WO2010075249A2 - A method for treating rheumatoid arthritis with b-cell antagonists - Google Patents

A method for treating rheumatoid arthritis with b-cell antagonists Download PDF

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WO2010075249A2
WO2010075249A2 PCT/US2009/068917 US2009068917W WO2010075249A2 WO 2010075249 A2 WO2010075249 A2 WO 2010075249A2 US 2009068917 W US2009068917 W US 2009068917W WO 2010075249 A2 WO2010075249 A2 WO 2010075249A2
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method
anti
antagonist
antibody
rheumatoid arthritis
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PCT/US2009/068917
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French (fr)
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WO2010075249A3 (en
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Michael J. Townsend
Zheng SU
Jay G. Wohlgemuth
Timothy W. Behrens
Preeti G. Lal
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Genentech, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Abstract

A method of treating rheumatoid arthritis in a patient is disclosed which comprises administering an effective amount of a B-cell antagaonist to the patient to treat the rheumatoid arthritis, provided that the rheumatoid arthritis is characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody in a sample from the patient. Further provided are kits and articles of manufacture useful for such methods.

Description

A METHOD FOR TREATING RHEUMATOID ARTHRITIS WITH B-CELL

ANTAGONISTS

Related Applications

This application claims priority under 35 USC 119(e) to U.S. Provisional Patent Application No. 61/203,442, filed 22 December 2008, the contents of which is incorporated herein by reference. Field of the Invention The present invention concerns methods for diagnosing and treating rheumatoid arthritis patients. In particular, the present invention is directed to methods for determining which patients will most benefit from treatment with B-cell antagonist therapies directed against B-cell surface markers or B-cell specific proliferation or survival factors, such as an antibody or immunoadhesin. Background of the Invention

Joint Destruction and Damage

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.

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-iil2 (2005)). Life expectancy is reduced by an average of 3-10 years (Alamanosa and Drosos, supra). Patients with a high titer of rheumatoid factor (RF) (approximately 80% of patients) have more aggressive disease (Bukhari et ah, 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)).

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(l):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 NFKB ligand (RANKL), and/or its soluble decoy receptor, osteoprotegerin (OPG), by osteoblast/stromal cells (Hossbauer et al, J. Bone Miner. Res., 15(1):2-12 (2000)). Tumor necrosis factor-alpha (TNF-α) 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-α 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-α. 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 fϊbroblast-like synoviocytes and macrophages that produce proinflammatory cytokines such as TNF-alpha and interleukin-1 (IL-I). 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.

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:Sl-6 (2003)). 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. 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)).

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.

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-I, lymphotoxin-alpha, interleukin-6 (IL-6), and interleukin-10 (IL-10) are amongst some of the cytokines that B cells may produce in the RA synovium.

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. Structural damage to joints is an important consequence of chronic synovial inflammation. Between 60% and 95% of patients with 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. 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., 43:495-505 (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)). 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 auranofm (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 ah, 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)).

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)).

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.

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.

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.

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:S204 (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)).

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.

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.

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.

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.

Etanercept is a fully human fusion protein that inhibits TNF and the subsequent 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 Qt 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. US 2007/0071747 discloses use of a TNF-alpha inhibitor for treatment of erosive polyarthritis.

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)). 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.

Further, reduction in radiographic progression in the hands and feet was observed in patients with early RA after receiving infliximab in combination with MTX(Van der Heijde et al, Annals Rheumatic Diseases 64:418-419 (2005)). Patients with earlyRA 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 MTX on radiographic progression in patients with early RA 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)). 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 Qt 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).

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 RA patients who had no clinical improvement, with a detailed subanalysis of data from the anti-TNF factor trial in RA 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 der Heijde 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 RA 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.

Autoimmune Disease Biomarkers

Autoantibodies are detected in a majority of patients with RA and predict more severe symptoms. The two major types of autoantibodies used clinically to create RA subsets are rheumatoid factor (RF), which is an immunoglobulin specific to the Fc region of IgG, and anti-cyclic citrullinated peptide (CCP) antibodies, which are antibodies directed against peptides in which arginine has been post-translationally modified to become citrulline (Schellekens et al, Arthritis Rheum., 43:155-163 (2000)). These autoantibodies are strongly correlated, but may represent distinct clinical subsets of RA.

Szodoray et al., Scandinavian J. of Immunol, 60:209-218 (2004) discloses the apoptotic effect of rituximab on peripheral blood B cells in RA, with the data suggesting that rituximab is less effective in RF-negative RA because B cells play a less significant role in RA pathogenesis in RF-negative patients. US 2005/0271658 discloses that anti-CD20 antibodies can be used in a subject at risk for experiencing one or more symptoms of RA, and further wherein the subject has abnormal levels of IgM RF antibodies directed against the Fc portion of IgG. DiFranco et al.,

"Relationship of rheumatoid factor isotype levels with joint lesions detected by magnetic resonance imaging in early rheumatoid arthritis" Rev. Rheum. Engl. Ed., 66(5):251-255 (1999) reported that quantitative RF isotype assays and magnetic resonance imaging evaluation of erosions of the hand and wrist may be useful for investigating patients with early RA.

Anti-CCP antibodies are highly specific for RA, can be detected years before the first clinical manifestations of RA (Rantapaa-Dahlqvist et al, Arthritis Rheum., 48:2741-2749 (2003)) and are reported to be a good predictor for the development of RA (van Gaalen et al., Arthritis Rheum., 50:709-715 (2004)). WO 2007/059188 discloses X-ray results regarding joint destruction in patients treated with anti-CD20 antibody. Tak et al. discloses the RF and anti-CCP markers in an abstract and poster entitled "Baseline autoantibody status (RF, Anti-CCP) and Clinical Response Following the First Treatment Course with Rituximab, poster 833 at ACR 2006. This publication showed that patients that lacked both of these autoantibodies had a lower response rate to rituximab.

WO 2005/085858 discloses a method of assessing RA by measuring anti-CCP and serum amyloid A (SAA). WO 2005/064307 and US 2007/0264673 assess RA by measuring anti-CCP and IL-6. WO 2007/000169 discloses a non-human mammalian disease model to test diseases associated with anti-CCP such as arthritis, e.g., RA. US 2006/263355 discloses treatment of bone disorders using an anti-CD20 antibody, wherein the change in anti-CCP, CRP, SlOO, and SAA serum levels suggests that a single, short course with rituximab has a profound effect on markers. WO 2005/029091 and US 2006/094056 provide methods to diagnose, treat, or evaluate inflammatory/autoimmune diseases such as RA by sampling fluids from a human with a suspected diagnosis for certain cytokines. CN 1796997 notes a kit for early RA diagnosis by detecting anti-CCP. US 2007/0148704 and WO 2007/039280 disclose use of anti-CCP and antinuclear antibodies as biomarkers in diagnosing RA. WO 2006/008183 discloses various biomarkers for RA. US 7244571 discloses a method for inducing a pro-asthma/pro-inflammatory-like state in a cell comprising contacting the cell with one or more cytokines. US 2007/0128626 discloses assessing response to anti-CD20 therapy by genotyping CIq components, e.g., the structure of the complement protein CIqA.

Scientific literature on anti-CCPs and/or RFs includes Li et al, "Inferring causal relationships among intermediate phenotypes and biomarkers: a case study of rheumatoid arthritis" Bioinformatics, 22(12): 1503-1507 (2006); Russell et al, "The role of anti-cyclic citrullinated peptide antibodies in predicting progression of palindromic rheumatism to rheumatoid arthritis" J. Rheumatol, 33(7): 1240-1242 (2006); Ota, "Immunologic laboratory testing in clinical practice for rheumatoid arthritis" Rinsho byori. Jap J. Clin. Pathol, 54(8):861-868 (2006); Avouac et al, "Diagnostic and predictive value of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a systematic literature review" Ann. Rheum. Dis., 65(7):845-851 (2006); Mewar and Wilson, "Autoantibodies in rheumatoid arthritis: a review" Biomed. & Pharmacother., 60(10):648-655 (2006); Nielen et al, "Simultaneous development of acute phase response and autoantibodies in preclinical rheumatoid arthritis" Ann. Rheum. Dis., 65(4):535-537 (2006); Nielen et al "Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors" Arthr. Rheum., 50:380-386 (2004)); Quinn et al, "Anti-CCP antibodies measured at disease onset help identify seronegative rheumatoid arthritis and predict radiological and functional outcome" Rheumatol, 45(4):478-480 (2006); Montano-Loza et al, "Frequency and significance of antibodies to cyclic citrullinated peptide in type 1 autoimmune hepatitis" Autoimmunity, 39(4):341-348 (2006); Griffiths, "Musculoskeletal disorders: an introduction" Medicine, 34(9):331-332 (2006); del VaI del Amo et al, "Anti-cyclic citrullinated peptide antibody in rheumatoid arthritis: relation with disease aggressiveness" Clin. Exper. Rheum., 24(3):281-286 (2006); Schulze-Koops and Manger, "Diagnostic and prognostic significance of antibodies against citrullinated peptides" Deutsche Medizinische Wochenschrift (1946), 131(6):269-271 (2006); Matsui, "Antibodies to citrullinated proteins in rheumatoid arthritis" Jap. J. Clin. Immunol, 29(2):49-56 (2006); van Venrooij et al, "Autoantibodies to citrullinated antigens in (early) rheumatoid arthritis" Autoimmun. Rev., 6(1):37-41 (2006); Saleem et al, "Biomarkers: Strategies to predict outcome of rheumatoid arthritis" Drug Discovery Today: Therapeutic Strategies, 3(1): 11-16 (2006); Meyer et al, "Serial determination of cyclic citrullinated peptide autoantibodies predicted five-year radiological outcomes in a prospective cohort of patients with early rheumatoid arthritis" Arthr. Res. & Ther, 8(2):R40 (2006); Johansson et al, "PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in combination strongly predicts future onset of rheumatoid arthritis and has a specificity of 100% for the disease" Arthr. Res. & Ther., 8(1):R19 (2006); de Seny et al, "Discovery of new rheumatoid arthritis biomarkers using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry ProteinChip approach" Arthr. & Rheum., 52(12):3801-12 (2005); Radstake et al, "Correlation of rheumatoid arthritis severity with the genetic functional variants and circulating levels of macrophage migration inhibitory factor" Arthr. & Rheum., 52(10):3020-3029 (2005); Sihvonen et al., "The predictive value of rheumatoid factor isotypes, anti-cyclic citrullinated peptide antibodies, and anti-neutrophil cytoplasmic antibodies for mortality in patients with rheumatoid arthritis" J. Rheumat, 32(11):2089-2094 (2005); Nell et al., "Autoantibody profiling as early diagnostic and prognostic tool for rheumatoid arthritis" Ann. Rheum. Dis., 64(12):1731-1736 (2005); van Gaalen et al, "A comparison of the diagnostic accuracy and prognostic value of the first and second anti-cyclic citrullinated peptides (CCPl and CCP2) autoantibody tests for rheumatoid arthritis" Ann. Rheum. Dis., 64(10):1510-1512 (2005); Nielen et al, "Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and prognostic value in early arthritis" Ann. Rheum. Dis., 64(8): 1199-1204 (2005); Mimori, "Clinical significance of anti-CCP antibodies in rheumatoid arthritis" Internal Medicine (Tokyo, Japan), 44(11):1122-1126 (2005); Fusconi et al, "Anti-cyclic citrullinated peptide antibodies in type 1 autoimmune hepatitis" Alimentary Pharmacol. & Therapeut., 22(10):951- 955 (2005); Hiura et al, "The examination of rheumatoid factor and other serum markers in rheumatoid arthritis" Yakugaku Zasshi, 125(11):881-887 (2005); Olivieri et al, "Management issues with elderly-onset rheumatoid arthritis: an update" Drugs & Aging, 22(10):809-822 (2005); Dai et al, "Significance of detecting anti-cyclic citrullinated peptide antibody in diagnosis of rheumatoid arthritis" Guangdong Yixue, 26(6):796-797 (2005); Yang et al, "Study on correlation between anti-cyclic citrullinated peptide antibody and erosion of bone in patients with rheumatoid arthritis" Huaxi Yixue 20(4):658-660 (2005); Boire et al, "Anti-Sa antibodies and antibodies against cyclic citrullinated peptide are not equivalent as predictors of severe outcomes in patients with recent-onset polyarthritis" Arthr. Res. & Ther.," 7(3):R592-603 (2005); Lienesch et al, "Absence of cyclic citrullinated peptide antibody in nonarthritic patients with chronic hepatitis C infection" J. Rheumatol., 32(3):489-493 (2005); Nakamura et al, "Clinical significance of anti-citrullinated peptide antibody in Japanese patients with established rheumatoid arthritis" Scandin. J. Rheumatol, 34(6):489-490 (2005); Yang et al, "Clinical utility of EDRA/CPA in diagnosis of rheumatoid arthritis" Tianjin Yiyao 7>7>(1)A22-A2A (2005); Momohara and Yamanaka, "Biomarker," Igaku to Yakugaku, 53(4):413-425 (2005); Healy and Helliwell, "Classification of the spondyloarthropathies" Curr. Opin. Rheumatol, 17(4):395-399 (2005); Vallbracht and Helmke, "Additional diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis" Autoimmun. Rev., 4(6):389-394 (2005); Kwok et al, "Anti-cyclic citrullinated peptide: diagnostic and prognostic values in juvenile idiopathic arthritis and rheumatoid arthritis in a Chinese population" Scandin. J. Rheumatol, 34(5):359-366 (2005); Senkpiehl et al, "HLA-DRBl and anti-cyclic citrullinated peptide antibody production in rheumatoid arthritis" Intern. Arch. Allergy & Immunol, 137(4):315-318 (2005); Greiner et al, "Association of anti-cyclic citrullinated peptide antibodies, anti-citrulline antibodies, and IgM and IgA rheumatoid factors with serological parameters of disease activity in rheumatoid arthritis" Ann. NY Acad. ScL, 1050(Autoimmunity):295-303 (2005); Raza et al, "Predictive value of antibodies to cyclic citrullinated peptide in patients with very early inflammatory arthritis" J. Rheum., 32(2):231-238 (2005); Tampoia et al,

"Proteomic: new advances in the diagnosis of rheumatoid arthritis" Clinica Chimica Acta; Intern. J. Clin. Chem., 357 (2):219-225 (2005); van Leeuwen et al, "Prognostic significance of anti-CCP in early RA, relationship with shared epitope and rheumatoid factor" Annals of the Rheumatic Diseases, 64(Suppl. 3):210 (2005); Annual European Congress of Rheumatology, Vienna, AT, June 8-11, 2005; Chaiamnuay and Bridges, "The role of B cells and autoantibodies in rheumatoid arthritis" Pathophysiology: The Official Journal of the International Society for Pathophysiology^^ , 12(3):203-216 (2005); Lindqvist et al, "Prognostic laboratory markers of joint damage in rheumatoid arthritis" Ann. Rheum. Dis., 64(2): 196-201 (2005); Vencovsky et al, "Antibodies against citrullinated proteins in rheumatoid arthritis" Ceska Revmatologie, 13(4): 164-175 (2005); Egerer et al, "A new powerful marker for the diagnosis and prognosis of rheumatoid arthritis- Anti-C VM (Anti-Citrullinated vimentin mutated) antibodies" Arthr. & Rheum., 52(9, Suppl. S):S118 (2005); 69th Annual Scientific Meeting of the American-College-of-Rheumatology/40th Annual Scientific Meeting of the Association-of-Rheumato logy-Health-Professionals, San Diego, CA, Nov. 12- 17, 2005; Kuribayashy et al, "Analysis of PADI4 gene polymorphisms in rheumatoid arthritis" J. Pharmacol. ScL, 97(No. Suppl. 1):86P (2005); 78th Annual Meeting of the Japanese-Pharmacological-Society, Yokohama, JP, March 22-24, 2005; Sedova et al., "Antibodies against cyclic citrullinated peptide (anti-CCP) in serum and synovial fluid from patients with rheumatoid arthritis and osteoarthritis" Ceska Revmatologie, 13(3):79-83 (2005); Boeckelmann et al., "Anti-cyclic citrullinated peptide antibodies occurring in psoriasis patients without arthritis" J. Invest. Derm., 125 (3, Suppl. S): A72 (2005); 35th Annual Meeting of the European-Society-for-Dermatological- Research, Tubingen, DE, Sep. 22-24 2005; Dubrous et al., "Value of anti-cyclic citrullinated peptides antibodies in comparison with rheumatoid factor for rheumatoid arthritis diagnosis" Pathologie Biologie 53(2):63-67 (2005); Yamato et al., "Evaluation of basic properties of reagents for measuring anti-CCP (cyclic citrullinated peptide) antibody" lryo to Kensa Kiki-Shiyaku 28(l):59-63 (2005); Vincent et al., "Autoantibodies to citrullinated proteins: ACPA" Autoimmunity,

38(1): 17-24 (2005); Hitchon et al., "A distinct multicytokine profile is associated with anti-cyclical citrullinated peptide antibodies in patients with early untreated inflammatory arthritis" J. Rheumatol, 31(12):2336-2346 (2004); Low et al., "Determination of anti-cyclic citrullinated peptide antibodies in the sera of patients with juvenile idiopathic arthritis" J. Rheumatol., 31(9):1829-1833 (2004); Forslind et al., "Prediction of radiological outcome in early rheumatoid arthritis in clinical practice: role of antibodies to citrullinated peptides (anti-CCP)" Ann. Rheum. Dis., 63(9): 1090-1095 (2004); Kastbom et al., "Anti-CCP antibody test predicts the disease course during 3 years in early rheumatoid arthritis (the Swedish TIRA project)," Ann. Rheum. Dis., 63(9): 1085-1089 (2004); Vallbracht et al., "Diagnostic and clinical value of anti-cyclic citrullinated peptide antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis" Ann. Rheum. Dis., 63(9): 1079-1084 (2004); Araki et al., "Usefulness of anti-cyclic citrullinated peptide antibodies (anti-CCP) for the diagnosis of rheumatoid arthritis" Rinsho Byori, 52(12):966-972 (2004); Kumagai et al., "Topics on immunological tests for rheumatoid arthritis" Rinsho byori. Jap. J.

Clin. Pathol, 52(10):836-843 (2004); Eguchi, "Early diagnosis of rheumatoid arthritis by serological markers" Igaku no Ayumi, 209(10): 802-808 (2004); Sawada, "New serum marker of rheumatoid arthritis" Gendai lryo, 36(3):718-722 (2004); van Gaalen et al, "Association between HLA class II genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis" Arthr. Rheum., 50(7):2113-2121 (2004); Sene et al, "Clinical utility of anti-cyclic citrullinated peptide antibodies in the diagnosis of hepatitis C virus associated- rheumatological manifestations" Hepatology, 40(4, Suppl. 1): 687A (2004); 55th Annual Meeting of the American- Association- for-the-Study-of-Liver-Diseases,

Boston, MA, Oct. 29 - Nov. 2, 2004; Bongi et al, "Anti-cyclic citrullinated peptide antibodies are highly associated with severe bone lesions in rheumatoid arthritis anti- CCP and bone damage in RA" Autoimmunity, 37(6-7):495-501 (2004); Feng and Yin, "Detection of anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis" Hebei Yike Daxue Xuebao, 25(6):371-373 (2004); Vossenaar and van Venrooij,

"Anti-CCP antibodies, a highly specific marker for (early) rheumatoid arthritis" Clin. & Appl. Immunol. Rev., 4(4):239-262 (2004); Erre et al, "Diagnostic and prognostic value of antibodies to cyclic citrullinated peptide (Anti-CCP) in rheumatoid arthritis" Reumatismo, 56(2):118-123 (2004); Bizzaro and Sebastiani "Laboratory diagnosis of rheumatoid arthritis" Progressi in Reumatologia, 5(l):82-88 (2004); Jansen et al.,

"The predictive value of anti-cyclic citrullinated peptide antibodies in early arthritis" J. Rheumatol., 30(8): 1691-1695 (2003); Hayashi and Kumagai, "New diagnostic tests for rheumatoid arthritis" Rinsho Byori, 51(10): 1030-1035 (2003); Salvador et al., "Prevalence and clinical significance of anti-cyclic citrullinated peptide and antikeratin antibodies in palindromic rheumatism. An abortive form of rheumatoid arthritis?" Rheumatology, 42(8):972-975 (2003); Okada and Kondo, "Early diagnosis and treatment of the bone and cartilage lesions in rheumatoid arthritis" Clinical Calcium, 13(6):729-733 (2003); Bas et al., "Anti-cyclic citrullinated peptide antibodies, IgM and IgA rheumatoid factors in the diagnosis and prognosis of rheumatoid arthritis" Rheumatology, 42 (Supplement 2):677-680 (2003); van Paassen et al., "Laboratory assessment in musculoskeletal disorders, Best practice & research" Clin. Rheumatol, 17(3):475-494 (2003); Vencovsky et al, "Autoantibodies can be prognostic markers of an erosive disease in early rheumatoid arthritis" Ann. Rheum. Dis., 62(5):427-430 (2003); Suzuki et al, "High diagnostic performance of ELISA detection of antibodies to citrullinated antigens in rheumatoid arthritis" Scand. J. Rheumatol, 32(4): 197-204 (2003); Vallbracht et al, "Additional diagnostic and clinical value of anti-citrullinated peptide antibodies in early rheumatoid arthritis compared to rheumatoid factor-isotypes" Ann. Rheum. Dis., 62 (No. Suppl. 1): 159 (2003); Annual European Congress of Rheumatology, Lisbon, PT, June 18, 2003; Marcelletti and Nakamura, "Assessment of serological markers associated with rheumatoid arthritis. Diagnostic autoantibodies and conventional disease activity markers" Clin. Appl. Immunol. Rev., 4(2): 109-123 (2003); Hromadnikova et al, "Anti-cyclic citrullinated peptide antibodies in patients with juvenile idiopathic arthritis" Autoimmunity, 35(6):397-401 (2002); Vasishta, "Diagnosing early-onset rheumatoid arthritis: the role of anti-CCP antibodies" Amer. Clin. Lab., 21(7):34-36 (2002); Kroot et at., "The prognostic value of anti-cyclic citrullinated peptide antibody in patients with recent-onset rheumatoid arthritis" Arthr. & Rheum., 43(8):1831-1835 (2000); van Jaarsveld et al, "The prognostic value of the antiperinuclear factor, anti-citrullinated peptide antibodies and rheumatoid factor in early rheumatoid arthritis" Clin. & Exper. Rheumatol, 17(6):689-697 (1999); Kroot et al., "The prognostic value of the antiperinuclear factor, determined by a recently developed peptide-based ELISA, using anti citrulline-containing peptide antibodies (anti-CCP) in patients with recent onset Rheumatoid Arthritis" Arthr. & Rheum., 42 (9 Suppl.):S179 (1999); 63rd Annual Scientific Meeting of the American College of Rheumatology and the 34th Annual Scientific Meeting of the Association of Rheumatology Health Professionals, Boston, MA, November 13-17, 1999. See also "Clinical Response Following the First Treatment Course with Rituximab: Effect of Baseline Autoantibody Status (RF, Anti-CCP)" Annals of the Rheumatic Diseases, Vol. 66, Supplement 2, p. 338 (July 2007). US 2007/0196835 discloses gene expression profiling for identification, monitoring, and treatment of RA. Ng et al, Ann Rheum. Dis., 66: 1259 (2007) discloses that autoantibody profiling may help identity SLE patients who will have a more sustained response to B-cell depletion therapy with rituximab and cyclophosphamide, and whether baseline parameters can predict the likelihood of disease flare.

Anti-CCP are present in the majority of patients with RA within the first year of disease onset, further confirming the role of citrullinated proteins in the initiation of the immune dysregulation of RA. In fact, anti-CCP could be detected up to 2.6 years before the clinical onset of RA (Berglin et al, Arthr. Rheum., 48(9):S678 (2003)). A study using the CCP2 assay (a second-generation assay) found progression from undifferentiated polyarthritis to RA in 93% of anti-CCP positive patients but only in 25% of anti-CCP negative patients after 3 years of follow-up. Jansen et al. "Peptides differentiate rheumatoid arthritis from undifferentiated polyarthritis in patients with early arthritis" J. Rheumatol, 29:2074-6 (2002). A decrease in anti-CCP titers was also observed in RA patients treated with anti-TNF-alpha therapy in combination with low-dose MTX (Alessandri et al "Decrease of anti-cyclic citrullinated peptide antibodies and rheumatoid factor following anti-TNF-alpha therapy (infliximab) in rheumatoid arthritis is associated with clinical improvement" Ann. Rheum. Dis., 63 : 1218- 1221 (2004)). In this study, changes in anti-CCP titers and clinical responses were correlated; patients with best clinical improvement during the therapy had the lowest anti-CCP titers at baseline and showed strongest decrease in titer upon therapy. Anti-CCP recognizes proteins containing citrulline, which is the product of posttranslational modification of arginine residues (Masson-Bessiere et al "The major synovial targets of the rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of the a- and P-chains of fibrin" J. Immunol., 166:4177-4184 (2001)). In RA, anti-CCP, anti-keratin antibodies (AKA) and IgM RFs have been suggested as markers for RA (Bas et al., Rheumatology, 41(7):809-14 (2002)). However, the value of such markers remains inconclusive (Scott, Rheumatology, 39(Supp) (2000)). See also US 2006/263783. Citrulline is the essential antigenic epitope target of anti-perinuclear, anti-keratin, anti-filaggrin, anti-CCP, and anti-Sa antibodies (van Venrooij and Pruijn "Citrullination: a small change for a protein with great consequences for rheumatoid arthritis" Arthritis Res., 2:249 (2000)).

One important genetic risk factor for RA is the HLA-class II alleles within the major histocompatibility complex (MHC) (Stastny and Fink, Transplant Proc,

9:1863-1866 (1977)). These alleles are likely to contribute to about one-third of the genetic risk in RA (Deighton et al, Clin. Genet., 36:178-182 (1989) and Rigby et al, Genet. Epidemiol, 8:153-175 (1991)). Although the MHC associations with RA are complex (Jawaheer et al, Am. J. Hum. Genet., 71 :585-594 (2002); Newton et al, Arthritis Rheum., 50:2122-2129 (2004)), the majority of the genetic signal from the MHC is explained by multiple alleles at the HLA-DRBl locus (Hall et al, QJM, 89:821-829 (1996); Jawaheer et al, supra, 2002; MacGregor et al, J. Rheumatol, 22:1032-1036 (1995)). These alleles are known collectively as "shared epitope" (SE) alleles because of their sequence similarity at positions 70-74 within the third hypervariable region of the HLA-DRBl alleles (Gregersen et al, Arthritis Rheum., 30:1205-1213 (1987)). This SE, also called rheumatoid epitope, can be found in approximately 80-90% of all Caucasian RA patients. However, most African- American patients with RA do not have the rheumatoid antigenic determinant (SE). McDaniel et al, Annals Int. Med., 123(3):181-187 (1995). Recently, it was observed both by linkage and association analyses that the SE alleles are a risk factor for only RA characterized by the presence of anti-CCP antibodies, and not for anti-CCP-negative RA (Huizinga et al., Arthritis Rheum., 52:3433-3438 (2005)). Van der Helm-van Mil et al, Arthritis and Rheumatism, 54: 1117-1121 (2006) discloses that the SE-containing HLA-DRBl alleles are primarily a risk factor for anti-CCP antibodies and are not an independent risk factor for development of RA. The human leukocyte antigen HLA-DRBl shared epitope haplotypes are associated with increased RA susceptibility risk.

PTPN22, also known as Lyp (see WO 1999/36548 and Cohen et al., Immunobiology, 93(6):2013-2024 (1999)), regulates the function of CbI and its associated protein kinases via its effect on the tyrosine protein kinase. Four proline- rich potential SH3 domain binding sites are located in the noncatalytic domain of PTNP22. PTPN22 regulates the function of CbI and its associated protein kinases. PTPN22 is an intracellular protein of about 105-kD with a single tyrosine phosphatase catalytic domain. Four pro line-rich potential SH3 domain binding sites are located in the noncatalytic domain of PTPN22. PTNP22 is localized to chromosome Ipl3. PTPN22 has an alternative spliced isoform, Lyp2. Lyp2 is a 85-kD protein having a different 7-amino acid C-terminus. PTPN22 is expressed in a number of cell types involved in the immune response and inflammation. PTNP22 is highly expressed in lymphoid tissues and cells, including both mature B and T cells and thymocytes. Phytohemagglutinin induces PTPN22 expression in peripheral T lymphocytes. PTNP22 is also constitutively associated with the proto-oncogene c-Cbl in thymocytes and T cells. CbI is a protein substrate of PTPN22, is critical in the regulation of diverse processes in a many cells and tissues. PTPN22 is expressed in myeloid cell lines as well as normal granulocytes and monocytes. PTPN22 is involved in CML. Erythroid and myeloid leukemic cell lines have distinct expression patterns of tyrosine phosphatases. In particular, the phosphorylation of multiple proteins in KCL22 chronic myeloid leukemia blast cells {e.g., CbI, Ber-Abl, Erkl/2, and CrkL PTPN221) is reduced by PTPN22 overexpression. Also, the phosphorylation of Bcr-Abl, Grb2, and Myc is reduced in Cos-7 cells co-expressing PTPN22 and Bcr-Abl. Also, anchorage-independent clonal growth of KCL22 cells is suppressed by PTPN22 overexpression. A negative regulatory role for Lyp in T-cell signaling is indicated by these interactions between Lyp and the adaptor Grb2. The ability of PTPN22 activity to reduce signaling by Bcr-Abl indicates PTPN22 is a potential tumor suppressor gone (Chien et al, J Biol. Chem., 278:27413-27420 (2003)). PTPN22 has a functional role, with the mutation being associated with autoimmune risk and disease, as is further illuminated by some of the literature discussed below. WO 2005/014622 discloses antigenic peptides binding to MHC Class II molecules with the shared epitope referred to as HLA-DR molecules and the proteins from which they are derived as markers for erosive and/or non-erosive RA. The antigenic peptides can be used as markers in diagnosis of RA and in therapy as anti- RA vaccines. These include citrullinated antigenic peptides with an increased affinity for HLA-DR molecules and associated with RA. US 2006/062859 discloses methods to measure genetic and metabolic contributing factors affecting disease diagnosis, stratification, and prognosis, and the metabolism, efficacy and/or toxicity associated with specific homeopathic ingredients. The DNA collected may be analyzed for polymorphisms of the Ras-Protein and HLA-DRBl *0404 and *010 lor PTPN22 R620W and IL-10 genes and the analysis may be used to adjust the dosage of Ganoderma Lucidum.

