IMMUNOBIOLOGY

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular

depletion and survival blockade
Wei Yu Lin,1 Qian Gong,1 Dhaya Seshasayee,1 Zhonghua Lin,1 Qinglin Ou,1 Shiming Ye,1 Eric Suto,1 Jean Shu,1
Wyne Pun Lee,1 Ching-Wei V. Lee,2 Germaine Fuh,2 Maya Leabman,3 Suhasini Iyer,3 Kathy Howell,3 Thomas Gelzleichter,3
Joseph Beyer,3 Dimitry Danilenko,4 Sherry Yeh,5 Laura E. DeForge,5 Allen Ebens,6 Jeffrey S. Thompson,7
Christine Ambrose,7 Mercedesz Balazs,1 Melissa A. Starovasnik,2 and Flavius Martin1
Departments of 1Immunology, 2Protein Engineering, 3Development Sciences, 4Pathology, 5Assay and Automation Technology, and 6Translational Oncology,
Genentech, South San Francisco, CA; and 7Department of Gene Discovery, Biogen IDEC, Cambridge Center, Cambridge, MA

Removal of pathogenic B lymphocytes by
depletion of monoclonal antibodies
(mAbs) or deprivation of B-cell survival
factors has demonstrated clinical benefit
in both oncologic and immunologic dis-
eases. Partial clinical responses and
emerging data demonstrating incomplete
B-cell depletion after immunotherapy fu-
els the need for improved therapeutic
modalities. Lessons from the first genera-
tion of therapeutics directed against B-
cell-specific antigens (CD20, CD22) are
being applied to develop novel antibodies
with additional functional attributes. We
describe the generation of a novel class
of B-cell-directed therapy (anti-BR3 mAbs)
that combines the depleting capacity of a
therapeutic mAb and blockade of B-cell-
activating factor (BAFF)–BR3 B-cell sur-
vival. In mice, treatment with antagonistic
anti-BR3 antibodies results in quantita-
tively greater reduction in some B-cell
subsets and qualitatively different effects
on bone marrow plasma cells compared
with BR3-Fc BAFF blockade or with anti-
CD20 treatment. Comparative analysis of
BR3-Fc and anti-BR3 mAb reveals a lower
B-cell dependence for BAFF-mediated
survival in nonhuman primates than in
mice. This novel class of B-cell-targeted
therapies shows species characteristics
in mice and primates that will guide trans-
lation to treatment of human disease.
(Blood. 2007;110:3959-3967)
© 2007 by The American Society of Hematology

Introduction
B-cell immunotherapy has shown clinical benefit in the treatment
of human B-cell malignancies and autoimmune diseases.1-4 Since
approval of a depleting anti-CD20 monoclonal antibody (mAb)
(rituximab [Genentech, South San Francisco, CA; Biogen-IDEC,
Cambridge, MA; Roche, Basel, Switzerland)] in 1997 for the
treatment of non-Hodgkin lymphoma (NHL), almost 1 million
patients have been treated with rituximab as a first or second line
therapy, either alone or in combination with chemotherapy. In
addition, rituximab maintenance therapy significantly prolongs
tumor remission and patient survival in patients with indolent
B-cell NHL or chronic lymphocytic leukemia (CLL).5,6 More
recently, rituximab has demonstrated clinical benefit in a variety of
autoimmune diseases including rheumatoid arthritis, pemphigus
vulgaris, immune thrombocytopenia and autoimmune hemolytic
anemia.4,7,8 As a result, understanding the contribution of B
lymphocytes to human autoimmune diseases received revived
interest, and several mechanisms have been postulated to partici-
pate in disease pathogenesis, including autoantibody production,
B-cell antigen presentation, cytokine generation, and lymphorgano-
genesis.2,3,9,10 Inhibition of different combinations of these mecha-
nisms is probably responsible for clinical benefit.
The success of anti-CD20 B-cell immunotherapy spearheaded a
large number of preclinical and clinical efforts to understand in
vivo mechanisms of drug activity. Direct elimination of malignant
B cells through antigen-dependent cell-mediated cytotoxicity
(ADCC), complement-mediated cytotoxicity (CDC), and apoptosis
have been demonstrated as the main mechanisms of action in a
variety of systems including mouse xenotumors and normal mouse
and nonhuman primate (NHP) B-cell subsets.11-15 Two major
determinants affecting normal mouse B-cell depletion have been
identified.13,16 First, the kinetics of B-cell recirculation determines
the speed and magnitude of anti-CD20 mAb-mediated B-cell
depletion. Cells with higher recirculatory kinetics from blood,
lymph nodes, and spleen follicular areas are depleted faster and
more completely than cells with lower recirculatory kinetics (eg,
peritoneal cavity [PEC], marginal zone [MZ], germinal centers
[GC]). Second, the local microenvironment influences the extent of
B-cell depletion. Marginal zone, Peyer patches (PP), germinal
center, memory, and peritoneal cavity B cells exhibit greater
resistance to depletion in mice and nonhuman primates. Reduced
recruitment of effector mechanisms in the peritoneal cavity as well
as intrinsic B1 B cells properties appear to cause the slower kinetics
and B-cell reduction after anti-CD20 mAb treatment.16 Differences
across mouse strains and epitopes recognized by anti-CD20
antibodies used for depletion might explain the differential effects
seen on mouse splenic marginal zone B cells.13,16 Small but
consistent numbers of residual B cells can be detected in most
lymphoid organs in mice and primates treated with anti-CD20
mAbs. One possibility of achieving a more complete B-cell
reduction would be to block B-cell survival signals in addition to

Submitted April 30, 2007; accepted July 31, 2007. Prepublished online as
Blood First Edition Paper, August 8, 2007; DOI 10.1182/blood-2007-
04-088088.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2007 by The American Society of Hematology

BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12 3959

3960 LIN et al
administration of a B-cell-depleting reagent. B-cell-activating
factor (BAFF) (also known as BLyS) and a proliferation-inducing
ligand (APRIL), 2 related tumor necrosis factor (TNF) family
members, and their receptors—BLyS receptor 3 (BR3; also known
as BAFFR), transmembrane activator and calcium modulator and
cyclophilin ligand interactor (TACI), and B-cell maturation antigen
(BCMA)—constitute a well characterized system involved in
B-cell development and survival.17-21 Of these, BAFF/BR3 is the
main axis transducing B-cell survival signals, and interference with
this pathway using blocking reagents (anti-BAFF antibodies or
BR3-, TACI-, or BCMA-Fc fusion proteins) results in B-cell
reductions in mice, nonhuman primates, and humans.22,23 Recent
clinical trials with an anti-BAFF antibody have shown clinical
benefit in rheumatoid arthritis (RA) and positive trends in retrospec-
tive subset analysis in patients with systemic lupus erythematosus
(SLE).24,25 In mice, BAFF blockade with BR3-Fc appears to act
synergistically with anti-CD20, resulting in more complete B-cell
depletion at doses suboptimal as monotherapy.13 It is noteworthy
that serum BAFF levels increase when B cells are lacking (ie,
rag-deficient mice) or after depletion with rituximab in patients
with RA, SLE, or Sjögren syndrome, probably because of a
decrease in receptor-mediated clearance and possibly other mecha-
nisms.26-30 Thus, increased serum BAFF could provide additional
survival signals for cells that were not removed during the
depletion phase and for emerging new B cells during the recovery
phases of B-cell immunotherapy.
