U.S. patent application number 16/866757 was filed with the patent office on 2021-01-28 for method for treating joint damage.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Biogen Inc., F. Hoffmann-La Roche AG, Genentech, Inc.. Invention is credited to Sunil Agarwal, Ariella Kelman, Timothy Mark Shaw, Mark Totoritis, David Yocum.
Application Number | 20210024643 16/866757 |
Document ID | / |
Family ID | 1000005138863 |
Filed Date | 2021-01-28 |
View All Diagrams
United States Patent
Application |
20210024643 |
Kind Code |
A1 |
Totoritis; Mark ; et
al. |
January 28, 2021 |
METHOD FOR TREATING JOINT DAMAGE
Abstract
Methods of treating joint damage in a subject eligible for
treatment are provided involving administering an antagonist that
binds to a B-cell surface marker, such as CD20 antibody, to the
subject in an amount effective to slow progression of the joint
damage as measured by radiography. Further provided are articles of
manufacture useful for such methods.
Inventors: |
Totoritis; Mark; (Rancho
Santa Fe, CA) ; Shaw; Timothy Mark; (Hertfordshire,
GB) ; Agarwal; Sunil; (Corte Madera, CA) ;
Yocum; David; (San Mateo, CA) ; Kelman; Ariella;
(Hillsborough, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc.
F. Hoffmann-La Roche AG
Biogen Inc. |
South San Francisco
Basel
Cambridge |
CA
MA |
US
CH
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000005138863 |
Appl. No.: |
16/866757 |
Filed: |
May 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16526727 |
Jul 30, 2019 |
10654940 |
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16866757 |
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16384333 |
Apr 15, 2019 |
10450379 |
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16526727 |
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15055485 |
Feb 26, 2016 |
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16384333 |
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11665525 |
Jun 4, 2009 |
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PCT/US06/44290 |
Nov 14, 2006 |
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15055485 |
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60737291 |
Nov 15, 2005 |
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60864463 |
Nov 6, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 47/6849 20170801; C07K 2317/56 20130101; C07K 16/2887
20130101; C07K 16/2896 20130101; C07K 2317/76 20130101; C07K
2317/24 20130101; A61K 39/3955 20130101; A61K 31/519 20130101; A61K
2039/505 20130101; C07K 2317/565 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/68 20060101 A61K047/68; A61K 31/519 20060101
A61K031/519; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method for treating joint damage in a subject comprising
administering a CD20 antibody to the subject, and giving the
subject, at least about one month after the administration, a
radiographic test that measures a reduction in the joint damage as
compared to baseline prior to the administration, wherein the
amount of CD20 antibody administered is effective in achieving a
reduction in the joint damage
2.-7. (canceled)
8. The method of claim 1 wherein the radiographic testing after
administering the CD20 antibody occurs at least about 52 weeks
after administering the antibody.
9. The method of claim 1 further comprising an additional
administration to the subject of a CD20 antibody in an amount
effective to achieve a continued or maintained reduction in joint
damage as compared to the effect of a prior administration of CD20
antibody.
10. The method of claim 9 wherein the CD20 antibody is additionally
administered to the subject even if there is no clinical
improvement in the subject at the time of the radiographic testing
after a prior administration.
11. (canceled)
12. The method of claim 1 wherein the test measures a total
modified Sharp score.
13. The method of claim 1 wherein the antibody is rituximab.
14.-18. (canceled)
19. The method of claim 4-g 19 wherein the joint damage is caused
by arthritis.
20. (canceled)
21. The method of claim 1 wherein the antibody is administered
intravenously.
22. The method of claim 1 wherein the antibody is administered
subcutaneously.
23.-52. (canceled)
53. The method of claim 1 wherein the subject is rheumatoid factor
positive.
54.-58. (canceled)
59. The method of claim 1 wherein the joint damage is caused by
rheumatoid arthritis and the subject has exhibited an inadequate
response to one or more anti-tumor necrosis factor (TNF)
inhibitors.
60. The method of claim 1 wherein concomitant methotrexate is
administered to the subject along with the CD20 antibody.
61. The method of claim 1 wherein the CD20 antibody is administered
at a dose of about 1000 mg.times.2 on days 1 and 15 intravenously
at the start of the treatment.
62.-66. (canceled)
67. A method for treating joint damage in a subject comprising
administering a CD20 antibody to the subject, and giving the
subject, at least about 52 weeks after the administration, a
radiographic test that measures a reduction in the joint damage as
compared to baseline prior to the administration, wherein the
amount of CD20 antibody administered is effective in achieving a
reduction in the joint damage.
68. The method of claim 67 wherein the test measures a total
modified Sharp score.
69. The method of claim 67 wherein the antibody is rituximab.
70.-103. (canceled)
104. A method for the treatment of joint damage in a subject,
wherein (a) the subject has exhibited an inadequate response to one
or more anti-tumor necrosis factor (TNF) inhibitors; (b) the
subject received at least one prior course of treatment with a CD20
antibody, and (c) the treatment comprises administering at least
one further course of treatment with a CD20 antibody.
105. The method of claim 104 wherein the subject responded to at
least one prior course of treatment with anti-CD20 antibody.
106. The method of claim 104 wherein the joint damage is caused by
rheumatoid arthritis.
107. (canceled)
108. The method of claim 104 wherein the antibody is rituximab.
109.-112. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a National Stage application filed under
35 USC .sctn. 371 of PCT Application No. PCT/2006/044290, filed
Nov. 14, 2006, which claims the benefit of priority under 35 U.S.C.
.sctn. 119(e) of provisional application Nos. 60/864,463, filed
Nov. 6, 2006, and 60/737,291, filed Nov. 15, 2005. The contents of
each application listed in this paragraph are fully incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] The present invention concerns methods for treating joint
damage in subjects suffering therefrom.
BACKGROUND OF THE INVENTION
[0003] Joint Destruction and Damage
[0004] Inflammatory arthritis is a prominent clinical manifestation
in diverse autoimmune disorders including rheumatoid arthritis
(RA), psoriatic arthritis (PsA), systemic lupus erythematosus
(SLE), Sjogren's syndrome and polymyositis. Most of these patients
develop joint deformities on physical examination but typically
only RA and PsA patients manifest bone erosions on imaging
studies.
[0005] RA is a chronic inflammatory disease that affects
approximately 0.5 to 1% of the adult population in northern Europe
and North America, and a slightly lower proportion in other parts
of the world. Alamonosa and Drosos, Autoimmun. Rev., 4: 130-136
(2005). It is a systemic inflammatory disease characterized by
chronic inflammation in the synovial membrane of affected joints,
which ultimately leads to loss of daily function due to chronic
pain and fatigue. The majority of patients also experience
progressive deterioration of cartilage and bone in the affected
joints, which may eventually lead to permanent disability. The
long-term prognosis of RA is poor, with approximately 50% of
patients experiencing significant functional disability within 10
years from the time of diagnosis. Keystone, Rheumatology, 44
(Suppl. 2): ii8-ii12 (2005). Life expectancy is reduced by an
average of 3-10 years. Alamanos and Rosos, supra. Patients with a
high titer of rheumatoid factor (RF) (approximately 80% of
patients) have more aggressive disease (Bukhari et al., Arthritis
Rheum. 46: 906-912 (2002)), with a worse long-term outcome and
increased mortality over those who are RF negative. Heliovaara et
al., Ann. Rheum. Dis. 54: 811-814 (1995)).
[0006] The pathogenesis of chronic inflammatory bone diseases, such
as RA, is not fully elucidated. Such diseases are accompanied by
bone loss around affected joints due to increased osteoclastic
resorption. This process is mediated largely by increased local
production of pro-inflammatory cytokines. Teitelbaum Science,
289:1504-1508 (2000); Goldring and Gravallese Arthritis Res.
2(1):33-37 (2000). These cytokines can act directly on cells in the
osteoclast lineage or indirectly by affecting the production of the
essential osteoclast differentiation factor, receptor activator of
NF<B ligand (RANKL), I and/or its soluble decoy receptor,
osteoprotegerin (OPG), by osteoblast/stromal cells. Hossbauer et
al. J. Bone Miner. Res. 15(1): 2-12 (2000). TNF-alpha is a major
mediator of inflammation, whose importance in the pathogenesis of
various forms of bone loss is supported by several lines of
experimental and clinical evidence (Feldmann et al. Cell
85(3):307-310 (1996). However, TNF-alpha is not essential for
osteoclastogenesis (Douni et al. J. Inflamm. 47:27-38 (1996)),
erosive arthritis (Campbell et al. J. Clin. Invest.
107(12):1519-1527 (2001)), or osteolysis (Childs et al. J. Bon.
Min. Res. 16:338-347 (2001)), as these can occur in the absence of
TNF-alpha.
[0007] In RA specifically, an immune response is thought to be
initiated/perpetuated by one or several antigens presenting in the
synovial compartment, producing an influx of acute inflammatory
cells and lymphocytes into the joint. Successive waves of
inflammation lead to the formation of an invasive and erosive
tissue called pannus. This contains proliferating fibroblast-like
synoviocytes and macrophages that produce proinflammatory cytokines
such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1
(IL-1). Local release of proteolytic enzymes, various inflammatory
mediators, and osteoclast activation contribute to much of the
tissue damage. There is loss of articular cartilage and the
formation of bony erosions. Surrounding tendons and bursa may
become affected by the inflammatory process. Ultimately, the
integrity of the joint structure is compromised, producing
disability.
[0008] The precise contributions of B cells to the
immunopathogenesis of RA are not completely characterized. However,
there are several possible mechanisms by which B cells may
participate in the disease process. Silverman and Carson, Arthritis
Res. Ther., 5 Suppl. 4: S1-6 (2003).
[0009] Historically, B cells were thought to contribute to the
disease process in RA predominantly by serving as the precursors of
autoantibody-producing cells. A number of autoantibody
specificities have been identified including antibodies to Type II
collagen, and proteoglycans, as well as rheumatoid factors. The
generation of large quantities of antibody leads to immune complex
formation and the activation of the complement cascade. This in
turn amplifies the immune response and may culminate in local cell
lysis. Increased RF synthesis and complement consumption has been
correlated with disease activity. The presence of RF itself is
associated with a more severe form of RA and the presence of
extra-articular features.
[0010] Recent evidence (Janeway and Katz, J. Immunol., 138:1051
(1998); Rivera et al., Int. Immunol., 13: 1583-1593 (2001)) shows
that B cells are highly efficient antigen-presenting cells (APC).
RF-positive B cells may be particularly potent APCs, since their
surface immunoglobulin would readily allow capture of any immune
complexes regardless of the antigens present within them. Many
antigens may thus be processed for presentation to T cells. In
addition, it has been recently suggested that this may also allow
RF-positive B cells to self-perpetuate. Edwards et al., Immunology,
97: 188-196 (1999).
[0011] For activation of T cells, two signals need to be delivered
to the cell; one via the T-cell receptor (TCR), which recognizes
the processed peptide in the presence of major histocompatibility
complex (MHC) antigen, and a second, via co-stimulatory molecules.
When activated, B cells express co-stimulatory molecules on their
surface and can thus provide the second signal for T-cell
activation and the generation of effector cells.
[0012] B cells may promote their own function as well as that of
other cells by producing cytokines. Harris et al., Nat. Immunol.,
1: 475-482 (2000). TNF-alpha and IL-1, lymphotoxin-alpha, IL-6, and
IL-10 are amongst some of the cytokines that B cells may produce in
the RA synovium.
[0013] Although T-cell activation is considered to be a key
component in the pathogenesis of RA, recent work using human
synovium explants in severe combined immunodeficiency disorders
(SCID) mice has demonstrated that T-cell activation and retention
within the joint is critically dependent on the presence of B
cells. Takemura et al., J. Immunol., 167: 4710-4718 (2001). The
precise role of B cells in this is unclear, since other APCs did
not appear to have the same effect on T cells.
[0014] Structural damage to joints is an important consequence of
chronic synovial inflammation. Between 60% and 95% of patients with
rheumatoid arthritis (RA) develop at least one radiographic erosion
within 3-8 years of disease onset (Paulus et al., J. Rheumatol.,
23: 801-805 (1996); Hulsmans et al., Arthritis Rheum. 43: 1927-1940
(2000)). In early RA, the correlation between radiographic damage
scores and functional capacity is weak, but after 8 years of
disease, correlation coefficients can reach as high as 0.68 (Scott
et al., Rheumatology, 39: 122-132 (2000)). In 1,007 patients
younger than age 60 years who had RA for at least four years, Wolfe
et al. (Arthritis Rheum, 43 Suppl. 9:S403 (2000)) found a
significant association between the rate of progression of the
Larsen radiographic damage score (Larsen et al., Acta Radiol.
Diagn. 18: 481-491 (1977)), increasing social security disability
status, and decreasing family income.
[0015] Prevention or retardation of radiographic damage is one of
the goals of RA treatment (Edmonds et al., Arthritis Rheum. 36:
336-340 (1993)). Controlled clinical trials of 6 or 12 months'
duration have documented that the progression of radiographic
damage scores was more rapid in the placebo group than in groups
that received methotrexate (MTX) (Sharp et al., Arthritis Rheum.
43: 30 495-505 (2000)), leflunomide (Sharp et al., supra),
sulfasalazine (SSZ) (Sharp et al., supra), prednisolone (Kirwan et
al., N. Engl. J. Med., 333: 142-146 (1995); Wassenburg et al.,
Arthritis Rheum, 42: Suppl 9:S243 (1999)), interleukin-1 receptor
antagonist (Bresnihan et al., Arthritis Rheum, 41: 2196-2204
(1998)), or an infliximab/MTX combination (Lipsky et al., N. Eng.
J. Med., 343: 1594-1604 (2000)), and that radiographic progression
following treatment with etanercept was less rapid than that
following treatment with MTX (Bathon et al., N. Engl. J. Med., 343:
1586-1593 (2000)).
[0016] Other studies have evaluated radiographic progression in
patients treated with corticosteroids (Joint Committee of the
Medical Research Council and Nuffield Foundation, Ann Rheum. Dis.
19: 331-337 (1960); Van Everdingen et al., Ann. Intern. Med., 136:
1-12 (2002)), cyclosporin A (Pasero et al., J. Rheumatol., 24:
2113-2118 (1997); Forre, Arthritis Rheum., 37: 1506-1512 (1994)),
MTX versus azathioprine (Jeurissen et al., Ann. Intern. Med., 114:
999-1004 (1991)), MTX versus auranofin (Weinblatt et al., Arthritis
Rheum., 36: 613-619 (1993)), MTX (meta-analysis) (Alarcon et al.,
J. Rheumatol., 19: 1868-1873 (1992)), hydroxychloroquine (HCQ)
versus SSZ (Van der Heijde et al., Lancet, 1: 1036-1038), SSZ
(Hannonen et al., Arthritis Rheum. 36: 1501-1509 (1993)), the COBRA
(Combinatietherapei Bij Reumatoide Artritis) combination of
prednisolone, MTX, and SSZ (Boers et al., Lancet, 350: 309-318
(1997); Landewe et al., Arthritis Rheum., 46: 347-356 (2002)),
combinations of MTX, SSZ, and HCQ (O'Dell et al., N. Engl. J. Med.,
334: 1287-1291 (1996); Mottonen et al., Lancet, 353: 1568-1573
(1999)), the combination of cyclophosphamide, azathioprine, and HCQ
(Csuka et al., JAMA, 255: 2115-2119 (1986)), and the combination of
adalimumab with MTX (Keystone et al., Arthritis Rheum., 46 Suppl.
9:S205 (2002)).
[0017] The FDA has now approved labeling claims that certain
medications, e.g., leflunomide, etanercept, and infliximab, slow
the progression of radiographic joint damage. These claims are
based on the statistically significant differences in progression
rates observed between randomly assigned treatment groups and
control groups. However, the progression rates in individuals
within the treatment and control groups overlap to a considerable
extent; therefore, despite significant differences between
treatment groups, these data cannot be used to estimate the
probability that a patient who is starting a treatment will have a
favorable outcome with respect to progression of radiographic
damage. Various methods have been suggested to categorize paired
radiographs from individual patients as not progressive, e.g.,
damage scores of 0 at both time points, no increase in damage
scores, no new joints with erosions, and a change in score not
exceeding the smallest detectable difference (i.e., 95% confidence
interval for the difference between repeated readings of the same
radiograph) (Lassere et al., J. Rheumatol., 26: 731-739
(1999)).
[0018] Determining whether there has been increased structural
damage in an individual patient during the interval between paired
radiographs obtained at the beginning and end of a 6- or 12-month
clinical trial has been difficult, for several reasons. The rate of
radiographic damage is not uniform within a population of RA
patients; a few patients may have rapidly progressing damage, but
many may have little or no progression, especially if the tie
interval is relatively short. The methods for scoring radiographic
damage, e.g., Sharp (Sharp et al., Arthritis Rheum., 14: 706-720
(1971); Sharp et al., Arthritis Rheum., 28: 1326-1335 (1985)),
Larsen (Larsen et al., Acta Radiol. Diagn., 18: 481-491 (1977)),
and modifications of these methods (Van der Heijde, J. Rheumatol.,
27: 261-263 (2000)), depend on the judgment and the interpretation
of the reader as to whether an apparent interruption of the
subchondral cortical plate is real, or whether a decrease in the
distance between the cortices on opposite sides of a joint is real
or is due to a slight change in the position of the joint relative
to the film and the radiographic beam, to a change in radiographic
exposure, or to some other technical factor.
[0019] Therefore, the recorded score is an approximation of the
true damage, and for many subjects, the smallest detectable
difference between repeat scores of the same radiographs is larger
than the actual change that has occurred during the interval
between the baseline and final radiographs. If the reader is
blinded to the temporal sequence of the films, these unavoidable
scoring errors may be in either direction, leading to apparent
"healing" when the score decreases or to apparent rapid progression
when reading error increases the difference between films. When the
study involves a sufficiently large population of patients who have
been randomly assigned to receive an effective treatment as
compared with placebo, the positive and negative reading errors
offset each other, and small but real differences between treatment
groups can be detected.
[0020] The imprecision of the clinical measures that are used to
quantitate RA disease activity has caused a similar problem;
statistically significant differences between certain outcome
measures from clinical trials were not useful for estimating the
probability of improvement for an individual who was starting the
treatment (Paulus et al., Arthritis Rheum., 33: 477-484 (1990)).
Attribution of individual improvement became practical with the
creation of the American College of Rheumatology (ACR) 20%
composite criteria for improvement (ACR20), which designated a
patient as improved if there was 20% improvement in the tender and
swollen joint counts and 20% improvement in at least 3 of 5
additional measures (pain, physical function, patient global health
assessment, physician global health assessment, and acute-phase
reactant levels) (Felson et al., Arthritis Rheum., 38: 727-735
(1995)). All of these measures have large values for the smallest
detectable difference, but by requiring simultaneous improvement in
5 of the 7 aspects of the same process (disease activity), the
randomness of the 7 measurement errors is constrained and it is
easier to attribute real improvement to the individual.
[0021] In RA, joint damage is a prominent feature. Radiologic
parameters of joint destruction are seen as a key outcome measure
in descriptions of disease outcome. In the recent OMERACT (Outcome
Measures in Rheumatology Clinical Trials) consensus meeting,
radiology was chosen as part of the core set of outcome measures
for longitudinal observational studies (Wolfe et al., Arthritis
Rheum., 41 Supp 9: 5204 (1998) abstract). Radiology is also part of
the WHO/ILAR (World Health Organization/International League of
Associations for Rheumatology) required core set of measures for
long-term clinical trials (Tugwell and Boers, J. Rheumatol., 20:
528-530 (1993)).
[0022] Available data on the outcome of radiologic damage in RA
have been obtained in both short-term and long-term studies. In
short-term studies of RA patients with recent-onset disease,
radiographs obtained every 6 months showed that after an initial
rapid progression, there was diminution of the progression rate of
radiologic damage in the hands and feet after 2-3 years (Van der
Heijde et al., Arthritis Rheum., 35: 26-34 (1992); Fex et al., Br.
J. Rheumatol., 35: 1106-1055 (1996)). In long-term studies with
radiographs taken less frequently, a constant rate of progression
was found, with relentless deterioration of damage up to 25 years
of disease duration (Wolfe and Sharp, Arthritis Rheum., 41:
1571-1582 (1998); Graudal et al., Arthritis Rheum., 41: 1470-1480
(1998); Plant et al., J. Rheumatol., 25: 417-426 (1998); Kaarela
and Kautiainen, J. Rheumatol., 24: 1285-1287 (1997)). Whether these
differences in radiographic progression pattern are due to
differences in the scoring techniques is not clear.
[0023] The scoring systems used differ in the number of joints
being scored, the presence of independent scores for erosions (ERO)
and joint space narrowing (JSN), the maximum score per joint, and
the weighing of a radiologic abnormality. As yet, there is no
consensus on the scoring method of preference. During the first 3
years of follow-up in a cohort study of patients with early
arthritis, JSN and ERO were found to differ in their contribution
to the measured progression in radiologic damage of the hands and
feet (Van der Heijde et al., Arthritis Rheum., 35: 26-34 (1992)).
Furthermore, methods that independently score ERO and JSN, such as
the Sharp and Kellgren scores, were found to be more sensitive to
change in early RA than methods using an overall measure, such as
the Larsen score (Plant et al., J. Rheumatol., 21: 1808-1813
(1994); Cuchacovich et al., Arthritis Rheum., 35: 736-739 (1992)).
The Sharp score is a very labor-intensive method (Van der Heijde,
Baillieres Clin. Rheumatol., 10: 435-533 (1996)). In late or
destructive RA, the Sharp and the Larsen methods were found to
provide similar information. However, the sensitivity to change of
the various scoring methods late in the disease has not yet been
investigated and it can be argued that the scoring methods that
independently measure ERO and JSN provide useful information
(Pincus et al., J. Rheumatol., 24: 2106-2122 (1997)). See also
Drossaers-Bakker et al., Arthritis Rheum., 43: 1465-1472 (2000),
which compared the three radiologic scoring systems for the
long-term assessment of RA.
[0024] Paulus et al., Arthritis Rheum., 50: 1083-1096 (2004)
categorized radiographic joint damage as progressive or
non-progressive in individuals with RA participating in clinical
trials, and concluded that RA joint damage in an observational
cohort can be classified as progressive or non-progressive with the
use of a composite definition that includes a number of imprecise
and related, but distinct, measures of structural joint damage. It
appears that in day-to-day clinical management of an RA patient, an
interval change between a pair of radiographs of at least five
Sharp radiographic damage score units should be present before one
considers the structural change to be real and uses it as the basis
for a treatment decision.
[0025] Over the past 10 years there have been major advances in the
treatment of RA. Combination use of existing disease-modifying
anti-rheumatic drugs (DMARDs), together with new biologic agents,
have provided higher levels of efficacy in a larger proportion of
patients, while the early diagnosis and treatment of the disease
has also improved outcomes.
[0026] Etanercept is a fully human fusion protein that inhibits
tumor necrosis factor (TNF) and the subsubsequent inflammatory
cytokine cascade. Etanercept has been shown to be safe and
effective in rapidly reducing disease activity in adults with RA
and in sustaining that improvement (Bathon et al., N. Eng. J. Med.,
343: 1586-1593 (2000); Moreland et al., N. Engl. J. Med., 337:
141-147 (1997); Moreland et al., Ann. Intern. Med., 130: 478-486
(1999); Weinblatt et al., N. Engl. J. Med., 340: 253-259 (1999);
Moreland et al., J. Rheumatol., 28: 1238-1244 (2001)). It is
equally effective in children with polyarticular juvenile RA
(Lovell et al., N. Engl. J. Med., 342: 763-769 (2000)). Etanercept
is approved for use as monotherapy, as well as combination therapy
with MTX, for the treatment of RA.
[0027] Loss of function and radiographic change occur early in the
course of the disease. These changes can be delayed or prevented
with the use of certain DMARDs. Although several DMARDs are
initially clinically effective and well tolerated, many of these
drugs become less effective or exhibit increased toxicity over
time. Based on its efficacy and tolerability, MTX has become the
standard therapy by which other treatments are measured (Bathon et
al., N. Eng. J. Med., 343: 1586-1593 (2000); Albert et al., J.
Rheumatol., 27: 644-652 (2000)).
[0028] Recent studies have examined radiographic progression in
patients with late-stage RA who have taken leflunomide, MTX, or
placebo (Strand et al., Arch. Intern. Med., 159: 2542-2550 (1999))
as well as patients who have taken infliximab plus MTX or placebo
plus MTX following a partial response to MTX (Lipsky et al., N.
Engl. J. Med., 343: 1594-1602 (2000); Maini et al., Lancet, 354:
1932-1939 (1999)). In the first year of the Enbrel ERA (early RA)
trial, etanercept was shown to be significantly more effective than
MTX in improving signs and symptoms of disease and in inhibiting
radiographic progression (Bathon et al., N. Eng. J. Med., 343:
1586-1593 (2000)). Genovese et al., Arthritis Rheum. 46: 1443-1450
(2002) reports results from the second year of the study,
concluding that etanercept as monotherapy was safe and superior to
MTX in reducing disease activity, arresting structural damage, and
decreasing disability over 2 years in patients with early,
aggressive RA.
[0029] Further, reduction in radiographic progression in the hands
and feet was observed in patients with early rheumatoid arthritis
after receiving infliximab in combination with methotrexate (Van
der Heijde et al., Annals Rheumatic Diseases 64: 418-419 (2005)).
Patients with early rheumatoid arthritis achieved a clinically
meaningful and sustained improvement in physical function after
treatment with infliximab (Smolen et al., Annals Rheumatic Diseases
64: 418 (2005)). The effect of infliximab and methotrexate on
radiographic progression in patients with early rheumatoid
arthritis is reported in Van der Heijde et al., Annals Rheumatic
Diseases 64: 417 (2005). Infliximab treatment of patients with
ankylosing spondylitis leads to changes in markers of inflammation
and bone turnover associated with clinical efficacy (Visvanathan et
al., Annals Rheumatic Diseases 64: 319 (2005)).
[0030] The effect of infliximab therapy on bone mineral density in
patients with ankylosing spondylitis (AS) resulting from a
randomized, placebo-controlled trial named ASSERT) is reported by
Van der Heijde et al., Annals Rheumatic Diseases 64: 319 (2005).
Infliximab was found to improve fatigue and pain in patients with
AS, in results from ASSERT (Van der Heijde et al., Annals Rheumatic
Diseases 64: 318-319 (2005)). Further, the efficacy and safety of
infliximab in patients with AS as a result of ASSERT are described
by van der Heijde et al., Arthritis Rheum. 5:582-591 (2005). The
authors conclude that infliximab was well tolerated and effective
in a large cohort of patients with AS during a 24-week study
period. In addition, the effect of infliximab therapy on spinal
inflammation was assessed by magnetic resonance imaging in a
randomized, placebo-controlled trial of 279 patients with AS (Van
der Heijde et al., Annals Rheumatic Diseases 64: 317 (2005)). The
manner in which the treatment effect on spinal radiographic
progression in patients with AS should be measured is addressed by
van der Heijde et al., Arthritis Rheum. 52: 1979-1985 (2005).
[0031] The results of radiographic analyses of the infliximab
multinational psoriatic arthritis controlled trial (IMPACT) after
one year are reported by Antoni et al., Annals Rheumatic Diseases
64: 107 (2005). Evidence of radiographic benefit of treatment with
infliximab plus MTX in rheumatoid arthritis patients who had no
clinical improvement, with a detailed subanalysis of data from the
anti-tumor necrosis factor trial in rheumatoid arthritis with
concomitant therapy study, is reported by Smolen et al., Arthritis
Rheum. 52:1020-1030 (2005). Radiographic progression as measured by
mean change in modified Sharp/van derHeijde score) was much greater
in patients receiving MTX plus placebo than in patients receiving
infliximab plus MTX. The authors conclude that even in patients
without clinical improvement, treatment with infliximab plus MTX
provided significant benefit with regard to the destructive
process, suggesting that in such patients these 2 measures of
disease are dissociated. The association between baseline
radiographic damage and improvement in physical function after
treatment of patients having rheumatoid arthritis with infliximab
is described by Breedveld et al., Annals Rheumatic Diseases
64:52-55 (2005). Structural damage was assessed using the van der
Heijde modification of the Sharp score. The authors conclude that
greater joint damage at baseline was associated with poorer
physical function at baseline and less improvement in physical
function after treatment, underlining the importance of early
intervention to slow the progression of joint destruction.
[0032] CD20 Antibodies and Therapy Therewith
[0033] Lymphocytes are one of many types of white blood cells
produced in the bone marrow during the process of hematopoiesis.
There are two major populations of lymphocytes: B lymphocytes (B
cells) and T lymphocytes (T cells). The lymphocytes of particular
interest herein are B cells.
[0034] B cells mature within the bone marrow and leave the marrow
expressing an antigen-binding antibody on their cell surface. When
a naive B cell first encounters the antigen for which its
membrane-bound antibody is specific, the cell begins to divide
rapidly and its progeny differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer
life span and continue to express membrane-bound antibody with the
same specificity as the original parent cell. Plasma cells do not
produce membrane-bound antibody, but instead produce the antibody
in a form that can be secreted. Secreted antibodies are the major
effector molecules of humoral immunity.
[0035] The CD20 antigen (also called human B-lymphocyte-restricted
differentiation antigen, Bp35, or B1) is a four-pass, glycosylated
integral membrane protein with a molecular weight of approximately
35 kD located on pre-B and mature B lymphocytes. Valentine et al.,
J. Biol. Chem. 264(19):11282-11287 (1989) and Einfeld et al., EVIBO
J. 7(3):711-717 (1988). The antigen is also expressed on greater
than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al.
Blood 63(6):1424-1433 (1984)), but is not found on hematopoietic
stem cells, pro-B cells, normal plasma cells, or other normal
tissues (Tedder et al. J. Immunol. 135(2):973-979 (1985)). CD20
regulates an early step(s) in the activation process for cell-cycle
initiation and differentiation (Tedder et al., supra), and possibly
functions as a calcium-ion channel Tedder et al., J. Cell. Biochem.
14D:195 (1990). CD20 undergoes phosphorylation in activated B cells
(Riley and Sliwkowski Semin Oncol, 27(12), 17-24 (2000)). CD20
appears on the surface of B-lymphocytes at the pre-B-cell stage and
is found on mature and memory B cells, but not plasma cells
(Stashenko et al. J Immunol 1980;125:1678-1685 (1980)); Clark and
Ledbetter Adv Cancer Res 52, 81-149 (1989)). CD20 has
calcium-channel activity and may have a role in the development of
B cells. The relationship between lysis of peripheral CD20' B cells
in vitro and rituximab activity in vivo is unclear. Rituximab
displays antibody-dependent cellular cytotoxicity (ADCC) in vitro
(Reff et al. Blood 83:435-445 (1994)). Potent complement-dependent
cytotoxic (CDC) activity has also been observed for rituximab on
lymphoma cells and cell lines (Reff et al., supra, 1994) and in
certain mouse xenograft models (Di Gaetano et al. J Immunol
171:1581-1587 (2003)). Several anti-CD20 antibodies, including
rituximab, have been shown to induce apoptosis in vitro when
crosslinked by a secondary antibody or by other means (Ghetie et
al. Proc Natl Acad Sci. 94,7509-7514 (1997)).
[0036] Given the expression of CD20 in B-cell lymphomas, this
antigen can serve as a candidate for "targeting" of such lymphomas.
In essence, such targeting can be generalized as follows:
antibodies specific to the CD20 surface antigen of B cells are
administered to a patient. These anti-CD20 antibodies specifically
bind to the CD20 antigen of (ostensibly) both normal and malignant
B cells; the antibody bound to the CD20 surface antigen may lead to
the destruction and depletion of neoplastic B cells. Additionally,
chemical agents or radioactive labels having the potential to
destroy the tumor can be conjugated to the anti-CD20 antibody such
that the agent is specifically "delivered" to the neoplastic B
cells. Irrespective of the approach, a primary goal is to destroy
the tumor; the specific approach can be determined by the
particular anti-CD20 antibody that is utilized, and thus, the
available approaches to targeting the CD20 antigen can vary
considerably.
[0037] The rituximab (RITUXAN.RTM.) antibody is a genetically
engineered chimeric murine/human monoclonal antibody directed
against the CD20 antigen. Rituximab is the antibody called "C2B8"
in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al.).
Rituximab is indicated for the treatment of patients with relapsed
or refractory low-grade or follicular, CD20-positive, B-cell
non-Hodgkin's lymphoma. In vitro mechanism-of-action studies have
demonstrated that rituximab binds human complement and lyses
lymphoid B-cell lines through CDC. Reff et al., Blood 83(2):435-445
(1994). Additionally, it has significant activity in assays for
ADCC. More recently, rituximab has been shown to have
anti-proliferative effects in tritiated thymidine-incorporation
assays and to induce apoptosis directly, while other anti-CD19 and
anti-CD20 antibodies do not. Maloney et al. Blood 88(10):637a
(1996). Synergy between rituximab and chemotherapies and toxins has
also been observed experimentally. In particular, rituximab
sensitizes drug-resistant human B-cell lymphoma cell lines to the
cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin,
and ricin (Demidem et al., Cancer Chemotherapy &
Radiopharmaceuticals 12(3):177-186 (1997)). In vivo preclinical
studies have shown that rituximab depletes B cells from the
peripheral blood, lymph nodes, and bone marrow of cynomolgus
monkeys, presumably through complement- and cell-mediated
processes. Reff et al., Blood 83:435-445 (1994).
[0038] Rituximab was approved in the United States in November 1997
for the treatment of patients with relapsed or refractory low-grade
or follicular CD20.sup.+ B-cell NHL at a dose of 375 mg/m.sup.2
weekly for four doses. In April 2001, the Food and Drug
Administration (FDA) approved additional claims for the treatment
of low-grade NHL: re-treatment (weekly for four doses) and an
additional dosing regimen (weekly for eight doses). There have been
more than 300,000 patient exposures to rituximab either as
monotherapy or in combination with immunosuppressant or
chemotherapeutic drugs. Patients have also been treated with
rituximab as maintenance therapy for up to 2 years. Hainsworth et
al., J. Clin. Oncol. 21:1746-1751 (2003); Hainsworth et al., J.
Clin. Oncol. 20:4261-4267 (2002). Also, rituximab has been used in
the treatment of malignant and nonmalignant plasma cell disorders.
Treon and Anderson, Semin. Oncol. 27: 79-85 (2000).
[0039] Rituximab has also been studied in a variety of
non-malignant autoimmune disorders, in which B cells and
autoantibodies appear to play a role in disease pathophysiology.
Edwards et al., Biochem Soc. Trans. 30:824-828 (2002). Rituximab
has been reported to potentially relieve signs and symptoms of, for
example, rheumatoid arthritis (RA) (Leandro et al., Ann. Rheum.
Dis. 61:883-888 (2002); Edwards et al., Arthritis Rheum., 46
(Suppl. 9): S46 (2002); Stahl et al., Ann. Rheum. Dis., 62 30
(Suppl. 1): OP004 (2003); Emery et al., Arthritis Rheum. 48(9):
5439 (2003)), lupus (Eisenberg, Arthritis. Res. Ther. 5:157-159
(2003); Leandro et al. Arthritis Rheum. 46: 2673-2677 (2002);
Gorman et al., Lupus, 13: 312-316 (2004)), immune thrombocytopenic
purpura (D'Arena et al., Leuk. Lymphoma 44:561-562 (2003); Stasi et
al., Blood, 98: 952-957 (2001); Saleh et al., Semin. Oncol., 27
(Supp 12):99-103 (2000); Zaia et al., Haematolgica, 87: 189-195
(2002); Ratanatharathorn et al., Ann. Int. Med., 133: 275-279
(2000)), pure red cell aplasia (Auner et al., Br. J. Haematol.,
116: 725-728 (2002)); autoimmune anemia (Zaja et al., Haematologica
87:189-195 (2002) (erratum appears in Haematologica 87:336 (2002)),
cold agglutinin disease (Layios et al., Leukemia, 15: 187-8 (2001);
Berentsen et al., Blood, 103: 2925-2928 (2004); Berentsen et al.,
Br. J. Haematol., 115: 79-83 (2001); Bauduer, Br. J. Haematol.,
112: 1083-1090 (2001); Damiani et al., Br. J. Haematol., 114:
229-234 (2001)), type B syndrome of severe insulin resistance (Coll
et al., N. Engl. J. Med., 350: 310-311 (2004), mixed
cryoglobulinemia (DeVita et al., Arthritis Rheum. 46 Suppl.
9:S206/S469 (2002)), myasthenia gravis (Zaja et al., Neurology, 55:
1062-63 (2000); Wylam et al., J. Pediatr., 143: 674-677 (2003)),
Wegener's granulomatosis (Specks et al., Arthritis & Rheumatism
44: 2836-2840 (2001)), refractory pemphigus vulgaris (Dupuy et al.,
Arch Dermatol., 140:91-96 (2004)), dermatomyositis (Levine,
Arthritis Rheum., 46 (Suppl. 9):51299 (2002)), Sjogren's syndrome
(Somer et al., Arthritis & Rheumatism, 49: 394-398 (2003)),
active type-II mixed cryoglobulinemia (Zaja et al., Blood, 101:
3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch.
Dermatol., 140: 91-95 (2004)), autoimmune neuropathy (Pestronk et
al., J. Neurol. Neurosurg. Psychiatry 74:485-489 (2003)),
paraneoplastic opsoclonus-myoclonus syndrome (Pranzatelli et al.
Neurology 60(Suppl. 1) PO5.128:A395 (2003)), and
relapsing-remitting multiple sclerosis (RRMS). Cross et al.
(abstract) "Preliminary Results from a Phase II Trial of Rituximab
in MS" Eighth Annual Meeting of the Americas Committees for
Research and Treatment in Multiple Sclerosis, 20-21 (2003).
