S13
ACR = American College of Rheumatology; BLyS = B lymphocyte stimulator; RA= rheumatoid arthritis; SLAM = Systemic Lupus Activity Measure;
SLE = systemic lupus erythematosus; SLEDAI = Systemic Lupus Erythematosus Disease Activity Index; TACI-Ig = transmembrane activator and
calcium-modulator and cyclophilin ligand interactor-immunoglobulin.
Available online http://arthritis-research.com/contents/7/S3/S13
Abstract
Although the precise pathogenesis of rheumatoid arthritis (RA)
remains unclear, many cell populations, including monocytes,
macrophages, endothelial cells, fibroblasts and B cells, participate
in the inflammatory process. Ongoing research continues to
evaluate the critical roles played by B cells in sustaining the
chronic inflammatory process of RA. These findings have
contributed to the development of targeted therapies that deplete
B cells, such as rituximab, as well as inhibitors of B lymphocyte
stimulation, such as belimumab. In a phase I trial, belimumab
treatment significantly reduced CD20+ levels in patients with
systemic lupus erythematosus. Phase I and phase II trials of
rituximab found that rituximab plus methotrexate achieved
significantly better American College of Rheumatology 50%
responses for patients with RA than those patients receiving
monotherapy with methotrexate. These clinical trial data present
promising evidence for B cell targeted therapies as future
therapeutic options for RA.
Introduction
Advances in our understanding of the pathogenesis of
rheumatoid arthritis (RA) have allowed clinicians to target
selectively the pathogenic elements of this disease. Until
recently, treatment focused on targeting cytokines such as
tumor necrosis factor-α and interleukin (IL)-1. However, it may
be more appropriate to move treatment upstream from the
cytokines to target the cellular processes that drive
rheumatoid disease. Recent studies have shown that
antibody-producing B cells not only generate antibodies but
also present antigen to T cells, resulting in many of the
cellular events and inflammatory processes of RA.
In an overview of studies of the roles played by B cells and B
cell depletion in RA, Tsokos [1] suggested that B cells
contribute to the expression of autoimmunity, and that
antibodies that target B cells, such as anti-CD20, may
substantially abrogate expression of disease. Tsokos
suggested four possible mechanisms of action by which anti-
CD20 could deplete B cells. First, after the antibody binds to
the extracellular domain of the CD20 antigen, it may activate
complement and lyse the targeted cell. Second, anti-CD20
antibody may permit antibody dependent, cell-mediated
cytotoxicity, which occurs after the Fc portion of the antibody –
the part of the antibody responsible for binding to cell
receptors – is recognized by appropriate receptors on
cytotoxic cells. Third, the antibody may alter the ability of B
cells to respond to antigen or other stimuli. Finally, anti-CD20
antibody may initiate programmed cell death or apoptosis of
B cells. All of these mechanisms of B cell depletion may be
involved to variable degrees, depending on which B cell pool
is affected. For example, Fc receptor mediated cytotoxicity is
prominent in the destruction of B cells in peripheral blood,
whereas complement activation is involved in the killing of B
cells in lymphoid organs [1].
Thus, the new concept is not only to target cytokines but also to
target the cellular elements, such as B cells, that cause or
perpetuate RA. How, then, may B cells be targeted? There are
several ways to target B cells. First, one may target cytokines
that promote B cell function and survival, such as IL-6 and B
lymphocyte stimulator (BLyS). This may be done in several
ways. One can produce an antibody to BLyS or use a soluble
receptor such as transmembrane activator and calcium-
modulator and cyclophilin ligand interactor-immunoglobulin
(TACI-Ig) to block positive signaling through BLyS receptors.
Second, B cells may be depleted by monoclonal antibodies that
inhibit CD19, CD20, CD21, or CD22 B cell surface antigen.
Finally, the co-stimulatory molecule may be targeted, preventing
B cells from contributing to the inflammatory process through
processing autoantigen and presenting it to T cells [2], as well
as by producing cytokines and autoantibody. Treatment with
anti-CD20 antibody destroys mature B cells in central lymphoid
organs, the synovium, and the peripheral blood.