WO 2006/010146 describes the human PTPN22 gene containing a single nucleotide polymorphism (SNP) at nucleotide 1858 in codon 620, encoding an arginine in both alleles of the PTPN22 gene (PTPN22*R1 858) for the wild-type protein in all published human and mouse LYP sequences, but encodes a tryptophan in at least one allele of the PTPN22 gene (PTPN22*T1 858) leading to a mutant LYP protein. The PTPN22*T1858 allele predisposes a person to develop type 1 diabetes (TlD). The PTPN22 gene resides at chromosomal region Ip 13, linked to SLE and RA. The in vivo component of the screen can be the PTPN22 gene, or nucleotides 1858- 1860 of the PTPN22 gene, or nucleotide 1858 of the PTPN22 gene, or a genotyping assay can be used to determine the nucleotide present at position 1858 in the PTPN22 gene. WO 2005/086872 describes methods for detecting polymorphisms of the PTPN22 genomic DNA; methods for associating polymorphisms of the PTPN22 gene with the occurrence of an immune disorder, inflammatory disorder or cell proliferation disorder; methods for identifying subjects at risk of an immune disorder, inflammatory disorder or cell proliferation disorder by determining if they have a polymorphism of the PTPN22 gene and treating such subjects with a tyrosine kinase inhibitor to prevent or delay the progression of such diseases; methods for identifying subjects having an immune disorder {e.g., RA), inflammatory disorder {e.g., Alzheimer's disease, arteriosclerosis), or cell proliferation disorder (e.g., cancer, CML) who are promising candidates for therapy with a tyrosine kinase inhibitor by determining if such subjects have a polymorphism of the PTPN22 gene; and methods of treating subjects having such disorder mediated by a polymorphism of the PTPN22 gene by administering to such subjects a tyrosine kinase inhibitor. A SNP of the PTPN22 gene is determined in a nucleic acid sample obtained from the subject and the presence of the nucleotide occurrence is associated with reduced PTPN22 tyrosine phosphatase activity and altered phosphorylation of regulatory proteins and an increased incidence of the disorders above. A sample of tissue from the subject can be assayed for PTPN22 tyrosine phosphatase activity and the amount of such activity can determine if the subject would have increased risk for developing such disorder. US 6,953,665 provides methods to classify a RA condition and to determine if a person suffering from a RA condition will develop severe disease. The method includes determining the level of a cytokine (e.g., IL-4, IL-IO, and IFN-γ) within a patient sample, comparing the level of the cytokine to a reference level to obtain information about the RA condition, and classifying the RA condition as diffuse, follicular, or granulomatous. US 2005/266410 and WO 2005/123951 disclose approaches to mapping the MHC region and provide methods to genotype the HLA loci A haplotype map of the region and methods of using it are disclosed. US 2003/232055 notes vaccines combining both signals needed to activate native T-cells- -a specific antigen and the co-stimulatory signal—leading to a robust and specific T- cell immune response. WO 2001/018240 notes a diagnostic method involving identifying a patient at risk of arthritis. The patient is tested to characterize a polymorphism in a first intron of the interferon-gamma gene. The polymorphisms may be distinguished based on a difference in the number of CA repeats in a portion of the first intron of the IFN-gamma gene. A patient may be tested for a polymorphism in an HLA protein (or gene), such as the HLA-DRBl protein. WO 2001/012848 notes a method to determine the tendency of a person to develop RA and/or severity thereof, by detecting or measuring the presence of an FcγR gene, gene fragment, or gene product. US 5,965,787 and WO 1998/08943 disclose HLA-DRBI peptides with specific binding affinity for HLA-DQ molecules. Transgenic mice carrying a human HLA-DQ gene deficient in mouse H-2 class II molecules are models to identify peptides to prevent or treat RA. US 2003/099943 notes a method for detecting non-responders to anti-TNF therapy comprising testing a person for homozygosity for a SNP in the gene encoding the TNF receptor II. Anti-TNF-α (infliximab) represents a treatment for steroid refractory Crohn's disease resulting in a remission rate of 30-50% after 4 weeks. Known SNPs within TNF Receptor I and TNF Receptor II were tested for association with response to therapy. As for joint diseases, HLA-DRB 1 <SUP>0</SUP>0401 , which is the allele of major histocompatibility complex (MHC), is reported to be associated with the development of chronic RA (J. Clin. Invest., 89:2033 (1992)). See also the following on HLA, Fc receptor-like 3, MHC, and PTP mutations: Dieude and Cornelis, "Genetic basis of rheumatoid arthritis" Joint, Bone, Spine: Revue Du Rhumatisme, 72(6):520-526 (2005); Batliwalla et al, "Peripheral blood gene expression profiling in rheumatoid arthritis" Genes & Immunity, 6(5):388-397 (2005); Harrison et al, "Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients" Rheumatology, 45(8): 1009-1011 (2006); Newman et al, Rheumatoid arthritis association with the FCRL3 -169C polymorphism is restricted to PTPN22 1858T-homozygous individuals in a Canadian population" Arthr & Rheum., 54(12):3820-3827 (2006); Barcellos et al., "Clustering of autoimmune diseases in families with a high-risk for multiple sclerosis: a descriptive study" Lancet Neurology, 5(11):924-931 (2006); Ikari et al, "Haplotype analysis revealed no association between the PTPN22 gene and RA in a Japanese population" Rheumatology, 45(11): 1345-1348 (2006); Wipff ef al, "Lack of association between the protein tyrosine phosphatase non-receptor 22 (PTPN22)*620W allele and systemic sclerosis in the French Caucasian population" Ann. Rheum. Dis., 65(9):1230-1232 (2006); Ray et al, "Protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene R620W variant and sporadic idiopathic hypoparathyroidism in Asian Indians" Intern. J. Immunogen., 33(4):237-240 (2006); Harrison et al, "Effects of PTPN22 C1858T polymorphism on susceptibility and clinical characteristics of British Caucasian rheumatoid arthritis patients" Rheumatology, 45(8):1009-1011 (2006); Pierer et al, "Association of PTPN22 1858 single-nucleotide polymorphism with rheumatoid arthritis in a German cohort: higher frequency of the risk allele in male compared to female patients" Arthr. Res. & Ther., 8(3):R75 (2006); Butt et al, "Association of functional variants of PTPN22 and tp53 in psoriatic arthritis: a case-control study" Arthr. Res. & Ther, 8(1):R27 (2006); Bottini et al, "Role of PTPN22 in type 1 diabetes and other autoimmune diseases" Seminars in Immunology, 18(4):207-213 (2006); Smyth et al, "Analysis of polymorphisms in 16 genes in type 1 diabetes that have been associated with other immune-mediated diseases" BMC Medical Genetics, 7:20 (2006); De Jager et al, Evaluating the role of the 620W allele of protein tyrosine phosphatase PTPN22 in Crohn's disease and multiple sclerosis" European J. Hum. Gen., 14(3):317-21 (2006); Oliver et al., "Genetic epidemiology of rheumatoid arthritis" Curr. Opin. Rheumatol, 18(2): 141-146 (2006); Burkhardt et al., "Association between protein tyrosine phosphatase 22 variant R620W in conjunction with the HLA-DRBl shared epitope and humoral autoimmunity to an immunodominant epitope of cartilage-specific type II collagen in early rheumatoid arthritis" Arthr. & Rheum., 54(l):82-89 (2006); Jagiello et al. , "The PTPN22 620W allele is a risk factor for Wegener's granulomatosis" Arthr. & Rheum., 52(12):4039-4043 (2005); Vang et al., "Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant" Nature Genetics, 37(12): 1317-1319 (2005); Worthington, "Investigating the genetic basis of susceptibility to rheumatoid arthritis" J. Autoimmunity, 25 Suppl:16- 20 (2005); Dieude et al., "Rheumatoid arthritis seropositive for the rheumatoid factor is linked to the protein tyrosine phosphatase nonreceptor 22-620 W allele" Arthr. Res. & Ther., 7(6):R1200-1207 (2005); Gomez et al., "PTPN22 C1858T polymorphism in Colombian patients with autoimmune diseases" Genes & Immun., 6(7):628-631 (2005); Wesoly et al., "Association of the PTPN22 C1858T single-nucleotide polymorphism with rheumatoid arthritis phenotypes in an inception cohort" Arthritis & Rheum., 52(9):2948-2950 (2005); Prescott et al., "A general autoimmunity gene (PTPN22) is not associated with inflammatory bowel disease in a British population" Tissue Antigens, 66(4):318-320 (2005); Carlton et al., "PTPN22 genetic variation: evidence for multiple variants associated with rheumatoid arthritis" Amer. J. Hum. Gen., 77(4):567-581 (2005); Zhernakova et al., "Differential association of the

PTPN22 coding variant with autoimmune diseases in a Dutch population" Genes & Immunity, 6(6):459-461 (2005); Hinks et al, "Association between the PTPN22 gene and rheumatoid arthritis and juvenile idiopathic arthritis in a UK population: further support that PTPN22 is an autoimmunity gene" Arthr. & Rheum., 52(6): 1694-1699 (2005); Simkins et al, "Association of the PTPN22 locus with rheumatoid arthritis in a New Zealand Caucasian cohort" Arthr. & Rheum., 52(7):2222-2225 (2005); Gregersen and Batliwalla, PTPN22 and rheumatoid arthritis: gratifying replication" Arthr. & Rheum., 52(7): 1952-1955 (2005); van Oene et al, "Association of the lymphoid tyrosine phosphatase R620W variant with rheumatoid arthritis, but not Crohn's disease, in Canadian populations" Arthr. & Rheumat., 52(7): 1993-1998 (2005); Mori et al, "Ethnic differences in allele frequency of autoimmune-disease- associated SNPs" J. Human Gen., 50(5):264-266 (2005); Viken et al, "Association analysis of the 1858C>T polymorphism in the PTPN22 gene in juvenile idiopathic arthritis and other autoimmune diseases" Genes & Immunity, 6(3):271-273 (2005); van der Helm- van Mil et al, "Understanding the genetic contribution to rheumatoid arthritis" Curr. Opinion Rheumatol, 17(3):299-304 (2005); Gregersen, "Pathways to gene identification in rheumatoid arthritis: PTPN22 and beyond" Immunological Rev., 204:74-86 (2005); Criswell et al, "Analysis of families in the multiple autoimmune disease genetics consortium (MADGC) collection: the PTPN22 620W allele associates with multiple autoimmune phenotypes" Amer. J. Hum. Gen., 76(4):561-571 (2005); Zheng and She, "Genetic association between a lymphoid tyrosine phosphatase (PTPN22) and type 1 diabetes" Diabetes, 54(3):906-908 (2005); Ladner et al, "Association of the single nucleotide polymorphism C1858T of the PTPN22 gene with type 1 diabetes" Human Immunol, 66(l):60-64 (2005); Orozco et al,

"Association of a functional single-nucleotide polymorphism of PTPN22, encoding lymphoid protein phosphatase, with rheumatoid arthritis and systemic lupus erythematosus" Arthr. & Rheum., 52(l):219-224 (2005); Brenner et al, "The non- major histocompatibility complex quantitative trait locus CialO contains a major arthritis gene and regulates disease severity, pannus formation, and joint damage" Arthr. & Rheum., 52(l):322-332 (2005); Begovich et al, "A missense single- nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis" Amer. J. Hum. Gen., 75(2):330-337 (2004); Reveille, "The genetic basis of autoantibody production" Autoimmun. Rev., 5(6):389-398 (2006); Mustelin "Allelic variation in signaling elements and autoimmunity" Seminars in Immunology, 18(4): 197-198 (2006); Brand et al, "HLA, CTLA-4 and PTPN22: The shared genetic master-key to autoimmunity?" Expert Rev. in Molec. Med., 7(23): 1-15 (2005); Vandiedonck et al, "Association of the PTPN22*R620W polymorphism with autoimmune myasthenia gravis" Ann. Neurol, 59(2):404-407 (2006); Pearce and Merriman, "Genetic progress towards the molecular basis of autoimmunity" Trends in Molec. Med., 12(2):90-98 (2006); Gregersen, "Gaining insight into PTPN22 and autoimmunity" Nat. Gen., 37(12):1300-1302 (2005); Gregersen and Batliwalla, "PTPN22 and rheumatoid arthritis: Gratifying replication" Arthr. & Rheum., 52(7):1952-1955 (2005). Alarcon-Riquelme, "The genetics of shared autoimmunity" Autoimmunity, 38(3):205-208 (2005); Steer et al., "Association of R602W in a protein tyrosine phosphatase gene with a high risk of rheumatoid arthritis in a British population: Evidence for an early onset/disease severity effect" Arthr. & Rheum., 52(l):358-360 (2005); Holmdahl, "Hereditary Basis of Rheumatic Diseases" Holmdahl, R., ed., Hereditary Basis of Rheumatic Diseases (2006), Pub. BIRKHAUSER VERLAG AG (Series: PROGRESS IN INFLAMMATION RESEARCH); Hueffmeier et al., "Male restricted genetic association of variant R620W in PTPN22 with psoriatic arthritis" J. Invest. Derm., 126(4):936-938 (2006); Anonymous, "1st Mexican-Canadian Congress of Rheumatology, Acapulco, MEXICO, February 17-21, 2006" J. Rheumatol., 33(2):405-428 (2006); Orozco et al., "Association of a functional single nucleotide polymorphism of PTPN22 with rheumatoid arthritis and systemic lupus erythematosus" Genes and Immunity, 6(Suppl. 1):S32 (April 2005), 19th European Immunogenetics and Histocompatibility Conference, Istanbul, TURKEY, April 23-26, 2005; Butt et al, "Association of functional variants of Ptpn22 and Tp53 PsA in Caucasian population" Arth. & Rheum., 52(9, Suppl. S):S642 (Sept. 2005), 69th Annual Scientific Meeting of the American- College-of-Rheumatology/40th Annual Scientific Meeting of the Association-of- Rheumatology-Health-Professionals, San Diego, CA November 12-17, 2005; Wyeth et al., Association analysis of rheumatoid arthritis candidate susceptibility genes in New Zealand Maori" Arthr. & Rheum., 52(9, Suppl. S):S582 (Sept. 2005), 69th

Annual Scientific Meeting of the American-College-of-Rheumatology/40th Annual Scientific Meeting of the Association-of-Rheumato logy-Health-Professionals, San Diego, CA, Nov. 12-17, 2005; Gomez et al., "Polymorphism in gene coding for LYP is a risk factor for primary Sjogren's syndrome and systemic lupus erythematosus" Arthr. & Rheum., 52(9, Suppl. S):S376 (Sept. 2005), 69th Annual Scientific Meeting of the American-College-of-Rheumatology/ 40th Annual Scientific Meeting of the Association-of-Rheumatology-Health-Professionals, San Diego, CA, Nov. 12-17, 2005; Burkhardt et al., "Protein tyrosine phosphatase 22 variant R620W in conjunction with HLA-DRBl shared epitope is associated with humoral autoimmunity to an immunodominant epitope of cartilage-specific type II collagen in early rheumatoid arthritis" Arthr. & Rheum., 52(9, Suppl. S):S146 (Sept. 2005), 69th Annual Scientific Meeting of the American-College-of-Rheumatology/40th Annual Scientific Meeting of the Association-of-Rheumato logy-Health-Professionals, San Diego, CA, November 12-17, 2005; Harrison et al., "The PTPN22 R620W polymorphism - Effects on susceptibility and clinical features on British Caucasian rheumatoid arthritis patients" Arthr. & Rheum., 52(9, Suppl. S):S145-S146 (Sept. 2005), 69th Annual Scientific Meeting of the American-College-of- Rheumatology/40th Annual Scientific Meeting of the Association-of-Rheumatology- Health-Professionals, San Diego, CA, Nov. 12-17, 2005; Costenbader et al, "The PTPN22 polymorphism and the risk of rheumatoid arthritis: Results from the Nurses' Health Study" Arthr. & Rheum., 52(9, Suppl. S):S145 (Sept. 2005), 69th Annual Scientific Meeting of the American-College-of-Rheumatology/40th Annual Scientific Meeting of the Association-of-Rheumato logy-Health-Professionals, San Diego, CA, Nov. 12-17, 2005; Wesoly et al, "PTPN22 1858T allele as rheumatoid arthritis susceptibility but not severity gene variant" Annals Rheumatic Diseases, 64:(No. Suppl. 3):78 (July 2005), Annual European Congress of Rheumatology. Vienna, AUSTRIA, June 8-11, 2005; Dieude et al, "The protein tyrosine phosphatase R620W polymorphism is linked and associated with rheumatoid arthritis seropositive for the rheumatoid factor in a Caucasian population" Ann. Rheum. Dis., 64(No. Suppl. 3):78 (July 2005), Annual European Congress of Rheumatology, Vienna, AT, June 8-11, 2005; Anonymous, Joint Meeting of the British-Society- for-

Rheumatology/Deutsche-Gesellschaft-fur-Rheumatologie and Spring Meeting of the British-Health-Pro fessionals-in-Rheumato logy, Birmingham, ENGLAND, April 19- 22, 2005, Rheumatology, 44(Suppl. 1):I2-I164 (March 2005); Matesanz et al,

"Protein tyrosine phosphatase gene (PTPN22) polymorphism in multiple sclerosis" J. Neurol, 252(8):994-995 (2005); Lee et al, "The PTPN22 C1858T functional polymorphism and autoimmune diseases-a meta-analysis" Rheumatology, 46(l):49-56 (2007); Gregersen and Plenge, "Emerging relationships: rheumatoid arthritis and the PTPN22 associated autoimmune disorders" Hereditary Basis of Rheumatic Diseases, 61-78 (2006), Ed.: Holmdahl, Publisher: Birkhaeuser Verlag, Basel, CH; van der Helm-van Mil and Huizinga, "Genetics and clinical characteristics to predict rheumatoid arthritis: where are we now and what are the future prospects?" Future Rheumatology l(l):79-89 (2006); Hueffmeier et al, "Male restricted genetic association of variant R620W in PTPN22 with psoriatic arthritis" J. Invest. Dermatol, 126(4):932-935 (2006); Yamada, "Large scale SNP LD mapping of rheumatoid arthritis-associated genes" Rinsho Men'eki 44(4):406-410 (2005); Kochi, "Recent findings on rheumatoid arthritis genetics" Igaku no Ayumi 215(4):259-260 (2005); Yamamoto et al, "Rheumatoid arthritis as multifactorial genetic diseases" Saishin Igaku 60(9, Zokango, Rinsho Idenshigaku O5):2111-2119 (2005); Yamada, Rheumatoid arthritis-associated genes" Saishin Igaku 60(9): 1935-1939 (2005); Velazquez-Cruz et al, "A functional SNP of PTPN22 is associated with childhood- onset systemic lupus erythematosus, but not with juvenile rheumatoid arthritis in Mexican population" 11th Intern. Cong, of Human Genetics (ICHG 2006), Brisbane Convention and Exhibition Centre, Brisbane, Queensland (Australia), 6-10 Aug 2006, Prof. Lyn Griffiths, Griffith University, Brisbane.

The non-synonymous SNP (R620W) in the PTPN22 gene is associated with increased susceptibility risk to RA, juvenile idiopathic arthritis, SLE, Addison's disease, systemic sclerosis, and Grave's disease type 1 diabetes. See, for example, Plenge et al, Am. J. Hum. Genet., 77:1044-1060 (2005) reporting that the R620W variant of PTPN22 is associated with the development of RF-positive and anti-CCP- positive RA and stating that the results provide support for an association of RA with variants in PAD14 and CTLA4. Further, see Lee et al, Genes and Immunity, 6:129- 133 (2004) discloses that the PTPN22 R620W polymorphism associates with RF- positive RA in a dose-dependent manner, but not with HLA-SE status. Seldin et al, Genes and Immunity, 6:720-722 (2005) discloses evidence that PTPN22 R620W polymorphism is a risk factor in RA, but suggest only minimal or no effect in juvenile idiopathic arthritis. Hinks et al, Rheumatology, 45(4):365-368 (2006) discloses the association of PTPN2 '2 with RA and juvenile idiopathic arthritis. See also the editorial in Rheumatology, 45:365-368 (2006) on the association of PTPN22 with RA and juvenile idiopathic arthritis. Hinks, Future Rheumatology, 1 : 153-158 (2006) explores whether PTPN22 is a confirmed RA susceptibility gene.

Kyogoku et al, Am. J. Hum. Genet., 75:504-507 (2004) discloses the genetic association of the R620W polymorphism of PTPN22 with human SLE. Kaufman et al, Arthritis Rheum., 54:2533-2540 (2006) reports that the 1858T allele of PTPN22 is associated with familial SLE but not with sporadic SLE in European Americans, thereby potentially explaining previous contradictory reports. Wu et al, Arthritis Rheum., 52:2396-2402 (2005) reports on the association analysis of the R620W polymorphism of PTPN22 in SLE families, specifically the increased t allele frequency in SLE patients with autoimmune thyroid disease.

Gourh et al, Arthritis Rheum., 54(12):3945-3953 (Dec. 2006) discloses an association of the PTPN22 R620W polymorphism with anti-topoisomerase I- and anti-centromere antibody-positive systemic sclerosis. However, Begovich et al, Am. J Hum. Genet., 76(1):184-187 (2005) discloses that the R620W polymorphism of the protein tyrosine phosphatase PTPN22 is not associated with multiple sclerosis. Gomez et al, Human Immunology, 66:1242-1247 (2005) discloses the genetic influence of PTPN22 R620W polymorphism in tuberculosis. Qu et al, J. Medical Genetics, 42:266-270 (2005) reports the confirmation of the association of the R620W polymorphism in the protein tyrosine phosphatase PTPN22 with type 1 diabetes in a family based study. Nistor et al., J. Invest. Dermatol, pp. 395-396 (Letter to the Editor) (2005) on lack of evidence for association of the PTPN22 polymorphism with psoriasis. See also Nistor et al., "Protein tyrosine phosphatase gene PTPN22 polymorphism is not associated with psoriasis" J. Invest. Derm., 124(4, Suppl. S):A80 (April 2005), 66th Annual Meeting of the Society- for-Investigative-Dermato logy, St Louis, MO, May 4-7, 2005.

Wagenleiter et al, Intern. J. Immunogenetics, 32(5):323-324 (2005) discloses a case-control study of tyrosine phosphatase (PTPN22) confirming the lack of association with Crohn's disease. Martin et al, Tissue Antigens, 66(4):314— 317 (2005) discloses that the functional genetic variation in the PTPN22 gene has a negligible effect on the susceptibility to develop inflammatory bowel disease.

TNF-α, IL-Ib, and IL-IRa gene polymorphisms are associated with increased RA susceptibility risk and disease severity. IL-Ib and TNF-α gene polymorphisms are associated with levels of anti-IL-lb and anti-TNF clinical responses, respectively.

The FcγRIIa (Val/Phe 158) and FcγRIIa (His/Arg 131) polymorphisms predicted rituximab clinical response in follicular lymphoma. The FcγRIIa (His/Arg 131) polymorphism predicted B-cell depletion efficacy in SLE. The FcγRIIb (-343 G/C) polymorphism is associated with increased SLE susceptibility. A method of assessing RA by analyzing biochemical markers is disclosed in

US 2007/0072237 involving measuring in a sample the concentration of RF and IL-6 and correlating the concentrations determined to the absence or presence of RA. The level of one or more additional markers may be determined together with RF and IL-6 and be correlated to the absence or presence of RA. B-CeIl Related Disclosure

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. 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. B-cell-related disorders include autoimmune diseases. Autoimmune diseases remain clinically important diseases in humans. As the name implies, autoimmune diseases act through the body's own immune system. While the pathological mechanisms differ among individual types of autoimmune diseases, one general mechanism involves the generation of antibodies (referred to herein as self-reactive antibodies or autoantibodies) directed against specific endogenous proteins.

Physicians and scientists have identified more than 70 clinically distinct autoimmune diseases, including RA, multiple sclerosis, vasculitis, immune-mediated diabetes, and lupus such as SLE. While many autoimmune diseases are rare-affecting fewer than 200,000 individuals-collectively, these diseases afflict millions of Americans, an estimated five percent of the population, with women disproportionately affected by most diseases. The chronic nature of these diseases leads to an immense social and financial burden.

Cytotoxic agents that target B-cell surface antigens are an important focus of B-cell-related cancer therapies. One such B-cell surface antigen is CD20, disclosed more in detail below. Other B-cell antigens, such as CD 19, CD22, and CD52, represent targets of therapeutic potential for treatment of lymphoma (Grillo-Lopez et al, Curr. Pharm. Biotechnol, 2:301-311 (2001)). CD22 is a 135-kDa B-cell- restricted sialoglycoprotein expressed on the B-cell surface only at the mature stages of differentiation (Dorken et al, J. Immunol, 136:4470-4479 (1986)). The predominant form of CD22 in humans is CD22β, which contains seven immunoglobulin superfamily domains in the extracellular domain (Figure 1 of Wilson et al, J. Exp. Med., 173:137-146 (1991)). A variant form, CD22α, lacks immunoglobulin superfamily domains 3 and 4 (Stamenkovic and Seed, Nature, 345:74-77 (1990)). Ligand-binding to human CD22 has been shown to be associated with immunoglobulin superfamily domains 1 and 2 (also referred to as epitopes 1 and 2) (Engel et al, J. Exp. Med., 181 :1581-1586 (1995).

In B-cell NHL, CD22 expression ranges from 91% to 99% in the aggressive and indolent populations, respectively (Cesano et al, Blood, 100:350a (2002)). CD22 may function both as a component of the B-cell activation complex (Sato et al, Semin. Immunol, 10:287-296 (1998)) and as an adhesion molecule (Engel et al, J. Immunol, 150:4719-4732 (1993)). The B cells of CD22-deficient mice have a shorter life span and enhanced apoptosis, which suggests a key role of this antigen in B-cell survival (Otipoby et al, Nature, 384:634-637 (1996)). After binding with its natural ligand(s) or antibodies, CD22 is rapidly internalized, providing a potent costimulatory signal in primary B cells andproapoptotic signals in neoplastic B cells (Sato et al, Immunity, 5:551-562 (1996)).

Anti-CD22 antibodies have been studied as potential therapies for B-cell cancers and other B-cell proliferative diseases. Such anti-CD22 antibodies include RFB4 (Mansfield et al, Blood, 90:2020-2026 (1997)), CMC-544 (DiJoseph, Blood, 103:1807-1814 (2004)) and LL2 (Pawlak-Byczkowska et al, Cancer Res., 49:4568- 4577 (1989)). The LL2 antibody (formerly called HPB-2) is an IgG2a mouse monoclonal antibody directed against the CD22 antigen (Pawlak-Byczkowska et al, 1989, supra). In vitro immunohisto logical evaluations demonstrated reactivity of the LL2 antibody with 50 of 51 B-cell NHL specimens tested, but not with other malignancies or normal nonlymphoid tissues (Pawlak-Byczkowska et al, 1989, supra; Stein et al, Cancer Immunol. Immunother., 37:293-298 (1993)).

The CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35, or Bl) 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, EMBOJ., 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, 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 Pr oc Natl Acad Sci. USA, 94:7509-7514 (1997)).

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.

The rituximab (RITUXAN®) antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody called "C2B8" in US 5736137 (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-CD 19 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)).

Rituximab was approved in the United States for the treatment of patients with relapsed or refractory low-grade or follicular CD20+ B-cell NHL at a dose of 375 mg/m2 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). Many patients have been exposed 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 two 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).

Rituximab has also been approved in the United States in combination with MTX to reduce signs and symptoms in adult patients with moderately- to severely- active RA who have had an inadequate response to at least one TNF antagonist.

Many studies address the use of rituximab in a variety of non-malignant autoimmune disorders, including RA, 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, 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 (Suppl. 1): OP004 (2003); Emery et al, Arthritis Rheum., 48(9): S439 (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); Zaja et al, Haematologica, 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, supra (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); Zaja et al, Br. J. Haematol, 115:232-233 (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, 55:1062-1063 (2000); Wylam et al, J. Pediatr., 141:614-611 (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):S1299 (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)).

A Phase II study (WA 16291) has been conducted in patients with RA, providing 48-week follow-up data on safety and efficacy of rituximab (Emery et al, Arthritis Rheum., 48(9):S439 (2003); Szczepanski et al, Arthritis Rheum., 48(9):S121 (2003)). A total of 161 patients were evenly randomized to four treatment arms: MTX, rituximab alone, rituximab plus MTX, 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)). 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 investigator-sponsored trial (1ST) of rituximab combined with interferon-beta (IFN-β) or glatiramer acetate in patients with RRMS (Cross et al, supra), one often 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 nine patients completed the four-infusion regimen without any reported adverse events.

Patents and patent publications concerning CD20 antibodies, CD20-binding molecules, and self-antigen vaccines include U.S. 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, US 2005/0186205, and WO 1994/11026 (Anderson et al); U.S. 6,455,043, US 2003/0026804, US 2003/0206903, and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); US 2004/0213784 and 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, US 2002/0028178, WO 2001/34194, and WO 2002/22212 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665, US 2005/0180975, WO 2001/74388, and U.S. 6,896,885 (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, US 2002/0039557, and U.S. 6,846,476 (White, C); US 2002/0128448 and WO 2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et a/.); WO 2002/096948 (Braslawsky et α/.);WO 2002/079255 (Reff and Davies); U.S. 6,171,586 and 6,991,790, and WO 1998/56418 (Lam et al); US 2004/0191256 and WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, U.S. 6,194,551, U.S. 6,242,195, 6,528,624 and 6,538,124 (Idusogie et al); U.S. 7,122,637, US 2005/0118174, US 2005/0233382, US 2006/0194291, US 2006/0194290, US 2006/0194957, and WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al); WO 2001/03734 (Grillo-Lopez et al); US 2002/0004587, US 2006/0025576, and WO 2001/77342 (Miller and Presta); US 2002/0197256 and WO 2002/078766 (Grewal, L); US 2003/0157108 and WO 2003/035835 (Presta, L.); U.S. 5,648,267, 5,733,779, 6,017,733, and 6,159,730, and WO 1994/11523 (Reff et al on expression technology); U.S. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721 (Kaminski et al); U.S. 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852, and 6,893,625 as well as WO 1988/04936 (Robinson et al); U.S. 6,410,391 (Zelsacher); U.S. 6,224,866 and WO 2000/20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); U.S. 7,074,403 (Goldenberg and Hansen); U.S. 7,151,164 (Hansen et al); 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 α/.);W0 2001/72333 (Wolin and Rosenblatt); U.S. 6,368,596 (Ghetie et al); U.S. 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. 5,849,898 and EP 330,191 (Seed et al); EP332,865A2 (Meyer and Weiss); U.S. 4,861,579 (Meyer et al); US 2001/0056066 (Bugelski et al); WO 1995/03770 (Bhat et al); US

2003/0219433 Al (Hansen et al); WO 2004/035607 and US 2004/167319 (Teeling et al); WO 2005/103081 (Teeling et al); US 2006/0034835, US 2006/0024300, and 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); US 2006/0251652 (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); US 2005/0123546 (Umana et al); US 2004/0072290 (Umana et al) ; US 2003/0175884 (Umana et al); and WO 2005/044859 (Umana et al); WO 2005/070963 (Allan et al); US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al); US 2003/118592 (Ledbetter et al); US 2003/133939 (Ledbetter and Hayden- Ledbetter); US 2005/0202012 (Ledbetter and Hayden-Ledbetter); US 2005/0175614 (Ledbetter and Hayden-Ledbetter); US 2005/0180970 (Ledbetter and Hayden- Ledbetter); US 2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/0202023 (Hayden-Ledbetter and Ledbetter); WO 2005/017148 (Ledbetter et al); WO 2005/037989 (Ledbetter et al); U.S. 6,183,744 (Goldenberg); U.S. 6,897,044 (Braslawski et al); WO 2006/005477 (Krause et al); US 2006/0029543 (Krause et al); US 2006/0018900 (McCormick et al); US 2006/0051349 (Goldenberg and Hansen); WO 2006/042240 (Iyer and Dunussi-Joannopoulos); US 2006/0121032 (Dahiyat et al); WO 2006/064121 (Teillaud et al); US 2006/0153838 (Watkins), CN 1718587 (Chen et al); WO 2006/084264 (Adams et al); US 2006/0188495 (Barron et al); US 2004/0202658 and WO 2004/091657 (Benynes, K.); US 2005/0095243, US 2005/0163775, WO 2005/00351, and WO 2006/068867 (Chan, A.); US

2006/0135430 and WO 2005/005462 (Chan et al); US 2005/0032130 and WO 2005/017529 (Beresini et al); US 2005/0053602 and WO 2005/023302 (Brunetta, P.); US 2006/0179501 and WO 2004/060052 (Chan et al); WO 2004/060053 (Chan et al); US 2005/0186206 and WO 2005/060999 (Brunetta, P.); US 2005/0191297 and WO 2005/061542 (Brunetta, P.); US 2006/0002930 and WO 2005/115453 (Brunetta et al); US 2006/0099662 and WO 2005/108989 (Chuntharapai et al); CN 1420129A (Zhongxin Guojian Pharmaceutical); US 2005/0276803 and WO 2005/113003 (Chan et al); US 2005/0271658 and WO 2005/117972 (Brunetta et al); US 2005/0255527 and WO 2005/11428 (Yang, J.); US 2006/0024295 and WO 2005/120437 (Brunetta, P.); US 2006/0051345 and WO 2005/117978 (Frohna, P.); US 2006/0062787 and WO 2006/012508 (Hitraya, E.); US 2006/0067930 and WO 2006/31370 (Lowman et al); WO 2006/29224 (Ashkenazi, A.); US 2006/0110387 and WO 2006/41680 (Brunetta, P.); US 2006/0134111 and WO 2006/066086 (Agarwal, S.); WO 2006/069403 (Ernst and Yansura); US 2006/0188495 and WO 2006/076651 (Dummer, W.); WO 2006/084264 (Lowman, H.); WO 2006/093923 (Quan and Sewell); WO 2006/106959 (Numazaki et al); WO 2006/126069 (Morawala); WO 2006/130458 (Gazit-Bornstein et al); US 2006/0275284 (Hanna, G.); US 2007/0014785 (Golay et al); US 2007/0014720 (Gazit-Bornstein et al); and US 2007/0020259 (Hansen et al); US 2007/0020265 (Goldenberg and Hansen); US 2007/0014797 (Hitraya); US 2007/0224189 (Lazar et al); WO 2007/014238 (Bruge and Bruger); and WO 2008/003319 (Parren and Baadsgaard).