[0040] A Phase II study (WA16291) has been conducted in patients
with rheumatoid arthritis (RA), providing 48-week follow-up data on
safety and efficacy of Rituximab. Emery et al. Arthritis Rheum
48(9):5439 (2003); Szczepanski et al. Arthritis Rheum 48(9):5121
(2003). A total of 161 patients were evenly randomized to four
treatment arms: methotrexate, rituximab alone, rituximab plus
methotrexate, and rituximab plus cyclophosphamide (CTX). The
treatment regimen of rituximab was one gram administered
intravenously on days 1 and 15. Infusions of rituximab in most
patients with RA were well tolerated by most patients, with 36% of
patients experiencing at least one adverse event during their first
infusion (compared with 30% of patients receiving placebo).
Overall, the majority of adverse events was considered to be mild
to moderate in severity and was well balanced across all treatment
groups. There were a total of 19 serious adverse events across the
four arms over the 48 weeks, which were slightly more frequent in
the rituximab/CTX group. The incidence of infections was well
balanced across all groups. The mean rate of serious infection in
this RA patient population was 4.66 per 100 patient-years, which is
lower than the rate of infections requiring hospital admission in
RA patients (9.57 per 100 patient-years) reported in a
community-based epidemiologic study. Doran et al., Arthritis Rheum.
46:2287-2293 (2002).
[0041] The reported safety profile of rituximab in a small number
of patients with neurologic disorders, including autoimmune
neuropathy (Pestronk et al., supra), opsoclonus-myoclonus syndrome
(Pranzatelli et al., supra), and RRMS (Cross et al., supra), was
similar to that reported in oncology or RA. In an ongoing
investigator-sponsored trial (IST) of rituximab in combination with
interferon-beta (IFN-3 .quadrature.) or glatiramer acetate in
patients with RRMS (Cross et al., supra), 1 of 10 treated patients
was admitted to the hospital for overnight observation after
experiencing moderate fever and rigors following the first infusion
of rituximab, while the other 9 patients completed the
four-infusion regimen without any reported adverse events.
[0042] Patents and patent publications concerning CD20 antibodies
and CD20-binding molecules include U.S. Pat. Nos. 5,776,456,
5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US
2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963, US
2003/0147885 (Anderson et al.); U.S. Pat. No. 6,455,043 and WO
2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and
White); WO 2000/27433 (Grillo-Lopez and Leonard); WO 2000/44788
(Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO 2001/10461
(Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US
2001/0018041, US 2003/0180292, WO 2001/34194 (Hanna and Hariharan);
US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US
2002/0012665, WO 2001/74388 and U.S. Pat. No. 6,896,885B5 (Hanna,
N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and
Hanna); US 2005/0123540 (Hanna et al.); US 2002/0009444 and WO
2001/80884 (Grillo-Lopez, A.); WO 2001/97858; US 2005/0112060, and
U.S. Pat. No. 6,846,476 (White, C.); US 2002/0128488 and WO
2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et al.);WO
2002/096948 (Braslawsky et al.);WO 2002/079255 (Reff and Davies);
U.S. Pat. No. 6,171,586 and WO 1998/56418 (Lam et al.); WO
1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642,
U.S. Pat. Nos. 6,194,551, 6,242,195, 6,528,624 and 6,538,124
(Idusogie et al.); WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd
et al.); WO 2001/03734 (Grillo-Lopez et al.); US 2002/0004587 and
WO 2001/77342 (Miller and Presta); US 2002/0197256 (Grewal, I.); US
2003/0157108 (Presta, L.); U.S. Pat. Nos. 6,565,827, 6,090,365,
6,287,537, 6,015,542, 5,843,398, and 5,595,721, (Kaminski et al.);
U.S. Pat. Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767,
6,652,852, 6,893,625 (Robinson et al.); U.S. Pat. No. 6,410,391
(Raubitschek et al.); U.S. Pat. No. 6,224,866 and WO00/20864
(Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO
2000/67795 (Goldenberg); US 2003/0133930; WO 2000/74718 and US
2005/0191300A1 (Goldenberg and Hansen); US 2003/0219433 and WO
2003/68821 (Hansen et al.); WO 2004/058298 (Goldenberg and Hansen);
WO 2000/76542 (Golay et al.);WO 2001/72333 (Wolin and Rosenblatt);
U.S. Pat. No. 6,368,596 (Ghetie et al.); U.S. Pat. No. 6,306,393
and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and
Hartmann); WO 2002/102312 (Engleman, E.); US 2003/0068664 (Albitar
et al.); WO 2003/002607 (Leung, S.); WO 2003/049694, US
2002/0009427, and US 2003/0185796 (Wolin et al.); WO 2003/061694
(Sing and Siegall); US 2003/0219818 (Bohen et al.); US 2003/0219433
and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.);
US 2002/0136719 (Shenoy et al.); WO 2004/032828 and US 2005/0180972
(Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter). See also U.S.
Pat. No. 5,849,898 and EP 330,191 (Seed et al.); EP332,865A2 (Meyer
and Weiss); U.S. Pat. No. 4,861,579 (Meyer et al.); US 2001/0056066
(Bugelski et al.); WO 1995/03770 (Bhat et al.); US 2003/0219433 A1
(Hansen et al.); WO 2004/035607 (Teeling et al.); WO 2005/103081
(Teeling et al.); WO 2004/056312 (Lowman et al.); US 2004/0093621
(shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901
(Tedder et al.); US 2005/0025764 (Watkins et al.); WO 2005/016969
(Carr et al.); US 2005/0069545 (Carr et al.); WO 2005/014618 (Chang
et al.); US 2005/0079174 (Barbera-Guillem and Nelson); US
2005/0106108 (Leung and Hansen); WO 2005/044859 and US 2005/0123546
(Umana et al.); WO 2005/070963 (Allan et al.); US 2005/0186216
(Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter
and Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter);
US 2005/0202023 (Hayden-Ledbetter and Ledbetter); U.S. Pat. No.
6,183,744 (Goldenberg); and U.S. Pat. No. 6,897,044 (Braslawski et
al.).
[0043] Publications concerning treatment with rituximab include:
Perotta and Abuel, "Response of chronic relapsing ITP of 10 years
duration to rituximab" Abstract #3360 Blood 10(1)(part 1-2): p. 88B
(1998); Perotta et al., "Rituxan in the treatment of chronic
idiopathic thrombocytopaenic purpura (ITP)", Blood, 94: 49
(abstract) (1999); Matthews, R., "Medical Heretics" New Scientist
(7 Apr. 2001); Leandro et al., "Clinical outcome in 22 patients
with rheumatoid arthritis treated with B lymphocyte depletion" Ann
Rheum Dis, supra; Leandro et al., "Lymphocyte depletion in
rheumatoid arthritis: early evidence for safety, efficacy and dose
response" Arthritis and Rheumatism 44(9): 5370 (2001); Leandro et
al., "An open study of B lymphocyte depletion in systemic lupus
erythematosus", Arthritis and Rheumatism, 46:2673-2677 (2002),
wherein during a 2-week period, each patient received two 500-mg
infusions of rituximab, two 750-mg infusions of cyclophosphamide,
and high-dose oral corticosteroids, and wherein two of the patients
treated relapsed at 7 and 8 months, respectively, and have been
retreated, although with different protocols; "Successful long-term
treatment of systemic lupus erythematosus with rituximab
maintenance therapy" Weide et al., Lupus, 12: 779-782 (2003),
wherein a patient was treated with rituximab (375
mg/m.sup.2.times.4, repeated at weekly intervals) and further
rituximab applications were delivered every 5-6 months and then
maintenance therapy was received with rituximab 375 mg/m.sup.2
every three months, and a second patient with refractory SLE was
treated successfully with rituximab and is receiving maintenance
therapy every three months, with both patients responding well to
rituximab therapy; Edwards and Cambridge, "Sustained improvement in
rheumatoid arthritis following a protocol designed to deplete B
lymphocytes" Rheumatology 40:205-211 (2001); Cambridge et al., "B
lymphocyte depletion in patients with rheumatoid arthritis: serial
studies of immunological parameters" Arthritis Rheum., 46 (Suppl.
9): 51350 (2002); Cambridge et al., "Serologic changes following B
lymphocyte depletion therapy for rheumatoid arthritis" Arthritis
Rheum., 48: 2146-2154 (2003); Edwards et al., "B-lymphocyte
depletion therapy in rheumatoid arthritis and other autoimmune
disorders" Biochem Soc. Trans., supra; Edwards et al., "Efficacy
and safety of rituximab, a B-cell targeted chimeric monoclonal
antibody: A randomized, placebo controlled trial in patients with
rheumatoid arthritis. Arthritis and Rheumatism 46(9): 5197 (2002);
Edwards et al., "Efficacy of B-cell-targeted therapy with rituximab
in patients with rheumatoid arthritis" N Engl. J. Med.
350:2572-2582 (2004); Pavelka et al., Ann. Rheum. Dis. 63:
(S1):289-290 (2004); Emery et al., Arthritis Rheum. 50 (S9):S659
(2004); Levine and Pestronk, "IgM antibody-related
polyneuropathies: B-cell depletion chemotherapy using rituximab"
Neurology 52: 1701-1704 (1999); Uchida et al., "The innate
mononuclear phagocyte network depletes B lymphocytes through Fc
receptor-dependent mechanisms during anti-CD20 antibody
immunotherapy" J. Exp. Med. 199: 1659-1669 (2004); Gong et al.,
"Importance of cellular microenvironment and circulatory dynamics
in B cell immunotherapy" J. Immunol. 174: 817-826 (2005); Hamaguchi
et al., "The peritoneal cavity provides a protective niche for B1
and conventional B lymphocytes during anti-CD20 immunotherapy in
mice" J. Immunol. 174: 4389-4399 (2005); Cragg et al. "The biology
of CD20 and its potential as a target for mAb therapy" Curr. Dir.
Autoimmun. 8:140-174 (2005); Eisenberg, "Mechanisms of
autoimmunity" Immunol. Res. 27: 203-218 (2003); DeVita et al.,
"Efficacy of selective B cell blockade in the treatment of
rheumatoid arthritis" Arthritis & Rheum 46:2029-2033 (2002);
Hidashida et al. "Treatment of DMARD-refractory rheumatoid
arthritis with rituximab." Presented at the Annual Scientific
Meeting of the American College of Rheumatology; Oct 24-29; New
Orleans, La. 2002; Tuscano, J. "Successful treatment of
infliximab-refractory rheumatoid arthritis with rituximab"
Presented at the Annual Scientific Meeting of the American College
of Rheumatology; Oct 24-29; New Orleans, La. 2002 and published
Tuscano, Arthritis Rheum. 46: 3420 (2002); "Pathogenic roles of B
cells in human autoimmunity; insights from the clinic" Martin and
Chan, Immunity 20:517-527 (2004); Silverman and Weisman, "Rituximab
therapy and autoimmune disorders, prospects for anti-B cell
therapy", Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz
and Isenberg, "Anti B cell therapy (rituximab) in the treatment of
autoimmune diseases", Current opinion in pharmacology, 4: 398-402
(2004); Virgolini and Vanda, "Rituximab in autoimmune diseases",
Biomedicine & pharmacotherapy, 58: 299-309(2004); Klemmer et
al., "Treatment of antibody mediated autoimmune disorders with a
AntiCD20 monoclonal antibody Rituximab", Arthritis And Rheumatism ,
48: (9) 9,S (SEP), page: S624-S624 (2003); Kneitz et al.,
"Effective B cell depletion with rituximab in the treatment of
autoimmune diseases", Immunobiology, 206: 519-527 (2002); Arzoo et
al., "Treatment of refractory antibody mediated autoimmune
disorders with an anti-CD20 monoclonal antibody (rituximab)"Annals
of the Rheumatic Diseases, 61 (10), p 922-924 (2002) Comment in Ann
Rheum Dis. 61: 863-866 (2002); "Future strategies in immunotherapy"
by Lake and Dionne, in Burger's Medicinal Chemistry and Drug
Discovery (2003 by John Wiley & Sons, Inc.) Article Online
Posting Date: Jan. 15, 2003 (Chapter 2 "Antibody-Directed
Immunotherapy"); Liang and Tedder, Wiley Encyclopedia of Molecular
Medicine, Section: CD20 as an Immunotherapy Target, article online
posting date: 15 January, 2002 entitled "CD20"; Appendix 4A
entitled "Monoclonal Antibodies to Human Cell Surface Antigens" by
Stockinger et al., eds: Coligan et al., in Current Protocols in
Immunology (2003 John Wiley & Sons, Inc) Online Posting Date:
May, 2003; Print Publication Date: February, 2003; Penichet and
Morrison, "CD Antibodies/molecules: Definition; Antibody
Engineering" in Wiley Encyclopedia ofMolecular Medicine Section:
Chimeric, Humanized and Human Antibodies; posted online 15 January,
2002.
[0044] Further, see Looney "B cells as a therapeutic target in
autoimmune diseases other than rheumatoid arthritis" Rheumatology,
44 Suppl 2: ii13-ii17 (2005); Chambers and Isenberg, "Anti-B cell
therapy (rituximab) in the treatment of autoimmune diseases" Lupus
14(3): 210-214 (2005); Looney et al., "B-cell depletion as a novel
treatment for systemic lupus erythematosus: a phase I/II
dose-escalating trial of rituximab" Arthritis Rheum. 50: 2580-2589
(2004); Looney, "Treating human autoimmune disease by depleting B
cells" Ann Rheum. Dis. 61: 863-866 (2002); Edelbauer et al.,
"Rituximab in childhood systemic lupus erythematosus refractory to
conventional immunosuppression Case report" Pediatr. Nephroi.
20(6): 811-813 (2005); D'Cruz and Hughes, "The treatment of lupus
nephritis" BMJ330(7488): 377-378 (2005); Looney, "B cell-targeted
therapy in diseases other than rheumatoid arthritis" J. Rheumatol.
Suppl. 73: 25-28; discussion 29-30 (2005); Sfikakis et al.,
"Remission of proliferative lupus nephritis following B cell
depletion therapy is preceded by down-regulation of the T cell
costimulatory molecule CD40 ligand: an open-label trial" Arthritis
Rheum. 52(2): 501-513 (2005); Rastetter et al., "Rituximab:
expanding role in therapy for lymphomas and autoimmune diseases"
Annu. Rev. Med. 55: 477-503 (2004); Silverman, "Anti-CD20 therapy
in systemic lupus erythematosus: a step closer to the clinic"
Arthritis Rheum. 52(2): 371-7 (2005), Erratum in: Arthritis Rheum.
52(4): 1342 (2005); Ahn et al., "Long-term remission from
life-threatening hypercoagulable state associated with lupus
anticoagulant (LA) following rituximab therapy" Am. J. Hematol.
78(2): 127-129 (2005); Tahir et al., "Humanized anti-CD20
monoclonal antibody in the treatment of severe resistant systemic
lupus erythematosus in a patient with antibodies against rituximab"
Rheumatology, 44(4): 561-562 (2005), Epub 2005 Jan. 11; Looney et
al., "Treatment of SLE with anti-CD20 monoclonal antibody" Curr.
Dir. Autoimmun. 8: 193-205 (2005); Cragg et al., "The biology of
CD20 and its potential as a target for mAb therapy" Curr. Dir.
Autoimmun. 8: 140-174 (2005); Gottenberg et al., "Tolerance and
short term efficacy of rituximab in 43 patients with systemic
autoimmune diseases" Ann. Rheum. Dis. 64(6): 913-920 (2005) Epub
2004 Nov. 18; Tokunaga et al., "Down-regulation of CD40 and CD80 on
B cells in patients with life-threatening systemic lupus
erythematosus after successful treatment with rituximab"
Rheumatology 44(2): 176-182 (2005), Epub 2004 Oct. 19. See also
Leandro et al., "B cell repopulation occurs mainly from naive B
cells in patient with rheumatoid arthritis and systemic lupus
erythematosus" Arthritis Rheum., 48 (Suppl 9): S1160 (2003).
[0045] Specks et al. "Response of Wegener's granulomatosis to
anti-CD20 chimeric monoclonal antibody therapy" Arthritis &
Rheumatism 44(12):2836-2840 (2001) discloses successful use of four
infusions of 375 mg/m.sup.2 of rituximab and high-dose
glucocorticoids to treat Wegener's granulomatosis. The therapy was
repeated after 11 months when the cANCA recurred, but therapy was
without glucocorticoids. At 8 months after the second course of
rituximab, the patients' disease remained in complete remission.
Further, in another study, rituximab was found to be a
well-tolerated, effective remission induction agent for severe
ANCA-associated vasculitis, when used in a dose of 375
mg/m.sup.2.times.4 along with oral prednisone 1 mg/kg/day, which
was reduced by week 4 to 40 mg/day, and to complete discontinuation
over the following 16 weeks. Four patients were re-treated with
rituximab alone for recurring/rising ANCA titers. Other than
glucocorticoids, no additional immunosuppressive agents seem to be
necessary for remission induction and maintenance of sustained
remission (6 months or longer). See online abstract submission and
invitation Keogh et al., "Rituximab for Remission Induction in
Severe ANCA-Associated Vasculitis: Report of a Prospective
Open-Label Pilot Trial in 10 Patients", American College of
Rheumatology, Session Number: 28-100, Session Title: Vasculitis,
Session Type: ACR Concurrent Session, Primary Category: 28
Vasculitis, Session 10/18/2004
(<www.abstractsonline.com/viewer/SearchResults.asp>). See
also Keogh et al., Kidney Blood Press. Res. 26:293 (2003), wherein
it is reported that eleven patients with refractory ANCA-associated
vasculitis were treated with four weekly doses of 375 mg/m.sup.2 of
rituximab and high-dose glucocortoicoids, resulting in
remission.
[0046] Patients with refractory ANCA-associated vasculitis were
administered rituximab along with immunosuppressive medicaments
such as intravenous cyclophosphamide, mycophenolate mofetil,
azathioprine, or leflunomide, with apparent efficacy. Eriksson,
"Short-term outcome and safety in 5 patients with ANCA-positive
vasculitis treated with rituximab", Kidney and Blood Pressure
Research, 26: 294 (2003) (five patients with ANCA-associated
vasculitis treated with rituximab 375 mg/m.sup.2 once a week for 4
weeks responded to the treatment); Jayne et al., "B-cell depletion
with rituximab for refractory vasculitis" Kidney and Blood Pressure
Research, 26: 294-295 (2003) (six patients with refractory
vasculitis receiving four weekly infusions of rituximab at 375
mg/m.sup.2 with cyclophosphamide along with background
immunosuppression and prednisolone experienced major falls in
vasculitic activity). A further report of using rituximab along
with intravenous cyclophosphamide at 375 mg/m.sup.2 per dose in 4
doses for administering to patients with refractory systemic
vasculitis is provided in Jayne, poster 88 (11.sup.th International
Vasculitis and ANCA workshop), 2003 American Society of Nephrology.
See also Stone and Specks, "Rituximab Therapy for the Induction of
Remission and Tolerance in ANCA-associated Vasculitis", in the
Clinical Trial Research Summary of the 2002-2003 Immune Tolerance
Network, http://www
immunetolerance.org/research/autoimmune/trials/stone.html, in which
a trial of rituximab in ANCA-associated vasculitis is proposed for
a total length of 18 months. See also Eriksson, J. Internal Med.,
257: 540-548 (2005) regarding nine patients with ANCA-positive
vasculitis who were successfully treated with two or four weekly
doses of 500 mg of rituximab, as well as Keogh et al., Arthritis
and Rheumatism, 52: 262-268 (2005), who reported that in 11
patients with refractory ANCA-associated vasculitis, treatment or
re-treatment with four weekly doses of 375 mg/m.sup.2 of rituximab
induced remission by B lymphocyte depletion, the study being
conducted between January 2000 and September 2002.
[0047] As to the activity of a humanized anti-CD20 antibody, see,
for example, Vugmeyster et al., "Depletion of B cells by a
humanized anti-CD20 antibody PRO70769 in Macaca fascicularis" J.
Immunother. 28: 212-219 (2005). For discussion of a human
monoclonal antibody, see Baker et al., "Generation and
characterization of LymphoStat-B, a human monoclonal antibody that
antagonizes the bioactivities of B lymphocyte stimulator" Arthritis
Rheum. 48: 3253-3265 (2003).
[0048] The findings of study WA17043, a phase IIb, randomized,
double-blind, dose-ranging study in rheumatoid arthritis patients
who have had an inadequate response to DMARDs (including anti-TNF
agents) (Emery et al., European League against Rheumatism (EULAR)
(June 2005) OP0008; Van Vollenhoven et al., EULAR (June 2005)
SAT0072), indicate that the combination of rituximab with MTX is
associated with a clinically and statistically significant
improvement in disease symptoms. This study identified doses of
rituximab in combination with MTX that require further
investigation and confirmation within the setting of a phase III
clinical study. See also World Pharmaceutical News,
www.scrippharma.com Scrip article dated 13 Jun. 2005, entitled
"Rituximab a future challenge for anti-TNFs?" describing the EULAR
studies and speculating whether x-ray data from Phase III REFLEX
study would show if rituximab can slow joint damage. In addition,
WO 2004/091657 published Oct. 28, 2004 discloses treating patients
having rheumatoid arthritis who exhibit an inadequate response to
TNF.alpha.-inhibitor therapies with CD20 antibodies, wherein the
patients may have radiographic evidence of at least one joint with
definite erosion attributable to rheumatoid arthritis, as
determined by the central reading site (any joint of the hands,
wrists or feet with the exception of the DIP joints of the hands).
A potential secondary endpoint includes change in modified Sharp
radiographic total score, erosion score, and joint space narrowing
score, which may be analyzed using continuous or categorical
methodology, as appropriate. Exploratory endpoints and analysis may
involve radiographic analyses including the proportion of patients
with no erosive progression, which may be assessed at weeks 24 and
beyond. See also US 2005/00001862 published Aug. 25, 2005
equivalent to WO 2005/060999 published Jul. 7, 2005 regarding
treatment of patients having rheumatoid arthritis with rituximab
wherein the potential secondary endpoint and exploratory endpoints
and analyses include those in WO 2004/091657, supra. Despite the
advances in treatment of joint disease, a significant number of
patients do not qualify for, are intolerant of, or experience an
insufficient response to current treatments. Therefore, new
treatment options, particularly those that may target different
aspects of the pathology of the disease and offer similar or better
levels of clinical benefit, are needed.
SUMMARY OF THE INVENTION
[0049] The present invention involves administration of a CD20
antagonist that provides a safe and active treatment regimen in
subjects with joint damage, including selection of an efficacious
dosing regimen and scheduled or unscheduled re-treatment. This
antagonist is effective both in initial therapy and in the
management of refractory disease.
[0050] Accordingly, the invention is as claimed. In a first aspect,
the present invention concerns a method for treating joint damage
in a subject comprising administering a CD20 antibody to the
subject and giving the subject, at least about one month after the
administration, a radiographic test that measures a reduction in
the joint damage as compared to baseline prior to the
administration, wherein the amount of CD20 antibody administered is
effective in achieving a reduction in the joint damage.
[0051] In another aspect, the invention relates to a method of
monitoring the treatment of joint damage in a subject comprising
administering an effective amount of a CD20 antibody to the subject
and measuring by radiography after at least about one month from
the administration whether the joint damage has been reduced over
baseline prior to the administration, wherein a decrease versus
baseline in the subject after treatment indicates the CD20 antibody
is having an effect on the joint damage. In a preferred embodiment,
the degree of reduction versus baseline is measured a second time
after the administration of the CD20 antibody.
[0052] In a further aspect, the invention provides a method of
determining whether to continue administering a CD20 antibody to a
subject with joint damage comprising measuring by radiography
reduction in joint damage in the subject after administration of
the CD20 antibody a first time, measuring by radiography reduction
in joint damage in the subject after administration of the CD20
antibody a second time, comparing the radiography scores in the
subject at the first time and at the second time, and if the score
is less at the second time than at the first time, continuing
administration of the CD20 antibody.
[0053] In yet another aspect, the invention is directed to an
article of manufacture comprising:
[0054] (a) a container comprising a CD20 antibody; and
[0055] (b) a package insert with instructions for treating joint
damage in a subject, wherein the instructions indicate that the
subject is administered the CD20 antibody and is then subjected, at
least one about month after the administration, to a radiographic
test that measures a reduction in the joint damage as compared to
baseline prior to the administration, wherein the amount of CD20
antibody administered is effective in achieving a reduction in the
joint damage.
[0056] In a preferred aspect, the article further comprises a
container comprising a second medicament, wherein the CD20 antibody
is a first medicament, further comprising instructions on the
package insert for treating the subject with an effective amount of
the second medicament.
[0057] In another embodiment of the invention, a method is provided
for treating joint damage in a subject comprising administering a
CD20 antibody to the subject, and giving the subject, at least
about 52 weeks after the administration, a radiographic test that
measures a reduction in the joint damage as compared to baseline
prior to the administration, wherein the amount of CD20 antibody
administered is effective in achieving a reduction in the joint
damage.
[0058] In a still further embodiment, the invention provides a
method of monitoring the treatment of joint damage in a subject
comprising administering an effective amount of a CD20 antibody to
the subject and measuring by radiography after at least about 52
weeks from the administration whether the joint damage has been
reduced over baseline prior to the administration, wherein a
decrease versus baseline in the subject after treatment indicates
the CD20 antibody is having an effect on the joint damage.
[0059] Further, the invention provides an article of manufacture
comprising:
[0060] (a) a container comprising a CD20 antibody; and
[0061] (b) a package insert with instructions for treating joint
damage in a subject, wherein the instructions indicate that the
subject is administered the CD20 antibody and is then subjected, at
least about 52 weeks after the administration, to a radiographic
test that measures a reduction in the joint damage as compared to
baseline prior to the administration, wherein the amount of CD20
antibody administered is effective in achieving a reduction in the
joint damage.
[0062] In a further aspect, the invention provides a method for the
treatment of joint damage in a subject, wherein (a) the subject has
exhibited an inadequate response to one or more anti-tumor necrosis
factor (TNF) inhibitors; (b) the subject received at least one
prior course of treatment with a CD20 antibody, and (c) the
treatment comprises administering at least one further course of
treatment with a CD20 antibody.
[0063] These and further aspects will be apparent from the rest of
the disclosure, including the examples and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIG. 1 shows the study design to treat RA patients with
control (placebo plus MTX) or rituximab (1000 mg.times.2) plus MTX
(Example 1 herein).
[0065] FIG. 2 shows the ACR responses at six months of RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0066] FIG. 3 shows the ACR20 responses over six months of RA
patients treated with control or with rituximab (1000 mg.times.2)
plus MTX.
[0067] FIG. 4 shows the changes in 328DS at six months of RA
patients treated with control or with rituximab (1000 mg.times.2)
plus MTX.
[0068] FIG. 5 shows EULAR responses at six months of RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0069] FIG. 6 shows EULAR remission or low disease at six months of
RA patients treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0070] FIG. 7 shows the median C-reactive protein (CRP) over six
months of RA patients treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0071] FIG. 8 shows the proportion of RA patients with clinically
relevant improvement in function at six months, wherein the
patients are treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0072] FIG. 9 shows the percentage change in ACR score set at six
months of RA patients treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0073] FIG. 10 shows the change in FACIT-F at six months of RA
patients treated with control or with rituximab (1000 mg.times.2)
plus MTX.
[0074] FIG. 11 shows the changes in SF-36 categories (mental and
physical health) at six months of RA patients treated with control
or with rituximab (1000 mg.times.2) plus MTX.
[0075] FIG. 12 shows the total rheumatoid factor at six months of
RA patients treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0076] FIG. 13 shows the mean change in Sharp-Genant total score at
six months of RA patients treated with control or with rituximab
(1000 mg.times.2) plus MTX.
[0077] FIG. 14 shows the mean change in Sharp-Genant erosion score
at six months of RA patients treated with control or with rituximab
(1000 mg.times.2) plus MTX.
[0078] FIG. 15 shows the mean change in Sharp-Genant joint space
narrowing (JSN) score at six months of RA patients treated with
control or with rituximab (1000 mg.times.2) plus MTX.
[0079] FIG. 16 shows the proportion of RA patients with no change
in erosion score at six months, wherein the patients are treated
with control or with rituximab (1000 mg.times.2) plus MTX.
[0080] FIG. 17 shows the change in radiographic endpoints at week
24 (exploratory endpoint) for RA patients treated with control or
with rituximab (1000 mg.times.2) plus MTX.
[0081] FIG. 18 shows mean percent change in ACR core set parameters
at week 24 for RA patients treated with control or with rituximab
(1000 mg.times.2) plus MTX.
[0082] FIG. 19 shows the median CD19 at six months of RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0083] FIG. 20 shows the most commonly reported adverse events in
the study of RA patients over six months, wherein the patients are
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0084] FIG. 21 shows adverse events leading to withdrawal in the
study of RA patients over six months, wherein the patients are
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0085] FIG. 22 shows events occurring during/within 24 hours of
infusions in the study of RA patients over six months, wherein the
patients are treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0086] FIG. 23 shows acute infusion reactions in the study of RA
patients over six months, wherein the patients are treated with
control or with rituximab (1000 mg.times.2) plus MTX.
[0087] FIG. 24 shows serious adverse events occurring during/within
24 hours of infusions in the study of RA patients over six months,
wherein the patients are treated with control or with rituximab
(1000 mg.times.2) plus MTX.
[0088] FIG. 25 shows system organ class--infections and
infestations in the study of RA patients over six months, wherein
the patients are treated with control or with rituximab (1000
mg.times.2) plus MTX.
[0089] FIG. 26 shows serious infections in the study of RA patients
over six months, wherein the patients are treated with control or
with rituximab (1000 mg.times.2) plus MTX.
[0090] FIG. 27 shows the infection rate in the study of RA patients
over six months, wherein the patients are treated with control or
with rituximab (1000 mg.times.2) plus MTX.
[0091] FIG. 28 shows HACA in the study of RA patients over six
months, wherein the patients are treated with control or with
rituximab (1000 mg.times.2) plus MTX.
[0092] FIG. 29 shows IgG levels over six months in RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0093] FIG. 30 shows IgA levels over six months in RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0094] FIG. 31 shows IgM levels over six months in RA patients
treated with control or with rituximab (1000 mg.times.2) plus
MTX.
[0095] FIG. 32A is a sequence alignment comparing the amino acid
sequences of the light chain variable domain (VL) of each of murine
2H7 (SEQ ID NO:1), humanized 2H7.v16 variant (SEQ ID NO:2), and the
human kappa light chain subgroup I (SEQ ID NO:3). The CDRs of
V.sub.L of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO:4),
CDR2 (SEQ ID NO:5), and CDR3 (SEQ ID NO:6).
[0096] FIG. 32B is a sequence alignment comparing the amino acid
sequences of the heavy chain variable domain (VH) of each of murine
2H7 (SEQ ID NO:7), humanized 2H7.v16 variant (SEQ ID NO:8), and the
human consensus sequence of the heavy chain subgroup III (SEQ ID
NO:9). The CDRs of V.sub.H of 2H7 and hu2H7.v16 are as follows:
CDR1 (SEQ ID NO:10), CDR2 (SEQ ID NO:11), and CDR3 (SEQ ID
NO:12).
[0097] In FIG. 32A and FIG. 32B, the CDR1, CDR2 and CDR3 in each
chain are enclosed within brackets, flanked by the framework
regions, FR1-FR4, as indicated. 2H7 refers to the murine 2H7
antibody. The asterisks in between two rows of sequences indicate
the positions that are different between the two sequences. Residue
numbering is according to Kabat et al. Sequences of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991), with insertions shown as a, b, c, d,
and e.
[0098] FIG. 33 shows the amino acid sequence of the mature 2H7.v16
L chain (SEQ ID NO:13)
[0099] FIG. 34 shows the amino acid sequence of the mature 2H7.v16
H chain (SEQ ID NO:14).
[0100] FIG. 35 shows the amino acid sequence of the mature 2H7.v31
H chain (SEQ ID NO:15). The L chain of 2H7.v31 is the same as for
2H7.v16.
[0101] FIG. 36 is a sequence alignment comparing the light-chain
amino acid sequences of the humanized 2H7.v16 variant (SEQ ID NO:2)
and humanized 2H7.v138 variant (SEQ ID NO:28).
[0102] FIG. 37 is a sequence alignment comparing the heavy-chain
amino acid sequences of the humanized 2H7.v16 variant (SEQ ID NO:8)
and humanized 2H7.v138 variant (SEQ ID NO:29).
[0103] FIG. 38 shows an alignment of the mature 2H7.v16 and
2H7.v511 light chains (SEQ ID NOS: 13 and 30, respectively), with
Kabat variable-domain residue numbering and Eu constant-domain
residue numbering.
[0104] FIG. 39 shows an alignment of the mature 2H7.v16 and
2H7.v511 heavy chains (SEQ ID NOS:14 and 31, respectively), with
Kabat variable-domain residue numbering and Eu constant-domain
residue numbering.
[0105] FIG. 40A shows the sequence of the humanized 2H7.v114
variable light-chain domain (SEQ ID NO:32); FIG. 40B shows the
sequence of the humanized 2H7.v114 variable heavy-chain domain (SEQ
ID NO:33); and FIG. 40C shows the sequence of the humanized
2H7.v114 full-length heavy chain (SEQ ID NO:34), with Kabat
variable-domain residue numbering and Eu constant-domain residue
numbering.
[0106] FIG. 41 illustrates the patient disposition of the REFLEX
clinical trial at 56 weeks, including ongoing treatments of
subgroups of patients selected from the treatment and placebo arms
of the Phase III REFLEX clinical trial.
[0107] FIG. 42. Change in radiographic endpoints at week 56.
[0108] FIG. 43. Mean change in total Sharp-Genant score over
time.
[0109] FIG. 44. Cumulative distribution of change in total
Sharp-Genant score.
[0110] FIG. 45. Sesitivity analyses: vhange in total Sharp-Genant
score.
[0111] FIG. 46. Patients with no radiographic changes at week
56.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0112] I. Definitions
[0113] A "B cell" is a lymphocyte that matures within the bone
marrow, and includes a naive B cell, memory B cell, or effector B
cell (plasma cells). The B cell herein is a normal or non-malignant
B cell.
[0114] A "B-cell surface marker" or "B-cell surface antigen" herein
is an antigen expressed on the surface of a B cell that can be
targeted with an antagonist that binds thereto. Exemplary B-cell
surface markers include the CD10, CD19, CD20, CD21, CD22, CD23,
CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,
CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86
leukocyte surface markers (for descriptions, see The Leukocyte
Antigen Facts Book, 2.sup.nd Edition. 1997, ed. Barclay et al.
Academic Press, Harcourt Brace & Co., New York). Other B-cell
surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X.sub.5,
HLA-DOB, CXCRS, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2,
ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. The B-cell surface
marker of particular interest is preferentially expressed on B
cells compared to other non-B-cell tissues of a mammal and may be
expressed on both precursor B cells and mature B cells. The
preferred B-cell surface markers herein are CD20 and CD22.
[0115] The "CD20" antigen, or "CD20," is an about 35-kDa,
non-glycosylated phosphoprotein found on the surface of greater
than 90% of B cells from peripheral blood or lymphoid organs. CD20
is present on both normal B cells as well as malignant B cells, but
is not expressed on stem cells. Other names for CD20 in the
literature include "B-lymphocyte-restricted antigen" and "Bp35".
The CD20 antigen is described in Clark et al., Proc. Natl. Acad.
Sci. (USA) 82:1766 (1985), for example
[0116] The "CD22" antigen, or "CD22," also known as BL-CAM or Lyb8,
is a type 1 integral membrane glycoprotein with molecular weight of
about 130 (reduced) to 140kD (unreduced). It is expressed in both
the cytoplasm and cell membrane of B-lymphocytes. CD22 antigen
appears early in B-cell lymphocyte differentiation at approximately
the same stage as the CD19 antigen. Unlike other B-cell markers,
CD22 membrane expression is limited to the late differentiation
stages comprised between mature B cells (CD22+) and plasma cells
(CD22-). The CD22 antigen is described, for example, in Wilson et
al., J. Exp. Med. 173:137 (1991) and Wilson et al., J. Immunol.
150:5013 (1993).
[0117] An "antagonist" is a molecule that, upon binding to CD20 on
B cells, destroys or depletes B cells in a mammal and/or interferes
with one or more B-cell functions, e.g. by reducing or preventing a
humoral response elicited by the B cell. The antagonist preferably
is able to deplete B cells (i.e. reduce circulating B-cell levels)
in a mammal treated therewith. Such depletion may be achieved via
various mechanisms such as ADCC and/or CDC, inhibition of B-cell
proliferation and/or induction of B-cell death (e.g. via
apoptosis). Antagonists included within the scope of the present
invention include antibodies, synthetic or native-sequence
peptides, immunoadhesins, and small-molecule antagonists that bind
to CD20, optionally conjugated with or fused to another molecule.
The preferred antagonist comprises an antibody.
[0118] An "antibody antagonist" herein is an antibody that, upon
binding to a B-cell surface marker on B cells, destroys or depletes
B cells in a mammal and/or interferes with one or more B-cell
functions, e.g., by reducing or preventing a humoral response
elicited by the B cell. The antibody antagonist preferably is able
to deplete B cells (i.e., reduce circulating B-cell levels) in a
mammal treated therewith. Such depletion may be achieved via
various mechanisms such as ADCC and/or CDC, inhibition of B-cell
proliferation and/or induction of B-cell death (e.g., via
apoptosis).
[0119] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies, polyclonal antibodies,
multispecific antibodies (e.g. bispecific antibodies) formed from
at least two intact antibodies, and antibody fragments so long as
they exhibit the desired biological activity.
[0120] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and
Fv fragments; diabodies; linear antibodies; single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0121] For the purposes herein, an "intact antibody" is one
comprising heavy and light variable domains as well as an Fc
region.