Strategies targeted at enhancing intrinsic properties of anti-
CD20 antibodies (eg, increased ADCC, CDC, and affinity to
antigen) represent one modality by which to achieve greater
clinical benefit compared with existing B-cell immunotherapy.31-35
Other efforts are driven toward testing antibodies directed against
other B-cell surface molecules (CD19, CD22).36-39 Finally, conju-
gates of radioactive isotypes (ibritumomab tiuxetan [Zevalin],
tositumomab [Bexxar]) and toxins (calicheamicin-antiCD22, anti-
CD79 antibody-drug conjugates) represent a third strategy to
enhance B-cell targeting.40-42 In addition to intrinsic molecule
improvements, combinations of B-cell-depleting agents with
molecules enhancing other arms of the immune system in-
cluding effector mechanisms for B-cell depletion (eg, interferon-␣,
granulocyte–colony-stimulating factor, granulocyte macrophage–
colony-stimulating factor, interleukin 2) are being attempted in
B-cell malignancies with variable success.43-46
Based on the demonstrated synergy between B-cell depletion
and blockade of B-cell survival through BR3, we developed
anti-BR3 antibodies that combine, in one molecule, the abilities to
deplete B cells and to block BAFF-mediated B-cell survival. These
antibodies combine 2 mechanisms of action and show increased in
vivo B-cell reduction in some subsets compared with anti-CD20
mAb-mediated B-cell depletion or BAFF-dependent survival block-
ade on their own. As a result, therapy with this novel type of
antibody has the potential to translate into increased clinical benefit
in diseases with B-cell-mediated pathogenic mechanisms.
Materials and methods
Anti-BR3 antibodies and in vivo dosing
mCB2 (mIgG2a) and mCB2 DANA, phage-displayed synthetic antibody,
were generated as described previously.47 PIH11 (anti-mouse BR3, nonblock-
ing antibody; Biogen-IDEC, Cambridge, MA), mBR3-Fc, and mouse
anti-human CD20 (mouse IgG2a version of anti-human CD20 mAb 2H7)
were described previously.13,48 Anti-ragweed mouse IgG2a was used as the
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12
isotype control in all mouse studies. Final endotoxin levels of antibodies for
all in vivo studies were less than 0.2 EU/mg. To be able to compare among
molecules, mice were dosed in all experiments to ensure maximal B-cell
reduction based on in vivo titrations of anti-BR3, BR3-Fc and anti-CD20
reagents. Dose regimes were determined to achieve similar trough concen-
trations for anti-BR3 and BR3-Fc in all experiments. Anti-BR3 at a 500-␮g
single dose achieves blood drug levels at day 15 similar to 200-␮g weekly
multidose trough concentrations. BR3-Fc at 150 ␮g, 3 times per week,
achieves a 72-hour trough in a similar range. Target trough concentrations
were determined to achieve maximal B-cell reduction in splenic B-cell
subsets based on a series of single-dose pharmacokinetic/pharmacodynamic
experiments. For example, at day 4, anti-BR3 blood concentrations reached
after a 20-␮g dose did not achieve maximal B-cell depletion, whereas those
reached after a 200-␮g dose did (Figure S1, available on the Blood website;
see the Supplemental Materials link at the top of the online article). The
half-life is 5 days for anti-CD20 and anti-BR3 antibodies and
3 days for mouse BR3-Fc. Mouse anti-human BR3 (clone 9.1; Biogen-
IDEC49) was humanized on a human IgG1 framework and used in NHP
studies. All in vitro properties (eg, BR3 binding, BAFF antagonism, ADCC)
were similar between the parent and humanized antibody.
Animals
Eight- to 12-week-old BALB/c mice (The Jackson Laboratory, Bar Harbor,
Maine) and C57BL/6 mice (Charles River Laboratories, Wilmington, MA)
were used for depletion studies. hCD20tgBAC FvB and C57BL/6 mice
were described previously.13 NZB/W F1 female mice (The Jackson
Laboratory), an SLE-prone model, were ⬃7 to 8 months old and weighed
35 to 50 g when they were enrolled in therapeutic studies. Urine was tested
for proteinuria by semiquantitatively dipsticks (Multistick; Thermo Fisher
Scientific, Waltham, MA) every 4 weeks. The baseline protein levels at the
start of the studies were between 30 and 300 mg/dL. All experimental
animal studies were conducted according to protocols that were reviewed
and approved by the Institutional Animal Care and Use Committees
(IACUC) of Genentech Lab Animal Research (LAR).