Review
B cell targeted therapies
Edward Keystone
Department of Medicine, University of Toronto, Ontario, Canada
Corresponding author: Edward Keystone, edkeystone@mtsinai.on.ca
Published: 18 May 2005 Arthritis Research & Therapy 2005, 7(Suppl 3):S13-S18
This article is online at http://arthritis-research.com/content/7/S3/S13 (DOI 10.1186/ar1738)
© 2005 BioMed Central Ltd

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Arthritis Research & Therapy June 2005 Vol 7 Suppl 3 Keystone
Silverman and Carson [2], in their analysis of B cells in RA,
found that B cells are also very efficient antigen presenting
cells, contributing to T cell activation through expression of co-
stimulatory molecules. In addition to being the precursors of
antibody-secreting plasma cells, the B cells play a critical role
in RA in terms of the afferent arm of the immune response.
Thus, B cells can act as highly efficient antigen presenting
cells, which assist in the activation of autoreactive T cells.
B cells both respond to and produce the chemokines and
cytokines that promote leucocyte infiltration into the joints,
formation of ectopic lymphoid structures, angiogenesis, and
synovial hyperplasia. The success of B cell depletion therapy
in RA may depend on disruption of some or all of these
diverse functions.
B lymphocyte stimulator inhibition: belimumab
Therapies that inhibit BLyS currently under development
include soluble receptors such as TACI-Ig and anti-BLyS
monoclonal antibodies. Furie and coworkers [3] conducted a
phase I investigation of belimumab (LymphoStat-B, Human
Genbome Sciences Inc., Rockville, MD, USA), a fully human
monoclonal antibody that inhibits soluble BLyS. This
randomized, double-blind study was designed to evaluate the
safety, tolerability, immunogenicity and pharmacology of four
different doses (1, 4, 10 and 20 mg/kg) of LymphoStat-B or
placebo, administered as a single intravenous infusion or two
infusions 21 days apart. Study participants had stable systemic
lupus erythematosus (SLE) of mild-to-moderate disease
activity and were receiving a stable standard-of-care SLE
treatment regimen for 2 months before enrollment.
Patients were followed for 84–105 days to assess peripheral
B cell concentrations, serologies, and disease activity (using
the Safety of Estrogens in Lupus Erythematosus: National
Assessment [SELENA] Systemic Lupus Erythematosus
Disease Activity Index [SLEDAI]), and to monitor adverse
events, pharmacokinetics, and safety. Data from placebo
patients (n = 13) in single-blind or double-dose cohorts were
pooled and compared with those receiving LymphoStat-B
(n = 57) in each of the four single or double dose cohorts.
The pharmacokinetics of single doses were dose propor-
tional. The lengthy half-life (13–17 days), slow clearance
(4.00 ± 1.56 ml/day per kg) and are consistent with a fully
human monoclonal antibody. All LymphoStat-B cohorts
exhibited significant reductions in CD20+ cells (12–47%) at
one or more visits from days 42–105 compared with
placebo.
Furie and coworkers found that LymphoStat-B was well
tolerated at all doses, with no study withdrawals. The overall
incidence of adverse events was similar between
LymphoStat-B and placebo groups. No increased incidence
of infections occurred in the treatment group, and none of the
infections reported was attributed to the study agent.
Six patients experienced serious adverse events, with similar
frequencies observed in the placebo and treatment groups.
None were deemed to be related to the study agent. Severe
(grade 3 and 4) laboratory abnormalities or adverse events
occurred infrequently. One patient experienced an infusion
reaction at the highest single dose, while another developed
neutralizing antibodies to LymphoStat-B.
Reductions in anti-dsDNA or immunoglobulin levels
compared with placebo were observed in some LymphoStat-
B cohorts. No change in SLE disease activity was observed
over this short exposure. The investigators concluded that
there was a significant reduction in peripheral B cells with
LymphoStat-B treatment, consistent with its ability to bind
and inhibit the biological activity of BLyS.
Rituximab
Primary analysis
Rituximab, a genetically engineered chimeric anti-CD20
monoclonal antibody, is currently approved for the treatment
of relapsed or refractory, low grade, or follicular CD20+ B cell
non-Hodgkin’s lymphoma. Rituximab selectively depletes B
cells that bear the CD20 surface marker via multiple
mechanisms that include antibody-dependent cellular
cytotoxicity and complement-dependent cytotoxicity, and via
the induction of apoptosis.
Edwards [4] hypothesized that depletion of B lymphocytes
could represent a new treatment for RA. In order to assess
better the efficacy of this B cell targeted therapy, a
randomized, double-blind, controlled study was carried out to
examine the effect of selective depletion of B cells with
rituximab in patients with RA [5].
