original article
The
new england journal
of
medicine
n engl j med
350;25
www.nejm.org june
17, 2004
2572
Efficacy of B-Cell–Targeted
Therapy with Rituximab in Patients
with Rheumatoid Arthritis
Jonathan C.W. Edwards, M.D., Leszek Szczepa´nski, M.D., Ph.D.,
Jacek Szechi´nski, M.D., Ph.D., Anna Filipowicz-Sosnowska, M.D., Ph.D.,
Paul Emery, M.D., David R. Close, Ph.D., Randall M. Stevens, M.D.,
and Tim Shaw, B.Sc.
From University College London, London
(J.C.W.E.); Medical University School of
Lublin, Lublin, Poland (L.S.); University
School of Wroclaw, Wroclaw, Poland (J.S.);
the Institute of Rheumatology, Warsaw,
Poland (A.F.-S.); Leeds Royal Infirmary,
Leeds, United Kingdom (P.E.); Roche Prod-
ucts, Welwyn Garden City, Hertfordshire,
United Kingdom (D.R.C., T.S.); and Roche,
Nutley, N.J. (R.M.S.). Address reprint re-
quests to Professor Edwards at the Centre
for Rheumatology, University College Lon-
don, 4th Fl., Arthur Stanley House, 40-50
Tottenham St., London W1P 9PG, United
Kingdom, or at jo.edwards@ucl.ac.uk.
N Engl J Med 2004;350:2572-81.
Copyright © 2004 Massachusetts Medical Society.
background
An open-label study indicated that selective depletion of B cells with the use of rituxi-
mab led to sustained clinical improvements for patients with rheumatoid arthritis. To
confirm these observations, we conducted a randomized, double-blind, controlled study.
methods
We randomly assigned 161 patients who had active rheumatoid arthritis despite treat-
ment with methotrexate to receive one of four treatments: oral methotrexate (≥10 mg per
week) (control); rituximab (1000 mg on days 1 and 15); rituximab plus cyclophospha-
mide (750 mg on days 3 and 17); or rituximab plus methotrexate. Responses defined
according to the criteria of the American College of Rheumatology (ACR) and the Euro-
pean League against Rheumatism (EULAR) were assessed at week 24 (primary analyses)
and week 48 (exploratory analyses).
results
At week 24, the proportion of patients with 50 percent improvement in disease symp-
toms according to the ACR criteria, the primary end point, was significantly greater with
the rituximab–methotrexate combination (43 percent, P=0.005) and the rituximab–
cyclophosphamide combination (41 percent, P=0.005) than with methotrexate alone
(13 percent). In all groups treated with rituximab, a significantly higher proportion
of patients had a 20 percent improvement in disease symptoms according to the ACR
criteria (65 to 76 percent vs. 38 percent, P≤0.025) or had EULAR responses (83 to 85
percent vs. 50 percent, P≤0.004). All ACR responses were maintained at week 48 in the
rituximab–methotrexate group. The majority of adverse events occurred with the first
rituximab infusion: at 24 weeks, serious infections occurred in one patient (2.5 percent)
in the control group and in four patients (3.3 percent) in the rituximab groups. Periph-
eral-blood immunoglobulin concentrations remained within normal ranges.
conclusions
In patients with active rheumatoid arthritis despite methotrexate treatment, a single
course of two infusions of rituximab, alone or in combination with either cyclophos-
phamide or continued methotrexate, provided significant improvement in disease
symptoms at both weeks 24 and 48.
abstract
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n engl j med
350;25
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17, 2004
rituximab for rheumatoid arthritis
2573
heumatoid arthritis is a systemic
autoimmune disease that affects approx-
imately 1 percent of the adult population.
1
It is characterized by chronic inflammation in the
synovial membrane of affected joints that ultimate-
ly leads to loss of daily function due to chronic pain
and fatigue. The majority of patients also have dete-
rioration of cartilage and bone in the affected joints,
which may eventually lead to permanent disability.
Rheumatoid arthritis is associated with increased
morbidity and mortality.