NHP cynomolgus monkey studies were conducted at contract research
organizations (CROs) according to their standard operating procedures and
in compliance with applicable regulations concerning the use of laboratory
animals. Naive monkeys, 2 to 5 years old, were acclimated to the study
rooms for 28 days before the initiation of the study.
Tissue collection
Whole spleen, lymph nodes, Peyer patches, and kidneys were collected for
analysis. Single-cell suspensions were prepared by grinding organs between
frosted glass slides. The total cell counts were measured using fluorescence
beads. Cells were filtered through a 70-␮m nylon cell strainer and used for
flow cytometry analysis and spot enzyme-linked immunosorbent assays.
Blood samples were collected from the retro-orbital sinus (100 ␮L) at
trough and by cardiac puncture (while the animals were anesthetized with
3% isoflurane anesthesia, 600⬃800 ␮L) before the mice were euthanized.
Flow cytometry and antibodies
Spleen, lymph nodes, Peyer patches, peritoneal cavity lavage, and periph-
eral blood cells were analyzed for B and T cell subsets using conjugated
antibodies as fluorescence-activated cell sorting (FACS) reagents: anti-
mouse CD3, CD4, CD8, CD5, CD23, CD21, CD69, B220, CD38, CD138,
CD44, CD62L, and anti-human CD19, CD5, and cynomolgus monkey
cross-reactive CD20, CD40, CD21, and CD27, all from Pharmingen (San
Diego, CA) and BD Biosciences (San Jose, CA). Polyclonal goat anti-
mouse IgM, IgG1, IgG2a, and IgA were purchased from Southern
Biotechnology Associates (Birmingham, AL). FACS analyses were con-
ducted on a FACSCalibur (BD Biosciences). B-cell subsets were identified
as described in each figure legend. Statistical analyses relative to the control
group were performed using the Dunnett test.
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12 IN VIVO B CELL EFFECTS OF ANTI-BR3 ANTIBODIES 3961

Figure 1. Anti-BR3 mAb reduces mouse B-cell subsets more efficiently than BR3-Fc. BALB/c mice were treated with anti-BR3 (500 ␮g), BR3-Fc (150 ␮g ⫻ 3/week) or
isotype control (500 ␮g); 15 days later, tissues were analyzed by flow cytometry (A,C) and immunofluorescence (B). Percentages of cells in B-cell subsets are displayed near
each gate as follows: Spleen: follicular (FO) B cells (CD23⫹CD21⫹), marginal zone (MZ) B cells (CD23⫺CD21hi), and plasma cells (PC, CD138⫹B220lo); Lymph nodes: B cells
(CD23⫹CD21⫹), blood (BL) B cells (CD23⫹CD21⫹), Peyer patches (PP), follicular B cells (B220⫹CD38⫹), and germinal center (GC) B cells (B220⫹CD38lo); peritoneal cavity
(PEC): B2 cells (B220⫹CD23⫹) and B1 cells (B220⫹CD23⫺) (A,C). B cells (blue) and T cells (green) are stained in spleen sections after treatment with control, BR3-Fc or
anti-BR3 mAb. Marginal zone and follicular B-cell areas are separated by the layer of metallophilic macrophages (MM [red]) (B). See “Immunofluorescence and
immunohistochemistry staining” for image acquisition details. Absolute B-cell numbers or percentage of lymphocyte gate belonging to a defined subset are shown as mean plus
or minus standard error. Statistical significance compared with the control treated group is indicated with asterisk (C). When 2 treatment groups are significantly different from
each other, the P value is displayed. These data are representative of more than 10 independent experiments done with several maximally depleting doses of anti-BR3, using
at least 5 mice/group.

BrdU labeling & staining
One week before take down, each mouse was received 2 mg of bromode-
oxyuridine (BrdU; Sigma, St. Louis, MO) in phosphate-buffered saline
(PBS) via intraperitoneal (i.p.) injection every other day for a week.
BrdU staining was performed using a BrdU flow kit (BD Biosciences).
First, B lineage surface antigens B220 and CD138 were stained, followed
by fixation and permeabilization of cells using BD Cytofix/Cytoperm
buffer. The cells were then treated with DNase to expose BrdU epitopes.
Cycling S phase cells were detected by staining cells with fluorochrome
conjugated anti-BrdU and anti-Ig isotype antibodies and then analyzing by
flow cytometry.
Immunofluorescence and immunohistochemistry staining
Five-micrometer cryosections of mouse spleen were stained with 7-amino-
4-methylcoumarin-3-acetic acid–anti-mouse IgM (Vector Laboratories,
Burlingame, CA), FITC-anti-mouse Thy1 (Pharmingen), and MOMA-1
mAb followed by horse anti–rat IgG/biotin and Cy3-Streptavidin (Jackson
Immunoresearch, West Grove, PA). Fluorescently labeled sections were
imaged on an Olympus BX-51 microscope (Olympus USA, Center Valley,
PA) equipped with filter cubes for DAPI/AMCA, FITC/Cy2 and TRITC/
Cy3. Twelve-bit monochrome images were acquired with a 10⫻ objective
using a Hamamatsu ORCA CCD camera (Olympus USA) driven by
Meta-Morph version 6.1 imaging software (Universal Imaging, Downing-
town, PA). Image files were transferred to Adobe Photoshop CS2 (Adobe,
Mountain View, CA), adjusted for contrast, and merged to create 24-bit
color images.
