD/CD56-PC7 served as A number of studies have shown that clinicala control. In addition, cells were labeled with scale NK cell product manufacture from nonappropriate combinations of fluorochrome- stimulated leukapheresis products, using a
conjugated antibodies (MAb) to monitor NK CD3+ cell depletion step followed by CD56+ cell
cell subsets, activating and inhibitory NK enrichment, leads to highly purified CD56+CD3
cell receptors, and activation status. MAbs NK cell products with a purity ranging from
used were CD16 (clone 3G8), CD45 (clone 89.7 to 98.6% (table1)11-,9-2. Purity of NK cell
J.33), CD56 (clone N901), HLA-DR (clone products was lower if the starting product was
Immu-357), CD69 (clone TP1.55.3), CD158a,h the negative fraction after CD34+ cell selection.
(KIR2DL1/S1, clone EB6B), CD158b1/b2,j The high NK cell purity and extensive T cell
(KIR2DL2/3/S2, clone GL183), CD158e1/e2 depletion is at the expense of a considerable loss
(KIRp70, KIR3DL1/S1, clone Z27.3.7), CD158i of NK cells during isolation. The final recovery
(KARp50.3, KIR2DS4, clone FES172), CD337 of CD3CD56+ NK cells ranged between 19.4%
(NKp30, clone Z25), CD336 (NKp44, clone and 58%. Overnight storage of the leukapheresis
Z231), CD335 (NKp46, clone BAB281), and product led to a greater loss of NK cells during
CD314 (NKG2D, clone ON72), all supplied by the NK cell enrichment process compared to
Beckman Coulter (Marseille, France), and CD3 processing of fresh harvests. A much higher
NK cell recovery was obtained by using only a
(clone SK7) supplied by BD Biosciences.
The cytotoxicity of the highly enriched NK CD3+ cell depletion step, without further CD56+
cells before and after IL-2 stimulation was cell enrichment. However, such a product
tested against the MHC class Inegative cell was associated with low purity and less T cell
line K562 or against the patients individual depletion. Similarly, the final T cell number
leukemic cells using an antibody-based flow was much higher if a CD56+ cell selection was
cytometric assay as described previously15,. used alone, but this kind of purification was
NK cells and leukemic cells were cocultured associated with an increased NK cell recovery
for four hours at effector-to-target ratios and purity. In contrast, the two-step NK cell
between 0.5:1 and 10:1. Absolute cell counts product manufacture described here led to
were determined using Flow-Count beads. efficient T cell depletion of 3.6 to 5.3 orders of
Cytotoxicity was defined as the loss of viable magnitude (table 1), and this could be further
target cells relative to the control. In other increased by performing the CD3+ cell depletion
studies NK cell functionality was measured by step twice11. A residual Tcell contamination
between 0.01 and 0.09% in the final product
a CD107a degranulation assay17,.
allows the infusion of NK cell products of more

