Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody.
ABSTRACT A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.
- [Show abstract] [Hide abstract]
ABSTRACT: Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.Nature Immunology 07/2014; 15(8):707-716. · 24.97 Impact Factor
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ABSTRACT: Monoclonal antibody (mAb) drugs that stimulate antitumor immunity are transforming cancer treatment but require optimization for maximum clinical impact. Here, we show that, unlike other immunoglobulin isotypes, human IgG2 (h2) imparts FcγR-independent agonistic activity to immune-stimulatory mAbs such as anti-CD40, -4-1BB, and -CD28. Activity is provided by a subfraction of h2, h2B, that is structurally constrained due its unique arrangement of hinge region disulfide bonds. Agonistic activity can be transferred from h2 to h1 by swapping their hinge and CH1 domains, and substitution of key hinge and CH1 cysteines generates homogenous h2 variants with distinct agonistic properties. This provides the exciting opportunity to engineer clinical reagents with defined therapeutic activity regardless of FcγR expression levels in the local microenvironment. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Cancer cell. 12/2014;
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ABSTRACT: Antibody agonists targeting tumor necrosis factor (TNF) superfamily receptors, including CD40, are being tested therapeutically as anticancer agents. Studies in mice have shown that anti-CD40 monoclonal antibody (mAb) requires Fc-receptor (FcR) engagement to activate antitumor immunity. In contrast, we have reported that clinically active anti-human CD40 mAb CP-870,893 does not require FcR crosslinking, a finding with translational implications.OncoImmunology 05/2014; 3(5):e28610. · 6.28 Impact Factor
The Journal of Immunology
Interaction with FcgRIIB Is Critical for the Agonistic Activity
of Anti-CD40 Monoclonal Antibody
Ann L. White,* H. T. Claude Chan,* Ali Roghanian,* Ruth R. French,* C. Ian Mockridge,*
Alison L. Tutt,* Sandra V. Dixon,* Daniel Ajona,* J. Sjef Verbeek,†Aymen Al-Shamkhani,*
Mark S. Cragg,* Stephen A. Beers,*,1and Martin J. Glennie*,1
A high activatory/inhibitory FcgR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack
cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcgR binding profiles of other anti-cancer mAb,
such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the
importance of isotype and FcgR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse
IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular
responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10–
100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcgR
knockout mice demonstrated a critical role for the inhibitory FcgRIIB in 3/23 activity, whereas activatory FcgR (FcgRI, -III, and
-IV) was dispensable. In vitro experiments established that the stimulatory effect of FcgRIIB was mediated through Ab cross-
linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcgR
provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcgRIIB in vivo reflects its cellular distribution
and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role
for the inhibitory FcgRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when
optimizing reagents for clinical use.The Journal of Immunology, 2011, 187: 1754–1763.
reagents function, it is agreed that interaction of the mAb Fc with
FcgR on immune effector cells is critical (3–6). Studies in pre-
clinical models show that binding to activatory FcgR promotes
cell killing by Ab-dependent cell-mediated cytotoxicity and phago-
cytosis, whereas interaction with the inhibitory FcgRII (FcgRII
or CD32 in mice, FcgRIIB or CD32B in humans; hereafter referred
to as FcgRIIB) is reported by some (3, 5, 7), but not all (8), to be
detrimental to activity. Thus, mAb, including mouse IgG2a and
human IgG1, with high activatory/inhibitory receptor binding (A/I)
ratios appear optimal (4, 7, 9). Genetic profiling studies also support
a critical role for activatory FcgR in mAb function in humans, as
individuals homozygous for allotypes of FcgRIIA (CD32A) or
FcgRIIIA (CD16A) with enhanced IgG Fc binding show better
clinical outcomes in response to therapy (10–13). Thus, a current
nti-cancer mAb, such as rituximab and alemtuzumab,
Although there is much debate about exactly how these
focus is mAb engineering to increase activatory FcgR binding and
A/I ratios (14, 15).
Other mechanisms, such as direct cell killing and complement-
mediated cytotoxicity, which doubtless play important roles with
different mAb (16, 17), may also influence isotype requirement.
This is exemplified by a recent study (18) that showed that the
in vivo tumoricidal activity of the anti-death receptor 5 (DR5)
mAb drozitumab was dependent upon FcgR interaction. However,
in this case, interaction was required for Ab cross-linking to
promote DR5-mediated apoptosis rather than for recruitment of
effector functions, and the effect could be mediated by either
activatory or inhibitory FcgR (18). Another class of mAb in de-
velopment as anticancer agents is immunostimulatory mAb as
exemplified by the anti–CTLA-4 mAb, ipilimumab, recently ap-
proved by the U.S. Food and Drug Administration for metastatic
melanoma (19). Immunostimulatory mAb bind either agonisti-
cally or antagonistically to receptors on immune effector cells and
provide therapy by stimulating immunity and potentially over-
coming tumor-induced immune tolerance (20–22). The isotype
requirements and the role of FcgR interaction for this type of mAb
have not been investigated but may also differ from those of
direct-targeting mAb, such as rituximab and alemtuzumab, that
rely on effector cell recruitment.
Our work has focused on immunostimulatory anti-CD40 mAb
(20, 23–25), which are also in clinical development (26, 27). Re-
agents targeting this molecule have been investigated for .20 y
and include both mAb and CD40L (20, 26). CD40 is a TNFR
superfamily member expressed on APC, such as B cells, macro-
phages, and dendritic cells (DC), as well as many nonimmune
cells and a wide range of tumors (28–30). Interaction with its
trimeric ligand on activated T cells results in APC activation,
required for the induction of adaptive immunity (28, 29). In pre-
clinical models, rat anti-mouse CD40 mAb show remarkable
*Division of Cancer Sciences, Faculty of Medicine, University of Southampton,
Southampton SO16 6YD, United Kingdom; and†Department of Human Genetics,
Leiden University Medical Center, Leiden 2333 ZA, The Netherlands
1S.A.B. and M.J.G. are the senior authors and contributed equally to this work.
Received for publication April 19, 2011. Accepted for publication June 12, 2011.
This work was supported by grants from the Tenovus Cancer Charity, Cancer Re-
search UK, and Leukaemia and Lymphoma Research.
Address correspondence and reprint requests to Prof. Martin J. Glennie and Dr. Ann
L. White, Tenovus Research Laboratory, Southampton General Hospital, Tremona
Road, Southampton, SO16 6YD, United Kingdom. E-mail addresses: firstname.lastname@example.org.
uk and email@example.com
Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; A/I, activatory/
inhibitory receptor binding; CHO, Chinese hamster ovary; DC, dendritic cells; DR5,
death receptor 5; IKK, IkB kinase; m1, mouse IgG1; m2a, mouse IgG2a; MHC II,
MHC class II; RU, resonance unit; WT, wild-type.
therapeutic activity in the treatment of CD40 positive B cell
lymphomas as well as various CD40-negative tumors (20, 24, 25).
