From bloodjournal.hematologylibrary.org at Newcastle University on July 4, 2013. For personal use only.

2002 100: 4067-4073

Prepublished online August 1, 2002;
doi:10.1182/blood-2002-03-0731

Human T cells resistant to complement lysis by bivalent antibody can be

efficiently lysed by dimers of monovalent antibody
Soren U. Nielsen and Edward G. Routledge

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From bloodjournal.hematologylibrary.org at Newcastle University on July 4, 2013. For personal use only.
IMMUNOBIOLOGY

Human T cells resistant to complement lysis by bivalent antibody

can be efficiently lysed by dimers of monovalent antibody
Soren U. Nielsen and Edward G. Routledge

We have previously shown that bivalent

human 1 CD3 monoclonal antibody
(mAb) is ineffective at mediating lysis of
human T cells with human complement.
In this paper we have used genetic engineering and sulfur chemistry to prepare 2
types of human 1 CD3 mAb dimer, with
the aim of improving complement lysis
activity. The IgG molecules forming the
dimers were linked together at their Ctermini by stable bismaleimide thioether
bridges. The first dimer was composed of
2 bivalent mAb molecules. This dimer
proved incapable of lysing human T-cell

blasts with human, rabbit, or guinea pig

complement. The second dimer consisted of 2 molecules of a monovalent
derivative (possessing a single Fab domain) of the bivalent mAb. This dimer was
highly lytic with human complement, with
a lytic titer 64-fold greater than that of the
nondimerized monovalent mAb. The maximum level of lysis of human T-cell blasts
achieved with this monovalent mAb dimer
was equal to that obtained with the therapeutic antilymphocyte mAb alemtuzumab,
but its lytic titer was 4-fold greater. The
monovalent mAb dimer was also found to

be lytic in the presence of rabbit and

guinea pig complement. Dimerization of
monovalent antibodies may provide a general strategy for improving the cytolytic
activity of other mAbs that are normally
unable to induce lysis with complement.
The monovalent CD3 mAb dimer may
have potential for development as an
agent for immunotherapy of T-cell leukemia. (Blood. 2002;100:4067-4073)

2002 by The American Society of Hematology

Introduction
Monoclonal antibodies (mAbs) specific for cell surface antigens
can be used to target unwanted cell types in vivo for destruction by
immune effector mechanisms.1,2 Although the most important
factor determining an antibodys in vivo cytolytic potency is its
interaction with Fc receptorbearing cells, complement lysis can
also play a significant role.3 Any modification that can be made to a
mAb to improve complement lysis activity is likely to have a
beneficial effect on its overall cytolytic activity, as long as its
interaction with Fc receptors is not compromised.
Human or humanized mAbs rather than rodent mAbs are the
preferred choice for human mAb therapy.4,5 They minimize the
likelihood of eliciting an antiglobulin response in the patient, which
may block the effect of the therapeutic antibody.5 Consequently,
when aiming to maximize cytolytic activity, attention has focused
on mAbs of the human IgG1 and 3 subclasses, because these
subclasses are able to recruit both complement and cytotoxic
FcR-bearing cells.6 However, in the case of complement, the
efficiency of cytolysis is not solely governed by mAb heavy-chain
isotype. The properties of the target cell surface antigen also play a
role.7-9 Some antigens, such as surface immunoglobulin on B
lymphocytes,10 CD33 on myeloid cells,11 and CD3 on T lymphocytes,12 redistribute and modulate when cross-linked by normal
bivalent mAbs. Antibodies to such antigens are generally poor
inducers of complement lysis, but the problem can be overcome to
some extent using enzymatic or genetic engineering techniques to
remove or inactivate one of the Fab domains of the mAb.1,13-15 This
renders the mAb monovalent, preventing it from cross-linking the

target antigen. This strategy has been shown to improve complement lysis activity, but the drawback is that it also reduces
mAb-binding avidity by a factor of 6,15 significantly increasing the
concentration of mAb that must be used (or dose that must be given
therapeutically) to achieve effective coating of the target cell.
Several groups of researchers have shown that tail-to-tail
dimerization of bivalent humanized IgG1 mAbs (mediated by
intermolecular disulfide bonds formed between cysteine residues,
introduced into position 444 of the heavy chain by genetic
engineering) can give dramatic (100- to 200-fold) improvements in
complement lysis activity.16-18 Although encouraging, some caution is needed in judging the implications of these studies for
human antibody therapy. In 2 of the studies,16,17 using mAbs
specific for a hapten and CD33, respectively, nonhuman sources of
complement were used in the cell lysis assays. The importance of
this is underlined by the observation that no lysis was observed
with the CD33 mAb dimer when tested with human complement in
the same assay. In a third study,18 dimers of the highly lytic CD52
mAb alemtuzumab (Campath-1H) only showed enhanced complement lysis activity when tested against a cell line expressing
abnormally low levels of the target antigen.
In this report, we describe the production and characterization
of dimers of bivalent and monovalent humanized IgG1 mAbs
specific for the human CD3 antigen. The dimers were created using
a bismaleimide cross-linker to form a nonreducible intermolecular
bridge between cysteine residues introduced at position 444 of the
heavy chains of the monomers. When tested for their lytic activity

From the Department of Microbiology and Immunology, University of

Newcastle upon Tyne, Medical School, Newcastle upon Tyne, England.

University of Newcastle upon Tyne, Medical School, Framlington Pl, Newcastle

upon Tyne, England, NE2 4HH; e-mail: s.u.nielsen@ncl.ac.uk.