Scientific publications concerning treatment with rituximab include: Perotta and Abuel, "Response of chronic relapsing ITP of 10 years duration to rituximab" Abstract # 3360 Blood, 10(l)(part 1-2):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 April, 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):S370 (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 two-week period, each patient received two 500-mg infusions of rituximab, two 750-mg infusions of CTX, and high- dose oral corticosteroids, and wherein two of the patients treated relapsed at seven and eight 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 x 4, repeated at weekly intervals) and further rituximab applications were delivered every five to six months and then maintenance therapy was received with rituximab 375 mg/m2 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): S1350 (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):S197 (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:(Sl):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 Bl 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); Higashida et al "Treatment of DMARD-refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology (Abstract #LB11), New Orleans, LA (Oct., 2002); Tuscano, "Successful treatment of infliximab-refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology, New Orleans, LA (Oct., 2002), published as 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) (SEP):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)"^4/?/?α/s of the Rheumatic Diseases, 61(10):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 (John Wiley & Sons, Inc., 2003) (Chapter 2 "Antibody-Directed Immunotherapy"); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target (2002); Appendix 4A entitled "Monoclonal Antibodies to Human Cell Surface Antigens" by Stockinger et al., eds: Coligan et al., in Current Protocols in Immunology (John Wiley & Sons, Inc., 2003); Penichet and Morrison, "CD Antibodies/molecules: Definition; Antibody Engineering" in Wiley Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies (2002). Further, see Looney "B cells as a therapeutic target in autoimmune diseases other than rheumatoid arthritis" Rheumatology, 44 Suppl 2:iil3-iil7 (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. Nephrol., 20(6): 811-813 (2005); D'Cruz and Hughes, "The treatment of lupus nephritis" BMJ, 330(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):371-377 (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 naϊve B cells in patient with rheumatoid arthritis and systemic lupus erythematosus" Arthritis Rheum., 48 (Suppl 9):S1160 (2003). Specks et al "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism, 44(12):2836-2840 (2001) disclosed successful use of four infusions of 375 mg/m 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 eight months after the second course of rituximab, the patients' disease remained in complete remission. 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/m2 x four along with oral prednisone at 1 mg/kg/day, which was reduced to 40 mg/day by week four, and to total 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 necessary for remission induction and maintenance of sustained remission (six months or longer). Keogh et al, Kidney Blood Press. Res., 26:293 (2003) reported that eleven patients with refractory ANCA-associated vasculitis went into remission upon treatment with four weekly 375 mg/m doses of rituximab and high-dose glucocorticoids.

Patients with refractory ANCA-associated vasculitis were administered rituximab along with immunosuppressive medicaments such as intravenous CTX, 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/m2 once a week for four 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 with CTX along with background immunosuppression and prednisolone experienced major falls in vasculitic activity). A further report of using rituximab along with intravenous CTX at 375 mg/m2 per dose in four doses for administering to patients with refractory systemic vasculitis is provided in Smith and Jayne, "A prospective, open label trial of B-cell depletion with rituximab in refractory systemic vasculitis" poster 998 (11th International Vasculitis and ANCA workshop), American Society of Nephrology, J. Am. Soc. Nephrol., 14:755A (2003). 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; and 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 375 mg/m2 doses of rituximab induced remission by B-lymphocyte depletion (study conducted from Jan. 2000 to Sept. 2002). 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). The MINT trial with rituximab was successful in treating aggressive non-Hodgkin's lymphoma in younger patients (Pfreundschuh et al, Lancet Oncology, 7(5):379-391 (2006)).

BLyS™ (also known as BAFF, TALL-I, THANK, TNFSF13B, or zTNF4) is a member of the TNFl ligand superfamily that is essential for B-cell survival and maturation. BAFF overexpression in transgenic mice leads to B-cell hyperplasia and development of severe autoimmune disease (Mackay et al, J. Exp. Med., 190:1697- 1710 (1999); Gross et al, Nature, 404:995-999 (2000); Khare et al, Proc. Natl. Acad. Sci. U.S.A, 97:3370-3375 (2000)). BAFF levels are elevated in human patients with a variety of autoimmune disorders, such as SLE, RA, and Sjogren's syndrome (Cheema et al, Arthritis Rheum., 44:1313-1319 (2001); Groom et al, J. Clin. Invest., 109:59-68 (2002); Zhang et al, J. Immunol, 166:6-10 (2001)). Furthermore, BAFF levels correlate with disease severity, suggesting that BAFF can play a direct role in the pathogenesis of these illnesses. BAFF acts on B cells by binding to three members of the TNF receptor superfamily, TACI, BCMA, and BR3 (also known as BAFF-R) (Gross et al, supra; Thompson et al, Science, 293:2108-2111 (2001); Yan et al, Curr. Biol 11 :1547-1552 (2001); Yan et al, Nat. Immunol, 1 :37-41 (2000); Schiemann et al, Science, 293:2111-2114 (2001)). Of the three, only BR3 is specific for BAFF; the other two also bind the related TNF family member, A proliferation-inducing ligand (APRIL). Comparison of the phenotypes of BAFF and receptor knockout or mutant mice indicates that signaling through BR3 mediates the B-cell survival functions of BAFF (Thompson et al, supra; Yan et al, supra, 2001; Schiemann et al, supra). In contrast, TACI ap- pears to act as an inhibitory receptor (Yan, Nat. Immunol, 2:638-643 (2001)), while the role of BCMA is unclear (Schiemann et al, supra). US 2007/0071760 discloses treating B-cell malignancies using a TACI-Ig fusion molecule in an amount sufficient to suppress proliferation-inducing functions of BIyS and APRIL.

BR3 is a 184-residue type III transmembrane protein expressed on the surface of B cells (Thompson et al, supra; Yan, Nat. Immun., supra). The intracellular region bears no sequence similarity to known structural domains or protein-protein interaction motifs. Nevertheless, BAFF-induced signaling through BR3 results in processing of the transcription factor NF-B2/pl00 to p52 (Claudio et al, Nat. Immunol, 3:958-965 (2002); Kayagaki et al, Immunity, 10:515-524 (2002)). The extracellular domain (ECD) of BR3 is also divergent. TNFR family members are usually characterized by the presence of multiple cysteine-rich domains (CRDs) in their extracellular region; each CRD is typically composed of about 40 residues stabilized by six cysteines in three disulfide bonds. Conventional members of this family make contacts with ligand through two CRDs interacting with two distinct patches on the ligand surface (Bodmer et al, Trends Biochem. Sci., 27: 19-26 (2002)). However, the BR3 ECD contains only four cysteine residues, capable of forming a partial CRD at most, raising the question of how such a small receptor imparts high- affinity ligand binding. It has been shown that the BAFF -binding domain of BR3 resides within a 26- residue core region (Kayagaki et al, supra). Six BR3 residues, when structured within a β -hairpin peptide (bhpBR3), were sufficient to confer BAFF binding and block BR3-mediated signaling. Others have reported polypeptides purported to interact with BAFF (e.g., WO 2002/24909, WO 2003/035846, WO 2002/16312, and WO 2002/02641).

For any given RA arthritis patient one frequently cannot predict or prognosticate which patient is likely to respond to a particular treatment, even with newer B-cell antagonist therapies, thus necessitating considerable trial and error, often at considerable risk and discomfort to the patient, in order to find the most effective therapy.

Thus, there is a need for more effective means for determining which patients will respond to which treatment and for incorporating such determinations into more effective treatment regimens for RA patients with B-cell antagonist therapies, whether used as single agents or combined with other agents to treat RA.

Summary of the Invention

In one aspect, the invention concerns a method of treating rheumatoid arthritis in a patient comprising administering an effective amount of a B-cell antagonist to the patient to treat the rheumatoid arthritis, provided that the rheumatoid arthritis is characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody in a sample from the patient.

In another aspect, the invention concerns a method of treating rheumatoid arthritis in a patient comprising first administering a B-cell antagonist to the patient to treat the rheumatoid arthritis, provided that the rheumatoid arthritis is characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody in a sample from the patient, and at least about 24 weeks after the first administration of the antagonist, re-treating the patient by administering an effective amount of the B-cell antagonist to the patient, wherein no clinical improvement is observed in the patient at the time of the testing after the first administration of the B-cell antagonist.

In yet another aspect, a method of treating rheumatoid arthritis in a patient comprising administering to the patient an effective amount of a B-cell antagonist, wherein before the administration, an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody was detected in a sample from the patient.

In a further aspect, the invention concerns a method of treating rheumatoid arthritis in a patient comprising administering to the patient an effective amount of a B-cell antagonist, wherein before the administration a sample from the patient was determined to contain an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody whereby the elevated amount and titer indicate that the patient will respond to treatment with the antagonist.

In a still further aspect, the invention concerns a method for advertising a B- cell antagonist or a pharmaceutically acceptable composition thereof comprising promoting, to a target audience, the use of the antagonist or pharmaceutical composition thereof for treating a patient or patient population with rheumatoid arthritis from which a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.

In all aspects, in one embodiment, the positive acute phase protein is selected from the group consisting of C-reactive protein (CRP), D-dimer protein, mannose- binding protein alpha 1 -antitrypsin, alpha 1-antichymotrypsin, alpha 2 macroglobulin, fibrinogen, prothrombin, Factor VIII, von Willebrand factor, plasminogen, complement factors, ferritin, serum amyloid A, ceruloplasmin, and haptoglobulin.

In another embodiment, the positive acute phase protein is CRP or serum amyloid A, preferably CRP. When the positive acute phase protein is CRP, the elevated level may, for example, be at least about 1 mg/dL CRP, or at least about 2 mg/dL, or at least about 3 mg/dL CRP, preferably at least about 3.9 mg/dL CRP in the sample from the patient.

In yet another embodiment, the rheumatoid arthritis-associated autoantibody is selected from the group consisting of anti-rheumatoid factor (anti-RF), anti-RA33, anti-calpastatin, anti-SA protein, anti-p68, anti-glucose-6-phosphate isomerase (GPI), and anti-CCP antibodies. In a particular preferred embodiment, the rheumatoid arthritis-associated autoantibody is one or more isotype of an anti-RF antibody, including IgA, IgG, IgM and/or IgE anti-RF antibodies.

In a further embodiment, the rheumatoid arthritis-associated autoantibody is an IgA anti-RF antibody, and the elevated titer is at least about 25 U/ml. In a still further embodiment, the sample is seropositive for at least one additional biomarker or rheumatoid arthritis, where the additional biomarker may, for example, be an anti-CCP antibody, such as an anti-CCP antibody is of the IgG isotype, or of the IgM isotype. In all embodiments, the B-cell antagonist may be an antibody or immunoadhesin, where the antibodies may be, without limitation, chimeric, humanized or human.

In other embodiments, the antagonist is to CD20, CD22, BAFF, or APRIL, or TACI-Ig. B-cell antagonist antibodies for use in according to the invention, include anti-

CD20 and anti-CD22 antibodies.

In one embodiment, the anti-CD20 antibody is rituximab.

In another embodiment, the anti-CD20 antibody is a 2H7 antibody.

In various further embodiments, the 2H7 antibody comprises the L-chain variable region sequence of SEQ ID NO: 1 and the H-chain variable region sequence of SEQ ID NO:2 or the L-chain variable region sequence of SEQ ID NO:3 and the H-chain variable region sequence of SEQ ID NO:4 or the L-chain variable region sequence of SEQ ID NO:3 and the H-chain variable region sequence of SEQ ID NO:5, or the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:7, or the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:8, or the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 10, or the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 11 , or the full-length L chain of SEQ ID NO:9 and the full- length H chain of SEQ ID NO: 12, or the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 13, or the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 14, or the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO: 15.

In a further embodiment, the antagonist is not conjugated with a cytotoxic agent. In a different embodiment, the antagonist is conjugated with a cytotoxic agent.

In various embodiments, the sample from the patient may, for example, be blood, synovial tissue, or synovial fluid.

In another embodiment, the patient has never been previously administered a medicament for the rheumatoid arthritis. In a different embodiment, the patient has been previously administered at least one medicament for the rheumatoid arthritis.

In a further embodiment, the patient was not responsive to at least one medicament that was previously administered. The previously administered medicament or medicaments may, for example, be an immunosuppressive agent, cytokine antagonist, integrin antagonist, corticosteroid, analgesic, a disease-modifying anti-rheumatic drug (DMARD), or a non-steroidal anti-inflammatory drug (NSAID).

In a particular embodiment, the previously administered medicament is a TNF-α inhibitor or methotrexate. In another embodiment, the previously administered medicament is a CD20 antagonist that is not rituximab or a 2H7 antibody.

In an alternative embodiment, the previously administered medicament is rituximab or a 2H7 antibody.

The B-cell antagonist may, for example, be administered intravenously, or subcutaneously.

In a further embodiment, at least about three months after the administration, an imaging test is given that measures a reduction in bone or soft tissue joint damage as compared to baseline prior to the administration and the amount of the B-cell antagonist administered is effective in achieving a reduction in the joint damage. In one embodiment, the test measures a total modified Sharp score.

In various embodiments, the antagonist is administered in a dose of about 0.2 to 4 grams, or about 0.2 to 3.5 grams, or about 0.4 to 2.5 grams, or about 0.5 to 1.5 grams.

In a particular embodiment, the antagonist is an anti-CD20 antibody and is administered in a dose of about 0.4 to 4 grams, or in a dose of about 0.4 to 1.3 grams, or in a dose of about 1.5 to 3.5 grams, or in a dose of about 1.5 to 2.5 grams.

In another embodiment, the antagonist is administered at a frequency of one to four doses within a period of about one month.

In yet another embodiment, the antagonist is an anti-CD20 antibody and the dose is about 200 mg to 1.2 grams, or about 200 mg to 1.1 grams.

In a further embodiment, the antagonist is administered in two to three doses and/or the antagonist is administered within a period of about 2 to 3 weeks.

In a specific embodiment, the antagonist is an anti-CD20 antibody and the dose is about 500 mg to 1.2 grams, or about 750 mg to 1.1 grams. In another embodiment, the B-cell antagonist is administered without any other medicament to treat the RA.

In yet another embodiment, the methods herein further comprise administering an effective amount of one or more second medicaments with the B-cell antagonist, wherein the B-cell antagonist is a first medicament.

In one embodiment, the second medicament is more than one medicament.

In another embodiment, the second medicament is 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 combination thereof.

In a particular embodiment, the second medicament is a DMARD, such as, for example, auranofm, chloroquine, D-penicillamine, injectable gold, oral gold, hydroxychloroquine, sulfasalazine, myocrisin and methotrexate.

In a different embodiment, the second medicament is a NSAID, such as, for example, fenbufen, naprosyn, diclofenac, etodolac, indomethacin, aspirin and ibuprofen.

In yet another embodiment, the immunosuppressive agent is selected from the group consisting of etanercept, infliximab, adalimumab, leflunomide, anakinra, azathioprine, and cyclophosphamide. In a still further embodiment, the second medicament is selected from the group consisting of anti-alpha4, etanercept, infliximab, etanercept, adalimumab, kinaret, efalizumab, osteoprotegerin (OPG), anti-receptor activator of NFKB ligand (anti-RANKL), anti-receptor activator of NFKB-FC (RANK-FC), pamidronate, alendronate, actonel, zolendronate, clodronate, methotrexate, azulfϊdine, hydroxychloroquine, doxycycline, leflunomide, sulfasalazine (SSZ), prednisolone, interleukin-1 receptor antagonist, prednisone, and methylprednisolone.

In an additional embodiment, the second medicament is selected from the group consisting of infliximab, an infliximab/methotrexate (MTX) combination, MTX, etanercept, a corticosteroid, cyclosporin A, azathioprine, auranofm, 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, where the corticosteroid may, for example, be prednisone, prednisolone, methylprednisolone, hydrocortisone, or dexamethasone. In another embodiment, the second medicament is MTX, which may be administered perorally or parenterally.

In a particular embodiment, the B-cell antagonist is an anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment.

In another particular embodiment, the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 600 mg.

In yet another particular embodiment, the patient has exhibited an inadequate response to one or more anti-tumor necrosis factor (TNF) inhibitors. In a further particular embodiment, the B-cell antagonist is an anti-CD20 antibody administered as a single dose or as two doses, with each dose being between about 200 mg and 1000 mg.

In another embodiment, the anti-CD20 antibody is administered at a dose of about 200 mg x 2, 500 mg x 2, or 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment.

In another embodiment, the methods herein further comprise re -treating the patient by administering an effective amount of the B-cell antagonist to the patient, wherein the re-treatment is commenced at at least about 24 weeks after the first administration of the antagonist. In yet another embodiment, the amount of the B-cell antagonist administered upon each administration thereof is effective to achieve a continued or maintained reduction in joint damage.

In a further embodiment, a further re-treatment is commenced with an effective amount of the B-cell antagonist. In various further embodiments, the further re-treatment is commenced at at least about 24 weeks after the second administration of the antagonist and/or joint damage has been reduced after the re-treatment and/or no clinical improvement is observed in the patient at the time of the testing after the re-treatment.

The clinical improvement may, for example, be determined by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity.

The clinical improvement may be determined, for example, by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity.

In one embodiment, the amount of the B-cell antagonist administered upon re- treatment is effective to achieve a continued or maintained reduction in joint damage as compared to the effect of a prior administration of the B-cell antagonist.

In another embodiment, before the administration, at least about 3.9 mg/dL CRP and an anti-RF antibody titer of at least about 25 U/ml was detected in the sample.

In another aspect, the invention concerns an article of manufacture comprising, packaged together, a pharmaceutical composition comprising a B-cell antagonist and a pharmaceutically acceptable carrier and a label stating that the antagonist or pharmaceutical composition is indicated for treating patients with rheumatoid arthritis from whom a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.

The article may further comprise a container comprising a second medicament, wherein the B-cell antagonist is a first medicament, further comprising instructions on the package insert for treating the patient with an effective amount of the second medicament. In a particular embodiment, the second medicament is methotrexate.

In yet another aspect, the invention concerns a method for manufacturing a B- cell antagonist or a pharmaceutical composition thereof comprising combining in a package the antagonist or pharmaceutical composition and a label stating that the antagonist or pharmaceutical composition is indicated for treating patients with rheumatoid arthritis from whom a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.

In a further aspect, the invention concerns a method of providing a treatment option for patients with rheumatoid arthritis comprising packaging a B-cell antagonist in a vial with a package insert containing instructions to treat patients with rheumatoid arthritis from whom a sample has been obtained that contains an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody In a still further aspect, the invention concerns a method for predicting whether a subject with rheumatoid arthritis will respond to a B-cell antagonist, the method comprising determining whether a sample from the subject contains an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody, wherein an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody indicates that the subject will respond to the antagonist.

In a different aspect, the invention concerns a method for marketing a B-cell antagonist for use in a rheumatoid arthritis patient subpopulation, the method comprising informing a target audience about the use of the antagonist for treating the patient subpopulation characterized by the presence, in samples from patients of such subpopulation, of an an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.

In another aspect, the invention concerns a method of assessing whether a sample from a patient with rheumatoid arthritis indicates responsiveness of the patient to treatment with a B-cell antagonist comprising: a. detecting in the sample whether an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody is present; b. implementing an algorithm to determine that the patient is responsive to said treatment; and c. recording a result specific to the sample being tested. The invention further concerns a system for analyzing susceptibility or responsiveness of a patient with rheumatoid arthritis to treatment with a B-cell antagonist comprising: a. reagents to detect in a sample from the patient an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody; b. hardware to perform detection of the biomarkers; and c. computational means to perform an algorithm to determine if the patient is susceptible or responsive to said treatment. Brief Description of the Drawings

FIG. 1 is an illustration of the threshold sensitivity analysis method used in Example 1. Plot for subgroup ACR50 efficacy differentials vs. biomarker threshold values corresponding to 20th, 25th, . . . 80th percentiles was generated. Logistic regression was used to characterize general biomarker-response relationship.

FIG. 2: REFLEX clinical trial exploratory biomarker analysis.

FIG. 3: Sensitivity analysis of CRP and IgA RF combination in the REFLEX clinical trial.

FIG. 4: Sensitivity analysis of CRP in the SERENE clinical trial. CRP>2.9 mg/dL defines an enhanced responder subgroup. Permutation P=O.26.

FIG. 5: Sensitivity analysis of IgA RF in the SERENE clinical trial. IgA titer>25 U/ml defines an enhanced responder subgroup. Permutation P=O.18.

FIG. 6: Sensitivity analysis of CRP and IgA combination in the SERENE clinical trial. CRP>2.9 mg/dL and IgA RF titer >25 U/ml defines an enhanced responder subgroup. Permutation P=O.02.

FIGS. 7A-7D: Association of the combination biomarker-defmed patient subgroup with alternative clinical efficacy endpoints in the SERENE clinical trial.

FIGS. 8A-8H: Effect of baseline autoantibody positivity on clinical efficacy after rituximab treatment. FIGS. 9A-9E: A subset of RA patients in the SERENE clinical trial with

CRP>2.9 mg/dL and who are seropositive for multiple autoantibody isotypes demonstrate enhanced clinical efficacy after rituximab treatment.

FIG. 10: Graphical representation of the clinical protocol of SERENE clinical trial. FIG. 11 : RITUXAN® SERENE clinical trial threshold sensitivity analysis for

CRP + IgA RF combination.

FIG. 12: 2H7 ACTION clinical trial week 24 ACR50 response rates.

FIGS. 13A-13H: Effect of RA autoantibodies on responsiveness to CD20 antibodies in the REFLEX and SERENE clinical trials. Autoantibody negativity and positivity is indicated below each group.

FIGS. 14A-14D: Combination of elevated CRP and positivity to any of the autoantibodies shown in FIGS. 13A-13H further enriches the responder subgroup - data based on the SERENE clinical trial. Detailed Description of the Preferred Embodiments A. Definitions

A "positive acute phase protein" is a protein whose plasma concentration increases in response to inflammation. Positive acute phase proteins include, without limitation, C-reactive protein (CRP), D-dimer protein, mannose-binding protein, alpha 1 -antitrypsin, alpha 1-antichymotrypsin, alpha 2-mactoglobulin, fibrinogen, prothrombin, Factor VIII, von Willebrand factor, plasminogen, complement factors, ferritin, serum amyloid P component, serum amyloid A, ceruplasmin, haptoglobulin. Preferred positive acute phase proteins include CRP and serum amyloid A. 1. A "rheumatoid arthritis-associated autoantibody" is an autoantibody produced by a rheumatoid arthritis patient to an antigen. In a particular embodiment, the autoantibody is specific for rheumatoid arthritis. In another embodiment, the autoantibody is non-specific but is produced also in other inflammatory and autoimmune diseases. Rheumatoid factor-associated autoantibodies include, without limitation, anti-rheumatoid factor (anti-RF), anti-RA33, anti-calpastatin, anti-SA protein, anti-p68, anti-glucose-6-phosphate isomerase (GPI), and anti-CCP antibodies. 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. A "B-cell malignancy" is a malignancy involving B cells. Examples include

Hodgkin's disease, including lymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicular center cell (FCC) lymphoma; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); hairy cell leukemia; plasmacytoid lymphocytic lymphoma; mantle cell lymphoma; AIDS or HIV-related lymphoma; multiple myeloma; central nervous system (CNS) lymphoma; post-transplant lymphoproliferative disorder (PTLD); Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma); mucosa-associated lymphoid tissue (MALT) lymphoma; and marginal zone lymphoma/leukemia.

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 CDlO, 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, 2nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers include RP 105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, BAFF, BLyS, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTAl, 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, CD22, CD23, CD40, BR3, BLyS, and BAFF.

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. 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 14OkD (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 CD 19 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).

An "B-cell antagonist" is a molecule that, upon binding to a B-cell surface marker or B-cell specific survival or proliferation factor, destroys or depletes B cells in a mammal and/or interferes with B-cell survival and/or 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 can be screened by various methods known in the art for apoptosis and other measurements for the depletion, and retardation or stopping of proliferation and growth of B cells or survival of B cells. For example, a method of screening can be employed as described in Sundberg et al, Cancer Research 66, 1775-1782 (2006) wherein a compound was screened for inhibition of B-cell proliferation by targeting c-myc protein for rapid and specific degradation. See also Mackay et al, Annual Review of Immunology, 21 : 231- 264 (2003) regarding BAFF, APRIL, and a tutorial on B-cell survival and screening, and Thangarajh et al., Scandinavian J. Immunol, 65(1):92 (2007) on B-cell proliferation and APRIL. In addition, Sakurai et al., European J. Immunol, 37(1): 110 (2007) discloses that TACI attenuates antibody production co-stimulated by BAFF-R and CD40. Further, Acosta-Rodriguez et al, European J. Immunol, 37(4):990 (2007) discloses that BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas-mediated cell death. Further screening methods can be found in Martin and Chan, "B Cell Immunobiology in Disease: Evolving Concepts from the Clinic Annual Review of Immunology," 24:467-496 (2006), Pillai et al, "Marginal Zone B Cells" Annual Review of Immunology, 23:161-196 (2005), and Hardy and Hayakawa, "B Cell Development Pathways," Annual Review of

Immunology, 19:595-621 (2001). From these and other references the skilled artisan can screen for the appropriate antagonists. Microarrays can be used for this purpose (Hagmann, Science, 290:82-83 (2000)), as well as RNA interference (RNAi) (Ngo et al, Nature, 441 : 106-110 (2006)). Antagonists included within the scope of the present invention include antibodies, synthetic or native-sequence peptides, immunoadhesins, and small- molecule antagonists that bind to a B-cell surface marker or a B-cell specific survival or proliferation factor, optionally conjugated with or fused to another molecule. The preferred antagonist comprises an antibody or immunoadhesin. It includes BLyS antagonists such as immunoadhesins, and is preferably anti-CD23 {e.g., lumiliximab), anti-CD20, anti-CD22, or anti-BR3 antibodies, APRIL antagonists, and/or BLyS immunoadhesins. The BLyS immunoadhesin preferably is selected from the group consisting of BR3 immunoadhesin comprising the extracellular domain of BR3, TACI immunoadhesin comprising the extracellular domain of TACI, and BCMA immunoadhesin comprising the extracellular domain of BCMA. The most preferred BR3 immunoadhesin is hBR3-Fc of SEQ ID NO:2 of WO 2005/00351 and US 2005/0095243. See also US 2005/0163775 and WO 2006/068867. Another preferred BLyS antagonist is an anti-BLyS antibody, more preferably wherein the anti-BLyS antibody binds BLyS within a region of BLyS comprising residues 162-275, or an anti-BR3 antibody, more preferably wherein the anti-BR3 antibody binds BR3 in a region comprising residues 23-38 of human BR3. Especially preferred immunoadhesins herein are TACI-Ig, or atacicept, and BR3-Ig. A preferred set of antagonists are to CD20, CD22, BAFF, or APRIL. The antagonist may be, in one aspect, an antibody or TACI-Ig.

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecifϊc antibodies (e.g. bispecifϊc antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater 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, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or 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.

"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 (VR) 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.

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). An "antibody that binds to a B-cell surface marker" or "antibody 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 antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement- dependent cytotoxicity (CDC), inhibition of B-cell proliferation, and/or induction of B-cell death (e.g. via apoptosis). The antibody that binds to a B-cell surface marker may be designated as follows: an antibody that binds to CD20 or CD22 is an "anti- CD20 antibody" or "anti-CD22 antibody," respectively. In a preferred embodiment, the antibody is an anti-CD20, anti-CD22, anti-CD23, anti-CD40, or anti-BR3 antibody. A more preferred antibody is an anti-CD20, anti-CD22, or anti-BR3 antibody. A particularly preferred embodiment is an anti-CD20 or anti-CD22 antibody, and most preferably the antibody is an anti-CD20 antibody. Examples of anti-CD20 antibodies include: "C2B8," which is now called

"rituximab" ("RITUXAN®/MABTHERA®") (U.S. 5,736,137); the yttrium-[90]- labelled 2B8 murine antibody designated "Y2B8" or "Ibritumomab Tiuxetan" (ZEV ALIN®) commercially available from Biogen Idee, Inc. (e.g., U.S. 5,736,137; 2B8 deposited with ATCC under accession no. HBl 1388 on June 22, 1993); murine IgG2a "Bl," also called "Tositumomab," optionally labelled with 131I to generate the "13 H-Bl" or "iodine 1131 tositumomab" antibody (BEXXAR™) commercially available from Corixa (see, also, e.g., U.S. 5,595,721); murine monoclonal antibody "1F5" (e.g., Press et al. Bloo d 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. 5,677,180); a 2H7 antibody (e.g., WO 2004/056312 (Lowman et al.) and as set forth below); HUMAX-CD20™ (ofatumumab) 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 ); a chimerized or humanized monoclonal antibody having a high binding affinity to an extracellular epitope of a CD20 antigen described in WO 2006/106959 (Numazaki et al, Biomedics Inc.); 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 including, but not limited to, TRU-015 (e.g., US 2005/0186216 (Ledbetter and Hayden-Ledbetter); US 2005/0202534 (Hayden-Ledbetter and Ledbetter); US

2005/0202028 (Hayden-Ledbetter and Ledbetter); US 2005/136049 (Ledbetter et al); US 2005/0202023 (Hayden-Ledbetter and Ledbetter) - Trubion Pharm Inc.); CD20- binding molecules such as the AME series of antibodies, e.g., AME-133™ antibodies as set forth, for example, in WO 2004/103404; US 2005/0025764; and US 2006/0251652 (Watkins et al, Applied Molecular Evolution, Inc.) and the anti-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 and other anti-CD20 antibodies as described, for example, in U.S. 7,151,164 (Hansen et al, Immunomedics; US 2005/0106108 (Leung and Hansen; Immunomedics); fully human antibodies against CD20 as described, e.g., in WO 2006/130458; Gazit et al, Amgen/AstraZeneca); antibodies against CD20 as described, for example, in WO 2006/126069 (Morawala, Avestha Gengraine Technologies Pvt Ltd.); chimeric or humanized B-LyI antibodies to CD20 (e.g., GA- 101) as described, for example, in WO 2005/044859; US 2005/0123546; US 2004/0072290; and US 2003/0175884 (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 orNU-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 anti-CD20 antibodies herein are chimeric, humanized, or human anti-CD20 antibodies, more preferably rituximab, a 2H7 antibody, chimeric or humanized A20 antibody (Immunomedics), and HUMAX-CD20™ human anti-CD20 antibody (Genmab).