[0122] An "antibody that binds to a B-cell surface marker" is a
molecule that, upon binding to a B-cell surface marker, destroys or
depletes B cells in a mammal and/or interferes with one or more
B-cell functions, e.g. by reducing or preventing a humoral response
elicited by the B cell. The antibody preferably is able to deplete
B cells (i.e. reduce circulating B-cell levels) in a mammal treated
therewith. Such depletion may be achieved via various mechanisms
such as ADCC and/or CDC, inhibition of B-cell proliferation and/or
induction of B-cell death (e.g. via apoptosis). In one preferred
embodiment, the B-cell surface marker is CD20 or CD22, so that the
antibody that binds to a B-cell surface marker is an antibody that
binds to CD20 or CD22, respectively, or a "CD20 antibody" or "CD22
antibody," respectively. Examples of CD22 antibodies include the
ones described in EP 1,476,120 (Tedder and Tuscano), EP 1,485,130
(Tedder), and EP 1,504,035 (Popplewell et al.), as well as those
described in US 2004/0258682 (Leung et al.). In a still more
preferred embodiment, the antibody is a CD20 antibody. A
particularly preferred embodiment is a CD20 or CD22 antibody,
preferably a CD20 antibody.
[0123] Examples of CD20 antibodies include: "C2B8," which is now
called "rituximab" ("RITUXAN.RTM./MABTHERA.RTM.") (U.S. Pat. No.
5,736,137); the yttrium-1901-labelled 2B8 murine antibody
designated "Y2B8" or "Ibritumomab Tiuxetan" (ZEVALINO) commercially
available from Biogen Idec, Inc. (e.g., U.S. Pat. No. 5,736,137;
2B8 deposited with ATCC under accession no. HB11388 on Jun. 22,
1993); murine IgG2a "B1," also called "Tositumomab," optionally
labelled with .sup.131I to generate the "131I-B1" or "iodine 1131
tositumomab" antibody (BEXXAR.TM.) commercially available from
Corixa (see, also, e.g., U.S. Pat. No. 5,595,721); murine
monoclonal antibody "1F5" (e.g., Press et al. Blood 69(2):584-591
(1987) and variants thereof including "framework patched" or
humanized 1F5 (e.g., WO 2003/002607, Leung, S.; ATCC deposit
HB-96450); murine 2H7 and chimeric 2H7 antibody (e.g., U.S. Pat.
No. 5,677,180); a humanized 2H7 (e.g., WO 2004/056312 (Lowman et
al.) and as set forth below); HUMAX-CD20.TM. fully human,
high-affinity antibody targeted at the CD20 molecule in the cell
membrane of B-cells (Genmab, Denmark; see, for example, Glennie and
van de Winkel, Drug Discovery Today 8: 503-510 (2003) and Cragg et
al., Blood 101: 1045-1052 (2003)); the human monoclonal antibodies
set forth in WO 2004/035607 and WO 2005/103081 (Teeling et al.,
GenMab/Medarex); the antibodies having complex N-glycoside-linked
sugar chains bound to the Fc region described in US 2004/0093621
(Shitara et al.); monoclonal antibodies and antigen-binding
fragments binding to CD20 (e.g., WO 2005/000901, Tedder et al.)
such as HB20-3, HB20-4, HB20-25, and MB20-11; single-chain proteins
binding to CD20 (e.g., US 2005/0186216 (Ledbetter and
Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and
Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US
2005/0202023 (Hayden-Ledbetter and Ledbetter)--Trubion Pharm Inc.);
CD20-binding molecules such as the AME series of antibodies, e.g.,
AME-33.TM. antibodies as set forth, for example, in WO 2004/103404
and US 2005/0025764 (Watkins et al., Applied Molecular Evolution,
Inc.) and the CD20 antibodies with Fc mutations as set forth, for
example, in WO 2005/070963 (Allan et al., Applied Molecular
Evolution, Inc.); CD20-binding molecules such as those described in
WO 2005/016969 and US 2005/0069545 (Carr et al.); bispecific
antibodies as set forth, for example, in WO 2005/014618 (Chang et
al.); humanized LL2 monoclonal antibodies as described, for
example, in US 2005/0106108 (Leung and Hansen; Immunomedics);
chimeric or humanized B-Ly1 antibodies to CD20 as described, for
example, in W02005/044859 and US 2005/0123546 (Umana et al.;
GlycArt Biotechnology AG); A20 antibody or variants thereof such as
chimeric or humanized A20 antibody (cA20, hA20, respectively) and
IMMUN-106 (e.g., US 2003/0219433, Immunomedics); and monoclonal
antibodies L27, G28-2, 93-1B3, B-Cl or NU-B2 available from the
International Leukocyte Typing Workshop (e.g., Valentine et al.,
In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University
Press (1987)). The preferred CD20 antibodies herein are chimeric,
humanized, or human CD20 antibodies, more preferably rituximab, a
humanized 2H7, chimeric or humanized A20 antibody (Immunomedics),
HUMAX-CD20.TM. human CD20 antibody (Genmab), and
immunoglobulins/proteins binding to CD20 (Trubion Pharm Inc.) .
[0124] The terms "rituximab" or "RITUXAN.RTM." herein refer to the
genetically engineered chimeric murine/human monoclonal antibody
directed against the CD20 antigen and designated "C2B8" in U.S.
Pat. No. 5,736,137, including fragments thereof which retain the
ability to bind CD20.
[0125] Purely for the purposes herein and unless indicated
otherwise, a "humanized 2H7" refers to a humanized CD20 antibody,
or an antigen-binding fragment thereof, comprising one, two, three,
four, five, or six of the following CDR sequences: [0126] CDR Ll
sequence RASSSVSYXH wherein X is M or L (SEQ ID NO:35), for
example, SEQ ID NO:4 (FIG. 32A), [0127] CDR L2 sequence of SEQ ID
NO:5 (FIG. 32A), [0128] CDR L3 sequence QQWXFNPPT wherein X is S or
A (SEQ ID NO:36), for example, SEQ ID NO:6 (FIG. 32A), [0129] CDR
H1 sequence of SEQ ID NO:10 (FIG. 32B), [0130] CDR H2 sequence of
AIYPGNGXTSYNQKFKG wherein X is D or A (SEQ ID NO:37), for example,
[0131] SEQ ID NO:11 (FIG. 32B), and [0132] CDR H3 sequence of
VVYYSXXYWYFDV wherein the X at position 6 is N, A, Y, W, or D, and
the X at position 7 is S or R (SEQ ID NO:38), for example, SEQ ID
NO:12 (FIG. 32B).
[0133] The humanized 2H7 antibodies herein include those with
heavy-chain amino acid sequences containing a C-terminal lysine and
those without. The CDR sequences above are generally present within
human variable light- and variable heavy-framework sequences, such
as substantially the human consensus FR residues of human
light-chain kappa subgroup I (V.sub.L6I), and substantially the
human consensus FR residues of human heavy-chain subgroup III
(V.sub.HIII). See also WO 2004/056312 (Lowman et al.).
[0134] The variable heavy region may be joined to a human IgG chain
constant region, wherein the region may be, for example, IgG1 or
IgG3, including native-sequence and non-native-sequence constant
regions.
[0135] In a preferred embodiment, such antibody comprises the
variable heavy-domain sequence of SEQ ID NO:8 (v16, as shown in
FIG. 32B), optionally also comprising the variable light-domain
sequence of SEQ ID NO:2 (v16, as shown in FIG. 32A), which
optionally comprises one or more amino acid substitution(s) at
positions 56, 100, and/or 100a, e.g., D56A, N100A, or N100Y, and/or
S100aR in the variable heavy domain and one or more amino acid
substitution(s) at positions 32 and/or 92, e.g. M32L and/or S92A,
in the variable light domain. Preferably, the antibody is an intact
antibody comprising the light-chain amino acid sequence of SEQ ID
NO:13 or 30, and heavy-chain amino acid sequence of SEQ ID NO:14,
15, 29, 31, 34, or 39, the sequence of SEQ ID NO:39 being given
below.
[0136] A preferred humanized 2H7 antibody is ocrelizumab
(Genentech, Inc.).
[0137] The antibody herein may further comprise at least one amino
acid substitution in the Fc region that improves ADCC activity,
such as one wherein the amino acid substitutions are at positions
298, 333, and 334, preferably S298A, E333A, and K334A, using Eu
numbering of heavy-chain residues. See also U.S. Pat. No.
6,737,056, L. Presta.
[0138] Any of these antibodies may comprise at least one
substitution in the Fc region that improves FcRn binding or serum
half-life, for example, a substitution at heavy-chain position 434,
such as N434W. See also U.S. Pat. No. 6,737,056, L. Presta.
[0139] Any of these antibodies may further comprise at least one
amino acid substitution in the Fc region that increases CDC
activity, for example, comprising at least a substitution at
position 326, preferably K326A or K326W. See also U.S. Pat. No.
6,528,624, Idusogie et al.
[0140] Some preferred humanized 2H7 variants are those comprising
the variable light domain of SEQ ID NO:2 and the variable heavy
domain of SEQ ID NO:8, including those with or without
substitutions in an Fc region (if present), and those comprising a
variable heavy domain with alteration in SEQ ID NO:8 of N100A; or
D56A and N100A; or D56A, N100Y, and S100aR; and a variable light
domain with alteration in SEQ ID NO:2 of M32L; or S92A; or M32L and
S92A.
[0141] M34 in the variable heavy domain of 2H7.v16 has been
identified as a potential source of antibody stability and is
another potential candidate for substitution.
[0142] In a summary of some various preferred embodiments of the
invention, the variable region of variants based on 2H7.v16
comprise the amino acid sequences of v16 except at the positions of
amino acid substitutions that are indicated in the table below.
Unless otherwise indicated, the 2H7 variants will have the same
light chain as that of v16.
TABLE-US-00001 Exemplary Humanized 2H7 Antibody Variants 2H7 Heavy
chain Light chain Version (V.sub.H) changes (V.sub.L) changes Fc
changes 16 for -- reference 31 -- -- S298A, E333A, K334A 73 N100A
M32L 75 N100A M32L S298A, E333A, K334A 96 D56A, N100A S92A 114
D56A, N100A M32L, S92A S298A, E333A, K334A 115 D56A, N100A M32L,
S92A S298A, E333A, K334A, E356D, M358L 116 D56A, N100A M32L, S92A
S298A, K322A, K334A, 138 D56A, N100A M32L, S92A S298A, K326A,
E333A, K334A, 477 D56A, N100A M32L, S92A S298A, K326A, E333A,
K334A, N434W 375 -- -- K334L 588 -- -- S298A, K326A, E333A, K334A
511 D56A, M32L, S92A S298A, K326A, E333A, N100Y, K334A S100aR
[0143] One preferred humanized 2H7 comprises 2H7.v16 variable
light-domain sequence:
TABLE-US-00002 (SEQ ID NO: 2)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG TKVEIKR;
[0144] and 2H7 vl 6 variable heavy-domain sequence:
TABLE-US-00003 (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSS.
[0145] Where the humanized 2H7.v16 antibody is an intact antibody,
it may comprise the light-chain amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 13)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC;
[0146] and the heavy-chain amino acid sequence of SEQ ID NO:14
or:
TABLE-US-00005 (SEQ ID NO: 15)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.
[0147] 20
[0148] Another preferred humanized 2H7 antibody comprises 2H7.v511
variable light-domain sequence:
TABLE-US-00006 (SEQ ID NO: 39)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG TKVEIKR
[0149] and 2H7.v511 variable heavy-domain sequence:
TABLE-US-00007 (SEQ ID NO: 40)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSYRYWYFDVWGQGTLVTVSS.
[0150] Where the humanized 2H7.v511 antibody is an intact antibody,
it may comprise the light-chain amino acid sequence:
TABLE-US-00008 (SEQ ID NO: 30)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0151] and the heavy-chain amino acid sequence of SEQ ID NO:31
or:
TABLE-US-00009 (SEQ ID NO: 41)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.
[0152] See FIGS. 38 and 39, which align the mature light and heavy
chains, respectively, of humanized 2H7.v511 with humanized 2H7.v16
using the C-terminal lysine sequence for the heavy chain
[0153] Where the humanized 2H7.v31 antibody is an intact antibody,
it may comprise the light-chain amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 13)
DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAP
SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[0154] and the heavy-chain amino acid sequence of SEQ ID NO:15
or:
TABLE-US-00011 (SEQ ID NO: 42)
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G or: (SEQ ID
NO: 43) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGA
IYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVV
YYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL
VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G.
[0155] A preferred embodiment herein is where the antibody is
humanized 2H7 comprising the variable domain sequences in SEQ ID
NOS:2 and 8 (version 16). Another preferred embodiment herein is
where the antibody is humanized 2H7 comprising the variable domain
sequences in SEQ ID NOS:39 and 40 (version 511). Further preferred
is where the antibody is humanized 2H7 comprising the variable
domain sequences in SEQ ID NOS:32 and 33 (see FIG. 40 re version
114), such as one comprising the variable light-chain domain in SEQ
ID NO:32 and the heavy-chain amino acid sequence of SEQ ID NO:34.
Further preferred is wherein the antibody is humanized 2H7
comprising a variable heavy-chain domain with alteration N100A, or
D56A and N100A, or D56A, N100Y, and S100aR in SEQ ID NO:8 and a
variable light-chain domain with alteration M32L, or S92A, or M32L
and S92A in SEQ ID NO:2.
[0156] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which nonspecific cytotoxic
cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) recognize bound antibody on a
target cell and subsequently cause lysis of the target cell. The
primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII
only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and
Fc.gamma.RIII. FcR expression on hematopoietic cells in summarized
is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol.
9:457-492 (1991). To assess ADCC activity of a molecule of
interest, an in vitro ADCC assay, such as that described in U.S.
Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 may be performed.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
[0157] "Human effector cells" are leukocytes that express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and carry out ADCC effector
function. Examples of human leukocytes that mediate ADCC include
peripheral blood mononuclear cells (PBMC), natural killer (NK)
cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and
NK cells being preferred.
[0158] The terms "Fc receptor" or "FcR" are used to describe a
receptor that binds to the Fc region of an antibody. The preferred
FcR is a native-sequence human FcR. Moreover, a preferred FcR is
one that binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RBI
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RII receptors include
Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an
"inhibiting receptor"), which have similar amino acid sequences
that differ primarily in the cytoplasmic domains thereof Activating
receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based
activation motif (ITAM) in its cytoplasmic domain. Inhibiting
receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based
inhibition motif (ITIM) in its cytoplasmic domain. (see Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol 9:457-492 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-341 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0159] "Complement-dependent cytotoxicity" or "CDC" refers to the
ability of a molecule to lyse a target in the presence of
complement. The complement activation pathway is initiated by the
binding of the first component of the complement system (Clq) to a
molecule (e.g. an antibody) complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0160] "Growth-inhibitory" antibodies are those that prevent or
reduce proliferation of a cell expressing an antigen to which the
antibody binds. For example, the antibody may prevent or reduce
proliferation of B cells in vitro and/or in vivo.
[0161] Antibodies that "induce apoptosis" are those that induce
programmed cell death, e.g. of a B cell, as determined by standard
apoptosis assays, such as binding of annexin V, fragmentation of
DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called
apoptotic bodies).
[0162] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VII) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end; the
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0163] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions both in the light chain and the heavy chain
variable domains. The more highly conserved portions of variable
domains are called the framework regions (FRs). The variable
domains of native heavy and light chains each comprise four FRs,
largely adopting a .beta.-sheet configuration, connected by three
hypervariable regions, which form loops connecting, and in some
cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in binding an antibody to an antigen, but
exhibit various effector functions, such as participation of the
antibody in ADCC.
[0164] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(abD2 fragment that has two antigen-binding sites and is
still capable of cross-linking antigen.
[0165] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and antigen-binding site. This region
consists of a dimer of one heavy chain and one light chain variable
domain in tight, non-covalent association. It is in this
configuration that the three hypervariable regions of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six hypervariable regions confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three
hypervariable regions specific for an antigen) has the ability to
recognize and bind antigen, although at a lower affinity than the
entire binding site.
[0166] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab').sub.2 antibody fragments originally were produced as
pairs of Fab' fragments that have hinge cysteines between them.
Other chemical couplings of antibody fragments are also known.
[0167] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (K) and lambda (i), based on the amino acid
sequences of their constant domains.
[0168] Depending on the amino acid sequence of the constant domain
of their heavy chains, antibodies can be assigned to different
classes. There are five major classes of intact antibodies: IgA,
IgD, IgE, IgG, and IgM, and several of these may be further divided
into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy chain constant domains that correspond to the
different classes of antibodies are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known.
[0169] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that enables the scFv to form the
desired structure for antigen binding. For a review of scFv see
Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0170] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (VL) in the same polypeptide chain (V.sub.H-V.sub.L). By
using a linker that is too short to allow pairing between the two
domains on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0171] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example
[0172] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies of interest herein include "primatized" antibodies
comprising variable domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, such as baboon, rhesus or
cynomolgus monkey) and human constant region sequences (U.S. Pat.
No. 5,693,780).
[0173] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence, except for FR
substitution(s) as noted above. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321:522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992).
[0174] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins
ofImmunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined.
[0175] A "naked antibody" is an antibody (as herein defined) that
is not conjugated to a heterologous molecule, such as a cytotoxic
moiety, polymer, or radiolabel.
[0176] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0177] "Joint damage" is used in the broadest sense and refers to
damage or partial or complete destruction to any part of one or
more joints, including the connective tissue and cartilage, where
damage includes structural and/or functional damage of any cause,
and may or may not cause joint pain/arthalgia. It includes, without
limitation, joint damage associated with or resulting from
inflammatory joint disease as well as non-inflammatory joint
disease. This damage may be caused by any condition, such as an
autoimmune disease such as lupus (e.g., systemic lupus
erythematosus), arthritis (e.g., acute and chronic arthritis,
rheumatoid arthritis including juvenile-onset rheumatoid arthritis,
juvenile idiopathic arthritis (JIA), or juvenile RA (JRA), and
stages such as rheumatoid synovitis, gout or gouty arthritis, acute
immunological arthritis, chronic inflammatory arthritis,
degenerative arthritis, type II collagen-induced arthritis,
infectious arthritis, septic arthritis, Lyme arthritis,
proliferative arthritis, psoriatic arthritis, Still's disease,
vertebral arthritis, osteoarthritis, arthritis chronica
progrediente, arthritis deformans, polyarthritis chronica primaria,
reactive arthritis, menopausal arthritis, estrogen-depletion
arthritis, and ankylosing spondylitis/rheumatoid spondylitis),
rheumatic autoimmune disease other than RA, significant systemic
involvement secondary to RA (including but not limited to
vasculitis, pulmonary fibrosis or Felty's syndrome), Sjogren's
syndrome, particular secondary such syndrome, secondary limited
cutaneous vasculitis with RA, seronegative spondyloarthropathy,
Lyme disease, inflammatory bowel disease, scleroderma, inflammatory
myopathy, mixed connective tissue disease, any overlap syndrome,
bursitis, tendonitis, osteomyelitis, infectious diseases, including
influenza, measles (rubeola), rheumatic fever, Epstein-Barr viral
syndrome, hepatitis, mumps, rebella (German measles), and varicella
(chickenpox), Chondromalacia patellae, collagenous colitis,
autoimmune disorders associated with collagen disease, joint
inflammation, unusual exertion or overuse such as sprains or
strains, injury including fracture, gout, especially found in the
big toe, as well as caused by neurological disorders, hemophilic
disorders (for example, hemophilic arthropathy), muscular
disorders, progressive disorders, bone disorders, cartilage
disorders, and vascular disorders. For purposes herein, joints are
points of contact between elements of a skeleton (of a vertebrate
such as an animal) with the parts that surround and support it and
include, but are not limited to, for example, hips, joints between
the vertebrae of the spine, joints between the spine and pelvis
(sacroiliac joints), joints where the tendons and ligaments attach
to bones, joints between the ribs and spine, shoulders, knees,
feet, elbows, hands, fingers, ankles and toes, but especially
joints in the hands and feet.
[0178] A "subject" herein is a human subject, including a patient,
eligible for treatment who is experiencing or has experienced one
or more signs, symptoms, or other indicators of joint damage, has
been diagnosed with joint damage, whether, for example, newly
diagnosed or previously diagnosed and now experiencing a recurrence
or relapse, or is at risk for developing joint damage, no matter
the cause. The subject may have been previously treated with CD20
antibody or not so treated. A subject eligible for treatment of
joint damage may optionally be identified as one who has been
screened, as in the blood, for elevated levels of infiltrating CD20
cells or is screened using an assay to detect auto-antibodies,
wherein autoantibody production is assessed qualitatively, and
preferably quantitatively. The subject may be naive to a second
medicament being used when the treatment is started, i.e., the
subject has not been previously treated with, for example, an
immunosuppressive agent such as methotrexate at "baseline" (i.e.,
at a set point in time before the administration of a first dose of
CD20 antibody in the treatment method herein, such as the day of
screening the subject before treatment is commenced). Such subjects
are generally considered to be candidates for treatment with such
second medicament.
[0179] "Treatment" of a subject herein refers to both therapeutic
treatment and prophylactic or preventative measures. Those in need
of treatment include those already with joint damage as well as
those in which the joint damage or the progress of joint damage is
to be prevented. Hence, the subject may have been diagnosed as
having the joint damage or may be predisposed or susceptible to the
joint damage, or may have limited joint damage, which is likely to
progress in the absence of treatment. Treatment is successful
herein if the joint damage is alleviated or healed, or progression
of joint or structural damage is halted or slowed down as compared
to prior to administration. Successful treatment further includes
complete or partial prevention of the development of joint damage.
For purposes herein, slowing down or reducing joint damage or the
progression of joint damage is the same as arrest, decrease, or
reversal in the joint damage.
[0180] "Clinical improvement" refers to prevention of further
progress of joint damage or any improvement in joint damage as a
result of treatment, as determined by other than radiographic
testing. Thus, clinical improvement may, for example, be determined
by assessing the number of tender or swollen joints, the Psoriasis
Assessment Severity Index, a global clinical assessment of the
subject, assessing erythrocyte sedimentation rate, or assessing the
amount of C-reactive protein level.
[0181] For purposes herein, a subject is in "remission" if he/she
has no symptoms of active joint damage, such as those detectable by
the methods disclosed herein, and has had no progression of joint
damage as assessed at baseline or at a certain point of time during
treatment. Those who are not in remission include, for example,
those experiencing a worsening or progression of joint damage. Such
subjects experiencing a return of symptoms, including active joint
damage, are those who have "relapsed" or had a "recurrence."
[0182] A "symptom" of joint damage is any morbid phenomenon or
departure from the normal in structure, function, or sensation,
experienced by the subject and indicative of joint damage, such as
those noted above, including tender or swollen joints.
[0183] The expression "effective amount" refers to an amount of the
antibody or antagonist that is effective for treating joint damage,
including an amount that is effective in achieving a reduction in
joint damage as compared to baseline prior to administration of
such amount as determined by radiographic testing. An effective
amount of other medicaments such as second medicaments is an amount
of such medicament effective to treat joint damage or other
undesirable effects, including side-effects or symptoms or other
conditions accompanying joint damage, including an underlying
disease or disorder.
[0184] "Total modified Sharp score" means a score obtained for
assessment of radiographs using the method according to Sharp, as
modified by Genant, Am. J. Med., 30: 35-47 (1983). The primary
assessment will be the change in the total Sharp-Genant score from
screening. The Sharp-Genant score combines an erosion score and a
joint space narrowing score of both hands and feet. Joint damage is
measured in this test scoring by a mean change of less than the
score at baseline (when patient is screened or tested before first
administration of CD20 antagonist herein).
[0185] As used herein, "rheumatoid arthritis" refers to a
recognized disease state which may be diagnosed according to the
2000 revised American Rheumatoid Association criteria for the
classification of rheumatoid arthritis, or any similar criteria.
Physiological indicators of RA include, symmetric joint swelling
which is characteristic though not invariable in rheumatoid
arthritis. Fusiform swelling of the proximal interphalangeal (PIP)
joints of the hands as well as metacarpophalangeal (MCP), wrists,
elbows, knees, ankles and metatarsophalangeal (MTP) joints are
commonly affected and swelling is easily detected. Pain on passive
motion is the most sensitive test for joint inflammation, and
inflammation and structural deformity often limits the range of
motion for the affected joint. Typical visible changes include
ulnar deviation of the fmgers at the MCP joints, hyperextension or
hyperflexion of the MCP and PIP joints, flexion contractures of the
elbows, and subluxation of the carpal bones and toes. The subject
with rheumatoid arthritis may be resistant to DMARDs, in that the
DMARDs are not effective or fully effective in treating symptoms.
Further, candidates for therapy according to this invention include
those who have experienced an inadequate response to previous or
current treatment with TNF inhibitors such as etanercept,
infliximab and/or adalimumab because of toxicity or inadequate
efficacy (for example, etanercept for 3 months at 25 mg twice a
week or at least 4 infusions of infliximab at 3 mg/kg). A patient
with "active rheumatoid arthritis" means a patient with active and
not latent symptoms of rheumatoid arthritis. Subjects with "early
active rheumatoid arthritis" are those subjects with active
rheumatoid arthritis diagnosed for at least 8 weeks but no longer
than four years, according to the revised 1987 ACR criteria for the
classification of RA.
[0186] Psoriatic arthritis (PsA) is an inflammatory joint disease
characterized by extensive bone resorption. Also disclosed herein,
blood samples from PsA patients, particularly those with bone
erosions on plain radiographs, exhibit a marked increase in
osteoclast precursors (OCP) compared to healthy controls.
[0187] "Antibody exposure" refers to contact with or exposure to
the antibody herein in one or more doses administered over a period
of time of about 1 day to about 5 weeks. The doses may be given at
one time or at a fixed or at irregular time intervals over this
period of exposure, such as, for example, one dose weekly for four
weeks or two doses separated by a time interval of about 13-17
days. Initial and later antibody exposures are separated in time
from each other as described in detail herein.
[0188] An exposure not being administered or provided until a
certain time "from the initial exposure" or from any prior exposure
means that the time for the second or later exposure is measured
from the time any of the doses from the prior exposure were
administered, if more than one dose was administered in that
exposure. For example, when two doses are administered in an
initial exposure, the second exposure is not given until at least
about 16-54 weeks as measured from the time the first or the second
dose was administered within that prior exposure. Similarly, when
three doses are administered, the second exposure may be measured
from the time of the first, second, or third dose within the prior
exposure. Preferably, "from the initial exposure" or from any prior
disclosure is measured from the time of the first dose.
[0189] The term "immunosuppressive agent" as used herein for
adjunct therapy refers to substances that act to suppress or mask
the immune system of the mammal being treated herein. This would
include substances that suppress cytokine production, down-regulate
or suppress self-antigen expression, or mask the MHC antigens.
Examples of such agents include 2-amino-6-aryl-5-substituted
pyrimidines (see U.S. Pat. No. 4,665,077); non-steroidal
anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus,
glucocorticoids such as cortisol or aldosterone, anti-inflammatory
agents such as a cyclooxygenase inhibitor, a 5-lipoxygenase
inhibitor, or a leukotriene receptor antagonist; purine antagonists
such as azathioprine or mycophenolate mofetil (MMF); alkylating
agents such as cyclophosphamide; bromocryptine; danazol; dapsone;
glutaraldehyde (which masks the MHC antigens, as described in U.S.
Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and
MHC fragments; cyclosporin A; steroids such as corticosteroids or
glucocorticosteroids or glucocorticoid analogs, e.g., prednisone,
methylprednisolone, including SOLU-MEDROL.RTM. methylprednisolone
sodium succinate, and dexamethasone; dihydrofolate reductase
inhibitors such as methotrexate (oral or subcutaneous);
anti-malarial agents such as chloroquine and hydroxychloroquine;
sulfasalazine; leflunomide; cytokine antagonists such as cytokine
antibodies or cytokine receptor antibodies including
anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor
necrosis factor (TNF)-alpha antibodies (infliximab (REMICADE.RTM.)
or adalimumab), anti-TNF-alpha immunoadhesin (etanercept),
anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and
anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6)
receptor antibodies and antagonists; anti-LFA-1 antibodies,
including anti-CD11 a and anti-CD18 antibodies; anti-L3T4
antibodies; heterologous anti-lymphocyte globulin; pan-T
antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies;
soluble peptide containing a LFA-3 binding domain (WO 90/08187
published 7/26/90); streptokinase; transforming growth factor-beta
(TGF-beta); streptodornase; RNA or DNA from the host; FK506;
RS-61443; , chlorambucil; deoxyspergualin; rapamycin; T-cell
receptor (Cohen et al., U.S. Pat. No. 5,114,721); T-cell receptor
fragments (Officer et al., Science, 251: 430-432 (1991); WO
90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); BAFF
antagonists such as BAFF antibodies and BR3 antibodies and zTNF4
antagonists (for review, see Mackay and Mackay, Trends Immunol.,
23:113-5 (2002)); biologic agents that interfere with T cell helper
signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154),
including blocking antibodies to CD4O-CD40 ligand (e.g., Durie et
al., Science, 261: 1328-30 (1993); Mohan et al., J. Immunol., 154:
1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7
(1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9.
Some immunosuppressive agents herein are also DMARDs, such as
methotrexate. Examples of preferred immunosuppressive agents herein
include cyclophosphamide, chlorambucil, azathioprine, leflunomide,
MMF, or methotrexate.
[0190] The term "cytokine" is a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines;
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, including
PROLEUKIN.RTM. rIL-2; a tumor necrosis factor such as TNF-.alpha.
or TNF-.beta.; and other polypeptide factors including LIF and kit
ligand (KL). As used herein, the term cytokine includes proteins
from natural sources or from recombinant cell culture and
biologically active equivalents of the native-sequence cytokines,
including synthetically produced small-molecule entities and
pharmaceutically acceptable derivatives and salts thereof. A
"cytokine antagonise" is a molecule that inhibits or antagonizes
such cytokines by any mechanism, including, for example, antibodies
to the cytokine, antibodies to the cytokine receptor, and
immunoadhesins.
[0191] The term "hormone" refers to polypeptide hormones, which are
generally secreted by glandular organs with ducts. Included among
the hormones are, for example, growth hormone such as human growth
hormone, N-methionyl human growth hormone, and bovine growth
hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; estradiol; hormone-replacement therapy; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
or testolactone; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); prolactin, placental lactogen, mouse
gonadotropin-associated peptide, gonadotropin-releasing hormone;
inhibin; activin; mullerian-inhibiting substance; and
thrombopoietin. As used herein, the term hormone includes proteins
from natural sources or from recombinant cell culture and
biologically active equivalents of the native-sequence hormone,
including synthetically produced small-molecule entities and
pharmaceutically acceptable derivatives and salts thereof.
[0192] The term "growth factor" refers to proteins that promote
growth, and include, for example, hepatic growth factor; fibroblast
growth factor; vascular endothelial growth factor; nerve growth
factors such as NGF-I.beta., platelet-derived growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-I.beta., insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -.beta., and -.gamma.; and colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF).
As used herein, the term growth factor includes proteins from
natural sources or from recombinant cell culture and biologically
active equivalents of the native-sequence growth factor, including
synthetically produced small-molecule entities and pharmaceutically
acceptable derivatives and salts thereof.
[0193] The term "integrin" refers to a receptor protein that allows
cells both to bind to and to respond to the extracellular matrix
and is involved in a variety of cellular functions such as wound
healing, cell differentiation, homing of tumor cells and apoptosis.
They are part of a large family of cell adhesion receptors that are
involved in cell-extracellular matrix and cell-cell interactions.
Functional integrins consist of two transmembrane glycoprotein
subunits, called alpha and beta, that are non-covalently bound. The
alpha subunits all share some homology to each other, as do the
beta subunits. The receptors always contain one alpha chain and one
beta chain Examples include Alpha6betal, Alpha3betal, Alpha7betal,
LFA-1 etc. As used herein, the term "integrin" includes proteins
from natural sources or from recombinant cell culture and
biologically active equivalents of the native-sequence integrin,
including synthetically produced small-molecule entities and
pharmaceutically acceptable derivatives and salts thereof.
[0194] For the purposes herein, "tumor necrosis factor alpha
(TNF-alpha)" refers to a human TNF-alpha molecule comprising the
amino acid sequence as described in Pennica et al., Nature, 312:721
(1984) or Aggarwal et al., JBC, 260:2345 (1985).A "TNF-alpha
inhibitor" herein is an agent that inhibits, to some extent, a
biological function of TNF-alpha, generally through binding to
TNF-alpha and neutralizing its activity. Examples of TNF inhibitors
specifically contemplated herein are etanercept (ENBREL.RTM.),
infliximab (REMICADE.RTM.), and adalimumab (HUMIRA.TM.).
[0195] Examples of "disease-modifying anti-rheumatic drugs" or
"DMARDs" include hydroxycloroquine, sulfasalazine, methotrexate,
leflunomide, etanercept, infliximab (plus oral and subcutaneous
methotrexate), azathioprine, D-penicillamine, gold salts (oral),
gold salts (intramuscular), minocycline, cyclosporine including
cyclosporine A and topical cyclosporine, staphylococcal protein A
(Goodyear and Silverman, J. Exp. Med., 197, (9), p1125-39 (2003)),
including salts and derivatives thereof, etc. A preferred DMARD
herein is methotrexate.
[0196] Examples of "non-steroidal anti-inflammatory drugs" or
"NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen,
flurbiprofen, naproxen, indomethacin, sulindac, tolmetin,
phenylbutazone, diclofenac, ketoprofen, benorylate, mefenamic acid,
methotrexate, fenbufen, azapropazone; COX-2 inhibitors such as
celecoxib (CELEBREX.RTM.;
4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)
benzenesulfonamide, valdecoxib (BEXTRA.RTM.), meloxicam
(MOBIC.RTM.), GR 253035 (Glaxo Wellcome); and MK966 (Merck Sharp
& Dohme), including salts and derivatives thereof, etc.
Preferably, they are aspirin, naproxen, ibuprofen, indomethacin, or
tolmetin.
[0197] Examples of "integrin antagonists or antibodies" herein
include an LFA-1 antibody, such as efalizumab (RAPTIVA.RTM.)
commercially available from Genentech, or an alpha 4 integrin
antibody such as natalizumab (ANTEGREN.RTM.) available from Biogen,
or diazacyclic phenylalanine derivatives (WO 2003/89410),
phenylalanine derivatives (WO 2003/70709, WO 2002/28830, WO
2002/16329 and WO 2003/53926), phenylpropionic acid derivatives (WO
2003/10135), enamine derivatives (WO 2001/79173), propanoic acid
derivatives (WO 2000/37444), alkanoic acid derivatives (WO
2000/32575), substituted phenyl derivatives (U.S. Pat. Nos.
6,677,339 and 6,348,463), aromatic amine derivatives (U.S. Pat. No.
6,369,229), ADAM disintegrin domain polypeptides (US2002/0042368),
antibodies to alphavbeta3 integrin (EP 633945), aza-bridged
bicyclic amino acid derivatives (WO 2002/02556), etc.
[0198] "Corticosteroid" refers to any one of several synthetic or
naturally occurring substances with the general chemical structure
of steroids that mimic or augment the effects of the naturally
occurring corticosteroids. Examples of synthetic corticosteroids
include prednisone, prednisolone (including methylprednisolone,
such as SOLU-MEDROL.RTM.methylprednisolone sodium succinate),
dexamethasone or dexamethasone triamcinolone, hydrocortisone, and
betamethasone. The preferred corticosteroids herein are prednisone,
methylprednisolone, hydrocortisone, or dexamethasone.
[0199] The terms "BAFF," "BAFF polypeptide," "TALL-1" or "TALL-1
polypeptide," and "BLyS" when used herein encompass
"native-sequence BAFF polypeptides" and "BAFF variants". "BAFF" is
a designation given to those polypeptides that have any one of the
amino acid sequences shown below:
TABLE-US-00012 Human BAFF sequence (SEQ ID NO: 16): 1
MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCC 61
LTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAP 121
GEGNSSQNSRNKRAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEE 181
KENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETL 241
PNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL Mouse BAFF sequence
(SEQ ID NO: 17): 1
MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLAATLLLALLSS 61
SFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPELTAGVKLLTPAAPRPHNSSR 121
GHRNRRAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNIIQDCLQLIADSDTP 181
TIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFIYSQVLYTDPIFAMGHVIQRKK 241
VHVFGDELSLVTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRNGDD 301
TFFGALKLL
and homologs and fragments and variants thereof, which have the
biological activity of the native BAFF. A biological activity of
BAFF can be selected from the group consisting of promoting B-cell
survival, promoting B-cell maturation and binding to BR3. Variants
of BAFF will preferably have at least 80% or any successive integer
up to 100% including, more preferably, at least 90%, and even more
preferably, at least 95% amino acid sequence identity with a native
sequence of a BAFF polypeptide.
[0200] A "native-sequence" BAFF polypeptide comprises a polypeptide
having the same amino acid sequence as the corresponding BAFF
polypeptide derived from nature. For example, BAFF exists in a
soluble form following cleavage from the cell surface by furin-type
proteases. Such native-sequence BAFF polypeptides can be isolated
from nature or can be produced by recombinant and/or synthetic
means.
[0201] The term "native-sequence BAFF polypeptide" or "native BAFF"
specifically encompasses naturally occurring truncated or secreted
forms (e.g., an extracellular domain sequence), naturally occurring
variant forms (e.g., alternatively spliced forms), and naturally
occurring allelic variants of the polypeptide. The term "BAFF"
includes those polypeptides described in Shu et al., J. Leukocyte
Biol., 65:680 (1999); GenBank Accession No. AF136293; WO 1998/18921
published May 7, 1998; EP 869,180 published Oct. 7, 1998; WO
1998/27114 published Jun. 25, 1998; WO 1999/12964 published Mar.
18, 1999; WO 1999/33980 published Jul. 8, 1999; Moore et al.,
Science, 285:260-263 (1999); Schneider et al., J. Exp. Med.,
189:1747-1756 (1999) and Mukhopadhyay et al., J. Biol. Chem.,
274:15978-15981 (1999).
