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Therapeutic control of B cell activation via recruitment of Fc╨Ю╤Ц receptor IIb CD32B inhibitory function with a novel bispecific antibody scaffold.

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Vol. 62, No. 7, July 2010, pp 1933–1943
DOI 10.1002/art.27477
© 2010, American College of Rheumatology
Therapeutic Control of B Cell Activation via Recruitment of
Fc␥ Receptor IIb (CD32B) Inhibitory Function
With a Novel Bispecific Antibody Scaffold
Maria-Concetta Veri, Stephen Burke, Ling Huang, Hua Li, Sergey Gorlatov, Nadine Tuaillon,
G. Jonah Rainey, Valentina Ciccarone, Tengfei Zhang, Kay Shah, Linda Jin, Lida Ning,
Tamara Minor, Paul A. Moore, Scott Koenig, Syd Johnson, and Ezio Bonvini
Objective. To exploit the physiologic Fc␥ receptor
IIb (CD32B) inhibitory coupling mechanism to control
B cell activation by constructing a novel bispecific
diabody scaffold, termed a dual-affinity retargeting
(DART) molecule, for therapeutic applications.
Methods. DART molecules were constructed by
pairing an Fv region from a monoclonal antibody (mAb)
directed against CD32B with an Fv region from a mAb
directed against CD79B, the ␤-chain of the invariant
signal-transducing dimer of the B cell receptor complex.
DART molecules were characterized physicochemically
and for their ability to simultaneously bind the target
receptors in vitro and in intact cells. The ability of the
DART molecules to negatively control B cell activation
was determined by calcium mobilization, by tyrosine
phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule
specific for the mouse ortholog of CD32B and CD79B
was also constructed and tested for its ability to inhibit
B cell proliferation in vitro and to control disease
severity in a collagen-induced arthritis (CIA) model.
Results. DART molecules were able to specifically
bind and coligate their target molecules on the surface
of B cells and demonstrated a preferential simultaneous
binding to both receptors on the same cell. DART
molecules triggered the CD32B-mediated inhibitory sig-
naling pathway in activated B cells, which translated
into inhibition of B cell proliferation and Ig secretion. A
DART molecule directed against the mouse orthologs
was effective in inhibiting the development of CIA in
DBA/1 mice.
Conclusion. This innovative bispecific antibody
scaffold that simultaneously engages activating and
inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and
potentially other autoimmune and inflammatory diseases in humans.
Activation-inhibition coupling, the pairing of a
positive signal with an inhibitory loop, controls the
magnitude and duration of many biologic processes
(1–3). In B lymphocytes, recognition of an antigen by the
clonotypic B cell receptor (BCR) induces a signal that
can direct clonal expansion, differentiation, the release
of cytokines, and, ultimately, Ig production. Uncontrolled activation is prevented by exhaustion of the
activating stimulus as well as by the triggering of a
negative feedback loop that involves the engagement of
an inhibitory Fc␥ receptor (Fc␥R), Fc␥RIIb (CD32B)
(4). The latter mechanism is triggered when the BCR
recognizes immune-complexed antigen, resulting in the
concomitant engagement of CD32B by the Fc domain of
the complex-bound IgG, thus preventing the expansion
of B cell clones that share the same specificity as that
recognized by the soluble IgG. Attesting to its critical
role in immune regulation, CD32B-knockout mice on
the Th1-prone C57BL/6 background develop a lupuslike glomerulonephritis (5,6). We have focused on
CD32B inhibitory signaling in B lymphocytes as a model
system for the development of an alternative class of
biologics that exploit activation-inhibition coupling for
the control of immune activation. In addition to Ig
Maria-Concetta Veri, PhD, Stephen Burke, BA, Ling Huang,
MD, Hua Li, MS, Sergey Gorlatov, PhD, Nadine Tuaillon, PhD, G.
Jonah Rainey, PhD, Valentina Ciccarone, PhD, Tengfei Zhang, BS,
Kay Shah, BS, Linda Jin, BS, Lida Ning, MS, Tamara Minor, BS,
Paul A. Moore, PhD, Scott Koenig, MD, PhD, Syd Johnson, PhD, Ezio
Bonvini, MD: MacroGenics, Inc., Rockville, Maryland.
All authors own stock or stock options in MacroGenics, Inc.
Address correspondence and reprint requests to Ezio Bonvini, MD, MacroGenics, Inc., 1500 East Gude Drive, Rockville, MD
20850. E-mail: [email protected]
Submitted for publication October 16, 2009; accepted in
revised form March 19, 2010.
production, B lymphocytes play a central immunologic
function as regulators of the adaptive immune response,
as shown by the clinical success of B cell–depleting
therapies, such as anti-CD20 monoclonal antibody
(mAb) interventions (e.g., rituximab), in autoimmune
diseases (7,8).
A successful negative regulatory strategy should
recapitulate the antigen-driven proximity of the activating and inhibitory receptors that form the molecular
basis for the negative signaling loop. We have developed
a platform of dual-affinity retargeting (DART) molecules in which the Fv regions of 2 distinct antibodies are
paired by heterodimerizing sequences and covalently
linked by a carboxyl-terminal disulfide bond. DART
molecules designed to modulate B cell function were
constructed by pairing an Fv region from a mAb directed
against CD32B (9) with an Fv region from a mAb
directed against CD79B, the ␤-chain of the invariant
signal-transducing dimer of the BCR complex (10). We
show here that coligation of activating and inhibitory
receptors by CD32B ⫻ CD79B DART molecules can
alter the response of human B lymphocytes to BCR
stimuli, resulting in signal disruption as well as in
inhibition of calcium mobilization, cell proliferation, and
Ig secretion in vitro. Furthermore, treatment of mice
with a DART molecule directed against the mouse
orthologs controls the development of collagen-induced
arthritis (CIA) in a susceptible strain. This novel approach to biologic response modulation has potential as
an intervention in autoimmune diseases.