than 1.0107 CD56+CD3 NK cells/kg BW with
less than 5.0104 CD3+ cells/kg BW and often
less than 2.5104 CD3+ Tcells/kg BW1.
The objective of NK cell purification is not
only to remove potentially unwanted T cells
but also to enable activation and expansion of
the NK cells. Indeed, enriched NK cells can be
infused without any additional manipulation,
or after overnight culture in high-dose
IL2. They can also be expanded in IL-2 or
other cytokines, such as IL-15, alone or in
combination, for two to several weeks in cell
culture bags or in a bioreactor11,,2. Similarly,
it is possible to expand single KIR+NKcells2.
Invitro expansion has two aims, to activate the
selected CD56+CD3 cells, and to increase the
total number of NK cells. Using CD69 as an
activation marker, activation of NK cells was
found to occur within 13days of incubation
with IL-29,2. When enriched CD56+CD3
NKcells were cultured with IL-2, a significant
expansion was observed although there was a
lag of 35 days before the NK cells started to
proliferate11. On day 5, expansion occurred and
led to a two- to tenfold increase of CD56+CD3
NK cells after 1014 days. Although NK cells
were viable immediately after purification
(>90%), the vital NK cell count decreased
by 3050% during the first three to five days
following IL-2 stimulation. Afterwards, cell
viability recovered to >98%, and by day 1014,
a maximal NK cell expansion was obtained.
No overgrowth of the remaining T cells was
observed during expansion and activation
Author
Method
Donors
(n)
NK cell purity
(%)
Recovery
(%)
Log T cell
depletion
Lang et al. (2002)
CD56 enrichment, followed by CD3 depletion
98.6 (m)
42.0 (m)
3.6104-fold
McKenna et al. (2007)
CD3 depletion
36
37.7 (x)
78.8
2.7
McKenna et al. (2007)
CD3 depletion, followed by CD56 enrichment
13
89.7 (x)
19.4
4.3
Koehl et al. (2004)
95.0 (m)
37.0 (m)
4.5 (m)
Koehl et al. (2005)
CD3 depletion twice, followed by CD56 enrichment
15
94.9 (m)
33.0 (m)
5.0 (m)
Iyengar et al. (2003)
12
91.0 (m)
48.7 (m)
5.3 (m)
Uharek et al. (2003)
CD34 neg fraction: CD3 depletion, CD56 enrichment
75.0 (m)
42.0 (m)
4.0
Passweg et al. (2004)
97.3 (m)
35.5 (m)
3.6
4.2 (m)
Meyer-Monard et al. (2009)
24
94.5 (m)
58.0 (m)
Rizzieri et al. (2010)
CD56 enrichment
51
96.5
80
m: median; x: mean
Table1 Clinical-grade NK cell enrichment using the CliniMACSSystem.
Vol 15 2/2013
MACS&more 27
Report
with IL-2. With a protocol that enables the be determined on the basis of this clinical
generation of NK cells on a clinical scale, data. The fact that (at least in some cases of
using a closed system that conforms to GMP GvHD) the T cell content was higher than
guidelines, the expanded NK cells were in cases without GvHD, seems to favor a T
highly cytotoxic against different leukemic cell effect. With regard to NK cell efficiency,
and tumor target cells9,2. Importantly, no non- Rubnitz et al. recently reported that NK
specific activation against normal allogeneic cell administration to ten pediatric patients
lymphocytes occurred1. In addition we were with AML in first complete remission led to
able to demonstrate that IL-2 stimulation led a two-year event-free survival of 100%, with
to up-regulation of all natural cytotoxicity all patients still in complete remission. An
receptors (NCRs) and the activating receptor earlier study demonstrated that patients with
NKG2D, which might explain the observed AML had a lower rate of leukemia relapse
increased cytotoxicity against MHC-I-negative compared to the expected rate, a lower rate of
targets9. There is evidence that a combination graft rejection, and a paradoxical reduction in
of cytokines, such as IL-2, IL-12, IL-15, and GvHD post-haploidentical SCT, when the NK
IL-21, may further increase cytotoxic activity cells possessed inhibitory KIRs for which the
of NKcells. In addition to NKcell enrichment recipient had no ligand3. We could show an
from leukapheresis products as summarized increased cytotoxic activity of stimulated NK
in table 1, NK cells can also be generated from cells against high-risk neuroblastoma (NB)
cord blood2.
due to IL-2mediated up-regulation of the
Clinical-scale collection, enrichment, activating receptors NKp30, NKp44, NKp46,
activation, and expansion of purified NK cells and NKG2D9. However, we have also been able
are feasible. Most of the technical aspects for to demonstrate tumor escape from immune
adoptive NKcell therapy have been developed surveillance by release of soluble MICA
for clinical applications. However, these (ligand MHC class Ichain-related gene A)
laboratory procedures are time consuming compromising NKG2D-dependent NK cell
and expensive, need particular skills, and must cytotoxicity in patients with NB. Elevated
be performed according to a GMP-compliant sMICA levels in patients plasma correlated
protocol.
significantly with impaired NK cellmediated
cytotoxicity of the infused donor NK cells3.
Clinical studies using freshly purified
Future perspectives
or IL-2activated NK cells
Previous trials and ongoing clinical phase I/II Future studies should improve NK cell
t herapy by increasing the
studies have shown the feasibility of using freshly immuno
purified or IL-2activated donor NK cells for understanding of the conditions leading to
the treatment of high-risk patients suffering tumor cell kill by NK cells, by increasing
from leukemia or tumors in both non-transplant the cytotoxicity of NK cells against various
settings and after haploidentical SCT as an malignancies, and by optimizing the schedule
additional immunotherapy2,2,2,2-. NK cell of the NK administration based on results
products were infused as a single dose rate or of ongoing phase I/II studies. Given the
as multiple applications with doses between plausible benefit of IL-2stimulated NKcells
0.2107 and 8.1107 CD56+ CD3 NK cells/ compared to freshly isolated, resting NKcells
kg BW, mostly with less than 2.5104 CD3+ with regard to cytotoxicity, it may be possible
T cells/kg BW
1,9,2,. These first to increase cytotoxicity by activation with
immunotherapy trials show that NK cells can cytokine combinations like IL-2/IL-15 or
be administered without immediate adverse by cross-talk with dendritic cells (DCs).
events, that they are well tolerated by the Additional investigation is necessary to
patients and do not induce GvHD>gradeII. develop strategies to overcome tumor immune
However, some cases of GvHD have been escape mechanisms. Options may encompass
observed after NK cell infusion. In some development of MAb against sMICA, genetic
instances this has been associated with a less engineering of NK cells by introduction of
efficient T cell depletion. Whether GvHD chimeric receptors for tumor retargeting, or
is attributable to contamination by T cells enhancing tumor cell recognition by using
or is due to the effects of NK cells cannot small interfering RNA to silence inhibitory
28 MACS&more
Vol 15 2/2013
receptors. Open issues in clinical studies