In our studies of therapeutic mAb, their potency is unprecedented,
clearing bulk tumors from mice with established disease and
providing immunity to rechallenge (20, 24). To date, four anti-
CD40 mAb [CP-870,893 (31), SGN-40 (32), HCD122 (26, 33)],
and Chi LOB7-4 (34) have been investigated in phase I/II trials.
These reagents show diverse activities ranging from antagonist
(HCD122) to strong agonist (CP-870,893) (26). Currently, there is
no satisfactory explanation for this heterogeneity, with little evi-
dence for epitope specificity being the determining factor.
In this study, we investigated the effect of mAb isotype on
the immunostimulatory activity of anti-CD40. Previous preclinical
studies have focused on rat [3/23 and FGK45 (20, 22)] and hamster
[IC10 (35)] anti-CD40 mAb. To address the role of isotype and
to avoid possible interference from mouse anti-rat Ig responses,
we engineered the epitope-binding (variable) regions of 3/23 onto
mouse IgG1 (m1) or mouse IgG2a (m2a) constant regions. Im-
portantly, both mAb retained equivalent binding to CD40 and
were biologically active in vivo and in vitro. However, we dis-
covered profound and unexpected differences in their immunos-
timulatory properties. When used to stimulate immunity to OVA,
3/23 m1 promoted a dramatic increase in both humoral and cell-
mediated responses, whereas 3/23 m2a had almost no stimulatory
effect. Strikingly, we found that interaction with the normally in-
hibitory FcgRIIB was required for 3/23 m1 stimulatory activity.
These data not only indicate that isotype is critical for anti-CD40
mAb efficacy, but also that its requirements are entirely different
from those of direct-targeting mAb such as rituximab.
Materials and Methods
C57BL/6, OTII TCR transgenic, and RAG2/2mice were sourced from
Charles River Laboratories (Kent, U.K.). Other genetically altered strains
used were FcgRIIB2/2, FcRg2/2, CD402/2(from Prof. Caetano Reis e
Sousa, London, U.K.), C57BL/6-Tg(IghelMD4)4Ccg/J (MD4) (from
Richard Cornall, Oxford, U.K.), and OTI TCR transgenic C57BL/6 mice
(from Dr. Matthias Merkenschlager, Imperial College, London, U.K.).
Animals were bred and housed in a local animal facility and were used
at ∼8–12 wk of age. All experiments were carried out according to local
ethical committee guidelines under United Kingdom Home Office license
Abs and reagents
The following hybridomas were used: rat anti-CD40 (clone 3/23; rat IgG2a)
was originally a gift from Gerry Klaus (National Institute of Medical Re-
search, London, U.K.); anti-rat CD4 (OX68) was a gift from Neil Barclay
(Oxford University, Oxford, U.K.); and anti-mouse FcgRII/III (2.4G2), anti-
mouse MHC class II (MHC II; M5/114.15.2), anti-mouse CD80 (1610A1),
and anti-mouse CD86 (GL1) were from LGC (Teddington, U.K.). Anti-
mouse FcgRI (AT152), FcgRIIB (AT130 and AT128), FcgRIII (AT154),
and FcgRIV (CD16-2; AT137) were produced in-house (A.L. Tutt, S. James,
S. Dixon, M. Ashton-Key, R. French, J. Teeling, E. Williams, A. Roghanian,
C.I. Mockridge, S.A. Beers, M.S. Cragg, and M.J. Glennie, manuscript in
preparation). Purified IgGs were prepared as described (16). All prepara-
tions were endotoxin low (,1 ng endotoxin/mg) as determined using the
Endosafe-PTS portable test system (Charles River Laboratories).
Polyclonal rabbit anti-OVA was from Millipore (Watford, U.K.). Anti-
CD19–PE (clone 1D3) and anti-F4/80–allophycocyanin were from AbD
Serotec (Oxford, U.K.). Anti-mouse CD23-PE was from BD Biosciences.
For OTII cell staining, anti-mouse Va2–TcR:FITC (clone B20.1), anti-
mouse Vb5.1,5.2 TcR:PE (clone MR9-4, IgG1), and allophycocyanin-
labeled anti-CD4 (clone RM4-5; all from BD Biosciences) were used.
For OTI cell staining, allophycocyanin-labeled anti-CD8a (clone 53-6.7;
BD Biosciences) and PE-labeled SIINFEKL tetramers were used. Tet-
ramers were produced essentially as described (36) by the Cancer Research
UK/Experimental Cancer Medicine Centre Protein Core Facility (Cancer
Sciences Division, University of Southampton, Southampton, U.K.) with
the following modifications: solubilized inclusion bodies were refolded in
the presence of SIINFEKL peptide (Peptide Synthetics, Fareham, U.K.) at
∼15 mM peptide, 4 mM mouse b2-microglobulin, and 2 mM H-2Kb. PE-
labeled streptavidin MHC class I tetramers were purified further on a
Superdex TM 200 10/300GL gel filtration column in PBS, dialyzed against
16% glycerol in PBS, and stored in the presence of 0.5% BSA and 0.1%
sodium azide at 220˚C.
Chicken OVA was purchased from Sigma-Aldrich (Poole, U.K.).
Endotoxin-free OVA was from Profos (Regensberg, Germany). 3/23
[Fab9 3 OVA] derivatives, consisting of a single molecule of OVA chemi-
cally linked to one, two, or three Fab9 fragments, were prepared as pre-
viously described (37) and contained ,0.5 ng endotoxin/mg conjugate.
Production of 3/23 chimeric mAb
DNA encoding the 3/23 H and L chain V regions was amplified from 3/23
hybridoma cDNA by PCR using Pfu DNA polymerase (Promega). The
(Invitrogen, Paisley, U.K.) containing the m1 or m2a constant regions via
HindIII/SpeI sites (H chain) and HindIII/BSiWI sites (L chain). The two
chains were then further subcloned into the expression vector pEE14.1
(Lonza). For mAb production, expression vectors were transfected into
Chinese hamster ovary (CHO)-K1 cells using GenePorter (Genlantis, San
Diego, CA) and cells secreting the highest amount of engineered Ab se-
lected. Ab was purified from cell-culture media on a protein A-Sepharose
(Sigma-Aldrich) column. All preparations were endotoxin low (see above).