Submitted March 8, 2002; accepted July 10, 2002. Prepublished online as

Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-03-0731.
Supported by the Leukaemia Research Fund, London, England.

The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 U.S.C. section 1734.

Reprints: Soren U. Nielsen, Department of Microbiology and Immunology,

2002 by The American Society of Hematology

BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

4067

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4068

NIELSEN and ROUTLEDGE

on human T-cell blasts using serum from the T-cell donor as the
source of complement, the monomers and dimers of the bivalent
CD3 mAb gave no lysis. Conversely, the dimers of the monovalent
mAb gave more lysis than the benchmark CD52 mAb alemtuzumab, an improvement of approximately 64-fold over the nondimerized monovalent mAb. Dimerization of monovalent mAbs
may provide a strategy for improving the cytolytic activity of
antibodies specific for other complement-resistant cell surface antigens.

BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

Murine NS0 cells were transfected20 with either the L H combination

vector, the L HCys444 combination vector, or a mixture of the L H combination vector and the PEE6-tHCys444 vector to produce cell lines constitutively
expressing wild-type bivalent, bivalentCys444, or monovalentCys444 1 CD3
mAbs, respectively. Colonies of transfected cells were screened for human
IgG secretion by enzyme-linked immunosorbent assay (ELISA); colonies
producing monovalent mAbs were identified by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).15
Purification and dimerization of antibodies

Materials and methods

Cloning and expression of the CD3 mAb H- and L-chain genes
The humanization of the H- and L-chain genes of the CD3 mAb YTH 12.5
(human 1 G1m (1,17) Kern Oz), and the construction of the gene encoding
the N-terminally truncated human 1 H chain (tH) polypeptide have been
described elsewhere.15 cDNA versions of the genes were used for antibody
production in this present study. A second version of the H-chain cDNA was
created, in which the serine codon (TCT) at position 444 (numbering based on
human myeloma protein EU)19 was changed to cysteine (TGC). The Ser444Cys
mutation, along with a Cys233Ser substitution (Kabat numbering),19 were also
introduced into the tH-chain gene. In total, 4 different immunoglobulin genes
were produced (H, L, HCys444, and tHCys444), encoding the 4 different polypeptides
that are components of the bivalent and monovalent CD3 mAb monomers
illustrated in Figure 1A.
The vectors PEE13 and PEE6 (Lonza Biologics PLC, Slough, England)20 were used for immunoglobulin expression. The L-chain gene was
inserted between the HindIII and EcoRI sites of PEE13. The H, HCys444, and
tHCys444 genes were separately inserted between the HindIII and EcoRI sites
of PEE6. PEE13/PEE6 combination vectors carrying either the L H, or
L HCys444 genes were then produced.

Figure 1. Structures of the humanized (1, ) CD3 mAb monomers and dimers
and the reaction scheme for their preparation. (A) WT indicates bivalent mAb not
carrying the Ser444Cys mutation; H, heavy-chain polypeptide; L, light-chain polypeptide; HCys444, heavy-chain polypeptide carrying the Ser444Cys mutation; tHCys444,
N-terminally truncated heavy-chain carrying the Cys233Ser and Ser444Cys double
mutation; mal, N,N-1,2-phenylenedimaleimide cross-linkage. (B) Outline of the
stages involved in the dimerization procedure. DTT indicates dithiothreitol; Py-SS-Py,
2,2-dipyridyl disulphide; o-PDM, N,N-1,2-phenylenedimaleimide; G, blocking groups
such as glutathione.