Cells and antibodies. B lymphocytes from healthy
donors and mouse splenic B cells were isolated with Dynal
B-Cell Negative Isolation Kits (Invitrogen). The CHO-CD32B
cell line was previously described (9). Daudi and RAMOS cell
lines were from American Type Culture Collection (ATCC).
Anti-human CD32B mAb, 2B6 and 3H7, were described
previously (9); mAb 8B5 was generated in a similar manner.
Monoclonal antibody CB3.1 (anti-human CD79B) (10) was
obtained from Dr. Max Cooper (University of Alabama at
Birmingham). Monoclonal antibodies 2.4G2 (anti-mouse
CD32/CD16), HM79 (anti-mouse CD79B), and 4-4-20 (antifluorescein) were from ATCC. Monoclonal antibodies 2B6,
8B5, and CB3.1 were humanized (IgG1) by complementaritydetermining region grafting. Monoclonal antibodies 2.4G2 and
HM79 were chimerized to mouse IgG1, and mAb 4-4-20 was
chimerized to a human IgG1.
DART molecules. DART molecules are bispecific diabodies (11) in which the 2 chains are linked via a disulfide bond
formed between Cys residues at the C-terminus of the 2
respective chains (Figure 1a). In some cases, we introduced
domains prior to the C-terminal cysteines to drive assembly of
the desired heterodimeric form. Control (CONTR) DART
molecules contained a 4-4-20 (antifluorescein) Fv region as the
irrelevant arm. The CD32B(2B6) ⫻ CD79B, CD32B(2B6) ⫻
CONTR, CD32B(8B5) ⫻ CONTR, and CONTR ⫻ CD79B
DART molecules were engineered with an E-coil domain
(chain A) and a K-coil domain (chain B). A second
CD32B(2B6) ⫻ CONTR DART molecule was constructed
with chain A appended with FNRGEC (residues 209–214 of
human C␬) and chain B appended with VEPKSC (residues
216–221 of human C␥1). CD32B(8B5) ⫻ CD79B and murine
(m) CD32 ⫻ mCD79B were built with both chains terminating
with LGGC. CD32B(2B6) ⫻ CONTR, CD32B(8B5) ⫻
CONTR, CONTR ⫻ CD79B, and CD32B(2B6) ⫻ CD79B
DART molecules were expressed by transient transfection of
the 2 chains, each driven by cytomegalovirus immediate early
promoter, into HEK 293H cells. CD32B(8B5) ⫻ CD79B and
mCD32 ⫻ mCD79B DART molecules were expressed using
the GS System (Lonza) in CHO-S cells.
All DART molecules were purified by antigen affinity
chromatography using Sepharose 4B (GE Healthcare) coupled
to one of the DART antigens: fluorescein-conjugated IgG,
human soluble (hs)CD79A/B-Fc-Agly (a fusion protein linking
the CD79A and CD79B ectodomains to an aglycosyl [N297Q]
human IgG1 domain), or a mouse soluble CD16-Fc fusion
protein (msCD16-Fc-Agly, the mouse CD16 ectodomain fused
to mouse N297Q IgG1 Fc; note that 2.4G2 cross-reacts with
mCD16). This was followed by size-exclusion chromatography.
The resulting proteins were ⬎95% monomeric heterodimer
with ⬍5% high molecular weight species. Dual antigen recognition was tested by enzyme-linked immunosorbent assay
(ELISA) using immobilized hsCD79A/B-Fc-Agly for capture
and biotinylated hsCD32B (hsCD32B-Fc) (9) plus horseradish
peroxidase (HRP)–conjugated streptavidin for detection. Data
were analyzed by using Prism software (GraphPad Software).
Surface plasmon resonance (SPR) analysis. Binding of
DART molecules to hsCD32B and hsCD79A/B was analyzed
by SPR using a BIAcore 3000 biosensor. Human soluble
CD32B or hsCD79A/B was immobilized on the CM-5 sensor
chip using the amine coupling kit (GE Healthcare). Briefly, the
carboxyl groups on the sensor chip surface were activated, and
the antigen was then injected over the activated CM-5 surface
in 10 mM sodium acetate, pH 5.0, at a flow rate of 5 ␮l/minute
until an immobilization level of ⬃500 reference units was
reached, followed by 1M ethanolamine for surface deactivation. Binding experiments were performed in HEPES buffered
saline with EDTA and Surfactant P20 (BIAcore). DART
molecules were injected in duplicate at concentrations of 0,
6.25, 12.5, 25, 50, and 100 nM and at a flow rate of 30 ␮l/minute
for 120 seconds, followed by a dissociation time of 180 seconds.
Data were analyzed using BIAevaluation 3.1 software (BIAcore) after subtraction of the buffer sensogram. Kinetic constants, ka and kd, were estimated by global fitting analysis of
the association/dissociation curves to the 1:1 Langmuir interaction model, with the equilibrium dissociation constant (KD)
calculated as KD ⫽ kd/ka.