A total of 161 patients with active RA who had failed to
respond to treatment with methotrexate (at least 10 mg/week
for at least 16 weeks) were randomly assigned to one of four
treatment regimens: oral methotrexate as a control arm;
intravenous rituximab alone (1000 mg on days 1 and 15);
intravenous rituximab plus cyclophosphamide (750 mg on days
3 and 17); or rituximab plus methotrexate. All patients received
100 mg methylprednisolone just before each treatment (or
intravenous placebo), as well as prednisone 60 mg/day on day
2 and days 4–7, and 30 mg/day on days 8–14.
Clinical assessments were performed at baseline (day 1) and at
weeks 12, 16, 20 and 24, using the American College of
Rheumatology (ACR) core set of disease activity measures:
swollen and tender joint counts (66 joints evaluated), patients’
evaluation of pain based on a scale of 0 (no pain) to 100
(unbearable pain), patients’ global assessment of disease activity
on a scale from 0 (disease inactivity) to 100 (maximal disease
activity), physicians’ assessment of disease activity, assessment
of physical function reported by patients utilizing a health
assessment questionnaire, and laboratory evaluation of serum C-
reactive protein levels and erythrocyte sedimentation rate.

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Patient responses were assessed at week 24 for the primary
analyses and at week 48 for the exploratory analyses. The
primary end-point was the proportion of patients with an
ACR50 response at week 24. Secondary end-points were
ACR20 (a 20% improvement according to ACR criteria) and
ACR70 (a 70% improvement according to ACR criteria)
responses, as well as a response based on the European
League Against Rheumatism criteria.
At week 24, the proportion of patients with an ACR50
response was significantly greater for the group of patients
taking the rituximab–methotrexate combination (43%;
P = 0.005) and the rituximab–cyclophosphamide group
(41%; P = 0.005) than for those receiving methotrexate alone
(13%). Investigators also noted that the number of patients
achieving an ACR50 response in the group receiving
rituximab alone was greater than that in the control group, but
this failed to reach statistical significance (P = 0.059).
Notably, the mean change from baseline in Disease Activity
Score at week 24 reflected significant improvement over
methotrexate alone in all rituximab groups (P = 0.002). The
clinical parameters employed in the calculation of the Disease
Activity Score include number of tender joints, number of
swollen joints, erythrocyte sedimentation rate, and the
patient’s subjective assessment of disease activity.
Exploratory analysis: a 48 week study
An exploratory analysis of ACR responses at 48 weeks found
that, of patients in the rituximab–methotrexate group, 35%
(P = 0.002) and 15% (P = 0.03) had ACR50 and ACR70
responses, respectively, as compared with 5% and 0% in the
methotrexate control group [5,6]. In addition, 27% of patients
in the rituximab–cyclophosphamide arm achieved an ACR50
response (P = 0.01; Fig. 1).
In terms of pharmacodynamic outcomes, treatment with
rituximab was associated with nearly complete depletion of
peripheral blood B cells throughout the entire 24-week
period. Although such a profound reduction in B cells may
suggest greater susceptibility to infection, the overall
incidence of infection was similar in the control and rituximab
groups at 24 and 48 weeks. By week 24, one patient in the
control group and four in the rituximab groups had suffered a
serious infection. An additional two serious infections –
including a fatal bronchopneumonia – were reported during
the extended 48-week period in the rituximab group. There
was no accumulation of any particular type of infection in
rituximab treated groups.
Despite B cell depletion, immunoglobulin levels did not
change substantially. Patients in the rituximab groups
experienced substantial and rapid reductions in rheumatoid
factor levels, whereas those in the methotrexate alone group
experienced only a moderate reduction in rheumatoid factor
levels [5] (Fig. 2).
Adverse events
Overall, 73–85% of patients in all treatment groups reported
at least one adverse event, with hypertension, hypotension,
nasopharyngitis, arthralgia, back pain, hyperglycaemia,
cough, flushing and headache reported most often. Of
patients in each group, 30–45% experienced events associa-
ted with initial infusion, although 85–95% of adverse events
related to rituximab infusions were characterized as mild or
moderate.