also include NK cell dose rate, time schedule,
appropriate selection of donor/recipients, and
also the types of tumors to be considered for
treatment, because it is already known that
certain types of malignant cells may be more
responsive to NK cell therapy than others.
Ultimately, expansion of tumor-reactive NK
cells within the patient might prove to be
feasible. It is possible to transfuse NK cells
simultaneously with the transplants, and the
first clinical trials indicate that an early NK
application post SCT may be most effective in
attacking minimal residual disease.
References
1. Passweg, J. R. et al. (2006) Best Pract. Res. Clin.
Haematol. 19: 811824.
2. Passweg, J. R. et al. (2005) Bone Marrow
Transplant. 35: 637643.
3. Farag, S. S. and Caligiuri, M. A. (2006) Blood Rev.
20: 123137.
4. Ljunggren, H. G. and Malmberg, K. J. (2007) Nat.
Rev. Immunol. 7: 329339.
5. Koehl, U. et al. (2007) Bone Marrow Transplant.
39: 269278.
6. Moretta, A. et al. (2001) Annu. Rev. Immunol. 19:
197223.
7. Passweg, J. R. et al. (2007) Trends Immunol. 28:
437441.
8. Lanier, L. L. (2005) Annu. Rev. Immunol. 23:
225274.
9. Huenecke, S. et al. (2010) J. Immunother. 33:
200210.
10. Karre, K. (2008) Nat. Immunol. 9: 477480.
11. Koehl, U. et al. (2005) Klin. Padiatr. 217: 345350.
12. Koehl, U. et al. (2013) Front. Oncol. 3: 118.
13. Meyer-Monard, S. et al. (2009) Transfusion 49:
362371.
14. Koehl, U. et al. (2008) Int. J. Hematol. 87: 98105.
15. Zimmermann, S. Y. et al. (2005 J. Immunol.
Methods 296: 6376.
16. Kloess, S. et al. (2007) J. Immunol. Methods, 325:
140147.
17. Penack, O. et al. (2005) Leukemia 19: 835840.
18. Fischer, L. et al. (2006) Exp. Hematol. 34: 753759.
19. Lang, P. et al. (2002) Bone Marrow Transplant.
29: 497502.
20. McKenna, D. H. et al. (2007) Transfusion 47:
520528.
21. Koehl, U. et al. (2004) Blood Cells Mol. Dis. 33:
261266.
22. Iyengar, R. et al. (2003) Cytotherapy 5: 479484.
23. Uharek, L. et al. (2003) Onkologie 26 (suppl. 5):
38.
24. Passweg, J. R. et al. (2004) Leukemia 18: 1835
1838.
25. Rizzieri, D. A. et al. (2010) Biol. Blood Marrow
Transplant. 16: 11071114.
26. Sutlu, T. et al. (2010) Cytotherapy 12: 10441055.
27. Siegler, U. et al. (2010) Cytotherapy 12: 750763.
28. Spanholtz, J. et al. (2010) PLoS One 5: e9221.
Report
29. Miller, J. S. et al. (2005) Blood 105: 30513057.