Cells were cultured at 37˚C in a humidified atmosphere under 5% CO2.
Mouse primary B cells and splenocytes were cultured in RPMI 1640
containing 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml
penicillin, 100 mg/ml streptomycin (all from Life Technologies, Invi-
trogen), and 50 mM 2-ME (Sigma-Aldrich). CHO cells were cultured in
GMEMS (First Link, Birmingham, U.K.) containing 5% dialyzed FCS, 2
mg/ml amphotericin B (Fungizone; Invitrogen), 100 U/ml penicillin, 100
mg/ml streptomycin, and 3 mM methionine sulfoxamine (Sigma-Aldrich).
Freestyle 293F cells (Life Technologies) were cultured according to the
manufacturer’s instructions. Mouse IIA1.6 B cell lymphoma cells trans-
fected with various forms of mouse FcgRIIB, some lacking intracellular
domains (A. Roghanian and M.S. Cragg, unpublished observations), were
maintained in RPMI 1640 as above, plus 1 mg/ml G418 (Geneticin; In-
Immunization and assessment of immune responses
Mice were immunized via tail vein injection in 200 ml saline as described
for individual experiments. Anti-OVA Ab levels in serum samples were
determined by ELISA as previously described (38). To analyze T cell
responses, mice were adoptively transferred with ∼1 3 106OVA-specific
CD8 (OTI) or CD4 (OTII) splenic T cells via tail vein injection the day
before immunization. Numbers of circulating OVA-specific T cells in
blood or in the spleen were determined by flow cytometry using an
FACSCalibur (BD Biosciences).
Bone marrow-derived macrophages (6) were plated into flat-bottomed 96-
well dishes (5 3 104per well) and left to adhere at 37˚C for 2 h. Mouse
splenic B cells were purified by negative selection (Miltenyi Biotec, Sur-
rey, U.K.) and labeled for 15 min at room temperature with 5 mM CFSE.
B cells were then coated with 3/23 m1 or m2a or isotype control mAb at 10
mg/ml for 20 min at room temperature, washed, and added to the mac-
rophages at a ratio of 5:1 B cells/macrophages. After 1 h at 37˚C, mac-
rophages were labeled with anti-F4/80–allophycocyanin (macrophage
marker) and the number of double-positive (CFSE+F4/80+) cells identified
by flow cytometry.
CD70 staining was performed as previously described (39). Briefly, 10-mm
frozen spleen sections were fixed in acetone, blocked with 5% normal goat
serum, and incubated with biotinylated rat anti-CD70 (BD Biosciences)
overnight at 4˚C. Tyramide signal amplification was used to enhance the
CD70 staining (TSA Kit No. 22; Invitrogen) followed by streptavidin-
conjugated Alexa Fluor 488 (Molecular Probes). Sections were mounted
in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA).
Images were collected on a Leica TCS SP2 confocal laser scanning mi-
croscope (Leica Microsystems) using argon (488 nm), green helium/neon
(543 nm), and red helium/neon (633 nm) lasers and a pinhole equivalent to
1 Airy disc. Image files (.tiff) were transferred to Adobe Photoshop CS2
The Journal of Immunology1755
B cell proliferation, activation, and survival
To analyze proliferation, total splenocytes or splenic B cells purified by
negative selection (Miltenyi Biotec) were plated into 96-well round-bottom
dishes at 1 3 105cells/well in a total volume of 200 ml containing various
concentrations of mAb as described for individual experiments. In some
cases, 1 3 105transfected cells (see below) were also added. Cells were
incubated at 37˚C for 5 d. During the final 16 h, (methyl-[3H]thymidine;
PerkinElmer, Cambridge, U.K.) was added (0.5 mCi/well). Cells were
harvested using a Packard Filtermate Harvester 96 (PerkinElmer) and
To analyze activation status, splenic B cells were purified and plated into
96-well dishes as described above. After 48 h at 37˚C, cells were harvested
and the expression of activation markers analyzed by flow cytometry.
Surviving B cells were assessed through exclusion of 7-aminoactinomycin
D (7AAD; BD Biosciences) staining.
For NF-kB signaling analysis, isolated wild-type (WT) C57BL/6 B cells
were plated in 12-well plates (∼2 3 106/ml/well) and stimulated with 10
mg/ml 3/23 m1 or m2a for 2, 5, 15, and 30 min at 37˚C. For caspase-8
analysis, isolated WT and FcgRIIB2/2splenic BALB/c B cells were
stimulated with 10 mg/ml mAb overnight at 37˚C. Cell lysates were pre-
pared for Western blotting as described (40) and 25–35 mg samples re-
solved by denaturing SDS-PAGE, transferred onto polyvinylidine difluoride
membranes, and then incubated with Abs to phospho-IkB kinase (IKK)
a/b (Ser176/180; clone 16A6), phospho–IkB-a (Ser32/36; clone 5A5),
anti-tubulin, b-actin (all from Cell Signaling Technologies), IkB-a (Santa
Cruz Biotechnology, Santa Cruz, CA), and caspase-8 (kindly provided
by Dr. Lorraine O’Reilly, Walter and Eliza Hall Institute, Melbourne,
Australia) followed by HRP-conjugated secondary Ab. Blots were visu-
alized using ECL reagents (GE Healthcare, Buckinghamshire, U.K.).
DNA fragments encoding mouse FcgRs were amplified from mouse
splenocytes and peripheral blood cDNA. After verifying their sequences,
they were subcloned into the expression vector pCIpuro via EcoRI and
NotI sites. The backbone vector pCI-puro was made by replacing the
neomycin resistance cassette from pCI-neo (Clontech, Basingstoke, U.K.)
with the puromycin resistance gene from pPUR (Clontech). Mouse g-chain
was subcloned into the expression vector pcDNA3 (Invitrogen) via HindIII
and EcoRI sites. Plasmids were transfected into 293F cells using the
293fectin transfection reagent (Invitrogen). A total of 10 mg DNAwas used
with 1 3 107cells. For the activatory receptors (I, III, and IV), 5 mg ap-
propriate a-chain and 5 mg g-chain DNA was used. FcgR expression was
assessed by flow cytometry 72 h later.
Surface plasmon resonance
A BiacoreT100(Biacore)wasusedto assaythe interactionbetweensoluble
FcgR and 3/23 m1 or m2a. Abs or BSA as a control were immobilized at
high (15,000 resonance units [RU]) and low (1,000 RU) densities to the
flow cells of CM5 sensor chips (Biacore) by standard amine coupling
according to the manufacturer’s instructions. Soluble Fc receptors (FcgRI,
-IIB, -III, and -IV; R&D Systems, Abingdon, U.K.) were injected through
the flow cell at 200, 100, 50, 25, 12.5, and 6.25 nM (50 nM point in du-
plicate) in HBS-EP running buffer (Biacore) at a flow rate of 30 ml/min.