[0202] The term "BAFF antagonist" as used herein is used in the
broadest sense, and includes any molecule that (1) binds a
native-sequence BAFF polypeptide or binds a native-sequence of BR3
to partially or fully block BR3 interaction with BAFF polypeptide,
and (2) partially or fully blocks, inhibits, or neutralizes
native-sequence BAFF activity. In one preferred embodiment the BAFF
receptor to be blocked is the BR3 receptor. Native BAFF activity
promotes, among other things, B-cell survival and/or B-cell
maturation. In one embodiment, the inhibition, blockage or
neutralization of BAFF activity results in a reduction in the
number of B cells. A BAFF antagonist according to this invention
will partially or fully block, inhibit, or neutralize one or more
biological activities of a BAFF polypeptide, in vitro and/or in
vivo. In one embodiment, a biologically active BAFF potentiates any
one or a combination of the following events in vitro and/or in
vivo: an increased survival of B cells, an increased level of IgG
and/or IgM, an increased numbers of plasma cells, and processing of
NF-.kappa.b2/100 to p52 NF-.kappa.b in splenic B cells (e.g.,
Batten et al., J. Exp. Med. 192:1453-1465 (2000); Moore et al.,
Science 285:260-263 (1999); Kayagaki et al. Immunity 17:515-524
(2002)).
[0203] As mentioned above, a BAFF antagonist can function in a
direct or indirect manner to partially or fully block, inhibit or
neutralize BAFF signaling, in vitro or in vivo. For instance, the
BAFF antagonist can directly bind BAFF. For example, BAFF
antibodies that bind within a region of human BAFF comprising
residues 162-275 and/or a neighboring residue of a residue selected
from the group consisting of 162, 163, 206, 211, 231, 233, 264 and
265 of human BAFF such that the antibody sterically hinders BAFF
binding to BR3 are contemplated, where such residue numbers refer
to SEQ ID NO:16. In another example, a direct binder is a
polypeptide comprising any portion of a BAFF receptor that binds
BAFF such as an extracellular domain of a BAFF receptor, or
fragments and variants thereof that bind native BAFF. In another
example, BAFF antagonists include the polypeptides having a
sequence of a polypeptide comprising the sequence of Formula I:
X.sub.1--C--X.sub.3--D--X.sub.5-L-X.sub.7--X.sub.8--X.sub.9--X.sub.10--X.-
sub.11--X.sub.12--C--X.sub.14--X.sub.15--X.sub.16--X.sub.17
(Formula I) (SEQ ID NO:18) wherein X.sub.1, X.sub.3, X.sub.5,
X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.14,
X.sub.15 and X.sub.17 are any amino acid except cysteine; and
wherein X.sub.16 is an amino acid selected from the group
consisting of L, F, I and V; and wherein the polypeptide does not
comprise a cysteine within seven amino acid residues N-terminal to
the most N-terminal cysteine C and C-terminal to the most
C-terminal cysteine C of Formula I.
[0204] In one embodiment, a polypeptide comprising the sequence of
Formula I has the two Cs joined by disulfide bonding;
X.sub.5LX.sub.7X.sub.8 forming the conformation of a type I beta
turn structure with the center of the turn between L and X.sub.7;
and has a positive value for the dihedral angle phi of X.sub.8. In
one embodiment, Xio is selected from the group consisting of W, F,
V, L, I, Y, M and a non-polar amino amino acid. In another
embodiment, X.sub.10 is W. In another embodiment, X.sub.3 is an
amino acid selected from the group consisting of M, V, L, I, Y, F,
W and a non-polar amino acid. In another embodiment, X.sub.5 is
selected from the group consisting of V, L, P, S, I, A and R. In
another embodiment, X.sub.7 is selected from the group consisting
of V, T, I and L. In another embodiment, X.sub.8 is selected from
the group consisting of R, K, G, N, H and a D-amino acid. In
another embodiment, X.sub.9 is selected from the group consisting
of H, K, A, R and Q. In another embodiment, X.sub.11 is I or V. In
another embodiment, X.sub.12 is selected from the group consisting
of P, A, D, E and S. In another embodiment, X.sub.16 is L. In one
specific embodiment, the sequence of Formula I is a sequence
selected from the group consisting of ECFDLLVRAWVPCSVLK (SEQ ID
NO:19), ECFDLLVRHWVPCGLLR (SEQ ID NO:20), ECFDLLVRRWVPCEMLG (SEQ ID
NO:21), ECFDLLVRSWVPCHMLR (SEQ ID NO:22), ECFDLLVRHWVACGLLR (SEQ ID
NO:23), and QCFDRLNAWVPCSVLK (SEQ ID NO:24). In a preferred
embodiment, the BAFF antagonist comprises any one of the amino acid
sequences selected from the group consisting of SEQ ID NO:19, 20,
21, 22, and 23.
[0205] In still another example, BAFF antagonists include the
polypeptides having a sequence of a polypeptide comprising the
sequence of Formula II:
X.sub.1--C-X.sub.3--D--X.sub.5--L--V--X.sub.8--X.sub.9--W--V--P--C--X.sub-
.14--X.sub.15--L--X.sub.17 (Formula II) (SEQ ID NO:25) wherein
X.sub.1, X.sub.3, X.sub.5, X.sub.8; X.sub.9, X.sub.14, X.sub.15 and
X.sub.17 are any amino acid, except cysteine; and wherein the
polypeptide does not comprise a cysteine within seven amino acid
residues N-terminal to the most N-terminal cysteine C and
C-terminal to the most C-terminal cysteine C of Formula II.
[0206] In one embodiment, a polypeptide comprising the sequence of
Formula II has a disulfide bond between the two Cs and has the
conformation of X.sub.5LX.sub.7X.sub.8 forming a type I beta turn
structure with the center of the turn between L and X.sub.7; and
has a positive value for the dihedral angle phi of X.sub.8. In
another embodiment of Formula II, X.sub.3 is an amino acid selected
from the group consisting of M, A, V, L, I, Y, F, W and a non-polar
amino acid. In another embodiment of Formula II, X.sub.5 is
selected from the group consisting of V, L, P, S, I, A and R. In
another embodiment of Formula II, X.sub.8 is selected from the
group consisting of R, K, G, N, H and D-amino acid. In another
embodiment of Formula II, X.sub.9 is selected from the group
consisting of H, K, A, R and Q.
[0207] In a further embodiment, the BAFF receptor from which the
extracellular domain or BAFF-binding fragment or BAFF-binding
variant thereof is derived is TACI, BR3 or BCMA. Alternatively, the
BAFF antagonist can bind an extracellular domain of a
native-sequence BR3 at its BAFF binding region to partially or
fully block, inhibit or neutralize BAFF binding to BR3 in vitro, in
situ, or in vivo. For example, such indirect antagonist is an
anti-BR3 antibody that binds in a region of BR3 comprising residues
23-38 of human BR3 as defined below (SEQ ID NO:26) or a neighboring
region of those residues such that binding of human BR3 to BAFF is
sterically hindered.
[0208] In some embodiments, a BAFF antagonist according to this
invention includes BAFF antibodies and immunoadhesins comprising an
extracellular domain of a BAFF receptor, or fragments and variants
thereof that bind native BAFF. In a further embodiment, the BAFF
receptor from which the extracellular domain or BAFF-binding
fragment or BAFF-binding variant thereof is derived is TACI, BR3 or
BCMA. In a still another embodiment, the immunoadhesin comprises an
amino acid sequence of that of Formula I or Formula II as set forth
above, including an amino acid sequence selected from any one of
the group consisting of SEQ ID NOS: 19, 20, 21, 22, 23, and 24.
[0209] According to one embodiment, the BAFF antagonist binds to a
BAFF polypeptide or a BR3 polypeptide with a binding affinity of
100nM or less. According to another embodiment, the BAFF antagonist
binds to a BAFF polypeptide or a BR3 polypeptide with a binding
affinity of 10nM or less. According to yet another embodiment, the
BAFF antagonist binds to a BAFF polypeptide or a BR3 polypeptide
with a binding affinity of 1nM or less.
[0210] The terms "BR3", "BR3 polypeptide" or "BR3 receptor" when
used herein encompass "native-sequence BR3 polypeptides" and "BR3
variants" (which are further defined herein). "BR3" is a
designation given to those polypeptides comprising the following
amino acid sequence and homologs thereof, and variants or fragments
thereof that bind native BAFF:
TABLE-US-00013 Human BR3 sequence (SEQ ID NO: 26): 1
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ 61
ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD 121
KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG 181
PEQQ.
[0211] The BR3 polypeptides of the invention can be isolated from a
variety of sources, such as from human tissue types or from another
source, or prepared by recombinant and/or synthetic methods. The
term BR3 includes the BR3 polypeptides described in WO 2002/24909
and WO 2003/14294.
[0212] A "native-sequence" BR3 polypeptide or "native BR3"
comprises a polypeptide having the same amino acid sequence as the
corresponding BR3 polypeptide derived from nature. Such
native-sequence BR3 polypeptides can be isolated from nature or can
be produced by recombinant and/or synthetic means. The term
"native-sequence BR3 polypeptide" specifically encompasses
naturally occurring truncated, soluble or secreted forms (e.g., an
extracellular domain sequence), naturally occurring variant forms
(e.g., alternatively spliced forms) and naturally occurring allelic
variants of the polypeptide. The BR3 polypeptides of the invention
include the BR3 polypeptide comprising or consisting of the
contiguous sequence of amino acid residues 1 to 184 of a human BR3
(SEQ ID NO:26).
[0213] A BR3 "extracellular domain" or "ECD" refers to a form of
the BR3 polypeptide that is essentially free of the transmembrane
and cytoplasmic domains. ECD forms of BR3 include a polypeptide
comprising any one of the amino acid sequences selected from the
group consisting of amino acids 1-77, 2-62, 2-71, 1-61, 7-71, 23-38
and 2-63 of human BR3. The invention contemplates BAFF antagonists
that are polypeptides comprising any one of the above-mentioned ECD
forms of human BR3 and variants and fragments thereof that bind a
native BAFF.
[0214] Mini-BR3 is a 26-residue core region of the BAFF-binding
domain of BR3, i.e., the amino acid sequence: TPCVPAECFD LLVRHCVACG
LLRTPR (SEQ ID NO:27)
[0215] "BR3 variant" means a BR3 polypeptide having at least about
80% amino acid sequence identity with the amino acid sequence of a
native-sequence, full-length BR3 or BR3 ECD and binds a
native-sequence BAFF polypeptide. Optionally, the BR3 variant
includes a single cysteine-rich domain. Such BR3 variant
polypeptides include, for instance, BR3 polypeptides wherein one or
more amino acid residues are added, or deleted, at the N- and/or
C-terminus, as well as within one or more internal domains, of the
full-length amino acid sequence. Fragments of the BR3 ECD that bind
a native sequence BAFF polypeptide are also contemplated. According
to one embodiment, a BR3 variant polypeptide will have at least
about 80% amino acid sequence identity, at least about 81% amino
acid sequence identity, at least about 82% amino acid sequence
identity, at least about 83% amino acid sequence identity, at least
about 84% amino acid sequence identity, at least about 85% amino
acid sequence identity, at least about 86% amino acid sequence
identity, at least about 87% amino acid sequence identity, at least
about 88% amino acid sequence identity, at least about 89% amino
acid sequence identity, at least about 90% amino acid sequence
identity, at least about 91% amino acid sequence identity, at least
about 92% amino acid sequence identity, at least about 93% amino
acid sequence identity, at least about 94% amino acid sequence
identity, at least about 95% amino acid sequence identity, at least
about 96% amino acid sequence identity, at least about 97% amino
acid sequence identity, at least about 98% amino acid sequence
identity or at least about 99% amino acid sequence identity with a
human BR3 polypeptide or a specified fragment thereof (e.g., ECD).
BR3 variant polypeptides do not encompass the native BR3
polypeptide sequence. According to another embodiment, BR3 variant
polypeptides are at least about 10 amino acids in length, at least
about 20 amino acids in length, at least about 30 amino acids in
length, at least about 40 amino acids in length, at least about 50
amino acids in length, at least about 60 amino acids in length, or
at least about 70 amino acids in length.
[0216] In one preferred embodiment, the BAFF antagonists herein are
immunoadhesins comprising a portion of BR3, TACI or BCMA that binds
BAFF, or variants thereof that bind BAFF. In other embodiments, the
BAFF antagonist is a BAFF antibody. A "BAFF antibody" is an
antibody that binds BAFF, and preferably binds BAFF within a region
of human BAFF comprising residues 162-275 of the human BAFF
sequence disclosed herein under the "BAFF" definition (SEQ ID
NO:16). In another embodiment, the BAFF antagonist is BR3 antibody.
A "BR3 antibody" is an antibody that binds BR3, and is preferably
one that binds BR3 within a region of human BR3 comprising residues
23-38 of the human BR3 sequence disclosed herein under the "BR3"
definition (SEQ ID NO:26). In general, the amino acid positions of
human BAFF and human BR3 referred to herein are according to the
sequence numbering under human BAFF and human BR3, SEQ ID NOS: 16
and 26, respectively, disclosed herein under the "BAFF" and "BR3"
definitions.
[0217] Other examples of BAFF-binding polypeptides or BAFF
antibodies can be found in, e.g., WO 2002/092620, WO 2003/014294,
Gordon et al., Biochemistry 42(20):5977-5983 (2003), Kelley et al.,
J. Biol. Chem. 279 (16):16727-16735 (2004), WO 1998/18921, WO
2001/12812, WO 2000/68378 and WO 2000/40716.
[0218] A "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications, other therapeutic
products to be combined with the packaged product, and/or warnings
concerning the use of such therapeutic products, etc.
[0219] A "medicament" is an active drug to treat the joint damage
or its symptoms or side effects.
[0220] II. Therapy
[0221] In one aspect, the present invention provides a method of
treating joint damage in a subject such as a patient comprising
administering an antagonist, preferably an antibody, that binds to
a B-cell surface marker (more preferably a CD20 antibody) to the
subject and assessing by radiographic x-ray if the subject has
shown a reduction in joint damage upon treatment.
[0222] Thus, the invention contemplates a method for treating joint
damage in a subject comprising administering an antagonist that
binds to a B-cell surface marker to the subject and giving the
subject, at least about one month after the administration, a
radiographic test that measures a reduction in the joint damage as
compared to baseline prior to the administration, wherein the
amount of antagonist administered is effective in achieving a
reduction in the joint damage, indicating that the subject has been
successfully treated for the joint damage.
[0223] The invention also contemplates a method for treating joint
damage in a subject comprising administering an antibody that binds
to a B-cell surface marker to the subject and giving the subject,
at least about one month after the administration, a radiographic
test that measures a reduction in the joint damage as compared to
baseline prior to the administration, wherein the amount of
antibody administered is effective in achieving a reduction in the
joint damage, indicating that the subject has been successfully
treated for the joint damage.
[0224] The invention further contemplates a method for treating
joint damage in a subject comprising administering a CD20 antibody
to the subject and giving the subject, at least about one month
after the administration, a radiographic test that measures a
reduction in the joint damage as compared to baseline prior to the
administration, wherein the amount of CD20 antibody administered is
effective in achieving a reduction in the joint damage, indicating
that the subject has been successfully treated for the joint
damage.
[0225] In a preferred embodiment, the radiographic testing after
administering the antagonist or antibody such as CD20 antibody
occurs at least about two months, more preferably at least about 10
weeks, still more preferably at least about three months, further
preferably at least about four months, still more preferably at
least about five months, further more preferably at least about 24
weeks, or at least about six months, and most preferably at least
about 52 weeks after administering the antagonist or antibody. In
another preferred embodiment, the test measures a total modified
Sharp score.
[0226] In another preferred embodiment, the subject is retreated in
that the method further comprises administering to the subject an
antagonist or antibody such as CD20 antibody in an amount effective
to achieve a continued or maintained reduction in joint damage as
compared to the effect of a prior administration of the antagonist
or antibody such as CD20 antibody. Thus, the subject may be
administered a second dosing of the antagonist or antibody and is
evaluated by radiographic testing at least about one month (and
preferably more than about two months, more preferably about 24
weeks or about 6 months) after such second dosing to determine if
the second dosing is effective (i.e., an effective amount of the
antagonist or antibody is administered) to maintain the effects of
the first dosing or improve the reduction in joint damage as
compared to the effect of the first dosing. This re-treatment
regimen can be repeated as long as desired or necessary to achieve
or maintain reduction in joint damage, which indicates successful
treatment of the joint damage.
[0227] In another embodiment, the antagonist or antibody such as
CD20 antibody is additionally (continued to be) administered to the
subject even if there is not a clinical improvement in the subject
at the time of the radiographic testing after a prior
administration, such as the first administration of the antagonist
or antibody. In the latter embodiment, preferably the clinical
improvement is determined by assessing the number of tender or
swollen joints, the Psoriasis Assessment Severity Index, a global
clinical assessment of the subject, assessing erythrocyte
sedimentation rate, or assessing the amount of C-reactive protein
level.
[0228] For purposes of this invention, the second antagonist or
antibody exposure for re-treatment is the next time the subject is
treated with the antagonist or, for example, CD20 antibody after
the initial antibody exposure, there being no intervening
antagonist or, e.g., CD20 antibody treatment or exposure between
the initial and second exposures. Such re-treatment may be
scheduled or unscheduled, but is preferably a scheduled redosing,
particularly to protect organs such as kidneys from damage. If an
antibody, especially a CD20 antibody, is employed, preferably the
second antibody exposure is about 0.5 to 4 grams, more preferably
about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5
grams, the second exposure not being provided until from about 20
to 35 weeks (preferably about 23 to 30, more preferably about 23 to
28 weeks) from the initial exposure.
[0229] The method contemplates administering to the subject an
effective amount of the antagonist or, for example, CD20 antibody
to provide a third antagonist or antibody exposure (if antibody,
more preferably CD20 antibody) preferably of about 0.5 to 4 grams,
more preferably about 1.5 to 3.5 grams, still more preferably about
1.5 to 2.5 grams), the third exposure not being provided until from
about 46 to 60 weeks (preferably about 46 to 55, more preferably
about 46 to 52 weeks) from the initial exposure. Preferably, no
further antagonist or antibody exposure is provided until at least
about 70-75 weeks from the initial exposure, and still more
preferably no further antagonist or antibody exposure is provided
until about 74 to 80 weeks from the initial exposure.
[0230] Where an antibody is employed, any one or more of the
antibody exposures herein may be provided to the subject as a
single dose of antibody, or as separate doses, for example, about
1-4 separate doses of the antibody (e.g., constituting a first and
second dose, or a first, second, and third dose, or a first,
second, third, and fourth dose, etc). The particular number of
doses (whether one, two or three or more) employed for each
antibody exposure is dependent, for example, on the type of joint
damage treated, the type of antibody employed, whether, what type,
and how much and how many of a second medicament is employed as
noted below, and the method and frequency of administration. Where
separate doses are administered, the later dose (for example,
second or third dose) is preferably administered from about 1 to 20
days, more preferably from about 6 to 16 days, and most preferably
from about 14 to 16 days from the time the previous dose was
administered. The separate doses are preferably administered within
a total period of between about 1 day and 4 weeks, more preferably
between about 1 and 20 days (e.g., within a period of 6-18 days).
In one such aspect, the separate doses are administered about
weekly, with the second dose being administered about one week from
the first dose and any third or subsequent dose being administered
about one week from the second dose. Each such separate dose of the
antibody is preferably about 0.5 to 1.5 grams, more preferably
about 0.75 to 1.3 grams.
[0231] In a most preferred embodiment, a method of treating joint
damage in a subject is provided comprising administering an
effective amount of an antibody that binds to a B-cell surface
marker (e.g., a CD20 antibody) to the subject to provide an initial
antibody exposure followed by a second antibody exposure, wherein
the second exposure is not provided until from about 16 to 54 weeks
from the initial exposure and each of the antibody exposures is
provided to the subject as a single dose or as two or three
separate doses of antibody. Preferably in such a method, the
antibody exposures are of about 0.5 to 4 grams each, and most
preferably the amounts given above.
[0232] In one embodiment, the subject is provided at least about
three exposures of the antibody, for example, from about 3 to 60
exposures, and more particularly about 3 to 40 exposures, most
particularly, about 3 to 20 exposures. Preferably, such exposures
are administered at intervals each of 24 weeks. In one embodiment,
each antibody exposure is provided as a single dose of the
antibody. In an alternative embodiment, each antibody exposure is
provided as separate doses of the antibody. However, not every
antibody exposure need be provided as a single dose or as separate
doses.
[0233] In one preferred embodiment, about 2-3 grams of the CD20
antibody is administered as the initial exposure. If about 3 grams
are administered, then about 1 gram of the CD20 antibody is
administered weekly for about three weeks as the initial exposure.
If about 2 grams of the CD20 antibody is administered as the
initial exposure, then about 1 gram of the CD20 antibody is
administered followed in about two weeks by another about 1 gram of
the antibody as the initial exposure. In a preferred aspect, the
second exposure is at about 24 weeks or six months from the initial
exposure and is administered in an amount of about 2 grams. In an
alternative preferred aspect, the second exposure is at about 24
weeks or six months from the initial exposure and is administered
as about 1 gram of the antibody followed in about two weeks by
another about 1 gram of the antibody.
[0234] In a preferred embodiment of the multi-exposure method
herein, the subject is in remission after the initial or any later
antagonist or antibody exposures. More preferably, the
multi-exposure method herein involves scheduled re-dosing or
re-treating such that the subject is in remission when provided the
second, and preferably all antagonist or antibody exposures. Such
re-dosing is scheduled to prevent any relapse, recurrence, or organ
damage, rather than to treat it therapeutically. Most preferably,
the subject is in remission for at least about 24 weeks or six
months, and still most preferably at least about nine months, and
even still most preferably at least about 52 weeks or one year
since the last antagonist or antibody exposure used in the
re-treatment method.
[0235] In yet another embodiment, the subject is treated with the
same antagonist or antibody, such as CD20 antibody, for at least
two antagonist or antibody exposures, and preferably for each
antagonist or antibody exposure. Thus, the initial and second
antagonist or antibody exposures are preferably with the same
antagonist or antibody, and more preferably all antagonist or
antibody exposures are with the same antagonist or antibody, i.e.,
treatment for the first two exposures, and preferably all
exposures, is with one type of antagonist or antibody that binds to
a B-cell surface marker, such as CD20 antibody, e.g., all with
rituximab or all with the same humanized 2H7.
[0236] In all the inventive methods set forth herein, the
antagonist (such as CD20 or B-cell surface marker antibody) may be
unconjugated, such as a naked antibody, or may be conjugated with
another molecule for further effectiveness, such as, for example,
to improve half-life. The preferred CD20 antibody herein is a
chimeric, humanized, or human CD20 antibody, more preferably
rituximab, a humanized 2H7 (e.g. comprising the variable domain
sequences in SEQ ID Nos. 2 and 8, or is humanized 2H7 comprising
the variable domain sequences in SEQ ID NOS:39 and 40, or
comprising the variable domain sequences in SEQ ID NOS:32 and 33,
or comprising a variable heavy-chain domain with alteration N100A,
or D56A and N100A, or D56A, N100Y, and S100aR in SEQ ID NO:8 and a
variable light-chain domain with alteration M32L, or S92A, or M32L
and S92A in SEQ ID NO:2), chimeric or humanized A20 antibody
(Immunomedics), HUMAX-CD20.TM. human CD20 antibody (Genmab), or
single-chain proteins binding to CD20 (Trubion Pharm Inc.). Still
more preferred is rituximab or a humanized 2H7.
[0237] In a further embodiment of all the methods herein, the
subject has never been previously treated with one or more drug(s),
such as with an anti-TNF-alpha inhibitor, e.g., an anti-TNF-alpha
or anti-TNF-alpha receptor antibody, to treat, for example,
arthritis, or with immunosuppressive agent(s) to treat the joint
damage or an underlying cause such as an autoimmune disorder,
and/or has never been previously treated with an antagonist (for
example, antibody) to a B-cell surface marker (e.g. never been
previously treated with a CD20 antibody). In one such embodiment,
the subject has never been previously treated with an anti-alpha 4
integrin antibody or co-stimulation modulator, a biologic agent, a
DMARD other than MTX, except for azathioprine and/or leflunomide, a
cell-depleting therapy, including investigational agents (e.g.,
CAMPATH, anti-CD4, anti-CD5, anti-CD3, anti-CD19, anti-CD1 1 a,
anti-CD22, or BLys/BAFF), a live/attenuated vaccine within 28 days
prior to baseline, or intra-articular or parenteral glucocorticoids
within 4 weeks prior to baseline.
[0238] In a still further aspect, the subject may have had a
relapse with the joint damage or suffered organ damage such as
kidney damage before being treated in any of the methods above,
including after the initial or a later antagonist or antibody
exposure. However, preferably, the subject has not relapsed with
the joint damage and more preferably has not had such a relapse
before at least the initial treatment.
[0239] In another embodiment, the antagonist (for example, CD20
antibody) is the only medicament administered to the subject to
treat the joint damage. In another embodiment, the antagonist
(e.g., CD20 antibody) is one of the medicaments used to treat the
joint damage. In a further embodiment, the subject does not have a
malignancy, including solid tumors, hematologic malignancies, or
carcinoma in situ (except basal cell and squamous cell carcinoma of
the skin that have been excised and cured). In a still further
embodiment, the subject does not have rheumatoid arthritis (RA). In
another aspect, the subject does not have rheumatic autoimmune
disease other than RA, or significant systemic involvement
secondary to RA (including but not limited to vasculitis, pulmonary
fibrosis or Felty's syndrome). In another embodiment, the subject
does have secondary Sjogren's syndrome or secondary limited
cutaneous vasculitis. In another embodiment, the subject does not
have functional class IV as defined by the ACR Classification of
Functional Status in RA. In a further embodiment, the subject does
not have inflammatory joint disease other than RA (including, but
not limited to, gout, reactive arthritis, psoriatic arthritis,
seronegative spondyloarthropathy, Lyme disease), or other systemic
autoimmune disorder (including, but not limited to, systemic lupus
erythematosus, inflammatory bowel disease, scleroderma,
inflammatory myopathy, mixed connective tissue disease, or any
overlap syndrome). In another embodiment, the subject does not have
juvenile idiopathic arthritis (JIA) or juvenile RA (JRA) and/or RA
before age 16. In another embodiment, the subject does not have
significant and/or uncontrolled cardiac or pulmonary disease
(including obstructive pulmonary disease), or significant
concomitant disease, including but not limited to, nervous system,
renal, hepatic, endocrine or gastrointestinal disorders., nor
primary or secondary immunodeficiency (history of, or currently
active), including known history of HIV infection. In another
aspect, the subject does not have any neurological (congenital or
acquired), vascular or systemic disorder which could affect any of
the efficacy assessments, in particular, joint pain and swelling
(e.g., Parkinson's disease, cerebral palsy, diabetic neuropathy).
In a still further embodiment, the subject does not have multiple
sclerosis. In a yet further embodiment, the subject does not have
lupus or Sjogren's syndrome. In still another embodiment, the
subject does not have an autoimmune disease. In yet another aspect
of the invention, the joint damage is not associated with an
autoimmune disease or with an autoimmune disease other than
arthritis, or with a risk of developing an autoimmune disease or an
autoimmune disease other than arthritis. For purposes of these
lattermost statements, an "autoimmune disease" herein is a disease
or disorder arising from and directed against an individual's own
tissues or organs or a co-segregate or manifestation thereof or
resulting condition therefrom. Without being limited to any one
theory, B cells demonstrate a pathogenic effect in human autoimmune
diseases through a multitude of mechanistic pathways, including
autoantibody production, immune complex formation, dendritic and
T-cell activation, cytokine synthesis, direct chemokine release,
and providing a nidus for ectopic neo-lymphogenesis. Each of these
pathways participates to different degrees in the pathology of
autoimmune diseases.
[0240] In a preferred embodiment, the joint damage is caused by
arthritis, aseptic joint loosening of orthopedic implants,
non-union of a fracture, spondyloarthropathies, psoriasis, or
Crohn's disease. More preferably, the joint damage is caused by
arthritis, which is more preferably rheumatoid arthritis,
osteoarthritis, ankylosing spondylitis, or psoriatic arthritis.
[0241] In still further embodiments, if the antagonist is an
antibody, the antibody is administered intravenously or
subcutaneously.
[0242] In further preferred aspects, if the antagonist is an
antibody, the antibody is administered in a dose of about 0.4 to 4
grams, more preferably about 1.5 to 3.5 grams, still more
preferably about 1.5 to 2.5 grams. In another aspect, the antibody
is preferably administered in a dose of about 0.4 to 1.3 grams at a
frequency of one to four doses within a period of about one month,
more preferably about 500 mg to 1.2 grams, still more preferably
about 500 mg or about 750 mg to about 1.1 grams, and more
preferably the antibody is administered in two to three doses.
Still more preferably, the antibody is administered within a period
of about 2 to 3 weeks.
[0243] In another preferred aspect, the subject is rheumatoid
factor negative. In another aspect, the subject is rheumatoid
factor positive.
[0244] In another preferred aspect of the above-described method,
the subject was administered methotrexate prior to the baseline or
start of treatment. More preferably, the methotrexate was
administered at a dose of about 10-25 mg/week. Also, preferably,
the methotrexate was administered for at least about 12 weeks prior
to the baseline, and still more preferably the methotrexate was
administered at a stable dose the last four weeks prior to the
baseline. In other embodiments, the methotrexate was administered
perorally or parenterally.
[0245] In particularly preferred embodiments of the
above-identified method, the joint damage is caused by rheumatoid
arthritis and the subject has exhibited an inadequate response to
one or more anti-tumor necrosis factor (TNF) inhibitors, and/or
methotrexate is administered to the subject along with the
antagonist, for example, CD20 antibody, and/or the antagonist is a
CD20 antibody that is administered at a dose of about 1000
mg.times.2 on days 1 and 15 intravenously at the start of the
treatment.
[0246] In still further embodiments, the invention provides a
method of monitoring the treatment of joint damage in a subject
comprising administering an effective amount of an antagonist to a
B-cell surface marker (such as an antibody thereto, including a
CD20 antibody) to the subject and measuring by radiography after at
least about one month from the administration whether the joint
damage has been reduced over baseline prior to the administration,
wherein a decrease versus baseline in the subject after treatment
indicates the antagonist or antibody such as CD20 antibody is
having an effect on the joint damage. Preferably, the degree of
reduction versus baseline is measured a second time after the
administration of the antagonist or antibody such as CD20 antibody.
Also, preferably the measurement is taken after at least about 24
weeks from the administration.
[0247] Also included herein is a method of monitoring the treatment
of joint damage in a subject comprising administering an effective
amount of an antagonist to a B-cell surface marker (such as an
antibody thereto, including a CD20 antibody) to the subject and
measuring by radiography after at least about 52 weeks from the
administration whether the joint damage has been reduced over
baseline prior to the administration, wherein a decrease versus
baseline in the subject after treatment indicates the antagonist or
antibody such as CD20 antibody is having an effect on the joint
damage. Preferably, the degree of reduction versus baseline is
measured a second time after the administration of the antagonist
or antibody such as CD20 antibody.
[0248] In yet another aspect, the invention provides a method of
determining whether to continue administering an antagonist to a
B-cell surface marker (such as an antibody thereto, including a
CD20 antibody) to a subject with joint damage comprising measuring
by radiography reduction in joint damage in the subject after
administration of the antagonist such as CD20 antibody a first
time, measuring by radiography reduction in joint damage in the
subject after administration of the antagonist such as CD20
antibody a second time, comparing the radiography scores in the
subject at the first time and at the second time, and if the score
is less at the second time than at the first time, continuing
administration of the antagonist or antibody such as CD20
antibody.
[0249] In a still further embodiment, a step is included in the
treatment method to test for the subject's response to treatment
after the administration step to determine that the level of
response is effective to treat the joint damage. For example, a
step is included to test the radiographic score after
administration and compare it to a baseline radiographic score
obtained before administration to determine if treatment is
effective by measuring if, and by how much, it has been changed.
This test may be repeated at various scheduled or unscheduled time
intervals after the administration to determine maintenance of any
partial or complete remission. Alternatively, the methods herein
comprise a step of testing the subject, before administration, to
see if one or more biomarkers or symptoms are present for joint
damage, as set forth above. In another method, a step may be
included to check the subject's clinical history, as detailed
above, for example, to rule out infections or malignancy as causes,
for example, primary causes, of the subject's condition, prior to
administering the antibody or antagonist to the subject.
Preferably, the joint damage is primary (i.e., the leading
disease), and is not secondary, such as secondary to infection or
malignancy, whether solid or liquid tumors.
[0250] In one embodiment of all the methods herein, no other
medicament than the antagonist such as CD20 antibody is
administered to the subject to treat the joint damage.
[0251] In any of the methods herein, preferably one may administer
to the subject along with the antagonist or antibody that binds a
B-cell surface marker an effective amount of a second medicament
(where the antagonist or antibody that binds a B-cell surface
marker (e.g., the CD20 antibody) is a first medicament). The second
medicament may be one or more medicaments, and include, for
example, an immunosuppressive agent, cytokine antagonist such as a
cytokine antibody, growth factor, hormone, integrin, integrin
antagonist or antibody, or any combination thereof. The type of
such second medicament depends on various factors, including the
type of joint damage, the severity of the joint damage, the
condition and age of the subject, the type and dose of first
medicament employed, etc.
[0252] Examples of such additional medicaments include an
interferon class drug such as interferon-alpha (e.g., from Amarillo
Biosciences, Inc.), IFN-beta-la (REBIF.RTM. and AVONEX.RTM.) or
IFN-beta-1b (BETASERON.RTM.), an oligopeptide such as glatiramer
acetate (COPAXONE.RTM.), an agent blocking CD40-CD40 ligand, an
immunosuppressive agent (such as mitoxantrone (NOVANTRONE.RTM.),
methotrexate, cyclophosphamide, chlorambucil, leflunomide, and
azathioprine), intravenous immunoglobulin (gamma globulin),
lymphocyte-depleting therapy (e.g., mitoxantrone, cyclophosphamide,
CAMPATH.TM. antibodies, anti-CD4, cladribine, a polypeptide
construct with at least two domains comprising a de-immunized,
autoreactive antigen or its fragment that is specifically
recognized by the Ig receptors of autoreactive B-cells (WO
2003/68822), total body irradiation, bone marrow transplantation),
integrin antagonist or antibody (e.g., an LFA-1 antibody such as
efalizumab/RAPTIVA.RTM. commercially available from Genentech, or
an alpha 4 integrin antibody such as natalizumab/ANTEGREN.RTM.
available from Biogen, or others as noted above), drugs that treat
symptoms secondary or related to joint damage such as those noted
herein, steroid such as corticosteroid (e.g., prednisolone,
methylprednisolone such as SOLU-MEDROL.TM. methylprednisolone
sodium succinate for injection, prednisone such as low-dose
prednisone, dexamethasone, or glucocorticoid, e.g., via joint
injection, including systemic corticosteroid therapy),
non-lymphocyte-depleting immunosuppressive therapy (e.g., MMF or
cyclosporine), cholesterol-lowering drug of the "statin" class
(which includes cerivastatin (BAYCOL.TM.), fluvastatin
(LESCOL.TM.), atorvastatin (LIPITOR.TM.), lovastatin (MEVACOR.TM.),
pravastatin (PRAVACHOL.TM.), and simvastatin (ZOCOR.TM.)),
estradiol, testosterone (optionally at elevated dosages; Stuve et
al. Neurology 8:290-301 (2002)), androgen, hormone-replacement
therapy, a TNF inhibitor such as an antibody to TNF-alpha, DMARD,
NSAID, plasmapheresis or plasma exchange,
trimethoprim-sulfamethoxazole (BACTRIM.TM., SEPTRA.TM.),
mycophenolate mofetil, H2-blockers or proton-pump inhibitors
(during the use of potentially ulcerogenic immunosuppressive
therapy), levothyroxine, cyclosporin A (e.g. SANDIMMUNE.RTM.),
somatastatin analogue, a DMARD or NSAID, cytokine antagonist such
as antibody, anti-metabolite, immunosuppressive agent,
rehabilitative surgery, radioiodine, thyroidectomy, BAFF antagonist
such as BAFF or BR3 antibodies or immunoadhesins, anti-CD40
receptor or anti-CD40 ligand (CD154), anti-IL-6 receptor
antagonist/antibody, another B-cell surface antagonist or antibody
such as a humanized 2H7 or other humanized or human CD20 antibody
with rituximab; IL-1 blockers, such as rHUIL-1Ra (Amgen-Synergen)
and tiaprofenic acid I-1B inhibitor (Hoechst); and co-stimulatory
modifiers, such as ORENCIA.RTM. (abatacept) (Bristol-Myers Squibb);
enlimomab (anti-ICAM-1 monoclonal antibody); CDO-855 (humanized
antibody, which binds specifically to a region of the Class II MHC
complex, Celltech); CH-3298 (Chiroscience); acemetacin (Merck);
GW353430 (anti-CD23 monoclonal antibody, Glaxo Wellcome); GR 252025
(COX02 inhibitor, Glaxo Wellcome); 4162W94 (anti-CD4 humanized
antibody; Glaxo Wellcome); azathioprine (DMARD, Glaxo Welcome);
penicilamine and fenoprofen (Eli Lilly); etc.
[0253] Preferred such medicaments are an antibiotic, anti-integrin,
gamma globulin, a pain-control agent, an integrin antagonist,
anti-CD4, cladribine, trimethoprimsulfamethoxazole, an H2-blocker,
a proton-pump inhibitor, cyclosporine, cholesterol-lowering drug of
the statin class, estradiol, testosterone, androgen,
hormone-replacement drug, a TNF inhibitor such as a TNF-alpha
inhibitor, DMARD, NSAID (to treat, for example, musculoskeletal
symptoms), levothyroxine, cyclosporin A, somatastatin analogue,
cytokine antagonist (including cytokine-receptor antagonist),
anti-metabolite, BAFF antagonist such as BAFF antibody or BR3
antibody, especially a BAFF antibody, immunosuppressive agent such
as methotrexate or a corticosteroid, a bisphosphonate, a hormone,
and another B-cell surface marker antibody, such as a combination
of rituximab and a humanized 2H7 or other humanized CD20
antibody.