Formalin-fixed, paraffin-embedded cynomolgus monkey tissues were
sectioned at 4 to 6 ␮m and processed by standard techniques for
immunohistochemistry. Immunohistochemistry for B cells was accom-
plished using mouse IgG2a anti-human CD20 (clone L26; Dako,
Carpinteria, CA) or an irrelevant IgG2a antibody (clone C1.18.4; BD
Pharmingen) and detection system using ABC-Peroxidase Elite with
Vector Blue (Vector Laboratories). Immunohistochemistry for T cells
was accomplished using mouse IgG1 anti-CD3 (clone SP34–2; BD
Pharmingen) or an irrelevant IgG1 antibody (clone MOPC-21; BD
Pharmingen) and detection system using ABD-Peroxidase Elite with
diaminobenzidine (Vector Laboratories).
Anti-DNA spot enzyme-linked immunosorbent assay
Sheared calf thymus DNA (Invitrogen, Carlsbad, CA) was coated
overnight onto 96-well microtiter plates (Dynex Technologies, Chan-
tilly, VA) at 10 ␮g/mL. After washing with PBS, plates were blocked for
1 hour with 0.5% bovine serum albumin in PBS at room temperature.
Mouse spleen cells (3 ⫻ 106 cells/well) or kidney suspension cells
(5 ⫻ 106 cells/well) in RPMI 1640 media with 10% fetal calf serum
were added into the first row of the plates and serially diluted at 1:2.
Plates were cultured overnight at 37°C, cells were aspirated, and plates
were washed 6 times followed by incubation with goat anti-mouse IgG
(Southern Biotechnology Associates) at 1 ␮g/mL for 2 hours at 37°C.
The plates were then washed 6 times with 0.05% Tween 20 in PBS.
Streptavidin-alkaline phosphatase was added to each well at 1:2000, and
plates were incubated at room temperature for 1 hour. After wash, the
plates were incubated with a mixture of 2-amino-2-methyl-1-propanol/5-
bromo-4-chloro-3 indolyl phosphate) and 0.6% agarose gel overnight at
4°C. Anti-ds/ssDNA IgG antibody-forming cells were enumerated as
blue spots using an inverted microscope and recorded as the number of
antibody-forming cells per spleen or per kidney.
3962 LIN et al
Results
Anti-BR3 mAb treatment resulted in greater mouse B-cell
reduction than BR3-Fc
Antibodies against mouse and human BR3 were generated by both
classic hybridoma and phage display technologies and selected
based on their ability to bind BR3 with high affinity, block BAFF
binding to BR3, and prevent BAFF-mediated B-cell survival and
proliferation in B-cell cultures.47,49 A phage-derived antibody
(CB2) was selected for further in vivo analysis.47 Fifteen days after
in vivo administration of anti-BR3 (CB2) mAb in BALB/c mice,
B cells were reduced in all lymphoid organs tested (Figure 1A-C).
In blood, splenic follicles (FO; CD23⫹CD21int), splenic MZ
(CD23loCD21hi), and lymph nodes, treatment with anti-BR3 mAb
resulted in profound B-cell reduction (90%-99%) compared with
isotype control-treated mice. In PP, FO cells (B220⫹CD38⫹) were
reduced similarly to other recirculating blood, spleen, and lymph
node compartments, whereas GC (B220⫹CD38lo) B cells were
reduced to a lesser extent (Figure 1A,C). In the peritoneal cavity
(PEC), B2 cells (B220⫹CD23⫹) and B1 cells (B220⫹CD23⫺) were
reduced by 90% and 70%–80%, respectively. Compared with the in
vivo effects of BR3-Fc fusion protein, anti-BR3 mAb treatment
caused a significantly higher level of B-cell reduction in blood,
spleen follicles, lymph nodes, and other compartments that contain
recirculating cells (PEC, PP follicles). In contrast, spleen marginal
zone B cells and plasma cells (PC) that do not recirculate and have
shown survival dependence on BAFF are reduced to similar levels
(Figure 1A,C). These findings were confirmed by immunofluores-
cence on spleen sections, which shows complete depletion of FO
and MZ regions in the anti-BR3 treated group with some residual
cells left in the follicles after BR3-Fc treatment (Figure 1B). Bright
plasma cells (IgM⫹) in the red pulp are reduced in both BR3-Fc-
and anti-BR3–treated groups.
Taken together, these data demonstrate that treatment with a
depleting antagonistic anti-BR3 antibody results in superior B-cell
reduction compared with BR3-Fc. These results were further
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12
Figure 2. Kinetics and comparative mouse B-cell
reduction after treatment with anti-BR3 mAb, BR3-Fc,
or anti-CD20 mAb. Mice ( ⱖ 5/group) treated with anti-
BR3 mAb (200 ␮g/week), BR3-Fc (150 ␮g ⫻ 3/week), or
anti-CD20 mAb (200 ␮g/week) were analyzed at different
times, with B-cell numbers and subsets evaluated by flow
cytometry. Anti-BR3 treatment reduced blood B cells
(expressed as percentage of baseline absolute cell counts)
faster and more profoundly than BR3-Fc but slower than
anti-CD20 treatment (A). By day 15 of treatment, the
extent of B-cell reduction in the lymph node in response
to anti-BR3 treatment appeared greater than that result-
ing from anti-CD20 mAb treatment (B). B-cell subsets
were quantified using similar definitions as in Figure 1.
Spleen and bone marrow plasma cells were defined as
CD138⫹B220lo/⫺. Analysis of different B-cell subsets after
8 weeks of continuous treatment (200 ␮g/week anti-BR3
and anti-CD20; 150 ␮g ⫻ 3/week BR3-Fc) shows signifi-
cantly higher MZ B-cell, spleen, and BM plasma cell
reduction in anti-BR3 mAb compared with anti-CD20
mAb treatment (C). Serum IgM and IgG1 were measured
by ELISA after 3 months of continuous treatment (D).