Examples of anti-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), U.S. 5,484,892 (Dana- Farber), U.S. 6,183,744 (Immunomedics, epratuzumab), and U.S. 7,074,403 (Goldenberg and Hansen).

Preferred specific examples of antibodies to B-cell surface markers include rituximab, a 2H7 antibody and variants thereof as defined herein, 2F2 (HUMAX- CD20™) (ofatumumab) human anti-CD20 antibody (an IgG LK- human MAb that binds to a different CD20 epitope than rituximab), humanized A20 antibody veltuzumab (IMMUN- 106™ or bA20), a humanized engineered antibody with complementarity-determining regions (CDRs) of murine origin and with 90% of the human framework regions identical to epratuzumab (a humanized anti-CD22 IgGl antibody); a small, modular immunopharmaceutical (SMIP) (herein called immunopharmaceutical) having SEQ ID NO : 16 (also known as TRU-015), a CD20- binding molecule that is an antibody comprising SEQ ID NOS: 17 and 18 (Lilly AME 33) or SEQ ID NOS: 19 and 20 (Lilly AME 133) or SEQ ID NO:21 (Lilly AME 133v, otherwise known as LY2469298, which binds with an increased affinity to the FcTRIIIa (CD16)), a humanized type II anti-CD20 antibody of the isotype IgGl with a glycoengineered Fc portion (bisected afucosylated carbohydrates in the Fc region) and a modified elbow hinge, known as GAlOl (see SEQ ID NOS:22-23 below), anti- BAFF antibody, anti-APRIL antibodies, anti-BR3 antibody, anti-BAFF receptor antibody, anti-BLyS antibody, anti-CD23 antibody such as lumiliximab, anti-CD37 antibody and antagonists including the small modular immunopharmaceutical drug TRUO 16™, anti-CD40 antibody, and anti-CD22 antibody such as epratuzumab, ABIOGEN™ anti-CD22 antibody, and IMPHERON™ anti-B cell antibody. Preferred examples of immunoadhesins herein include BR3-Ig, BR3-Fc, and APRIL immunoadhesins such as TACI-Ig, anti-BAFF peptibody, BCMA-Ig, and BAFF-R-Ig (US 2006/0263349). The TRU-015 polypeptide sequence is:

Met Asp Phe GIn VaI GIn He Phe Ser Phe Leu Leu He Ser Ala Ser VaI He Met Ser Arg GIy GIn He VaI Leu Ser GIn Ser Pro Ala He Leu Ser Ala Ser Pro GIy GIu Lys VaI Thr Met Thr Cys Arg Ala Ser Ser Ser VaI Ser Tyr Met His Trp Tyr GIn GIn Lys Pro GIy Ser Ser Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser GIy VaI Pro Ala Arg Phe Ser GIy Ser GIy Ser GIy Thr Ser Tyr Ser Leu Thr He Ser Arg VaI GIu Ala GIu Asp Ala Ala Thr Tyr Tyr Cys GIn GIn Trp Ser Phe Asn Pro Pro Thr Phe GIy Ala GIy Thr Lys Leu GIu Leu Lys Asp GIy GIy GIy Ser GIy GIy GIy GIy Ser GIy GIy GIy GIy Ser Ser GIn Ala Tyr Leu GIn GIn Ser GIy Ala GIu Ser VaI Arg Pro GIy Ala Ser VaI Lys Met Ser Cys Lys Ala Ser GIy Tyr Thr Phe Thr Ser Tyr Asn Met His Trp VaI Lys GIn Thr Pro Arg GIn GIy Leu GIu Trp He GIy Ala He Tyr Pro GIy Asn GIy Asp Thr Ser Tyr Asn GIn Lys Phe Lys GIy Lys Ala Thr Leu Thr VaI Asp Lys Ser Ser Ser Thr Ala Tyr Met GIn Leu Ser Ser Leu Thr Ser GIu Asp Ser Ala VaI Tyr Phe Cys Ala Arg VaI VaI Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp VaI Trp GIy Thr GIy Thr Thr VaI Thr VaI Ser Asp GIn GIu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Ser Ala Pro GIu Leu Leu GIy GIy Pro Ser VaI Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro GIu VaI Thr Cys VaI VaI VaI Asp VaI Ser His GIu Asp Pro GIu VaI Lys Phe Asn Trp Tyr VaI Asp GIy VaI GIu VaI His Asn Ala Lys Thr Lys Pro Arg GIu GIu GIn Tyr Asn Ser Thr Tyr Arg VaI VaI Ser VaI Leu Thr VaI Leu His GIn Asp Trp Leu Asn GIy Lys GIu Tyr Lys Cys Lys VaI Ser Asn Lys Ala Leu Pro Ala Pro He GIu Lys Thr He Ser Lys Ala Lys GIy GIn Pro Arg GIu Pro

GIn VaI Tyr Thr Leu Pro Pro Ser Arg Asp GIu Leu Thr Lys Asn GIn VaI Ser Leu Thr Cys Leu VaI Lys GIy Phe Tyr Pro Ser Asp He Ala VaI GIu Trp GIu Ser Asn GIy GIn Pro GIu Asn Asn Tyr Lys Thr Thr Pro Pro VaI Leu Asp Ser Asp GIy Ser Phe Phe Leu Tyr Ser Lys Leu Thr VaI Asp Lys Ser Arg Trp GIn GIn GIy Asn VaI Phe Ser Cys Ser VaI Met His GIu Ala Leu His Asn His Tyr Thr GIn Lys Ser Leu Ser Leu Ser Pro GIy Lys (SEQ ID NO: 16)

See also US 2007/0059306.

The polypeptide representing the light-chain variable region of the AME 33 antibody has the following sequence: GIu He VaI Leu Thr GIn Ser Pro GIy Thr Leu Ser Leu Ser Pro GIy GIu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser VaI Pro Tyr He His Trp Tyr GIn GIn Lys Pro GIy GIn Ala Pro Arg Leu Leu He Tyr Ala Thr Ser Ala Leu Ala Ser GIy He Pro Asp Arg Phe Ser GIy Ser GIy Ser GIy Thr Asp Phe Thr Leu Thr He Ser Arg Leu GIu Pro GIu Asp Phe Ala VaI Tyr Tyr Cys GIn GIn Trp Leu Ser Asn Pro Pro Thr Phe GIy GIn GIy Thr Lys Leu GIu He Lys (SEQ ID NO: 17)

The polypeptide representing the heavy-chain variable region of the AME 33 antibody has the following sequence: GIu VaI GIn Leu VaI GIn Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Lys He Ser Cys Lys GIy Ser GIy Arg Thr Phe Thr Ser Tyr Asn Met His Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Ala He Tyr Pro Leu Thr GIy Asp Thr Ser Tyr Asn GIn Lys Ser Lys Leu GIn VaI Thr He Ser Ala Asp Lys Ser He Ser Thr Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Ser Thr Tyr VaI GIy GIy Asp Trp GIn Phe Asp VaI Trp GIy Lys GIy Thr Thr VaI Thr VaI Ser Ser (SEQ ID NO: 18)

See also Figures 2-3 as well as SEQ ID NOS:59-62 of US 2005/0025764 and US 2006/0251652, for light- and heavy-chain variable region nucleotide and amino acid AME 33 sequences. The polypeptide representing the light-chain variable region of the AME 133 antibody has the following sequence:

GIu He VaI Leu Thr GIn Ser Pro GIy Thr Leu Ser Leu Ser Pro GIy GIu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser VaI Pro Tyr He His Trp Tyr GIn GIn Lys Pro GIy GIn Ala Pro Arg Leu Leu He Tyr Ala Thr Ser Ala Leu Ala Ser GIy He Pro Asp Arg Phe Ser GIy Ser GIy Ser GIy Thr Asp Phe Thr Leu Thr He Ser Arg Leu GIu Pro GIu Asp Phe Ala VaI Tyr Tyr Cys GIn GIn Trp Leu Ser Asn Pro Pro Thr Phe GIy GIn GIy Thr Lys Leu GIu He Lys Arg Thr VaI Ala Ala Pro Ser VaI Phe He Phe Pro Pro Ser Asp GIu GIn Leu Lys Ser GIy Thr Ala Ser VaI VaI Cys Leu Leu Asn Asn Phe Tyr Pro Arg GIu Ala Lys VaI GIn Trp Lys VaI Asp Asn Ala Leu GIn Ser GIy Asn Ser GIn GIu Ser VaI Thr GIu GIn Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr GIu Lys His Lys VaI Tyr Ala Cys GIu VaI Thr His GIn GIy Leu Ser Ser Pro VaI Thr Lys Ser Phe Asn Arg GIy GIu Cys (SEQ ID NO: 19) The polypeptide representing the heavy-chain variable region of the AME 133 antibody has the following sequence: GIu VaI GIn Leu VaI GIn Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser Leu Lys He Ser Cys Lys GIy Ser GIy Arg Thr Phe Thr Ser Tyr Asn Met His Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Ala He Tyr Pro Leu Thr GIy Asp Thr Ser Tyr Asn GIn Lys Ser Lys Leu GIn VaI Thr He Ser Ala Asp Lys Ser He Ser Thr Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Ser Thr Tyr VaI GIy GIy Asp Trp GIn Phe Asp VaI Trp GIy Lys GIy Thr Thr VaI Thr VaI Ser Ser (SEQ ID NO:20)

See also US 2005/0136044.

The polypeptide representing AME 133v, a fusion protein prepared from the AME 133 Fab region fused to modified BChE variant L530, has the following sequence: GIu VaI GIn Leu VaI GIn Ser GIy Ala GIu VaI Lys Lys Pro GIy GIu Ser

Leu Lys He Ser Cys Lys GIy Ser GIy Arg Thr Phe Thr Ser Tyr Asn Met His Trp VaI Arg GIn Met Pro GIy Lys GIy Leu GIu Trp Met GIy Ala He Tyr Pro Leu Thr GIy Asp Thr Ser Tyr Asn GIn Lys Ser Lys Leu GIn VaI Thr He Ser Ala Asp Lys Ser He Ser Thr Ala Tyr Leu GIn Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Ser Thr Tyr VaI GIy GIy Asp

Trp GIn Phe Asp VaI Trp GIy Lys GIy Thr Thr VaI Thr VaI Ser Ser Ala Ser Thr Lys GIy Pro Ser VaI Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GIy GIy Thr Ala Ala Leu GIy Cys Leu VaI Lys Asp Tyr Phe Pro GIu Pro VaI Thr VaI Ser Trp Asn Ser GIy Ala Leu Thr Ser GIy VaI His Thr Phe Pro Ala VaI Leu GIn Ser Ser GIy Leu Tyr Ser Leu Ser Ser VaI VaI Thr VaI Pro Ser Ser Ser Leu GIy Thr GIn Thr Tyr He Cys Asn VaI Asn His Lys Pro Ser Asn Thr Lys VaI Asp Lys Lys Ala GIu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Lys Leu GIu Asp Asp He He He Ala Thr Lys Asn GIy Lys VaI Arg GIy Met Asn Leu Thr VaI Phe GIy GIy Thr VaI Thr Ala Phe Leu GIy He Pro Tyr Ala GIn Pro Pro Leu GIy Arg Leu Arg Phe Lys Lys

Pro GIn Ser Leu Thr Lys Trp Ser Asp He Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys Cys GIn Asn He Asp GIn Ser Phe Pro GIy Phe Phe GIy Ser GIu Met Trp Asn Pro Asn Thr Asp Leu Ser GIu Asp Cys Leu Tyr Leu Asn VaI Trp He Pro Ala Pro Lys Pro Lys Asn Ala Thr VaI Leu He Trp He Tyr GIy GIy GIy Phe GIn Thr GIy Thr Ser Ser Leu His VaI Tyr Asp GIy Lys Phe Leu Ala Arg VaI GIu Arg VaI He VaI VaI Ser Met Asn Tyr Arg VaI GIy Ala Leu GIy Phe Leu Ala Leu Pro GIy Asn Pro GIu Ala Pro GIy Asn Met GIy Leu Phe Asp GIn GIn Leu Ala Leu GIn Trp VaI GIn Lys Asn He Ala Ala Phe GIy GIy Asn Pro Lys Ser VaI Thr Leu Phe GIy GIu Ser Ala GIy Ala Ala Ser VaI Ser Leu His Leu Leu Ser Pro GIy Ser His Ser Leu Phe Thr Arg Ala He Leu GIn Ser GIy Ser Ala Asn Ala Pro Trp Ala VaI Thr Ser Leu Tyr GIu Ala Arg Asn Arg Thr Leu Asn Leu Ala Ly s Leu Thr GIy Cys Ser Arg GIu Asn GIu Thr GIu He He Lys Cys Leu Arg Asn Lys Asp Pro GIn GIu He Leu Leu Asn GIu Ala Phe VaI VaI Pro Tyr GIy Thr Asn Leu Ser VaI Asn Phe GIy Pro Thr VaI Asp GIy Asp Phe Leu Thr Asp Met Pro Asp He Leu Leu GIu Leu GIy GIn Phe Lys Lys Thr GIn He Leu VaI GIy VaI Asn Lys Asp GIu GIy Thr Ala Phe Leu Ala Tyr GIy Ala Pro GIy Phe Ser Lys Asp Asn Asn Ser He He Thr Arg Lys GIu Phe GIn GIu GIy Leu Lys He Phe Phe Pro

GIy VaI Ser GIu Phe GIy Lys GIu Ser He Leu Phe His Tyr Thr Asp Trp VaI Asp Asp GIn Arg Pro GIu Asn Tyr Arg GIu Ala Leu GIy Asp VaI VaI GIy Asp Tyr Asn Phe He Cys Pro Ala Leu GIu Phe Thr Lys Lys Phe Ser GIu Trp GIy Asn Asn Ala Phe Phe Tyr Tyr Phe GIu His Arg Ser Ser Lys Leu Pro Trp Pro GIu Trp Met GIy VaI Met His GIy Tyr GIu He GIu Phe VaI Phe GIy Leu Pro Leu GIu Arg Arg Asp Asn Tyr Thr Lys Ala GIu GIu He Leu Ser Arg Ser He VaI Lys Arg Trp Ala Asn Phe Ala Lys Tyr GIy Asn Pro Asn GIu Thr GIn Asn Asn Ser Thr Ser Trp Pro VaI Phe Lys Ser Thr GIu GIn Lys Tyr Leu Thr Leu Asn Thr GIu Ser Thr Arg He Met Thr Lys Leu Arg Ala GIn GIn Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys VaI (SEQ ID NO:21)

See also SEQ ID NO:202 and Fig. 19 from US 2005/0136044.

The polypeptide representing the light-chain variable region of the humanized type II anti-CD20 IgGl antibody (GAlOl) has the following sequence: Asp He VaI Met Thr GIn Thr Pro Leu Ser Leu Pro VaI Thr Pro GIy GIu Pro Ala Ser He Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn GIy He Thr Tyr Leu Tyr Trp Tyr Leu GIn Lys Pro GIy GIn Ser Pro GIn Leu Leu He Tyr GIn Met Ser Asn Leu VaI Ser GIy VaI Pro Asp Arg Phe Ser GIy Ser GIy Ser GIy Thr Asp Phe Thr Leu Lys He Ser Arg VaI GIu Ala GIu Asp VaI GIy VaI Tyr Tyr Cys Ala GIn Asn Leu GIu Leu Pro Tyr Thr Phe GIy GIy GIy Thr Lys VaI GIu He Lys Arg Thr VaI (SEQ ID NO:22)

The polypeptide representing the heavy-chain variable region of the humanized type II anti-CD20 IgGl antibody (GAlOl) has the following sequence: GIn VaI GIn Leu VaI GIn Ser GIy Ala GIu VaI Lys Lys Pro GIy Ser Ser VaI Lys VaI Ser Cys Lys Ala Ser GIy Tyr Ala Phe Ser Tyr Ser Trp Met Asn Trp VaI Arg GIn Ala Pro GIy GIn GIy Leu GIu Trp Met GIy Arg He Phe Pro GIy Asp GIy Asp Thr Asp Tyr Asn GIy Lys Phe Lys GIy Arg VaI Thr He Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met GIu Leu Ser Ser Leu Arg Ser GIu Asp Thr Ala VaI Tyr Tyr Cys Ala Arg Asn VaI Phe Asp GIy Tyr Trp Leu VaI Tyr Trp GIy GIn GIy Thr Leu VaI Thr VaI Ser Ser (SEQ ID NO:23)

See also US 2005/0123546 regarding BHH2-KV1-GE (GAlOl), which was humanized by grafting CDR sequences from murine B-IyI on framework regions with fully human IgGl-kappa germline sequences. Figure 7 of US 2005/0123546 lists a selection of predicted CDR regions of B-IyI . The sequence for the BHH2 component of GAlOl (the heavy-chain variable region) is presented in Tables 2 and 3 thereof as SEQ ID NOS:31 (nucleotide) and 32 (amino acid). The KVl component (the light- chain variable region) is presented in Tables 2 and 3 thereof as SEQ ID NOS :75 (nucleotide) and 76 (amino acid). The apparent variable heavy-chain and light-chain signal sequences are also set forth in these Tables as SEQ ID NOS :73 (variable heavy-chain, nucleotide), 74 (variable heavy-chain, amino acid), 77 (variable light- chain, nucleotide), and 76 (variable light-chain, amino acid).

The "variable region" or "variable domain" of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

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 (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et at., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. 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 (λ), based on the amino acid sequences of their constant domains.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and MoI. Immunology, 4th ed. (W. B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

A "naked antibody" for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel. "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')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. 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(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment which contains a complete antigen- binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHl) 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 CHl 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 a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthϋn, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer- Verlag, New York: 1994), pp 269-315.

The term "diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). 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 may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med., 9:129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med., 9: 129-134 (2003). 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 except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal-antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal-antibody preparations are advantageous in that they are typically 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 a variety of techniques, including, for example, the hybridoma method {e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14(3) :253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas, 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. 4,816,567), phage-display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. MoI. Biol, 222:581-597 (1992); Sidhu et al., J. MoI. Biol, 338(2):299-310 (2004); Lee et al, J. MoI Biol, 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA,

101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods, 284(1-2):119- 132(2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al, Nature, 362: 255-258 (1993); Bruggemann et al, Year in Immunol, 7:33 (1993); U.S. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio/Technology, 10: 779-783 (1992); Lonberg et al, Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al, Nature Biotechnol, 14:845-851 (1996); Neuberger, Nature Biotechnol, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol, 13:65-93 (1995).

The monoclonal antibodies herein specifically include "chimeric" antibodies 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 (e.g., U.S. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity. In some instances, 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 may be made to further refine antibody performance. In general, a 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. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., 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). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol., 1 :105-115 (1998); Harris, Biochem. Soc. Transactions, 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994); and U.S. 6,982,321 and 7,087,409. A "human antibody" is one which possesses an amino-acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries.

Hoogenboom and Winter, J. MoI. Biol, 227:381 (1991); Marks et al, J. MoI Biol, 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J. Immunol, 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody-variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (Hl, H2, H3), and three in the VL (Ll, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity, 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology, 248: 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature, 363:446-448 (1993) and Sheriff s al., Nature Struct. Biol., 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. The HVRs that are Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody-modeling software. The "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.

Figure imgf000070_0001

HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (Ll), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (Hl), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-HVR definitions.

"Framework" or "FR" residues are those variable-domain residues other than the HVR residues as herein defined. The expression "variable-domain residue-numbering as in Kabat" or "amino- acid-position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al, supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues {e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

An "affinity-matured" antibody is one with one or more alterations in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). In one embodiment, an affinity-matured antibody has nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. For example, Marks et al, Bio/Technology, 10:779-783 (1992) describes affinity maturation by VH- and VL-domain shuffling. Random mutagenesis of HVR and/or framework residues is described by, for example: Barbas et al, Proc Nat. Acad. Sci. USA, 91 :3809-3813 (1994); Schier et al, Gene, 169: 147- 155 (1995); Yelton et al, J. Immunol, 155:1994-2004 (1995); Jackson et al, J. Immunol, 154(7):3310-3319 (1995); and Hawkins et al, J. MoI Biol, 226:889-896 (1992). "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.

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).

Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native-sequence Fc region or amino-acid-sequence-variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: CIq binding and complement- dependent cytotoxicity (CDC); Fc- receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell-surface receptors (e.g. B-cell receptor); and B- cell activation. The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The "EU index as in Kabat" refers to the residue numbering of the human IgGl EU antibody.

A "functional Fc region" possesses an "effector function" of a native-sequence Fc region. Exemplary "effector functions" include CIq binding; CDC; Fc-receptor binding; ADCC; phagocytosis; down-regulation of cell-surface receptors (e.g. B-cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody-variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein.

A "native-sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native-sequence human Fc regions include a native-sequence human IgGl Fc region (non-A and A allotypes); native-sequence human IgG2 Fc region; native-sequence human IgG3 Fc region; and native-sequence human IgG4 Fc region, as well as naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence which differs from that of a native- sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native- sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native-sequence Fc region and/or with an Fc region of a parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

The term "Fc-region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to this invention can comprise an antibody with K447, with all K447 removed, or a mixture of antibodies with and without the K447 residue.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native-human FcR. In some embodiments, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcγRIIA contains an immunoreceptor tyrosine -based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et ah, Immunomethods 4:25-34 (1994); and de Haas et ah, J. Lab. CHn. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

The term "Fc receptor" or "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et ah, J. Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)) and regulation of homeostasis of immunoglobulins. Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunology Today, l% (12):592-8 (1997); Ghetie et al, Nature Biotechnology, 15 (7):637-40 (1997); Hinton et al, J. Biol. Chem.,279(β):6213-6 (2004); WO 2004/92219 (Hinton et al.). Binding to human FcRn in vivo and serum half-life of human FcRn high- affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides with a variant Fc region are administered. WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See, also, for example, Shields et al., J. Biol. Chem., 9(2): 6591-6604 (2001).

"Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector function(s). Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural-killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. The effector cells may be isolated from a native source, e.g., from blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in 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. 5,500,362 or 5,821,337 or U.S. 6,737,056 (Presta), may be performed. Useful effector cells for such assays include PBMC and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al, Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

"Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to antibodies (of the appropriate subclass), which are bound to their 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. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased CIq binding capability are described, e.g., in U.S. 6,194,551 and WO 1999/51642. See, also, e.g., Idusogie et al, J. Immunol. 164:4178-4184 (2000).

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule {e.g. , an antibody) and its binding partner {e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair {e.g. , antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described in the following.

In one embodiment, the "Kd" or "Kd value" according to this invention is measured by a radiolabeled antigen-binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Solution-binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. MoI. Biol, 293:865-881 (1999)). To establish conditions for the assay, microtiter plates (DYNEX Technologies, Inc.) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ I]-antigen are mixed with serial dilutions of a Fab of interest {e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al, Cancer Res., 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% TWEEN-20™ surfactant in PBS. When the plates have dried, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays. According to another embodiment, the Kd or Kd value is measured by using surface-plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'- (3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately ten response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN 20™ surfactant (PBST) at 25°C at a flow rate of approximately 25 μl/min. Association rates (kon) and dissociation rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, e.g., Chen et al., J. MoI. Biol, 293:865-881 (1999). If the on-rate exceeds 106 M-1S"1 by the surface-plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence-emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow-equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. An "on-rate," "rate of association," "association rate," or "kon" according to this invention can also be determined as described above using a BIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc., Piscataway, NJ).

The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody of the invention and the other associated with a reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.

The phrase "substantially reduced," or "substantially different," as used herein, denotes a sufficiently high degree of difference between two numeric values (generally one associated with a molecule and the other associated with a reference/comparator molecule) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the value for the reference/comparator molecule.

The terms "rituximab" or "RITUXAN®" herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated "C2B8" in U.S. 5,736,137, including fragments thereof which retain the ability to bind CD20.

Purely for the purposes herein and unless indicated otherwise, "2H7" or "2H7 antibody" refers to a humanized anti-CD20 antibody with the sequences provided immediately below and/or described in US 2006/0034835 and WO 2004/056312 (both Lowman et al.); US 2006/0188495 ( Barron et al); and US 2006/0246004 (Adams et al). Briefly, humanization of the murine anti-human CD20 antibody, 2H7 (also referred to herein as m2H7, m for murine), was carried out in a series of site- directed mutagenesis steps. The murine 2H7 antibody variable region sequences and the chimeric 2H7 with the mouse V and human C have been described, e.g., in U.S. Pat. Nos. 5,846,818 and 6,204,023. The CDR residues of 2H7 were identified by comparing the amino acid sequence of the murine 2H7 variable domains (disclosed in U.S. 5,846,818) with the sequences of known antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, Ed. 5 (Public Health Service, National Institutes of Health, Bethesda, MD, 1991)). The CDR regions for the light and heavy chains were defined based on sequence hypervariability (Kabat et al., supra). Using synthetic oligonucleotides, site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. (USA), 82:488-492 (1985)) was used to introduce all six of the murine 2H7 CDR regions into a complete human Fab framework corresponding to a consensus sequence VKI, VHIII (VL kappa subgroup I, VH subgroup III) contained on plasmid pVX4 (see Fig. 2 in WO 2004/056312). Further modifications of the V regions (CDR and/or FR) were made in the phagemid pVX4 by site-directed mutagenesis. Plasmids for expression of full-length IgG 's were constructed by subcloning the VL and VH domains of chimeric 2H7 Fab as well as humanized Fab versions 2 to 6 into previously described pRK vectors for mammalian cell expression (Gorman et al, DNA Prot. Eng. Tech., 2:3-10 (1990)).

The following 2H7 antibodies are included within the definition herein: (I) A humanized antibody comprising the VL sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNL ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO :1); and the VH sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSS (SEQ ID NO:2). (2) A humanized antibody comprising the VL sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNL ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO:3); and the VH sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SASYWYFDVWGQGTLVTVSS (SEQ ID NO:4). (3) A humanized antibody comprising the VL sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNL ASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO:3); and the VH sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SYRYWYFDVWGQGTLVTVSS (SEQ ID NO:5).

(4) A humanized antibody comprising a full length light (L) chain having the sequence of SEQ ID NO:6, and a full length heavy (H) chain having the sequence of one of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:15, wherein the sequences are indicated below.

(5) A humanized antibody comprising a full length light (L) chain having the sequence of SEQ ID NO:9, and a full length heavy (H) chain having the sequence of one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO: 14, wherein the sequences are indicated below.

SEQ ID NO:6:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNL

ASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSV

FIFPPS

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

YSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 7:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSL

SLSPGK

SEQ ID NO:8:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:9:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNL ASGVPSR

FSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSV FIFPPS

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST

YSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 10:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSL

SLSPGK

SEO ID NO:! ! :

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCAVSNKALPAPIEATISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSL SLSPGK

SEO ID NO: 12:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY

SASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN

HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP

EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMT

KNQVSLTCLVKGFYPSDIAVEW

ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

NHYTQKSL

SLSPGK

SE0 ID N0:13:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEW

ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH

WHYTQKSL

SLSPGK

SEQ ID NO: 14:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI

YPGNGATSY

NQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDV

WGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

TFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQ

YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREP

QVYTLPPSR

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEO ID NO:15:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAI YPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYY SNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTP EVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLT VLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The murine anti-human CD20 antibody, m2H7 has the sequences: VL sequence: QIVLSQSPAI LSASPGEKVT MTCRASSSVS YMHWYQQKPG SSPKPWIYAP SNLASGVPAR

FSGSGSGTSY SLTISRVEAE DAATYYCQQW SFNPPTFGAG TKLELK (SEQ ID NO:24) VH sequence: QAYLQQSGAE LVRPGASVKM SCKASGYTFT SYNMHWVKQT

PRQGLEWIGA IYPGNGDTSY NQKFKGKATL TVDKSSSTAY MQLSSLTSED SAVYFCARVV YYSNSYWYFD VWGTGTTVTV S (SEQ ID NO:25)

In the B-cell-surface marker-binding antibodies that comprise an Fc region, the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the Ab or by recombinant engineering the nucleic acid encoding the antibody polypeptide. For example, 2H7 or another humanized antibody herein can comprise an Fc region including the K447 residue, or with all the K447 residues removed, or a mixture of antibodies having Fc regions with and without the K447 residue. In certain embodiments, the humanized antibody useful herein further comprises amino acid alterations in the IgG Fc and exhibits increased binding affinity for human FcRn over an antibody having wild-type IgG Fc, by at least 60 fold, at least 70 fold, at least 80 fold, more preferably at least 100 fold, preferably at least 125 fold, even more preferably at least 150 fold to about 170 fold. The N-glycosylation site in IgG is at Asn297 in the CH2 domain. Included for use in therapy herein are compositions of any humanized antibodies having an Fc region, wherein about 80-100% (and preferably about 90-99%) of the antibody in the composition comprises a mature core carbohydrate structure that lacks fucose, attached to the Fc region of the glycoprotein, or has reduced fucose content. A "bispecific humanized antibody" encompasses an antibody wherein one arm of the antibody has at least the antigen binding region of the H and/or L chain of a humanized antibody of the invention, and the other arm has V-region binding specificity for a second antigen. In specific embodiments, the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD 16, NKG2D, or other NK-activating ligands.

The terms "BAFF," "BAFF polypeptide," "TALL-I" or "TALL-I polypeptide," "BLyS", and "THANK" when used herein encompass "native-sequence BAFF polypeptides" and "BAFF variants." "BAFF" is a designation given to those polypeptides that have the human BAFF sequence as set forth in, for example, US 2006/0110387, and homo logs and fragments and variants thereof, which have the biological activity of the native-sequence 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. The term "BAFF" includes those polypeptides described in Shu et ah, J. Leukocyte Biol., 65:680 (1999); GenBank Accession No. AF136293; WO 1998/18921; EP 869,180; WO 1998/27114; WO 1999/12964; WO 1999/33980; 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).

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 BR3 polypeptide to block, partially or fully, BR3 interaction with BAFF polypeptide, and (2) partially or fully blocks, inhibits, or neutralizes native- sequence BAFF signaling. Native-sequence BAFF polypeptide signaling promotes, among other things, B-cell survival and B-cell maturation. The inhibition, blockage, or neutralization of BAFF signaling results in, inter alia, a reduction in the number of B cells. A BAFF antagonist as defined herein will partially or fully block, inhibit, or neutralize one or more biological activities of a BAFF polypeptide, in vitro or in vivo. In one embodiment, a biologically active BAFF potentiates any one or a combination of the following events in vitro 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-κb2/100 to p52 NF-κβ in splenic B cells {e.g., Batten et ah, J. Exp. Med. 192:1453-1465 (2000); Moore et al, Science 285:260-263 (1999); and Kayagaki et al, Immunity, 10:515-524 (2002)).