2
Although the precise pathogenesis of rheuma-
toid arthritis remains unclear, it has been postulated
that multiple exogenous or endogenous antigenic
triggers, or both, act in the presence of a back-
ground genetic predisposition to initiate a self-
perpetuating series of autoimmune responses in the
synovial compartment.
3,4
Many cell populations, in-
cluding monocytes, macrophages, B cells, T cells,
endothelial cells, and fibroblasts, participate in the
ongoing inflammatory process.
3
The precise con-
tribution of B cells to the immunopathogenesis of
rheumatoid arthritis is not fully understood, al-
though a number of mechanisms have been pro-
posed.
4-6
However, strong evidence for a critical role
of B cells in rheumatoid arthritis came from a small
open-label study of rituximab in combination with
cyclophosphamide and corticosteroids.
7
Rituximab is a genetically engineered chimeric
anti-CD20 monoclonal antibody that is approved
for the treatment of relapsed or refractory, low-
grade or follicular, CD20+ B-cell non-Hodgkin’s
lymphoma. CD20 is a B-cell surface antigen that is
expressed only on pre-B and mature B cells. It is not
present on stem cells and is lost before differentia-
tion of B cells into plasma cells. Therefore, rituxi-
mab causes a selective transient depletion of the
CD20+ B-cell subpopulation.
7
To confirm the role
of B cells in rheumatoid arthritis, we evaluated the
effect of rituximab in patients with active rheuma-
toid arthritis in a multicenter, randomized, double-
blind, controlled study.
patients
Patients were recruited from 26 rheumatology cen-
ters in 11 countries (Australia, Canada, Israel, and
8 European countries). Eligible patients were at least
21 years of age, fulfilled the revised 1987 American
Rheumatism Association criteria,
1
and had active
disease despite treatment with at least 10 mg of
methotrexate per week. Active disease was defined
by the presence of at least eight swollen and eight
tender joints and at least two of the following: a
serum C-reactive protein level of at least 15 mg per
liter, an erythrocyte sedimentation rate of at least
28 mm per hour, or morning stiffness lasting longer
than 45 minutes. In addition, eligible patients were
seropositive for rheumatoid factor, as defined by a
plasma rheumatoid factor level of at least 20 IU per
milliliter.
Patients were excluded if they had an auto-
immune disease other than rheumatoid arthritis
(except concurrent Sjögren’s syndrome), American
Rheumatism Association functional class IV dis-
ease, active rheumatoid vasculitis, a history of sys-
temic diseases associated with arthritis, chronic
fatigue syndrome, serious and uncontrolled coexist-
ing diseases, active infection, a history of recurrent
clinically significant infection or of recurrent bac-
terial infections with encapsulated organisms, pri-
mary or secondary immunodeficiency, or a history
of cancer (except basal-cell carcinoma of the skin
that had been excised).
Patients were allowed to receive nonsteroidal
antiinflammatory drugs at stable doses or cortico-
steroids at doses that did not exceed 12.5 mg per
day of prednisolone (or the equivalent). Concurrent
treatment with any disease-modifying antirheumat-
ic drug or any anti–tumor necrosis factor
a
therapy
during the trial was prohibited.
The study was approved by the institutional re-
view board or the ethics committee at each study
site. All patients gave written informed consent.
study protocol
At study entry, all patients had received methotrex-
ate (as a single disease-modifying antirheumatic
drug) for at least 16 weeks. Therapy with methotrex-
ate during the last four weeks before baseline was at
a stable dose. Patients were randomly assigned to
receive one of four treatments: oral methotrexate at
a dose of 10 mg or more per week plus placebos for
rituximab and cyclophosphamide (control group),
rituximab plus placebos for methotrexate and cyclo-
phosphamide, rituximab plus cyclophosphamide in
an intravenous infusion of 750 mg on days 3 and 17
plus placebo for methotrexate, and rituximab plus
methotrexate at a dose of 10 mg or more a week
plus placebo for cyclophosphamide. In all three
groups that received rituximab (MabThera, Roche;
Rituxan, Genentech and IDEC Pharmaceuticals),
rituximab was administered as a 1000-mg intrave-
r
methods
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n engl j med
350;25
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17
,
2004
The
new england journal
of
medicine
2574
nous infusion on days 1 and 15. Investigators and
patients remained blinded to the assigned study
medications.