30. Stern, M. et al. (2013) Bone Marrow Transplant.
48: 433438.
31. Brehm, C. et al. (2011) PLoS One 6: e27351.
32. Rubnitz, J. E., et al. (2010) J. Clin. Oncol. 28:
955959.
33. Ruggeri, L. et al. (2002) Science 295: 20972100.
34. Kloess, S. et al. (2010) Eur. J. Immunol. 40: 3255
3267.
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miltenyibiotec.com/wholeblood
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MACS&more 29
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Clinical-grade separation and expansion of

regulatory T cells for clinical studies on cell
therapy in solid organ transplantation
Henrieta Fazekasova, Sarah Thirkell, Katie Lowe, Andrew Bushell, and Giovanna Lombardi
Immunoregulation laboratory, MRC Centre for Transplantation, Kings College London, Guys Hospital, London, UK
Introduction
Organ transplantation is the treatment of
choice for patients with end-stage organ
failure (e.g. kidney). The use of broadspectrum immunosuppressive drugs results
in accelerated mortality, is toxic to transplants,
and does not prevent chronic rejection. Thus,
the establishment of clinical tolerance to
engrafted tissues to minimize or eliminate
immunosuppression is a key research goal.
Naturally occurring, thymus-derived,
CD4+CD25+ FoxP3+ regulatory T (Treg) cells
play a critical role in shaping many types of
immune responses. They are important not
only in maintaining peripheral tolerance to
self-antigens and controlling autoimmune
diseases, they can also limit immune responses
to foreign antigens such as alloantigens. A great
deal of experimental data suggests that adoptive
therapy of ex vivo freshly isolated or in vitro
expanded Treg cells may successfully promote
tolerance. Although numerous preclinical
animal models have shown that Treg cells
can successfully prevent graft rejection, it still
remains to be proved that this approach can be
successfully translated into the clinical setting.
A number of clinical trials using Treg cells are
underway to control GvHD following bone
marrow transplantation and as a potential
treatment for type 1 diabetes. The application
30 MACS&more
Vol 15 2/2013
by centrifugation. Cells were resuspended

in 100 mL of buffer in a bag, which was then
attached to a CliniMACS Tubing Set LS. CD8+
cells were removed using the depletion 2.1
program of the CliniMACS Plus Instrument.
The cell fraction depleted of CD8+ cells was
then diluted with cold CliniMACS Buffer to
380mL and incubated with CliniMACS CD25
Reagent (Miltenyi Biotec) for 15 min at 4 C
with constant agitation. Excess beads were
again removed by centrifugation, and the cells
were resuspended in 100 mL of buffer. The
cell suspension was enriched for CD25+ cells
using the CliniMACS Tubing Set and the
enrichment 3.2 program. All reagents and
consumables used were of clinical grade and
Separation of Treg cells
processing steps were performed in a closed
200 mL of whole blood from a healthy system using bags, which were attached and
donor and a renal failure patient was volume detached by means of a sterile welder.
reduced using the Sepax 2 device (Biosafe).
CD4+CD25+ T cells were separated on a Purity and identity of separated Treg cells
CliniMACS Plus Instrument (Miltenyi Biotec) Analysis of cell surface and intracellular
in two steps: depletion of CD8+ cells followed molecules to confirm the identity and purity
by enrichment of CD25+ cells.
of the separated cell population was performed
Buffy coat was diluted to 100 mL with by multicolor staining using the following
CliniMACS PBS/EDTA buffer supplemented monoclonal anti-human antibodies: CD4with 0.5% human serum albumin and incubated PerCP/Cy5.5, CD25-PE, CD8-APC, and
with CliniMACS CD8 Reagent (Miltenyi FOXP3-FITC. Cells were incubated 30 min
Biotec) for 30 min at room temperature with with antibodies against the cell surface
constant agitation. Excess beads were removed markers (CD4, CD8, and CD25), washed,
of Treg cells is also a promising strategy for the