In some embodiments, a BAFF antagonist as defined herein includes anti- BAFF antibodies, BAFF-binding polypeptides (including immunoadhesins and peptides), and BAFF-binding small molecules. BAFF antagonists include, for example, the BAFF-binding antibodies described in WO 2002/02641 {e.g., antibodies comprising the amino acid sequence of any of SEQ ID NOS: 1-46, 321-329, 834-872, 1563-1595, 1881-1905 of Table 1 thereof). In a further embodiment, the immunoadhesin comprises a BAFF-binding region of a BAFF receptor (e.g., an extracellular domain of BR3, BCMA, or TACI). In a still further embodiment, the immunoadhesin is BR3-Fc. Other examples of BAFF-binding Fc proteins can be found in WO 2002/66516, WO 2000/40716, WO 2001/87979, WO 2003/024991, WO 2002/16412, WO 2002/38766, WO 2002/092620, and WO 2001/12812. Methods of making BAFF antagonists are described, for example, in US 2005/0095243 and US 2005/0163775. The terms "BR3", "BR3 polypeptide" or "BR3 receptor" when used herein encompass native- sequence BR3 polypeptides and BR3 variants, as defined hereinbelow. "BR3" is a designation given to those polypeptides comprising, for example, the human BR3 sequence set forth in WO 2003/14294 and US 2005/0070689. 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, WO 2003/14294, and US 2005/0070689. Anti-BR3 antibodies can be prepared in accordance with methods set for in, for example, WO 2003/14294 and US 2005/0070689.

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 (see WO 2003/14294 and US 2005/0070689).

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.

"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.

The term "APRIL antagonist" as used herein is used in the broadest sense, and includes any molecule that (1) binds a native-sequence APRIL polypeptide or binds a native-sequence ligand to APRIL to block, partially or fully, the ligand's interaction with APRIL polypeptide, and (2) partially or fully blocks, inhibits, or neutralizes native-sequence APRIL signaling. Native-sequence APRIL polypeptide signaling promotes, among other things, B-cell survival and B-cell maturation. APRIL (a proliferation-inducing ligand) is a TNF family member with a shared receptor to BAFF. Examples of preferred APRIL antagonists include atacicept (same as TACI-Ig immunoadhesin) and a BAFF/ APRIL antagonist (soluble BCMA-Fc).

As used herein, "rheumatoid arthritis" or "RA" refers to a recognized disease state that may be diagnosed according to the 2000 revised American Rheumatoid Association criteria for the classification of RA, or any similar criteria. The term includes not only active and early RA, but also incipient RA, as defined below. Physiological indicators of RA include, symmetric joint swelling which is characteristic though not invariable in RA. 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 fingers 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 RA 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 RA. Subjects with "early active rheumatoid arthritis" are those subjects with active RA diagnosed for at least 8 weeks but no longer than four years, according to the revised 1987 ACR criteria for the classification of RA. Subjects with "early rheumatoid arthritis" are those subjects with RA diagnosed for at least eight weeks but no longer than four years, according to the revised 1987 ACR criteria for classification of RA. RA includes, for example, juvenile-onset RA, juvenile idiopathic arthritis (JIA), or juvenile RA (JRA). Patients with "incipient RA" have early polyarthritis that does not fully meet ACR criteria for a diagnosis of RA, in association with the presence of RA-specifϊc prognostic biomarkers such as anti-CCP and shared epitope. They include patients with positive anti-CCP antibodies who present with polyarthritis, but do not yet have a diagnosis of RA, and are at high risk for going on to develop bonaβde ACR criteria RA (95% probability).

"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, especially arthritis, and most especially RA. Exemplary such conditions include acute and chronic arthritis, RA including juvenile-onset RA, JIA, or 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, and significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Felty's syndrome). 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. "Treatment" of a subject herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with RA or joint damage as well as those in which the RA or joint damage or the progress of RA or joint damage is to be prevented. Hence, the subject may have been diagnosed as having the RA or joint damage or may be predisposed or susceptible to the RA or joint damage, or may have RA or joint damage that is likely to progress in the absence of treatment. Treatment is successful herein if the RA or joint damage is alleviated or healed, or progression of RA or joint damage, including its signs and symptoms and structural damage, is halted or slowed down as compared to the condition of the subject prior to administration. Successful treatment further includes complete or partial prevention of RA or of the development of joint or structural damage. For purposes herein, slowing down or reducing RA or joint damage or the progression of joint damage is the same as arrest, decrease, or reversal of the RA or joint damage. As used herein, the term "patient" refers to any single animal, more preferably a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. Most preferably, the patient herein is a human.

A "subject" herein is any single human subject, including a patient, eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of RA or joint damage, whether, for example, newly diagnosed or previously diagnosed and now experiencing a recurrence or relapse, or is at risk for RA or joint damage, no matter the cause. Intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects once used as controls. The subject may have been previously treated with a medicament for RA or joint damage, including a B-cell antagonist, or not so treated. The subject may be naϊve to a second medicament being used when the treatment herein is started, i.e., the subject may not have been previously treated with, for example, an immunosuppressive agent such as MTX at "baseline" (i.e., at a set point in time before the administration of a first dose of antagonist in the treatment method herein, such as the day of screening the subject before treatment is commenced). Such "naϊve" subjects are generally considered to be candidates for treatment with such second medicament.

"Clinical improvement" refers to prevention of further progress of RA or joint damage or any improvement in RA or joint damage as a result of treatment, as determined by various testing, including 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.

For purposes herein, a subject is in "remission" if he/she has no symptoms of RA or active joint damage, such as those detectable by the methods disclosed herein, and has had no progression of RA or 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 RA or joint damage. Such subjects experiencing a return of symptoms, including active RA or joint damage, are those who have "relapsed" or had a "recurrence." A "symptom" of RA or joint damage is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the subject and indicative of RA or joint damage, such as those noted above, including tender or swollen joints.

The expression "effective amount" refers to an amount of a medicament that is effective for treating RA or joint damage. This would include an amount that is effective in achieving a reduction in RA or joint damage as compared to baseline prior to administration of such amount as determined, e.g., by radiographic or other testing. An effective amount of a second medicament may serve not only to treat the RA or joint damage in conjunction with the antagonist herein, but also serve to treat undesirable effects, including side-effects or symptoms or other conditions accompanying RA or joint damage, including a concomitant or underlying disease or disorder.

"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 the antagonist herein). A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CTX (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as MTX and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, MTX, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); CTX; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhόne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-I l; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. 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. 4,665,077); 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 CTX; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. 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- MEDRO L® methylprednisolone sodium succinate, and dexamethasone; dihydrofolate reductase inhibitors such as MTX (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-TNF-alpha antibodies (infliximab (REMICAD E®) or adalimumab), anti-TNF-alpha immunoadhesin (etanercept), anti- TNF-beta antibodies, anti- IL-2 antibodies and anti-IL-2 receptor antibodies, and anti- IL-6 receptor antibodies and antagonists (such as ACTEMRA™ (tocilizumab)); anti- LFA-I antibodies, including anti-CDl la and anti-CD 18 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 1990/08187); 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. 5,114,721); T-cell receptor fragments (Offner et al, Science, 251 :430-432 (1991); WO 1990/11294; Ianeway, Nature, 341 :482 (1989); and WO 1991/01133); BAFF antagonists such as anti-BAFF antibodies and anti-BR3 antibodies and zTNF4 antagonists (for review, see Mackay and Mackay, Trends Immunol., 23:113-115 (2002)); biologic agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD 154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al, Science, 261 :1328-1330 (1993); Mohan et al, J. Immunol, 154: 1470-1480 (1995)) and CTLA4-Ig (Finck et ah, Science, 265:1225-1227 (1994)); and T-cell receptor antibodies (EP 340,109) such as T10B9. Some immunosuppressive agents herein are also DMARDs, such as MTX. Examples of preferred immunosuppressive agents herein include CTX, chlorambucil, azathioprine, leflunomide, MMF, or MTX. 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-I, IL-lα, IL-Ib, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, including PROLEUKIN® rIL-2; a tumor necrosis factor such as TNF-α or TNF-β; 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 antagonist" 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.

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, which 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 alphaόbetal, alpha3betal, alpha7betal, the alpha4 chain such as alpha4betal, the beta7 chain such as the beta7 integrin subunit of alpha4beta7 and/or alphaEbeta7, LFA-I 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.

An "integrin antagonist" is a molecule that inhibits or antagonizes such integrins by any mechanism, including, for example, antibodies to the integrin. Examples of "integrin antagonists or antibodies" herein include an LFA-I antibody, such as efalizumab (RAPTIV A®) commercially available from Genentech, or other CDl 1/1 Ia and CD 18 antibodies, or an alpha 4 integrin antibody such as natalizumab (ANTEGREN®) available from Biogen-IDEC, 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. 6,677,339 and 6,348,463), aromatic amine derivatives (U.S. 6,369,229), ADAM disintegrin domain polypeptides (US 2002/0042368), antibodies to alphavbeta3 integrin (EP 633945), anti-beta7 antibodies such as rhuMAb Beta7 (US 2006/0093601) and MLN-02 (Millennium Pharmaceuticals), anti-alpha4 antibodies such as TYSABRI® (Biogen-IDEC-Elan), T0047 (GSK/Tanabe), CDP- 323 (oral) (UCB), aza-bridged bicyclic amino acid derivatives (WO 2002/02556), etc.

For the purposes herein, "tumor necrosis factor alpha" or "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®), infliximab (REMICADE®), and adalimumab (HUMIRA™). Examples of "disease-modifying anti-rheumatic drugs" or "DMARDs" include hydroxycloroquine, sulfasalazine, MTX, leflunomide, etanercept, infliximab (plus oral and subcutaneous MTX), 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): 1125-1139 (2003)), including salts and derivatives thereof, etc. A preferred DMARD herein is MTX.

Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, tolmetin, COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3- (trifluoromethyl)-lH-pyrazol-l-yl) benzenesulfonamide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®), including salts and derivatives thereof, etc. Preferably, they are aspirin, naproxen, ibuprofen, indomethacin, or tolmetin.

"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-MEDRO L® methylprednisolone sodium succinate), dexamethasone or dexamethasone triamcinolone, hydrocortisone, and betamethasone. The preferred corticosteroids herein are prednisone, methylprednisolone, hydrocortisone, or dexamethasone.

A "medicament" is an active drug to treat RA or joint damage or the signs or symptoms or side effects of RA or joint damage.

The term "pharmaceutical formulation" refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

A "sterile" formulation is aseptic or free from all living microorganisms and their spores. A "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products or medicaments, 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 or medicaments, etc. A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g., a medicament for treatment of RA or joint damage, or a probe for specifically detecting a biomarker gene or protein of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention. A "target audience" is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.

The term "sample" shall generally mean any biological sample obtained from an individual, body fluid, body tissue, cell line, tissue culture, or other source. Body fluids are, e.g., lymph, sera, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid and spinal fluid. Samples also include synovial tissue, skin, hair follicle, and bone marrow. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the term "sample" is used alone, it shall still mean that the "sample" is a "biological sample", i.e., the terms are used interchangeably. The term "serum sample" shall generally mean any serum sample obtained from an individual. Methods for obtaining sera from mammals are well known in the art.

The term "biomarker" as used in the present application refers generally to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in a subject's sample can be detected by standard methods (or methods disclosed herein) and is predictive or prognostic of the effective responsiveness or sensitivity of a mammalians subject with RA to a B-cell antagonist. Such biomarkers contemplated by the present invention include, but are not limited to positive acute phase proteins, such as, for example, C-reactive protein (CRP) and serum amyloid A, and rheumatoid arthritis-associated autoantibodies, such as, for example, anti-rheumatoid factor (anti-RF) antibodies. One or more of the positive acute phase protein biomarkers herein are present in a higher amount than a certain threshold level in a sample taken from a patient with RA (a control sample). One or more of the autoantibodies herein have a higher titer than a certain threshold level in a sample taken from a patient with RA. Any genetic biomarkers assessed (e.g., specific mutations and/or SNPs) are present in such a sample, but not in a control sample, or certain biomarkers are seropositive in the sample, but seronegative in a control sample. Also, optionally, expression of such a biomarker may be determined to be higher than that observed for a control sample. The terms "marker" and "biomarker" are used herein interchangeably. The terms "predictive" and "prognostic" as used herein are also interchangeable, in the sense of meaning that the methods for prediction or prognostication are to allow the person practicing the method to select patients that are deemed (usually in advance of treatment, but not necessarily) more likely to respond to treatment with a B-cell antagonist. The term "serum cytokine" as used herein means IL-Ib, TNF-alpha, and/or IL-

6.

The verbs "determine" and "assess" shall have the same meaning and are used interchangeably throughout the application. "Seropositivity" as used herein means showing a positive reaction to a test on blood serum indicated by the presence of a certain autoantibody in the blood sample.

An "effective response" of a patient or a patient's "responsiveness" to treatment with a B-cell antagonist and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for or suffering from RA from or as a result of the treatment with the antagonist, such as an anti-CD20, anti-CD22, or anti- BR3 antibody or BR3-Fc immunoadhesin. Such benefit includes cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from or as a result of the treatment with the antagonist. For example, an effective response can be a higher ACR50 in a patient diagnosed with a lower amount of at least one of the serum cytokines herein versus a patient not diagnosed with lower amounts of one or more of the biomarkers. The incidence of biomarker(s) herein effectively predicts, or predicts with high sensitivity, such effective response. The expression "not responsive to," as it relates to the reaction of subjects or patients to one or more of the medicaments that were previously administered to them, describes those subjects or patients who, upon administration of such medicament(s), did not exhibit any or adequate signs of treatment of the disorder for which they were being treated, or they exhibited a clinically unacceptably high degree of toxicity to the medicament(s), or they did not maintain the signs of treatment after first being administered such medicament(s), with the word treatment being used in this context as defined herein. The phrase "not responsive" includes a description of those subjects who are resistant and/or refractory to the previously administered medication(s), and includes the situations in which a subject or patient has progressed while receiving the medicament(s) that he or she is being given, and in which a subject or patient has progressed within 12 months (for example, within six months) after completing a regimen involving the medicament(s) to which he or she is no longer responsive. The non-responsiveness to one or more medicaments thus includes subjects who continue to have active disease following previous or current treatment therewith. For instance, a patient may have active disease activity after about one to three months of therapy with the medicament(s) to which they are non-responsive. Such responsiveness may be assessed by a clinician skilled in treating the disorder in question. For purposes of non-response to medicament(s), a subject who experiences "a clinically unacceptably high level of toxicity" from previous or current treatment with one or more medicaments experiences one or more negative side-effects or adverse events associated therewith that are considered by an experienced clinician to be significant, such as, for example, serious infections, congestive heart failure, demyelination (leading to multiple sclerosis), significant hypersensitivity, neuropathological events, high degrees of autoimmunity, a cancer such as endometrial cancer, non-Hodgkin's lymphoma, breast cancer, prostate cancer, lung cancer, ovarian cancer, or melanoma, tuberculosis (TB), etc. By "reducing the risk of a negative side effect" is meant reducing the risk of a side effect resulting from treatment with the antagonist herein to a lower extent than the risk observed resulting from treatment of the same patient or another patient with a previously administered medicament. Such side effects include those set forth above regarding toxicity, and are preferably infection, cancer, heart failure, or demyelination.

By "correlate" or "correlating" is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to various embodiments herein, one may use the results of an analytical assay to determine whether a specific therapeutic regimen using a B-cell antagonist, such as anti-CD20 antibody, should be performed. The word "label" when used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent such as a nucleic acid probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable {e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

The "amount" or "level" of a biomarker associated with an increased clinical benefit to a RA patient or patient with joint damage is a detectable level in a biological sample. These can be measured by methods known to the expert skilled in the art and also disclosed by this invention. The expression level or amount of biomarker assessed can be used to determine the response to the treatment.

The terms "level of expression" or "expression level" in general are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post-translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the protein, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a protein, and also those that are transcribed into RNA but not translated into a protein (for example, transfer and ribosomal RNAs).

An "algorithm" as used in the methods and systems herein is a specific set of instructions or a definite list of well-defined instructions for carrying out a procedure, typically proceeding through a well-defined series of successive states, and eventually terminating in an end-state, in this case, a binary answer of yes or no to the amount(s) of the biomarker(s) herein.

As used herein, the term "covariate" refers to certain variables or information relating to a patient. The clinical endpoints are frequently considered in regression models, where the endpoints represent the dependent variable and the biomarkers represent the main or target independent variables (regressors). If additional variables from the clinical data pool are considered, they are denoted as (clinical) covariates.

The term "clinical covariate" is used herein to describe all clinical information about the patient, which is in general available at baseline. These clinical covariates comprise demographic information like sex, age, etc., other anamnestic information, concomitant diseases, concomitant therapies, results of physical examinations, common laboratory parameters obtained, known properties of the RA or joint damage, information quantifying the extent of RA disease, clinical performance scores like ECOG or Karnofsky index, clinical disease staging, timing and result of pretreatments, disease history, as well as all similar information that may be associated with the clinical response to treatment.

As used herein, the term "raw analysis" or "unadjusted analysis" refers to regression analyses, wherein besides the considered biomarkers, no additional clinical covariates are used in the regression model, neither as independent factors nor as stratifying covariate.

As used herein, the term "adjusted by covariates" refers to regression analyses, wherein besides the considered biomarkers, additional clinical covariates are used in the regression model, either as independent factors or as stratifying covariate.

As used herein, the term "univariate" refers to regression models or graphical approaches wherein, as an independent variable, only one of the target biomarkers is part of the model. These univariate models can be considered with and without additional clinical covariates.

As used herein, the term "multivariate" refers to regression models or graphical approaches wherein, as independent variables, more than one of the target biomarkers is part of the model. These multivariate models can be considered with and without additional clinical covariates. B. Modes for Carrying Out the Invention:

The present invention provides a method for identifying patients whose RA or joint damage is likely to be responsive to B-cell antagonist therapy. The method is useful, inter alia, for increasing the likelihood that administration of a B-cell antagonist to a patient with RA or joint damage will be efficacious.

The methods and assays disclosed herein are directed to the examination of the amount of one or more biomarkers in a biological sample, wherein the determination of that amount of one or more such biomarkers is predictive or indicative of whether the sample will be sensitive to B-cell antagonists such as antibodies or immunoadhesins.

The disclosed methods and assays provide for convenient, efficient, and potentially cost-effective means to obtain data and information useful in assessing appropriate or effective therapies for treating patients. For example, for the CRP level determination, a patient having been diagnosed with RA could provide a blood sample and the sample could be examined by way of various in vitro assays to determine whether the patient's cells would be sensitive to a therapeutic agent that is a B-cell antagonist, such as an anti-CD20, anti-CD22, or anti-BR3 antibody.

I. Diagnostics The invention provides methods for predicting the sensitivity of a sample to a

B-cell antagonist. The methods may be conducted in a variety of assay formats, including assays detecting protein expression (such as enzyme immunoassays) and biochemical assays detecting appropriate activity. Determination of quantities of such biomarker(s) in the samples is predictive that the patient providing the sample will be sensitive to the biological effects of a B-cell antagonist. The invention herein is that the presence of a higher than threshold or baseline amount/titer of one or more of the acute phase protein and./or autoantibody biomarkers herein in a sample such as a serum sample from a RA patient (the baseline amount being different for each biomarker) would correlate with the observed treatment efficacy of such a patient to a B-cell antagonist.

In one aspect, this invention provides a method of predicting whether a patient with RA will respond effectively to treatment with a B-cell antagonist, comprising assessing, as a biomarker, the level(s) of one or more C-reactive proteins, alone or in combination with seropositivity for one or more rheumatoid arthritis- associated autoantibodies. In a preferred embodiment, the acute phase protein is a C- reactive protein and the autoantibody is an anti-RF antibody. According to the present invention, patients with an elevated level or an acute phase protein (indicative of elevated inflammation) and higher autoantibody (e.g. anti-RF antibody) titer (indicative of B cell involvement) will respond effectively to treatment with the antagonist.

In another embodiment, the present invention provides a method of predicting the sensitivity of a RA patient to a B-cell antagonist. This method comprises assessing the level(s) of one or more of positive acute phase proteins and the titer of a rheumatoid arthritis-associated autoantibody from a patient sample relative to predetermined threshold levels, wherein a level of greater than the predetermined threshold level correlates with high sensitivity of the patient to effective treatment with a B-cell antagonist. According to this method, a serum sample is obtained from the patient and subjected to an assay to evaluate how much of the biomarkers are present in the sample. In one preferred alternative, the level(s) of the biomarkers of the present invention is/are evaluated without any other biomarkers.

In a further aspect a method is provided for predicting whether a subject with RA will respond to a B-cell antagonist, the method comprising determining whether a sample from the subject contains an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody, wherein elevated levels indicate that the subject will respond to the antagonist.

The invention also supplies a method of specifying a B-cell antagonist for use in a RA patient subpopulation, the method comprising providing instruction to administer the B-cell antagonist to a patient subpopulation characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.

The present invention further provides a method of determining the likelihood that a patient with RA will show relatively long symptom-free benefit from therapy with a B-cell antagonist comprising determining the levels of the biomarkers herein. In further expressed embodiments, the present invention provides a method of predicting the sensitivity of a RA patient to effective response to treatment with a B- cell antagonist, or predicting whether a RA patient will respond effectively to treatment with a B-cell antagonist, comprising assessing the level of one or more of the biomarkers identified herein present in the sample; and predicting the sensitivity of the patient to inhibition by a B-cell antagonist, wherein a higher amount than predetermined threshold level(s) of one or more of these biomarkers correlates with high sensitivity of the patient to effective response to treatment with a B-cell antagonist. In these and other embodiments, the positive acute phase protein preferably is a

C-reactive protein (CRP), which is preferably present at a level of at least about 1 mg/dL, or at least about 1.5 mg/dL, or at least about 2 mg/dL, or at least about 2.5 mg/dL, or at least about 3 mg/dL or at least about 3.5 mg/dL, or at least about 3.9 mg/dL, or at least about 4 mg/dL. Preferably, for optimal sensitivity, CRP is present in an amount of at least about 3.9 mg/dL.

In these and other embodiments, the autoantibody preferably is an anti- rheumatoid factor (anti-RF) antibody, where the antibody can be of any isotype (i.e. IgA, IgG, IgM, IgE), or a mixture of one or more different isotypes, including total anti-RF present in the sample. In a preferred embodiment, the titer of the anti-RF antibody is at least about 15 U/ml, or about 20 U/ml, or at least about 25 U/ml, or at least about 30 U/ml, preferably at least about 24 U/ml.

In a preferred embodiment, both the CRP amount and the anti-RF antibody titer are at present at the indicated levels. In another embodiment, the methods herein additionally include determination of the anti-CCP3 antibody titer, where a titer below about 900 U/ml, or below about 870 U/ml, or below about 850 U/ml, combined with the other biomarkers, predicts responsiveness to B-cell antagonist treatment.

In a further embodiment, the methods herein additionally include determination of soluble CD25 concentration, where a concentration below about 4000 pg/ml, or below about 3900 pg/ml, or below about 3800 pg/ml, preferably below 3926 pg/ml, combined with the other biomarkers, predicts responsiveness to B-cell antagonist treatment.

In yet another embodiment, the methods herein additionally include determination of DAS-ESR, where DAS-ESR greater than about 5, or greater than about 6, or greater than about 7, preferably greater than 6.3, combined with the other biomarkers, predicts responsiveness to B-cell antagonist treatment.

Further biomarkers that can be used for predicting and/or monitoring effective response of a patient to a B-cell antagonist treatment include serum amyloid A (SAA), SlOO (e.g. S100A12), osteopontin, matrix metalloprotease 1 (MMP-I), anti- agalactosyl IgG antibodies (CARF), a pro-form of MMP-I such as pro-MMP, matrix metalloprotease 3 (MMP-3), HA, sCD14, antinuclear autoantibodies (ANA), anti- double-stranded DNA antibodies, antibodies to extractable nuclear antigens (ENA), and anti-neutrophil cytoplasmic autoantibodies (ANCA), anti-keratin antibodies (AKA), anti-fϊlaggrin antibody (AFA), angiogenesis markers, and products of bone, cartilage or synovium metabolism. In addition cytokines can be biomarkers, such as one or more of IL-Ib, TNF-alpha, IL-6, IFN-γ, G-CSF, GM-CSF, IL-4, IL-10, IL-13, IL-5, CCL4/MIP-lβ, IL-7, IL-2, GM-CSF, G-CSF, CCL2/MCP-1, EGF, VEGF, CXCL8/IL-8, IL- 12, IL- 17, as well as erythrocyte sedimentation rate and joint counts compared to the severe RA groups.

The reagents to detect the biomarker(s) may be, for example, antibodies, polynucleotides, and other molecules that bind to the biomarkers herein. The hardware is preferably a machine or computer to perform the detection step, and the computational means may be by, for example, computer or machine. Any additional biomarker being measured may be assessed from the same sample or a different biological sample. If the sample is different, it is preferably blood, synovial tissue, or synovial fluid, more preferably blood or synovial fluid, and most preferably blood. One of skill in the medical arts, particularly pertaining to the application of diagnostic tests and treatment with therapeutics, will recognize that biological systems are somewhat variable and not always entirely predictable, and thus many good diagnostic tests or therapeutics are occasionally ineffective. Thus, it is ultimately up to the judgment of the attending physician to determine the most appropriate course of treatment for an individual patient, based upon test results, patient condition and history, and his or her own experience. There may even be occasions, for example, when a physician will choose to treat a patient with a B-cell antagonist even when a patient is not predicted to be particularly sensitive to B-cell antagonists, based on data from diagnostic tests or from other criteria, particularly if all or most of the other obvious treatment options have failed, or if some synergy is anticipated when given with another treatment. The fact that the anti-CD20 antibodies as a class of drugs are relatively well tolerated compared to more traditional immunosuppressive agents used in the treatment of RA makes this a more viable option.

In all the methods described herein the sample is taken from a patient who is suspected to have, or is diagnosed to have RA, and hence is likely in need of treatment. For assessment of marker expression, patient samples, such as those containing cells, or proteins or nucleic acids produced by these cells, may be used in the methods of the present invention. In the methods of this invention, the level of a biomarker can be determined by assessing the amount (e.g. absolute amount or concentration) of the markers in a sample, preferably assessed in bodily fluids or excretions containing detectable levels of biomarkers. Bodily fluids or secretions useful as samples in the present invention include, e.g., blood, urine, saliva, stool, pleural fluid, lymphatic fluid, sputum, ascites, prostatic fluid, cervical vaginal fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivative thereof. The word blood is meant to include whole blood, plasma, serum, or any derivative of blood. Assessment of a biomarker in such bodily fluids or excretions can sometimes be preferred in circumstances where an invasive sampling method is inappropriate or inconvenient. However, the sample to be tested herein is preferably blood/serum, synovial tissue, or synovial fluid, most preferably blood/serum. The sample may be frozen, fresh, fixed (e.g. formalin fixed), centrifuged, and/or embedded (e.g. paraffin embedded), etc. The cell sample can, of course, be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc) prior to assessing the amount of the marker in the sample. Likewise, biopsies may also be subjected to post-collection preparative and storage techniques, e.g., fixation.

Measurement of biomarker expression or protein levels may be performed by using a software program executed by a suitable processor. Suitable software and processors are well known in the art and are commercially available. The program may be embodied in software stored on a tangible medium such as CD-ROM, a floppy disk, a hard drive, a DVD, or a memory associated with the processor, but persons of ordinary skill in the art will readily appreciate that the entire program or parts thereof could alternatively be executed by a device other than a processor, and/or embodied in firmware and/or dedicated hardware in a well known manner. Following the measurement of the protein levels of biomarkers identified herein, or expression of gene biomarkers or their expression products, and the determination that a subject is likely or not likely to respond to treatment with a B-cell antagonist, the assay results, findings, diagnoses, predictions and/or treatment recommendations are typically recorded and communicated to technicians, physicians and/or patients, for example. In certain embodiments, computers will be used to communicate such information to interested parties, such as patients and/or the attending physicians. In some embodiments, the assays will be performed or the assay results analyzed in a country or jurisdiction that differs from the country or jurisdiction to which the results or diagnoses are communicated.

In a preferred embodiment , a diagnosis, prediction, and/or treatment recommendation based on the expression or protein level in a test subject of one or more of the biomarkers herein is communicated to the subject as soon as possible after the assay is completed and the diagnosis and/or prediction is generated. The results and/or related information may be communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, electronic forms of communication, such as email, or telephone. Communication may be facilitated by use of a computer, such as in case of e-mail communications. In certain embodiments, the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software that will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in US 6,283,761; however, the present invention is not limited to methods that utilize this particular communications system. In certain embodiments of the methods of the invention, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

As to physical and quantitative tests for detection of protein biomarkers various protein assays are available. For example, the sample may be contacted with an antibody specific for said biomarker under conditions sufficient for an antibody- biomarker complex to form, and then detecting said complex. The presence of the protein biomarker may be accomplished in a number of ways, such as by Western blotting (with or without immunoprecipitation), 2-dimensional SDS-PAGE, immunoprecipitation, fluorescence activated cell sorting (FACS), flow cytometry, and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These include both single-site and two-site or "sandwich" assays of the noncompetitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.

Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate, and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of biomarker.

Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In a typical forward sandwich assay, a first antibody having specificity for the biomarker is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g., from room temperature to 400C such as between 25° C and 32° C inclusive) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the biomarker. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the molecular marker.

An alternative method involves immobilizing the target biomarkers in the sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule. By "reporter molecule", as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e., radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase, and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of biomarker which was present in the sample. Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic color visually detectable with a light microscope. As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody-molecular marker complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the molecular marker of interest. Immunofluorescence and EIA techniques are both very well established in the art. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

Anti-CCP antibodies, in particular, can be analyzed by an enzyme immunoassay (EIA) and serological assay, including a second-generation ELISA (IMMUNOSCAN HA™), as well as an agglutination assay (Latex and Waaler-Rose) and specific ELISA (IgM, IgG and IgA). For example, the presence of anti-CCP in sera may be measured using anti-CCP-ELISA (CCPl test, cf. Schellekens et al., Arthr. Rheum, 43:155-163 (2000)). Commercially available ELISAs can be used, including IMMUNOSCAN RA™ (Eurodiagnostica, The Netherlands), Inova Diagnostics and Axis-Shield Diagnostics. Detection can be using 3 synthetic citrullinated peptide variants. Anti-CCP2 concentrations can be measured using a second-generation ELISA. A third-generation ELISA for anti-CCP marketed by Inova Diagnostics, may also be used. Associations between anti-CCP antibodies and clinical and laboratory parameters can be determined by Fisher's exact test. Anti-CCP can be measured as described by van Venroij et al. in WO 2003/050542. The assay may be set up by using one or more CCP as antigen and detecting the binding of anti-CCP antibodies comprised in a sample to the CCP antigen by appropriate means. Anti-CCP antibodies may be detected by homogeneous assays formats, e.g., by agglutination of latex particles coated with CCP. Also, a heterogeneous immunoassay may be used to measure anti-CCP. Such heterogeneous measurement is based on directly or indirectly coating CCP to a solid phase, incubating the solid phase with a sample known or suspected to comprise anti-CCP antibodies under conditions allowing for binding of anti-CCP antibodies to CCP, and directly or indirectly detecting the anti- CCP antibody bound. A further assay format is the so-called double antigen bridge assay, wherein in case of an anti-CCP measurement, CCPs are used both at the solid phase side as well as at the detection side of this immunoassay.