The CD3 mAbs were concentrated and purified from culture supernatant by
protein A-Sepharose affinity chromatography.21
Homodimers of mAbs carrying the Cys444 mutation were prepared using a
variation of the protocols described by Glennie et al,22 Stevenson et al,23 and
Stalteri and Mather,24 as outlined in Figure 1B. BivalentCys444 CD3 mAb at a
protein concentration of approximately 2 mg/mL was reduced by the addition of
dithiothreitol (DTT; Sigma, code D9779, Poole, England) to a final concentration
of 20 mM (Figure 1B, step 1), followed by incubation for 20 minutes at 37C and
40 minutes at 25C. The DTT was removed by dialysis against phosphatebuffered saline (PBS) 1.0 mM EDTA (ethylenediaminetetra-acetic acid), then
the reduced interchain disulfide bonds were reconstituted by treatment with
2,2-dipyridyl disulfide (Py-SS-Py; Sigma, code D5767) as follows. Dimethylformamide (DMF, Sigma, code D8654) was first added to the dialyzed antibody to a
final concentration of 15% vol/vol. Py-SS-Py from a 20-mM stock solution in
anhydrous DMF (Aldrich, code 22705-6, Poole, England) was also added, to a
final molar concentration of 0.7 times the molar concentration of free sulfhydryl
(-SH) groups in the reduced antibody (estimated as 10 for the bivalentCys444
mAb). The mixture was incubated at 37C for 1 hour, then cooled on ice for 15
minutes. A one-tenth volume of ice-cold 0.5 M sodium acetate, pH 4.6, was
added to adjust the pH of the solution to 4.7; this was followed by DTT to a final
concentration of 1.0 mM, and incubation was continued at 4C for 1 hour. Under
these conditions DTT selectively reduces cysteine-SS-pyridyl disulfide bonds
(Cys-SS-Py) bonds, while leaving the re-formed interchain disulfide bonds intact.
The antibody was dialyzed as before, then put through a repeat round of disulfide
bond reconstitution with Py-SS-Py (but not DTT reduction) followed by dialysis,
to ensure that the interH-chain disulfide bonds in the IgG hinge had re-formed.
At this point the antibody was reconcentrated to approximately 2 mg/mL using
a Millipore ultrafree centrifugal device (Fisher, code FDR-582-050R, Loughborough, England), and stored at 80C prior to the final steps in the crosslinking procedure.
To begin the final steps in the dimerization process, up to 15 mg
antibody, which had been through the disulfide bond reduction and
reconstitution process, was thawed and again selectively reduced with DTT
at pH 4.7 as described earlier. The DTT was removed by dialysis against 50
mM sodium acetate, 1 mM EDTA, pH 4.6, at 4C for 4 hours, followed by
dialysis against the same buffer at pH 5.3 at 4C, overnight. (All sodium
acetate buffer used for these dialysis steps, and for the buffer exchange on
Sephadex G25, see below, were degassed under vacuum then purged with
N2 gas before use.) The mAb solution was then split into 2 portions in the
ratio of 0.55:0.45 and held on ice. The bismaleimide cross-linking reagent
N, N-1,2-phenylenedimaleimide (o-PDM, Aldrich, code 10459-0) dissolved in anhydrous DMF was added to the larger portion of antibody to
give final concentrations of o-PDM and DMF of 4.5 mM and 25% vol/vol,
respectively. After 40 minutes on ice followed by centrifugation at 12 000
rpm for 2 minutes at 4C, this sample was buffer-exchanged into 20 mM
sodium acetate, 5 mM EDTA, using an XK16/40 column of Sephadex G25
(Pharmacia, Bucks, England) equipped with a cooling mantle of circulating
water kept at 0C. This relatively long column of Sephadex G25 (40 cm)
was necessary to ensure complete removal of surplus N, N-1,2phenylenedimaleimide24 and was run at 12 mL/min to achieve a fast and
efficient buffer exchange. Fractions containing antibody were identified by
OD 280 measurement and collected on ice, pooled, and then mixed with the
remaining portion of reduced, dialyzed antibody. The reaction mixture was
reconcentrated as before to approximately 2 mg/mL, then incubated on a
rotary shaker at 4C for 3 days to allow dimers to form. At the end of this
time N-acetyl-L-cysteine (Sigma, code A8199) was added at a final
concentration of 1 mM to block any free maleimide groups, and the mixture
was held at room temperature for 10 minutes.

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BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

The dimers were separated from nondimerized monomers by gel

filtration on an XK26/100 column of Superdex 200 pg (Pharmacia, code
17-1043-01) equilibrated with 25 mM Tris (tris(hydroxymethyl)aminomethane), pH 7.5, 0.5 M NaCl, 0.1% wt/vol betaine, 2 mM EDTA, and 0.05%
sodium azide. Dimer and monomer peak fractions were separately pooled
and concentrated to approximately 1 mg/mL. They were then dialyzed
extensively against PBS, filter sterilized, and stored at 80C prior to
assessment for antigen binding and cell lysis activity (see Competitive
binding assay and Complement lysis assay).
The monovalentCys444 mAb was dimerized in essentially the same way
as the bivalent mAb, except for the first stage. The protein Apurified
immunoglobulin from the NS0 cells transfected with the combination of H,
L, and tHCys444 genes actually contains 3 species of molecules due to the
random association of H- and tH-chain polypeptides within the cell.15 These
species are bivalent CD3 mAb (170 kDa), monovalent CD3 mAb (120
kDa), and Fc molecules (70 kDa), consisting of 2H 2L, H L
tHCys444, and 2tHCys444 polypeptides, respectively. Prior to dimerization, the
monovalent species first had to be separated from the other 2. This was
achieved by gel filtration using the system described above for separation of
the bivalent mAb monomers and dimers. Before loading the protein
Apurified antibody onto the column, it was first reduced with 20 mM DTT
as described in the first step of the bivalent mAb dimerization procedure.
After gel filtration, the purified, reduced monovalent mAb fraction collected from the column was then immediately treated with Py-SS-Py in the
disulfide bond reconstitution step, without prior dialysis into PBS. When
calculating the quantity of Py-SS-Py to use, the number of -SH groups in
the monovalentCys444 mAb was assumed to be 7.
The buffers used in the above procedures were prepared with deionized
water containing 0.12 endotoxin units (EU)/mL as measured using a
Limulus amoebocyte assay (Sigma, code 210-A1). Final antibody preparations contained 1 to 3 103 EU/mg mAb.