treatment of other autoimmune diseases and in
the prevention of transplant rejections in solid
organ transplantation. Development of cellular
therapies exploiting the immunoregulatory
attributes of Treg cells and their introduction
to clinical practice would significantly increase
the chance for long-term graft survival without
need of life-long immunosuppression.
The aim of this study was to develop an optimal
GMP-compatible protocol for the generation
and expansion of Treg cells that will be used
in clinical studies to prevent graft rejection in
solid organ transplantation.
Report
to generate doses for patient treatment plus

reference samples for quality control purposes.
Data shown here are generated using samples
from one healthy donor and one patient
suffering from kidney failure.
2, 3
Stimulation 1
0.5 106
Expansion
Purity
Beads (4:1) or (1:1)
Suppression
IL-2 (500 U)
Rapamycin (100 nM)
Feed 1
Figure 1 Stimulation and feeding regime for expansion of Treg cell lines. For details see the text.
Expansion of Treg lines

Human CD4+CD25+ T cells were seeded in
MACS GMP Cell Expansion Bags at 0.5106
cells/mL in TexMACS GMP Medium (both
Miltenyi Biotec) supplemented with 5%
human serum containing 100 nM rapamycin
(Rapamune). Cells were activated with antiCD3- and anti-CD28-coated beads (MACS
GMP ExpAct Treg Kit, Miltenyi Biotec) at
a bead-to-cell ratio of 4:1. IL-2 (500 IU/mL,
Proleukin) was added at feed 3 after activation
and replenished at feed 4 for cycle 1 and feeds
14 for cycles 2 and 3. Cells were re-stimulated
23 days after the fifth feed. The incubation
time between each feed was 23 days. At the day
of restimulation, cells were pooled, fresh beads
(1:1), rapamycin, and IL-2 were added, and
the suspension seeded into bigger expansion
bags (250, 500, or 1000 mL). Expanded cells
were harvested at the end of cycle 3 and used
for further analysis. The expansion process is
depicted in figure 1.
Suppression assay
The functionality of the expanded Treg lines
was assessed by an in vitro suppression assay.
CD4+CD25 T cells (Teff) were labeled with
2.5 nM CFSE and cocultured at different ratios
with Treg cell lines. For cell activation antiCD3/CD28-coated beads were used. Cells
were harvested after 5 days. Proliferation of
CFSE-labeled Teff cells was analyzed by flow
cytometry. The suppressive ability of Treg cell
lines was assessed as the percentage of decrease
of Teff cell proliferation in the presence of Treg
cells.
Results and discussion

The results shown in this report are a part of
the scale-up process in the context of a clinical
study. The goal was to obtain GMP-compatible
Treg cell populations that are highly pure,
functional, and available in a number sufficient
1013
1012
Number of cells
and then incubated 30 min with fixation/

permeabilization buffer. Subsequently, cells
were washed with permeabilization buffer,
incubated with an antibody against the
intracellular marker FOXP3 for another
30min, and analyzed by flow cytometry using
the FlowJo software.
Recovery and expansion of Treg cells