Abreu et a\., "Multiplexed immunoassay for detection of rheumatoid factors by FIDIS Technology" Annals of the New York Academy of Sciences 1050(Autoimmunity), 357-363 (2005) compares FIDIS RHEUMA™, a multiplexed immunoassay designed for simultaneous detection of IgM class RF directed against Fc determinants of IgG from humans and animals, with agglutination and ELISA and evaluates the clinical sensitivity and specificity of biological markers for RA. FIDIS technology was employed using the LUMINEX™ system and consisted of distinct color-coded microsphere sets, a flow cytometer, and digital signal processing hardware and software. Agglutination and ELISA tests can be performed with commercial kits. For human specificity, FIDIS was compared with latex agglutination and ELISA. For animal specificity, FIDIS was compared with Waaler- Rose and ELISA. Detection of IgG anti-CCP by ELISA by immunofluorescence was also determined. Dubois-Galopin et al., "Evaluation of a new fluorometric immunoassay for the detection of anti-cyclic citrullinated peptide autoantibodies in rheumatoid arthritis" Annales de Biologie Clinique, 64(2): 162- 165 (2006) evaluated the measurement of anti-CCP antibodies by a new fluorescent-enzyme immunoassay, called EIiA CCP™, fully automated onto UniCAP lOOε. This compares well with an ELISA method (Euroimmun).

RFs can be analyzed by, for example, latex-enhanced turbidimetry or latex agglutination and two isotype-specific (IgM and IgA) EIAs that are commercially available, or ELISAs. Isotypes of anti-CCPs can be detected by similar means. Methods for detecting any genetic biomarkers desired to be assessed in addition to the biomarker(s) herein (for example, polymorphisms) include protocols that examine the presence and/or expression of a SNP, for example, in a sample. Tissue or cell samples from mammals can be conveniently assayed for, e.g., genetic- marker mRNAs or DNAs using Northern, dot-blot, or polymerase chain reaction (PCR) analysis, array hybridization, RNase protection assay, or using DNA SNP chip microarrays, which are commercially available, including DNA microarray snapshots. For example, real-time PCR (RT-PCR) assays such as quantitative PCR assays are well known in the art. In an illustrative embodiment of the invention, a method for detecting a SNP mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a SNP polynucleotide as sense and antisense primers to amplify SNP cDNAs therein; and detecting the presence of the amplified SNP cDNA. In addition, such methods can include one or more steps that allow one to determine the levels of SNP mRNA in a biological sample {e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member). Optionally, the sequence of the amplified SNP cDNA can be determined.

In one specific embodiment, genotyping of a polymorphism can be performed by RT-PCR technology, using the TAQMAN™ 5'-allele discrimination assay, a restriction fragment-length polymorphism PCR-based analysis, or a PYROSEQUENCER™ instrument. In addition, the method of detecting a genetic variation or polymorphism set forth in U.S. 7,175,985 may be used. In this method a nucleic acid is synthesized utilizing the hybridized 3'- end, which is synthesized by complementary strand synthesis, on a specific region of a target nucleotide sequence existing as the nucleotide sequence of the same strand as the origin for the next round of complementary strand synthesis.

Probes used for PCR may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator, or enzyme. Such probes and primers can be used to detect the presence of a SNP in a sample and as a means for detecting a cell expressing SNP-encoded proteins. As will be understood by the skilled artisan, a great many different primers and probes may be prepared based on known sequences and used effectively to amplify, clone, and/or determine the presence and/or levels of SNP mRNAs. Other methods include protocols that examine or detect mRNAs in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes that have potential to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Differential gene expression analysis of disease tissue can provide valuable information. Microarray technology utilizes nucleic acid hybridization techniques and computing technology to evaluate the mRNA expression profile of thousands of genes within a single experiment (see, e.g., WO 2001/75166). See, for example, U.S. 5,700,637, U.S. 5,445,934, and U.S. 5,807,522, Lockart, Nature Biotechnology, 14:1675-1680 (1996); and Cheung et al., Nature Genetics, 21(Suppl): 15-19 (1999) for a discussion of array fabrication.

In addition, the DNA profiling and SNP detection method utilizing microarrays described in EP 1,753,878 may be employed. This method rapidly identifies and distinguishes between different DNA sequences utilizing short tandem repeat (STR) analysis and DNA microarrays. In an embodiment, a labeled STR target sequence is hybridized to a DNA microarray carrying complementary probes. These probes vary in length to cover the range of possible STRs. The labeled single-stranded regions of the DNA hybrids are selectively removed from the microarray surface utilizing a post- hybridization enzymatic digestion. The number of repeats in the unknown target is deduced based on the pattern of target DNA that remains hybridized to the microarray. One example of a microarray processor is the Affymetrix GENECHIP® system, which is commercially available and comprises arrays fabricated by direct synthesis of oligonucleotides on a glass surface. Other systems may be used as known to one skilled in the art. Other methods for determining the level of the biomarker besides RT-PCR or another PCR-based method include proteomics techniques, as well as individualized genetic profiles that are necessary to treat RA based on patient response at a molecular level. The specialized microarrays herein, e.g., oligonucleotide microarrays or cDNA microarrays, may comprise one or more biomarkers having expression profiles that correlate with either sensitivity or resistance to one or more anti-CD20 antibodies. Additionally, SNPs can be detected using electronic circuitry on silicon microchips, as disclosed, for example, in WO 2000/058522.

Identification of biomarkers that provide rapid and accessible readouts of efficacy, drug exposure, or clinical response is increasingly important in the clinical development of drug candidates. Embodiments of the invention include measuring changes in the levels of secreted proteins, or plasma biomarkers, which represent one category of biomarker. In one aspect, plasma samples, which represent a readily accessible source of material, serve as surrogate tissue for biomarker analysis.

Many references are available to provide guidance in applying the above techniques (Kohler et ah, Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); Campbell, Monoclonal Antibody Technology (Elsevier, Amsterdam, 1984); Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); and Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)). Northern blot analysis is a conventional technique well known in the art and is described, for example, in Molecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook, Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview, NY 11803-2500. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunob lotting) and 18 (PCR Analysis).

I l l For use in detection of the biomarkers, kits or articles of manufacture are also provided by the invention. Such kits can be used to determine if a subject with RA will be effectively responsive to a B-cell antagonist. These kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a probe that is or can be detectably labeled. Such probe may be an antibody or polynucleotide specific for a protein or autoantibody marker or a gene or message, respectively. Where the kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, e.g. , avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. Such kit will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use, such as those described above.

The kits of the invention have a number of embodiments. A typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container, wherein the composition includes a primary antibody that binds to a protein or autoantibody biomarker, and the label on said container indicates that the composition can be used to evaluate the presence of such proteins or antibodies in a sample, and wherein the kit includes instructions for using the antibody for evaluating the presence of biomarker proteins in a particular sample type. The kit can further comprise a set of instructions and materials for preparing a sample and applying antibody to the sample. The kit may include both a primary and secondary antibody, wherein the secondary antibody is conjugated to a label, e.g., an enzymatic label.

Another embodiment is a kit for detecting the biomarkers herein along with a genetic polymorphism biomarker that comprises a first container, a label on said container, and a composition contained within said container, wherein the composition includes a reagent to detect the biomarkers as noted above, a second container, a label on said container, and a composition contained within said second container, wherein the composition includes one or more polynucleotides that hybridize to a complement of the polynucleotide polymorphism being detected under stringent conditions, and the label on said first container indicates that the composition can be used to evaluate the presence of one or more of the biomarkers herein in a serum sample, and the label on said second container indicates that the composition can be used to evaluate the presence of a SNP in a sample (the sample being the same or different from the one containing the biomarker(s)), and wherein the kit includes instructions for using the reagent for detecting the amount(s) of biomarker (s) in a particular serum sample and instructions for using the polynucleotide(s) for evaluating the presence of the SNP RNA or DNA in a particular sample type.

Other optional components of the kit include one or more buffers (e.g., block buffer, wash buffer, substrate buffer, etc.), other reagents such as substrate (e.g., chromogen) that is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s), etc. Kits can also include instructions for interpreting the results obtained using the kit.

In further specific embodiments, for antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a biomarker protein; and, optionally, (2) a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label.

For kits that also detect genes (oligonucleotide-based kits), the kit can also comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a biomarker protein or (2) a pair of primers useful for amplifying a biomarker nucleic acid molecule. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. II. Statistics

As used herein, the general form of a prediction rule consists in the specification of a function of one or multiple biomarkers potentially including clinical covariates to predict response or non-response, or more generally, predict benefit or lack of benefit in terms of suitably defined clinical endpoints.

The simplest form of a prediction rule consists of a univariate model without covariates, wherein the prediction is determined by means of a cutoff or threshold. This can be phrased in terms of the Heaviside function for a specific cutoff c and a biomarker measurement x, where the binary prediction A or B is to be made, then IfH (jc-c)=O, then predict A. If H (jc-c)=l , then predict B.

This is the simplest way of using univariate biomarker measurements in prediction rules. If such a simple rule is sufficient, it allows for a simple identification of the direction of the effect, i.e., whether high or low expression levels are beneficial for the patient.

The situation can be more complicated if clinical covariates need to be considered and/or if multiple biomarkers are used in multivariate prediction rules. The two hypothetical examples below illustrate the issues involved: Covariate Adjustment (Hypothetical Example): For a biomarker X it is found in a clinical trial population that high expression levels are associated with a worse clinical response (univariate analysis). A closer analysis shows that there are two types of RA clinical response in the population, one of which possesses a worse response than the other one and at the same time the biomarker expression for this overall RA group is generally higher. An adjusted covariate analysis reveals that for each of the RA types the relation of clinical benefit and clinical response is reversed, i.e., within the RA types, lower expression levels are associated with better clinical response. The overall opposite effect was masked by the covariate RA type—and the covariate adjusted analysis as part of the prediction rule reversed the direction. Multivariate Prediction (Hypothetical Example):

For a biomarker X it is found in a clinical trial population that high expression levels are slightly associated with a worse clinical response (univariate analysis). For a second biomarker Y a similar observation was made by univariate analysis. The combination of X and Y revealed that a good clinical response is seen if both biomarkers are low. This makes the rule to predict benefit if both biomarkers are below some cutoffs (AND— connection of a Heaviside prediction function). For the combination rule, a simple rule no longer applies in a univariate sense; for example, having low expression levels in X will not automatically predict a better clinical response.

These simple examples show that prediction rules with and without covariates cannot be judged on the univariate level of each biomarker. The combination of multiple biomarkers plus a potential adjustment by covariates does not allow assigning simple relationships to single biomarkers. Since the marker genes, in particular in serum, may be used in multiple-marker prediction models potentially including other clinical covariates, the direction of a beneficial effect of a single marker gene within such models cannot be determined in a simple way, and may contradict the direction found in univariate analyses, i.e., the situation as described for the single marker gene. III. Treatment with the Antagonist

Once the patient or patient population most responsive to treatment with a B- cell antagonist has been identified, treatment with the antagonist herein, alone or in combination with other medicaments, results in an improvement in the RA or joint damage, including signs or symptoms thereof. For instance, such treatment may result in an improvement in ACR measurements relative to a patient treated with the second medicament only (e.g., an immunosuppressive agent such as MTX), and/or may result in an objective response (partial or complete, preferably complete) as measured by ACR. Moreover, treatment with the combination of an antagonist herein and at least one second medicament preferably results in an additive, more preferably synergistic (or greater than additive) therapeutic benefit to the patient. Preferably, in this method the timing between at least one administration of the second medicament and at least one administration of the antagonist herein is about one month or less, more preferably, about two weeks or less.

It will be appreciated by one of skill in the medical arts that the exact manner of administering to the patient a therapeutically effective amount of a B-cell antagonist following a diagnosis of a patient's likely responsiveness to the antagonist will be at the discretion of the attending physician. The mode of administration, including dosage, combination with other anti-RA agents, timing and frequency of administration, and the like, may be affected by the extent of the diagnosis of the patient's likely responsiveness to such antagonist (for example, higher seropositivity of anti-CCP or RF than normal), as well as the patient's condition and history.

The composition comprising an antagonist will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular type of RA being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the RA, the site of delivery of the antagonist, possible side-effects, the type of antagonist, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The effective amount of the antagonist to be administered will be governed by such considerations.

A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required, depending on such factors as the particular antagonist type and safety profile. For example, the physician could start with doses of such antagonist, such as an anti-CD20 or anti- CD22 antibody or immunoadhesin, employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect to assess safety, and gradually increase the dosage until the desired effect (without compromising safety) is achieved. The effectiveness of a given dose or treatment regimen of the antagonist can be determined, for example, by assessing signs and symptoms in the patient using the standard RA measures of efficacy.

As a general proposition, the effective amount of the antagonist administered parenterally per dose will be in the range of about 20 mg to about 5000 mg, by one or more dosages. Exemplary dosage regimens for intact antibodies such as anti-CD20 antibodies and anti-CD22 antibodies, and BAFF and APRIL antagonists, include 375 mg/m2 weekly x 4 (e.g., on days 1, 8, 15, and 22); or 500 mg x 2 (e.g., on days 1 and 15), or 1000 mg x 2 (e.g., on days 1 and 15); or 1 gram x 3 (e.g., on days 1, 15, and 21); or 200 mg x 1-4; or 300 mg x 1-4, or 400 mg x 1-4; or 500 mg x 3-4; or 1 gram x 4.

Preferably, the antagonist is administered in a dose of about 0.2 to 4 grams, more preferably about 0.2 to 3.5 grams, more preferably about 0.4 to 2.5 grams, more preferably about 0.5 to 1.5 grams, and even more preferably about 0.7 to 1.1 gram. More preferably, such doses apply to antagonists that are antibodies or immunoadhesins. Alternatively, the antagonist is anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment. In another alternative preferred embodiment, the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 1.2 g, more preferably about 200 mg to 1.1 g, and still more preferably about 200 mg to 900 mg.

In another preferred embodiment the B-cell antagonist is an anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment. Preferably the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 600 mg. In a preferred aspect, the antagonist is administered at a frequency of one to four doses within a period of about one month. The antagonist is preferably administered in two to three doses. In addition, the antagonist is preferably administered within a period of about two to three weeks.

As noted above, however, these suggested amounts of antagonist and frequency of dosing are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and schedule is the result obtained, as indicated above. For example, relatively higher doses may be needed initially for the treatment of ongoing and acute RA. To obtain the most efficacious results, once antagonist therapy is predicted by the biomarkers herein the antagonist is administered as close to the first sign, diagnosis, appearance, or occurrence of the RA as possible or during remissions of the RA.

In all the inventive methods set forth herein, the antagonist (such as an antibody that binds to a B-cell surface marker) 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 most preferred antagonist is a CD20, CD22, CD23, CD40, or BAFF antagonist, more preferably antibodies or immunoadhesins such as a BR3-Fc or TACI-Ig fusion molecule (same as TACI-Ig or atacicept available from ZymoGenetics; see also Gross et ah, Immunity, 15:289-291 (2001) and US 2007/0071760). The preferred antagonist antibody herein is a chimeric, humanized, or human antibody, more preferably, an anti-CD20, anti-CD22, or anti-BR3 antibody, and most preferably rituximab, epratuzumab, a 2H7 antibody (including one that comprises the L-chain variable region sequence of SEQ ID NO: 1 and the H-chain variable region sequence of SEQ ID NO:2, one that comprises the L-chain variable region sequence of SEQ ID NO:3 and the H-chain variable region sequence of SEQ ID NO:4, one that comprises the L-chain variable region sequence of SEQ ID NO: 3 and the H- chain variable region sequence of SEQ ID NO: 5, one that comprises the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:7, one that comprises the full-length L chain of SEQ ID NO: 6 and the full-length H chain of SEQ ID NO:8, one that comprises the full-length L chain of SEQ ID NO:9 and the full- length H chain of SEQ ID NO: 10, one that comprises the full-length L chain of SEQ ID NO: 9 and the full-length H chain of SEQ ID NO: 11 , one that comprises the full- length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 12, one that comprises the full-length L chain of SEQ ID NO: 9 and the full-length H chain of SEQ ID NO: 13, one that comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 14, or one that comprises the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:15), chimeric or humanized A20 antibody (Immunomedics), HUMAX-CD20™ human anti-CD20 antibody (Genmab), single-chain proteins binding to CD20 (a small modular immunopharmaceutical (SMIP™) drug candidate (e.g., TRU-015; Trubion Pharm Inc.; Wyeth), an AME antibody against CD20 (Lilly) such as those set forth above (e.g., AME-33, AME-133, or AME-133v), or a humanized type II CD20 IgGl antibody called GAlOl (GlyArt Biotechnology AG; Roche) (see, e.g., US 2005/0123546). Still more preferred is an anti-CD20 antibody selected from the group consisting of rituximab, HUMAX-CD20™, epratuzumab, TRU-015, GAlOl, or a 2H7 antibody, such as those set forth above.

In a further embodiment of the methods herein, the subject has never been previously treated with one or more drugs, such as with a TNF-α inhibitor, e.g., TNFR-Ig or an anti-TNF-α or anti-TNF-α receptor antibody, to treat, for example, RA, or with immunosuppressive agent(s) to treat joint damage or an underlying cause such as an autoimmune disorder, and/or has never been previously treated with a B-cell antagonist (e.g., antibody to a B-cell surface marker such as an anti-CD20, anti-CD22, or anti-BR3 antibody). In another embodiment, the subject has never been previously treated with an integrin antagonist such as anti-α4 integrin antibody or co-stimulation modulator, an immunosuppressive agent, a cytokine antagonist, an anti-inflammatory agent such as a NSAID, 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-CD 19, anti-CD 11a, anti-CD22, or BLys/BAFF), a live/attenuated vaccine within 28 days prior to baseline, or a corticosteroid such as an intra-articular or parenteral glucocorticoid within 4 weeks prior to baseline. More preferably, the subject has never been treated with an immunosuppressive agent, cytokine antagonist, integrin antagonist, corticosteroid, analgesic, a DMARD, or a NSAID. Still more preferably, the subject has never been treated with an immunosuppressive agent, cytokine antagonist, integrin antagonist, corticosteroid, DMARD, or NSAID.

In a further aspect, the subject may have had a relapse with the RA or 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 RA or joint damage and more preferably has not had such a relapse before at least the initial treatment.

In a further embodiment, the subject does not have a malignancy, including a B-cell malignancy, 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 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, or Lyme disease), or other systemic autoimmune disorder (including, but not limited to, SLE, 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), 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 that could affect any of the efficacy assessments, in particular, joint pain and swelling (e.g., Parkinson's disease, cerebral palsy, or diabetic neuropathy). In a still further embodiment, the subject does not have MS. In a yet further aspect, the subject does not have lupus or Sjogren's syndrome. In still another aspect, the subject does not have an autoimmune disease other than RA. In yet another aspect of the invention, any joint damage in the subject is not associated with an autoimmune disease or with an autoimmune disease other than RA, or with a risk of developing an autoimmune disease or an autoimmune disease other than RA. 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. In many of these autoimmune and inflammatory disorders, a number of clinical and laboratory markers may exist, including, but not limited to, hypergammaglobulinemia, high levels of autoantibodies, antigen-antibody complex deposits in tissues, benefit from corticosteroid or immunosuppressive treatments, and lymphoid cell aggregates in affected tissues. Without being limited to any one theory regarding B-cell mediated autoimmune disease, it is believed that 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 may participate to different degrees in the pathology of autoimmune diseases. "Autoimmune disease" can be an organ-specific disease (i.e., the immune response is specifically directed against an organ system such as the endocrine system, the hematopoietic system, the skin, the cardiopulmonary system, the gastrointestinal and liver systems, the renal system, the thyroid, the ears, the neuromuscular system, the central nervous system, etc.) or a systemic disease that can affect multiple organ systems (for example, SLE, RA, polymyositis, etc.). Preferred such diseases include autoimmune rheumatologic disorders (such as, for example, RA, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti- phospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neurological disorders (such as, for example, MS, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)). More preferred such diseases include, for example, RA, ulcerative colitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

In another preferred aspect of the above-described method, the subject was administered MTX prior to the baseline or start of treatment. More preferably, the MTX was administered at a dose of about 10-25 mg/week. Also, preferably, the MTX was administered for at least about 12 weeks prior to the baseline, and still more preferably the MTX was administered at a stable dose the last four weeks prior to the baseline. In other embodiments, the MTX was administered perorally or parenterally.

In a particularly preferred embodiment of the above-identified methods, the subject has exhibited an inadequate response to one or more TNF-α inhibitors or to MTX. In another aspect, the subject has been refractory to a B-cell antagonist, such as those other than rituximab or a 2H7 antibody. However, the subject may also have been refractory to rituximab or a 2H7 antibody.

In another preferred aspect, MTX is administered to the subject along with the antagonist, for example, anti-CD20 antibody. In another aspect, the antagonist is an anti-CD20 antibody that is administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment or is administered a dose of about 400 to 800 mg as a single dose or as two doses, such as infusions.

Also included herein, after the diagnosis step, is a method of monitoring the treatment of bone or soft tissue joint damage in a subject comprising administering an effective amount of a B-cell antagonist (such as an antibody thereto, including an anti- CD20, anti-CD22, or anti-BR3 antibody) to the subject and measuring by imaging techniques such as MRI or radiography after at least about three months, preferably about 24 weeks, from the administration whether the bone or soft tissue joint damage has been reduced over baseline prior to the administration, wherein a decrease versus baseline in the subject after treatment indicates the antagonist such as an anti-CD20, anti-CD22, or anti-BR3 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 such as an antibody or immunoadhesin.

In other aspects, at least about three months after the administration, an imaging test (radiographic and/or MRI) is given that measures a reduction in bone and soft tissue joint damage as compared to baseline prior to the administration, and the amount of antagonist administered is effective in achieving a reduction in the joint damage. Preferably, the test measures a total modified Sharp score. In other preferred embodiments, the method further comprises an additional administration to the patient of a B-cell antagonist 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. In preferred aspects, the antagonist is additionally administered to the patient even if there is no clinical improvement in the patient at the time of the radiographic testing after a prior administration. Preferably, the clinical improvement is determined by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity (disease response), such as the DAS-28, ACR- 20, -50, or -70 scores. In yet another aspect, the invention provides, after the diagnosis step, a method of determining whether to continue administering a B-cell antagonist (such as an antibody thereto or immunoadhesin, including an anti-CD20 antibody) to a subject with bone or soft tissue joint damage comprising measuring reduction in joint damage in the subject, using imaging techniques, such as radiography and/or MRI, after administration of the antagonist a first time, measuring reduction in joint damage in the subject, using imaging techniques such as radiography and/or MRI after administration of the antagonist a second time, comparing imaging findings 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.

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 bone or soft tissue joint damage. For example, a step is included to test the imaging (radiographic and/or MRI) score after administration and compare it to baseline imaging results 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 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.

In one embodiment of all the methods herein, the antagonist (for example, anti-CD20 antibody) is the only medicament administered to the subject to treat the RA, i.e., no other medicament than the antagonist is administered to the subject to treat the RA.

In any of the methods herein, preferably the antagonist is one of the medicaments used to treat the RA. Thus, one may administer to the subject along with the B-cell antagonist an effective amount of a second medicament (where the B- cell antagonist (e.g., an anti-CD20 antibody or BR3-Fc) is a first medicament). The second medicament may be one or more medicaments, and includes, for example, an immunosuppressive agent, a cytokine antagonist such as a cytokine antibody, an integrin antagonist (e.g. , antibody), a corticosteroid, or any combination thereof. The type of such second medicament depends on various factors, including the type of RA and/or joint damage, the severity of the RA and/or joint damage, the condition and age of the subject, the type and dose of the first medicament employed, etc.

Examples of such additional medicaments include an immunosuppressive agent (such as mitoxantrone (NOVANTRONE®), MTX, cyclophosphamide, chlorambucil, leflunomide, and azathioprine), intravenous immunoglobulin (gamma globulin), lymphocyte-depleting therapy {e.g., mitoxantrone, cyclophosphamide, CAMPATH™ 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, and bone marrow transplantation), integrin antagonist or antibody {e.g., an LFA-I antibody such as efalizumab/RAPTIVA® commercially available from Genentech, or an alpha 4 integrin antibody such as natalizumab/ANTEGREN® available from Biogen, or others as noted above), drugs that treat symptoms secondary or related to RA and/or joint damage such as those noted herein, steroids such as corticosteroid {e.g., prednisolone, methylprednisolone such as SOLU-MEDROL™ 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), a TNF-α inhibitor such as an antibody to TNF-α or its receptor or TNFR-Ig {e.g., etanercept), DMARD, NSAID, plasmapheresis or plasma exchange, trimethoprim-sulfamethoxazole (BACTRIM™, SEPTRA™), MMF, H2 -blockers or proton-pump inhibitors (during the use of potentially ulcerogenic immunosuppressive therapy), levothyroxine, cyclosporin A {e.g., SANDIMMUNE®), somatostatin analogue, a DMARD or NSAID, cytokine antagonist such as antibody, anti-metabolite, immunosuppressive agent, rehabilitative surgery, radioiodine, thyroidectomy, anti-IL-6 receptor antagonist/antibody {e.g. , ACTEMRA™ (tocilizumab)), or another B-cell antagonist such as BR3-Fc, TACI-Ig, anti-BR3 antibody, anti-CD40 receptor or anti-CD40 ligand (CD 154), agent blocking CD40-CD40 ligand, epratuzumab (anti-CD22 antibody), lumiliximab (anti-CD23 antibody), or anti-CD20 antibody such as rituximab or 2H7 antibody.

Preferred such medicaments include gamma globulin, an integrin antagonist, anti-CD4, cladribine, trimethoprimsulfamethoxazole, an H2 -blocker, proton-pump inhibitor, cyclosporine, TNF-α inhibitor, DMARD, NSAID (to treat, for example, musculoskeletal symptoms), levothyroxine, cytokine antagonist (including cytokine - receptor antagonist), anti-metabolite, immunosuppressive agent such as MTX or a corticosteroid, bisphosphonate, and another B-cell antagonist, such as an anti-CD20 antibody, anti-CD22 antibody, anti-BR3 antibody, lumiliximab (anti-CD23 antibody), BR3-Fc, or TACI-Ig. The more preferred such medicaments are an immunosuppressive agent such as MTX or a corticosteroid, a DMARD, an integrin antagonist, a NSAID, a cytokine antagonist, a bisphosphonate, or a combination thereof.

In one particularly preferred embodiment, the second medicament is a DMARD, which is preferably selected from the group consisting of auranofm, chloroquine, D-penicillamine, injectable gold, oral gold, hydroxychloroquine, sulfasalazine, myocrisin, and MTX.

In another such embodiment, the second medicament is a NSAID, which is preferably selected from the group consisting of: fenbufen, naprosyn, diclofenac, etodolac and indomethacin, aspirin, and ibuprofen. 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, MTX, and cyclophosphamide.

In other preferred aspects, the second medicament is selected from the group consisting of anti-α4, etanercept, infliximab, etanercept, adalimumab, kinaret, efalizumab, OPG, RANK-Fc, anti-RANKL, pamidronate, alendronate, actonel, zolendronate, clodronate, MTX, azulfϊdine, hydroxylchloroquine, doxycycline, leflunomide, SSZ, prednisolone, IL-I receptor antagonist, prednisone, and methylprednisolone. In still preferred embodiments, the second medicament is selected from the group consisting of infliximab, an infliximab/ MTX combination, etanercept, a corticosteroid, cyclosporin A, azathioprine, auranofϊn, hydroxychloroquine (HCQ), a combination of prednisolone, MTX, and SSZ, a combination of MTX, SSZ, and HCQ, a combination of cyclophosphamide, azathioprine, and HCQ, and a 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 antagonist is not administered to a subject treated with a corticosteroid as standard-of-care therapy. Most preferably, the second medicament is MTX. 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.

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.

The combined administration of a second medicament includes coadministration (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 antagonist herein is administered by any suitable means, including parenteral, topical, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. Parenteral infusions include intramuscular, intravenous (i.v.), intraarterial, intraperitoneal, or subcutaneous (s.c.) administration. Intrathecal administration is also suitable (see, e.g., US 2002/0009444, Grillo-Lopez, concerning intrathecal delivery of an anti-CD20 antibody). Also the antagonist may suitably be administered by pulse infusion, e.g., with declining doses of the antagonist. Preferably if the antagonist is an antibody or immunoadhesin, the dosing is given by i.v. or s.c. means, and more preferably by i.v. infusion(s) or injection(s).

In one embodiment, the antagonist such as an anti-CD20 antibody is administered as a slow i.v. infusion rather than an i.v. push or bolus. For example, in one aspect a steroid such as prednisolone or methyl-prednisolone (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 an anti-CD20 antibody. The anti-CD20 antibody is, for example, infused through a dedicated line.

For the initial dose of a multi-dose exposure to anti-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 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 anti-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. If more than one infusion (dose) of anti-CD20 antibody is given to achieve the total exposure, the second or subsequent anti-CD20 antibody infusions in this 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 anti-CD20 antibody (e.g., an about 1000-mg total dose) is completed by about 195 minutes (3 hours 15 minutes).

Aside from administration of antagonists to the patient by traditional routes as noted above, the present invention includes administration by gene therapy. Such administration of nucleic acids encoding the antagonist is encompassed by the expression "administering an effective amount of an antagonist". See, for example, WO 1996/07321 concerning the use of gene therapy to generate intracellular antibodies. There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells, in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antagonist is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g. US 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retrovirus. The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno- associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example). In some situations it is desirable to provide the nucleic acid source with an agent specific for the target cells, such as an antibody specific for a cell-surface membrane protein on the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins that undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et ah, J. Biol. Chem., 262:4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA, 87:3410-3414 (1990). Gene-marking and gene-therapy protocols are described, for example, in Anderson et ah, Science, 256:808-813 (1992) and WO 1993/25673.

In another embodiment, a method is provided for treating joint damage in a subject eligible for treatment based on the biomarker analysis herein comprising administering a B-cell antagonist, such as an antibody thereto, for example, anti- CD20 antibody, to the subject, and giving the subject, at least about 52 weeks after the administration, an imaging test that measures a reduction in the joint damage as compared to baseline prior to the administration, wherein the amount of antagonist such as anti-CD20 antibody administered is effective in achieving a reduction in the joint damage, indicating that the subject has been successfully treated.

In this method, preferably the test measures a total modified Sharp score. In another preferred embodiment of this joint-treatment method, the antagonist is an anti-CD20, anti-CD22, or anti-BR-3 antibody or BR3-Fc. More preferably, the anti- CD20 antibody is the preferred such antibodies set forth above, including rituximab, GAlOl, TRU-015, and a 2H7 antibody as set forth above. In another preferred embodiment, the joint damage is caused by arthritis, preferably RA, and more preferably early or incipient RA. In all the methods herein, the RA is preferably early or incipient RA. The subject herein may be RF negative or positive. In another aspect, such method further comprises re-treating the subject by providing an additional administration to the subject of the antagonist such as an anti- CD20 antibody in an amount effective to treat RA or achieve a continued or maintained reduction in joint damage as compared to the effect of a prior administration of the antagonist. The re-treatment may be commenced at least about 24 weeks (preferably at about 24 weeks) after the first administration of the antagonist, and one or more further re-treatments is optionally commenced. In another embodiment, the further re-treatment is commenced at least about 24 weeks after the second administration of the antagonist.

In one aspect the antagonist is additionally administered to the subject even if there is no clinical improvement in the subject at the time of RA testing or another imaging testing after a prior administration.

In a further preferred aspect, RA or joint damage has been reduced after the re -treatment as compared to the extent of RA or joint damage after the first assessment such as imaging assessment. If multiple exposures of antagonist are provided as in re-treatment, each exposure may be provided using the same or a different administration means. In one embodiment, each exposure is by i.v. administration. In another embodiment, each exposure is given by s.c. administration. In yet another embodiment, the exposures are given by both i.v. and s.c. administration. Preferably the same antagonist, such as anti-CD20, anti-CD22, or anti-BR3 antibody, BR3-Fc, or TACI-Ig, is used for at least two antagonist exposures, and preferably for each antagonist exposure. Thus, the initial and second antagonist exposures are preferably with the same antagonist, and more preferably all antagonist exposures are with the same antagonist, i.e., treatment for the first two exposures, and preferably all exposures, is with one type of B-cell antagonist, e.g., an antagonist that binds to a B-cell surface marker, such as an anti-CD20 antibody, e.g. , all with rituximab or all with the same 2H7 antibody.