[0254] The more preferred such medicaments are an antibiotic, an
immunosuppressive agent such as methotrexate or a corticosteroid, a
DMARD, a pain-control agent, an integrin antagonist, a NSAID, a
cytokine antagonist, a bisphosphonate, or a hormone, or a
combination thereof.
[0255] In one particularly preferred embodiment, the second
medicament is a DMARD, which is preferably selected from the group
consisting of auranofin, chloroquine, D-penicillamine, injectable
gold, oral gold, hydroxychloroquine, sulfasalazine, myocrisin and
methotrexate.
[0256] In another such embodiment, the second medicament is a
NSAID, which is preferably selected from the group consisting of:
pentasa, mesalazine, asacol, codeine phosphate, benorylate,
fenbufen, naprosyn, diclofenac, etodolac and indomethacin, aspirin
and ibuprofen.
[0257] In another such embodiment, the second medicament is a
pain-control agent, which is preferably selected from the group
consisting of: paracetamol and dextropropoxyphene.
[0258] In a further such embodiment, the second medicament is an
immunosuppressive agent, which is preferably selected from the
group consisting of etanercept, infliximab, adalimumab,
leflunomide, anakinra, azathioprine, methotrexate, and
cyclophosphamide.
[0259] In other preferred embodiments, the second medicament is
selected from the group consisting of OPG, etanercept, infliximab,
etanercept, adalimumab, kinaret, raptiva, osteoprotegerin (OPG),
RANKFc, anti-RANKL, pamidronate, alendronate, actonel,
zolendronate, clodronate, methotrexate, azulfidine,
hydroxychloroquine, doxycycline, leflunomide, sulfasalazine (SSZ),
prednisolone, interleukin-1 receptor antagonist, prednisone and
methylprednisolone.
[0260] In still preferred embodiments, the second medicament is
selected from the group consisting of infliximab, an
infliximab/methotrexate (MTX) combination, etanercept, a
corticosteroid, cyclosporin A, azathioprine, auranofin,
hydroxychloroquine (HCQ), combination of prednisolone, MTX, and
SSZ, combinations of MTX, SSZ, and HCQ, the combination of
cyclophosphamide, azathioprine, and HCQ, and the combination of
adalimumab with MTX. If the second medicament is a corticosteroid,
preferably it is prednisone, prednisolone, methylprednisolone,
hydrocortisone, or dexamethasone. Also, preferably, the
corticosteroid is administered in lower amounts than are used if
the CD20 antibody is not administered to a subject treated with a
corticosteroid. Most preferably, the second medicament is
methotrexate.
[0261] All these second medicaments may be used in combination with
each other or by themselves with the first medicament, so that the
expression "second medicament" as used herein does not mean it is
the only medicament besides the first medicament, respectively.
Thus, the second medicament need not be one medicament, but may
constitute or comprise more than one such drug.
[0262] These second medicaments as set forth herein are generally
used in the same dosages and with administration routes as used
hereinbefore or about from 1 to 99% of the heretofore-employed
dosages. If such second medicaments are used at all, preferably,
they are used in lower amounts than if the first medicament were
not present, especially in subsequent dosings beyond the initial
dosing with the first medicament, so as to eliminate or reduce side
effects caused thereby.
[0263] For the re-treatment methods described herein, where a
second medicament is administered in an effective amount with an
antagonist or antibody exposure, it may be administered with any
exposure, for example, only with one exposure, or with more than
one exposure. In one embodiment, the second medicament is
administered with the initial exposure. In another embodiment, the
second medicament is administered with the initial and second
exposures. In a still further embodiment, the second medicament is
administered with all exposures. It is preferred that after the
initial exposure, such as of steroid, the amount of such second
medicament is reduced or eliminated so as to reduce the exposure of
the subject to an agent with side effects such as prednisone,
prednisolone, methylprednisolone, and cyclophosphamide.
[0264] The combined administration of a second medicament includes
co-administration (concurrent administration), using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
(medicaments) simultaneously exert their biological activities. The
antibody or antagonist herein is administered by any suitable
means, including parenteral, topical, subcutaneous,
intraperitoneal, intrapulmonary, intranasal, and/or intralesional
administration. Parenteral infusions include intramuscular,
intravenous (i.v.), intraarterial, intraperitoneal, or subcutaneous
administration. Intrathecal administration is also contemplated
(see, e.g., US 2002/0009444, Grillo-Lopez, A concerning intrathecal
delivery of a CD20 antibody). In addition, the antibody or
antagonist may suitably be administered by pulse infusion, e.g.,
with declining doses of the antibody or antagonist. Preferably, the
dosing is given intravenously or subcutaneously, and more
preferably by intravenous infusion(s).
[0265] If multiple exposures of antibody are provided, each
exposure may be provided using the same or a different
administration means. In one embodiment, each exposure is by
intravenous administration. In another embodiment, each exposure is
given by subcutaneous administration. In yet another embodiment,
the exposures are given by both intravenous and subcutaneous
administration.
[0266] In one embodiment, the CD20 antibody is administered as a
slow intravenous infusion rather than an intravenous push or bolus.
For example, a steroid such as prednisolone or methylprednisolone
(e.g., about 80-120 mg i.v., more specifically about 100 mg i.v.)
is administered about 30 minutes prior to any infusion of the CD20
antibody. The CD20 antibody is, for example, infused through a
dedicated line.
[0267] For the initial dose of a multi-dose exposure to CD20
antibody, or for the single dose if the exposure involves only one
dose, such infusion is preferably commenced at a rate of about 50
25 mg/hour. This may be escalated, e.g., at a rate of about 50
mg/hour increments every about 30 minutes to a maximum of about 400
mg/hour. However, if the subject is experiencing an
infusion-related reaction, the infusion rate is preferably reduced,
e.g., to half the current rate, e.g., from 100 mg/hour to 50
mg/hour. Preferably, the infusion of such dose of CD20 antibody
(e.g., an about 1000-mg total dose) is completed at about 255
minutes (4 hours 15 min.). Optionally, the subjects receive a
prophylactic treatment of acetaminophen/paracetamol (e.g., about 1
g) and diphenhydramine HCl (e.g., about 50 mg or equivalent dose of
similar agent) by mouth about 30 to 60 minutes prior to the start
of an infusion.
[0268] If more than one infusion (dose) of CD20 antibody is given
to achieve the total exposure, the second or subsequent CD20
antibody infusions in this infusion embodiment are preferably
commenced at a higher rate than the initial infusion, e.g., at
about 100 mg/hour. This rate may be escalated, e.g., at a rate of
about 100 mg/hour increments every about 30 minutes to a maximum of
about 400 mg/hour. Subjects who experience an infusion-related
reaction preferably have the infusion rate reduced to half that
rate, e.g., from 100 mg/hour to 50 mg/hour. Preferably, the
infusion of such second or subsequent dose of CD20 antibody (e.g.,
an about 1000-mg total dose) is completed by about 195 minutes (3
hours 15 minutes).
[0269] In another embodiment, a method is provided for treating
joint damage in a subject comprising administering an antagonist to
a B-cell surface marker, such as an antibody thereto, for example,
CD20 antibody, to the subject, and giving the subject, at least
about 52 weeks after the administration, a radiographic test that
measures a reduction in the joint damage as compared to baseline
prior to the administration, wherein the amount of antagonist or
antibody such as CD20 antibody administered is effective in
achieving a reduction in the joint damage, indicating that the
subject has been successfully treated.
[0270] In this method, preferably the test measures a total
modified Sharp score. In another preferred embodiment, the
antagonist is a CD20 antibody. More preferably, the CD20 antibody
is rituximab or is humanized 2H7 comprising the variable domain
sequences in SEQ ID Nos. 2 and 8, or is humanized 2H7 comprising
the variable domain sequences in SEQ ID NOS:39 and 40, or is
humanized 2H7 comprising the variable domain sequences in SEQ ID
NOS:32 and 33, or is humanized 2H7 comprising a variable
heavy-chain domain with alteration N100A, or D56A and N100A, or
D56A, N100Y, and S100aR in SEQ ID NO:8 and a variable light-chain
domain with alteration M32L, or S92A, or M32L and S92A in SEQ ID
NO:2.
[0271] In another preferred embodiment, the joint damage is caused
by arthritis, preferably RA, and more preferably early active RA.
In another preferred embodiment, the subject has not been
previously treated with an immunosuppressive agent before the
administration of a first dose of antagonist or antibody such as
CD20 antibody in the treatment method. In a preferred embodiment,
the antagonist or antibody is administered in a dose of about 0.4
to 4 grams, and more preferably the antagonist or antibody is
administered in a dose of about 0.4 to 1.3 grams at a frequency of
one to four doses within a period of about one month. Still more
preferably, the dose is about 500 mg to 1.2 grams, and in other
embodiments is about 750 mg to 1.1 grams. In such aspects, the
antagonist or antibody is preferably administered in two to three
doses, and/or is administered within a period of about 2 to 3
weeks.
[0272] In another aspect, such method further comprises re-treating
the subject by providing an additional administration to the
subject of the antagonist such as a CD20 antibody in an amount
effective to achieve a continued or maintained reduction in joint
damage as compared to the effect of a prior administration of the
antagonist or antibody such as CD20 antibody. In one aspect of this
embodiment, the antagonist or antibody such as CD20 antibody is
additionally administered to the subject even if there is no
clinical improvement in the subject at the time of the radiographic
testing after a prior administration. The re-treatment may be
commenced at at least about 24 weeks after the first administration
of the antagonist such as CD20 antibody, and one or more further
re-treatments is optionally commenced. In another embodiment, the
further re-treatment is commenced at at least about 24 weeks after
the second administration of the antagonist such as CD20 antibody.
In a further preferred aspect, joint damage has been reduced after
the re-treatment as compared to the joint damage extent after the
first radiographic assessment.
[0273] Preferably, in this method regarding the about 52-week
assessment, a second medicament is administered in an effective
amount, wherein the antagonist or antibody such as CD20 antibody is
a first medicament. In one aspect, the second medicament is more
than one medicament. In another aspect, the second medicament is an
antibiotic, an immunosuppressive agent, a disease-modifying
anti-rheumatic drug (DMARD), a pain-control agent, an integrin
antagonist, a non-steroidal anti-inflammatory drug (NSAID), a
cytokine antagonist, a bisphosphonate, or a hormone, or a
combination thereof, most preferably methotrexate. The subject may
be rheumatoid factor negative or positive. Also, preferably, the
antagonist such as CD20 antibody is administered intravenously or
subcutaneously, most preferably intravenously.
[0274] A discussion of methods of producing, modifying, and
formulating such antibodies follows.
[0275] III. Production of Antibodies
[0276] The methods and articles of manufacture of the present
invention use, or incorporate, an antibody that binds to a B-cell
surface marker, especially one that binds to CD20. Accordingly,
methods for generating such antibodies will be described here.
[0277] CD20 antigen to be used for production of, or screening for,
antibody(ies) may be, e.g., a soluble form of CD20 or a portion
thereof, containing the desired epitope. Alternatively, or
additionally, cells expressing CD20 at their cell surface can be
used to generate, or screen for, antibody(ies). Other forms of CD20
useful for generating antibodies will be apparent to those skilled
in the art.
[0278] A description follows as to exemplary techniques for the
production of the antibodies used in accordance with the present
invention.
[0279] (i) Polyclonal Antibodies
[0280] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (s.c.) or intraperitoneal (i.p.) injections
of the relevant antigen and an adjuvant. It may be useful to
conjugate the relevant antigen to a protein that is immunogenic in
the species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0281] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0282] (ii) Monoclonal Antibodies
[0283] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical and/or bind the
same epitope except for possible variants that arise during
production of the monoclonal antibody, such variants generally
being present in minor amounts. Thus, the modifier "monoclonal"
indicates the character of the antibody as not being a mixture of
discrete or polyclonal antibodies.
[0284] For example, the monoclonal antibodies may be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).
[0285] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as hereinabove described to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable
fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103
(Academic Press, 1986)).
[0286] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0287] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X.sub.63-Ag8-653 cells available
from the American Type Culture Collection, Rockville, Md. USA.
Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0288] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0289] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis of Munson et al.,
Anal. Biochem., 107:220 (1980).
[0290] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium.
In addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
[0291] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-SEPHAROSE.TM., hydroxylapatite chromatography,
gel electrophoresis, dialysis, or affinity chromatography.
[0292] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding the antibody include Skerra et al., Curr. Opinion in
Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs.,
130:151-188 (1992).
[0293] In a further embodiment, antibodies or antibody fragments
can be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage
libraries.
[0294] Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0295] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0296] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0297] In addition, antibodies comprising a variant Fc region with
high affinity for Fc.gamma.R are useful for treating diseases where
an enhanced efficacy of effector cell function is desired, such as
autoimmune diseases, as set forth, for example, in US 2005/0037000
and WO 2004/63351 (Macrogenics, Inc. STAVENHAGEN et al.).
[0298] (iii) Humanized Antibodies
[0299] Methods for humanizing non-human antibodies have been
described in the art. Preferably, a humanized antibody has one or
more amino acid residues introduced into it from a source that is
non-human These non-human amino acid residues are often referred to
as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some hypervariable region residues and possibly
some FR residues are substituted by residues from analogous sites
in rodent antibodies.
[0300] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence that is closest to that of the rodent
is then accepted as the human framework region (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chain variable regions. The same framework may be used for several
different humanized antibodies (Carter et al., Proc. Natl. Acad.
Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623
(1993)).
[0301] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to a
preferred method, humanized antibodies are prepared by a process of
analysis of the parental sequences and various conceptual humanized
products using three-dimensional models of the parental and
humanized sequences. Three-dimensional immunoglobulin models are
commonly available and are familiar to those skilled in the art.
Computer programs are available that illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0302] (iv) Human Antibodies
[0303] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and
5,545,807.
[0304] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson et al., Current Opinion in
Structural Biology 3:564-571 (1993). Several sources of V-gene
segments can be used for phage display. Clackson et al., Nature,
352:624-628 (1991) isolated a diverse array of anti-oxazolone
antibodies from a small random combinatorial library of V genes
derived from the spleens of immunized mice. A repertoire of V genes
from unimmunized human donors can be constructed and antibodies to
a diverse array of antigens (including self-antigens) can be
isolated essentially following the techniques described by Marks et
al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.
12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0305] Human antibodies may also be generated by in vitro activated
B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
[0306] (v) Antibody Fragments
[0307] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992) and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
For example, the antibody fragments can be isolated from the
antibody phage libraries discussed above. Alternatively, Fab'-SH
fragments can be directly recovered from E. coli and chemically
coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
Fab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner. In other
embodiments, the antibody of choice is a single chain Fv fragment
(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.
5,587,458. The antibody fragment may also be a "linear antibody",
e.g., as described in U.S. Pat. No. 5,641,870 for example Such
linear antibody fragments may be monospecific or bispecific.
[0308] (vi) Bispecific Antibodies
[0309] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of the
CD20 antigen. Other such antibodies may bind CD20 and further bind
a second B-cell surface marker. Alternatively, an anti-CD20 binding
arm may be combined with an arm that binds to a triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or
CD3), or Fc receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so as to focus
cellular defense mechanisms to the B cell. Bispecific antibodies
may also be used to localize certain agents to the B cell. These
antibodies possess a CD20-binding arm and an arm that binds the
agent (e.g. methotrexate). Bispecific antibodies can be prepared as
full-length antibodies or antibody fragments (e.g.
F(ab').sub.2bispecific antibodies).
[0310] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0311] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first heavy chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0312] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0313] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers that are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the CH3 domain of an antibody constant
domain. In this method, one or more small amino acid side chains
from the interface of the first antibody molecule are replaced with
larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size to the large side chain(s)
are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g.
alanine or threonine). This provides a mechanism for increasing the
yield of the heterodimer over other unwanted end-products such as
homodimers.
[0314] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0315] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0316] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy chain variable domain (V.sub.H) connected to a light chain
variable domain (V.sub.L) by a linker that is too short to allow
pairing between the two domains on the same chain Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0317] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tat et al., J.
Immunol. 147: 60 (1991).
[0318] IV. Conjugates and Other Modifications of the Antibody
[0319] Modifications of the antibody are contemplated herein. Thus,
in one embodiment, the antibody may be conjugated to another
molecule, for example, to increase half-life or stability or
otherwise improve the pharmacokinetics of the antibody. For
example, the antibody may be linked to one of a variety of
non-proteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol. Antibody fragments,
such as Fab', linked to one or more PEG molecules are an especially
preferred embodiment of the invention.
[0320] The antibodies disclosed herein may also be formulated as
liposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO 97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0321] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of an antibody of the present invention
can be conjugated to the liposomes as described in Martin et al. J.
Biol. Chem. 257: 286-288 (1982) via a disulfide interchange
reaction.
[0322] Amino acid sequence modification(s) of protein or peptide
antibodies described herein are contemplated. For example, it may
be desirable to improve the binding affinity and/or other
biological properties of the antibody Amino acid sequence variants
of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody nucleic acid, or by peptide
synthesis.
[0323] Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid changes also may alter
post-translational processes of the antibody, such as changing the
number or position of glycosylation sites.
[0324] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells, Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0325] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a polypeptide
or polymer. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody of an
enzyme, or a polypeptide that increases the serum half-life of the
antibody.
[0326] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by different residue. The sites of
greatest interest for substitutional mutagenesis of antibodies
include the hypervariable regions, but FR alterations are also
contemplated. Conservative substitutions are shown in the table
below under the heading of "preferred substitutions". If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in the
table below, or as further described below in reference to amino
acid classes, may be introduced and the products screened.
TABLE-US-00014 Original Exemplary Preferred Residue Substitutions
Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys
Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)
Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala
Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Leu
Phe; Norleucine Leu (L) Norleucine; Ile; Val; Ile Met; Ala; Phe Lys
(K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu;
Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val;
Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V)
Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0327] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain Amino acids may be grouped according
to similarities in the properties of their side chains (in A. L.
Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0328] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0329] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (Q)
[0330] (3) acidic: Asp (D), Glu (E)
[0331] (4) basic: Lys (K), Arg (R), His(H)
[0332] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0333] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0334] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0335] (3) acidic: Asp, Glu;
[0336] (4) basic: His, Lys, Arg;
[0337] (5) residues that influence chain orientation: Gly, Pro;
[0338] (6) aromatic: Trp, Tyr, Phe.
[0339] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0340] Any cysteine residue not involved in maintaining the proper
conformation of the antibody also may be substituted, generally
with serine, to improve the oxidative stability of the molecule and
prevent aberrant crosslinking Conversely, cysteine bond(s) may be
added to the antibody to improve its stability (particularly where
the antibody is an antibody fragment such as an Fv fragment).
[0341] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody. Generally, the resulting variant(s) selected for
further development will have improved biological properties
relative to the parent antibody from which they are generated. A
convenient way for generating such substitutional variants is
affinity maturation using phage display. Briefly, several
hypervariable region sites (e.g. 6-7 sites) are mutated to generate
all possible amino substitutions at each site. The antibody
variants thus generated are displayed in a monovalent fashion from
filamentous phage particles as fusions to the gene III product of
M13 packaged within each particle. The phage-displayed variants are
then screened for their biological activity (e.g. binding affinity)
as herein disclosed. In order to identify candidate hypervariable
region sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or in
additionally, it may be beneficial to analyze a crystal structure
of the antigen-antibody complex to identify contact points between
the antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0342] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. Such altering
includes deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0343] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0344] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites).
[0345] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody are described in US Pat Appl No US
2003/0157108 (Presta, L.). See also US 2004/0093621 (Kyowa Hakko
Kogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine
(GlcNAc) in the carbohydrate attached to an Fc region of the
antibody are referenced in WO 2003/011878, Jean-Mairet et al. and
U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least one
galactose residue in the oligosaccharide attached to an Fc region
of the antibody are reported in WO 1997/30087, Patel et al. See,
also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)
concerning antibodies with altered carbohydrate attached to the Fc
region thereof. See also US 2005/0123546 (Umana et al.) on
antigen-binding molecules with modified glycosylation.
[0346] The preferred glycosylation variant herein comprises an Fc
region, wherein a carbohydrate structure attached to the Fc region
lacks fucose. Such variants have improved ADCC function.
Optionally, the Fc region further comprises one or more amino acid
substitutions therein which further improve ADCC, for example,
substitutions at positions 298, 333, and/or 334 of the Fc region
(Eu numbering of residues). Examples of publications related to
"defucosylated" or "fucose-deficient" antibodies include: US
2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US
2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US
2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO
2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol.
Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng.
87: 614 (2004). Examples of cell lines producing defucosylated
antibodies include Lec13 CHO cells deficient in protein
fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545
(1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO
2004/056312 A1, Adams et al., especially at Example 11), and
knockout cell lines, such as alpha-1,6-fucosyltransferase gene,
FUT8,knockout CHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87:
614 (2004)).
[0347] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0348] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g. so as to enhance ADCC
and/or CDC of the antibody. This may be achieved by introducing one
or more amino acid substitutions in an Fc region of an antibody.
Alternatively or additionally, cysteine residue(s) may be
introduced in the Fc region, thereby allowing interchain disulfide
bond formation in this region. The homodimeric antibody thus
generated may have improved internalization capability and/or
increased complement-mediated cell killing and ADCC. See Caron et
al., J. Exp Med. 176:1191-1195 (1992) and Shopes, J. Immunol.
148:2918-2922 (1992). Homodimeric antibodies may also be prepared
using heterobifunctional cross-linkers as described in Wolff et al.
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered that has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al. Anti-Cancer
[0349] Drug Design 3:219-230 (1989).
[0350] WO 00/42072 (Presta, L.) describes antibodies with improved
ADCC function in the presence of human effector cells, where the
antibodies comprise amino acid substitutions in the Fc region
thereof. Preferably, the antibody with improved ADCC comprises
substitutions at positions 298, 333, and/or 334 of the Fc region.
Preferably the altered Fc region is a human IgG1 Fc region
comprising or consisting of substitutions at one, two or three of
these positions.
[0351] Antibodies with altered Clq binding and/or CDC are described
in WO 99/51642, U.S. Pat. No. 6,194,551B1, U.S. Pat. No.
6,242,195B1, U.S. Pat. No. 6,528,624B1 and U.S. Pat. No. 6,538,124
(Idusogie et al.). The antibodies comprise an amino acid
substitution at one or more of amino acid positions 270, 322, 326,
327, 329, 313, 333 and/or 334 of the Fc region thereof.
[0352] To increase the serum half-life of the antibody, one may
incorporate a salvage receptor binding epitope into the antibody
(especially an antibody fragment) as described in U.S. Pat. No.
5,739,277, for example. As used herein, the term "salvage receptor
binding epitope" refers to an epitope of the Fc region of an IgG
molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
Antibodies with substitutions in an Fc region thereof and increased
serum half-lives are also described in WO00/42072 (Presta, L.).
[0353] Engineered antibodies with three or more (preferably four)
functional antigen binding sites are also contemplated (US Appin
No. US 2002/0004587 A1, Miller et al.).
[0354] V. Pharmaceutical Formulations
[0355] Therapeutic formulations of the antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0356] Exemplary anti-CD20 antibody formulations are described in
WO98/56418. This publication describes a liquid multidose
formulation comprising 40 mg/mL rituximab, 25 mM acetate, 150 mM
trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that
has a minimum shelf life of two years storage at 2-8.degree. C.
Another anti-CD20 formulation of interest comprises 10 mg/mL
rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate
dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for
Injection, pH 6.5.
[0357] Lyophilized formulations adapted for subcutaneous
administration are described in U.S. Pat. No. 6,267,958 (Andya et
al.). Such lyophilized formulations may be reconstituted with a
suitable diluent to a high protein concentration and the
reconstituted formulation may be administered subcutaneously to the
mammal to be treated herein.
[0358] Crystallized forms of the antibody are also contemplated.
See, for example, US 2002/0136719A1 (Shenoy et al.).
[0359] The formulation herein may also contain more than one active
compound (a second medicament as noted above) as necessary,
preferably those with complementary activities that do not
adversely affect each other. The type and effective amounts of such
medicaments depend, for example, on the amount of antibody present
in the formulation, and clinical parameters of the subjects. The
preferred such medicaments are noted above.
[0360] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0361] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0362] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0363] VI. Articles of Manufacture
[0364] In another embodiment of the invention, articles of
manufacture containing materials useful for the treatment of joint
damage described above are provided. The invention, in particular,
provides an article of manufacture comprising: (a) a container
comprising an antagonist such as an antibody that binds to a B-cell
surface marker (e.g., a CD20 antibody) (preferably the container
comprises the antibody and a pharmaceutically acceptable carrier or
diluent within the container); and (b) a package insert with
instructions for treating joint damage in a subject, wherein the
instructions indicate that the subject is administered the
antagonist or antibody (e.g., CD20 antibody) and is then subjected,
at least about one month after the administration, to a
radiographic test that measures a reduction in the joint damage as
compared to baseline prior to the administration, wherein the
amount of antagonist or antibody such as CD20 antibody administered
is effective in achieving a reduction in the joint damage,
indicating that the subject has been successfully treated.
[0365] In a preferred embodiment of this inventive aspect, the
article of manufacture herein further comprises a container
comprising a second medicament, wherein the antagonist or antibody
is a first medicament, and which article further comprises
instructions on the package insert for treating the subject with
the second medicament, in an effective amount. The second
medicament may be any of those set forth above, with an exemplary
second medicament being those set forth above, including an
antibiotic, an immunosuppressive agent, a disease-modifying
anti-rheumatic drug (DMARD), a pain-control agent, an integrin
antagonist, a non-steroidal anti-inflammatory drug (NSAID), a
cytokine antagonist, bisphosphonate, or a hormone, or a combination
thereof, more preferably a DMARD, NSAID, pain-control agent, or
immunosuppressive agent. Most preferably, the second medicament is
methotrexate.
[0366] In this aspect, the package insert is on or associated with
the container. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds or
contains a composition that is effective for treating the joint
damage and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). At least one
active agent in the composition is the antagonist or antibody. The
label or package insert indicates that the composition is used for
treating joint damage in a subject eligible for treatment with
specific guidance regarding dosing amounts and intervals of
antagonist or antibody and any other medicament being provided. The
article of manufacture may further comprise an additional container
comprising a pharmaceutically acceptable diluent buffer, such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution, and/or dextrose solution. The article of
manufacture may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0367] In more specific embodiments, an article of manufacture
comprises: (a) a container comprising an antagonist such as an
antibody that binds to a B-cell surface marker (e.g., a CD20
antibody) (preferably the container comprises the antibody and a
pharmaceutically acceptable carrier or diluent within the
container); and (b) a package insert with instructions for treating
joint damage in a subject, wherein the instructions indicate that
the subject is administered the antagonist or antibody (e.g., CD20
antibody) and is then subjected, at least about 52 weeks after the
administration, to a radiographic test that measures a reduction in
the joint damage as compared to baseline prior to the
administration, wherein the amount of CD20 antibody administered is
effective in achieving a reduction in the joint damage, indicating
that the subject has been successfully treated. In a preferred
embodiment, the article comprises a container comprising a second
medicament, wherein the antagonist or antibody (such as CD20
antibody) is a first medicament, further comprising instructions on
the package insert for treating the subject with the second
medicament in an effective amount. Preferably, this second
medicament is methotrexate.
[0368] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
EXAMPLE 1
[0369] Efficacy and safety of rituximab in patients with an
inadequate response or lack of tolerance to prior anti-TNF
therapy
[0370] This is a Phase III randomized, double-blind, parallel-group
multicenter clinical trial, called: Randomised Evaluation oF
Long-term Efficacy of Rituximab in RA (REFLEX). The study design is
shown in FIG. 1.
[0371] The objectives of this study were: [0372] To determine the
efficacy and safety of rituximab when used in combination with
methotrexate (MTX) in 517 patients with active rheumatoid arthritis
who have an inadequate response to one or more anti-TNF therapies.
[0373] To explore the pharmacokinetics and pharmacodynamics of
rituximab in this patient population (e.g. extent of duration of
B-cell depletion and effects on immunoglobulins and rheumatoid
factor).
[0374] The outcomes of this study were: [0375] Primary endpoint
[0376] The proportion of patients with an ACR20 response at Week
24. [0377] Secondary Endpoints [0378] Proportion of patients with
ACR50 and ACR70 responses at Week 24. [0379] Change in DAS28 from
baseline to Week 24. [0380] EULAR response at Week 24. [0381]
Changes from baseline in ACR core set. [0382] Changes from baseline
in SF-36. [0383] Change in Genant-modified Sharp radiographic total
at week 56. [0384] Change in Genant-modified Sharp radiographic
score at week 24 (exploratory); [0385] Change in erosion score, and
joint space narrowing score.
[0386] The key inclusion criteria of this study were: [0387]
Experienced an inadequate response to previous or current treatment
with etanercept, infliximab or adalimumab because of toxicity or
inadequate efficacy [0388] Etanercept for .gtoreq.3 months at 25 mg
twice a week [0389] Infliximab at least 4 infusions of at 3 mg/kg
[0390] Adalimumab for .gtoreq.3 months at 40 mg every other week.
[0391] Must have received MTX at a dose 10-25 mg/week (peroral
(p.o.) or parenteral) for at least 12 weeks, with the last 4 weeks
prior to screening at a stable dose. [0392] All other
DMARDs/biological response modifiers withdrawn at least 4 weeks
prior to randomization)8 weeks for infliximab, leflunomide and
adalimumab) [0393] Prednisone equivalent .ltoreq.10 mg/day. [0394]
Swollen joint count (SJC) .gtoreq.8 (66 joint count), and tender
joint count (TJC) .gtoreq.8 (68 joint count). [0395] Either CRP
.gtoreq.1.5 mg/dL (15 mg/L) or ESR .gtoreq.28 mm/h [0396]
Radiographic evidence of at least one joint with a definite erosion
attributable to rheumatoid arthritis.
[0397] The study treatment of the study was: [0398] Group A: [0399]
Rituximab two i.v. infusions of 1000 mg on days 1 and 15 [0400]
Group B [0401] Placebo i.v. infusions on days 1 and 15 [0402] Both
Groups [0403] 100 mg i.v. methylprednisolone prior to each
rituximab/placebo infusion [0404] 60 mg/d prednisone on days 2-7
and 30 mg/d on days 8-14
[0405] 6-month results:
[0406] Patient Populations:
TABLE-US-00015 No of Pts Placebo Rituximab Enrolled 209 308 ITT 201
298 Vial Breaks 4 3 Audited Site 1 4 Treated Prior 3 3
Randomisation ITT By Region 201 298 US 116 (58%) 172 (58%) Non-US
85 (42%) 126 (42%) ITT By RF 201 298 RF+ve 160 (80%) 234 (79%)
RF-ve 41 (20%) 64 (21%) Per Protocol 161 259 Exclusions 48 (24%) 49
(16%) Safety 209 308
[0407] ITT [0408] Randomised [0409] Received part of infusion
[0410] Analysed as randomised [0411] Per protocol [0412] As above
but adhered to protocol [0413] Safety [0414] Randomised [0415]
Received part of infusion [0416] Analysed as received
[0417] Patient Demographics:
TABLE-US-00016 Rituximab Placebo 2 .times. 1000 mg (n = 201) (n =
298) Sex Female 82% 81% Male 18% 19% Age (Mean, Yrs) 53 52
Methotrexate dose (mg/wk) 15 15 Previous DMARDs (mean) 2.5 2.6
Number of prior anti-TNF therapies 1.5 1.5 Pts receiving
concomitant 78% 74% Corticosteroids* Pts receiving NSAIDs or COX2*
75% 67%
[0418] Patients' Baseline Disease Characteristics
TABLE-US-00017 Rituximab Placebo 2 .times. 1000 mg (n = 201) (n =
298) Disease Duration (yrs) 11.7 12.2 SJC (mean) 23 23 TIC (mean)
33 34 RF Positive 80% 79% RF (mean, IU/L) 320 328 CRP (mean, mg/dL)
3.8 3.8 ESR (mean, mm/h) 48 48 DAS28 6.8 6.9
[0419] Patient Disposition
TABLE-US-00018 Rituximab Placebo 2 .times. 1000 mg Randomised 209
308 Withdrawn 97 (46%) 54 (18%) Adverse events 1 (<1%) 8 (3%)
Death -- -- Insufficient response 83 (40%) 36 (12%) Refused
Treatment 5 (2%) 5 (2%) Others 8 (4%) 5 (2%) Completed 24 weeks 112
(54%) 254 (82%)
[0420] The efficacy outcomes of the study are shown in FIGS.
1-12.
[0421] The radiographic outcomes of the study are shown in FIGS.
13-17.
[0422] The mean change in ACR core set parameters is shown in FIG.
18.
[0423] The safety analysis of the study is shown in FIGS.
19-31.
[0424] The conclusions from REFLEX study were: [0425] Rituximab was
associated with a significant increase in ACR20 response rate over
placebo (primary endpoint) [0426] All secondary and exploratory
endpoints (DAS, EULAR, ACR core set) supported primary analysis
[0427] The radiographic outcomes indicate that the treatment arm
showed a lower Sharp-Genant total score over the 24 weeks versus
the placebo arm (FIG. 13), a lower Sharp-Genant erosion score over
the 24 weeks versus the placebo arm (FIG. 14), a lower Sharp-Genant
JSN score over the 24 weeks versus the placebo arm (FIG. 15), a
much larger proportion of patients with no change in erosion score
at 24 weeks of the treatment versus the placebo arm (FIG. 16), with
a summary of the radiographic endpoints at week 24 for placebo and
treatment arms given in FIG. 17. [0428] Rituximab was generally
well tolerated. [0429] Infusion related events [0430] Rate of all
infections comparable to placebo [0431] Slight increase in
rate/incidence of serious infections [0432] No significant impact
on immunoglobulins [0433] Low HACA
[0434] 56-week results:
TABLE-US-00019 No. of patients (%)* Placebo Rituximab (n = 186) (n
= 277) Completed 56 weeks 141 (76) 202 (73) Withdrew 46 (25) 75
(27) Withdrew and received treatment 12 (6.5) 29 (10.5) with TNF
inhibitor *Patients with radiographic data available at 56
weeks.
[0435] FIG. 41 further illustrates the patient disposition of the
REFLEX clinical trial at 56 weeks, including ongoing treatments of
subgroups of patients selected from the treatment and placebo arms
of the Phase III REFLEX clinical trial.
[0436] As shown in FIG. 42, at week 56 all of the mean change in
total Genant-modified Sharp score (Genant, Am. J. Med., 30: 35-47
(1983)), in joint space narrowing (JSN) and in erosion score showed
a statistically significant improvement over placebo.
[0437] The efficacy of rituximab treatment is further illustrated
by the mean change in total Sharp-Genant score over time. As shown
in FIG. 43, the improvement has continued from week 24 to week
56.
[0438] FIG. 44 shows the cumulative distribution of change in total
Sharp-Genant score.
[0439] FIG. 45 shows the results of sensitivity analyses, expressed
by the change in total Sharp-Genant score. The rituximab+MTX
treatment arm has been consistently superior over the placebo +MTX
treatment arm.
[0440] FIG. 46 shows the percentage of patients who showed no
radiographic changes at the week 56 observation point in their
joint condition, as measured by erosion score and Genant-modified
Sharp score, respectively.
[0441] In conclusion, the results of this clinical trial show that
rituximab significantly inhibits radiographic progression in
rheumatoid arthritis (RA) patients with an inadequate response or
intolerance to one or more TNF inhibitors. In addition, this study
provides the first indication that a B cell-targeted therapy can
inhibit radiographic progression.
EXAMPLE 2
Rituximab in RA: Phase III Program
[0442] An important outcome to assess in RA includes the inhibition
of progression of structural joint damage and the improvement in
physical function. This outcome is particularly important for
patients who have recently been diagnosed with RA, since early
impact on these has potentially higher long-term benefit. Genovese
et al., Arthritis Rheum. 46: 1443-1450 (2002). A population of
early RA patients would therefore be an appropriate population to
study for these important outcomes.
[0443] With regard to a suitable control treatment for these
patients, the current gold standard therapy for early RA is MTX.
Consequently, selecting a population of MTX-naive patients and
comparing rituximab plus MTX to MTX alone provides a comparison of
this new treatment approach against the current gold standard for
these patients.
[0444] This study is a randomized Phase III controlled
soluble-blind, parallel-group multicenter study to evaluate the
safety and efficacy of rituximab in combination with MTX compared
to MTX alone, in MTX-naive patients with active RA.
[0445] Primary Objectives: [0446] 1. To determine the efficacy of
rituximab in the prevention of progression in structural joint
damage and to evaluate the safety of rituximab in patients with
active RA initiating treatment with MTX. [0447] 2. To evaluate the
efficacy of rituximab in improving the patient's physical function
and signs and symptoms of RA. [0448] 3. To investigate by a
population analysis approach the pharmacokinetics (PK) of rituximab
in the target RA patient population and the influence of covariates
on the PK parameters. [0449] 4. To explore the long-term efficacy
and safety of further courses of rituximab.
[0450] First Course--Study Design: [0451] Group A: rituximab 500 mg
i.v. x2 plus MTX (7.5 mg escalated to 20 mg p.o.) [0452] Group B:
rituximab 1000 mg i.v. x 2 plus MTX (7.5 mg escalated to 20 mg
p.o.) [0453] Group C: Placebo rituximab i.v. x 2 plus MTX (7.5 mg
escalated to 20 mg p.o.)
[0454] For Group C patients, from week 104, re-treatment with
courses of rituximab 500 mg i.v. x 2 plus MTX will be available for
eligible patients.