These data are representative of 3 independent experi-
ments. Data are shown as mean plus or minus standard
error. Statistical significance compared with the control
treated group is indicated with an asterisk. When 2
treatment groups are significantly different from each
other, the P value is displayed.
confirmed by more frequent and longer-term treatments (data not
shown).
Anti-BR3 treatment reduced mouse B cells more slowly but
more profoundly in certain subsets than anti-CD20
To compare the effects of anti-BR3 and anti-CD20 mAbs on B-cell
depletion, we used human CD20BACTg mice.13 Analysis of the
kinetics of blood B-cell depletion with anti-BR3 revealed a 75%
reduction within 1 day and 99% over 8 days (Figure 2A). In
contrast, treatment with anti-CD20 mAb was faster at reducing
blood B cells (75% reduction within 1 hour and 99% in 1 day),
whereas the effect of BR3-Fc was slower than both anti-BR3 and
anti-CD20 (Figures 2A, S2). In lymph nodes, B-cell reduction
achieved by anti-BR3 mAb occurred at a rate slower than that
induced by anti-CD20 but became superior in magnitude by day 15
(Figures 2B, S2).
To evaluate maximal B-cell reduction reached with these
therapies in various B-cell subsets, we compared courses of
8 weeks of continuous treatment with each reagent. Blood B cells
were reduced by 99% by both anti-CD20 and anti-BR3 mAbs and
by 90%-95% in the BR3-Fc–treated mice (Figure 2C). In spleen
and lymph nodes, FO cells were virtually eliminated by anti-BR3
mAb, whereas small residual subsets were seen after treatment with
either anti-CD20 mAb (mostly MZ, few FO) or BR3-Fc (few FO).
Reduction of PP GC cells and spleen plasma cells were comparable
in mice treated with either anti-BR3 mAb or BR3-Fc (⬃75% and
50%, respectively). In contrast, no significant reduction in PP GC
or spleen plasma cells was observed after treatment with anti-CD20
mAb, suggesting that decrease in these 2 subsets was probably due
to BAFF blockade.
The numbers of bone marrow (BM) PCs, which are enriched in
long-lived PCs, were not altered by long-term treatment with either
anti-CD20 mAb or BR3-Fc. However, BM PCs showed a signifi-
cant decrease (⬃50%) with anti-BR3 mAb treatment. These effects
on BM plasma cells translated into significant decreases in serum
IgM and IgG after anti-BR3 mAb treatment, whereas only IgG1
changed after 3 months of BR3-Fc treatment (Figure 2D and data
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12 IN VIVO B CELL EFFECTS OF ANTI-BR3 ANTIBODIES 3963

Figure 3. NHP B-cell reduction effects of anti-BR3

mAb, BR3-Fc, and anti-CD20 mAb. Comparison of
blood B cell numbers from cynomolgus monkeys treated
with anti-BR3 mAb (20 mg/kg/week: days 0-29, n ⫽ 16;
days 29-60, n ⫽ 10; terminal d29, n ⫽ 6), BR3-Fc (20 mg/
kg/week: days 0-29, n ⫽ 12; days 29-60, n ⫽ 4; terminal
d29, n ⫽ 8), anti-CD20 (2 ⫻ 50 mg/kg: days 0 and 14,
n ⫽ 4), and vehicle control (same numbers as active agent
groups) (A). Anti-BR3 appeared to reduce blood and
spleen B cells faster and more profoundly than BR3-Fc but
slower and less completely compared with anti-CD20
mAb. Naive lymph node B cells (CD20⫹CD21⫹CD27⫺)
were reduced similarly by anti-BR3 and anti-CD20 treat-
ments and less by BR3-Fc. Other lymph node B-cell
subsets—memory (CD20⫹CD21⫹CD27⫹), marginal zone
(CD20⫹CD21hiCD27⫺/⫹), germinal center (CD20⫹CD21⫺)
cells—were reduced similarly by all treatments (germinal
center cells were not assayed in the BR3-Fc-treated
experiment) (B). Statistical significance compared with the
control treated group is indicated with an asterisk. Because
treatment groups were in separate experiments, statistical
analysis was always performed against internal vehicle
control group. Treatment with anti-BR3 or BR3-Fc result in
spleen B-cell reduction measured by immunohistochemis-
try. B cells, blue (anti-CD20); T cells, orange (anti-CD3)
(C). See “Immunofluorescence and immunohistochemistry
staining” for image acquisition details.

not shown). Anti-CD20 mAb caused no reduction in serum
immunoglobulin even after 1 year of continuous treatment.13
Because bone marrow plasma cells express low levels of BR3 but
not CD2050 in both mice and humans (Figure S3), anti-BR3 mAbs
may provide a therapeutic modality to target BM PCs.
The B-cell reduction profile of anti-BR3 was distinct from those
of anti-CD20 and BR3-Fc in NHP
B-cell reduction profiles elicited by BAFF-targeting reagents from
mice and primates are different.17,19,20 Although mouse B cells are
exquisitely sensitive to BAFF targeting and more than 90% are
eliminated within 2 weeks of treatment, NHP and human studies
have shown a maximum of only 50% overall reduction after 1 to
6 months of treatment. This is due in part to different B-cell subset
sensitivities for BAFF blockade (memory B cells, which comprise
a larger part of the primate repertoire, appear less sensitive) but also
to other unknown factors that might differ between rodents and
primates. To evaluate and help translate the anti-BR3 findings from
mouse to humans, we performed an NHP study in cynomolgus
monkeys with a humanized antagonistic anti-BR3 antibody (h9.1).