Preferably, in this re-treatment method, a second medicament is administered in an effective amount, wherein the antagonist is a first medicament. In one aspect, the second medicament is more than one medicament. In another aspect, the second medicament is one of those set forth above, including an immunosuppressive agent, a DMARD, an integrin antagonist, a NSAID, a cytokine antagonist, a bisphosphonate, or a combination thereof, most preferably MTX. For the re-treatment methods described herein, where a second medicament is administered in an effective amount with an antagonist 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. In one embodiment of the re-treatment method, the subject has never been previously administered any drug(s), such as immunosuppressive agent(s), to treat the RA or joint damage. In another aspect, the subject or patient is responsive to previous therapy for the RA or joint damage.

In another aspect of re-treatment, the subject or patient has been previously administered one or more medicaments(s) to treat the RA or joint damage. In a further embodiment, the subject or patient was not responsive to one or more of the medicaments that had been previously administered. Such drugs to which the subject may be non-responsive include, for example, chemotherapeutic agents, immunosuppressive agents, cytokine antagonists, integrin antagonists, corticosteroids, analgesics, or B-cell antagonists such as antagonists to B-cell surface markers, for example, anti-CD20 antibody. More particularly, the drugs to which the subject may be non-responsive include immunosuppressive agents or B-cell antagonists such as anti-CD20 antibodies. Preferably, such antagonists are not antibodies or immunoadhesins, and are, for example, small-molecule inhibitors, or anti-sense oligonucleotides, or antagonistic peptides, as noted, for example, in the background section. In a further aspect, such antagonists include an antibody or immunoadhesin, such that re-treatment is contemplated with one or more antibodies or immunoadhesins of this invention to which the subject was formerly non-responsive. Most preferably, the subject or patient is not responsive to previous therapy with MTX or a TNF-α inhibitor.

In another aspect the invention provides a method of treating RA in a patient comprising first administering a B-cell antagonist to the patient to treat the RA, provided that a sample from the patient contains one or more biomarkers identified in accordance with the present invention over a predetermined threshold level, and at least about 24 weeks after the first administration of the antagonist, re -treating the patient by administering an effective amount of the B-cell antagonist to the patient, wherein no clinical improvement is observed in the patient at the time of the testing after the first administration of the B-cell antagonist. In a preferred aspect of this method the clinical improvement is determined by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity. In another preferred aspect the amount of the B-cell antagonist administered upon re-treatment is effective to achieve a continued or maintained reduction in joint damage as compared to the effect of a prior administration of the B-cell antagonist.

In another embodiment, a method is provided for treating joint damage in a subject comprising administering a B-cell antagonist, such as an antibody thereto, for example, anti-CD20 antibody, to the subject, and giving the subject, at least about 52 weeks after the administration, an imaging test that measures a reduction in the joint damage as compared to baseline prior to the administration, wherein the amount of antagonist such as anti-CD20 antibody administered is effective in achieving a reduction in the joint damage, indicating that the subject has been successfully treated. In this method, preferably the test measures a total modified Sharp score. In another preferred embodiment of this joint-treatment method, the antagonist is an anti-CD20, anti-CD22, or anti-BR-3 antibody or BR3-Fc. More preferably, the anti- CD20 antibody is rituximab or a 2H7 antibody as set forth above.

Preferably, in this method regarding the about 52-week assessment, a second medicament is administered in an effective amount, wherein the antagonist such as anti-CD20 antibody is a first medicament. In one aspect, the second medicament is more than one medicament. In another aspect, the second medicament is one of those set forth above, including an immunosuppressive agent, a DMARD, an integrin antagonist, a NSAID, a cytokine antagonist, a bisphosphonate, or a combination thereof, most preferably MTX.

In a further aspect, the invention involves a method of reducing the risk of a negative side effect in a subject (e.g., selected from the group consisting of an infection, cancer, heart failure, and demyelination) comprising administering to the subject an effective amount of a B-cell antagonist if the subject has one or more of the biomarkers herein.

A discussion of methods of producing, modifying, and formulating such antagonists follows. IV. Production of Antagonists

The methods and articles of manufacture of the present invention use, or incorporate, a B-cell antagonist such as an antibody or immunoadhesin. Methods for screening for such antagonists are noted above. Methods for generating such antagonists are well within the skill of the art, and include chemical synthesis, recombinant production, hybridoma production, peptide synthesis, oligonucleotide synthesis, phage-display, etc., depending on the type of antagonist being produced.

B-cell surface antigens or B-cell specific proliferation or survival factors to be used for production of, or screening for, antagonist(s) may be, e.g., a soluble form of the antigen or proliferation/survival factor or a portion thereof, containing the desired epitope. Alternatively, or additionally, cells expressing the antigen at their surface, or expressing the B-cell specific survival/proliferation factor, can be used to generate, or screen for, antagonist(s). Other forms of B-cell surface markers and proliferation/survival factors useful for generating antagonists will be apparent to those skilled in the art. While the preferred antagonist is an antibody or immunoadhesin, other antagonists are contemplated herein. For example, the antagonist may comprise a small-molecule antagonist optionally fused to, or conjugated with, a cytotoxic agent. Libraries of small molecules may be screened against the B-cell surface antigen or survival/proliferation factor of interest herein to identify a small molecule that binds to that antigen or factor. The small molecule may further be screened for its antagonistic properties and/or conjugated with a cytotoxic agent.

The antagonist may also be a peptide generated by rational design or by phage display (see, e.g., WO 1998/35036). In one embodiment, the molecule of choice may be a "CDR mimic" or antibody analogue designed based on the CDRs of an antibody. While such peptides may be antagonistic by themselves, the peptide may optionally be fused to a cytotoxic agent so as to add or enhance antagonistic properties of the peptide.

A description follows as to exemplary techniques for the production of the antibody antagonists used in accordance with the present invention.

(i) Polyclonal antibodies

Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) 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, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg 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.

(U) Monoclonal antibodies

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. 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. 4,816,567).

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)).

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.

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 SaIk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland 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)).

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). 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).

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. 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, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

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 Plϋckthun, Immunol. Revs., 130:151-188 (1992). 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. MoI Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. 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.

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. 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.

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. (Hi) Humanized antibodies 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 which 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 ah, Nature, 321 : 522-525 (1986); Riechmann et ah, 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. 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.

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 which 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. MoI 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)).

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 which 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.

(iv) Human antibodies

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 (JH) 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. 5,591,669, 5,589,369 and 5,545,807. 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 M 13 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 and Chiswell, 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. MoI. Biol., 222:581- 597 (1991), or Griffith et al., EMB0 J., 12:725-734 (1993). See, also, U.S. 5,565,332 and 5,573,905.

Human antibodies may also be generated by in vitro activated B cells (see U.S. 5,567,610 and 5,229,275).

(v) Antibody fragments

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., J. Biochem. Biophys. 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')2 fragments (Carter et al, Bio/Technology, 10:163- 167 (1992)). According to another approach, F(ab')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 1993/16185; U.S. 5,571,894; and U.S. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in US Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific. (vi) Bispecific antibodies

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 or B-cell specific proliferation/survival factor. 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 (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD 16) so as to focus cellular defense mechanisms to the B cell. Bispecific antibodies may also be used to localize cytotoxic agents to the B cell. These antibodies possess a CD20-binding arm and an arm which binds the cytotoxic agent {e.g. saporin, anti-interferon-α, vinca alkaloid, ricin A chain, MTX or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments {e.g. F(ab')2 bispecific antibodies).

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 ah, 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 1993/08829, and in Traunecker et al, EMBOJ., 10:3655-3659 (1991).

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 (CHl) 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. 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 1994/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121 :210 (1986). According to another approach described in U.S. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which 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.

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. 4,676,980), and for treatment of HIV infection (WO 1991/00360, WO 1992/200373, 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. 4,676,980, along with a number of cross-linking techniques. 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')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.

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 (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH 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). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J. Immunol, 147:60 (1991). V. Modifications of the Antagonist

Modifications of the antagonist are contemplated herein. For example, the antagonist may be linked to one of a variety of nonproteinaceous 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 a therapeutic embodiment of the invention. The antagonists disclosed herein may also be formulated as liposomes. Liposomes containing the antagonist 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. 4,485,045 and 4,544,545; and WO 1997/38731. Liposomes with enhanced circulation time are disclosed in U.S. 5,013,556. 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. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al, J. National Cancer Inst., 81(19):1484 (1989).

Amino acid sequence modification(s) of protein or peptide antagonists described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antagonist. Amino acid sequence variants of the antagonist are prepared by introducing appropriate nucleotide changes into the antagonist nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antagonist. 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 antagonist, such as changing the number or position of glycosylation sites. A useful method for identification of certain residues or regions of the antagonist 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 antagonist variants are screened for the desired activity.

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 antagonist with an N-terminal methionyl residue or the antagonist fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include the fusion to the N- or C- terminus of the antagonist of an enzyme, or a polypeptide which increases the serum half-life of the antagonist.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antagonist molecule replaced by different residue. The sites of greatest interest for substitutional mutagenesis of antibody antagonists include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened. Table 1

Figure imgf000145_0001
Substantial modifications in the biological properties of the antagonist 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. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the proper conformation of the antagonist 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 antagonist to improve its stability (particularly where the antagonist is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one or more HVR 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 HVR 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 M 13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.

Alanine-scanning mutagenesis can be performed to identify candidate HVR residues contributing significantly to antigen binding for possible modification. Alternatively, or in addition, 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. Another type of amino acid variant of the antagonist alters the original glycosylation pattern of the antagonist. Such altering includes deleting one or more carbohydrate moieties found in the antagonist, and/or adding one or more glycosylation sites that are not present in the antagonist.

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.

Addition of glycosylation sites to the antagonist is typically 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 antagonist (for O-linked glycosylation sites). 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 2003/0157108 (Presta). See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine (GIcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in WO 2003/011878, Jean- Mairet et al. and U.S. 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) and WO 1999/22764 (Raju) concerning antibodies with altered carbohydrate attached to the Fc region thereof. See also US 2005/0123546 (Umana et al); US 2004/0072290 (Umana et al); US 2003/0175884 (Umana et al); and WO 2005/044859 (Umana et al) on antigen-binding molecules with modified glycosylation, including antibodies with an Fc region containing N-linked oligosaccharides.

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; US 2006/0063254; US 2006/0064781; US 2006/0078990; US 2006/0078991; Okazaki et al, J. MoI. Biol, 336: 1239-1249 (2004); Yamane-Ohnuki et al, Biotech. Bioeng., 87:614 (2004). Examples of cell lines producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al, Arch. Biochem. Biophys., 249:533-545 (1986); US 2003/0157108 Al (Presta) and WO 2004/056312 Al (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)). See also Kanda et al, Biotechnol. Bioeng., 94:680- 688 (2006). US 2007/0048300 (Biogen-IDEC) discloses a method of producing aglycosylated Fc-containing polypeptides, such as antibodies, having desired effector function, as well as aglycosylated antibodies produced according to the method and as methods of using such antibodies as therapeutics.

See also US 2006/024304 (Gerngross et al); U.S. 7,029,872 (Gerngross); US 2004/018590 (Gerngross et al); US 2006/034828 (Gerngross et al); US 2006/034830 (Gerngross et al); US 2006/029604 (Gerngross et al); WO 2006/014679 (Gerngross et al); WO 2006/014683 (Gerngross et al); WO 2006/014685 (Gerngross et al); WO 2006/014725 (Gerngross et al); and WO 2006/014726 (Gerngross et al) on recombinant glycoproteins and glycosylation variants.

Nucleic acid molecules encoding amino-acid-sequence variants of the antagonist 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 antagonist.

It may be desirable to modify the antagonist used herein with respect to effector function, e.g. so as to enhance ADCC and/or CDC of the antagonist. This may be achieved by introducing one or more amino acid substitutions in an Fc region of an antibody antagonist. 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 ah, Cancer Research, 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989). WO 2000/42072 (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. Antibodies with altered CIq binding and/or CDC are described in WO

1999/51642 and U.S. 6,194,551, 6,242,195, 6,528,624, and 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. To increase the serum half life of the antagonist, one may incorporate a salvage receptor binding epitope into the antagonist (especially an antibody fragment) as described in U.S. 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 WO 2000/42072 (Presta, L.).

Engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US 2002/0004587, Miller et al). VI. Pharmaceutical Formulations

Therapeutic formulations of the antagonists used in accordance with the present invention are prepared for storage by mixing the antagonist having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. For general information concerning formulations, see, e.g., Gilman et al. , (eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, (1990), Mack Publishing Co., Eastori, Pennsylvania.; Avis et al, (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman et al.., (eds.) (1990)

Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman et al., (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, Kenneth A. Walters (ed.) (2002) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), VoI 119, Marcel Dekker. Acceptable carriers, excipients, or stabilizers are non-toxic 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 T WEEN™, PLURONIC S™, or polyethylene glycol (PEG). Exemplary anti-CD20 antibody formulations are described in WO 1998/56418, which 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°C. Another anti-CD20 formulation of interest comprises 10 mg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/niL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5.

Lyophilized formulations adapted for subcutaneous administration are described, for example, in US Pat No. 6,267,958 (Andya et ah). 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.

Crystallized forms of the antagonist are also contemplated. See, for example, US 2002/0136719Al (Shenoy et al). The formulation herein may also contain more than one active compound (a second medicament as noted above), 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 and type of B-cell antagonist present in the formulation, and clinical parameters of the subjects. The preferred such second medicaments are noted above.

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, micro emulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, 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. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. VII. Articles of Manufacture

Articles of manufacture containing materials useful for the treatment of the RA described above are provided herein. The article of manufacture comprises a container and a label or package insert on or associated with the container. 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 the antagonist that is effective for treating the RA or 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 B-cell antagonist. The label or package insert indicates that the composition is used for treating joint damage or RA in a subject eligible for treatment with specific guidance regarding dosing amounts and intervals of antagonist and any other medicament being provided. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and 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.

The kits and articles of manufacture of the present invention also include information, for example in the form of a package insert or label, indicating that the composition is used for treating RA or joint damage where levels of one or more of the biomarkers herein no greater than predetermined threshold levels for each biomarker are detected in a serum sample from the patient with the disease.

Optionally, the label or package insert may indicate that seropositivity for anti-CCP and/or RF can be detected in addition to the presence of one or more of the other biomarkers. The insert or label may take any form, such as paper or electronic media, for example, a magnetically recorded medium {e.g., floppy disk) or a CD-ROM. The label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture.

Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding the antagonist may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references and patent information. VIII. Methods of Advertising

The invention herein also encompasses a method for advertising a B-cell antagonist or a pharmaceutically acceptable composition thereof comprising promoting, to a target audience, the use of the antagonist or pharmaceutical composition thereof for treating a patient or patient population with RA from which a serum sample has been obtained showing an elevated amount of a positive acute phase protein, preferably CRP and an elevated titer of a rheumatoid arthritis- associated autoantibody, preferably anti-RF antibody.

Also provided is a method for marketing a B-cell antagonist for use in a RA patient subpopulation, the method comprising informing a target audience about the use of the antagonist for treating the patient subpopulation characterized by the presence, in samples from patients of such subpopulation, of an elevated amount of a positive acute phase protein, preferably CRP and an elevated titer of a rheumatoid arthritis-associated autoantibody, preferably anti-RF antibody.

Advertising is generally paid communication through a non-personal medium in which the sponsor is identified and the message is controlled. Advertising for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. This term also includes sponsored informational public notices appearing in any of the print communications media designed to appeal to a mass audience to persuade, inform, promote, motivate, or otherwise modify behavior toward a favorable pattern of purchasing, supporting, or approving the invention herein.

The advertising and promotion of the diagnostic method herein may be accomplished by any means. Examples of advertising media used to deliver these messages include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercials, which are messages appearing in the broadcast media. Advertisements also include those on the seats of grocery carts, on the walls of an airport walkway, and on the sides of buses, or heard in telephone hold messages or in-store PA systems, or anywhere a visual or audible communication can be placed. More specific examples of promotion or advertising means include television, radio, movies, the internet such as webcasts and webinars, interactive computer networks intended to reach simultaneous users, fixed or electronic billboards and other public signs, posters, traditional or electronic literature such as magazines and newspapers, other media outlets, presentations or individual contacts by, e.g., e-mail, phone, instant message, postal, courier, mass, or carrier mail, in-person visits, etc.

The type of advertising used will depend on many factors, for example, on the nature of the target audience to be reached, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and the relevant jurisdictional laws and regulations governing advertising of medicaments and diagnostics. The advertising may be individualized or customized based on user characterizations defined by service interaction and/or other data such as user demographics and geographical location.

Many alternative experimental methods known in the art may be successfully substituted for those specifically described herein in the practice of this invention, as for example described in many of the excellent manuals and textbooks available in the areas of technology relevant to this invention (e.g. Using Antibodies, A Laboratory Manual, edited by Harlow, E. and Lane, D., 1999, Cold Spring Harbor Laboratory Press, (e.g. ISBN 0-87969-544-7); Roe B. A. et. al. 1996, DNA Isolation and Sequencing (Essential Techniques Series), John Wiley & Sons (e.g. ISBN 0-471- 97324-0); Methods in Enzymology: Chimeric Genes and Proteins, 2000, ed. J. Abelson, M. Simon, S. Emr, J. Thomer. Academic Press; Molecular Cloning: a Laboratory Manual, 2001, 3rd Edition, by Joseph Sambrook and Peter MacCallum, (the former Maniatis Cloning manual) (e.g. ISBN 0-87969-577-3); Current Protocols in Molecular Biology, Ed. Fred M. Ausubel, et. al. John Wiley & Sons (e.g. ISBN 0- 471-50338-X); Current Protocols in Protein Science, Ed. John E. Coligan, John Wiley & Sons (e.g. ISBN 0-471-11184-8); and Methods in Enzymology: Guide to protein Purification, 1990, Vol. 182, Ed. Deutscher, M.P., Academic Press, Inc. (e.g. ISBN 0-12-213585-7)), or as described in the many university and commercial websites devoted to describing experimental methods in molecular biology. 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. EXAMPLES

Statistical Methods

The statistical tasks can comprise the following steps:

• 1. Pre-selection of candidate biomarkers » 2. Pre-selection of relevant clinical efficacy response predictive covariates

• 3. Selection of biomarker prediction functions at a univariate level

• 4. Selection of biomarker prediction functions including clinical covariates at a univariate level

• 5. Selection of biomarker prediction functions at a multivariate level • 6. Selection of biomarker prediction functions including clinical covariates at a multivariate level

The following text details the different steps:

1 : Pre-selection of candidate biomarkers: The statistical pre-selection of candidate biomarkers is oriented towards the strength of association with measures of clinical benefit. For this purpose the different clinical endpoints may be transformed in derived surrogate scores, as, e.g., an ordinal assignment of the degree of clinical benefit scores regarding TTP that avoid censored observations. These surrogate transformed measures can be easily used for simple correlation analysis, e.g. by the non-parametric Spearman rank correlation approach. An alternative is to use the biomarker measurements as metric covariates in time-to-event regression models, as, e.g., Cox proportional hazard regression. Depending on the statistical distribution of the biomarker values, this step may require some pre-processing, as, for example, variance-stabilizing transformations and the use of suitable scales or, alternatively, a standardization step such as using percentiles instead of raw measurements. A further approach is inspection of bivariate scatter plots, for example, by displaying the scatter of (x-axis=biomarker value, y-axis=measure of clinical benefit) on a single-patient basis. Some non-parametric regression line as achieved, for example, by smoothing splines can be useful to visualize the association of biomarker and clinical benefit. The goal of these different approaches is the pre-selection of biomarker candidates that show some association with clinical benefit in at least one of the benefit measures employed, while results for other measures are not contradictory. When there are available control groups, then differences in association of biomarkers with clinical benefit in the different arms could be a sign of differential prediction that makes the biomarker(s) eligible for further consideration.

2: Pre-selection of relevant clinical efficacy response predictive covariates: The statistical pre-selection of clinical covariates as defined herein parallels the approaches for pre-selecting biomarkers and is also oriented towards the strength of association with measures of clinical benefit. So in principle the same methods apply as considered under 1 above. In addition to statistical criteria, criteria from clinical experience and theoretical knowledge may apply to pre-select relevant clinical covariates. The predictive value of clinical covariates could interact with the predictive value of the biomarkers. They will be considered for refined prediction rules, if necessary.

3: Selection of biomarker prediction functions at a univariate level: The term "prediction function" will be used in a general sense to mean a numerical function of a biomarker measurement that results in a number scaled to imply the target prediction. A simple example is the choice of the Heaviside function for a specific cutoff c and a biomarker measurement x, where the binary prediction A or B is to be made, then If H (x-c)=0, then predict A. If H (x-c)= 1 , then predict B.

This is probably the most common way of using univariate biomarker measurements in prediction rules. The definition of "prediction function" as noted above includes referral to an existing training data set that can be used to explore the prediction possibilities. Different routes can be taken to achieve a suitable cutoff c from the training set. First, the scatterplot with smoothing spline mentioned under 1 can be used to define the cutoff. Alternatively, some percentile of the distribution could be chosen, e.g., the median or a quartile. Cutoffs can also be systematically extracted by investigating all possible cutoffs according to their prediction potential with regard to the measures of clinical benefit. Then, these results can be plotted to allow for an either manual selection or to employ some search algorithm for optimality. This can be realized based on certain clinical endpoints using a Cox model, wherein at each test cutoff the biomarker is used as a binary covariate. Then the results for the clinical endpoints can be considered together to chose a cutoff that shows prediction in line with both endpoints. Another uncommon approach for choosing a prediction function can be based on a fixed-parameter Cox regression model obtained from the training set with biomarker values (possibly transformed) as covariate. A further possibility is to base the decision on some likelihood ratio (or monotonic transform of it), where the target probability densities are pre-determined in the training set for separation of the prediction states. Then the biomarker would be plugged into some function of predictive criteria.

4: Selection of biomarker prediction functions including clinical covariates at a univariate level: Univariate refers to using only one biomarker —with regard to clinical covariates, this can be a multivariate model. This approach parallels the search without clinical covariates, except that the methods should allow for incorporating the relevant covariate information. The scatterplot method of choosing a cutoff allows only a limited use of covariates, e.g., a binary covariate could be color coded within the plot. If the analysis relies on some regression approach, then the use of covariates (also many of them at a time) is usually facilitated. The cutoff search based on the Cox model described under 3 above allows for an easy incorporation of covariates and thereby leads to a covariate adjusted univariate cutoff search. The adjustment by covariates may be done as covariates in the model or via the inclusion in a stratified analysis. Also the other choices of prediction functions allow for the incorporation of covariates.

This is straightforward for the Cox model choice as prediction function. This includes the option to estimate the influence of covariates on an interaction level, which means that, e.g., for different age groups different predictive criteria apply. For the likelihood ratio type of prediction functions, the prediction densities must be estimated including covariates. For this purpose, the methodology of multivariate pattern recognition can be used or the biomarker values can be adjusted by multiple regression on the covariates (prior to density estimation).

The CART technology (Classification and Regression Trees; Breiman et al. (Wadsworth, Inc.: New York, 1984) can be used for this purpose, employing a biomarker (raw measurement level) plus clinical covariates and utilizing a clinical benefit measure as response. Cutoffs are searched and a decision-tree type of function will be found involving the covariates for prediction. The cutoffs and algorithms chosen by CART are frequently close to optimal and may be combined and unified by considering different clinical benefit measures.

5: Selection of biomarker prediction functions at a multivariate level: When there are several biomarker candidates that maintain their prediction potential within the different univariate prediction function choices, then a further improvement may be achieved by combinations of biomarkers, i.e., considering multivariate prediction functions.

Based on the simple Heaviside function model, combinations of biomarkers may be evaluated, e.g., by considering bivariate scatterplots of biomarker values where optimal cutoffs are indicated. Then a combination of biomarkers can be achieved by combining different Heaviside function by the logical "AND" and "OR" operators to achieve an improved prediction.

The CART technology can be used for this purpose, employing multiple biomarkers (raw measurement level) and a clinical benefit measure as response, to achieve cutoffs for biomarkers and decision-tree type of functions for prediction. The cutoffs and algorithms chosen by CART are frequently close to optimal and may be combined and unified by considering different clinical benefit measures.

The Cox-regression can be employed on different levels. A first way is to incorporate the multiple biomarkers in a binary way (i.e., based on Heaviside functions with some cutoffs). The other option is to employ biomarkers in a metric way (after suitable transformations), or a mixture of the binary and metric approach. The evolving multivariate prediction function is of the Cox type as described under 3 above.

The multivariate likelihood ratio approach is difficult to implement, but presents another option for multivariate prediction functions.

6: Selection of biomarker prediction functions including clinical covariates at a multivariate level: When there are relevant clinical covariates, then a further improvement may be achieved by combining multiple biomarkers with multiple clinical covariates. The different prediction function choices will be evaluated with respect to the possibilities to include clinical covariates.

Based on the simple logical combinations of Heaviside functions for the biomarkers, further covariates may be included to the prediction function based on the logistic regression model obtained in the training set. The CART technology and the evolving decision trees can be easily used with additional covariates, which would include these in the prediction algorithm.

All prediction functions based on the Cox-regression can use further clinical covariates. The option exists to estimate the influence of covariates on an interaction level, which means that, e.g. , for different age groups different predictive criteria apply.

The multivariate likelihood ratio approach is not directly extendible to the use of additional covariates.

EXAMPLE 1 Predictive biomarkers for response to anti-CD20 therapy in rheumatoid arthritis

REFLEX and SERENE trials

INTRODUCTION RA is a chronic systemic autoimmune disease characterized by symmetric inflammation of affected joints (Sangha, Rheumatology (Oxford), 39 Suppl 2:3-12 (2000); Smith and Haynes, Ann. Intern. Med., 136(12):908 (2002)). Previous clinical experience with rituximab, a chimeric monoclonal anti-CD20 antibody, has demonstrated clinical benefit for moderate-severe RA, thereby establishing the important role of B cells in RA disease pathogenesis (Edwards et al, 2004, supra; Emery et al, Arthr. and Rheum., 54: 1390-1400 (2006) and Cohen et al, Arthr. and Rheum., 54:2793-2806 (2006)). Several critical B-cell functions may contribute to this effect, including autoantibody secretion, autoantigen presentation, proinflammatory cytokine production, and regulation of dendritic cell function (Olsen and Stein, N EnglJ Med, 350(21):2167 (2004); Dorner and Burmester, Curr. Opin. Rheumatol, 15(3):246-52 (2003); Edwards et al, 1999, supra; Metlay et al, Adv. Immunol, 47:45 (1989); Pistoia and Corcione, Stem Cells, 13(5):487 (1995); Takemura et al, J. Immunol, 167(8):4710 (2001)).

Ocrelizumab is a humanized, monoclonal anti-CD20 antibody (a 2H7 antibody) constructed with recombinant DNA techniques (Clark et al, Proc Natl Acad Sd USA, 82(6):1766-1770 (1985)), to selectively target CD20+ B cells. Ocrelizumab has the variable light-chain domain of SEQ ID NO:26 and the variable heavy-chain domain of SEQ ID NO:29 of US 2006/0088523. The aim of the work described in the present example was to identify and verify biomarkers that could predict response to anti-CD20 therapy in rheumatoid arthritis patients. A threshold sensitivity analysis method was used to determine optimal thresholds and effect sizes of baseline analytes and clinical features associated with clinical efficacy. We conducted an analysis of the REFLEX clinical trial (Rituximab treatment of RA patients that had an inadequate response to one or more TNFα-blockade therapies) and identified pre -treatment biomarker profiles that enriched a patient subgroup with an enhanced clinical response versus the unselected population or patients without the biomarker profile. Then, these biomarkers were tested prospectively in a verification study (SERENE clinical trial, Rituximab treatment of RA patients that had an inadequate response to DMARD therapies) both individually and in combination. A biomarker combination comprising elevated levels of C-reactive protein (CRP) and elevated levels of IgA Rheumatoid Factor (RF) antibodies defines an RA patient subset with enriched clinical response to anti-CD20 treatment. The data also shows that these findings can be extended to patients that have elevated CRP levels and are seropositive for other RF antibody isotypes as well as anti-CCP antibodies. Therefore, a combination of inflammation and autoantibody positivity at baseline defines an RA patient subgroup with enhanced clinical response to anti-CD20 therapy. MATERIALS AND METHODS

Threshold Sensitivity Analysis

We defined clinical response as a 50% improvement or better in ACRn score (ACR50) at 24 weeks. We examined pre-treatment biomarkers comprising clinical features, blood components, and serum proteins and assessed their correlation with clinical response in patients receiving either anti-CD20 therapy or placebo. A threshold sensitivity analysis method was used such that we examined the range of each biomarker between the 20th and 80th percentiles in increments of 5%, and for each cut of the range we examined the efficacy response of the biomarker-defmed population above and below the threshold. Logistic regression was then utilized to examine the biomarker-response relationship and determine the optimal cutoff point for each biomarker. An illustration of this process is given below (FIG. 1). REFLEX Clinical Trial

This trial evaluated primary efficacy and safety at 24 weeks in patients enrolled in the Randomized Evaluation of Long-Term Efficacy of Rituximab in RA (REFLEX) Trial, a 2-year, multicenter, randomized, double-blind, placebo-controlled, phase III study of rituximab therapy. Patients with active RA and an inadequate response to one or more anti-TNF agents were randomized to receive intravenous rituximab (1 course, consisting of 2 infusions of 1,000 mg each) or placebo, both with background MTX. The primary efficacy end point was a response on the American College of Rheumatology 20% improvement criteria (ACR20) at 24 weeks. Secondary end points were responses on the ACR50 and ACR70 improvement criteria, the Disease Activity Score in 28 joints, and the European League against Rheumatism (EULAR) response criteria at 24 weeks. Additional end points included scores on the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F), Health Assessment Questionnaire (HAQ) Disability Index (DI), and Short Form 36 (SF-36) instruments, as well as Genant-modified Sharp radiographic scores at 24 weeks.

Patients assigned to placebo (n = 209) and rituximab (n = 311) had active, longstanding RA. At week 24, significantly more (P < 0.0001) rituximab-treated patients than placebo-treated patients demonstrated ACR20 (51% versus 18%), ACR50 (27% versus 5%), and ACR70 (12% versus 1%) responses and moderate-to- good EiULAR. responses (65% versus 22%). AU ACR response parameters were significantly improved in rituximab-treated patients, who also had clinically meaningful improvements in fatigue, disability, and health-related quality of life (demonstrated by FACIT-F, HAQ DL and SF-36 scores, respectively) and showed a trend toward less progression in radiographic end points. Rituximab depleted peripheral CD20+ B cells, but the mean immunoglobulin levels (IgG, IgM, and IgA) remained within normal ranges. Most adverse events occurred with the first rituximab infusion and were of mild-to-moderate severity. The rate of serious infections was 5.2 per 100 patient-years in the rituximab group and 3.7 per 100 patient-years in the placebo group.

At 24 weeks, a single course of rituximab with concomitant MTX therapy provided significant and clinically meaningful improvements in disease activity in patients with active, longstanding RA who had an inadequate response to 1 or more aπti-TNF therapies

For further details of the REFLEX trial see, Cohen et al., Arthritis & Rheumatism 54(9):2793-2806 (2006). SERENE Clinical Trial

This is a phase III, randomized, placebo controlled, double-blind, parallel group, international study in approximately 500 patients with active RA who have had an inadequate clinical response to MTX therapy.