[0455] The following rules for re-treatment, in line with the above
regimens, apply:
[0456] Second Course
[0457] A second course of rituximab plus MTX or placebo plus MTX
will be administered as soon as feasibly possible when the
following is achieved: [0458] 1. Minimum of 24 weeks has passed
since the first infusion of the last course of study medication.
[0459] 2. DAS28-ESR >2.6 [0460] 3. Eligibility criteria for
absolute neutrophil count (not below 1.5.times.10.sup.3 .mu.L), IgG
(not below 5.0 mg/mL) and IgM (not below 0.40 mg/mL) were met at
the last blood sample analysis.
[0461] In addition, the patient must meet the eligibility exclusion
criteria 8, 10 and 11 as described below: [0462] 1. Significant
cardiac or pulmonary disease (including obstructive pulmonary
disease) [0463] 2. Primary or secondary immunodeficiency (history
of, or currently active), including known history of HIV infection.
[0464] 3. Known active infection of any kind (excluding fungal
infections of nail beds), or any major episode of infection
requiring hospitalization or treatment with i.v. anti-infectives
within 4 weeks of infusion or completion of oral anti-infectives
within 2 weeks prior to infusion.
[0465] Patients who do not meet the criteria for a second course of
rituximab/placebo at week 24 will be followed every 4 to 8 weeks
and will subsequently receive the second course at the time they
become eligible based on the above criteria.
[0466] Further courses:
[0467] From week 48 patients may become eligible to receive a third
course of rituximab/placebo. The third course and beyond may only
be administered to patients who have met the above criteria for the
second course and in whom the investigator deems there has been a
relevant clinical response following either the first or the second
course of rituximab. Patients who have never had a clinical
response should be withdrawn into safety follow-up (SFU).
[0468] Patients who are deemed to have had a clinical response, but
do not currently meet the criteria for further courses of rituximab
will be followed every 4 weeks from week 48 to week 56 and then
every 8 weeks and will subsequently receive further courses at the
time they become eligible based on the above criteria.
[0469] All patients will receive pre-medication with 100 mg i.v.
methylprednisolone prior to each infusion. All patients will also
receive a stable dose of folate (at least 5 mg/week) given as
either a single dose or as a divided weekly dose.
[0470] All patients should continue to receive any background
corticosteroid (at least 10 mg/day prednisone or equivalent) or
oral nonsteroidal anti-inflammatory drugs (NSAIDs) at a stable
dose. Randomization will be stratified by region (US or ROW) and
rheumatoid factor (positive or negative) to ensure balanced
allocation of patients across region and RF status between
treatment arms.
[0471] Patients will attend the clinic once every 4 weeks for the
first 24 weeks and every 8 weeks thereafter (except for weeks 48-56
where visits will be every 4 weeks) for efficacy, safety,
immunology, and quality of life assessments. Radiographic
assessments will be conducted at screening, week 24, and week 52.
Radiographic assessments will also be conducted at 2 and 3 years
after the first dose of study medication.
[0472] Evaluation of the primary endpoint (change in total modified
Sharp score) will occur at 52 weeks. Secondary and exploratory
endpoints will include further radiographic endpoints and signs and
symptoms, physical function and remission endpoints.
[0473] At any time after radiographic assessment at week 52,
patients who do not have a 20% improvement in both swollen and
tender joint counts may receive rescue therapy with an increased
dose of MTX or one non-biologic DMARD.
[0474] All patients who withdraw from the study or at any point or
complete the total treatment period should return for SFU
assessments at weeks 4, 12, 24, 36, and 48 after withdrawal or
completion. This effectively follows the patient for one year after
the patient withdraws from/completes the study. If a patient's
peripheral B-cell count (CD19) has not returned to their baseline
level or to within the normal range, whichever is lower, after one
year, safety follow-up visits should continue to be performed at
12-week intervals until B-cell repletion occurs.
[0475] For all serious infectious adverse events reported CBC,
differentials, platelets, quantitative Ig and CD19 counts should be
determined within one week of the infectious adverse event becoming
serious.
[0476] Patients withdrawing into SFU are to be strongly encouraged
to return for all scheduled radiographic assessments (at weeks 24,
52, 104, and 152) irrespective of their point of withdrawal from
the study. Radiographs for these patients are to be taken in line
with the original schedule of assessments, relative to the original
day of randomization.
[0477] Approximately 852 patients will be recruited into this study
and will be randomized equally into three treatment groups.
Patients will be stratified by region (US or Rest of World (ROW))
and RF status (positive RF at least 20 IU/mL or negative RF less
than 20 IU/mL). The overall proportion of RF-negative patients will
be limited to 20% of the total sample size. Recruitment will be
competitive with no more than 70% and no less than 30% being
enrolled in either region. There will be no replacement of patients
should a patient's treatment be discontinued for any reason.
[0478] Target population:
[0479] The target population for this study is patients with early
active RA, who are naive to MTX.
[0480] Inclusion criteria:
[0481] Patients must meet the following criteria to be eligible for
study entry: [0482] 1. Able and willing to give written informed
consent and comply with the requirements of the study protocol.
[0483] 2. Patients with RA diagnosed for at least 8 weeks, but no
more than 4 years, according to the revised 1987 ACR criteria for
the classification of RA. [0484] 3. Patients naive to, and
considered to be candidates for, treatment with MTX. [0485] 4.
Swollen joint count (SJC) at least 8 (66 joint count), and tender
joint count (TJC) at least 8 (68 joint count) at screening and
baseline. [0486] 5. At screening CRP at least 1.2 mg/dL (12 mg/L).
[0487] 6. Age 18-80 years. [0488] 7. Glucocorticoids at least 10
mg/day prednisolone or equivalent is permitted if stable for at
least four weeks prior to baseline. [0489] 8. Use of NSAIDs is
permitted if stable for at least two weeks prior to baseline.
[0490] 9. For patients of reproductive potential (males and
females), use of a reliable means of contraception (e.g., hormonal
contraceptive, patch, intrauterine device, physical barrier)
throughout study participation. [0491] 10. Must be willing to
receive oral folate. [0492] 11. For RF-negative patients only,
radiographic evidence of at least one joint with definite erosion
attributable to RA. [0493] 12. Patients who are to receive, or who
are current receiving, treatment on an outpatient basis.
[0494] Exclusion criteria: [0495] Exclusions related to RA [0496]
1. Rheumatic autoimmune disease other than RA, or significant
systemic involvement secondary to RA (including but not limited to
vasculitis, pulmonary fibrosis or Felty's syndrome). Secondary
Sjogren's syndrome or secondary limited cutaneous vasculitis with
RA is permitted. [0497] 2. Functional class IV as defined by the
ACR Classification of Functional Status in RA. [0498] 3. History
of, or current, inflammatory joint disease other than RA
(including, but not limited to, gout, reactive arthritis, psoriatic
arthritis, seronegative spondyloarthropathy, Lyme disease), or
other systemic autoimmune disorder (including, but not limited to,
systemic lupus erythematosus, inflammatory bowel disease,
scleroderma, inflammatory myopathy, mixed connective tissue
disease, or any overlap syndrome). [0499] 4. Diagnosis of juvenile
idiopathic arthritis (JIA) or juvenile RA (JRA) and/or RA before
age 16.
[0500] Exclusions Related to General Health [0501] 5. Any surgical
procedure, including bone/joint surgery/synovectomy (including
joint fusion or replacement) within 12 weeks prior to baseline or
planned during the study. [0502] 6. Lack of peripheral venous
access. [0503] 7. Pregnancy or breast feeding. [0504] 8.
Significant and/or uncontrolled cardiac or pulmonary disease
(including obstructive pulmonary disease). [0505] 9. Evidence of
significant concomitant disease, including but not limited to,
nervous system, renal, hepatic, endocrine or gastrointestinal
disorders which, in the investigator's opinion, would preclude
patient participation. [0506] 10. Primary or secondary
immunodeficiency (history of, or currently active), including known
history of HIV infection. [0507] 11. Known active infection of any
kind (excluding fungal infections of nail beds), or any major
episode of infection requiring hospitalization or treatment with
i.v. anti-infectives within 4 weeks of baseline or completion of
oral anti-infectives within 2 weeks prior to baseline. [0508] 12.
History of deep space/tissue infection (e.g. fasciitis, abscess,
osteomyelitis) within 52 weeks prior to baseline. [0509] 13.
History of serious recurrent or chronic infection (for screening
for a chest infection a chest radiograph will be performed at
screening if not performed within 12 weeks prior to screening).
[0510] 14. History of cancer, including solid tumors, hematologic
malignancies and carcinoma in situ (except basal cell and squamous
cell carcinoma of the skin that have been excised and cured).
[0511] 15. Any neurological (congenital or acquired), vascular or
systemic disorder which could affect any of the efficacy
assessments, in particular, joint pain and swelling (e.g.,
Parkinson's disease, cerebral palsy, diabetic neuropathy). [0512]
16. Currently active alcohol or drug abuse or history of alcohol or
drug abuse within 24 weeks prior to baseline.
[0513] Exclusions Related to Medications [0514] 17. History of a
severe allergic or anaphylactic reaction to a biologic agent or
known hypersensitivity to any component of rituximab or to murine
proteins. [0515] 18. Previous treatment with any approved or
investigational biologic agent for RA. [0516] 19. Previous
treatment with an anti-alpha 4 integrin antibody or co-stimulation
modulator. [0517] 20. Concurrent treatment with any biologic agent
or DMARD other than MTX. Treatment must be discontinued 14 days
prior to baseline, except for the following: azathioprine for at
least 28 days; leflunomide for at least 8 weeks (or at least 14
days after 11 days of standard cholestyramine or activated charcoal
washout). [0518] 21. Previous treatment with any cell-depleting
therapies, including investigational agents (e.g., CAMPATH,
anti-CD4, anti-CDS, anti-CD3, anti-CD19, anti-CD11 a, anti-CD22,
BLys/BAFF, and anti-CD20).
[0519] 1 22. Treatment with any investigational agent within 28
days of baseline or five half-lives of the investigational drug
(whichever is the longer). [0520] 23. Receipt of a live/attenuated
vaccine within 28 days prior to baseline (it is recommended that a
patient's vaccination record and the need for immunization prior to
receiving rituximab/placebo should be carefully investigated).
[0521] 24. Intra-articular or parenteral glucocorticoids within 4
weeks prior to baseline. [0522] 25. Intolerance or
contra-indications to i.v. glucocorticoids.
[0523] Exclusions Related to Laboratory Findings [0524] 26.
Positive serum human chorionic gonadotropin (hCG) measured prior to
the first rituximab infusion. [0525] 27. Positive tests for
hepatitis B surface antigen (HBsAg), hepatitis B core antibody
(HBsAb) or hepatitis C serology. [0526] 28. Hemoglobin less than
8.0 g/dL. [0527] 29. Concentrations of serum IgG and/or IgM below
5.0 and 0.40 mg/mL, respectively. [0528] 30. Absolute neutrophil
count (ANC) less than 1.5.times.10.sup.3/.mu.L. [0529] 31. AST or
ALT greater than 2.5 times upper limit of normal.
[0530] The end of treatment is defined as the 3-year time point,
following which there will be an additional period of at least a
year of SFU.
[0531] Rituximab (500 mg or 1000 mg) plus MTX or placebo plus MTX
will be administered by IV infusion on days 1 and 15. Patients may
be eligible for re-treatment (two doses 14 days apart) with a
maximum frequency of one re-treatment every 24 weeks. Patients
originally randomized to receive rituximab plus MTX will receive
re-treatment at the same dose throughout the study. From week 104
patients originally randomized to placebo plus MTX may be eligible
to receive rituximab (500 mg) plus MTX. Premedication with 100 mg
i.v. methylprednisolone is to be administered prior to each
infusion.
[0532] Methotrexate tablets (7.5 mg/week escalated to 20 mg/week)
will be administered orally to all groups.
[0533] It is recommended that all patients should be pre-medicated
with paracetamol/acetaminophen (1 gm p.o.) and diphenhydramine HCl
(100 mg i.v. or oral equivalent antihistamine) 30 to 60 minutes
prior to the start of an infusion to reduce the potential for
infusion reactions.
[0534] Patients will receive folate or equivalent (at least 5
mg/week) given as either a single dose or as a divided weekly
dose.
[0535] Patients may continue to receive any background
glucocorticoid (at least 10 mg/day of prednisone or equivalent).
Analgesics may be used for pain as required.
[0536] The primary endpoint is the change from screening in total
modified Sharp score at week 52 using the modified intent-to-treat
(ITT) population.
[0537] The radiographic secondary endpoints are: [0538] 1. a change
in modified total Sharp score at Weeks 24 and 104 [0539] 2. a
change in modified Sharp erosion score at Week 52 [0540] 3. a
change in modified joint space narrowing score at Week 52 [0541] 4.
The proportion of patients without radiographic progression at Week
52 (defined as change in total modified Sharp score of less than or
equal to 0). In addition, the proportion of patients without
radiographic progression at Weeks 24 and 104 will be analyzed.
[0542] The radiographic exploratory endpoints are: [0543] 1. a
change in modified Sharp scores will be presented over time. [0544]
2. Proportion of patients without radiographic progression at Weeks
24, 52, and 104 will be further presented in the following
sub-categories: [0545] a. Proportion of patients with no change in
modified Sharp erosion score from screening [0546] b. Proportion of
patients with no change in modified joint space narrowing score
from screening [0547] c. Proportion of patients with no newly
eroded joints.
[0548] Radiographic assessments will be made as follows: separate
radiographs of each hand posterior-anterior (PA) and each foot
anterior-posterior (AP) will be taken as per schedule of
assessments. At the screening visit the readability and quality of
the radiographs (as can be found in a procedure manual for
radiographic examinations of the hands, wrist and feet) must be
confirmed before the patient leaves the site. Radiographs for
RF-negative patients at the screening visit will be checked for
radiographic evidence of at least one joint with a definite erosion
attributable to RA by the central reading site. All radiographs
will be assessed using the method according to Sharp, as modified
by Genant, Am. J. Med., 30: 35-47 (1983). The primary assessment
will be the change from screening in the total modified Sharp score
at week 52. The total modified Sharp score combines an erosion
score and a joint space narrowing score of both hands and feet. The
maximum total erosion score in the hands is 100 and in the feet 42,
the maximum scores for joint space narrowing in the hands is 100
and in the feet 48. The maximum total modified Sharp score
achievable is 290. The change in score at week 52 is to be
calculated as:
Change=week 52 score minus screening score.
[0549] Radiographs will be taken of the hands and feet to compute a
total modified Sharp score. Before starting the trial all radiology
departments will participate in training sessions to standardize
radiographs. This will include standardization for validation
procedures for equipment, films, cassettes, placement of hands/feet
and procedures for obtaining consistent radiographs.
[0550] The change in total modified Sharp score is expected to be
skewed and hence not normally distributed. Therefore, the change in
total modified Sharp score at week 52 will be tested between
treatment groups using a non-parametric test statistic stratifying
for region and RF status. However, if the data are shown to be
approximately normally distributed, the data will be analyzed using
an analysis of variance (ANOVA) model with region, RF status, and
treatment groups as exploratory terms in the model.
[0551] Closure principle will be used to adjust multiple
comparisons in primary endpoint. The first comparison will be
between each of the three treatment arms using a Kruskal-Wallis
test statistic.
[0552] The three treatment arms will be considered different if
there is sufficient statistical evidence to reject the following
null hypothesis: [0553] H.sub.0:.mu..sub.1=.mu..sub.2=.mu..sub.1
i.e., no evidence that there is any difference in the change in
total modified
[0554] Sharp score in any of the treatment arms and accept the
alternative hypothesis: [0555] H.sub.1: .mu..sub.1 does not equal
.mu..sub.2, or .mu..sub.1 does not equal .mu.3, or .mu..sub.2 does
not equal .mu..sub.3 [0556] i.e., there is a difference in the
change in total modified Sharp score in at least one of the
pairwise treatment comparisons.
[0557] If the test result is statistically significant at
.A-inverted.=0.05 level, it is concluded that there is a difference
in the change from baseline in total modified Sharp score at week
52, between the treatment arms.
[0558] Subsequently, each of the rituximab groups are compared with
the placebo group, at the .A-inverted.=0.05 level, as described
below. The primary comparisons will be considered to be the
individual rituximab dose group vs. the placebo group, using the
Van Elteren test statistic.
[0559] Each of the rituximab-treated arms will be considered
superior to placebo if there is sufficient statistical evidence to
reject the following null hypothesis: [0560] H.sub.0:
.mu..sub.1=.mu..sub.2 i.e., no evidence that the change in total
modified Sharp score in the rituximab arm is superior to the
placebo arm [0561] and accept the alternative hypothesis: [0562]
H.sub.1: .mu..sub.1 does not equal .mu..sub.2 i.e., the change in
total modified Sharp score in the rituximab arm
[0563] is superior to the placebo arm.
[0564] If the test result is statistically significant at
.A-inverted.=0.05 level, it will be concluded that the rituximab
arm demonstrated a superior change from baseline in total modified
Sharp score at week 52 when compared to the placebo arm.
[0565] Every effort will be made to ensure, wherever possible, that
patients return for their radiographic visits, even if withdrawing
from study drug. Missing week 52 data will be imputed using the
following methods:
[0566] If week 52 radiographic data is missing, then week 24
radiographic data will be used to linearly extrapolate that
patient's week 52 result. Those patients who withdraw prematurely
from the study prior to week 52 will be included as part of the
week 52 analysis of radiographic data. Any patient who has no post
screening radiographic data will be excluded from the modified ITT
population and hence from the primary endpoint analysis.
[0567] The influence of potential imbalances in treatment
allocations within particular sites will be investigated.
Sensitivity analyses will be performed to assess the impact of
grossly imbalanced sites on the primary analysis.
[0568] As to the radiographic secondary endpoints, change in
modified total Sharp score at weeks 24 and 104 will be analyzed in
the same manner as specified for the primary endpoint. The change
in modified Sharp erosion score at week 52 will be analyzed in the
same manner as specified for the primary endpoint. In addition,
change in modified Sharp erosion score at weeks 24 and 104 will be
analyzed. Change in modified joint space narrowing score at week 52
will be analyzed in the same manner as specified for the primary
endpoint. In addition, change in modified joint space narrowing
score at weeks 24 and 104 will be analyzed. The proportion of
patients without radiographic progression at week 52 (defined as a
change in total modified Sharp score of less than or equal to 0)
will be assessed as follows: The difference in the proportions will
be tested using a Cochran-Mantel Haenszel (CMH) test statistic
stratifying for region and RF status. In addition, the proportion
of patients without radiographic progression at weeks 24 and 104
will be analyzed.
[0569] As to radiographic exploratory endpoints, the change in
modified Sharp scores will be presented over time. The proportion
of patients without radiographic progression at weeks 24, 52, and
104 will be further presented in the following sub-categories:
[0570] Proportion of patients with no change in modified Sharp
erosion score from screening [0571] Proportion of patients with no
change in modified joint space narrowing score from screening
[0572] Proportion of patients with no newly eroded joints.
[0573] Details of the joints for radiographic assessment and
grading scales (Genant, Am. J. Med., 30: 35-47 (1983)) are as
follows: [0574] Grading Scales:
[0575] 1. Joint Space Narrowing (JSN) [0576] Grade 0--Normal [0577]
Grade 0.5--Subtle JSN or equivocal findings. [0578] Grade 1.0--Mild
JSN (focal or minor). [0579] Grade 1.5--Mild-to-moderate JSN.
[0580] Grade 2.0--Moderate JSN. [0581] Grade
2.5--Moderate-to-severe JSN. [0582] Grade 3.0--Severe JSN. [0583]
Grade 3.5--Severe JSN close to ankylosis. [0584] Grade 4.0--Defmite
ankylosis.
[0585] 2. Erosions (discrete interruption of cortical surface)
[0586] Grade 0--Normal [0587] Grade 0.5--Subtle loss of cortical
continuity or equivocal findings of bone erosion. [0588] Grade
1.0--Mild. Definite but small erosions of one or both articular
bones, usually at the bare areas involving less than 25% of the
articular surfaces. [0589] Grade 1.5--Mild-to-moderate.
Small-medium erosions involving less than 25% of the articular
surface of one or both articular bones. [0590] Grade 2.0--Moderate.
Medium-large erosions involving 26-50% of the articular surface of
both articular bones. [0591] Grade 2.5--Moderate-to-severe.
Erosions of 51-75% of the articular surfaces. [0592] Grade
3.0--Severe. Erosions of 76-90% of the articular surfaces. [0593]
Grade 3.5--Very severe. Erosions of 100% of the articular surfaces
(total destruction of the articular surfaces).
Joint Evaluation:
[0594] 1. Joint Space Narrowing in the hand (13 joints per hand)
[0595] a. All proximal interphalangeal (PIP) joints on digits II-V.
[0596] b. The interphalangeal joint on digit I. [0597] c. All
metacarpophalangeal (MCP) joints. [0598] d. The carpometacarpal
(CMC) joints of digits III-V as a single unit. [0599] e. The
pericapitate (scaphoid-capitate and lunate-capitate combined)
space. [0600] f. The radiocarpal joint.
[0601] 2. Erosion Scores in the hand (14 joints per hand) [0602] a.
All proximal interphalangeal (PIP) joints on digits II-V. [0603] b.
The interphalangeal joint on digit I. [0604] c. All
metacarpalphalangeal (MCP) joints [0605] d. The carpometacarpal
(CMC) joints of digit I [0606] e. The scaphoid bone. [0607] f. The
distal radius. [0608] g. The distal ulnar.
[0609] The scores will be summed separately (14.times.3.5 maximum
per joint x2=98 for erosions and 13.times.4 maximum per joint x2
for JSN). Each sum will be normalized to a scale of 0-100. Both
scores will be added to obtain a total score (scale 0-200).
[0610] 3. Joint Space Narrowing and Erosions (6 joints per foot)
[0611] a. All metatarsolphalangeal (MTP) joints. [0612] b. All
interphalangeal joints on digit I.
[0613] The scores will be summed separately (6.times.3.5 maximum
per joint x2=42 for erosions and 6.times.4 maximum per joint x2 =48
for JSN. Both scores will be added to obtain a total score with a
scale 0-90).
[0614] Original radiographs will be sent to the central reading
site, where they will be quality controlled. Should the initial
radiograph be of unacceptable quality, the center will be advised
as to what amendments are needed and instructed to obtain a second
radiograph.
[0615] All patients randomized in the study who have received at
least part of a dose of rituximab (including those who have
withdrawn into safety follow-up) will have radiographs of
hands/wrists and feet taken at weeks 24, 52, 104, and 152. Patients
are to be strongly encouraged to return for all radiographic
assessments irrespective of their point of withdrawal from the
study. Radiographs of these patients are to be taken in line with
the original schedule of assessments, relative to the original day
of randomization.
[0616] Additional secondary and exploratory endpoints will be for
signs and symptoms, physical function, remission, and
patient-reported outcomes.
[0617] The change in total modified Sharp score at week 52 will be
tested between treatment groups using a non-parametric test
statistic stratifying for region and RF status. However, if the
data is shown to be approximately normally distributed, the data
will be analyzed using an analysis of variance (ANOVA) model with
region, RF status and treatment groups as exploratory terms in the
model.
[0618] It is clearly desirable to maintain patients in a state of
low disease activity by limiting disease flares and potentially
limiting progression of structural damage. In the DANCER study
referred to above (Emery et al., EULAR, supra, and Van Vollenhoven
et al., EULAR, supra), at week 24, approximately 90% of patients
treated with rituximab had not achieved EULAR (DAS28)
remission.
[0619] In the study that is the subject of this Example, such
patients would be eligible to receive their first re-treatment of
rituximab at week 24. Mandatory re-treatment based on DAS28-ESR
provides objective information concerning a disease activity-based
re-treatment paradigm. The minimum period of 24 weeks between
courses is recommended based on the pharmacokinetics and
pharmacodynamics of rituximab. Without being limited to any one
theory, the pharmacokinetics and pharmacodynamics of rituximab
appear to demonstrate that, at this time (week 24), drug levels are
below the level of detection and there is evidence of returning
peripheral CD19+cells. This is paralleled by an apparent increase
in disease activity after this time point, and therefore represents
a reasonable point from which further courses could be given.
[0620] It is expected that rituximab (or a humanized 2H7 antibody
substituted for rituximab) in combination with MTX will be
efficacious in meeting the primary endpoint in the prevention of
progression in structural joint damage as set forth in this
Example, It is also expected that the regimen of this study will
meet one or more of the secondary radiographic endpoints. Thus, it
is expected that administration of a first dose of rituximab or
humanized 2H7 (at 500 mg.times.2 or 100 mg.times.2) with
methotrexate) will reduce joint damage from baseline (before first
administration of CD20 antibody), as measured by the modified total
Sharp score, at least about one month from baseline or start of
treatment, preferably at least about 24 weeks from baseline, more
preferably at least about 52 weeks from baseline, and at further
time points up to 104 weeks from baseline. It is also expected that
the patients can be efficaciously re-treated at 24 weeks or 52
weeks from baseline to maintain this prevention of progression of
joint damage.
[0621] It is predicted and expected that administration of
rituximab or a humanized 2H7 to the subject in the scheduled
re-dosing protocol set forth above will be effective in preventing
progression of structural joint damage at week 52 or later. These
results are expected to be significantly better than those of the
control.
[0622] It is also expected that at about week 48-54, another 1-g or
2-g dose of the CD20 antibody (e.g., rituximab or a humanized 2H7)
given all at once or spread out over about 14-16 days in 0.5- or
1-gram amounts would be effective to treat joint damage for the
entire second year, with or without one or more second medicaments
such as immunosuppressive agents. Thus, the CD20 antibody would be
administered initially within about the 2-week time period,
followed by another treatment at about 4-8 months, followed by
another treatment at about one year from initial treatment
(measured from the time any one of the doses was given), followed
by treatment at about two years from initial treatment, with
expected success, in about one-half-gram or one-gram x 2-4 dosing
for each treatment, administered together, about weekly, or about
every other week over about two to four weeks. The results of this
treatment would be expected to be much better than those of the
control with placebo. This re-treatment protocol is expected to be
successfully used for several years with little or no adverse
effects.
[0623] It is also contemplated that rituximab or another CD20
antibody will meet the primary endpoint as a monotherapy using the
same regimen at the same dose or a higher dose without a second
medicament such as MTX, and that re-treatment using the same
regimen at the same dose or a higher dose would be successful as a
monotherapy.
EXAMPLE 3
Study of Efficacy of Retreatment with Rituximab in Patients with
Rheumatoid Arthritis
[0624] This example describes a phase III randomized double-blind,
placebo-controlled multicenter study of retreatment with rituximab
in subjects with RA receiving background methotrexate.
[0625] The primary objective of this study is to evaluate the
efficacy of retreatment with rituximab in subjects with active RA
who are receiving MTX and who have had an inadequate response to
TNF inhibitors.
[0626] The secondary objectives of this study are as follows:
[0627] To evaluate the safety of retreatment with rituximab in
subjects with active RA who are receiving MTX and who have had an
inadequate response to TNF inhibitors [0628] To evaluate the safety
of rituximab in subjects with active RA who are receiving MTX and
who have had an inadequate response to TNF inhibitors
[0629] This is a Phase III, randomized, double-blind,
placebo-controlled, multicenter study evaluating the efficacy of
retreatment with rituximab in subjects with active RA who are
receiving MTX. The study consists of four parts: screening,
treatment period (open-label rituximab for first course and, for
eligible subjects, double-blind, randomized retreatment), safety
follow-up (SFU), and B-cell follow-up. Subjects must have had
inadequate response to treatment with one or more TNF inhibitors
because of toxicity or inadequate efficacy. Approximately 555
subjects will enter the treatment period at approximately 150
investigational sites in the United States. RF-positive and
RF-negative subjects will be enrolled and will be allocated equally
between treatment arms, with the overall proportion of RF-negative
subjects limited to 20% of the total sample size.
[0630] Prior to Day 1, subjects will be discontinued from all
DMARDs except MTX (for leflunomide, adalimumab, and infliximab for
.gtoreq.8 weeks and etanercept for .gtoreq.4 weeks). All subjects
will continue to receive MTX 10-25 mg/wk, at a stable dose for the
study duration. The screening visit can occur up to 56 days prior
to receiving the first dose of study treatment depending on washout
requirements.
[0631] All subjects who meet eligibility criteria and are enrolled
in the trial will receive rituximab for the first course of
treatment. A course of rituximab is defined as two 1000 mg
intravenous (IV) doses given 14 days apart, with pre-medication
with methylprednisolone 100 mg IV prior to each dose of rituximab.
All subjects will also receive a stable dose of folate .gtoreq.5
mg/wk). All subjects should continue to receive any background
corticosteroids (.ltoreq.10 mg/day prednisone or equivalent) or
oral nonsteroidal anti-inflammatory drugs (NSAIDs) at a stable
dose.
[0632] The first dose of rituximab should be given within 24 hours
following baseline assessments. However, if necessary, up to 72
hours will be allowed between baseline assessments and the first
dose of study drug.
[0633] During Weeks 24-40, subjects who have active disease based
on a disease activity score in 28 joints (DAS 28-erythrocyte
sedimentation rate [ESR]) of .gtoreq.2.6 will be considered
eligible for retreatment and will be randomized in a 2:1 ratio to
receive retreatment with one additional course of rituximab (Group
A) or placebo (Group B). Subjects who do not meet the criteria for
a second course of rituximab during Weeks 24-40 will continue to be
followed for safety and efficacy. Subjects who meet the criteria
for retreatment during Weeks 24-40 and refuse retreatment, for any
reason, will be withdrawn from the treatment period and will enter
into the SFU period.
[0634] Standard arthritis and safety assessments will be performed.
Pharmacodynamic measures will include CD19.sup.+B cells,
immunoglobulins, and autoantibodies. DAS 28-ESR scores will be
calculated by the investigator at regularly scheduled visits
(Prevoo et al. Arthritis Rheum 38:44-48 (1995); DAS-score.n1 2005
DAS-score.n1 2005: home of the DAS. Department of Rheumatology
University Medical Center Nijmegan--the Netherlands. [cited 1 Sep.
2005]. Available from:
http://www.das-score.nl/www.das-score.nl/index.html).
[0635] The treatment period is 72 weeks long (Day 1 to Week 72).
All subjects who withdraw from the treatment period at any time or
who receive retreatment with rituximab/placebo between Weeks 24-40
and complete the treatment period should return for SFU assessments
at SFU Weeks 4, 12, 24, 36, and 48 after withdrawal or completion.
All subjects will be followed for at least 48 weeks after their
last dose of rituximab. All subjects who receive only one course of
rituximab treatment (i.e., those that do not qualify for
retreatment during the study) and complete the treatment period
will not return for SFU.
[0636] Subjects whose peripheral B-cell counts have not recovered
by the end of the treatment period or the SFU period will continue
to be followed for laboratory evaluations and the occurrence of
serious adverse events every 12 weeks until B-cell recovery. B-cell
recovery is defined as peripheral B-cell counts that have returned
to baseline values or the lower limit of normal (LLN), whichever is
lower.
[0637] At or following 16 weeks after retreatment, subjects who
have not achieved a 20% improvement in both tender joint counts
(TJCs) and swollen joint counts (SJCs) compared with baseline may
initiate rescue treatment with one non-biologic DMARD, the choice
of which is at the discretion of their treating physician.
[0638] In a retreatment study of rituximab in RA, subjects achieved
sustained American College of Rheumatology (ACR)20, 50, and 70
responses (Pavelka et al. "Efficacy and safety following repeated
courses of rituximab in patients with active rheumatoid arthritis."
Abstract presented at EULAR 2005). However, because this study is
an open-label study, there are no controlled data evaluating the
efficacy and safety of retreatment with rituximab in RA. This
present study is designed to evaluate the efficacy of retreatment
in a placebo-controlled trial with a single additional course of
rituximab in subjects with active RA. Subjects with active disease,
as characterized by a DAS 28-ESR .gtoreq.2.6, will be treated with
a second course of study drug (rituximab or placebo) during Weeks
24-40.
[0639] The purpose of retreatment with rituximab is to prevent
flare, promote sustained control of disease, and potentially
prevent disease progression. The criterion of DAS 28-ESR
.gtoreq.2.6 for retreatment will ensure that subjects with
clinically significant disease activity are retreated. The DAS
28-ESR will be calculated using the number of swollen and tender
joints, the ESR, and the Patient's Global Assessment of Disease
Activity (on a 100 mm visual analog scale [VAS]).
[0640] The primary endpoint of this study is the proportion of
retreated subjects with an ACR20 at Week 48 relative to baseline
(Day 1), not relative to time of retreatment. Baseline (Day 1), not
time of retreatment, was chosen in order to evaluate the overall
benefit of retreatment with rituximab in subjects with moderate to
severe RA. Without being limited to any one theory, it is
hypothesized that retreatment with RTX results in maintenance of
effect to a slight improvement at Week 48 relative to Week 24, in
comparison with a deterioration in placebo-treated subjects.
Although there may be a clear treatment benefit in retreated
subjects, ACRresponses at Week 48 relative to a Week 24 baseline
for both groups may be insignificant, Thus, this study is not
designed to evaluate improvement from the time of retreatment.
[0641] Based on Week 24 data in the Phase III study (REFLEX,
WA17042/U2646s/IDEC102-20), approximately 91% of subjects treated
with rituximab would have met the retreatment criterion (DAS 28-ESR
.gtoreq.2.6) at Week 24 of this protocol. Mandatory retreatment
based on DAS 28-ESR will provide objective efficacy information
using a disease activity-based retreatment paradigm. The minimum
period of 24 weeks is based on the pharmacokinetics and
pharmacodynamics of rituximab. This is paralleled by an apparent
increase in disease activity after this timepoint and, therefore,
represents a reasonable time to retreat.
[0642] The primary outcome measure is the proportion of retreated
subjects with an ACR20 response at Week 48 relative to baseline
(Day 1).
[0643] The secondary outcome measures are as follows: [0644]
Proportion of retreated subjects with an ACR50 and ACR70 response
at Week 48 relative to baseline (Day 1). [0645] Change in DAS
28-ESR at Week 48 compared with baseline (Day 1) for retreated
subjects [0646] Proportion of retreated subjects who achieve a
European League Against Rheumatism (EULAR) response (good or
moderate) at Week 48 relative to baseline (Day 1) [0647] Change in
ACR core set at Week 48 compared with baseline (Day 1) for
retreated subjects (SJC, TJC, Health Assessment Questionnaire
[HAQ], patient and physician global assessments, patient pain
assessment, C-reactive protein [CRP], and ESR) [0648] ACR-N at Week
48 in retreated subjects [0649] Change in SF-36 subscale and
summary scores from baseline (Day 1) to Week 48 in retreated
subjects [0650] Change in Functional Assessment of Chronic Illness
Therapy-Fatigue (FACIT-F) assessment from baseline (Day 1) to Week
48 in retreated subjects [0651] Proportion of ACR20, ACR50, and
ACR70 response at Week 48 in all subjects
[0652] The exploratory outcome measures are: [0653] Proportion of
all subjects who achieve an ACR20, ACR50, and ACR70 response at
Week 72 compared with baseline [0654] Proportion of subjects with
DAS 28-ESR remission (DAS 28-ESR <2.6) at Week 48 [0655]
Proportion of subjects with DAS 28-ESR low disease (DAS 28-ESR
.ltoreq.3.2) at Week 48 [0656] Proportion of subjects with DAS
28-ESR remission (DAS 28-ESR <2.6) at Week 72 [0657] Proportion
of subjects with DAS 28-ESR low disease (DAS 28-ESR .ltoreq.3.2) at
Week 72
[0658] Eligible subjects who have active RF-positive 20 IU/mL) or
RF-negative RA, are receiving MTX, and have had a previous or
current inadequate response to one or more TNF inhibitors will be
screened for study participation. RF-negative subjects will be
limited to 20% of the total enrolled population.