COMPLEMENT LYSIS BY DIMERS OF MONOVALENT ANTIBODY

4069

lyophilized rabbit HLA-ABC serum (Sigma, code S7764) or guinea pig

serum (Sigma, code S1639) was used as a source of rabbit or guinea pig
complement, respectively. Diluent (Iscove modified Dulbecco medium
[IMDM]; Gibco BRL, Paisley, Scotland; code 42200-048 containing 1%
BSA) was used in no antibody and no complement control wells. The
plate was incubated at 37C for 1 hour; then the extent of 51Cr release was
determined by gamma counting.
Antibody-dependent cellular cytotoxicity
Human CD56 natural killer (NK) cells were isolated from peripheral
blood mononuclear cells (PBMCs) using CD56 antibody (BectonDickinson, code 347740) and M-450 Dynabeads (Dynal, code 110.01;
Bromborough, England) as described by Naume et al.26 The CD56 cells
were cultured for 15 days as described by Miller et al.27 On day 10 the small
percentage of CD56 CD3 cells that copurified with the CD56 CD3
cells were removed by complement lysis using 5 g/mL of the monovalent
1 CD3 mAb dimer, and homologous human serum at a final concentration
of 33% vol/vol. After 1 hour at 37C the cells were washed and returned to
culture. On day 15 the cells were harvested for use as effector cells in
antibody-dependent cellular cytotoxicity (ADCC) assays.
Target T cells were freshly isolated from the NK cell donor on the day of
the assay. PBMCs isolated on lymphoprep gradients were passed down a
nylon wool column and then depleted of CD56 cells using magnetic
beads. This population was typically 65% CD3 and nearly 100% CD52.
The lymphocytes were labeled with 51Cr (Amersham, code CJS4; Bucks,
England), washed, then incubated with dilutions of test mAb (in RPMI
1640 1% BSA) in a 96-well round-bottomed microtray (2 104 target
cells/well). After 90 minutes the cells were washed 3 times to remove
nonbound mAb, then effector cells were added (effector-target [E/T] ratio
of 5:1). After a further 4 hours at 37C the extent of 51Cr release was
determined.

Competitive binding assay

The relative binding avidity of the CD3 mAb monomers and dimers were
assessed in a competitive binding assay using cells from a human T-cell
leukemia cell line (HPBALL), and 1 g/mL biotinylated wild-type bivalent
humanized CD3 mAb as the labeled antibody, as described previously.15
The control mAb alemtuzumab25 (humanized 1 G1m (1,17) , CD52
mAb) was kindly provided by Dr G. Hale (Therapeutic Antibody Centre,
Heddington, Oxford, England).
Assessment of CD3 antigen modulation
Human T-cell blasts1 were incubated with a saturating concentration (3
g/mL) of test CD3 antibody (or nonmodulating control mAb, alemtuzumab) in culture medium at 37C in a CO2 incubator. At 6 time points
between 1 and 24 hours after beginning the incubation, samples of cells
were taken and prepared for fluorescence-activated cell sorting (FACS)
analysis to assess the surface expression of CD3 antigen. This was done by
rinsing the cells in FACS diluent (PBS with 1% bovine serum albumin
[BSA], 5% heat-inactivated normal rabbit serum, 0.1% sodium azide), then
staining them for 1 hour at 4C with 1 g/mL R-phycoerythrinlabeled goat
antihuman IgG (Sigma, code P 9170) in FACS diluent. The mean
fluorescence of approximately 5000 cells/sample was then determined
using a Becton Dickinson FACScan (Oxford, England). To indicate the
maximum level of staining that could be produced by the test CD3 mAbs in
the absence of modulation, T-cell blasts were also incubated with each mAb
(3 g/mL) for 1 hour at 37C in cell culture medium in the presence of 0.1%
sodium azide to minimize modulation. These cells were then also stained
with R-phycoerythrinlabeled goat antihuman IgG and analyzed by FACS.
Complement lysis assay
Human T-cell blasts1 were labeled with 51Cr, washed, then added (2 105
cells/well in a 25-L volume) to 50-L dilutions of test mAb in a 96-well
round-bottomed microtray. After mixing, 25 L defibrinated homologous
human serum was added to each well as a source of human complement,
and the plate mixed again. Alternatively, 25 L freshly reconstituted

Results
Dimerization of antibodies using the Shopes
mutation (Ser444Cys)

The C-terminally linked IgG dimers that have been described

previously16-18 have been held together by disulfide bonds, formed
between cysteine residues introduced at H-chain position 444. In
this study the strategy of introducing a cysteine at position 444 was
combined with the use of a bismaleimide cross-linking reagent.
The aim was to produce dimers held together by a nonreducible
covalent linkage, the stability of which has been confirmed in
vivo.28 Two types of bismaleimide-linked dimer were assembled
from a humanized IgG1 mAb specific for the human CD3 antigen
(Figure 1). The first (the bivalentCys444 dimer) was composed of 2
bivalent IgG molecules, each with 2 Fab domains. The second (the
monovalentCys444 dimer) consisted of 2 monovalent IgG molecules,
each possessing only a single Fab domain.
The monovalent IgG molecules used to make the second dimer
were generated using a truncated H-chain strategy. This enabled
IgG carrying only one Cys444 per molecule to be produced,
preventing the formation of high-order multimers during the
dimerization process.
The 2 dimers, along with their respective nondimerized monomers (bivalentCys444 monomer and monovalentCys444 monomer) and
the original wild-type humanized IgG1 CD3 mAb (bivalent WT
monomer) were analyzed for purity, antigen-binding avidity, and in
vitro cytolytic activity.
Analysis of cross-linked CD3 antibodies by SDS-PAGE

The SDS-PAGE performed under nonreducing conditions (Figure

2) revealed multiple bands in each sample. The major band in each

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4070

NIELSEN and ROUTLEDGE

BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

nature of the bismaleimide cross-linkage. HCys444 polypeptide

dimers could also be seen in the bivalentCys444 monomer preparation, indicating that many of the monomer molecules have an
intramolecular bismaleimide cross-linkage. This linkage is probably between the 2 Cys444 residues rather than between 2 of the
hinge -SH groups, because corresponding tHCys444 polypeptide
dimers were absent in the monovalentCys444 monomer preparation
(the monovalentCys444 mAb has the same potential to form intramolecular linkages in the hinge as the bivalentCys444 mAb, but because
it only has a single Cys444 residue, the formation of a linkage
between the C-termini of its H and tH polypeptides is not possible).
The effect of cross-linking on CD3 antibody-binding avidities