This clone is a surrogate of the mouse antibody (high affinity for
human and cynomolgus monkey BR3, BAFF-blocking in vitro
cellular assays, capable of ADCC, data not shown) while binding a
different epitope on BR3.47 Monkeys were treated with anti-BR3,
BR3-Fc, or anti-CD20 (in parallel experiments), and their B cells
were compared with a control group. In contrast to mice, in which
99% of blood B cells were eliminated within 1 to 2 weeks after
anti-BR3 mAb treatment, blood B cells in cynomolgus monkeys
decreased by approximately 50% after 2 weeks (Figure 3A).
Maximum blood B-cell reduction of approximately 75% was
reached after 1 month and remained near this level for the rest of
the treatment. Lymphoid organ analysis at a 4-week timepoint
using flow cytometry and immunohistochemistry showed that in
spleen and lymph nodes, B cells were decreased by approximately
80% (Figure 3B,C). Reduction of naive B cells in blood and lymph
nodes (both FO and MZ) was faster and more profound compared
with memory B cells (Figure 3B and data not shown), suggesting a
differential sensitivity for BAFF blockade for these subsets in
primates. Consistent with this lower primate sensitivity to BAFF
blockade compared with mice, serum immunoglobulins did not
significantly change after the first 3 months of anti-BR3 mAb
treatment (data not shown).
Compared with the anti-CD20 mAb and BR3-Fc studies,
anti-BR3 mAb was intermediate between the 2 in both kinetics and
degree of B-cell reduction in blood and spleen (Figure 3A-C).
BR3-Fc does not reduce blood B cells significantly in the first
4 weeks, whereas anti-BR3 and anti-CD20 mAbs do, suggesting
that cellular depletion, not survival blockade, is the main mecha-
nism responsible in this time frame for blood B-cell reduction.
Reduction of lymph node B cells by anti-BR3 treatment seems to
be similar to that induced by anti-CD20 mAb (Figure 3B). These
findings in NHP show clear differences of the BAFF/BR3 system
compared with rodents and have important consequences in
guiding translatability of studies in rodents to treatment of human
diseases.
Anti-BR3 reduced some B-cell subsets more profoundly than
BR3-Fc in the NZB/W lupus-prone mouse strain
To examine the effects of anti-BR3 immunotherapy, we evaluated
the potency on autoimmune B-cell reduction of this dual targeting
agent after treatment in the lupus-prone NZB/W mouse strain.
Seven- to 8-month-old NZB/W mice with established proteinuria
(30-100 mg/dL) were treated with control, anti-BR3 mAb, or
BR3-Fc continuously for up to 5.5 months. Treatment with
anti-BR3 mAb decreased total B cells in blood and spleen to a
greater extent compared with BR3-Fc (Figure S5). Although
overall B-cell depletion in diseased NZB/W mice was efficient, the
reduction was less than that observed in normal healthy mice used
as control group in the same experiment (data not shown). As
early as 2 weeks (data not shown), and maximal at 6 weeks,
anti-BR3 mAb and BR3-Fc treatments decreased spleen anti-
DNA-producing plasma cells, total plasma cells, and kidney-
infiltrating B and T cells but left naive T cells unchanged (Figure
4A). Spleen germinal center B cells and noncycling IgG1
3964 LIN et al
plasma cells were reduced more with anti-BR3 mAb than
BR3-Fc treatment (Figure 4B,C; detailed below).
To evaluate the impact of anti-BR3 mAb or BR3-Fc treat-
ment on cycling versus noncycling B-cell compartments, we
performed BrdU pulse experiments. In contrast to cyclophospha-
mide (Cytoxan), which reduces mainly cycling cells, both
therapies decreased cycling and noncycling B-cell compart-
ments effectively after 11 days (Figures 4C, S5). Likewise, both
cycling and noncycling plasma cells were reduced; the isotype-
switched plasma cells were more profoundly decreased than
IgM-positive plasma cells after therapy. Trends toward greater
effects from anti-BR3 mAb compared with BR3-Fc treatment
were observed in all compartments, but statistical significance
between the 2 groups was reached only for IgG1 plasma cells.
Effects of treatment on the plasma cell compartment were
paralleled by decreases in total serum IgM and IgG isotypes, as
well as of IgG anti-DNA antibodies (data not shown). In all
these readouts, anti-BR3 mAb was superior to BR3-Fc, suggest-
ing that, similar to normal mice, effects on the autoimmune
plasma cell compartment are more pronounced with anti-BR3
mAb than BR3-Fc.
This series of experiments showed that anti-BR3 mAb reduced
certain subsets (GC B cells, IgG1 plasma cells) more profoundly
than BR3-Fc, although in all other B-cell subsets, the 2 therapies
have similar effects.
Figure 5. Anti-BR3 combines 2 mechanisms of action. BALB/c mice (5/group) were treated with the wild-type BAFF-blocking anti-BR3 mAb (CB2), anti-BR3 Fc mutant mAb
(CB2-DANA), anti-BR3 nonblocking mAb (PIH11), or BR3-Fc and evaluated for B-cell reduction at days 1 and 6. In blood at day 1, only treatments that had the ability to engage
Fc␥ receptors and induce ADCC showed B-cell reduction, whereas at day 6, ADCC and survival blockade appear to combine for maximal effects (A). In spleen FO cells, both
mechanisms appeared to contribute to B-cell reduction, whereas in MZ, the dominant mechanism appears to be BAFF/BR3-dependent survival blockade. At day 6, the spleen
plasma cell compartment was reduced only by ADCC competent molecules (B). Statistical significance compared with the control-treated group is indicated with an asterisk; P
values between groups are shown numerically.