All groups, including the control group, also re-
ceived a 17-day course of treatment with cortico-
steroids. This consisted of intravenous infusions of
100 mg of methylprednisolone before infusions
of either rituximab (or the placebo for rituximab)
or cyclophosphamide (or the placebo for cyclophos-
phamide), together with 60 mg per day of oral pred-
nisone on day 2 and days 4 to 7 and 30 mg per day on
days 8 to 14. All patients also received leucovorin
calcium (folinic acid) orally as a single 10-mg dose
on day 1, to counter any undesired effects of metho-
trexate. During the course of the study, all treat-
ments for rheumatoid arthritis remained stable.
To prevent potential unblinding due to nausea
and vomiting associated with the use of cyclophos-
phamide, it was recommended that all patients be
administered an antiemetic agent (granisetron, 2 mg
orally) one hour before the infusions of cyclophos-
phamide or the placebo for cyclophosphamide.
Clinical assessments were performed at baseline
(day 1) and at weeks 12, 16, 20, and 24 according to
the American College of Rheumatology (ACR) core
set of disease-activity measures. These consisted
of a count of swollen joints (66 joints evaluated), a
count of tender joints (68 joints evaluated), patient’s
assessment of pain on a scale from 0 (no pain) to
100 (unbearable pain), patient’s global assessment
of disease activity on a scale from 0 (disease inac-
tive) to 100 (maximal disease activity), physician’s
assessment of disease activity, patient’s assessment
of physical function (by means of a health-assess-
ment questionnaire
8
), and laboratory evaluation of
acute-phase reactants (serum C-reactive protein lev-
el and erythrocyte sedimentation rate).
Laboratory assessments (including complete
blood counts and serum biochemical analyses) were
performed at screening (three weeks before base-
line), on days 1, 3, 15, and 17, and at weeks 4, 8, 12,
16, 20, and 24. At selected visits, levels of CD19+
B cells and CD3+, CD4+, and CD8+ T cells were
measured by fluorescence-activated cell sorting, and
immunoglobulin (IgG, IgA, and IgM) concentra-
tions and rheumatoid factor levels were measured by
nephelometry. Once rituximab is present in the plas-
ma, it interferes with the flow cytometry in the as-
sessment of CD20. Consequently, in order for the
levels of B cells to be assessed, a separate marker is
required. CD19 is a surface antigen and is present
on B cells and was the marker used for B cells after
the exposure of plasma to rituximab. To investigate
the effect of selective B-cell depletion on acquired
immunity, antitetanus antibody titers were assessed
at baseline and at week 24. Samples were taken at
baseline (before the dose of study medication, on
day 1) and at week 24 for analysis of human anti-
chimeric antibodies against rituximab with the use
of an enzyme-linked immunosorbent bridging as-
say (Genentech).
Adverse events were recorded at each visit. A se-
rious adverse event was defined as an event that was
fatal, was life-threatening, required hospitalization
or prolongation of existing hospitalization (with the
exception of hospitalization for exacerbations of ar-
ticular or periarticular manifestations of rheuma-
toid arthritis), resulted in persistent or substantial
disability or incapacity or in a congenital anomaly or
birth defect, or was medically significant or required
intervention to prevent any of the outcomes men-
tioned.
Additional follow-up of patients, including as-
sessments of efficacy, laboratory values, and safety,
was conducted at regular intervals up to 48 weeks.