[0659] Subjects must meet the following criteria to be eligible for
study entry: [0660] Signed informed consent form [0661] Ability and
willingness to comply with the requirements of the study protocol
[0662] Age 18-80 [0663] Diagnosis of RA for at least 6 months,
according to the revised 1987 ACR criteria for the classification
of RA (Hochberg et al. Arthritis Rheum 35:498-502 (1992)):
[0664] Class I
[0665] Complete functional capacity with ability to carry on all
usual duties without handicaps
[0666] Class II
[0667] Functional capacity adequate to conduct normal activities
despite handicap of discomfort or limited mobility of one or more
joints
[0668] Class III
[0669] Functional capacity adequate to perform only few or none of
the duties of usual occupation or self-care
[0670] Class IV
[0671] Largely or wholly incapacitated with subject bedridden or
confined to wheel chair, permitting little or no self-care [0672]
Receiving treatment for RA on an outpatient basis [0673] Documented
moderate to severe active RA activity at screening as follows:
[0674] TIC .gtoreq.8 (68 joint count), and [0675] SJC .gtoreq.8 (66
joint count), and [0676] Abnormal CRP of .gtoreq.0.6 mg/dL, or ESR
of .gtoreq.28 mm/hr [0677] Documented inadequate response to
previous or current treatment with one or more of the following:
etanercept, infliximab, and/or adalimumab because of toxicity or
inadequate efficacy [0678] Inadequate efficacy consists of
treatment with etanercept for .gtoreq.3 months at doses of 25 mg
twice weekly or 50 mg weekly, at least four infusions of .gtoreq.3
mg/kg infliximab, or 40 mg adalimumab every other week for
.gtoreq.3 months. [0679] Use of MTX 10-25 mg/wk for .gtoreq.12
weeks prior to Day 1 at a stable dose for .gtoreq.4 weeks [0680]
Willingness to receive oral folic acid [0681] If taking a
background corticosteroid 10 mg/day prednisone or equivalent), use
of the corticosteroid must be at a stable dose during the 4 weeks
prior to Day 1 [0682] Use of one NSAID is permitted if the dose is
stable for 2 weeks prior to Day 1 [0683] For men and women of
reproductive potential, willingness to use a reliable means of
contraception (e.g., hormonal contraceptive, intrauterine device,
physical barrier) for 30 days prior to Day 1 and for the study
duration or the duration that the subject's peripheral CD19+B cells
are depleted, whichever is longer
[0684] Subjects who meet any of the following criteria will be
excluded from study entry:
[0685] a. General [0686] Rheumatic autoimmune disease other than RA
or significant systemic involvement secondary to RA (e.g.,
vasculitis, pulmonary fibrosis, or Felty's syndrome) [0687]
Secondary Sjogren's syndrome with RA is permitted. [0688] History
of or current inflammatory joint disease other than RA (e.g., gout,
reactive arthritis, psoriatic arthritis, seronegative
spondyloarthropathy, or Lyme disease) or other systemic rheumatic
disorder (e.g., systemic lupus erythematosus, inflammatory bowel
disease, scleroderma, inflammatory myopathy, or overlap syndrome)
[0689] Functional Class IV, as defined by the ACR Classification of
Functional Status in Rheumatoid Arthritis) [0690] Any surgical
procedure, including bone/joint surgery/synovectomy (including
joint fusion or replacement), within 12 weeks prior to Day 1 or
planned within 48 weeks after Day 1 [0691] Known hypersensitivity
to any component of a humanized or murine monoclonal antibody
[0692] Receipt of a live vaccination within 4 weeks prior to Day 1
[0693] Significant cardiac or pulmonary disease, including
obstructive pulmonary disease [0694] Evidence of significant
uncontrolled concomitant disease, such as, but not limited to
nervous system, renal, hepatic, endocrine, or gastrointestinal
disorders [0695] Known active bacterial, viral, fungal,
mycobacterial, or other infection (including tuberculosis or
atypical mycobacterial disease but excluding fungal infections of
the nail beds) or any major episode of infection requiring
hospitalization or treatment with IV antibiotics within 4 weeks of
Day 1 or oral antibiotics within 2 weeks of Day 1 [0696] History of
serious recurrent or chronic infection (for screening for a chest
infection a chest radiograph will be performed at screening if not
performed 12 weeks prior to screening) [0697] History of or
currently active primary or secondary immunodeficiency, including
HIV infection [0698] History of cancer, including solid tumors and
hematologic malignancies (except basal cell or squamous cell
carcinoma of the skin that has been excised and cured) [0699]
History of significant cytopenias or other bone marrow disorders
[0700] History of alcohol, drug, or chemical abuse within 24 weeks
prior to Day 1 [0701] Pregnancy or lactation [0702] Neuropathies
and neurovasculopathies that might interfere with pain evaluation
[0703] Poor peripheral venous access [0704] Intolerance or
contraindications to oral or IV corticosteroids
[0705] b. Laboratory Exclusion Criteria [0706] Hemoglobin <8.0
g/dL [0707] Absolute neutrophil count <1.5.times.10.sup.3/.mu.L
[0708] IgM <0.40 mg/mL [0709] IgG <5.0 mg/mL [0710] A
Aspartate aminotransferase (AST) or alanine aminotransferase (ALT)
>2.5.times. the upper limit of normal [0711] Positive hepatitis
B surface antigen or hepatitis C antibody serology [0712] For women
of childbearing potential (including those who have had a tubal
ligation), a positive serum pregnancy test at screening [0713]
Excluded Previous or Concomitant Medications [0714] Current use of
any DMARD other than MTX [0715] Concurrent treatment with any
biologic agent [0716] Treatment must be discontinued at least 4
weeks prior to Day 1, except for the following: leflunomide for
.gtoreq.8 weeks (or .gtoreq.14 days after 11 days of standard
cholestyramine washout), infliximab .gtoreq.8 weeks, and adalimumab
.gtoreq.8 weeks [0717] Treatment with any investigational agent
within 4 weeks prior to Day 1 or five half-lives of the
investigational drug (whichever is longer) [0718] Any previous
treatment with rituximab or other cell-depleting therapies,
including CAMPATH, anti-CD4, anti-CDS, anti-CD3, anti-CD19,
anti-CD11 a, anti-CD22, BLys/BAFF, and other anti-CD20 agents
[0719] Previous treatment with an anti-a 4 integrin agent,
including natalizumab [0720] Previous treatment within 6 months of
screening with IV .gamma. globulin or the Prosorba.RTM. Column
[0721] Upon completion of all screening evaluations and
verification that the subject has met all inclusion and exclusion
criteria, site personnel will contact the interactive voice
response system (ivrs) to obtain the subject number and confirm
subject enrollment for study drug inventory management. all
enrolled subjects will receive rituximab as their initial course of
treatment.
[0722] During Weeks 24-40, subjects who are eligible for
retreatment will be randomized in a 2:1 ratio to either Group A
(rituximab retreatment) or Group B (placebo; see Table 1). If a
subject meets the eligibility criteria for retreatment during Weeks
24-40, site personnel will contact IVRS to initiate randomization
of the subject and to obtain the study drug kit number.
[0723] An independent IVRS provider will conduct the randomization
and hold the treatment assignment codes. Randomization will be
stratified by study center, baseline RF status (RF positive, RF
negative), and Week 24 improvements in tender and swollen joint
count (.gtoreq.20% improvement or <20% improvement). At the time
of the unblinding, the treatment assignment codes and the kit
treatment codes will be requested from the IVRS provider.
Documentation of the transfer and the data included in the transfer
will be kept on file.
TABLE-US-00020 TABLE 1 Study Treatment Groups Group Rituximab: 1000
mg IV on Days 1 and 15 for the first A course 1000 mg IV on Days 1
and 15 for the retreatment course for eligible subjects (DAS 28-ESR
.gtoreq. 2.6 and meets the retreatment criteria herein) during
Weeks 24-40 Corticosteroids: 100 mg IV methylprednisolone prior to
each infusion MTX: 10-25 mg/wk Folate: .gtoreq.5 mg/wk Group
Rituximab: 1000 mg IV on Days 1 and 15 for the first B course
Placebo IV on Days 1 and 15 for the retreatment course for eligible
subjects (DAS 28-ESR .gtoreq. 2.6 and meets the retreatment
criteria herein) during Weeks 24-40 Corticosteroids: 100 mg IV
methylprednisolone prior to each infusion MTX: 10-25 mg/wk Folate:
.gtoreq.5 mg/wk
[0724] During retreatment, treatment group assignment will be
blinded to site personnel and Genentech. To prevent potential
unblinding because of observed efficacy or laboratory changes
during retreatment, a dual assessor approach will be used to
evaluate efficacy and safety.
[0725] Peripheral CD19 B-cell counts will be blinded to site
personnel and Genentech until the time of database lock for the
primary endpoint at Week 48 for all subjects enrolled in the trial.
Therefore, all subjects who have completed the treatment period and
SFU prior to database lock will be assumed to be peripherally
depleted and will remain in B-cell follow-up.
[0726] The Efficacy Assessor (or designee) should be a
rheumatologist or skilled arthritis assessor. The Efficacy Assessor
must not be the Principal Investigator. The Efficacy Assessor will
only have access to efficacy data and will be responsible for
completing the joint counts and Physician's Global Assessment of
disease activity VAS only. To ensure consistent joint evaluation
throughout the trial, individual subjects should be evaluated by
the same joint assessor for all study visits. During a study visit,
all subject-reported outcomes should be completed prior to all
other assessments.
[0727] The Safety Assessor (or designee) should be a rheumatologist
and will have access to both safety and efficacy data. The Safety
Assessor may be the Principal Investigator. The Safety Assessor
will have access to source documents, laboratory results, and Case
Report Forms (CRFs) and will be responsible for calculating the DAS
28-ESR and making treatment decisions based on a subject's clinical
response and laboratory parameters. The Safety Assessor will not
complete any efficacy assessments or record the results of any
efficacy assessments on behalf of the Efficacy Assessor.
[0728] Rituximab for use in this study is a sterile, clear,
colorless, preservative-free liquid concentrate for IV
administration. Rituximab is supplied at a concentration of 10
mg/mL in 500 mg (50 mL) single-use vials. The product is formulated
for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL
sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile
Water for Injection. The pH is adjusted to 6.5.
[0729] All enrolled subjects will receive a course of rituximab
(1000 mg rituximab by IV infusion on Days 1 and 15) as their
initial treatment. During Weeks 24-40, subjects eligible for
retreatment will be randomized to Group A to receive an additional
course (two doses) of rituximab 1000 mg IV 14 days apart or to
Group B to receive a course (two doses) of placebo 1000 mg IV 14
days apart.
[0730] Premedication with 1000 mg oral acetaminophen and 50 mg oral
diphenhydramine is recommended for all subjects, and 100 mg IV
methylprednisolone to be completed within 30-60 minutes prior to
each rituximab/placebo infusion is required.
[0731] All subjects will continue to receive MTX at a stable dose
of 10-25 mg/wk and receive a stable dose of folate 5 mg/week) given
as either a single dose or as a divided weekly dose.
[0732] Rituximab/placebo infusions should be administered to
subjects under the close supervision of the investigator or
designee in a hospital or clinic where full resuscitation
facilities are immediately available. Although rituximab may be
administered on an outpatient basis, subjects may be hospitalized
for observation at the discretion of the investigator.
Rituximab/placebo should be administered as a slow IV infusion. Do
not administer as an IV push or bolus. At the end of each infusion,
the IV line should remain in place for at least 1 hour to allow
administration of IV drugs if necessary. If no adverse event occurs
during this time, the IV line may be removed.
[0733] Using appropriate aseptic technique, withdraw the necessary
amount of rituximab/placebo and dilute to a final concentration of
4 mg/mL into an infusion bag containing either 0.9% Sodium
Chloride, USP, or 5% Dextrose in Water, USP. Gently invert the bag
to mix the solution. Rituximab vials are biologically and
chemically stable at 2.degree. C-8.degree. C. (36.degree.
F-46.degree. F.). Vials must not be used beyond the expiration
date. Rituximab should be protected from exposure to direct
sunlight.
[0734] Once reconstituted in IV bags, rituximab solutions for
infusions may be stored at 2.degree. C-8.degree. C. (36.degree.
F-46.degree. F.) for 24 hours. Rituximab solutions for infusion
have been shown to be stable for an additional 24 hours at room
temperature (23.degree. C. or 73.degree. F.). However, since
rituximab solutions do not contain a preservative, diluted
solutions should be refrigerated (2.degree. C-8.degree. C.). No
incompatibilities between rituximab and polyvinyl chloride or
polyethylene bags have been observed.
[0735] As to retreatment, DAS 28-ESR will be calculated using the
joint count and Patient's Global Assessment of Disease Activity
(VAS) from the current visit and the ESR from the current or the
previous visit only if the ESR from the current visit is
unavailable. Subjects who meet the following criteria at any visit
during Weeks 24-40 are eligible to receive retreatment with study
drug (rituximab or placebo): [0736] DAS 28-ESR .gtoreq.2.6 [0737]
Negative urine pregnancy test for women of childbearing potential
(including those who have had a tubal ligation)
[0738] In addition, subjects who qualify for retreatment by DAS
28-ESR .gtoreq.2.6 during Weeks 24-40 must also meet the following
criteria in order to be retreated based on results from the
previous visit if current results are unavailable: [0739]
Hemoglobin .gtoreq.8.0 g/dL [0740] Absolute neutrophil count
.gtoreq.1.5 .mu. 10.sup.3/.mu.L [0741] IgM .gtoreq.0.40 mg/mL
[0742] IgG .gtoreq.5.0 mg/mL [0743] No significant cardiac or
pulmonary disease (including obstructive pulmonary disease) [0744]
No primary or secondary immunodeficiency, including known history
of HIV infection [0745] No evidence of significant uncontrolled
concomitant disease, such as, but not limited to nervous system,
renal, hepatic, endocrine, or gastrointestinal disorders [0746] No
active infection of any kind, (excluding fungal infections of nail
beds), or any major episode of infection requiring hospitalization
or treatment with IV antibiotics within 4 weeks of infusion or
completion of oral antibiotics within 2 weeks prior to infusion
[0747] Rituximab dose modification is not permitted during this
study. In the event of an infusion-related reaction, the rate of
infusion may be adjusted. If a subject experiences an
infusion-related reaction requiring an interruption in the infusion
and the investigator determines that the infusion should not be
restarted, the subject should be withdrawn from the study treatment
period and enrolled into the SFU.
[0748] All subjects will receive concomitant 10-25 mg/wk MTX, as
prescribed by their treating physician. Subjects must have been
treated with MTX for .gtoreq.12 weeks prior to entering the study
and must remain on a stable dose of MTX for .gtoreq.4 weeks before
Day 1 and during the study, unless modification is necessary for
toxicity.
[0749] Certain adverse events that are commonly associated with MTX
treatment may occur. In order to minimize MTX toxicity, all
subjects treated with MTX will also receive a stable dose of folic
acid 5 mg/week) given as either a single weekly dose or as a
divided daily dose. The dosing regimen is at the investigator's
discretion.
[0750] Daily treatment with .ltoreq.10 mg prednisone or equivalent
is allowed if the dose is stable for .gtoreq.4 weeks prior to Day
1. The dose should remain stable throughout the study, unless
modification is required for toxicity.
[0751] Use of one NSAID is allowed if the dose is stable for 2
weeks prior to Day 1. The dose should remain stable throughout the
study, unless modification is required for toxicity.
[0752] In addition, daily treatment with 325 mg acetylsalicylic
acid is allowed for cardiovascular prophylaxis.
[0753] Additional analgesics may be used for pain as required.
However, subjects should not take analgesics within 12 hours prior
to a visit where efficacy assessments will be performed.
Adjustments to the analgesic regimen may be made, but the change
must be documented in the appropriate CRFs.
[0754] Intra-articular injections of corticosteroid are
discouraged, particularly during the first 48 weeks; however, they
may be used on a limited basis to manage a subject's RA activity
during the study. No more than one joint per 24-week period should
be injected, and the same joint should not be injected more than
once in any 48-week period. No single injection should exceed 40 mg
triamcinolone (or equivalent), and the total dose of corticosteroid
should not exceed 80 mg triamcinolone (or equivalent) during any
48-week period.
[0755] During the study, subjects may continue to receive a
background corticosteroid with a dose of .ltoreq.10 mg/day of
prednisone (or equivalent). In the case of a disease flare or
non-RA condition, such as asthma, requiring treatment with oral
corticosteroids, appropriate doses should be administered for a
maximum of 2 weeks and should be tapered down to the previous level
as rapidly as medically possible.
[0756] Increasing the dose of corticosteroids will be considered
worsening of a subject's condition from baseline and should be
recorded as an adverse event on the CRF.
[0757] IV or intramuscular corticosteroids are not permitted in the
study, other than those specified in the protocol treatments.
[0758] Increasing the dose of corticosteroids will be considered
worsening of a subject's condition from baseline and should be
recorded as an adverse event on the CRF.
[0759] Subjects must remain on a stable dose of MTX for .gtoreq.4
weeks before Day 1 and during the study, unless modification is
necessary for toxicity.
[0760] At or following 16 weeks after retreatment, subjects who
have not achieved a 20% improvement in both TJCs and SJCs compared
with baseline may initiate rescue treatment with one non-biologic
DMARD, the choice of which is at the discretion of their treating
physician. Subjects who receive rescue will not be withdrawn from
the study.
[0761] Laboratory samples must be drawn prior to infusions of
methylprednisolone IV and rituximab/placebo.
[0762] Subject-reported data (e.g., Patient's Global Assessment of
Disease Activity, Patient's Assessment of Pain) may only be
recorded by the study nurse/investigator on behalf of the subject
if the subject has difficulty writing during the visit or is unable
to read. This must be documented clearly in the subject notes.
[0763] To prevent potential unblinding, a dual assessor (Efficacy
Assessor and Safety Assessor) approach will be used to evaluate
efficacy and safety. It is essential that assessments completed by
the subject and the Efficacy Assessor are made before those by the
Safety Assessor.
[0764] The subject's overall assessment of his or her disease
activity during the last 24 hours should be described using the
100-mm horizontal VAS where the left-hand extreme of the line
represents no disease activity (symptom free and no arthritis
symptoms) and the right-hand extreme represents maximum disease
activity (maximum arthritis disease activity).
[0765] The subject's assessment of his or her level of pain during
the last 24 hours should be described using the 100-mm horizontal
VAS where the left-hand extreme of the line represents no pain and
the right-hand extreme represents unbearable pain.
[0766] The Stanford HAQ disability index is a subject-reported
questionnaire specific for RA. It consists of 20 questions
referring to eight component sets: dressing/grooming, arising,
eating, walking, hygiene, reach, grip, and activities. The
questionnaire will be scored based on instructions from Stanford
University Medical Center (Fries et al. Arthritis Rheum 23:137-145
(1980)).
[0767] The FACIT-F will be used to assess fatigue. It is a 13-item
questionnaire in which subjects are requested to score each
question on a 0-4 scale. The assessment was originally developed
for chronic illnesses and is now validated for subjects with RA
(Cella et al. J Rheumatology 32(5):811-819 (2005)).
[0768] The SF-36 is a generic health-related quality of life
instrument that has been widely tested for its psychometric
properties and is widely used in clinical and epidemiological
studies (Ware et al. How to Score Version Two of the SF-36 Health
Survey. Lincoln, RI: Qualitymetric Incorporated, 2000). The SF-36
(Version 2) is provided by the Medical Outcomes Trust (Boston,
Mass., USA).
[0769] An evaluation of 66 joints for swelling and 68 joints for
tenderness should be performed by a rheumatologist or skilled joint
assessor. Joints will be evaluated and classified as swollen or not
swollen and tender or not tender based on pressure and joint
manipulation upon physical examination. Joints with total joint
prosthesis or arthrodesis should not be evaluated; however, all
other joints should be evaluated. The joints to be evaluated for
swelling and tenderness are provided below: [0770]
Temporomandibular joint [0771] Sternoclavicular joint [0772]
Acromioclavicular joint [0773] Shoulders .sup.a [0774] Elbows*
[0775] Wrists* [0776] Interphalangeal on digit 1.sup.a [0777]
Distal interphalangeal joints on digits 2-5 [0778] Proximal
interphalangeal joints on digits 2-5 .sup.a [0779]
Metacarpophalangeal joints on digits 1-5 .sup.a [0780] Hips
(tenderness only) [0781] Knees .sup.a [0782] Ankles [0783]
Metatarsals [0784] Interphalangeal joints on toes 1-5 [0785]
Metatarsophalangeal joints on toes 1-5 [0786] .sup.a Includes the
28 joints used to calculate the Disease Activity Score (DAS)28.
[0787] Joints that have undergone a procedure should be evaluated
as follows: [0788] Surgery: Any joint that has been replaced or
fused at any time prior to or during the study should be documented
as nonevaluable (NE) for the duration of the study. [0789] Any
joint that has undergone synovectomy (including chemical and
radiological synovectomy) should be documented as not done (ND) for
24 weeks following synovectomy. After this time, the joint may be
evaluated again. [0790] Intra-articular injection: Any joint that
has received an intra-articular injection of a corticosteroid
should be documented as ND for the following 12 weeks. After this
time, the joint may be evaluated again. [0791] Arthrocentesis: Any
joint that undergoes synovial fluid aspiration will not be
evaluated at the following scheduled visit and will be graded as
ND. After this time, the joint may be evaluated again.
[0792] The physician's assessment of a subject's disease activity
during the last 24 hours should be described using the 100-mm
horizontal VAS where the left-hand extreme of the line represents
no disease activity (symptom free and no arthritis symptoms) and
the right-hand extreme represents maximum disease activity. This
should be completed by the Efficacy Assessor who may or may not be
a physician.
[0793] CRP will be analyzed at a central laboratory. ESR will be
determined using the Westergren method at the local laboratory.
[0794] A DAS 28-ESR .gtoreq.2.6 has been selected as a threshold
for retreatment in this trial. Subjects with a DAS 28-ESR
.gtoreq.2.6 during Weeks 24-40 may be eligible to receive a second
course of study drug (rituximab or placebo).
[0795] A general physical examination (including the
cardiovascular, respiratory, gastrointestinal and neurological
systems) should be performed at the times indicated in the Schedule
of Assessments. Any persisting abnormalities should be stated each
time the examination is performed. Diagnosis of new abnormalities
should be recorded as an adverse event if appropriate.
[0796] Vital signs (heart rate, systolic and diastolic blood
pressure, and temperature) will be taken at the times indicated in
the Schedule of Assessments. Assessments should be taken after the
subject has been in a semi-supine position for at least 5
minutes.
[0797] Twelve lead electrocardiograms (ECGs) should be performed at
the times indicated in the Schedule of Assessments.
[0798] Posterior-anterior and lateral chest radiographs should be
obtained at screening and reviewed by the investigator or designee.
At screening, if chest radiographs have been taken within the past
12 weeks that show no clinically significant abnormality and there
are no signs or symptoms suggestive of pulmonary disease that would
exclude the subject from the trial, then chest radiograph does not
need to be repeated.
[0799] Hematology, serology, chemistry, urinalysis, serum pregnancy
test, flow cytometry, immunology, and CRP analyses will be
performed by a central laboratory; pharmacokinetic and HACA
analyses will be performed by Genentech; and urine pregnancy test
and ESR assessments will be performed locally at the study sites.
Instruction manuals and supply kits, including pharmacokinetic and
HACA supplies, will be provided for all laboratory assessments.
Laboratory assessments will include the following: [0800]
Hematology/CBC: Hemoglobin, hematocrit, red blood cells (RBC),
white blood cells (WBC) with differential, and platelet counts.
[0801] Serology: Hepatitis B surface antigen (HBsAg) and hepatitis
C virus (HCV) antibody. [0802] Serum chemistries: AST/SGOT,
ALT/SGPT, alkaline phosphatase, total protein, albumin, total
bilirubin, blood urea nitrogen (BUN), uric acid, creatinine, random
glucose, potassium, sodium, chloride, calcium, and phosphorous.
[0803] Urinalysis: Blood, protein, and glucose (microscopic
examination, if abnormal and applicable). [0804] Pregnancy test:
All women of childbearing potential (including those who have had a
tubal ligation) will have a serum pregnancy test at screening. In
addition, regular urine pregnancy tests will be administered at all
other visits. If a urine pregnancy test is positive, it must be
confirmed by a serum pregnancy test. [0805] Serum C3 and C4
complement levels. [0806] Immunologic assessments: Quantitative
immunoglobulins (Total Ig, IgG, IgA, and IgM), RF (total and
isotype concentrations), and anti-cyclic-citrullinated peptide
(CCP) antibody (IgG). [0807] Expanded fluorescent-activated cell
sorter (FACS) analysis: Cell populations assessed will include
monocytes (CD14 and CD16); NK cells (CD56); T-cell subsets (CD3,
CD4, CD8, CD45RO, and CD45RA); and B-cell subsets (CD19, CD27,
CD38, and IgD). Activation markers may also be evaluated (CD25,
CD69, CD4OL, and CD80). [0808] B cell FACS analysis: absolute B
cells (CD19) only. [0809] Analysis for HACA response will be
performed using an ELISA for all enrolled subjects. [0810]
Pharmacokinetic assays: Serum samples will be obtained for
pharmacokinetic assay at the visits indicated in the SOA and also
at the same timepoints as HACA. Serum rituximab concentrations are
required to accurately interpret HACA results. [0811] Optional
biomarker samples: [0812] For subjects who consent separately,
optional research samples (whole blood, serum) will be collected
during the course of the study for exploratory biomarker
assessments. Whole blood samples, collected using PAXgene.TM. RNA
tubes, will be used for gene expression profiling. Serum sample
assessments of markers related to RA or rituximab may include, but
not be limited to, cytokine/chemokine measurements and quantitation
of bone and cartilage turnover markers. All samples will be
collected at the same timepoints for optimal data comparability.
[0813] It is anticipated that information from this analysis will
promote and facilitate individualized health care via better
understanding of rituximab's mode of action, related efficacy and
safety, predictors for good response, and possibly determinants of
RA (and other autoimmune) disease progression. These samples will
be stored for up to 15 years after database closure.
[0814] All subjects must provide written informed consent before
any study-specific procedures or assessments are performed,
including changes to a subject's current treatment regimen.
Screening evaluations may be performed during a 56-day period prior
to the first infusion of rituximab on Day 1. Subject reported
assessments should be performed prior to other clinical
assessments.
[0815] If a subject fails a laboratory exclusion criterion at
screening, the investigator may repeat the test up to twice within
the screening period. If the subject fails the laboratory criterion
a third time, they will be considered a screen failure. A blood
sample or laboratory test will not be considered as re-tested if
the sample was redrawn because of sample handling problems,
breakage, or sample integrity. A subject who fails screening may be
re-screened.
[0816] A subject may be re-screened if they did not meet all the
eligibility criteria within 56 days of the original screening
visit. A subject who undergoes re-screening must repeat the entire
screening process and be re-consented prior to any study-specific
procedures. Subjects may be re-screened only once. [0817] a.
Screening Visit (Day--56) [0818] Written Informed Consent [0819]
Review of inclusion and exclusion criteria [0820] Contact IVRS to
obtain assignment of subject screening number [0821] Demographic
data (e.g., sex, age, race/ethnicity) [0822] Complete medical
history (including vaccination history) [0823] Concomitant
medications taken within 12 weeks before screening, including
vaccines, all prior DMARDs, and biologic agents [0824] Vital signs
(heart rate, blood pressure, and temperature) [0825] Complete
physical examination, including height and weight measurements
[0826] Joint evaluation [0827] 12-Lead ECG [0828] Chest X-ray
[0829] If a chest X-ray has been performed within 12 weeks before
screening and showed no clinically significant abnormality, a chest
X-ray is not required at screening. [0830] Central laboratory
assessments [0831] Hematology/CBC [0832] Hepatitis B surface
antigen and hepatitis C antibody [0833] Serum pregnancy test for
women of childbearing potential (including those who have had a
tubal ligation) [0834] Serum chemistries [0835] Urinalysis [0836]
CRP [0837] IgG and IgM [0838] Total RF [0839] ESR (local
laboratory; Westergren method)
[0840] All assessments during the treatment period should be
performed within the specified time window for each visit.
Assessments and procedures scheduled on days when rituximab is
administered should be performed prior to the rituximab infusion,
unless otherwise indicated. For this study, Day 1 is the day of
initial rituximab infusion. The Day 1 visit should be performed on
a day that will allow subsequent visits (e.g., Day 15 visit) to
occur without delay. Subject-reported assessments should be
performed prior to other clinical assessments. For subjects
eligible for retreatment, the visit at which the subject meets the
eligibility criteria for retreatment is considered the qualifying
visit. [0841] a. Day 1
[0842] All assessments will be performed 30 minutes pre-infusion
unless otherwise specified. [0843] Review of inclusion and
exclusion criteria [0844] Contact IVRS for subject enrollment
number and study drug inventory [0845] Patient's Global Assessment
of Disease Activity (VAS) [0846] Patient's Assessment of Pain (VAS)
[0847] HAQ [0848] FACIT-F [0849] SF-36 [0850] Vital signs (heart
rate, blood pressure, and temperature): pre-infusion, during
infusion (every 15 minutes for 1 hour, and then every 30 minutes
until the end of infusion), and post-infusion (every 30 minutes for
1 hour post-infusion) [0851] Physical examination, including
measurement of body weight [0852] Joint evaluation [0853]
Physician's Global Assessment of Disease Activity (VAS) [0854]
Urine pregnancy test for women of childbearing potential (including
those who have had a tubal ligation) [0855] Central laboratory
assessments [0856] Hematology/CBC (within 30 minutes pre-and
post-infusion) [0857] Serum chemistries [0858] Urinalysis [0859]
Expanded FACS (within 30 minutes pre- and post-infusion) [0860]
CD19 B cells (within 30 minutes pre-and post-infusion) [0861] CRP
[0862] Immunoglobulins [0863] RF [0864] Anti-CCP antibody [0865]
C3, C4 [0866] Pharmacokinetic sample (30 minutes pre- and
post-infusion) [0867] HACA sample [0868] ESR (local laboratory;
Westergren method) [0869] Optional research biomarker samples
(whole blood and serum samples) [0870] Methylprednisolone
administration [0871] Rituximab administration [0872] Adverse
events [0873] Concomitant medications [0874] b. Day 15 (.+-.1
day)
[0875] All assessments will be performed 30 minutes pre-infusion
unless otherwise specified. [0876] Urine pregnancy test for women
of childbearing potential (including those who have had a tubal
ligation) [0877] Vital signs (heart rate, blood pressure, and
temperature): pre-infusion, during infusion (every 15 minutes for 1
hour, and then every 30 minutes until end of infusion), and
post-infusion (every 30 minutes for 1 hour post-infusion) [0878]
Central laboratory assessments [0879] Hematology/CBC (within 30
minutes pre- and post-infusion of rituximab) [0880] Serum
chemistries [0881] Urinalysis [0882] C3, C4 [0883] Pharmacokinetic
sample (30 minutes pre- and post-infusion) [0884]
Methylprednisolone administration [0885] Rituximab administration
[0886] Adverse events [0887] Concomitant medications [0888] c.
Weeks 4, 12, and 20 (Days 28, 84, and 140, respectively; .+-.3
days) [0889] Patient's Global Assessment of Disease Activity (VAS)
[0890] Patient's Assessment of Pain (VAS) [0891] HAQ [0892] FACIT-F
(Week 12 only) [0893] Joint evaluation [0894] Physician's Global
Assessment of Disease Activity (VAS) [0895] Urine pregnancy test
for women of childbearing potential (including those who have had a
tubal ligation) [0896] Central laboratory assessments [0897]
Hematology/CBC [0898] Serum chemistries [0899] Urinalysis [0900]
Expanded FACS (Week 12 only) [0901] CD19 B cells (Weeks 4 and 20
only) [0902] CRP [0903] C3, C4 (Week 4 only) [0904] Pharmacokinetic
sample [0905] Immunoglobulins [0906] ESR (local laboratory;
Westergren method) [0907] Optional research biomarker samples
(whole blood and serum samples) (Week 12 only) [0908] Adverse
events [0909] Concomitant medications [0910] d. Week 24 (Day 168
.+-.3 days) [0911] Patient's Global Assessment of Disease Activity
(VAS) [0912] Patient's Assessment of Pain (VAS) [0913] HAQ [0914]
FACIT-F [0915] SF-36 [0916] Joint evaluation [0917] Physician's
Global Assessment of Disease Activity (VAS) [0918] Calculate DAS
28-ESR using the joint count and Patient's Global Assessment of
Disease Activity (VAS) from this visit and the ESR from the
previous visit, if ESR result is not available [0919] Evaluate
subject's eligibility for retreatment based on the calculated DAS
28-ESR and laboratory results from the previous visit. [0920] If
the subject is eligible for retreatment, proceed to Retreatment Day
1 and complete assessments as specified. [0921] Urine pregnancy
test for women of childbearing potential (including those who have
had a tubal ligation) [0922] Central laboratory assessments [0923]
Hematology/CBC [0924] Serum chemistries [0925] Urinalysis [0926]
Expanded FACS [0927] CRP [0928] Immunoglobulins [0929] RF [0930]
Anti-CCP antibody [0931] Pharmacokinetic sample [0932] HACA sample
[0933] ESR (local laboratory; Westergren method) [0934] Optional
research biomarker samples (whole blood and serum samples) [0935]
Adverse events [0936] Concomitant medications [0937] e. Week 28
(Day 196 .+-.3 days) [0938] Patient's Global Assessment of Disease
Activity (VAS) [0939] Patient's Assessment of Pain (VAS) [0940] HAQ
[0941] Joint evaluation [0942] Physician's Global Assessment of
Disease Activity (VAS) [0943] Complete only if the subject has not
been retreated. Calculate DAS 28-ESR using the joint count and
Patient's Global Assessment of Disease Activity (VAS) from this
visit and the ESR from the previous visit, if ESR result is not
available. [0944] Evaluate subject's eligibility for retreatment
based on the calculated DAS 28-ESR and laboratory results from the
previous visit. [0945] If the subject is eligible for retreatment,
proceed to Retreatment Day 1 and complete assessments as specified.
[0946] Urine pregnancy test for women of childbearing potential
(including those who have had a tubal ligation) [0947] Central
laboratory assessment [0948] Hematology/CBC [0949] Serum
chemistries [0950] Urinalysis [0951] CD19 B cells [0952] CRP [0953]
Immunoglobulins [0954] Pharmacokinetic sample [0955] ESR (local
laboratory; Westergren method) [0956] Adverse events [0957]
Concomitant medications [0958] f. Weeks 32, 40, and 44 (Days 224,
280, and 308, respectively; .+-.3 days) [0959] Patient's Global
Assessment of Disease Activity (VAS) [0960] Patient's Assessment of
Pain (VAS) [0961] HAQ [0962] FACIT-F (Week 32 only) [0963] Joint
evaluation [0964] Physician's Global Assessment of Disease Activity
(VAS) [0965] Complete only if the subject has not been retreated.
Calculate DAS 28-ESR using the joint count and Patient's Global
Assessment of Disease Activity (VAS) from this visit and the ESR
from the previous visit, if ESR result is not available (Weeks 32
and 40 only) [0966] Evaluate subject's eligibility for retreatment
based on the calculated DAS 28-ESR and laboratory results from the
previous visit. [0967] If the subject is eligible for retreatment,
proceed to Retreatment Day 1 and complete assessments as specified.
[0968] Urine pregnancy test for women of childbearing potential
(including those who have had a tubal ligation) [0969] Central
laboratory assessments [0970] Hematology/CBC [0971] Serum
chemistries [0972] Urinalysis [0973] Expanded FACS (perform only if
subject has not been retreated; Week 44 only) [0974] CD19 B cells
[0975] Immunoglobulins [0976] CRP [0977] Pharmacokinetic sample
[0978] ESR (local laboratory; Westergren method) [0979] Adverse
events [0980] Concomitant medications [0981] g. Week 48 (Day 336
.+-.3 days) [0982] Patient's Global Assessment of Disease Activity
(VAS) [0983] Patient's Assessment of Pain (VAS) [0984] HAQ [0985]
FACIT-F [0986] SF-36 [0987] Joint evaluation [0988] Physician's
Global Assessment of Disease Activity (VAS) [0989] Urine pregnancy
test for women of childbearing potential (including those who have
had a tubal ligation) [0990] Central laboratory assessments [0991]
Hematology/CBC [0992] Serum chemistries [0993] Urinalysis [0994]
Expanded FACS [0995] CRP [0996] Immunoglobulins [0997] RF [0998]
Anti-CCP antibody [0999] Pharmacokinetic sample [1000] HACA sample
[1001] ESR (local laboratory; Westergren method) [1002] Optional
research biomarker samples (whole blood and serum samples) [1003]
Adverse Events [1004] Concomitant Medications [1005] h. Week 60
(Day 420 .+-.7 days) [1006] Patient's Global Assessment of Disease
Activity (VAS) [1007] Patient's Assessment of Pain (VAS) [1008] HAQ
[1009] FACIT-F [1010] Joint evaluation [1011] Physician's Global
Assessment of Disease Activity (VAS) [1012] Urine pregnancy test
for women of childbearing potential (including those who have had a
tubal ligation) [1013] Central laboratory assessments [1014]
Hematology/CBC [1015] Serum chemistries [1016] Urinalysis [1017]
Expanded FACS [1018] CRP [1019] Immunoglobulins [1020] Optional
research biomarker sample (whole blood and serum samples) [1021]
ESR (local laboratory; Westergren method) [1022] Adverse events
[1023] Concomitant medications [1024] i. Week 72 (Day 504 .+-.7
days)
[1025] Subjects whose peripheral B-cell (CD19.sup.+) counts have
not recovered at the completion of this visit will enter the B-cell
follow-up period. B-cell recovery is defined as peripheral
CD19.sup.+counts that have returned to baseline values or the LLN,
whichever is lower. [1026] Patient's Global Assessment of Disease
Activity (VAS) [1027] Patient's Assessment of Pain (VAS) [1028] HAQ
[1029] FACIT-F [1030] SF-36 [1031] Vital signs (heart rate, blood
pressure, and temperature) [1032] Physical examination, including
weight p0 Joint evaluation [1033] Physician's Global Assessment of
Disease Activity (VAS) [1034] 12-Lead ECG [1035] Urine pregnancy
test for women of childbearing potential (including those who have
had a tubal ligation) [1036] Central laboratory assessments [1037]
Hematology/CBC [1038] Serum chemistries [1039] Urinalysis [1040]
Expanded FACS [1041] CRP [1042] Immunoglobulins [1043] RF [1044]
Anti-CCP antibody [1045] ESR (local laboratory; Westergren method)
[1046] Optional research biomarker samples (whole blood and serum
samples) [1047] Adverse events [1048] Concomitant medications
[1049] Subjects eligible for retreatment during Weeks 24-40 will be
randomized to receive an additional course of study drug. Subjects
should complete all Retreatment Day 1 assessments that have not
been performed during the qualifying visit. Assessments performed
from the qualifying visit do not need to be repeated.
[1050] If the infusion cannot be given on the same day as the
qualifying visit, the subject should return for the infusion within
72 hours of the qualifying visit.