Flow cytometric analysis. For the detection of DART
molecule–mediated cell clustering, CHO-CD32B cells,
RAMOS cells (CD79B⫹) (a B cell lymphoma line), Daudi
cells (CD32B⫹CD79B⫹) (a Burkitt’s lymphoma cell line), or
purified human B cells were labeled with CellTrace 5,6carboxyfluorescein succinimidyl ester (Invitrogen) or with
PKH26 (Sigma) following the manufacturers’ instructions.
Cells (5 ⫻ 106/ml) were resuspended in phosphate buffered
Figure 1. Structure, biochemical characterization, and binding properties of CD32B ⫻ CD79B dual-affinity retargeting (DART) molecules. a,
Schematic representation of linear sequences assembled into covalently linked DART molecules. The 2 polypeptide chains are depicted with an
N-terminal VL domain, an amino acid linker, a mismatched C-terminal VH domain, and a Cys residue for covalent linkage. b, Analysis of purified
DART molecules by sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Lane 1,
CD32B(2B6) ⫻ CONTR (control); lane 2, CD32B(8B5) ⫻ CONTR; lane 3, CONTR ⫻ CD79B; lane 4, CD32B(2B6) ⫻ CD79B; lane 5,
CD32B(8B5) ⫻ CD79B; lane 6, murine (m) CD32 ⫻ mCD79B. c, Kinetic parameters obtained by surface plasmon resonance analysis of DART
molecules binding to the corresponding immobilized receptor ectodomains. Binding constants were measured in 2 independent experiments. The
difference between measurements was ⬍20%. d, Results of CD32B-CD79B bispecific enzyme-linked immunosorbent assay. Data were fitted to a
sigmoidal dose-response curve by using Prism software (GraphPad Software) and are representative of 3 independent experiments. Values are the
mean ⫾ SEM. RLU ⫽ relative luminescence units.
saline, and pairs were combined in a 1:1 ratio in the presence
of 2 nM DART molecules for 1 hour at room temperature.
Cells were analyzed by flow cytometry on a FACSCalibur flow
cytometer (Becton Dickinson).
Expression of CD32B and CD79B was detected with
Alexa 488–conjugated 2B6 and phycoerythrin-conjugated
CB3.1, respectively. Cell-bound DART molecules, used at a
concentration of 1 ␮g/ml, were detected using a polyclonal
rabbit antibody directed against the E-coil/K-coil domain
located within the DART molecule heterodimerization domain (anti-DART linker) followed by detection with 0.2 ␮g/ml
of allophycocyanin-conjugated F(ab⬘)2 fragments of donkey
anti-rabbit IgG (Jackson ImmunoResearch).
Western blotting. CD32B immunoprecipitation from
cleared lysates (from 1 ⫻ 107 cells) was performed with 2B6
prebound to protein A/G beads. Washed immunoprecipitates
or whole-cell lysates (from 1 ⫻ 106 cells) were resolved by
9–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes,
blotted with 1 ␮g/ml of anti-CD32B (3H7), anti–phosphoCD32B, anti–phospho-Syk, anti–phospho–ZAP-70, anti–
phospho-Akt, anti–phospho–ERK-1/ERK-2 (all from Cell Signaling Technology), anti–phospho-SHIP (Stem Cell
Technology), or anti-GAPDH (Sigma), and visualized using
enhanced chemiluminescence.
Intracellular calcium mobilization. B cells were loaded
for 30 minutes at 37°C with 4 ␮M Fluo-4AM (Invitrogen) in
Hanks’ balanced salt solution containing 0.02% Pluronic F-127
and 1 mM probenecid (Invitrogen). Changes in intracellular
Ca2⫹ levels were recorded as Fluo-4 fluorescence emission in
fluorescence channel 1 of a FACSCalibur instrument (BD
Bioscience) running Flow-Jo software (Tree Star).
B cell proliferation and Ig production. Human or
mouse B cells (5 ⫻ 104/ml) were seeded in complete RPMI
1640 medium in 96-well plates (100 ␮l/well). For human B cell
proliferation assay, cells either were activated with 2.5 ␮g/ml of
mouse monoclonal anti-human ␮-chain (Southern Biotechnology) and 10 ␮g/ml of goat anti-mouse antibodies in the
presence of increasing concentrations of DART molecules or
were treated with DART molecules or with control antibodies
alone. For mouse B cell proliferation assay, cells were activated with goat anti-mouse ␮-chain (30 ␮g/ml) in the presence
of increasing concentrations of DART molecules or control
antibodies. One microcurie per well of 3H-thymidine was
added to the wells after 48 hours for a 16–18-hour pulse before
harvesting. Incorporation of 3H-thymidine was measured by
liquid scintillation counting. For Ig measurements, resting
human B cells were left untreated or were activated with goat
anti-human ␮-chain (30 ␮g/ml) in the presence of 20 nM
DART molecules or control antibodies. Cells were incubated
for 72 hours at 37°C, supernatants were harvested, and secreted Ig were measured by ELISA. Briefly, microtiter plates
were coated with 2 ␮g/ml of goat anti-human Ig to capture the
secreted Ig. Bound Ig were detected using HRP-conjugated
goat anti-human ␬-chain antibody (Invitrogen) with subsequent colorimetric development.