Infusion reactions occurred during the first infusion in
approximately one-third of patients in the groups receiving
rituximab and the placebo group (36% and 30%,
respectively). The rate of first infusion reaction in patients with
Available online http://arthritis-research.com/contents/7/S3/S13
Figure 1
Rituximab in rheumatoid arthritis. Phase IIa: American College of Rheumatology (ACR) responses at 48 weeks (NRI). *P < 0.0001; †P = 0.003;
‡P = 0.03; §P = 0.01; P values using Fisher’s exact test, comparing MTX with each RTX group. CTX, cyclophosphamide; MTX, methotrexate;
NRI, nonresponder imputation; RTX, rituximab. With permission from Edwards and coworkers [5]. Copyright © 2004 Massachusetts Medical
Society. All rights reserved.
%
ACR20 ACR50 ACR70
80
70
60
50
40
30
20
10
0
MTX
RTX
RTX + CTX
RTX + MTX
20
33
49§
65*
5
15
27§
35†
0
10 10
15‡

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RA was considerably lower than rates seen in patients with
non-Hodgkin’s lymphoma (36% versus 70–80%). According
to the 48-week data, the most common infusion related
reactions reported by patients were hypotension (14%
versus 10% in placebo group), hypertension (9% versus
5%), flushing (6%), pruritus (6%), and rash (6%). Six patients
during the primary 24-week trial period and an additional
three at 48 weeks withdrew from the trial because of adverse
events, including exacerbation of ongoing RA, hypotension
and bronchopneumonia, staphylococcal septicaemia, renal
impairment and rash.
Rituximab in systemic lupus erythematosus
Albert and coworkers [7] conducted a phase I study to
determine the safety and efficacy of B cell depletion with the
anti-CD20 monoclonal antibody rituximab as treatment for
SLE. Seven patients with active and persistent SLE who had
failed at least one immunosuppressive agent enrolled in this
pilot study. Each patient received 4-weekly infusions of
rituximab at 375 mg/m2.
Six out of seven patients exhibited a clinical response,
defined as improvement in SLEDAI. All but one patient had
greater than 99% B cell depletion lasting more than
3 months. The patient who exhibited only 95% B cell
depletion had no clinical response. Interestingly, this patient
also showed responses to immunizations, whereas two other
patients with complete B cell depletion (and clinical
response) failed to achieve adequate immune responses.
Among clinical responders, 50% had a brief remission
(6 weeks to 6 months), and the other 50% had a more
prolonged remission (6–9 months).
Among longer term responders, the steroid dose was either
stabilized or lowered; one patient had been in remission for
14 months without the need for steroid therapy. All brief
responders returned to an immunosuppressive regimen.
There was a trend toward a decrease in the percentage of B
cells expressing CD86 among all patients. There was no
distinctive pattern of other cell surface marker expression
among responders and nonresponders. No consistent
changes were found in serum chemistry, serum complement
levels, autoantibody titres and immunoglobulin levels.
A study conducted by Anolik and coworkers [8] found that,
compared with normal control individuals, lupus patients
exhibited several abnormalities in peripheral B cell homeo-
stasis at baseline, including naïve lymphopenia, and
expansion of circulating plasmablasts. Remarkably, these
abnormalities partially resolved after effective B cell depletion
with rituximab and immune reconstitution [8].
The frequency of autoreactive VH4.34 memory B cells also
decreased 1 year following treatment, despite the presence
of low levels of residual memory B cells at the point of
maximal B cell depletion and persistently elevated serum
autoantibody titres in most patients. This study demonstrates
that, in SLE patients, specific B cell depletion therapy with
rituximab dramatically improves the abnormalities in B cell
homeostasis that are characteristic of this disease.
Looney and coworkers [9], in a phase I/II study, examined the
potential use of rituximab in B cell depletion for patients with
SLE. In order to establish the safety and efficacy of rituximab
in this patient population, those investigators performed a
Arthritis Research & Therapy June 2005 Vol 7 Suppl 3 Keystone
Figure 2
Rituximab in rheumatoid arthritis. Phase IIa: changes in total rheumatoid factor (median). CTX, cyclophosphamide; MTX, methotrexate; RTX,
rituximab. Data from Edwards and coworkers [5] and from Emery and coworkers [6].
M
ed
ia
n
ch
an
ge
(IU
/L)
20
0
–20
–40
–60
–80
–100
–120
–140
–160
Steroids given on days 1, 3, 15 and 17
0 4 8 12 16 20 24 28 32 36 40 44 48
MTX
RTX
RTX + CTX
RTX + MTX

S17
dose escalation trial of rituximab added to ongoing therapy in
SLE. They administered rituximab as a single infusion of
100 mg/m2 (low dose), a single infusion of 375 mg/m2
(intermediate dose), or as four infusions (1 week apart) of
375 mg/m2 (high dose). CD19+ lymphocytes were measured
to determine the effectiveness of B cell depletion. The
Systemic Lupus Activity Measure (SLAM) score was used as
the primary outcome for clinical efficacy.