[1051] After the retreatment course (Retreatment Days 1 and 15),
the subject should return to their next scheduled visit based on
the schedule of assessments from the point at which the subject
qualified for retreatment. For example, if a subject qualified for
retreatment at the Week 32 visit, after the retreatment course
(Retreatment Days 1 and 15), the subject's next scheduled visit
should be the Week 40 visit. [1052] a. Retreatment Day 1 (R1)
[1053] All assessments will be performed 30 minutes pre-infusion
unless otherwise specified. [1054] Randomization and assignment of
study drug through IVRS [1055] Patient's Global Assessment of
Disease Activity (VAS) [1056] Patient's Assessment of Pain (VAS)
[1057] HAQ [1058] FACIT-F [1059] SF-36 [1060] Vital signs (heart
rate, blood pressure, and temperature): pre-infusion, during
infusion (every 15 minutes for 1 hour, and then every 30 minutes
until the end of infusion), and post-infusion (every minutes for 1
hour post-infusion) [1061] Physical examination, including
measurement of body weight [1062] Joint evaluation [1063]
Physician's Global Assessment of Disease Activity (VAS) [1064]
Urine pregnancy test for women of childbearing potential (including
those who have had a tubal ligation) [1065] Central laboratory
assessments [1066] Hematology/CBC (within 30 minutes pre-and
post-infusion) [1067] Serum chemistries [1068] Urinalysis [1069]
CRP [1070] Expanded FACS (within 30 minutes pre- and post-infusion)
[1071] CD19 B cells (within 30 minutes pre- and post-infusion)
[1072] Immunoglobulins [1073] RF [1074] Anti-CCP antibody [1075]
C3, C4 [1076] Pharmacokinetic sample (30 minutes pre- and
post-infusion) [1077] HACA sample [1078] ESR (local laboratory;
Westergren method) [1079] Optional research biomarker samples
(whole blood and serum samples) [1080] Methylprednisolone
administration [1081] Study drug administration [1082] Adverse
events [1083] Concomitant medications [1084] b. Retreatment Day 15
(R15; .+-.1 day)
[1085] All assessments will be performed 30 minutes pre-infusion
unless otherwise specified. [1086] Vital signs (heart rate, blood
pressure, and temperature): pre-infusion, during infusion (every 15
minutes for 1 hour, and then every 30 minutes until the end of
infusion), and post-infusion (every minutes for 1 hour
post-infusion) [1087] Urine pregnancy test for women of
childbearing potential (including those who have had a tubal
ligation) [1088] Central laboratory assessments [1089]
Hematology/CBC (within 30 minutes pre-and post-infusion) [1090]
Serum chemistries [1091] Urinalysis [1092] C3, C4 [1093]
Pharmacokinetic sample (30 minutes pre- and post-infusion) [1094]
Methylprednisolone administration [1095] Study drug administration
[1096] Adverse events [1097] Concomitant medications
[1098] Subjects who withdraw early from the treatment period for
any reason will be asked to return for an early treatment
withdrawal visit (up to 14 days after withdrawal) and then to
return at 4 weeks after the withdrawal visit and then every 12
weeks for at least 48 weeks from the time the subject withdraws.
The following assessments will be performed at the early treatment
withdrawal visit: [1099] Patient's Global Assessment of Disease
Activity (VAS) [1100] Patient's Assessment of Pain (VAS) [1101] HAQ
[1102] FACIT-F [1103] SF-36 [1104] Vital signs (heart rate, blood
pressure, and temperature) [1105] Physical examination, including
weight [1106] Joint evaluation [1107] Physician's Global Assessment
of Disease Activity (VAS) [1108] 12-Lead ECG [1109] Urine pregnancy
test for women of childbearing potential (including those who have
had a tubal ligation) [1110] Central laboratory assessments [1111]
Hematology/CBC [1112] Serum chemistries [1113] Urinalysis [1114]
CRP [1115] CD19 B cells [1116] Immunoglobulins [1117] RF [1118]
Anti-CCP antibody [1119] Pharmacokinetic sample [1120] HACA sample
[1121] Optional research biomarker sample (whole blood and serum
samples) [1122] ESR (local laboratory; Westergren method) [1123]
Adverse events [1124] Concomitant medications
[1125] Safety follow up (SFU) assessments will be performed at SFU
Weeks 4, 12, 24, 36, and 48 after early withdrawal or completion of
the treatment period as defined for the following subjects: [1126]
Subjects who were retreated during Weeks 24-40 and completed the
treatment period (i.e., the Week 72 visit) [1127] Subjects who
withdrew early from the treatment period
[1128] The following assessments will be performed at each SFU
visit: [1129] All adverse events up to week 4 [1130] Serious
adverse events and infectious adverse events after week 4 [1131]
Concomitant medications used to treat these events [1132] Central
laboratory assessments [1133] Hematology/CBC [1134] Immunoglobulins
[1135] Pharmacokinetic sample [1136] HACA sample [1137] CD19 B
cells
[1138] At the last SFU visit, subjects whose peripheral B-cell
(CD19.sup.+) counts have not recovered will enter the B-cell
follow-up period. B-cell recovery is defined as peripheral
CD19.sup.+counts that have returned to baseline values or the LLN,
whichever is lower. For all serious infectious adverse events
reported, CBC with differentials, quantitative Ig and CD19 counts
should be determined within 1 week of onset.
[1139] Subjects whose peripheral B-cell (CD19.sup.+) counts have
not recovered at the last protocol defined visit (Week 72 or at end
of SFU) will enter the B-Cell Follow-Up Period and return for study
visits every 12 weeks from last visit until B-cell recovery. B-cell
recovery is defined as peripheral CD19.sup.+counts that have
returned to baseline values or the LLN, whichever is lower.
[1140] The following evaluations and procedures will be performed
at each B-cell follow-up visit: [1141] Serious adverse events and
infectious adverse events [1142] Concomitant medications used to
treat these events [1143] Central laboratory assessments [1144]
Hematology/CBC [1145] Immunoglobulins [1146] Flow cytometry: B-cell
(CD19.sup.+) count [1147] For all serious infectious adverse events
reported, CBC withdifferentials, quantitative Ig and CD19 counts
should be determined within 1 week of onset.
[1148] Blood, serum, and urine samples will be obtained at
specified timepoints. Instructions for processing, storing, and
shipping samples to Genentech and the central clinical laboratory
will be provided in the laboratory manuals. Standard assays will be
used for hematology, serology, chemistry, serum .beta.-hCG, flow
cytometry, immunology, and urinalysis.
[1149] Rituximab pharmacokinetic ELISA measures the rituximab
levels in human serum samples. It uses affinity-purified polyclonal
goat anti-rituximab as the capture reagent and goat antibody to
mouse IgG F(ab).sub.2 conjugated to horseradish peroxidase (HRP) as
the detection reagent.
[1150] The anti-rituximab HACA ELISA is a bridging assay, which
uses rituximab as the capture reagent and biotinylated rituximab
and streptavidin-HRP for detection. The assay uses a calibrator
curve prepared with affinity-purified polyclonal goat antibodies to
rituximab and is confirmed by immunodepletion with rituximab.
Results from this assay will be reported relative to this
polyclonal antibody in terms of relative units (RU)/mL.
[1151] Subjects may withdraw or be discontinued from the treatment
period at any time, but should return to the study center within 14
days for an early treatment withdrawal visit, SFU and potentially
B-cell follow-up. Subjects who have discontinued from the treatment
period will be followed for safety assessments. Every effort should
be made to obtain these assessments for subjects who discontinue
early. The primary reason for early discontinuation must be
recorded on the appropriate CRF page.
[1152] Reasons for subject discontinuation by the investigator
include, but are not limited to, the following: [1153] Voluntary
withdrawal of consent [1154] Any medical condition that may
jeopardize the subject's safety if he or she continues in the
study, as determined by the investigator [1155] Investigator
determination that it is not in the subject's best interest to
continue
[1156] Subjects who have a positive urine or serum pregnancy test
at any time during the study.
[1157] Subjects who withdraw early will not be replaced. [1158]
Genentech has the right to terminate this study at any time.
Reasons for terminating the study may include, but are not limited
to, the following: [1159] The incidence or severity of adverse
events in this or other studies indicates a potential health hazard
to subjects. [1160] Subject enrollment is unsatisfactory. [1161]
Data recording is inaccurate or incomplete.
[1162] This study is designed to evaluate the efficacy of one
course of retreatment with rituximab or placebo for eligible
subjects and the safety of rituximab treatment in subjects with
active RA who are receiving MTX and who had a previous or current
inadequate response to one or more anti-TNF therapies. The
eligibility of a subject for retreatment is based on the DAS 28-ESR
remission criterion (DAS 28-ESR .gtoreq.2.6). Approximately 555
subjects will be enrolled. The study is open-label for the first
course of treatment (with rituximab) and blinded for subjects
eligible for a retreatment course (study drug). During Weeks 24-40,
subjects who meet the retreatment criteria will be randomized to a
2:1 ratio to either rituximab or placebo.
[1163] Unless otherwise specified, all statistical tests are two
sided and will be performed at the .quadrature.=0.05 level of
significance.
[1164] The primary endpoint is the proportion of subjects with an
ACR20 response at Week 48 relative to baseline (Day 1). The
analysis of unblinded data will commence once the Week 48
assessments from all subjects are in the database and the database
is cleaned and frozen.
[1165] For the open-label treatment segment with rituximab (first
24 weeks of the study), the number of subjects enrolled will be
tabulated by center. For subjects who discontinue from the
open-label treatment segment, reasons for discontinuation will be
summarized. Key eligibility criteria violations, other major
protocol deviations, and the number of subjects who complete each
scheduled dose will be summarized.
[1166] For the blinded retreatment segment with study drug, the
number of subjects randomized will be tabulated by center and
treatment group. The disposition of subjects will be summarized by
treatment group with respect to subject randomization, treatment,
and completion of the study. Key eligibility criteria violations,
other major protocol deviations, and the number of subjects who
complete each scheduled dose will be summarized by treatment group.
For subjects who discontinue early from the placebo-controlled,
retreatment segment, reasons for discontinuation will be summarized
and listed by treatment group.
[1167] Baseline of the treatment groups will be assessed for
comparability with respect to demographics (i.e., age, sex,
race/ethnicity) and baseline characteristics (e.g., body weight,
duration of RA, baseline RF status, baseline SJC/TJC, baseline DAS
28-ESR, and number of prior TNF therapies). The baseline value of
any variable will be defined as the last available value prior to
the first administration of rituximab (Day 1).
[1168] Safety will be assessed through the summary of adverse
events, deaths, laboratory test results, vital signs, and HACAs.
These summaries will be produced as overall summary for the
open-label treatment segment and by treatment group for the
placebo-controlled, retreatment segment. Safety analyses will be
based on subjects who received any amount of study drug. Subjects
will be analyzed according to the actual treatment received.
[1169] The following safety summaries will be included in the
safety analyses.
[1170] Verbatim descriptions of treatment-emergent adverse events
will be mapped to MedDRA thesaurus terms. Adverse events will be
tabulated according to system organ class, treatment arm, and NCI
CTCAE grade. Adverse event tabulations for serious adverse events,
infection-related adverse events, rituximab-related infusion
reactions, and adverse events leading to study drug discontinuation
will be based on all treated subjects and tabulated following the
first infusion of the initial rituximab course and the retreatment
course.
[1171] In addition, serious adverse events will be summarized as
incidence per 100 patient-years following the first infusion of the
initial rituximab course and retreatment course as well as for the
entire observation period.
[1172] Subject deaths and primary cause of death will be listed
and/or summarized.
[1173] Descriptive summaries of laboratory values and changes from
baseline throughout the study will be provided. The proportion of
subjects experiencing treat-emergent laboratory abnormalities will
be summarized.
[1174] Descriptive summaries of vital signs and changes from
baseline throughout the study will be provided by treatment
group.
[1175] The proportion of subjects with a measurable antibody
response to rituximab will be summarized.
[1176] The intent-to-treat (ITT) population consists of all
subjects who are randomized into the placebo-controlled retreatment
segment and receive any study drug. The ITT population is the
primary analysis population for the primary and secondary
endpoints. Subjects will be analyzed according to their randomized
treatment.
[1177] The primary endpoint is the proportion of retreated subjects
with an ACR20 response at Week 48 compared with baseline (Day
1).
[1178] To achieve an ACR20 requires an 20% improvement compared
with baseline in both TJCs and SJCs as well as an 20% improvement
in three of five additional measurements as follows: [1179]
Physician's Global Assessment of disease activity [1180] Patient's
Global Assessment of Disease Activity (VAS) [1181] Patient's
Assessment of Pain (VAS) [1182] HAQ [1183] Acute phase reactant
(CRP or ESR)
[1184] CRP will be used in the calculation of ACR20. If CRP is
missing or not performed, ESR will be used.
[1185] The primary analysis of the difference in ACR20 response
rates between placebo retreatment and rituximab retreatment arm
will be presented using the Cochran-Mantel Haenszel test statistic,
stratified by baseline RF status. Results will be summarized
descriptively by treatment group and expressed as proportions, with
the corresponding adjusted 95% confidence intervals (CIs) of the
difference between response rates and p-values.
[1186] ACR20 response rates will also be analyzed using logistic
regression and testing for an association between ACR20 response at
Week 48 and treatment arm while controlling for baseline RF status
using a logistic regression model. The parameter estimates from the
model will be examined by tabulating the standard errors, Wald
statistics, and odds ratios with the corresponding 95% CIs and
p-values.
[1187] Sensitivity analyses will also be performed adjusting for
potential explanatory terms for ACR20 response (e.g., baseline
SJC).
[1188] The secondary endpoints will be analyzed as follows: [1189]
Proportion of subjects with ACR50 and ACR70 response at Week 48
will be analyzed in the same manner as specified for the primary
endpoint. [1190] Change in DAS 28-ESR from baseline to Week 48 will
be assessed using an analysis of variance (ANOVA) model, with
placebo/rituximab retreatment group and baseline RF status as
explanatory terms in the model. [1191] Ordered category of ACR
response (ACR70 responders, ACR50-70 responders, ACR20-50
responders, and ACR20 non-responders) at Week 48 will be analyzed
using the cumulative logits model, stratified by baseline RF
status. The parameter estimates from the model will be examined by
tabulating the standard errors, Wald statistics, and odds ratios
with the corresponding 95% CIs and p-values. [1192] EULAR response
rates (good or moderate) at Week 48 will be analyzed in the same
manner as specified for the primary endpoint. [1193] Change in ACR
core set (SJC, TJC, HAQ, patient's and physician's global
assessments, pain, CRP, and ESR) from baseline to Week 48 will be
analyzed in an ANOVA model, with placebo/rituximab retreatment
group and baseline RF status as explanatory terms in the model.
[1194] Week 48 ACRn and AUC of ACRn at week 48 will be assessed
using an analysis of variance (ANOVA) model, with placebo/rituximab
retreatment group and baseline RF status as explanatory terms in
the model. [1195] Change in SF-36 subscale and summary scores from
baseline to Week 48 will be reported for the eight domain scores
and the mental and physical component scores using analyzed in an
ANOVA model, with placebo/rituximab retreatment group and baseline
RF status as explanatory terms in the model. In addition, the
mental and physical component scores will be further categorized
and analyzed. [1196] Change in FACIT-F assessment from baseline to
Week 48 will be analyzed using analyzed in an ANOVA model, with
placebo/rituximab retreatment group and baseline RF status as
explanatory terms in the model. [1197] Proportion of subjects
achieving DAS 28-ESR remission (DAS 28-ESR <2.6) at Week 48 will
be analyzed in the same manner as specified for the primary
endpoint. [1198] Proportion of subjects achieving DAS 28-ESR low
disease (DAS 28-ESR .ltoreq.3.2) at Week 48 will be analyzed in the
same manner as specified for the primary endpoint.
[1199] Pharmacokinetic (PK) parameters derived from serum
concentrations of rituximab, including maximum serum concentration
(C.sub.max), time of maximum serum concentration (T.sub.max), area
under the concentration-time curve (AUC), systemic clearance (CL),
volume of distribution (V) and half-life (t.sup.1/2), will be
estimated for all subjects using population PK model. Due to the
sparse sampling, the distribution phase may not be well
characterized. PK data from this study will be combined with data
from other studies for population PK analysis.
[1200] For the population PK analysis, overall mean PK parameters
will be estimated for the entire study population along with
estimates of intra- and inter-subject variance and an estimate of
random error. Individual subject parameter estimates will be
computed using post hoc analysis procedures. A prospective analysis
plan will be prepared and the population PK analysis will be
presented in a separate report from the clinical study report.
[1201] The population PK analyses will include an exploratory
analysis to identify baseline covariates that affect the
pharmacokinetics of rituximab in this patient population. Baseline
covariates to be examined will include demographics and other
subject characteristics, such as disease severity and selected
laboratory measures.
[1202] Data will be summarized using descriptive statistics,
including mean, standard deviation, geometric mean, coefficient of
variation, median, and range.
[1203] Potential pharmacodynamic markers from blood samples,
including B-cell counts, quantitative Ig levels, lymphocyte
subtypes, and HACA concentration, will be summarized descriptively.
For these analyses, the baseline values measured from the Day 1
predose sample will be used to calculate the change from baseline
at each sampling timepoint.
[1204] Exploratory analyses will be performed to assess the
possible relationship between pharmacodynamic markers, PK measures,
and clinical response and will be specified in the Statistical
Analysis Plan.
[1205] Approximately 555 subjects will be enrolled. Assuming a
dropout rate of up to 25% during the open-label treatment segment
and up to 10% subjects who achieve DAS 28-ESR remission at Week 24
and are not retreated, with a 2:1 randomization ratio, this sample
size will have 80% power to detect a 16% difference in ACR20
response rates from baseline to Week 48 between the rituximab
retreatment group (50% ACR20 responders) and placebo group (34%
ACR20 responders) using Fisher's exact test.
[1206] Safety assessments will consist of monitoring and recording
protocol-defined adverse events (AEs) and serious adverse events
(SAEs); measurement of protocol-specified laboratory (hematology,
clinical chemistry, and urinalysis) variables; measurement of
protocol-specified vital signs; and other protocol-specified tests
that are deemed critical to the safety evaluation of the study
drug.
[1207] To monitor infusion-related reactions, vital signs will be
obtained immediately pre-infusion, every 15 minutes for the first
hour during the infusion, then every 30 minutes for the remainder
of the infusion, and every 30 minutes for one hour post-infusion,
on days of study drug administration. Additional readings may be
obtained in the event of an infusion-related reaction (e.g.,
hypotension and/or fever).
[1208] An AE is any unfavorable and unintended sign, symptom, or
disease temporally associated with the use of an investigational
(medicinal) product or other protocol-imposed intervention,
regardless of attribution.
[1209] This includes the following: [1210] AEs not previously
observed in the subject that emerge during the protocol-specified
AE reporting period, including signs or symptoms associated with RA
that were not present prior to the AE reporting period [1211]
Complications that occur as a result of protocol-mandated
interventions (e.g., invasive procedures such as biopsies) [1212]
If applicable, AEs that occur prior to assignment of study
treatment associated with medication washout, no treatment run-in,
or other protocol-mandated intervention [1213] Preexisting medical
conditions (other than the condition being studied) judged by the
investigator to have worsened in severity or frequency or changed
in character during the protocol-specified AE reporting period
[1214] An AE should be classified as an SAE if it meets the
following criteria: [1215] It results in death (i.e., the AE
actually causes or leads to death). [1216] It is life threatening
(i.e., the AE, in the view of the investigator, places the subject
at immediate risk of death. It does not include an AE that, had it
occurred in a more severe form, might have caused death.). [1217]
It requires or prolongs inpatient hospitalization. [1218] It
results in persistto have worsened in severity or frequency or
changed in character during the protocol-specified AE reporting
period
[1219] An AE should be classified as an SAE if it meets the
following criteria: [1220] It results in death (i.e., the AE
actually causes or leads to death). [1221] It is life threatening
(i.e., the AE, in the view of the investigator, places the subject
at immediate risk of death. It does not include an AE that, had it
occurred in a more severe form, might have caused death.). [1222]
It requires or prolongs inpatient hospitalization. [1223] It
results in persistent or significant disability/incapacity (i.e.,
the AE results in substantial disruption of the subject's ability
to conduct normal life functions). [1224] It results in a
congenital anomaly/birth defect in a neonate/infant born to a
mother exposed to the investigational product. [1225] It is
considered a significant medical event by the investigator based on
medical judgment (e.g., may jeopardize the subject or may require
medical/surgical intervention to prevent one of the outcomes listed
above).
[1226] All AEs that do not meet any of the criteria for serious
should be regarded as nonserious AEs.
[1227] The terms "severe" and "serious" are not synonymous.
Severity (or intensity) refers to the grade of a specific AE, e.g.,
mild (Grade 1), moderate (Grade 2), or severe (Grade 3) myocardial
infarction. "Serious" is a regulatory definition and is based on
subject or event outcome or action criteria usually associated with
events that pose a threat to a subject's life or functioning.
Seriousness (not severity) serves as the guide for defining
regulatory reporting obligations from the Sponsor to applicable
regulatory authorities.
[1228] Severity and seriousness should be independently assessed
when recording AEs and SAEs on the CRF.
[1229] Investigators will assess the occurrence of AEs and SAEs at
all subject evaluation timepoints during the study. All AEs and
SAEs whether volunteered by the subject, discovered by study
personnel during questioning, or detected through physical
examination, laboratory test, or other means will be recorded in
the subject's medical record and on the appropriate AE or SAE CRF
page.
[1230] Each recorded AE or SAE will be described by its duration
(i.e., start and end dates), severity (see Table 2), regulatory
seriousness criteria if applicable, suspected relationship to the
investigational product (see following guidance), and actions
taken.
[1231] The AE grading (severity) scale found in the NCI CTCAE,
V3.0, will be used for AE reporting.
TABLE-US-00021 TABLE 2 Adverse Event Grading (Severity) Scale Grade
Severity Alternate Description .sup.a 1 Mild (apply event-specific
Transient or mild discomfort NCI CTCAE grading criteria) (<48
hours); no interference with the subject's daily activities; no
medical intervention/therapy required 2 Moderate (apply
event-specific Mild to moderate interference NCI CTCAE grading
criteria) with the subject's daily activities; no or minimal
medical intervention/therapy required 3 Severe (apply
event-specific Considerable interference with NCI CTCAE grading
criteria) the subject's daily activities; medical
intervention/therapy required; hospitalization possible 4 Very
severe, life threatening, Extreme limitation in activity; or
disabling (apply event-specific significant medical NCI CTCAE
grading criteria) intervention/therapy required, hospitalization
probable 5 Death related to AE Note: Regardless of severity, some
events may also meet regulatory serious criteria. Refer to
definitions of an SAE. .sup.a Use these alternative definitions for
Grade 1, 2, 3, and 4 events when the observed or reported AE is not
in the NCI CTCAE listing.
[1232] To ensure consistency of AE and SAE causality assessments,
investigators should apply the following general guideline: [1233]
Yes [1234] There is a plausible temporal relationship between the
onset of the AE and administration of the investigational product,
and the AE cannot be readily explained by the subject's clinical
state, intercurrent illness, or concomitant therapies; and/or the
AE follows a known pattern of response to the investigational
product; and/or the AE abates or resolves upon discontinuation of
the investigational product or dose reduction and, if applicable,
reappears upon re-challenge. [1235] No [1236] Evidence exists that
the AE has an etiology other than the investigational product
(e.g., preexisting medical condition, underlying disease,
intercurrent illness, or concomitant medication); and/or the AE has
no plausible temporal relationship to administration of the
investigational product (e.g., cancer diagnosed 2 days after first
dose of study drug).
[1237] Note: The investigator's assessment of causality for
individual AE reports is part of the study documentation process.
Regardless of the "Yes" or "No" causality assessment for individual
AE reports, the Sponsor will promptly evaluate all reported SAEs
against cumulative product experience to identify and expeditiously
communicate possible new safety findings to investigators and
applicable regulatory authorities.
[1238] RA should be recorded as an AE or SAE only if judged by the
investigator to have unexpectedly worsened in severity and/or
frequency or changed in nature any time during the study. When
recording an unanticipated worsening of rheumatoid arthritis on an
AE or SAE CRF page, it is important to convey the concept that the
condition has changed by including applicable descriptors (e.g.,
"accelerated rheumatoid arthritis").
[1239] It is expected that re-treatment under the protocol herein
(or with a different CD20 antibody) will be effective in preventing
or slowing down the progression in structural joint damage and
erosion caused by RA. It is contemplated that efficacious results
would occur if MTX is not employed, i.e., monotherapy with the CD20
antibody is used.
EXAMPLE 4
Study of Efficacy of Rituximab in Patients with Psoriatic
Arthritis
[1240] The protocol in Example 2 is followed except that the
patients are treated for joint damage caused by psoriatic
arthritis. It is expected that similar results will be observed
(using rituximab or a humanized 2H7 antibody) as for rheumatoid
arthritis, i.e., that progression in structural joint damage will
be prevented upon a first dose of CD20 antibody (with or without
methotrexate depending on , e.g., dosages as can be adjusted
appropriately) at at least about one month from baseline or start
of treatment, preferably at least 24 weeks from baseline, more
preferably at least 52 weeks from baseline, and at further time
points up to 104 weeks from baseline.
[1241] These rituximab-based regimens challenge the current
standard of care, and are expected to demonstrate improved net
clinical benefit, with the primary objective to prevent progression
in joint damage. These results are expected to be significantly
better than those of the control arm.
[1242] It is predicted and expected that administration of
rituximab or a humanized 2H7 to the subject in the scheduled
re-dosing protocol set forth in Example 2 will be effective in
preventing progression of structural joint damage at week 52 or
later. These results are expected to be significantly better than
those of the control.
[1243] It is also expected that at about week 48-54, another 1-g or
2-g dose of the CD20 antibody (e.g., rituximab or a humanized 2H7)
given all at once or spread out over about 14-16 days in 0.5-gram
or 1-gram amounts would be effective to treat joint damage for the
entire second year, with or without one or more second medicaments
such as immunosuppressive agents. Thus, the CD20 antibody would be
administered initially within about the 2-week time period,
followed by another treatment at about 4-8 months, followed by
another treatment at about one year from initial treatment
(measured from the time any one of the doses was given), followed
by treatment at about two years from initial treatment, with
expected success, in about one-half-gram or one-gram .times.2-4
dosing for each treatment, administered together, about weekly, or
about every other week over about two to four weeks. The results of
this treatment would be expected to be much better than those of
the control with placebo. This re-treatment protocol is expected to
be successfully used for several years with little or no adverse
effects.
EXAMPLE 5
Treatment Study of Efficacy of Rituximab in Patients with
Osteoarthritis and Crohn's Disease
[1244] It is expected that Example 2 results would be successful if
the patients have joint damage as a result of osteoarthritis or
Crohn's disease, whether rituximab or another CD20 antibody such as
humanized 2H7 is used, and whether an immunosuppressive agent is
used or not, with adjustment of doses as would be known to those
skilled in the art. Further, if such patients are initially treated
with CD20 antibody and then re-treated with such antibody about six
months or about one year after first being treated, otherwise using
the same dosing and other protocol of Example 2, it is expected
that they will continue to experience prevention of progression of
joint damage for an extended period of time.
[1245] It is also expected that rituximab or other CD20 antibody
will be at least as effective as the conventional treatment regimen
for induction and maintenance of joint damage remission, offering
substantial advantages over standard therapy by virtue of its
superior side-effect profile, e.g., much less toxic than steroids,
and better at restoring tolerance.
[1246] It is expected that the patients in the treatment arm will
tolerate rituximab infusions well and that their B cells will be
depleted swiftly. Other than methotrexate, if needed, no additional
immunosuppressive agents are expected to be necessary for induction
of remission and maintenance of sustained remission (6 months or
longer) in the CD20-antibody-treated patients.
Sequence CWU 1
1
471107PRTMus musculus 1Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu
Ser Ala Ser Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Arg Ala Ser
Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Ser
Ser Pro Lys Pro Trp Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly
Val Pro Ala Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser Arg Val Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr
Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg 100 1052213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu
Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp
Ser Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150 155
160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys 2103108PRTHomo
sapiens 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Asn Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Leu Pro Trp 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg 100 105410PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Arg Ala Ser Ser Ser Val
Ser Tyr Met His1 5 1057PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Ala Pro Ser Asn Leu Ala Ser1
569PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gln Gln Trp Ser Phe Asn Pro Pro Thr1 57122PRTMus
musculus 7Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro
Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Ser Tyr 20 25 30Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly
Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser
Tyr Asn Gln Lys Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95Ala Arg Val Val Tyr Tyr Ser
Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Thr Gly Thr Thr
Val Thr Val Ser Ser 115 1208452PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 8Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 4509119PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile
Ser Gly Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Thr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Gly Arg Val Gly Tyr Ser Leu Tyr Asp Tyr Trp Gly Gln
Gly 100 105 110Thr Leu Val Thr Val Ser Ser 1151010PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Gly
Tyr Thr Phe Thr Ser Tyr Asn Met His1 5 101117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Ala
Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10
15Gly1213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp
Val1 5 1013213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 13Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr 85 90 95Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105
110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205Asn Arg Gly
Glu Cys 21014452PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 14Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe
Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345
350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
Lys 45015452PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 15Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe
Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Ala
Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345
350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
Lys 45016285PRTHomo sapiens 16Met Asp Asp Ser Thr Glu Arg Glu Gln
Ser Arg Leu Thr Ser Cys Leu1 5 10 15Lys Lys Arg Glu Glu Met Lys Leu
Lys Glu Cys Val Ser Ile Leu Pro 20 25
30Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly Lys Leu Leu
35 40 45Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys Leu Thr Val
Val 50 55 60Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp Leu Ala Ser
Leu Arg65 70 75 80Ala Glu Leu Gln Gly His His Ala Glu Lys Leu Pro
Ala Gly Ala Gly 85 90 95Ala Pro Lys Ala Gly Leu Glu Glu Ala Pro Ala
Val Thr Ala Gly Leu 100 105 110Lys Ile Phe Glu Pro Pro Ala Pro Gly
Glu Gly Asn Ser Ser Gln Asn 115 120 125Ser Arg Asn Lys Arg Ala Val
Gln Gly Pro Glu Glu Thr Val Thr Gln 130 135 140Asp Cys Leu Gln Leu
Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys145 150 155 160Gly Ser
Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser 165 170
175Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr
180 185 190Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr
Ala Met 195 200 205Gly His Leu Ile Gln Arg Lys Lys Val His Val Phe
Gly Asp Glu Leu 210 215 220Ser Leu Val Thr Leu Phe Arg Cys Ile Gln
Asn Met Pro Glu Thr Leu225 230 235 240Pro Asn Asn Ser Cys Tyr Ser
Ala Gly Ile Ala Lys Leu Glu Glu Gly 245 250 255Asp Glu Leu Gln Leu
Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu 260 265 270Asp Gly Asp
Val Thr Phe Phe Gly Ala Leu Lys Leu Leu 275 280 28517309PRTMus
musculus 17Met Asp Glu Ser Ala Lys Thr Leu Pro Pro Pro Cys Leu Cys
Phe Cys1 5 10 15Ser Glu Lys Gly Glu Asp Met Lys Val Gly Tyr Asp Pro
Ile Thr Pro 20 25 30Gln Lys Glu Glu Gly Ala Trp Phe Gly Ile Cys Arg
Asp Gly Arg Leu 35 40 45Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser
Ser Ser Phe Thr Ala 50 55 60Met Ser Leu Tyr Gln Leu Ala Ala Leu Gln
Ala Asp Leu Met Asn Leu65 70 75 80Arg Met Glu Leu Gln Ser Tyr Arg
Gly Ser Ala Thr Pro Ala Ala Ala 85 90 95Gly Ala Pro Glu Leu Thr Ala
Gly Val Lys Leu Leu Thr Pro Ala Ala 100 105 110Pro Arg Pro His Asn
Ser Ser Arg Gly His Arg Asn Arg Arg Ala Phe 115 120 125Gln Gly Pro
Glu Glu Thr Glu Gln Asp Val Asp Leu Ser Ala Pro Pro 130 135 140Ala
Pro Cys Leu Pro Gly Cys Arg His Ser Gln His Asp Asp Asn Gly145 150
155 160Met Asn Leu Arg Asn Ile Ile Gln Asp Cys Leu Gln Leu Ile Ala
Asp 165 170 175Ser Asp Thr Pro Thr Ile Arg Lys Gly Thr Tyr Thr Phe
Val Pro Trp 180 185 190Leu Leu Ser Phe Lys Arg Gly Asn Ala Leu Glu
Glu Lys Glu Asn Lys 195 200 205Ile Val Val Arg Gln Thr Gly Tyr Phe
Phe Ile Tyr Ser Gln Val Leu 210 215 220Tyr Thr Asp Pro Ile Phe Ala
Met Gly His Val Ile Gln Arg Lys Lys225 230 235 240Val His Val Phe
Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys 245 250 255Ile Gln
Asn Met Pro Lys Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala 260 265
270Gly Ile Ala Arg Leu Glu Glu Gly Asp Glu Ile Gln Leu Ala Ile Pro
275 280 285Arg Glu Asn Ala Gln Ile Ser Arg Asn Gly Asp Asp Thr Phe
Phe Gly 290 295 300Ala Leu Lys Leu Leu3051817PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(1)Any amino acid except CysMOD_RES(3)..(3)Any
amino acid except CysMOD_RES(5)..(5)Any amino acid except
CysMOD_RES(7)..(12)Any amino acid except CysMOD_RES(14)..(15)Any
amino acid except CysMOD_RES(16)..(16)Leu, Phe, Ile or
ValMOD_RES(17)..(17)Any amino acid except Cyssee specification as
filed for detailed description of substitutions and preferred
embodiments 18Xaa Cys Xaa Asp Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Cys
Xaa Xaa Xaa1 5 10 15Xaa1917PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Glu Cys Phe Asp Leu Leu Val
Arg Ala Trp Val Pro Cys Ser Val Leu1 5 10 15Lys2017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Glu
Cys Phe Asp Leu Leu Val Arg His Trp Val Pro Cys Gly Leu Leu1 5 10
15Arg2117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Glu Cys Phe Asp Leu Leu Val Arg Arg Trp Val Pro
Cys Glu Met Leu1 5 10 15Gly2217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Glu Cys Phe Asp Leu Leu Val
Arg Ser Trp Val Pro Cys His Met Leu1 5 10 15Arg2317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Glu
Cys Phe Asp Leu Leu Val Arg His Trp Val Ala Cys Gly Leu Leu1 5 10
15Arg2416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Gln Cys Phe Asp Arg Leu Asn Ala Trp Val Pro Cys
Ser Val Leu Lys1 5 10 152517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(1)..(1)Any amino acid
except CysMOD_RES(3)..(3)Any amino acid except
CysMOD_RES(5)..(5)Any amino acid except CysMOD_RES(8)..(9)Any amino
acid except CysMOD_RES(14)..(15)Any amino acid except
CysMOD_RES(17)..(17)Any amino acid except Cyssee specification as
filed for detailed description of substitutions and preferred
embodiments 25Xaa Cys Xaa Asp Xaa Leu Val Xaa Xaa Trp Val Pro Cys
Xaa Xaa Leu1 5 10 15Xaa26184PRTHomo sapiens 26Met Arg Arg Gly Pro
Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro1 5 10 15Thr Pro Cys Val
Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His Cys 20 25 30Val Ala Cys
Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro Ala Gly Ala 35 40 45Ser Ser
Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln Glu Ser Val Gly 50 55 60Ala
Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro Gly Leu Leu Phe Gly65 70 75
80Ala Pro Ala Leu Leu Gly Leu Ala Leu Val Leu Ala Leu Val Leu Val
85 90 95Gly Leu Val Ser Trp Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala
Ser 100 105 110Ser Ala Glu Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu
Pro Leu Asp 115 120 125Lys Val Ile Ile Leu Ser Pro Gly Ile Ser Asp
Ala Thr Ala Pro Ala 130 135 140Trp Pro Pro Pro Gly Glu Asp Pro Gly
Thr Thr Pro Pro Gly His Ser145 150 155 160Val Pro Val Pro Ala Thr
Glu Leu Gly Ser Thr Glu Leu Val Thr Thr 165 170 175Lys Thr Ala Gly
Pro Glu Gln Gln 1802726PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Thr Pro Cys Val Pro Ala Glu
Cys Phe Asp Leu Leu Val Arg His Cys1 5 10 15Val Ala Cys Gly Leu Leu
Arg Thr Pro Arg 20 2528213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 28Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ala Phe Asn Pro Pro Thr
85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 21029452PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 29Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly Lys 45030213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 30Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Leu 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ala Phe Asn Pro Pro Thr
85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 21031452PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 31Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu 435 440 445Ser Pro Gly Lys 45032107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Leu 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu
Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp
Ala Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 10533122PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 33Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro
Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe
Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Val Val Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12034451PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly
Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser
Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val
Tyr Tyr Ser Ala Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Ala Ala
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly
4503510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(9)..(9)Met or Leu 35Arg Ala Ser Ser Ser
Val Ser Tyr Xaa His1 5 10369PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(4)..(4)Ser or Ala
36Gln Gln Trp Xaa Phe Asn Pro Pro Thr1 53717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(8)..(8)Asp or Ala 37Ala Ile Tyr Pro Gly Asn Gly Xaa
Thr Ser Tyr Asn Gln Lys Phe Lys1 5 10 15Gly3813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(6)..(6)Asn, Ala, Tyr, Trp or AspMOD_RES(7)..(7)Ser
or Arg 38Val Val Tyr Tyr Ser Xaa Xaa Tyr Trp Tyr Phe Asp Val1 5
1039107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Ser Ser Val Ser Tyr Leu 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser Asn Leu Ala Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Trp Ala Phe Asn Pro Pro Thr 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 10540122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
40Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr Ser Tyr Arg
Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 12041451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45042451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 42Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45043451PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 43Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile
Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly
Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Val Val Tyr Tyr Ser Tyr Arg Tyr Trp Tyr Phe Asp Val
Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu225 230 235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu 245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser 260 265 270His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Ala Leu Pro Ala Pro
325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 340 345 350Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val 355 360 365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 370 375 380Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro385 390 395 400Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly 45044107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr 35 40 45Ala Pro Ser
Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr
85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
10545122PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 45Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly
Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser
Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val
Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 12046356PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
46Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr Asn
Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val Tyr Tyr Ser Ala Ser
Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu225 230 235 240Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280
285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ala Thr
290 295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn305 310 315 320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 325 330 335Ile Ala Ala Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 340 345 350Val Tyr Thr Leu
35547451PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Asn Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Ala Ile Tyr Pro Gly Asn Gly
Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Phe Thr Ile Ser
Val Asp Lys Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Val
Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp 100 105 110Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr145 150 155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro 165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200 205His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210 215 220Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 260 265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr 290 295 300Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn305 310 315 320Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360
365Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro385 390 395 400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr 405 410 415Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val 420 425 430Met His Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445Ser Pro Gly 450
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References