Pharmacokinetic analysis. The level of mCD32 ⫻
mCD79B in mouse serum was determined in a bispecific
ELISA using msCD79A/B-Fc-Agly for capture and msCD16Fc-Agly-biotin (2.4G2 binds mouse Fc ␥ RIII) plus
streptavidin–alkaline phosphatase for detection. Concentrations of mCD32 ⫻ mCD79B were calculated using a 4-parameter algorithm fitting the data against a standard curve, and
pharmacokinetic calculations were performed using WinNonlin Professional 5.1 (Pharsight). Parameters were determined
by noncompartmental analysis based on an intravenous (IV)
injection model (Model 201) and the linear trapezoidal
Mouse model of CIA. The mouse strains used in the
CIA study were generated from CD16–/–, hCD32A-transgenic,
and hCD16A-transgenic mice obtained from Dr. Jeffrey
Ravetch (Rockefeller University, New York, NY). Arthritis in
DBA/1 mouse strains was induced with a single intradermal
(ID) injection of 200 ␮l of a 1-mg/ml suspension of bovine type
II collagen in modified Freund’s complete adjuvant (CFA)
(both from Chondrex), and, when indicated, the animals were
immunized with 2 additional ID injections of bovine type II
collagen in Freund’s incomplete adjuvant (Sigma) on days 21
and 42. Disease severity was scored as footpad swelling
recorded with an electronic caliper 3 times a week. Mice from
the same litter were randomly assigned to treatment and
control groups and were generated from ⬎10 backcrosses. All
experiments were conducted under the approval of the Institutional Animal Care and Use Committee.
Construction, characterization, and binding
properties of CD32B- and CD79B-specific DART molecules. CD32B ⫻ CD79B DART molecules were constructed by using humanized variable chains from 2
anti-CD32B mAb and those from a single anti-BCR Fv
region (Figures 1a and b). One DART molecule incorporated the humanized version of 2B6, a high-affinity
anti-human CD32B mAb capable of blocking immune
complex binding to the inhibitory receptor (9). A second
DART molecule was built on humanized 8B5, an antibody that binds a linear epitope outside of the IgG
binding site of CD32B and does not compete for immune complex binding. Control DART molecules were
built by substituting one or the other arm with an
antifluorescein Fv region from mAb 4-4-20. Each purified DART molecule migrates as a single species under
nonreducing conditions and can be resolved into its
constituent 2 chains of the predicted molecular mass
(based on its Fv regions and linker components) under
reducing conditions (Figure 1b).
The affinity of the CD32B arm for its antigen was
comparable with that of the CD79B arm, as in the case
of the CD32B(8B5) ⫻ CD79B DART molecule, while
the CD32B(2B6)-based DART molecules showed
greater affinity for CD32B than the CD32B(8B5)-based
versions, as anticipated (Figure 1c). In both cases, the
on-rate for CD32B binding was faster than that for
CD79B binding. Both CD32B ⫻ CD79B DART molecules interacted with their antigens, as shown in a
dual-specificity ELISA in which soluble recombinant
CD32B was immobilized on the plate and soluble
CD79B was used as detecting reagent for bound DART
molecules (Figure 1d). The shift in the CD32B(8B5) ⫻
CD79B DART molecule binding curve compared with
that of the CD32B(2B6) ⫻ CD79B DART molecule was
consistent with the lower affinity of its CD32B arm.
While control DART molecules reacting with
only 1 of the 2 antigens went undetected in this ELISA
format, fluorescence-activated cell sorting (FACS) analysis demonstrated binding to the corresponding antigens on live cells for all DART molecules (Figure 2a).
CHO cells transfected with the human inhibitory receptor (CHO-CD32B) were used to detect CD32B-specific
binding, while RAMOS cells, a B cell lymphoma line
that expresses undetectable levels of CD32B, were used
to detect CD79B-specific binding. Daudi cells, a Burkitt’s lymphoma cell line that expresses both CD32B and
CD79B, were used as double-positive cells, while nontransfected CHO cells represented the negative control.
Bound DART molecules were detected by using a
polyclonal rabbit antibody raised against a linker region
shared by DART molecules. CHO-CD32B cells were
stained exclusively by DART molecules that included
the anti-CD32B specificity (CD32B ⫻ CONTR and
CD32B ⫻ CD79B); conversely, only anti-CD79B–
bearing DART molecules (CONTR ⫻ CD79B and
CD32B ⫻ CD79B) bound RAMOS cells, while all
DART molecules bound Daudi cells, confirming that
each molecule performed as expected.
A critical requirement for signal modulation via
inhibitory coupling is that both receptors be coligated on
the same cell rather than on adjacent cells; consistent
with this notion, independent crosslinking of CD32B,
even if simultaneous with BCR activation mediated by
CD79B, was found to be insufficient to trigger negative
signaling (further information is available at http:// Given
that DART molecules are monovalent binders for each
target, we anticipated that avidity would favor binding
to cells coexpressing both antigens (cis-binding mode)
rather than binding either antigen on separate cells
(trans-binding mode).