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12
Figure 4. Anti-BR3 mAb decreases pathogenic B-cell
subsets in the NZB/W F1 SLE mouse model. Therapeu-
tic treatment of proteinuric NZB/W F1 mice (15/group) for
6 weeks with anti-BR3 mAb (600 ␮g/week) or BR3-Fc
(300 ␮g ⫻ 3/week) reduced splenic anti-DNA-producing
cells, kidney-infiltrating B cells (B220⫹), and T cells (CD3⫹)
but not naive spleen T cells (A). Splenic germinal center B
cells (B220⫹CD38lo) were decreased only by anti-BR3 mAb
treatment, whereas plasma cells (CD138⫹B220lo) were de-
creased by both treatments (B). After 1 week, anti-BR3
mAb and BR3-Fc treatment showed significant reduction
in both cycling and noncycling splenic B cells (B220⫹)
and IgG1 plasma cells (cytoplasmic IgG1⫹CD138⫹), but
only anti-BR3 mAb treatment resulted in significant reduc-
tion in IgM plasma cells (cytoplasmic IgM⫹CD138⫹) (C).
Statistical significance compared with the control treated
group is indicated with an asterisk. When 2 treatment
groups are significantly different from each other, the P
value is displayed.
Fc-mediated B-cell depletion and survival blockade contributed
to the mechanism of anti-BR3 mAb action
To further dissect in vivo the mechanisms by which anti-BR3
(CB2) mAb mediates B-cell reduction, we compared the wild-type
mAb with a D265A/N297A anti-BR3 mutant mAb incapable of Fc
receptor binding and ADCC but still capable of BAFF/BR3
blockade.51 This anti-BR3 Fc mutant molecule had the same
affinity for BR3 and blocked BAFF-BR3 indistinguishably from
the wild-type molecule (data not shown). We further compared the
effects due to treatment with a nonblocking anti-BR3 mAb (PIH11)
capable of inducing ADCC, but that does not prevent BAFF/BR3,
whereas a final comparison group was treated with BR3-Fc, which
blocks BAFF binding to all its receptors without any B-cell-
directed ADCC. One day after therapeutic administration, both
blocking and nonblocking anti-BR3 mAbs showed significant
peripheral B-cell reduction, whereas the anti-BR3 mutant mAb and
BR3-Fc did not (Figure 5A). These data indicate that early
peripheral B-cell reduction appeared to result solely from Fc-
mediated mechanisms. After 6 days of treatment, a greater degree
of peripheral B-cell reduction was observed in mice treated with
wild-type anti-BR3 (CB2) mAb than in all other groups. Hence,
both Fc-mediated cell killing and BAFF/BR3 survival blockade
function in vivo and contribute to B-cell reduction.
For spleen and lymph node FO B cells, the best reduction was
again achieved with the blocking anti-BR3 (CB2) mAb, whereas
BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12
all other groups showed lower but significant decreases in B-cell
populations (Figure 5B). In contrast, the MZ compartment, which
is extremely BAFF dependent for survival but does not recirculate,
demonstrated comparable degrees of B-cell reduction with anti-
BR3 (CB2) mAb, mutant anti-BR3 (mutant CB2), and BR3-Fc
(Figure 5B). That BAFF blockade represents a major mechanism of
B-cell reduction by anti-BR3 (CB2) mAb is further supported by
the lesser reduction in MZ B cells by a nonblocking anti-BR3
(PIH11) mAb.
Finally, at day 6, spleen plasma cells were decreased by
treatment with depleting blocking and nonblocking anti-BR3
(CB2 and PIH11) but not by mutant anti-BR3 or BR3-Fc (Figure
5B). This suggests that Fc-mediated depletion functions to
decrease tissue-resident plasma cells. Because longer treatments
with BR3-Fc reduce this compartment in the spleen (Figures
1C,2C) but not in bone marrow, Fc-mediated cell depletion
confers additional qualitative properties distinct from BAFF
survival blockade.
These data, taken together, demonstrate that a blocking anti-
BR3 antibody reaches maximal efficiency to reduce B cells by
combining Fc-mediated cell killing with survival blockade of the
BAFF/BR3 pathway.
Discussion
In an attempt to create a therapeutic strategy combining B-cell
depletion with survival factor blockade, we generated anti-BR3
mAbs with dual attributes—depletion and blockade of survival
signals. In both mice and primates, these antibodies were shown
to use a dual mechanism in reducing different B-cell subsets. In
mice, the effect of anti-BR3 was slower than anti-CD20, faster
than BR3-Fc, and ultimately resulted in a maximal B-cell
reduction profile superior to anti-CD20 in particular B-cell
subsets. This was evident in subsets that are relatively resistant
to anti-CD20 depletion (eg, spleen marginal zone, Peyer patch
germinal centers, spleen, and bone marrow plasma cells). It is
important to mention that although these conclusions reflect
comparisons between representative anti-BR3 and anti-CD20
mAbs, other specific antibodies against different CD20 epitopes
and with additional or enhanced depleting properties could
behave somewhat differently. In NHP, similar to mice, anti-BR3
mAb B-cell depletion was faster than BR3-Fc and slower than
anti-CD20. However, the maximal B-cell reduction obtained
after 1 month of treatment was approximately 80% in certain
subsets (naive, follicular, and marginal zone) and less in others
(memory, germinal center, and plasma cells). This reduction was
superior to previously reported anti-BAFF reagents (anti-BAFF
antibodies, BR3-Fc, TACI-Fc), showing that anti-BR3 mAb, in
addition to blocking survival, is also depleting B cells in NHP.
Compared with anti-CD20, which entirely depletes NHP blood
and spleen B cells and incompletely depletes lymph node B
cells, anti-BR3 B-cell reductions appear less in blood and spleen
and comparable in lymph nodes.52 The faster kinetics of B-cell
reduction of anti-CD20 is probably due to the higher density of
CD20 surface expression compared with BR3 as well as capping
differences between these molecules after mAb binding (data
not shown). Although anti-CD20 depletion translates relatively
well from rodents to primates, blockade of the BAFF/BR3
system does not. It is likely that this is due in part to a reduced
dependence for BAFF survival signals of memory, germinal
center B cells, and plasma cells compared with naive B cells.