Soluble Fc receptors were injected for 5 min, and dissociation was mon-
itored for 10 min. Background binding to the control flow cell was mon-
itored automatically. Affinity constants were derived from the data by
equilibrium binding analysis (FcgRIV and FcgRI to m2a) and/or analysis
of association and dissociation using a 1:1 binding model as indicated
using Biacore Bioevaluation software (Biacore).
Student t tests (unpaired) were performed using GraphPad Prism software
(GraphPad Software, La Jolla, CA). For comparison of Ab responses, data
were log-transformed before analysis. Significance was accepted when p ,
Characterization of m1 and m2a anti-mouse CD40 mAb
To examine the importance of mAb isotype in the immunosti-
mulatory activity of anti-CD40, we cloned the variable regions of
the rat anti-mouse CD40 mAb 3/23 (3/23 r2a) onto m1 and m2a
constant regions and expressed the chimeric mAb in CHO cells.
The purified mAb showed equivalent Ag-binding function, with
similar binding curves to cell-expressed mouse CD40 and com-
petition with the parent 3/23 r2a for binding to B cells (Fig. 1A).
The function of the Fc portion of each mAb was assessed through
comparison of their binding to each of the mouse FcgR (I, IIB, III,
and IV) using surface plasmon resonance (Fig. 1B). Consistent
with reports in the literature (41), 3/23 m2a showed high-affinity
binding to FcgRI and FcgRIV and bound much less well to
FcgRIIB and -III (Fig. 1B). In contrast, 3/23 m1 showed no de-
tectable binding to FcgRI and -IV and bound to FcgRIIB and -III
(Fig. 1B) with relatively low affinity. Fig. 1C gives estimated KD
values for each interaction, which are similar to those previously
reported (41). We also examined the ability of the mAb to enhance
phagocytosis of opsonized B cells by macrophages in vitro (Fig.
1D). As expected from their FcgR binding profiles (42), 3/23 m2a
promoted phagocytosis, whereas m1 did not (Fig. 1D). Impor-
tantly, both mAb were functional in vivo, with a 100-mg dose of
either isotype causing a transient reduction in circulating B cells in
mice and, in the case of 3/23 m1, causing an ∼2-fold increase in
spleen weight by day 7 after treatment (Fig. 1E), which is similar
to that reported for 3/23 r2a (Fig. 1E) (20).
3/23 m1 but not m2a is an immunostimulator
To assess the influence of isotype on immunostimulatory function,
we examined the ability of the 3/23 m1 and m2a mAb to stimulate
adaptive immune responses (Fig. 2). A 100-mg dose of each mAb,
or the parent r2a, was injected i.v. into mice along with the model
Ag, OVA. For Ab responses, the OVA was administered either as
immune complexes, free soluble protein (Sigma-Aldrich; con-
taminated with ∼50 IU/mg endotoxin), or endotoxin-free soluble
protein. For all forms of the Ag, 3/23 m1 caused a dramatic (from
10- to .100-fold) increase in circulating anti-OVA Ab levels by
day 14. For immune complexes, this enhancement was greater
than that observed for the parent r2a (Fig. 2A). In contrast, 3/23
m2a had no effect on the Ab response (Fig. 2A). Further experi-
ments demonstrated that as little as 25 mg 3/23 m1 provided
maximal stimulation of the Ab response, whereas as much as 500
mg m2a had no stimulatory effect (data not shown). In similar
studies, 3/23 m1 but not m2a also increased anti–4-hydroxy-3-
nitrophenyl Ab responses against OVA–4-hydroxy-3-nitrophenyl
(data not shown).
ToassessT cellresponses,micewereadoptivelytransferred with
OVA-specific CD4 (OTII) or CD8 (OTI) T cells then immunized
as above with endotoxin-free OVA plus 3/23 mAb. Circulating
numbers of OTII and OTI cells were followed over time. In ad-
dition, T cell numbers in the spleen were determined 4 d after
immunization (Fig. 2B, 2C). For both CD4 and CD8 T cells, a
small, transient increase in circulating numbers was observed in
control mice that was not influenced by the presence of 3/23 m2a.
In contrast, 3/23 m1 promoted a 2- and 11-fold increase in cir-
culating CD4 and CD8 T cells, respectively. Similarly, 3/23 m2a
had no effect on numbers of OVA-specific CD8 T cells in the
spleen, although it caused a small, but significant, increase in
splenic CD4 T cell numbers. In contrast, 3/23 m1 caused a dra-
matic 5- and 21-fold increase in OVA-specific splenic CD4 and
CD8 T cells, respectively (Fig. 2B, 2C, insets). Thus, for both
arms of the adaptive immune response, 3/23 m1, but not 3/23 m2a,
was able to provide stimulation.
Differential effects of anti-CD40 mAb on activation of DC and
Ligation of CD40 on APC plays an essential role in the initiation
of adaptive immune responses (28, 29). Two key APC that express
CD40 are DC and B cells. We therefore examined the effect of the
mAb on these APC in vivo and in vitro. Consistent with their
1756 ESSENTIAL ROLE FOR FcgRIIB IN ANTI-CD40 ACTIVITY
differential effects on immune responses (Fig. 2), 3/23 r2a and m1
increased the expression of CD70 on splenic DC (Fig. 3A) and
promoted proliferation of B cells (Fig. 3B) in vivo. In contrast,
although a small increase in CD70 expression on DC was ob-
served in response to 3/23 m2a (Fig. 3A), this mAb had no effect
on B cell proliferation in vivo (Fig. 3B). In addition, 3/23 r2a and
m1, but not m2a, promoted robust proliferation of isolated B cells
in vitro (Fig. 3C), an assay commonly used to assess the agonistic
activity of anti-CD40 mAb. 3/23 m1 also promoted greater up-
regulation of activation markers on B cells as assessed by ex-
pression of CD23, CD80, CD86, and MHC II (Fig. 3D) and a
higher phosphorylation of IKKa/b and IkB-a proteins than m2a
(Fig. 3E), leading to reduced levels of IkB in a time-dependent
manner, which is suggestive of higher NF-kB activation in these
cells (43). Interestingly, both forms of the Ab were equally able to
promote B cell survival in vitro (Fig. 3F), consistent with studies
showing differential requirements for anti-CD40–induced B cell
proliferation and rescue from apoptosis (44). Thus, as measured
by several different approaches, 3/23 m1 and r2a promoted much
greater activation of APC in vitro and in vivo than m2a, which
may explain their differential effects on immune responses.