Looney and coworkers demonstrated that rituximab was well
tolerated in this patient population, with most experiencing
no significant adverse effects. Three patients had serious
adverse events requiring hospitalization (one
Staphylococcus aureus abscess of the thigh, one localized
case of herpes zoster and a transient ischaemic attack),
although none was deemed related to rituximab
administration. The majority of patients (11 out of 17) had
profound B cell depletion (CD19+ B cell count < 5/µl). In
these patients the SLAM score was significantly improved at
2 and 3 months compared with baseline (P = 0.0016 and
P = 0.0022, respectively). This improvement persisted for
12 months, despite the absence of a significant change in
anti-dsDNA antibody and complement levels. Six patients
developed human antichimeric antibodies at a level of
100 ng/ml or greater (range of peak levels
631–9930 ng/ml). These human antichimeric antibody titres
were associated with African American ancestry, higher
baseline SLAM scores, reduced B cell depletion and lower
levels of rituximab at 2 months after initial infusion.
Conclusion
Recent reports underscored the critical role played by
peripheral blood B cells in self-sustaining chronic inflam-
matory processes. A novel B cell targeted approach, via
inhibition of BlyS, has shown early promise in the treatment of
SLE. A recent phase I study of 70 patients found that
treatment with the BlyS inhibitor belimumab reduced CD20+
cells to between 12% and 47% compared with placebo [3].
Moreover, several clinical trials have provided a substantial
body of evidence that the B cell depleting agent rituximab,
alone or in combination with other disease-modifying anti-
rheumatic drugs, produces profound and prolonged
depletion of B cells and confers clinically meaningful benefits
to patients with RA and SLE.
Edwards and colleagues [5] showed that, at 24 weeks,
significantly more patients receiving rituximab plus metho-
trexate and rituximab plus cyclophosphamide had ACR50
responses than patients receiving methotrexate alone [5].
Moreover, in a phase I trial of rituximab conducted in seven
patients with SLE [7], six (85%) showed an improvement in
SLEDAI, as well as a 99% depletion of B cells for more than
3 months. Notably, of the clinical responders in long term
remission (6–9 months), the steroid dose remained stable or
was lowered. In fact, one patient was in remission for
14 months without the need for any steroid therapy.
Important questions remain regarding this emerging form of
therapy (Table 1). Research suggests that there is restoration
of B cells, usually 6–18 months after depletion therapy. Long-
term rituximab treatment for RA is likely to involve
maintenance doses, possibly in combination with immuno-
suppressive or immunomodulatory agents (e.g. biologic
response modifiers). Further studies are needed to address
the need for a regimen that can be used for maintenance
therapy. Therefore, with B cell targeted therapies, as with
other biologics, close monitoring of immunocompetence and
risks for serious adverse events and opportunistic infections
is required in patients undergoing treatment for RA or other
autoimmune disorders.
Taken together, the data presented suggest that B cell
targeted therapies are likely to be promising additions to our
therapeutic armamentarium, in the treatment both of RA and
other autoimmune diseases.
Competing interests
EK has received financial support from Amgen, Bristol-Myers
Squibb, Centocor Inc., Hoffmann-LaRoche Ltd (Canada),
Abbott Laboratories, Schering Plough Inc., and Wyeth
Pharmaceuticals. EK is a consultant or on the advisory board
for the following: Abbott Laboratories, Amgen, Bristol-Myers
Squibb, Celltech, Centocor Inc., Genentech, Hoffmann-
LaRoche Ltd, Scering Plough Inc., and Wyeth Ayerst.
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Available online http://arthritis-research.com/contents/7/S3/S13
Table 1
Unanswered questions about B cell therapies
What is the duration of clinical efficacy after a single course of
rituximab? Belimumab?
Is there a need for concomitant corticosteroids with each course of
corticosteroids?
What are the long-term implications of CD20+ B cell depletion?
Is there a risk for increased occurrence of serious infections with B cell
targeted therapies? Opportunistic infections?
What role might biologics play in combination with B cell targeted
therapies?

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Arthritis Research & Therapy June 2005 Vol 7 Suppl 3 Keystone