Figure 2. SDS-PAGE analysis of CD3 mAb monomers and dimers performed

under nonreducing conditions. A 1.5-g quantity of each sample was run on a 5%
to 18% gradient polyacrylamide gradient gel possessing a 4% stacking gel, under
nonreducing conditions. The gel was stained with Coomassie blue. Lane 1 (BM),
bivalentCys444 monomer; lane 2 (BD), bivalentCys444 dimer; lane 3 (MM), monovalentCys444
monomer; lane 4 (MM), monovalentCys444 dimer; lane 5 (BMWT), bivalentWT monomer;
MWM, molecular weight markers. The positions of the bands corresponding to the antibody
monomers and dimers are illustrated on the gel.

lane is the relevant mAb monomer or dimer. The minor 70-kDa

band in the monovalentCys444 monomer and dimer preparations
(lanes 3 and 4, respectively) is probably residual Fc protein
produced by the monovalent mAb cell line that was not separated
from the monovalent antibody (by the gel filtration step), prior to
the beginning of the dimerization process. The minor 140-kDa
band in the monovalentCys444 dimer preparation (lane 4) is probably
some of this Fc protein that has cross-linked to produce Fc dimers.
Some of the other minor bands with molecular weights smaller than
the major bands represent partially dissociated IgG molecules,
present because a proportion of the IgG in the purified mAb
preparations exists naturally in a partially reduced state. This is
normal and is seen in mAb that has not been through the
dimerization process (the BMWT mAb, lane 5).
Other bands of greater size than the major bands are also present
in some of the preparations. In the bivalentCys444 dimer sample (lane
2), 2 minor bands are visible above the main dimer band. It is not
clear what these particular 2 bands are, but one possibility is that
they represent species of dimer in which the mAb molecules are
held together by only 1, rather than 2, bismaleimide linkages. The
existence of single-linkage dimers in this preparation was
suggested by SDS-PAGE analysis under reducing conditions
(not shown), where a faint band of noncross-linked H chains
was visible. Under nonreducing conditions, the 2 faint highmolecular-weight bands in the bivalentCys444 monomer preparation indicate that it is contaminated by a small quantity of
bivalentCys444 dimer from the gel filtration procedure (lane 1).
Likewise, the monovalentCys444 monomer contains a small amount
of its corresponding dimer (lane 3).
When SDS-polyacrylamide gels were run under reducing
conditions (not shown), HCys444 and tHCys444 polypeptide dimers
could be seen in the dimerized mAbs, confirming the nonreducible

The relative binding avidities of the CD3 mAb monomers and

dimers were compared by measuring their ability to block the
binding of biotinylated wild-type CD3 mAb to cells expressing the
human CD3 antigen (Figure 3). The results obtained with the
bivalentCys444 monomer and bivalentWT monomer suggest that the
chemical manipulations involved in the cross-linking procedure
may have slightly reduced the antibodys binding activity (by a
factor of 1.3). Dimerization of the bivalentCys444 mAb produced
only a 1.3-fold rise in avidity; this is in line with the increases
reported by others16,17 for disulfide bondlinked bivalent mAb
dimers. The avidity of the monovalentCys444 monomer was 4.7-fold
less than that of the bivalentCys444 monomer on a microgram per
milliliter basis; this is equivalent to a 3.5-fold difference on a molar
Fab basis. This difference is not as great as the 6-fold difference
reported previously between monovalent and bivalent CD3 mAbs.15
It is probably due to the presence of traces of monovalentCys444
dimer protein as seen by SDS-PAGE. The presence of small
amounts of dimerized antibody would increase the apparent avidity
of the monovalentCys444 monomer preparation. Dimerization of the
monovalentCys444 mAb increased its avidity by a factor of at least
2.8 (possibly more, if the estimate for the monovalentCys444
monomer was too high). This raised its avidity to just under that of
the bivalentCys444 monomer (1.2-fold less on a molar Fab basis).

Figure 3. Comparison of relative binding avidities of cross-linked CD3 antibodies. (, BM), bivalentCys444 monomer; (, BD), bivalentCys444 dimer; (E, MM),
monovalentCys444 monomer; (, MD), monovalentCys444 dimer; (, BMWT), bivalentWT
monomer; (, alem), CD52 mAb alemtuzumab. Fluorescence values obtained in the
absence of competitor mAb (, 100% fluorescence), or in the absence of competitor
mAb and biotinylated CD3 mAb (f, 0% fluorescence) are indicated. Each point is the
mean of duplicate tests.

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BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

Figure 4. Modulation of CD3 antigen expression on T cells after incubation with

CD3 antibodies. (, BMWT), bivalentWT monomer; (, BD), bivalentCys444 dimer; (,
MD), monovalentCys444 dimer; (, alem), CD52 mAb alemtuzumab. Each point is the
mean of duplicate samples. The mean fluorescence values have been corrected
using a no first antibody control.