Personnel at all sites remained blinded to treatment
during this follow-up.
clinical outcome measures
The primary end point of the study was the propor-
tion of patients with an ACR 50 response at week
24. An ACR 50 response was defined as an improve-
ment of at least 50 percent from baseline in counts
of both tender and swollen joints, as well as in three
of the five remaining disease-activity measures of
the ACR core set: physician’s assessment of disease
activity, patient’s assessment of disease activity, pa-
tient’s assessment of pain, patient’s assessment of
physical function (by means of the health-assess-
ment questionnaire), and the value for one acute-
phase reactant (either serum C-reactive protein level
or erythrocyte sedimentation rate).
9
Secondary outcomes included ACR 20 and ACR
70 responses (20 percent and 70 percent improve-
ment, respectively, according to the ACR criteria), a
change in the disease-activity score (which includes
the physician’s assessment of 28 joints and the pa-
tient’s self-assessment of disease activity),
10
and the
response according to the criteria of the European
League against Rheumatism (EULAR response).
11
statistical analysis
Sample-size calculations were based on the assump-
tion that the proportion of patients continuing to
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n engl j med
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17, 2004
rituximab for rheumatoid arthritis
2575
receive only methotrexate and achieving an ACR 50
response at week 24 would be 5 percent and that
the proportion of patients in any of the rituximab
treatment groups would be 30 percent. On the basis
of these assumptions and with the use of Fisher’s
exact test with a two-sided significance level of 0.05,
we calculated that a sample of 40 patients per treat-
ment group would provide the study with 82 percent
power to detect a difference between the two pro-
portions.
The primary analyses were based on the inten-
tion-to-treat principle. For patients who withdrew
before week 24, a last-observation-carried-forward
method of imputation was applied. Statistical analy-
ses (with the two-sided Fisher’s exact test) were
performed only for comparisons of each rituximab
group with the control group. Exploratory second-
ary analyses were performed for ACR response rates
at week 48 with use of a nonresponder imputed
rule for all patients who withdrew before that time.
Roche was the study sponsor and was responsi-
ble for data collection. Statistical analyses were con-
ducted by suitably qualified statisticians who were
employees of the sponsor. The trial protocol was
designed jointly by the lead clinical investigator
(Dr. Edwards) and the sponsor. All the authors had
access to and involvement in the interpretation of
the data, as well as input into and control over the
content of this manuscript (supervised by Dr. Ed-
wards).
characteristics of the patients
A total of 161 patients were recruited into the study.
The baseline characteristics and measures of dis-
ease activity were similar in the four treatment
groups (Table 1). The patients had long-standing
and highly active disease, as shown by a high mean
number of swollen and tender joints, elevated val-
ues for acute-phase reactants, and a high mean dis-
ease-activity score. The median dose of methotrex-
ate at study entry was 12.5 to 15 mg per week.
All patients who underwent randomization re-
ceived at least one dose of their assigned medica-
tion and had at least one follow-up assessment after
baseline. Therefore, the intention-to-treat and safety
populations were identical and consisted of all 161
patients who entered the study. Ten patients with-
drew from the study before week 24 (Fig. 1). Nine
of these 10 patients did not have ACR responses;
1 patient in the rituximab–cyclophosphamide group
had an ACR 20 response before an adverse event
led to early withdrawal.
clinical efficacy
On the basis of the primary end point of an ACR 50
response at week 24, the regimens of rituximab in
combination with either methotrexate or cyclophos-
phamide resulted in levels of response that were
significantly higher (P=0.005) than the levels in
results
* Plus–minus values are means ±SD. The disease-activity score was defined according to the European League against
Rheumatism criteria.
Table 1. Baseline Characteristics of the Patients.*
Characteristic
Methotrexate Group
(N=40)
Rituximab Group
(N=40)
Rituximab–
Cyclophosphamide
Group
(N=41)
Rituximab–
Methotrexate
Group
(N=40)
Age (yr)
54±11 54±10 53±10 54±12
Female sex (%) 80 73 83 75
Duration of disease (yr) 11±7 9±6 10±6 12±7
Previous disease-modifying anti-
rheumatic drugs (no.)