Essential role for FcgRIIB in the immunostimulatory activity
FcgR mediate many Ab functions in vivo, and activatory FcgR,
particularly FcgRIIA and -IIIA in humans [FcgRIII and IV in
mice (45)], are crucial in providing the efficacy for rituximab and
other anti-cancer mAb. To determine the role of FcgR in the ag-
onistic activity of 3/23 and whether isotype differences in FcgR
interaction were responsible for the contrasting effects of 3/23 m1
and m2a on immune responses, we analyzed anti-OVA Ab
responses in FcgR2/2mice. Two strains were used; FcgRIIB2/2
mice, which lack the inhibitory receptor but have all of the acti-
vatory FcgR (I, III, and IV) intact (46); and FcRg2/2mice, which
lack all activatory FcgR but retain the inhibitory FcgRIIB (47).
Despite some variation in response among animals, 3/23 m1
caused a very large and significant increase in Ab titers in both
WTand FcRg2/2mice, whereas this effect was lost in FcgRIIB2/2
mice (Fig. 4A).
Consistent with the in vivo data, 3/23 m1 stimulated robust
proliferation of B cells in splenocyte cultures in vitro (Fig. 4B).
Loss of activatory receptors in FcRg2/2splenocyte cultures did
not impact the ability of 3/23 m1 to stimulate B cell proliferation
in vitro, whereas loss of FcgRIIB abrogated its effect (Fig. 4B). In
contrast, 3/23 m2a allowed only a low level of proliferation in WT,
FcRg2/2, and FcgRIIB2/2cultures (Fig. 4B).
Results in Fig. 4A and 4B suggest that FcgRIIB plays an es-
sential role in delivering the agonistic activity of 3/23 and that
activatory FcgR are not required. Thus, isotype differences in anti-
CD40 activity may be explained by differences in the affinity of
3/23 m1 and m2a for FcgRIIB (Fig. 1B, 1C) (41).
To examine the role of FcgRIIB further, we determined the
effects of the 3/23 mAb on purified B cells in vitro where FcgRIIB
is the only FcgR expressed. Consistent with the data in Fig. 4B,
3/23 m1 stimulated robust proliferation of WT B cells but not
FcgRIIB2/2B cells, whereas 3/23 m2a did not stimulate pro-
liferation of either (Fig. 4C). LPS stimulation resulted in pro-
liferation of both WT and FcgRIIB2/2B cells and to a similar
extent (Fig. 4C, inset), demonstrating that the FcgRIIB2/2B cells
were capable of dividing. Furthermore, two different anti-mouse
centrations of 3/23 m1 or m2a followed by goat anti-mouse IgG Fc-FITC (left panel) or in the presence of 1 mg/ml 3/23 rat IgG2a-FITC (right panel). Cells
were analyzed by flow cytometry for FITC labeling. Results are presented as mean fluorescence intensity 6 SE of triplicate samples and represent one of at
least two experiments. B, Surface plasmon resonance analysis of 3/23 m1 and m2a binding to mouse FcgR. Recombinant, soluble FcgR proteins (0, 6.25,
12.5, 25, 50, 50, 100, and 200 nM) were passed over 3/23 mAb immobilized at 1000 RU. Sensograms are shown. Similar results were obtained with mAb
immobilized at 15,000 RU. C, Calculated KDvalues for interactions shown in B. D, Phagocytosis of fluorescently labeled mouse B cells, uncoated (C) or
coated with 3/23 m1 or m2a, by bone marrow-derived macrophages. Results are mean 6 SEM for quadruplicate samples and represent one out of two
experiments. E, Mice were injected with a 100-mg dose of 3/23 mIgG1 or mIgG2a in PBS or with PBS alone (C). Left panel, Numbers of circulating B cells
were followed over time (mean 6 SD of three mice). Right panel, Spleen weights were determined on day 7. Results are representative of one out of three
experiments. n.d., not determined, as binding level too low.
Characterization of m1 and m2a forms of 3/23 anti-mouse CD40. A, Mouse B cells [p-BCL1(54)] were incubated with increasing con-
The Journal of Immunology1757
FcgRIIB mAb blocked the ability of 3/23 m1 to stimulate pro-
liferation of WT B cells (Fig. 4D). Consistent with the isotype-
independent role of 3/23 in promoting B cell survival (Fig. 3F),
the loss of FcgRIIB did not prevent 3/23 m1 from increasing cell
survival (Fig. 4E). In contrast, activation of B cells, as measured
by increased expression of MHC II (Fig. 4F) or caspase-8 acti-
vation (Fig. 4G), was reduced.
The role of FcgRIIB is in Ab cross-linking
Using B cell proliferation as a readout of activity, we took a num-
ber of approaches to determine the role that FcgRIIB plays in 3/23
activity. First, we compared the ability of immobilized 3/23 m1
and m2a to promote the proliferation of WT and FcgRIIB2/2
B cells. Under these conditions, both mAb provided equivalent
and effective stimulation, with levels of proliferation similar in
WT and FcgRIIB2/2cultures (Fig. 5A). Thus, when immobilized
to simulate cross-linking, the dependence on FcgRIIB interaction
was lost, and both mAb became effective agonists.
Second, we determined the ability of a truncated FcgRIIB that
lacked its intracellular tail to enhance anti-CD40 activity. The
intracellular domain of FcgRIIB contains an ITIM signaling motif
that mediates its regulatory effects on immune responses (48).
Control-transfected FcgR-negative mouse IIA1.6 B cell lym-
phoma cells and IIA1.6 cells transfected with either full-length
mouse FcgRIIB (b1 isoform) or its tailless equivalent (FcgRIIB-
SLV) were treated with 3/23 m1 or m2a and their activation status
examined. 3/23 m1 was more potent than 3/23 m2a at inducing
activation of both forms of FcgRIIB-transfected IIA1.6 cell lines,
as suggested by higher upregulation of MHC II (not shown) and
CD23 cell-surface expression (Fig. 5B). Thus, intracellular sig-
naling through FcgRIIB was not required for this activity.
Third, we examined the ability of 3/23 [Fab9 3 OVA] conju-
gates (37, 38) to stimulate B cell proliferation. [Fab9 3 OVA] con-
jugates consisted of a single molecule of OVA chemically cross-
linked to one, two, or three 3/23 Fab9 fragments (3/23 [F(ab9)13
OVA], [F(ab9)2 3 OVA], and [F(ab9)33 OVA], respectively).