Antibody dimers modulate CD3 antigen expression

The relative antigen-modulating activities of the dimerized CD3

mAbs was assessed by incubating human T-cell blasts in saturating
concentrations of the mAbs over a 24-hour period. Cell samples
were taken at different time points to determine the level of cell
surface CD3 antigen expression. The results (Figure 4) indicated
that the modulating activity of the monovalentCys444 dimer was very
similar to that of the bivalentWT monomer; both reduced the level of
CD3 antigen on the surface of the T-cell blasts by three fourths after
24 hours. The bivalentCys444 dimer modulated the CD3 antigen to a
similar extent, but its initial modulation rate was more rapid. The
mAb alemtuzumab, which was used as a nonmodulating control
antibody, caused little change in the cell surface level of its target
antigen CD52, except in the first 1-hour period of the experiment.
The brighter fluorescence given by this antibody is due to the fact
that CD52 is expressed at a higher level on the cell surface
compared to CD3.8

COMPLEMENT LYSIS BY DIMERS OF MONOVALENT ANTIBODY

4071

guinea pig serum as sources of complement, but the results were

again negative (data not shown).
The complement lysis result obtained with the monovalentCys444
dimer was completely different. Although its binding avidity was
slightly less than that of the bivalentCys444 dimer, the maximum
level of lysis produced with human complement was equal to the
maximum produced by alemtuzumab, and its lytic titer was 4-fold
greater (Figure 5). Dimerization of the monovalentCys444 CD3 mAb
had therefore produced a 64-fold increase in lytic titer. This was
consistently observed in 4 lysis experiments. Interestingly, when
rabbit complement was used (data not shown) the difference
between the monovalentCys444 dimer and alemtuzumab increased to
16-fold, but when guinea pig complement was used there was a
marked reversal in the relative lytic activities of the 2 antibodies
(alemtuzumab 60% lysis, monovalentCys444 dimer 10% lysis, at
12.5 g/mL mAb with guinea pig complement). Despite the poor
level of lysis seen with the monovalentCys444 dimer with guinea pig
complement, it was the only form of CD3 mAb to show any
activity with this complement type.
The complement source-dependent difference in the relative
lytic activities of alemtuzumab and the monovalentCys444 dimer
suggests that the activity of complement from different species is
influenced to different extents by the antigen specificity of the
activating antibody. It implies that an antibody with an antigenic
specificity that works well with one species of complement will not
necessarily be good with another. This is an important consideration if antibodies are being selected for therapeutic applications
based on their lytic activity with nonhuman complement.
CD3 antibody dimers are effective in ADCC

When tested in an ADCC assay (Figure 6), the monovalentCys444

dimer and the bivalentCys444 dimer produced similar levels of
cytotoxicity, giving levels of lysis above those of their nondimerized counterparts. This indicates that the use of the bismaleimide
cross-linker has not destroyed the ability of dimers to interact with
FcR-bearing cytotoxic cells, an important feature because it
correlates strongly with in vivo cell depletion activity.3 Whereas
complement-dependent lysis was detected down to a concentration
of 200 ng/mL monovalentCys444 dimer, ADCC was detectable at

Complement-mediated lytic activities of the CD3

antibody dimers

The pattern of complement lysis (Figure 5) observed with the

monovalent and bivalent monomers using human complement
was the same as that reported previously.15 The bivalentWT
monomer and bivalentCys444 monomer gave no lysis, whereas the
monovalentCys444 monomer produced 22% lysis at a concentration of
25 g/mL, a lytic titer 16-fold lower than that seen with the
benchmark CD52 mAb, alemtuzumab. Some of the activity of the
monovalentCys444 monomer preparation is probably due to the
presence of the dimer contaminant seen in SDS-PAGE.
In contrast to previous studies which described dimerized
bivalent mAbs specific for a hapten and CD33,16,17 no improvement
in complement lysis activity was observed with dimerized bivalent
CD3 mAb. One possible reason for the difference (other than the
different target antigens involved) was that the previous studies
used nonhuman sources of complement. Human cells are generally
more resistant to lysis by human complement because they are
protected by anticomplement defense proteins, such as CD59,
which inhibit the formation of homologous complement membrane
attack complexes.29 To explore this possibility, we also tested the
lytic activity of the dimerized bivalent mAb using rabbit serum and

Figure 5. Lysis of human T-cell blasts with human complement. (, BMWT),

bivalentWT monomer; (, BM), bivalentCys444 monomer; (, BD), bivalentCys444 dimer;
(E, MM), monovalentCys444 monomer; (, MD), monovalentCys444 dimer; (, alem),
CD52 mAb alemtuzumab. Each point is the mean of duplicate tests. The range of
lysis observed with all mAbs at their maximum test concentration in the absence of
complement is indicated (NC).

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4072

NIELSEN and ROUTLEDGE

Figure 6. ADCC of human PBMCs by homologous CD56 effector cells. (, BM),

bivalentCys444 monomer; (, BD), bivalentCys444 dimer; (E, MM), monovalentCys444
monomer; (, MD), monovalentCys444 dimer; (, alem), CD52 mAb alemtuzumab.
Each point is the mean of duplicate tests. The range of lysis observed with all mAbs at
their maximum test concentration in the absence of effector cells (NE) and the level of
lysis given by effector cells in the absence of mAb (NA) are indicated.

concentrations of CD3 mAb dimers as low as 5 to 50 ng/mL

(compare Figures 5 and 6).