isolated from blood of a healthy donor and
a patient
The final goal of this study is the use of a single
dose of Treg cells (derived from the patient) to
be injected in vivo to induce prolongation of
organ allograft survival. To ensure a successful
preparation of a Treg cell dose a sufficient
starting number of Treg cells needed to be
purified and expanded. In this study Treg cells
were separated from 200 mL of blood from
a healthy donor and a patient in a waiting
list for a kidney transplant (ESKF). The two
preparations were compared with regard to cell
recovery and the cells' ability to expand. While
a total of 29106 Treg cells were obtained from
the blood of the healthy donor a considerably
lower number of Treg cells was recovered
from the patient blood (1106). We think that
this may be an isolated case and is probably
patient dependent. However, we were able to
demonstrate that the expansion ability of Treg
cells obtained was comparable between the
patient (1109) and the healthy donor (11011).
For both preparations a sufficient number of
Treg cells was obtained to create a cell dose of
3106/kg of patients body weight and quality
1011
1010
109
108
107
106
Freshly
isolated
Expansion
cycle 1
Healthy donor
Expansion
cycle 2
Expansion
cycle 3
Patient
Figure 2 Expansion of Treg cell lines generated from blood of a healthy donor and a patient. Cells were
cultured as described in materials and methods. Cell numbers were determined prior to cell culture (freshly
isolated) and after one, two, and three expansion cycles.
Vol 15 2/2013
MACS&more 31
Report
control samples. The expansion profile is

shown in figure 2.
Healthy donor
Freshly isolated Treg cells
Q1
2.46%
Q2
88.9%
Patient
Expanded Treg cells

Q1
2.72%
Q2
96.3%
Expanded Treg cells

Q1
9.43%
Q2
88.5%
CD25
FoxP3
88.5%
FoxP3
95.9%
Q4
1.21%
FoxP3
88.5%
Q3
7.45%
Q4
0.234%
Q3
0.796%
Q4
0.769%
Q3
1.28%
Q9
17.5%
Q10
68.2%
Q9
0.830%
Q10
99.2%
Q5
0.372%
Q6
99.6%
Q12
3.23%
Q11
11.0%
Q12
0.00%
Q11
0.00%
Q8
0.00%
Q7
0.00%
Q5
85.7%
Q6
0.036%
Q5
98.1%
Q5
0.831%
Q9
97.1%
Q10
1.44%
Q8
14.2%
Q7
0.036%
Q8
1.02%
Q7
0.009%
Q12
1.48%
Q11
0.00%
CD25
FoxP3
CD25
CD4
CD8
Figure 3 Identity and purity of Treg cells generated from blood of a healthy donor and a patient. Freshly
isolated Treg cells from a healthy donor and expanded Treg cell lines from a healthy donor and a patient were
labeled with fluorochrome-conjugated antibodies and analyzed by flow cytometry.
100
% of suppression
80
60
40
20
0
1:1
1:5
Ratio Treg:Teff cells
Healthy donor
Freshly isolated Treg cells
Healthy donor
Expanded Treg cells
1:10
Patient
Expanded Treg cells
Figure 4 Suppressive ability of freshly isolated and expanded Treg cells from healthy donor and patient.
The assay was performed as described in the materials and methods section. Three different ratios of Treg:Teff
were used. Data indicate the percentage of suppression, i.e., the decrease of Teff cell proliferation in the
presence of Treg cells.
32 MACS&more
Vol 15 2/2013
Identity and purity of freshly isolated and