Addition of [3/23 F(ab9)3 3 OVA] stimulated proliferation of
WT B cells to a similar extent as 3/23 m1 (Fig. 5C), whereas [3/23
F(ab9)13 OVA] and [3/23 F(ab9)23 OVA] were much less ef-
fective and showed similar activity to 3/23 m2a. This is interesting,
as CD40-driven responses are initiated following binding to a tri-
meric CD40L (CD154) (49). [3/23 F(ab9)33 OVA] was also ef-
fective in stimulating the proliferation and activation of both
FcgRIIB2/2B cells and MyD882/2/Toll/IL-1R domain-containing
adapter inducing IFN-b2/2B cells (data not shown).
Finally, we determined whether FcgRIIB needed to be ex-
pressed on the same cell as CD40 to provide cross-linking. For
this, we prepared purified CD402/2and FcgRIIB2/2B cells and
analyzed their ability to proliferate in response to 3/23 m1 when
cultured alone or mixed together. As expected, neither cell type
proliferated when cultured alone. However, when mixed together,
robust proliferation was observed (Fig. 5D). As expected, pro-
liferation was attributable to the FcgRIIB2/2(CD40+/+) cells, as
prior irradiation of the FcgRIIB2/2cells prevented radiolabel
incorporation in mixed cultures, whereas irradiation of the
CD402/2cells did not (Fig. 5D). Interestingly, the level of pro-
liferation observed in the mixed cultures was similar to that seen
for WT B cells, suggesting that FcgRIIB is maximally effective
when present on neighboring cells (Fig. 5D).
In summary, the results presented in Fig. 5 demonstrate that the
role of FcgRIIB in anti-CD40 activity is one of cross-linking, that
intracellular signaling through FcgRIIB is not required, and that
its presence on a neighboring cell is sufficient to provide activity.
Other FcgR can also provide cross-linking for anti-CD40
The cross-linking role of FcgRIIB raises the question as to why
other FcgR do not provide effective cross-linking for 3/23 m2a
in vivo, as this binds to FcgRI and -IV with much higher affinity
than 3/23 m1 does to FcgRIIB (Fig. 1B, 1C). Thus, the dominant
role of FcgRIIB in anti-CD40 activity in vivo must either reflect
specific properties of this receptor that make it particularly adept
at cross-linking anti-CD40 mAb or its specific pattern and/or level
of expression. To address this issue, we assessed the ability of all
four mouse FcgR to provide effective cross-linking for 3/23 m1
and m2a when expressed in vitro. 293F cells transfected with each
receptor (Fig. 6A) were cocultured with 3/23 mAb and FcgRIIB2/2
B cells and the level of B cell proliferation assessed (Fig. 6B).
Consistent with the surface plasmon resonance data (Fig. 1B, 1C),
FcgRIIB and -III stimulated proliferation of B cells in the pres-
ence of 3/23 m1 to a similar extent, whereas FcgRI and IV
mice were injected with 100 mg OVA immune complexes (IC), 0.5 mg
soluble OVA (Sigma-Aldrich), or 50 mg endotoxin-free (endo) OVA, plus
100 mg isotype control m1 (C), 3/23 m1, m2a, or r2a, as indicated. Cir-
culating levels of anti-OVA Ab were determined 7 (OVA IC) or 14 d later.
Results for individual mice are shown and represent one out of three
experiments for each Ag. Mice were adoptively transferred with OVA-
specific CD4 (OTII) (B) or CD8 (OTI) (C) T cells on day 1, then immu-
nized with 50 mg endoOVA plus 100 mg control m1, 3/23 m1, or m2a on
day 0. Circulating numbers of OTII and OTI cells were determined over
time in serial bleeds. Results are mean 6 SD for three animals and rep-
resent one out of three (B) or four (C) experiments. Insets show numbers of
OTII/OTI T cells in the spleens day 4 after immunization and are mean 6
SD for three animals. *p , 0.05, **p , 0.001 versus control.
3/23 m1 but not m2a is immunostimulatory. A, C57BL/6
1758ESSENTIAL ROLE FOR FcgRIIB IN ANTI-CD40 ACTIVITY
provided no stimulation (Fig. 6B). In contrast, all four FcgR were
able to cross-link 3/23 m2a to stimulate proliferation (Fig. 6B).
Surprisingly, the order of effectiveness did not correlate with the
relative FcgR binding affinity, as although FcgRI was the most
effective, FcgRIV was the least despite similar high-affinity
binding (Fig. 6B). In addition, FcgRIIB was able to provide ef-
fective cross-linking for 3/23 m2a in these experiments (Fig. 6B).
In contrast, cell lines expressing lower levels of FcgRIIB (data not
shown) or WT B cells that express FcgRIIB (Fig. 2C; level of
FcgRIIB expression shown in the inset in Fig. 6A) were unable to
cross-link m2a, suggesting that the level of FcgR expression is
These experiments thus suggest that it is the bioavailability of
FcgRIIB in vivo, rather than specific properties of this receptor,
that dictates its dominant role in anti-CD40 activity.
In this study, we have shown that interaction with the normally
inhibitory FcgR, FcgRIIB, is a prerequisite for the in vivo
immunostimulatory activity of anti-CD40 mAb. This is in stark
contrast to the requirement of direct-targeting anti-cancer mAb,
such as rituximab and alemtuzumab, in which activatory FcgR are
required for efficacy and interaction with FcgRIIB may be detri-
mental to activity (2). This differential FcgR dependence is
reflected in the optimally active isotype of each type of mAb in
mouse models; thus, IgG2a is optimal for direct-targeting mAb,
whereas in this study, we show that IgG1 is optimal for anti-CD40.
Current emphasis in the design of therapeutic mAb is focused
upon the engineering of variants with high affinity for activatory
FcgR (14, 15). Our data indicate this approach may not be ap-
propriate for immunostimulatory mAb and thus has significant
implications for the design of immunotherapeutic agents.
The essential role of FcgRIIB in the immunostimulatory acti-
vity of anti-CD40 is surprising, as this receptor normally plays
an inhibitory role in the immune system (48). Detailed in vitro
studies, however, showed that intracellular signaling through
FcgRIIB was not required for mediating the effects of anti-CD40;
rather the role of the receptor was in mAb cross-linking. As other
FcgR bind mAb Fc with higher affinity than FcgRIIB (this study
and Refs. 41, 50), we investigated whether they could also provide
effective cross-linking in vitro. Indeed, the ability of the different
mouse FcgR to effectively cross-link 3/23 m1 and m2a correlated
with their Fc binding profiles. Thus, FcgRI, -II, -III, and -IV all
provided cross-linking for 3/23 m2a, whereas only FcgRIIB and
-III were effective for 3/23 m1. Interestingly, however, their rel-
ative effectiveness did not correlate with measured Fc affinities.