Discussion
The reason the monovalentCys444 dimer is so active in complement
lysis while the bivalentCys444 dimer is ineffective is not clear. One
hypothesis used in the past to explain the superior performance of
(nondimerized) monovalent CD3 mAbs compared to bivalent CD3
mAbs is that monovalent mAbs induce less antigenic modulation.
However, the monovalentCys444 dimer has 2 Fab domains and is
able to cross-link antigen on the cell surface, and we have found
that it modulates antigen to a similar extent as the original nonlytic
bivalentWT monomer. If reduced modulation was the only mechanism behind the superior lytic activity of monovalent CD3 mAbs,
dimerization would be predicted to inhibit their complement lysis
activity. This was not the case.
Tail-to-tail antibody dimers have been proposed to form naturally (via noncovalent interactions) after the binding of IgG to cell
surface antigens,30 facilitating C1q binding. This idea is supported
by the studies of Shopes16 and Caron et al,17 in which human IgG1
dimers formed artificially using engineered disulfide binds were
found to have much higher lytic activity than their monomeric
counterparts. A second hypothesis that has been used to explain the
superior lytic activity of monovalent compared to bivalent CD3
mAbs is that monovalent binding to the cell surface might permit
greater mobility in the Fc region, making it easier for adjacent Fc
regions to interact and achieve a configuration favorable for
complement activation.15,31 If binding antigen by both Fabs
inhibits suitable Fc interactions in bivalent CD3 mAbs, then
prefixing their Fc regions in a favorable configuration (ie, by
covalent tail-to-tail dimerization) prior to reaction with antigen
would be predicted to overcome the problem. However, the
bivalentCys444 dimer did not show improved lytic activity. This
observation also suggests that the cross-linking of the 2 Fc regions
in the monovalentCys444 dimer is not the only factor responsible for
its potency.
One major difference in the structure of the 2 CD3 mAb dimers
is that the monovalentCys444 dimer is held together by a single

BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

cross-link, whereas the bivalentCys444 dimer has the potential to be

held together by 2. It is unlikely that this could be solely
responsible for their different lytic activities, because SDS-PAGE
carried out under reducing conditions (not shown) indicated that a
proportion of the dimers in the bivalentCys444 dimer preparation are
in fact only held together by one intermolecular cross-linkage.
Therefore, if the single cross-link was the determining factor for
the observed lytic differences, some lytic activity would probably
have been seen with this dimer. Nevertheless, it could make a
beneficial contribution to dimer flexibility by permitting a greater
degree of bending and rotation of the dimer.
Perhaps the important factor influencing the outcome of complement activation by 1 CD3 mAbs is the distance or freedom of
movement between adjacent cross-linked CD3 antigen complexes,
rather than the occurrence or absence of antigen cross-linking per
se. The 2 antigen-binding sites on the monovalentCys444 dimer are
further apart than those on the bivalentCys444 monomer. Furthermore, the monovalentCys444 dimer is potentially very flexible. When
both Fab domains of this dimer are bound to a cell surface, the Fc
domains should still be totally free to rotate around their longitudinal axes. This is not true for the bivalent mAb or its dimer, where
crossover of the H-chain polypeptides between the hinge and the 2
Fab domains will restrict Fc rotation. These features of the
monovalentCys444 dimer (illustrated in Figure 7) may give the
complement membrane attack complex easier access to the cell
membrane. Some support for this idea is given by previous work1
showing that monovalent and bivalent CD3 mAbs (of the rat 2b
isotype) fix similar levels of C1q and cell-bound C3. This indicates
that their complement-activating properties are closely matched
and indirectly implies that a later step in the complement cascade is
the limiting factor. A correlation between segmental flexibility and
the complement-fixing activity of antibodies has been suggested in
the past.31

Figure 7. Models to explain the improved lytic activity of the monovalentCys444

dimer (MD). (A) Representation of the different cross-linked antigen lattices that
might be formed on the cell surface by wild-type bivalent and dimerized monovalent
antibody. MAC indicates membrane attack complex. (B) Probable differences in the
relative rotational freedom of the Fc regions of the bivalent and monovalent antibody
dimers, when bound to CD3 antigen on a cell surface.

From bloodjournal.hematologylibrary.org at Newcastle University on July 4, 2013. For personal use only.
BLOOD, 1 DECEMBER 2002 VOLUME 100, NUMBER 12

In conclusion, we have shown that if bivalent 1 mAb

monomers and dimers of a particular antigenic specificity are
unable to mediate complement lysis of target cells, it is possible to
achieve efficient lysis using dimers of a monovalent derivative of
the mAb. Effective lysis of human T cells has also been achieved
using an antigen that is less abundantly expressed than the CD52
antigen recognized by the highly lytic alemtuzumab antibody.
These observations create opportunities to improve the lytic
function of other bivalent monoclonal antibodies with poor lytic
abilities. The results also provide an insight into why cells may be
resistant to lysis by mAbs to certain antigens. The next step is to
determine whether or not the enhanced in vitro complement lysis
activity of the monovalentCys444 dimer translates into superior

COMPLEMENT LYSIS BY DIMERS OF MONOVALENT ANTIBODY

4073

cell-depleting activity in vivo. The stability of the bismaleimide

cross-linker used to make the dimers should allow this assessment
to be made.

Acknowledgments
The cross-linking protocol was developed based on initial work by
Dr Sonya Patterson. Dr B. Shenton is acknowledged for help with
FACS analysis. The region genes of the humanized YTH 12.5
mAbs were kindly provided by Prof H. Waldmann (University of
Oxford, England).