IN VIVO B CELL EFFECTS OF ANTI-BR3 ANTIBODIES 3965
Because the primate immune system is populated at steady state
with a much larger proportion of memory, germinal center, and
plasma cells, compared with that in pathogen-free mice, pri-
mates could be expected to be less affected by BAFF blockade
therapy than mice. Supporting this hypothesis, B-cell reduction
after treatment with anti-BR3 mAb in lupus-prone mice, which
have an activated immune system, is inferior to that seen in
normal BALB/c, C57BL/6, or FvB mice. In addition, unknown
primate B-cell survival characteristics could also contribute to
this apparent discrepancy between species. Initial results from
mice deficient in members of the BAFF/BR3-induced alterna-
tive nuclear factor ␬B (NF-␬B) 2 pathway suggested that this
pathway was indispensable for B-cell development and survival.
However, more recent studies have demonstrated that the classic
and alternative NF-␬B pathways can replace each other in
certain conditions during B-cell ontogeny.53,54 Considering the
large number of cues feeding into these pathways during B-cell
development (eg, BCR, BR3, CD40, TLR, interleukins), it is
possible that during evolution, mice and primates developed
different optimal combinations of survival and maturation
signals. Further research in dissecting the developmental cues in
various B-cell subsets in mice versus primates should shed light
on this question.
Another aspect of therapeutical targeting relates to the B-cell
subsets that are affected differentially with various strategies.
Clinical and preclinical data shows that marginal zone B cells, as
well as memory, germinal center, and plasma cells, are deregulated
in human rheumatologic disorders. Marginal zone B cells associ-
ated with high levels of circulating BAFF have been described in
both human Sjögren syndrome salivary gland infiltrates as well as
in a Sjögren syndrome mouse model.28,55 Likewise, subsets of
patients with memory B cells, germinal center, and plasma cells
with different organization patterns in the joint have been described
in patients with rheumatoid arthritis.56 Recent data from arthritis
patients treated with anti-CD20 therapy show that B-cell depletion
in the bone marrow and joints is incomplete, although the
correlation of this finding with clinical response has not been
studied yet.57,58 A more direct parallel between incomplete malig-
nant B-cell depletion by immunotherapy and clinical relapse is
better understood in NHL.5 In this light, for clinical benefit,
different human diseases with B-cell deregulation could require
distinct levels of B-cell reduction better achieved through an
appropriate but distinct B-cell immunotherapy strategy.
Regardless of B-cell depletion level, because of the demon-
strated dual mechanism of action of anti-BR3 mAbs, surviving
residual B cells will be in an environment without a functional
BAFF/BR3 pathway. This is important for potential therapy
because both in oncology (CLL, NHL) and immunology (RA, SLE,
Sjögren syndrome), increased BAFF levels signaling through its
receptors contribute to pathogenic B-cell survival.59,60
BR3 was expressed at different levels on the surface of both the
malignant and normal residual B cells in a series of 16 patients with
CLL who varied widely with respect to CD20 expression (3/16
were negative as evaluated by flow cytometry; Figure S6). It was
suggested that some of the in vitro BAFF survival effects on CLL B
cells result through non-BR3 receptor (TACI) signaling and classic
NF-␬B activation.59 However, a functional BR3 survival pathway
does exist in CLL B cells, and its in vivo inhibition could be
beneficial in addition to the anti-BR3 mAb cellular depletion
mechanism. Compared with normal B cells, CLL B cells have
lower levels of CD20 expression, which probably contributes to the
relative lower efficacy of anti-CD20 immunotherapy in CLL
3966 LIN et al BLOOD, 1 DECEMBER 2007 䡠 VOLUME 110, NUMBER 12

compared with NHL.61 Patients with negligible CD20 on CLL
B cells that express significant BR3 constitute prime candidates for
potential therapeutic benefit from anti-BR3 treatment. It is impor-
tant to point out that our data show properties of anti-BR3 mAb
largely in normal and mouse autoimmune B cells. Additional
experiments will be required to evaluate this therapy in the context
of malignant B cells.
Finally, reduction of BM plasma cells after anti-BR3 mAb
treatment in mice parallels the synergistic effect seen with combin-
ing anti-CD20 mAb and BR3-Fc.13 If this effect translates even
partially to humans, it should have therapeutic value in subsets of
autoimmune patients with pathogenic long-lived autoantibody
components. Future clinical trials in both oncology and immunol-
ogy will test the translatability and therapeutic value of B-cell
reduction through anti-BR3 mAbs in human disease.
Acknowledgments
We acknowledge the hard work and dedication of the in vivo
immunology group in dosing, tracking down, and analyzing a very
large number of studies, Wesley Cheng and Ronald Ferrando for
immunofluorescence, and the development sciences FACS labora-
tory for the NHP FACS.
Authorship
Contribution: W.Y.L, Q.G., D.S., Z.L., W.P.L., K.L., S.Y.,
L.E.D., Q.O., S.Y., E.S., J.S., M.B., M.A.S., and F.M. performed
research, collected, analyzed and interpreted data; K.H., M.L.,
S.I., J.B., D.D., and T.G. analyzed and interpreted data; CW.L.,
J.F., J.S.T., C.A., and A.E. contributed vital new reagents; T.G.,
M.B., M.A.S., and F.M. designed research; and FM drafted the
manuscript.
Conflict-of-interest disclosure: The authors are employees of
Genentech, with the exception of J.S.T. and C.A., who are
employees of Biogen-IDEC.
Correspondence: Flavius Martin, Department of Immunology,
Genentech, Inc, 1 DNA Way, MS34, 12-286, South San Francisco,
CA 94080; e-mail: flavius@gene.com.

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