Figure 2. Cell binding properties of CD32B ⫻ CD79B DART molecules. a, Left, CD32B and CD79B were detected on the indicated cell lines (see
Materials and Methods) expressing CD32B, CD79B, or both CD32B and CD79B, using Alexa 488–conjugated 2B6 and phycoerythrin-conjugated
CB3.1, respectively. Right, DART molecules were detected using the anti-DART linker rabbit antibody and allophycocyanin-conjugated F(ab⬘)2
fragments of donkey anti-rabbit IgG. b, Cells were labeled with 5,6-carboxyfluorescein succinimidyl ester (CFSE) or with PKH26, and pairs were
combined as indicated in the leftmost diagram in a 1:1 ratio in the presence of the indicated DART molecules. Cell–cell clustering was detected in
the upper right quadrants, and the results are representative of at least 3 experiments. PBS ⫽ phosphate buffered saline (see Figure 1 for other
Detection of DART molecule–mediated cell
clustering of cells bearing one, the other, or both specificities with different combinations of ligands was performed by FACS analysis of mixtures of cells loaded with
2 independent fluorochromes. When mixtures of CHOCD32B cells and RAMOS cells (CD79B⫹) were used as
targets, the CD32B ⫻ CD79B DART molecule mediated at most 26% clustering of the 2 cell types under
optimal conditions (2 nM) (Figure 2b), while no clustering was observed with control DART molecules (Figure
2b). In contrast, when Daudi cells (CD32B⫹CD79B⫹)
were used as targets, the CD32B ⫻ CD79B DART
molecule did not exhibit trans binding activity. Furthermore, the CD32B ⫻ CD79B DART molecule did not
mediate trans binding even when added to a mixture of
Daudi and CHO-CD32B or RAMOS cells, indicating
that the cis-bound DART molecule is not available to
mediate heterologous cell clustering. The preferential
cis-binding mode of the CD32B ⫻ CD79B DART
molecule also occurred with human peripheral blood B
cells (Figure 2b).
Treatment with a CD32B ⴛ CD79B DART molecule disrupts BCR-induced calcium mobilization and
signal transduction. CD32B inhibitory function is the
result of the phosphorylation of an immunoreceptor
tyrosine–based inhibition motif (ITIM) in its intracellular tail (12–15). Treatment of human peripheral blood
B lymphocytes with the CD32B(8B5) ⫻ CD79B DART
molecule resulted in the phosphorylation of the CD32B
ITIM motif (Tyr292) in a B cell activation–dependent
manner (aggregation of cell surface IgM by goat antihuman ␮-chain was used as a surrogate for antigenmediated BCR engagement) (Figure 3a, left panels),
confirming that CD32B phosphorylation is dependent
upon the activation of BCR-linked kinases (16). Importantly, the DART molecule did not trigger significant
Figure 3. CD32B ⫻ CD79B DART molecules activate an inhibitory pathway in human B cells. a, Left, Lysates from B cells treated with goat
anti-human ␮-chain (GAH␮; anti-␮) alone or in combination with CD32B(8B5) ⫻ CD79B DART molecules for 10 minutes were subjected to
immunoprecipitation (IP) with anti-CD32B (clone 2B6), resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, immunoblotted
with pCD32B Y292 (top), and stripped and reprobed with anti-CD32B (clone 3H7) (bottom). Right, Western blot (WB) analysis of whole-cell lysates
from purified B cells that were treated as shown at left at different time points. The blots were probed with pCD32B Y292 (top) and anti–pSHIP1
Y1020 (middle). Equivalent loading was confirmed by stripping and reprobing with anti-GAPDH (bottom). b, Ca2⫹ mobilization was measured as
mean fluorescence emission of Fluo-4AM–loaded B lymphocytes treated with phosphate buffered saline (PBS) or 1 ␮g/ml of the indicated DART
molecules and stimulated for 140 seconds with 30 ␮g/ml of goat anti-human ␮-chain followed by 1 ␮M ionomycin for an additional 140 seconds. The
ionomycin response indicates equivalent loading. c and d, Whole-cell lysates from human B cells treated as described and for the indicated time
intervals were immunoblotted with anti–pAkt S473 (c) or with anti–pZAP-70 Y319, anti–pSyk Y525/526, or anti–pERK-1/2 T202/Y204 (d). Immunoblots
were stripped and reprobed with anti-GAPDH as a loading control. All results are representative of 3 independent experiments. See Figure 1 for
other definitions.
ITIM phosphorylation in the absence of BCR activation;
therefore, coligation of CD79B with CD32B by a bispecific ligand whose arms are each functionally monovalent for their respective antigens is insufficient to trigger
a signal. The phosphorylated CD32B ITIM recruits the
cytoplasmic SHIP1, which in turn acts as a kinase
substrate (13).
Consistent with CD32B phosphorylation, phosphorylation of SHIP1 Tyr1020, a docking site for the Ras
inhibitory adaptor, DOK1, was observed only in the
presence of the CD32B ⫻ CD79B DART molecule and
exclusively in response to BCR stimulation (Figure 3a,
right panels). SHIP1 counteracts phosphatidylinositol
3-kinase (PI 3-kinase) signaling by dephosphorylating
phosphatidylinositol 3,4,5-trisphosphate, a phospholipid
that enables membrane translocation of proteins possessing pleckstrin homology domains. One such molecule, phospholipase C␥, generates a second messenger
that controls calcium mobilization (17). Consistent with
SHIP1 activation, both CD32B ⫻ CD79B DART molecules attenuated BCR-induced Ca2⫹ mobilization (Figure 3b), while control DART molecules had no effect.