2.6±1.3 2.5±1.6 2.6±1.4 2.5±1.4
Swollen joints (no.) 19±10 21±11 19±10 23±13
Tender joints (no.) 32±13 34±15 33±14 32±16
Serum C-reactive protein (mg/liter) 32±43 26±22 40±40 29±32
Erythrocyte sedimentation rate
(mm/hr)
52±32 47±23 55±29 53±23
Disease-activity score 6.9±0.75 6.8±0.97 6.9±0.84 6.8±0.92
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n engl j med
350;25
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17
,
2004
The
new england journal
of
medicine
2576
the control group (Fig. 2). The ACR 50 response in
the rituximab-monotherapy group was numerical-
ly higher than the response in the control group
(which received only methotrexate) but the differ-
ence did not reach statistical significance (P=0.059).
The proportions of patients with ACR 20 and ACR
70 responses at week 24 were higher in the rituxi-
mab groups than in the control group, with statis-
tically significant increases in the frequency of ACR
20 responses in all rituximab groups (P≤0.025) and
ACR 70 responses in the rituximab–methotrexate
group (P=0.048).
The mean change from baseline in the disease-
activity score at week 24 showed significant im-
provement over methotrexate alone in all rituximab
groups (P≤0.002) (Table 2). Furthermore, a signif-
icantly higher proportion of patients in the rituxi-
mab groups than in the control group responded to
treatment, as defined by EULAR criteria (P≤0.004).
In addition, 20 to 24 percent of patients receiving
rituximab had a good EULAR response at week 24,
as compared with 5 percent of patients receiving
methotrexate alone.
Exploratory analyses of ACR responses at week
48 (Fig. 2) showed ACR 70, ACR 50, and ACR 20 re-
sponses in 0 percent, 5 percent, and 20 percent of
patients in the methotrexate control group, respec-
tively, as compared with 15 percent, 35 percent, and
65 percent of patients in the rituximab–methotrex-
ate group (P=0.03, P=0.002, and P<0.001, respec-
tively). In the rituximab–cyclophosphamide group,
27 percent and 49 percent of patients had ACR 50
and ACR 20 responses, respectively (P=0.01 for
both comparisons). All other comparisons of ACR
responses at week 48 favored rituximab therapy but
did not reach statistical significance.
pharmacodynamic outcomes at week 24
Rituximab treatment was associated with nearly
complete depletion of peripheral-blood B cells,
which lasted throughout the 24-week study period
(Fig. 3A). Conversely, numbers of B cells (CD19+
cells) first declined and then remained stable in the
control group. An initial decline and subsequent re-
bound were also seen in T-cell populations (CD3+,
CD4+, and CD8+) in all treatment groups. Such ef-
fects are most likely associated with the use of corti-
costeroids during the initial phase of the study.
7
Despite depletion of B cells, levels of immunoglob-
ulins did not change substantially (mean values re-
mained within normal ranges for IgG, IgM, and IgA
isotypes), and there was no effect on antitetanus
antibody titers (Table 2).
Rituximab treatment was associated with a large
Figure 1. Disposition of All Randomized Patients at 24 and 48 Weeks.
Patients who did not complete the study for unknown reasons or for reasons other than an adverse event, lack of response, or withdrawal
of consent are classified as “other.”
161 Underwent randomization and received at least 1 dose of assigned treatment
40 Treated with methotrexate
alone
1 Had an adverse event
2 Had a lack of response
40 Treated with rituximab
alone
2 Had an adverse event
41 Treated with rituximab
and cyclophosphamide
2 Had an adverse event
2 Withdrew consent
40 Treated with rituximab
and methotrexate
1 Had an adverse event
37 Completed 24 wk
1 Had an adverse event
5 Had a lack of response
1 Withdrew consent
4 Other
38 Completed 24 wk
2 Had an adverse event
2 Had a lack of response
2 Other
37 Completed 24 wk
2 Had a lack of response
1 Other
39 Completed 24 wk
1 Had a lack of response
26 Completed 48 wk 32 Completed 48 wk 34 Completed 48 wk 38 Completed 48 wk
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