expanded Treg cells
The identity and purity of freshly isolated
and expanded Treg cells was confirmed by
flow cytometry. Cells were stained for the cell
surface markers CD4, CD25, and CD8, and
intracellular staining was performed to show
expression of FOXP3.
The quality of the cell separation process
was very good, and the percentages of
contaminating non-Treg cells were low in both
preparations. Flow cytometric analysis of the
Treg cells obtained from the healthy donor
demonstrated that the percentage of cells
expressing CD25 and FOXP3 cells was 89%;
68% of the cell population were CD4+CD25+
(fig. 3, left). However, during the course
of expansion the proportion of CD4 cells
decreased and by the time of final harvest
they were all eliminated (fig. 3, middle). The
contamination with CD8+ cells in freshly
isolated Treg cells was minimal at 0.04% (fig. 3
left). We did not perform staining of Treg cells
from the patient sample prior to cell culture as
the number of freshly isolated cells was too low.
The gating strategy for the analysis of cells after
the final harvest was slightly different (fig. 3).
The identity of the cells and the purity of the
final formulation product complied with set
release criteria for a Treg cell therapy product,
which are 60% CD4+CD25+FoxP3+cells and
10% CD8+ cells: The results demonstrated
that at the end of the culture 96% of the total
live cells were CD25+FoxP3+ in the cell line
from the healthy donor and 89% from the
patient, and of these cells 99% were also CD4+.
The percentage of contaminating CD8+ cells in
the sample from the healthy donor was 0.8%
and from the patient 1.4% (fig. 3, middle and
right). Purities were well within the range of
the release criteria.
Functionality of freshly isolated and
expanded Treg cell lines
We have tested the suppression effect of
freshly isolated and expanded Treg cells from
a healthy donor and a patient sample. Our data
show that the percentage of suppression in the
sample from the healthy donor considerably
increased from 66% (before expansion) to
94% (after expansion) at a 1:1 ratio of Treg:Teff
Report
cells. The same results were seen also at other

Treg:Teff cell ratios. This increased suppressive
ability directly correlated with increased cell
purity at the end of expansion. Due to the cell
number restriction at the start of cell culture,
we were unable to test the suppressive function
of freshly isolated Treg cells from the patient
sample. However, expanded Treg cell lines
from the patient showed the same suppressive
ability as Treg cell lines from the healthy donor
with percentages of suppression between 85%
and 96% at all Treg:Teff cell ratios (fig. 4).
Conclusion
Using the GMP-compatible protocol for
the separation and expansion of regulatory
Tcells we were able to isolate Treg cells of
high purity.
Treg cells were expanded to a clinically
relevant number while maintaining their
functional ability.
The expanded Treg cells fulfill all the
requirements of set release criteria and are
therefore suitable for use as cell therapy
product in a phase I clinical trial.
MACS Product
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CliniMACS PBS/EDTA
Buffer
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162-01
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170-076-306
MACS GMP ExpAct Treg Kit
170-076-119
MACS GMP Expansion or

Cell Differentiation Bags
Visit miltenyibiotec.com/gmp
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MACS&more 33
Report
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MACS&more 35
Report
Translating discoveries
into therapies is never easy.
Translational research is a complex endeavor.

The steps are many, the target can be distant. Miltenyi Biotec provides the instruments,
tools, and reagents that best prepare scientists for successful translational research. From
research- to GMP-grade reagents and cell separation instruments for both basic and clinical
research, our integrated portfolio supports you at every stage.
Learn more at www.miltenyibiotec.com
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NK cells for cancer

Treg cells in solid

Supporting the quest

MACS&more (ISSN 1610-4994) is published by

Seamless translation with MACS GMP Products 

An optimized method for the manufacture of a dendritic cellbased 

Enrichment, stimulation, and viral transduction of naive and 

Clinical-grade purification and expansion of CD56+CD3 NK cells 

Clinical-grade separation and expansion of regulatory T cells 

Editorial board: Anthony Allen, Adrian Arechiga, Volker Arps,

The CliniMACS System components: Reagents, Tubing Sets,

Cover image: The picture shows mature plasmacytoid dendritic cells

naive and central memory CD8+ T cells

Jurjen Tel et al. report on the first clinical

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Figure 1 The Miltenyi Biotec GMP facility in Teterow, Germany.

MACS GMP Products are manufactured

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GMP guidelines and designed following

Figure 2 The CliniMACS Prodigy.

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MACS GMP Cytokines

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Figure 1 pDC isolation based on the CliniMACS

(Lonza) containing 2% pooled human serum,

In-oxinate labeling and

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Results and discussion

Clinical study with natural pDCs

Although the initial endpoint of this study

control patients: 22.0 months versus 7.6 months.

we observed increases in the expression of

7. de Vries, I.J. et al. (2003) Clin. Cancer Res. 9:

CliniMACS Plus Instrument

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Seamless translation with MACS GMP Products

An optimized method for the manufacture of a dendritic cellbased

Enrichment, stimulation, and viral transduction of naive and

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