Treatment with a CD32B ⫻ CD79B DART molecule
did not affect BCR-induced phosphorylation of ZAP-70
or Syk (Figure 3d), 2 CD79B proximal kinases. In
contrast, the phosphorylation of ERK-1/2 and Akt, 2
molecules downstream of Ca2⫹ mobilization and PI
3-kinase activation, was reduced compared with that of
samples activated in the absence of the DART molecule
(Figures 3c and d). These data indicate that by recruiting
Figure 4. B cell treatment with CD32B ⫻ CD79B DART molecules inhibits B cell receptor–induced proliferation and Ig secretion. a, Purified
human B cells were left untreated (phosphate buffered saline [PBS]) (top) or stimulated with mouse monoclonal anti-human ␮-chain and goat
anti-mouse antibodies (MAH␮ ⫹ GAM) together with increasing concentrations of the indicated DART molecules (top) or with increasing
concentrations of the indicated DART molecules and chimeric (Ch) monoclonal antibodies (mAb) (bottom). Values are the mean ⫾ SEM
H-thymidine (3H-TdR) incorporation. b, The indicated DART molecules (40 nM ⫽ 1 ␮g/ml) were added to purified B cells at the indicated time
points before or after stimulation. After 48 hours, proliferation was assessed as 3H-thymidine incorporation during an 18-hour pulse. Values are the
mean and SEM of triplicate cultures. c, Purified B cells were left untreated (PBS) or incubated for 72 hours with 30 ␮g/ml of F(ab⬘)2 fragments of
goat anti-human ␮-chain (GAH␮) in the presence of 20 nM of the indicated DART molecules or mAb (mouse mAb were used in the assay to avoid
interference with the detection assay). Total Ig secretion in the supernatants was detected by enzyme-linked immunosorbent assay. Values are the
mean and SEM of triplicate cultures. All results are representative of at least 3 independent experiments. See Figure 1 for other definitions.
CD32B within the activation complex, treatment with a
CD32B ⫻ CD79B DART molecule biases B cells toward
inhibitory coupling of BCR activation.
Treatment with CD32B ⴛ CD79B DART molecules inhibits BCR-induced proliferation and Ig production. Treatment with CD32B ⫻ CD79B DART molecules inhibited BCR-induced proliferation of human
peripheral blood B cells in a dose-dependent manner
(Figure 4a, upper and lower panels). Proliferation was
unaffected by control DART molecules or the antiCD79B or anti-CD32B mAb. Omitting the anti–␮-chain
mAb resulted in no proliferation; under these conditions, the addition of CD32B ⫻ CD79B DART molecules did not result in detectable activation (Figure 4a,
upper panel). Delaying DART molecule addition to
prestimulated cultures for up to 24 hours resulted in
nearly maximal attenuation of proliferation (Figure 4b,
upper and lower panels). B cells treated with CD32B ⫻
CD79B DART molecules also showed greatly diminished Ig secretion upon stimulation with goat antihuman ␮-chain compared with cells treated with antibodies of the individual specificities (Figure 4c, upper
and lower panels). In multiple experiments, the 2B6based DART molecule exhibited higher potency than
the 8B5-based DART molecule, consistent with the
differential affinity for the inhibitory receptor (Figure
1c). Inhibition was not associated with increased cell
death or apoptosis (data not shown). These data show
that CD32B ⫻ CD79B DART molecule–mediated
coligation of the activating and inhibitory receptors
biased the cells toward hyporesponsiveness via negative
Table 1. Role of mCD16 and the hCD16A or the hCD32A activating
Fc␥ receptor transgene in the induction of CIA in DBA/1 mice*
Mouse line
hCD16A⫹/⫹ DBA/1†
hCD32A⫹/⫹ DBA/1‡
hCD16A⫹/⫹ hCD32A⫹/⫹ DBA/1‡
* Arthritis was induced and assessed in mice as described in Materials
and Methods. mCD16 ⫽ murine CD16; hCD16A ⫽ human CD16A;
CIA ⫽ collagen-induced arthritis.
† Animals received 3 immunizations with bovine type II collagen.
‡ Animals received 1 immunization with bovine type II collagen.
Treatment with CD32B ⴛ CD79B DART molecules reduces disease severity in a mouse model of CIA.
To determine whether a CD32B ⫻ CD79B DART
molecule–based intervention could control autoimmunity, we engineered a surrogate molecule directed
against murine CD32B and CD79B, since the antihuman CD32B and anti-human CD79B mAb do not
cross-react with the corresponding mouse antigens (data
not shown). Mice express only the inhibitory form of
Fc␥RII (mCD32), which is homologous to human
CD32B. As in humans, mouse B lymphocytes express
Figure 5. A mouse-specific CD32 ⫻ CD79B DART molecule reduces disease severity of murine collagen-induced arthritis. a, Proliferation of
purified splenic mCD16–/– hCD32A⫹/⫹ DBA/1 mouse B cells stimulated in the presence of increasing concentrations of mCD32 ⫻ mCD79B DART
molecules or anti-mCD32 or anti-mCD79B monoclonal antibodies (mAb). The inset shows mCD32 and mCD79B expression in the B cell population
used in the assay. Values are the mean ⫾ SEM 3H-thymidine (3H-TdR) incorporation. Data are representative of 3 separate experiments. b,
Pharmacokinetics analysis of mCD32 ⫻ mCD79B DART molecules in mCD16–/– C57BL/6 mice following a single intravenous injection at 2 ␮g/gm.