For example, FcgRIIB and -III bound 3/23 m2a with .10-fold
lower affinity than FcgRIVand yet provided more effective cross-
linking in vitro, suggesting perhaps that some FcgR are more
adept at cross-linking anti-CD40 than others. This could reflect
properties of the receptors themselves or of the 3/23 mAb.
Whether this is also true for other immunostimulatory mAb (e.g.,
anti-CD27, anti–4-1BB, and anti–CTLA-4) is a question we are
or 3/23 m1, m2a, or r2a as indicated. Two days later, spleens were removed and sections stained for CD70 (green) and the DC marker miDC8 (red). RAG2/2
mice were used as CD70 expression is more easily visualized in these mice; however, similar results were obtained in WTanimals (not shown). Scale bars,
50 mm. B, Mice injected with CFSE-labeled hen egg lysozyme (HEL)-specific purified splenic B cells received 100 mg indicated mAb. Four days later,
spleens were removed and CFSE-labeled B cells analyzed by flow cytometry after intracellular staining with HEL-FITC to visualize the transferred cells.
Plots are gated on CD19+lymphocytes and are representative of one out of four experiments performed in duplicate. Inset arrow in Con panel indicates
region containing divided cells. Similar results were obtained with WT B cells (not shown). C, Purified splenic B cells were incubated with increasing
concentrations of the indicated mAb for 5 d. Incorporation of [3H]thymidine was assessed during the final 16 h. Results are mean 6 SEM of triplicate
samples and show results from 1 out of .10 experiments. D, Expression of activation markers on purified splenic B cells incubated with 1 mg/ml m1 or m2a
isotype control mAb (filled histogram) or 3/23 m1 or m2a (black line) for 48 h. Results from one out of five experiments are shown. E, Western blot of
lysates from B cells treated for the indicated times with 3/23 m1 or m2a probed with Ab specific for phospho-IKKa/b, phospho-IkB-a, or IkB-a. Anti-
tubulin was used as a loading control. F, B cells incubated as in D were analyzed by staining with 7AAD and flow cytometry to determine the percentage of
live (7AAD-negative) cells. Results are mean 6 SE of quadruplicate samples and are representative of four experiments. *p , 0.01 versus control.
Differential effects of anti-CD40 mAb on activation of APC. A, C57BL/6 RAG2/2mice were injected with 100 mg isotype control m1 (Con)
The Journal of Immunology1759
In vitro studies also revealed that high levels of FcgRIIB ex-
pressed on transfected human fibroblasts were able to cross-link 3/
23 m2a in vitro. Lower levels of FcgRIIB found on WT B cells
(this study) or stable cell lines expressing the receptor (not
shown), however, did not. Although we could detect minimal
binding of 3/23 m2a to FcgRIIB, this was too low to calculate an
accurate KDvalue, consistent with the reported lower affinity of
m2a versus m1 for FcgRIIB (41). The difference in activity be-
tween 3/23 m1 and m2a in vivo may thus reflect a threshold of
affinity for FcgRIIB required for functional activity. These data
suggest that expression levels and biodistribution of FcgRIIB, as
well as Fc binding affinity, are important for its cross-linking
Interestingly, we have found that the parent 3/23 r2a mAb has
a similar mouse FcgR binding profile to 3/23 m1 and that its
ability to stimulate B cell proliferation in vitro is also dependent
upon the presence of FcgRIIB (A.L. White, unpublished obser-
vations). More detailed analyses will be required to determine
whether its reduced ability compared with 3/23 m1 to stimulate
immunity is due to a lower affinity for FcgRIIB.
Wilson et al. (18) recently reported that FcgR interaction was
required for the therapeutic activity of drozitumab (anti-DR5
mAb) in mice. As we have shown in this study for anti-CD40,
the role of FcgR in this case was also in mAb cross-linking rather
than effector cell recruitment (18). However, in contrast to the
current study, either activatory or inhibitory FcgR could provide
cross-linking for drozitumab in vivo. Interestingly, these authors
also demonstrated that the ability of the rat anti-mouse CD40 mAb
FGK-45 to stimulate NF-kB activation in isolated mouse B cells
was lost in cells from FcgRIIB2/2mice (18). From these com-
bined data, we hypothesize that for mAb that require FcgR-
mediated cross-linking the location of target expression may de-
termine the profile of FcgR dependence. DR5 is ubiquitously
expressed. CD40 is also expressed on many cell types. However,
in terms of its role in adaptive immune responses, perhaps the
most important locations are on the surface of DC and B cells.
Interestingly, FcgRIIB is the only FcgR expressed on B cells,
whereas both activatory and inhibitory FcgR are present on mouse
DC. Thus, it is tempting to speculate that the in vivo activity of
anti-CD40 is mediated through B cells, in which a locally high
munized with 100 mg OVA IC plus 100 mg isotype control m1 (C) or 3/23 m1 or m2a. Day 7 anti-OVA Ab titers are shown for individual animals. *p ,
0.001, **p , 0.05 versus control. B, B cell proliferation in splenocyte cultures from WT, FcgRIIB2/2, and FcRg2/2mice in response to 3/23 m1 or m2a
analyzed as in Fig. 2C. Isotype m1 control is shown for WT splenocytes only. Results are mean 6 SEM for triplicate samples and representative of two
experiments. C, Proliferation of purified splenic B cells from WTor FcgRIIB2/2mice. Results are mean 6 SEM of triplicate samples from one out of five
experiments. Inset shows proliferation in response to 10 mg/ml LPS. D, Proliferation of WT B cells in response to 1 mg/ml control m1 (C) or 3/23 m1 in the
absence or presence of 10 mg/ml two different anti-FcgRIIB mAbs, AT128 and AT130-5. Results are mean 6 SEM of triplicate samples from one of two
experiments. *p , 0.001 versus 3/23 m1 alone. E, Percent of surviving WTand FcgRIIB2/2B cells after 48-h culture in the presence of isotype control (C)
or 3/23 m1 or m2a mAb at 1 mg/ml. Data are mean 6 SD of six samples from two different experiments. *p , 0.01 versus isotype control. F, MHC II
expression on WTand FcgRIIB2/2B cells incubated for 48 h with isotype control (filled gray histograms) or 3/23 m1 or m2a (black lines). Results are from
one out of two experiments. G, WT and FcgRIIB2/2purified spenic B cells were not treated (NT) or incubated overnight with 10 mg/ml anti-mIgM, 3/23
m1, or m2a. The following day, cell lysates were analyzed by Western blotting for caspase-8 or b-actin as loading control.