References
1. Clark M, Bindon C, Dyer M, et al. The improved
lytic function and in vivo efficacy of monovalent
monoclonal antibodies. Eur J Immunol. 1989;19:
381-388.
2. Dyer M, Hale G, Marcus R, Waldmann H. Remission induction in patients with lymphoid malignancies using unconjugated CAMPATH-1 monoclonal antibodies. Leuk Lymphoma. 1990;2:179-193.
3. Dyer MJS, Hale G, Hayhoe FGJ, Waldmann H.
Effects of CAMPATH-1 antibodies in vivo in patients with lymphoid malignancies: influence of
antibody isotype. Blood. 1989;73:1431-1439.
4. Gorman SD, Clark MR. Humanization of monoclonal antibodies for therapy. Semin Immunol.
1990;2:457-466.
5. Isaacs JD. The antiglobulin response to therapeutic antibodies. Semin Immunol. 1990;2:449456.
6. Bruggemann M, Williams GT, Bindon CI, et al.
Comparison of the effector functions of human
immunoglobulins using a matched set of chimeric
antibodies. J Exp Med. 1987;166:1351-1361.
7. Circolo A, Battista P, Borso T. Efficiency of activation of complement by anti-hapten antibodies at
the red cell surface; effect of patchy vs random
distribution of hapten. Mol Immunol.
1985;22:207-214.
8. Bindon CI, Hale G, Waldmann H. Importance of
antigen specificity for complement-mediated lysis
by monoclonal antibodies. Eur J Immunol. 1988;
18:1507-1514.
9. Michaelsen TE, Garred P, Aase A. Human IgG
subclass pattern of inducing complement-mediated cytolysis depends on antigen concentration,
and to a lesser extent on epitope patchiness, antibody affinity and complement concentration. Eur
J Immunol. 1991;21:11-16.
10. Gordon J, Stevenson GT. Antigenic modulation of
lymphocyte surface immunoglobulin yielding resistance to complement-mediated lysis, II: relationship to redistribution of the antigen. Immunology. 1981;42:13-17.

11. Scheinberg DA, Lovett D, Divgi CR, et al. A phase

I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide. J Clin Oncol. 1991;9:478-490.
12. Henell KR, Bakke A, Kenny TA, Kimball JA, Barry
JM, Norman DJ. Degree of modulation of cellsurface CD3 by anti-lymphocyte therapies. Transplant Proc. 1991;23:1070-1071.
13. Glennie MJ, Stevenson GT. Univalent antibodies
kill tumour cells in vitro and in vivo. Nature. 1982;
295:712-714.
14. Cobbold SP, Waldmann H. Therapeutic potential
of monovalent monoclonal antibodies. Nature.
1984;308:460-462.
15. Routledge EG, Lloyd I, Gorman SD, Clark M,
Waldmann H. A humanized monovalent CD3 antibody which can activate homologous complement. Eur J Immunol. 1991;21:2717-2725.
16. Shopes B. A genetically engineered human IgG
mutant with enhanced cytolytic activity. J Immunol. 1992;148:2918-2922.
17. Caron PC, Laird W, Co MS, Avdalovic NM,
Queen C, Scheinberg DA. Engineered humanized dimeric forms of IgG are more effective antibodies. J Exp Med. 1992;176:1191-1195.
18. Greenwood J, Gorman SD, Routledge EG, Lloyd
IS, Waldmann H. Engineering multiple-domain
forms of the therapeutic antibody CAMPATH-1H:
effects on complement lysis. Ther Immunol. 1994;
1:247-255.
19. Kabat EA, Wu TT, Reid-Miller M, Perry HM,
Gottesman KS. Sequences of proteins of Immunological Interest. 4th ed. Bethesda, MD: US Department of Health and Human Services, Public
Health Service, National Institutes of Health;
1987.
20. Bebbington C. Use of vectors based on gene amplification for the expression of cloned genes in
mammalian cells. In: Glover DM, Hames BD, eds.
DNA Cloning 4. 2nd ed. Oxford, England: IRL
Press; 1995:85-112.

21. Harlow E, Lane L. Antibodies, a Laboratory

Manual. New York; NY: Cold Spring Harbor Laboratory Press; 1988.
22. Glennie MJ, McBride HM, Worth AT, Stevenson
GT. Preparation and performance of bispecific
F(ab)2 antibody containing thioether-linked
Fab fragments. J Immunol. 1987;139:23672375.
23. Stevenson GT, Pindar A, Slade CJ. A chimaeric
antibody with dual Fc regions (bis FabFc) prepared by manipulations of the IgG hinge. Anticancer Drug Des. 1989;3:219-230.
24. Stalteri MA, Mather SJ. A cross-linked monoclonal antibody fragment for improved tumor targeting. Bioconj Chem. 1995;6:179-186.
25. Riechmann L, Clark M, Waldmann H, Winter G.
Reshaping human antibodies for therapy. Nature.
1988;332:323-327.
26. Naume B, Nonstad U, Steinkjer B, Funderud S,
Smeland E, Espevik T. Immunomagnetic isolation
of NK and LAK cells. J Immunol Methods. 1991;
136:1-9.
27. Miller JS, Oelkers S, Verfaillie C, McGlave P. Role
of monocytes in the expansion of human activated natural killer cells. Blood. 1992;80:22212229.
28. Glennie MJ, Brennand DM, Bryden F, et al.
Bispecific F(ab)2 antibody for the delivery of
saporin in the treatment of lymphoma. J Immunol.
1988;141:3662-3670.
29. Meri S, Morgan BP, Davies A, et al. Human protectin (CD59), an 18,000-20,000 MW complement lysis restricting factor, inhibits C5b-8 catalyzed insertion of C9 into lipid bilayers.
Immunology. 1990;71:1-9.
30. Burton DR. Is IgM-like dislocation a common feature of antibody function? Immunol Today. 1986;
7:165-167.
31. Oi VT, Vuong TM, Hardy R et al. Correlation between segmental flexibility and effector function.
Nature. 1984;307:136-140.