Blood was collected from 5 male and 5 female mice at the indicated time points, and mCD32 ⫻ mCD79B DART molecule serum levels were
determined by bispecific enzyme-linked immunosorbent assay. Values are the mean ⫾ SEM. Pharmacokinetic (PK) parameters are shown in the
inset. T ⁄ ⫽ half-life; Tmax ⫽ time to maximum concentration of drug in serum; AUC ⫽ area under the curve; Cmax ⫽ maximum concentration. c
and d, Induction and recording of arthritis in female mCD16–/– hCD32A⫹⫹ DBA/1 mice as described in Materials and Methods. Treated mice
received 2 ␮g/gm of mCD32 ⫻ mCD79B DART molecules intravenously daily for 5 consecutive days (arrows). Values are the mean ⫾ SEM of the
average of all 4 paws of 11 phosphate buffered saline–treated mice (untreated) or 12 DART molecule–treated mice per group. The difference
between untreated mice and mice treated on days 0–4 (c) or on days 14–18 (d) was significant (P ⬍ 0.05 by analysis of variance followed by paired
2-tailed t-test). The difference in d remained significant after Bonferroni correction for multiple comparisons. See Figure 1 for other definitions.
mCD32, where it functions as a negative regulator of
BCR-induced activation. The mAb 2.4G2 is a rat mAb
that binds mCD32; however, 2.4G2 also reacts with
mCD16 (Fc␥RIII), an activating Fc receptor (18). To
eliminate mCD16 reactivity, a confounding factor, mice
deficient for mCD16 were generated on the autoimmune DBA/1 background (mCD16–/– DBA/1 mice) to
generate a CIA model that could be used to test the
therapeutic effect of DART molecules, since this model
has been shown to be dependent on B cells, because B
cell–deficient mice do not develop arthritis (19). Contrary to what occurs in wild-type DBA/1 mice, however,
collagen immunization of these mice did not result in
arthritis (20) (Table 1), expanding the observation of a
previous study that showed that DBA/1 mice lacking the
activating Fc␥R common ␥-chain are resistant to CIA
In order to reconstitute CIA in mCD16–/– DBA/1
mice, the human CD16A or the human CD32A activating Fc␥R was incorporated as a transgene, resulting
in mCD16–/– hCD32A⫹/⫹ DBA/1 mice, mCD16–/–
hCD16A⫹/⫹ DBA/1 mice, and mCD16–/– hCD16A⫹/⫹
hCD32A⫹/⫹ DBA/1 mice. The human CD16A was
unable to reconstitute CIA in mCD16–/– DBA/1 mice
even after 3 injections of collagen; in contrast, when the
hCD32A transgene was incorporated in mCD16–/–
DBA/1 mice or in mCD16–/– hCD16A⫹/⫹ DBA/1 mice,
severe arthritis was observed after a single injection with
bovine type II collagen (Table 1). Human CD16A does
not bind murine IgG (22), and this characteristic may
explain the inability to reconstitute CIA in mCD16–/–
DBA/1 mice. The human activating CD32A receptor
binds murine IgG (23) (data not shown), and it can
substitute for the mouse CD16 in the mCD16–/– DBA/1
The anti-CD79B arm of the mCD32 ⫻ mCD79B
DART molecule was engineered from HM79, a hamster
anti-mouse CD79B (24). The DART molecule was
qualified for performance characteristics consistent with
those of its human-reactive counterpart. These included
the ability of the mCD32 ⫻ mCD79B DART molecule
to inhibit BCR-induced proliferation of murine splenic
B cells (Figure 5a) and a preferential cis-binding modality with double-positive cells (data not shown). Pharmacokinetic analysis following a single IV injection of
mCD32 ⫻ mCD79B DART molecules in mCD16–/–
C57BL/6 mice showed single elimination kinetics with a
serum half-life of 9.1 hours (Figure 5b).
To ascertain the DART molecule therapeutic
activity, arthritis was induced in female mCD16–/–
hCD32A⫹/⫹ DBA/1 mice with an ID injection of bovine
type II collagen in modified CFA on day 0. Approxi-
mately 2 weeks after the induction, untreated mice
developed signs of arthritis (inflamed paws and reduced
mobility) that were associated with increased paw thickness (Figures 5c and d). Mice that received 2 ␮g/gm of
mCD32 ⫻ mCD79B DART molecules IV daily for 5
consecutive days starting on day 0 (Figure 5c) showed
delayed arthritis onset. More importantly, mice treated
with 2 ␮g/gm of mCD32 ⫻ mCD79B DART molecules
IV for 5 consecutive days starting on day 14 after
induction, which is around the time of disease onset,
showed disease regression with diminished disease severity compared with untreated mice (Figure 5d). In a
separate experiment, a DART molecule reacting only
with CD32B failed to ameliorate disease (data not
shown). These data indicate that treatment with an
inhibitory DART molecule capable of coligating CD32B
and the BCR may be used to control autoimmune
The physiologic role of inhibitory receptors in
limiting cell activation makes them appealing targets for
controlling pathogenic processes underlying autoimmune diseases. In B lymphocytes, the antigen itself
serves as a scaffold for the formation of an immune
complex that can link the stimulatory BCR and the
inhibitory Fc␥R (25). We transduced this paradigm into
a tool with pharmacologic potential by engineering a
new class of bispecific diabodies, DART molecules, with
specificities that directly ligate inhibitory and stimulatory receptors on the B cell surface. Treatment of B
lymphocytes with a DART molecule recognizing CD32B
and CD79B negatively regulated B cell activation with
disruption of signal transduction and attenuation of
BCR-induced calcium mobilization, proliferation, and Ig
secretion. Furthermore, a mouse-specific CD32B ⫻
CD79B DART molecule demonstrated activity in a
murine arthritis model. To our knowledge, this is the
first example of pharmacologic manipulation of inhibitory signaling in autoimmunity.