Essential role for FcgRIIB in the immunostimulatory activity of anti-CD40. A, WT, FcgRIIB2/2, and FcRg2/2C57BL/6 mice were im-
1760 ESSENTIAL ROLE FOR FcgRIIB IN ANTI-CD40 ACTIVITY
concentration of FcgRIIB would be present (e.g., in B cell fol-
licles). Its direct action on B cells could explain its potent ability
to stimulate humoral immunity and also T cell responses, as ac-
tivated B cells have been demonstrated to act as APC for both
CD4 and CD8 T cells (51, 52). Indeed, preliminary data suggest
that prior depletion of B cells with anti-CD20 mAb prevents the
stimulatory effect of 3/23 m1 on CD8 T cell expansion (not
shown). Future experiments, involving mouse models in which
FcgRIIB is selectively knocked out on B cells or DC, will be used
to address the site of anti-CD40 action in vivo.
A possible explanation for the lack of immunostimulatory ac-
tivity of 3/23 m2a was that this mAb depleted CD40-positive cells,
much as anti-CD20 m2a depletes B cells in mice (6). We believe
this was not the case, however, as: 1) the transient decrease in
numbers of circulating B cells was similar in 3/23 m1- and m2a-
treated animals (Fig. 1E); 2) there was no evidence that 3/23 m2a
depleted adoptively transferred CFSE-labeled B cells from the
spleen (Fig. 3B); 3) numbers of B cells and DC in the spleen were
similar in 3/23 m2a-treated and control mice (Figs. 1E, 3A and
data not shown); and 4) 3/23 m2a did not inhibit B or T cell
responses (Fig. 2), as might be expected if this mAb depleted
CD40-positive cells. In fact, our data suggested some limited
stimulatory activity of 3/23 m2a, as it caused a detectable, albeit
small and reproducible, increase in CD70 expression on splenic
DC and also a small, but significant, increase in OVA-specific
CD4 T cell expansion, consistent with the expression of activa-
tory FcgR on DC.
A crucial question is how our results may be translated to the
human system, in which there are clear differences from mice in
terms of mAb isotypes and their interaction with FcgR (41, 50).
The positive role of activatory receptors (FcgRIIa and -IIIa) in
the activity of direct binding anti-cancer mAb in humans has
been confirmed through analysis of drug responses in patients with
polymorphisms in these receptors (10–13). As a result, much re-
search has been focused on the engineering of mAb that have
enhanced activatory FcgR binding and A/I ratios (14, 15). Our
studies suggest that this may not be the best approach for anti-
CD40 mAb and by extension possibly other immunostimulatory
agents and cancer-binding mAb. Once these agents reach the
clinic, similar genetic studies to those conducted for rituximab,
alemtuzumab, and cetuximab (10–13) in humans would be re-
quired to address this. It would be of particular interest to examine
responses in patients with different alleles of FcgRIIB shown to
influence either levels of receptor expression or incorporation into
concentrations of 3/23 m1 and m2a or m1 isotype control (con) were
coated onto plastic overnight before incubation with WTor FcgRIIB2/2B
cells. B cell proliferation was assessed as in Fig. 3C. Results are mean 6
SEM of triplicate wells and show one out of two independent experiments.
B, Control (Con) mouse B cell lymphoma cells or cells transfected with
full-length mouse FcgRIIB1 or a truncated FcgRIIB lacking its in-
tracellular tail (FcgRIIB-SLV) were incubated for 24 h with or without
(NT) 10 mg/ml 3/23 m1 or m2a. Surface expression of CD23 was analyzed
by flow cytometry. C, WT B cell proliferation in response to isotype con-
trol mAb (Con), 3/23 m1, or m2a [3/23 F(ab9)33 OVA], [3/23 F(ab9)23
OVA], [3/23 F(ab9)13 OVA], or [F(ab9)33 OVA] targeted to MHC II
(con [F(ab9)33 OVA]) was assessed as described in Fig. 3C. Results are
mean 6 SEM for triplicate samples from one out of four independent
experiments. D, WT, FcgRIIB2/2and CD402/2B cells either untreated
or irradiated (Irr) to prevent proliferation were incubated with increasing
concentrations of 3/23 m1. A single cell type or two cell types were mixed
as indicated. B cell proliferation was assessed as described for Fig. 3C.
Results are counts per 105CD40+cells/well, except when FcgRIIB2/2
cells were used alone, for which results for 105total cells are shown.
Results are mean 6 SEM for triplicate samples and are from one out of
two independent experiments.
The role of FcgRIIB is in Ab cross-linking. A, Different
CD40. A, 293F cells were transfected with the different mouse FcgRs as
indicated and levels of expression analyzed by flow cytometry. Histograms
represent untransfected (gray filled histogram) and FcgR-transfected cells
(black line) stained with the relevant FITC-labeled mAb. Inset in the
FcgRIIB histogram shows levels of FcgRIIB expression on WT splenic B
cells. B, FcgRIIB2/2B cells were incubated with untransfected 293 cells
(C) or cells transfected as in A plus 625 ng/ml 3/23 m1 or m2a and pro-
liferation assessed as in Fig. 3C. Results are mean 6 SEM for triplicate
samples and represent one out of three independent experiments. *p #
0.001 versus control.
Other Fc receptors can also provide cross-linking for anti-
The Journal of Immunology1761
lipid rafts, which may influence cross-linking function (53). In-
terestingly, of the anti-human CD40 mAb currently in clinical
trials, CP870,893 is the most agonistic. This mAb has a human
IgG2 Fc region, whereas the others have IgG1. This is intriguing,
as human IgG2 is reported to bind less well than IgG1 to all FcgR
(50). Clearly, further studies are required to determine the con-
tribution of epitope specificity, mAb isotype, and FcgR interaction
in the activity of anti-human CD40 mAb.
in the activity of immunostimulatory anti-CD40 mAb. However,
in stark contrast to direct-targeting anti-cancer mAb that require
interaction with activatory FcgR (6), we demonstrate an essential
role for the inhibitory receptor, FcgRIIB, in anti-CD40 activity.
Future studies will be required to determine whether our obser-
vations can be extended to humans or to other immunostimulatory
agents, some of which are already in clinical trials (19). These
findings have important implications when considering the design
of optimally active mAb based therapeutics.
We thank Christine Penfold, Jinny Kim, Kerry Cox, Vallari Shah, and the
staff of the Tenovus Biomedical Research facility for technical support and
Leon Douglas from the Cancer Research UK/Experimental Cancer Medi-
cine Centre Protein Core Facility for making SIINFEKL tetramers.
The authors have no financial conflicts of interest.
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