Attempts to manipulate CD32B negative signaling have previously focused on approaches with a common Fc domain–based strategy. To attenuate basophil
and mast cell antigenic responses (26,27), proteins were
engineered with an IgE Fc (the GE2 molecule) or with
FelD, a cat allergen (the GFD molecule) fused to an IgG
Fc domain. More recently, Chu et al (28) developed an
anti-CD19 antibody with an Fc domain engineered with
increased affinity for CD32B as a tool to coengage this
receptor. Compared with its wild-type Fc domain counterpart, treatment with this antibody resulted in apopto-
sis of primary human B cells. Either strategy corecruited
CD32B to the activating receptor by means of an IgG1
Fc domain. This approach is limited for applications in
vivo, since IgG1 Fc-based fusion proteins will likely bind
FcR other than CD32B. Even in the case of the Fcengineered anti-CD19, binding to FcR other than
CD32B is likely to occur, since the mutant demonstrated
affinities for CD64 (Fc␥RI) and the H131 polymorphism
of CD32A (Fc␥RIIA) that were identical to those of a
native IgG1 (28). Furthermore, the interaction with the
inhibitory receptor in an Fc domain–based strategy will
be driven by proximity via primary binding to the
activating receptors.
By engineering our scaffold with anti-CD32B
mAb, we have obviated the limitations of Fc domain–
based strategies together with increasing the specificity
for the intended inhibitory target. Furthermore, the
relative affinity of either arm of the scaffold can be
selected or engineered in ways to introduce bias for one
or the other antigen. To favor inhibitory receptor recognition, CD32B arms were engineered with faster
on-rates and affinities equal to or greater than those of
the CD79B arm, properties expected to favor primary
recognition of CD32B. Furthermore, in selecting 8B5 as
an alternate component of the CD32B ⫻ CD79B DART
molecule, we have shown the feasibility of employing a
binding entity that does not block the inhibitory receptor, which may be preferable to avoid interference with
physiologic inhibitory mechanisms mediated through
CD32B. Future studies will address the ability of
CD32B ⫻ CD79B DART molecules to inhibit B cell
responses in patients with autoimmune diseases, including ascertaining the inhibitory activity in relation to the
CD32B Ile232Thr signaling polymorphisms (29).
Several reports have suggested that B cell apoptosis can be triggered by homoligation of CD32B.
Accounting for up to 10% apoptosis of ex vivo mouse
plasma cells or in vitro plasmablasts (30) or for up to
⬃15% apoptosis of splenic mouse B cells (31), this
phenomenon is physiologically important but of limited
pharmacologic magnitude. Following treatment with antiCD32B mAb and secondary crosslinking, we have observed apoptosis of human peripheral blood B cells of a
magnitude similar to that reported (⬃15%), although no
effect on BCR-induced proliferation was seen (data not
shown). Conversely, even extensive CD32B crosslinking
in the absence of coligation with CD79B was ineffective
in attenuating B cell proliferation regardless of the
concomitant activation status (data not shown), and no
apoptosis was observed after CD32B ⫻ CD79B DART
molecule treatment irrespective of BCR activation (data
not shown). Therefore, simultaneous coengagement of
CD32B with the activating receptor is an absolute
requirement for B cell inhibition.
This requirement is driven by a molecular constraint exploited by the CD32B ⫻ CD79B DART
molecule—the need for CD32B to leverage a kinase
associated with the activating receptor in order to phosphorylate its intracellular tail, since the inhibitory receptor is not coupled to a kinase (14). CD32B inhibitory
function is therefore conditional on its proximity to, and
the activation status of, the tyrosine kinase–coupled
receptor. By loading the BCR complex with CD32B in
resting B cells, a CD32B ⫻ CD79B DART molecule
sensitizes the antigen receptor to deliver an inhibitory
signal if antigen recognition occurs. Furthermore, in
stimulated cells the inhibitory signal predominates over
the activation signal, effectively dampening ongoing
activation, as shown by inhibition of proliferation after
delayed DART molecule addition and the DART molecule’s ability to control ongoing inflammation in mouse
CIA, a model that displays immunologic and histopathologic similarities to rheumatoid arthritis and the severity
of which correlates with CD32B expression (32,33).
The exploitation of CD32B inhibition coupling by
DART molecules is defined by a unique set of features.
By ligating each component in an essentially monovalent
fashion, DART molecules appear to have no intrinsic
activation properties. Furthermore, these DART molecules function as activation-dependent inhibitors, exerting their activity only in the context of antigen receptor
signaling. CD32B-based DART proteins are simple to
engineer, capable of faithfully preserving the binding
properties of the parental Fv regions, and potent; these
characteristics make them suitable not only as investigational tools but also for pharmacologic applications.
We would like to thank Robert Whitener and Amanda
Zhang for technical assistance with antibody and DART
molecule purification, Weili Wang, Arin Whiddon, and Wanhua Yan for help with protein expression, Wenjun Zhang for in
vitro assays, and Shelley Butler for assistance with animal
modeling. We thank Jeffrey L. Nordstrom, Kathryn E. Stein,
and Timothy J. Mayer for discussion and critical review of the
manuscript and Melinda Hanson for editorial assistance.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Bonvini had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
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Acquisition of data. Veri, Burke, Huang, Li, Gorlatov, Tuaillon,
Rainey, Ciccarone, Zhang, Shah, Jin, Ning, Minor.
Analysis and interpretation of data. Veri, Tuaillon, Koenig, Johnson,
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