The individual treatment groups are: Group A: Rituximab 500 mg i.v. x2

Group B: Rituximab 1000 mg i.v. x2

Group C: Placebo Rituximab i.v. χ2

An overview of the SERENE study treatment is shown in FIG. 10. All patients receive concomitant MTX 10-25 mg/week at a stable dose (p.o. or parenteral) as prescribed by the treating physician and in accordance with the local label.

Rituximab (500 mg, 1000 mg or placebo) will be administered by intravenous infusion on days 1 and 15. 100 mg i.v. methylprednisolone is to be administered by slow infusion to be completed at least 30 minutes prior to each infusion of rituximab/placebo. 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. Since transient hypotension may occur during rituximab infusion, the investigator may wish to withhold anti-hypertensive medications 12 hours prior to rituximab infusion. All patients also receive a stable dose of folate (> 5 mg/week) given as either a single dose or as a divided weekly dose. All patients should continue to receive any background glucocorticoids (< 10 mg/day prednisone or equivalent) or oral nonsteroidal anti-inflammatory drugs (NSAIDs) at a stable dose. Treatment with all DMARDs, except MTX, must be discontinued 14 days prior to baseline, except for the following: azathioprine for > 28 days; leflunomide for > 8 weeks (or > 28 days after 11 days of standard cholestyramine or activated charcoal washout).

RF-positive and RF-negative patients are enrolled and allocated equally between treatment arms, with the overall proportion of RF-negative patients being limited to 20% of the total sample size. The screening visit can occur up to 28 days prior to receiving the first dose of study treatment. However, for patients requiring washout from leflunomide, the screening period may be extended to 56 days.

Patient eligibility for this study is determined at the baseline visit, at which time the patient is randomized. The first dose of study medication occurs within 24 hours following the baseline assessments. However, when necessary, up to 72 hours will be allowed between baseline assessments being performed, and when the first dose of study medication is administered.

After receiving study medication on day 1 and day 15 the patients return for safety and efficacy assessments at weeks 4, 8, 12, 16, 20 and 24.

Evaluation of the primary endpoint (ACR20) occurs at week 24. Between week 16 and week 23, patients who do not have a 20% improvement in both tender and swollen joint counts compared to baseline are allowed to initiate "rescue"treatment with one non-biologic DMARD, the choice of which is at the discretion of their treating physician.

After week 24 all patients are followed every 8 weeks and may receive further courses of active rituximab, based on the disease activity.

All patients who withdraw from the study at any point or complete the total 3- year treatment period should return for safety follow up (SFU) assessments at weeks 4, 12, 24, 36 and 48 after withdrawal or completion. This effectively follows the patient for 1 year after the patient withdraws from/completes the study. If a patient's peripheral B cell count has not returned to their baseline level or to within the normal range, whichever is lower, after 1 year, safety follow-up visits should be performed at 12 week intervals until B cell repletion occurs.

Each patient is eligible to receive further courses of rituximab for up to a total treatment period of 3 years from their initial treatment, at which point it is expected that the drug will be commercially available in this population. The end of treatment is therefore defined as the 3 year time point, following which there will be an additional period of at least a year of SFU as described above. Biomarker measurement methodology

C-reactive protein was measured using standard or high sensitivity lab tests (Roche Modular immuno-turbimetry polyethylene glycol assay and Dade Behring high sensitivity assay, respectively). IgA RF, IgM RF and IgG RF were measured using the EL-RF/3 immunoassay (TheraTest labs). IgG anti-CCP3 was measured using the CCP3 immunoassay (Inova Diagnostics). Soluble CD25 was measured using Duo Set ELISA development reagents from R&D systems according to the manufacturer's protocol.

RESULTS

Examination of the training set

The REFLEX study (Phase III study of rituximab treatment of active RA patients that have failed one or more therapies against TNFalpha) was used as the training set for these analyses. Biomarkers such as clinical features, serum proteins, and autoantibodies were measured at baseline (pre-treatment) and their association with clinical response after rituximab or placebo (defined as ACR50 at 24 weeks) was determined using the statistical methodology described above. The biomarkers shown in FIG. 2 were found to be associated with a responsive subgroup of patients: C- reactive protein (CRP) greater than 3.9 mg/dL, IgG anti-CCP3 antibody titer below 870 U/ml, soluble CD25 concentration below 3926 pg/ml, IgA Rheumatoid Factor antibody titer above 25 U/ml, and DAS-ESR greater than 6.3.

These biomarkers were additionally examined to determine whether a combination of two of them could derive further predictive power. We found that a combination of CRP (greater than 2.9 mg/dL) and IgA RF (titer greater than 25 U/ml) had further power in defining a subgroup of RA patients with increased clinical response to rituximab (see FIG. 3).

Verification of biomarker profiles

We used the SERENE study (phase III clinical trial of rituximab in active RA patients who have an inadequate response to DMARDs) to prospectively test these biomarkers and the biomarker combination described above. We again measured the candidate biomarkers identified in the REFLEX study and applied the same statistical analysis to ascertain their relationship to clinical response after rituximab or placebo treatement. This analysis confirmed that elevated CRP level and elevated IgA RF titer was associated with improved placebo-corrected clinical efficacy to rituximab. In addition, we noted that the combination of these two biomarkers had further power to define an enhanced rituximab-responsive subgroup of RA patients. The data are shown in FIGS. 4-6 with the placebo corrected delta between the two biomarker- defined subgroups stated.

We then examined the association of this combination CRP & IgA RF biomarker signature with other efficacy endpoints besides ACR50 (ACR20, ACR70, EULAR response and delta DAS at 24 weeks). As shown in FIGS. 7A-7D, the signature-defined RA subgroup performed better with all of these endpoints versus the negative subgroup.

We next examined the data set to see whether one could define an enhanced responder population additionally on the basis of the respective autoantibody positivity threshold as defined by the antibody test manufacturer, and whether this in combination with CRP further refined this subpopulation. FIGS. 8A-8H shows that in both the REFLEX and SERENE trials, patients who were positive for IgM RF, IgG RF, IgA RF, total RF, and IgG anti-CCP3 antibodies at baseline had an enhanced response to rituximab versus patients who were negative for these antibodies.

We then used the combination of CRP>2.9 mg/dL and positivity for IgM RF, IgG RF, IgA RF, total RF, and IgG anti-CCP3 antibodies to subset the patient population in the SERENE study. FIGS. 9A-9E shows that these combinations further enrich patient subgroups with improved clinical efficacy versus patients with CRP<2.9 mg/dL and seronegative for these autoantibodies. Thus, it holds that a combination of CRP and autoantibody positivity at baseline further defines an RA patient subset with enhanced clinical efficacy after rituximab treatment more than autoantibody positivity alone.

The results of the RITUXAN® SERENE trial threshold sensitivity analysis for CRP + IgA RF combination are shown in FIG. 11.

By examining a training set (REFLEX) and a verification set (SERENE) of placebo-controlled phase III clinical trials of RA patients treated with rituximab, we found that biomarkers pertaining to elevated inflammation (CRP) and elevated autoantibodies (IgA RF) defined a pre-treatment subset of RA patients that showed enhanced clinical response after treatment with rituximab. These biomarkers have greater power when used in combination. These findings can be extended to seropositivity for other RF isotypes and IgG anti-CCP antibodies in combination with CRP. The implication of these results is that rituximab works best in RA patients whose disease is characterized by elevated inflammation and is accompanied by B cell involvement. Patients with low inflammatory disease can still benefit from rituximab, but the response rate in this subgroup is lower. EXAMPLE 2

Predictive biomarkers for response to anti-CD20 therapy in rheumatoid arthritis -

ACTION clinical trial

STUDY DESIGN

The ACTION trial was a randomized, placebo-controlled, blinded Phase I/II, dose-ranging study evaluating the safety of ocrelizumab in combination with MTX. All other DMARDs were withdrawn at least four weeks prior to randomization (eight weeks for etanercept, infliximab, adalimumab, and leflunomide). Patients had received 10-25 mg MTX weekly for at least 12 weeks (stable dose for at least 4 weeks) before treatment. Stable doses of oral glucocorticosteroids (prednisone equivalent up to 10 mg daily) and NSAIDs were permitted.

In Phase I, 45 patients were treated in sequential cohorts with escalating doses of ocrelizumab or placebo and evaluated for safety. The first cohort received 10 mg x 2. Patients began enrolling into the next higher dosing cohort 72 hours after the last patient in the prior dose cohort received the second infusion (FIG. 1). An interim safety analysis was conducted after Phase I, and subsequently Phase II was opened for an additional 192 patients randomized in parallel to receive one infusion on Day 1 and one infusion on Day 15 of placebo or 10, 50, 200, 500, 1000 mg of ocrelizumab. Patients were randomized according to a fixed block design in Phase I, while a dynamic allocation algorithm was applied in Phase II to balance for baseline TJC and study center. Patients and investigators were blinded to study medication, but unblinded to dose level due to different infusion times. Oral antihistamine and acetaminophen were recommended before the infusion, but no intravenous (IV) corticosteroids were given as pre-medication. Assessments Descriptions of adverse events (AEs) reported during the study period were defined according to MedDRA terms and graded according to the National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 3.0. All treatment-emergent AEs as well as serious AEs (SAEs) were listed and summarized by treatment group. Clinical activity was evaluated as a secondary endpoint via ACR20, ACR50, and ACR70 responses at Week 24 after treatment initiation with placebo or ocrelizumab. Efficacy assessments performed after Week 24 were considered to be of limited utility due to permitted use of alternative DMARDs after Week 24. Disease activity scores based on 28 joints (DAS28) were also determined.

Based on these data, DAS remission responses (DAS28 score <2.6) and EULAR Good responses (DAS28 <3.2 and improvement of > 1.2) were assessed. In addition, the proportion of patients exhibiting a swollen joint count of zero (SJC=O) was examined.

Serum samples were collected for determination of human anti-human antibodies (HAHAs) and assayed in an electrochemiluminescence assay. Circulating B cells were measured by fluorescence-activated cell sorting (FACS) using labeled antibodies against CD 19, a marker also present on B cells.

Data analysis

The sponsor was responsible for data collection, and statistical analyses were conducted by statisticians who were employees of the sponsor.

All data analyses were conducted using descriptive statistics, which were based on the intent-to-treat (ITT) principle, unless stated otherwise. Results were summarized separately for the placebo group (pooled across all dose levels) and each of the five ocrelizumab groups. For categorical efficacy response analyses, a non- responder imputation was applied to all patients who did not complete the Week 24 visit, required an increase in the dose of MTX or corticosteroids, or required additional therapeutic interventions before Week 24. Statistical analyses were performed using SAS® or S-P LUS® software.

The sample size at the dose-escalation phase (n = 5, 10, 10, 10, 10 at the protocol-specified dose levels) was chosen to detect major intolerability signals. The combined Phase I/II sample size of approximately 40 patients per dose level was selected to provide adequate characterization of the relationship between dose level and the rates of safety events and efficacy responses. Consistent with the nature of Phase II trials, this study was not designed to provide statistical confirmation of efficacy.

Relevant lab parameters

C-Reactive Protein (CRP)

To assess the effect of ocrelizumab on CRP, data were excluded from patients who had received excluded medications before Week 24. At Week 24, the median percent CRP reduction ranged from 31-38% at doses of 200 mg and higher, compared to 16% in the placebo group and 11-15% in the 10-mg and 50-mg dose groups.

Immunoglobulins

Median IgM levels were reduced by 10% to 23% at Week 24 among the 5 ocrelizumab-treated groups compared with 2% in the placebo group. A total of 8

(4.1%) ocrelizumab patients demonstrated a drop in IgM levels below the lower limit of normal at any time during the study. The lowest IgM level recorded was 0.29 mg/mL in a patient receiving 500-mg ocrelizumab. There was no association between IgM levels and observed infection-related AEs. There were no differences in median IgG or IgA levels observed between ocrelizumab-treated and placebo groups.

RESULTS

Examining data from the above ACTION 2H7 RA trial, we combined the high dose 2H7 groups (200mg, 500mg and lOOOmg), and examined the response profile in the placebo and active arms using the combination biomarker profile of elevated CRP (>2.0 mg/dL) and IgA RF antibodies (>25 U/ml). The data shown in FIG. 12 confirms our observations from the REFLEX and SERENE trials that this biomarker-defmed RA subgroup (CRP>2.9 mg/dL & IgA RF>25 U/ml (23% patients)) demonstrates increased ACR50 response rates at 24 weeks versus the biomarker-negative subgroup.

EXAMPLE 3 Effect of further autoantibodies - REFLEX and SERENE clinical trials

In addition to IgA RF antibodies, we also examined the effect of other prototypical RA autoantibodies on response to anti-CD20 antagonism in both the REFLEX and SERENE trials. As shown in FIGS. 13A-13H, we found that patients who were positive for IgM RF, IgG RF, IgA RF and IgG anti-CCP3 antibodies demonstrated higher ACR50 responses at 24 weeks versus patients who were negative for these autoantibodies.

Further, we observed that a combination of elevated CRP plus positivity for any of the autoantibodies shown in FIGS. 13A-13H also enriches a responder subgroup similar to that seen with CRP plus IgA RF as previously described. Data illustrating this from the SERENE trial is shown in FIGS. 14A-14D CRP1" = >2.9 mg/dL.

Therefore the combinations of CRP plus positivity for all RF isotypes and IgG anti-CCP3 antibodies are useful as a baseline predictive biomarker signature. Of these, the combination of CRP1" and IgA RF+ gave the best discrimination for ACR50 response rate at 24 weeks.

The experimental data shows that increasing amount of the biomarkers and biomarker combinations herein are associated with enhanced clinical response. The thresholds shown have been defined statistically as the optimal cut points under the circumstances, but these can be altered while still enriching a responder subgroup.

While the invention is illustrated by reference to specific embodiments, it is not so limited. One of ordinary skill will understand that various modifications are possible and can be readily made without any inventive activity or undue experimentation. All such variants are modifications are intended to be within the scope of the invention herein.

Claims

WHAT IS CLAIMED IS:
1. A method of treating rheumatoid arthritis in a patient comprising administering an effective amount of a B-cell antagonist to the patient to treat the rheumatoid arthritis, provided that the rheumatoid arthritis is characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody in a sample from the patient.
2. The method of claim 1 wherein the positive acute phase protein is selected from the group consisting of C-reactive protein (CRP), D-dimer protein, mannose- binding protein alpha 1 -antitrypsin, alpha 1-antichymotrypsin, alpha 2 macroglobulin, fibrinogen, prothrombin, Factor VIII, von Willebrand factor, plasminogen, complement factors, ferritin, serum amyloid A, ceruloplasmin, and haptoglobulin.
3. The method of claim 2 wherein the positive acute phase protein is CRP or serum amyloid A.
4. The method of claim 3 wherein the positive acute phase protein is CRP.
5. The method of claim 4 wherein the rheumatoid arthritis is characterized by at least about 1 mg/dL CRP in said sample.
6. The method of claim 5 wherein the rheumatoid arthritis is characterized by at least about 2 mg/dL CRP in said sample.
7. The method of claim 6 wherein the rheumatoid arthritis is characterized by at least about 3 mg/dL CRP in said sample.
8. The method of claim 7 wherein the rheumatoid arthritis is characterized by at least about 3.9 mg/dL CRP in said sample.
9. The method of any one of claims 1 -8 wherein the rheumatoid arthritis- associated autoantibody is selected from the group consisting of anti-rheumatoid factor (anti-RF), anti-RA33, anti-calpastatin, anti-SA protein, anti-p68, anti-glucose- 6-phosphate isomerase (GPI), and anti-CCP antibodies.
10. The method of claim 9 wherein the rheumatoid arthritis-associated autoantibody is one or more isotype of an anti-RF antibody.
11. The method of claim 10 wherein the rheumatoid arthritis-associated autoantibody is an IgA, IgG, IgM and/or IgE anti-RF antibody.
12. The method of claim 11 wherein the rheumatoid arthritis-associated autoantibody is an IgA anti-RF antibody.
13. The method of claim 12 wherein the titer of the IgA anti-RF antibody is at least about 25 U/ml.
14. The method of claim 13 wherein the sample is seropositive for at least one additional biomarker or rheumatoid arthritis.
15. The method of claim 14 wherein the additional biomarker is an anti-CCP antibody.
16. The method of claim 15 wherein the anti-CCP antibody is of the IgG isotype.
17. The method of claim 15 wherein the anti-CCP antibody is of the IgM isotype.
18. The method of claim 1 wherein the antagonist is an antibody or immunoadhesin.
19. The method of claim 1 wherein the antagonist is to CD20, CD22, BAFF, or APRIL.
20. The method of claim 1 wherein the antagonist is an antibody or TACI-Ig.
21. The method of claim 1 wherein the antagonist is an antibody.
22. The method of claim 21 wherein the antibody is a chimeric, humanized, or human antibody.
23. The method of claim 22 wherein the antagonist is anti-CD20 antibody or anti- CD22 antibody.
24. The method of claim 23 wherein the antagonist is anti-CD20 antibody.
25. The method of claim 24 wherein the anti-CD20 antibody is rituximab.
26. The method of claim 24 wherein the anti-CD20 antibody is a 2H7 antibody.
27. The method of claim 26 wherein the 2H7 antibody comprises the L-chain variable region sequence of SEQ ID NO: 1 and the H-chain variable region sequence of SEQ ID NO:2.
28. The method of claim 26 wherein the 2H7 antibody comprises the L-chain variable region sequence of SEQ ID NO:3 and the H-chain variable region sequence of SEQ ID NO:4.
29. The method of claim 26 wherein the 2H7 antibody comprises the L-chain variable region sequence of SEQ ID NO:3 and the H-chain variable region sequence of SEQ ID NO:5.
30. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:7.
31. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO:8.
32. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 10.
33. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 11.
34. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 12.
35. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 13.
36. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:9 and the full-length H chain of SEQ ID NO: 14.
37. The method of claim 26 wherein the 2H7 antibody comprises the full-length L chain of SEQ ID NO:6 and the full-length H chain of SEQ ID NO: 15.
38. The method of claim 1 wherein the antagonist is not conjugated with a cytotoxic agent.
39. The method of claim 1 wherein the antagonist is conjugated with a cytotoxic agent.
40. The method of claim 1 wherein the sample is blood, synovial tissue, or synovial fluid.
41. The method of claim 40 wherein the sample is blood.
42. The method of claim 1 wherein the patient has never been previously administered a medicament for the rheumatoid arthritis.
43. The method of claim 1 wherein the patient has been previously administered at least one medicament for the rheumatoid arthritis.
44. The method of claim 43 wherein the patient was not responsive to at least one medicament that was previously administered.
45. The method of claim 44 wherein the previously administered medicament or medicaments are an immunosuppressive agent, cytokine antagonist, integrin antagonist, corticosteroid, analgesic, a disease-modifying anti-rheumatic drug (DMARD), or a non-steroidal anti-inflammatory drug (NSAID).
46. The method of claim 45 wherein the previously administered medicament or medicaments are an immunosuppressive agent, cytokine antagonist, integrin antagonist, corticosteroid, DMARD, or NSAID.
47. The method of claim 45 wherein the previously administered medicament is a TNF-α inhibitor or methotrexate.
48. The method of claim 45 wherein the previously administered medicament is a CD20 antagonist that is not rituximab or a 2H7 antibody.
49. The method of claim 45 wherein the previously administered medicament is rituximab or a 2H7 antibody.
50. The method of claim 1 wherein the B-cell antagonist is administered intravenously.
51. The method of claim 1 wherein the B-cell antagonist is administered subcutaneously.
52. The method of claim 1 wherein at least about three months after the administration, an imaging test is given that measures a reduction in bone or soft tissue joint damage as compared to baseline prior to the administration, and the amount of the B-cell antagonist administered is effective in achieving a reduction in the joint damage.
53. The method of claim 52 wherein the test measures a total modified Sharp score.
54. The method of claim 1 wherein the antagonist is administered in a dose of about 0.2 to 4 grams.
55. The method of claim 54 wherein the dose is about 0.2 to 3.5 grams.
56. The method of claim 55 wherein the dose is about 0.4 to 2.5 grams.
57. The method of claim 56 wherein the dose is about 0.5 to 1.5 grams.
58. The method of claim 1 wherein the antagonist is an anti-CD20 antibody and is administered in a dose of about 0.4 to 4 grams.
59. The method of claim 58 wherein the antibody is administered in a dose of about 0.4 to 1.3 grams.
60. The method of claim 59 wherein the dose is about 1.5 to 3.5 grams.
61. The method of claim 60 wherein the dose is about 1.5 to 2.5 grams.
62. The method of claim 1 wherein the antagonist is administered at a frequency of one to four doses within a period of about one month.
63. The method of claim 62 wherein the antagonist is an anti-CD20 antibody and the dose is about 200 mg to 1.2 grams.
64. The method of claim 63 wherein the dose is about 200 mg to 1.1 grams.
65. The method of claim 62 wherein the antagonist is administered in two to three doses.
66. The method of claim 62 wherein the antagonist is administered within a period of about 2 to 3 weeks.
67. The method of claim 66 wherein the antagonist is an anti-CD20 antibody and the dose is about 500 mg to 1.2 grams.
68. The method of claim 67 wherein the dose is about 750 mg to 1.1 grams.
69. The method of claim 1 wherein the B-cell antagonist is administered without any other medicament to treat the RA.
70. The method of claim 1 further comprising administering an effective amount of one or more second medicaments with the B-cell antagonist, wherein the B-cell antagonist is a first medicament.
71. The method of claim 70 wherein the second medicament is more than one medicament.
72. The method of claim 70 wherein the second medicament is 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 combination thereof.
73. The method of claim 72 wherein the second medicament is a DMARD.
74. The method of claim 73 wherein the DMARD is selected from the group consisting of auranofm, chloroquine, D-penicillamine, injectable gold, oral gold, hydroxychloroquine, sulfasalazine, myocrisin and methotrexate.
75. The method of claim 72 wherein the second medicament is a NSAID.
76. The method of claim 75 wherein the NSAID is selected from the group consisting of: fenbufen, naprosyn, diclofenac, etodolac, indomethacin, aspirin and ibuprofen.
77. The method of claim 72 wherein the immunosuppressive agent is selected from the group consisting of etanercept, infliximab, adalimumab, leflunomide, anakinra, azathioprine, and cyclophosphamide.
78. The method of claim 72 wherein the second medicament is selected from the group consisting of anti-alpha4, etanercept, infliximab, etanercept, adalimumab, kinaret, efalizumab, osteoprotegerin (OPG), anti-receptor activator of NFKB ligand (anti-RANKL), anti-receptor activator of NFKB-FC (RANK-FC), pamidronate, alendronate, actonel, zolendronate, clodronate, methotrexate, azulfϊdine, hydroxychloroquine, doxycycline, leflunomide, sulfasalazine (SSZ), prednisolone, interleukin-1 receptor antagonist, prednisone, and methylprednisolone.
79. The method of claim 72 wherein the second medicament is selected from the group consisting of infliximab, an infliximab/methotrexate (MTX) combination, MTX, etanercept, a corticosteroid, cyclosporin A, azathioprine, auranofϊn, 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.
80. The method of claim 79 wherein the corticosteroid is prednisone, prednisolone, methylprednisolone, hydrocortisone, or dexamethasone.
81. The method of claim 79 wherein the second medicament is MTX.
82. The method of claim 81 wherein the MTX is administered perorally or parenterally.
83. The method of claim 1 wherein the B-cell antagonist is an anti-CD20 antibody administered at a dose of about 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment.
84. The method of claim 1 wherein the anti-CD20 antibody is administered as a single dose or as two infusions, with each dose at about 200 mg to 600 mg.
85. The method of claim 1 wherein the patient has exhibited an inadequate response to one or more anti-tumor necrosis factor (TNF) inhibitors.
86. The method of claim 1 wherein the B-cell antagonist is an anti-CD20 antibody administered as a single dose or as two doses, with each dose being between about
200 mg and 1000 mg.
87. The method of claim 86 wherein the anti-CD20 antibody is administered at a dose of about 200 mg x 2, 500 mg x 2, or 1000 mg x 2 on days 1 and 15 intravenously at the start of the treatment.
88. The method of claim 1 further comprising re -treating the patient by administering an effective amount of the B-cell antagonist to the patient, wherein the re -treatment is commenced at at least about 24 weeks after the first administration of the antagonist.
89. The method of claim 88 wherein the amount of the B-cell antagonist administered upon each administration thereof is effective to achieve a continued or maintained reduction in joint damage.
90. The method of claim 88 wherein a further re -treatment is commenced with an effective amount of the B-cell antagonist.
91. The method of claim 90 wherein the further re-treatment is commenced at at least about 24 weeks after the second administration of the antagonist.
92. The method of claim 88 wherein joint damage has been reduced after the re- treatment.
93. The method of claim 88 wherein no clinical improvement is observed in the patient at the time of the testing after the re-treatment.
94. The method of claim 93 wherein the clinical improvement is determined by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity.
95. A method of treating rheumatoid arthritis in a patient comprising first administering a B-cell antagonist to the patient to treat the rheumatoid arthritis, provided that the rheumatoid arthritis is characterized by an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody in a sample from the patient, and at least about 24 weeks after the first administration of the antagonist, re-treating the patient by administering an effective amount of the B-cell antagonist to the patient, wherein no clinical improvement is observed in the patient at the time of the testing after the first administration of the B- cell antagonist.
96. The method of claim 95 wherein the clinical improvement is determined by assessing the number of tender or swollen joints, conducting a global clinical assessment of the patient, assessing erythrocyte sedimentation rate, assessing the amount of C-reactive protein level, or using composite measures of disease activity.
97. The method of claim 95 wherein the amount of the B-cell antagonist administered upon re-treatment is effective to achieve a continued or maintained reduction in joint damage as compared to the effect of a prior administration of the B- cell antagonist.
98. A method of treating rheumatoid arthritis in a patient comprising administering to the patient an effective amount of a B-cell antagonist, wherein before the administration, an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody was detected in a sample from the patient.
99. The method of claim 98 wherein before the administration, at least about 3.9 mg/dL CRP and an anti-RF antibody titer of at least about 25 U/ml was detected in the sample.
100. A method of treating rheumatoid arthritis in a patient comprising administering to the patient an effective amount of a B-cell antagonist, wherein before the administration a sample from the patient was determined to contain an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis- associated autoantibody whereby the elevated amount and titer indicate that the patient will respond to treatment with the antagonist.
101. The method of claim 100 wherein the elevated amount of acute phase protein is at least about 3.9 mg/dL CRP and the antibody is an anti-RF antibody having a titer of at least about 25 U/ml.
102. A method for advertising a B-cell antagonist or a pharmaceutically acceptable composition thereof comprising promoting, to a target audience, the use of the antagonist or pharmaceutical composition thereof for treating a patient or patient population with rheumatoid arthritis from which a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.
103. An article of manufacture comprising, packaged together, a pharmaceutical composition comprising a B-cell antagonist and a pharmaceutically acceptable carrier and a label stating that the antagonist or pharmaceutical composition is indicated for treating patients with rheumatoid arthritis from whom a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.
104. The article of claim 103 further comprising a container comprising a second medicament, wherein the B-cell antagonist is a first medicament, further comprising instructions on the package insert for treating the patient with an effective amount of the second medicament.
105. The article of claim 104 wherein the second medicament is methotrexate.
106. A method for manufacturing a B-cell antagonist or a pharmaceutical composition thereof comprising combining in a package the antagonist or pharmaceutical composition and a label stating that the antagonist or pharmaceutical composition is indicated for treating patients with rheumatoid arthritis from whom a serum sample has been obtained showing an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.
107. A method of providing a treatment option for patients with rheumatoid arthritis comprising packaging a B-cell antagonist in a vial with a package insert containing instructions to treat patients with rheumatoid arthritis from whom a sample has been obtained that contains an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.
108. A method for predicting whether a subject with rheumatoid arthritis will respond to a B-cell antagonist, the method comprising determining whether a sample from the subject contains an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody, wherein an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis- associated autoantibody indicates that the subject will respond to the antagonist.
109. A method for marketing a B-cell antagonist for use in a rheumatoid arthritis patient subpopulation, the method comprising informing a target audience about the use of the antagonist for treating the patient subpopulation characterized by the presence, in samples from patients of such subpopulation, of an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody.
110. A method of assessing whether a sample from a patient with rheumatoid arthritis indicates responsiveness of the patient to treatment with a B-cell antagonist comprising: a. detecting in the sample whether an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody is present; d. implementing an algorithm to determine that the patient is responsive to said treatment; and e. recording a result specific to the sample being tested.
111. The method of claim 110 wherein a computer or machine is used to record the result specific to the sample being tested.
112. A system for analyzing susceptibility or responsiveness of a patient with rheumatoid arthritis to treatment with a B-cell antagonist comprising: a. reagents to detect in a sample from the patient an elevated level of a positive acute phase protein and an elevated titer of a rheumatoid arthritis-associated autoantibody; b. hardware to perform detection of the biomarkers; and c. computational means to perform an algorithm to determine if the patient is susceptible or responsive to said treatment.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2482074A1 (en) 2011-01-27 2012-08-01 Medizinische Hochschule Hannover Methods and means for diagnosing vasculitis
US9260517B2 (en) 2009-11-17 2016-02-16 Musc Foundation For Research Development Human monoclonal antibodies to human nucleolin
US9352021B2 (en) 2013-08-28 2016-05-31 Cytonics Corporation Systems, compositions, and methods for transplantation and treating conditions
US10265388B2 (en) 2012-02-21 2019-04-23 Cytonics Corporation Systems, compositions, and methods for transplantation

Citations (294)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4016043A (en) 1975-09-04 1977-04-05 Akzona Incorporated Enzymatic immunological method for the determination of antigens and antibodies
US4018653A (en) 1971-10-29 1977-04-19 U.S. Packaging Corporation Instrument for the detection of Neisseria gonorrhoeae without culture
US4120649A (en) 1975-04-10 1978-10-17 Israel Schechter Transplants
EP0003089A1 (en) 1978-01-06 1979-07-25 Bernard David Drier for silkscreen printed sheets
US4424279A (en) 1982-08-12 1984-01-03 Quidel Rapid plunger immunoassay method and apparatus
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4665077A (en) 1979-03-19 1987-05-12 The Upjohn Company Method for treating rejection of organ or skin grafts with 6-aryl pyrimidine compounds
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
WO1988004936A1 (en) 1987-01-08 1988-07-14 International Genetic Engineering, Inc. Chimeric antibody with specificity to human b cell surface antigen
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4861579A (en) 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
EP0330191A2 (en) 1988-02-25 1989-08-30 The General Hospital Corporation DNA encoding CD40
EP0340109A2 (en) 1988-04-28 1989-11-02 The Board Of Trustees Of The Leland Stanford Junior University Anti-T-cell receptor determinants as autoimmune disease treatment
US4892538A (en) 1987-11-17 1990-01-09 Brown University Research Foundation In vivo delivery of neurotransmitters by implanted, encapsulated cells
WO1990008187A1 (en) 1989-01-19 1990-07-26 Dana Farber Cancer Institute Soluble two domain cd2 protein
WO1990011294A1 (en) 1989-03-21 1990-10-04 The Immune Response Corporation Vaccination and methods against diseases resulting from pathogenic responses by specific t cell populations
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
WO1991000360A1 (en) 1989-06-29 1991-01-10 Medarex, Inc. Bispecific reagents for aids therapy
WO1991001133A1 (en) 1989-07-19 1991-02-07 Arthur Allen Vandenbark T cell receptor peptides as therapeutics for autoimmune and malignant disease
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
US5114721A (en) 1988-03-15 1992-05-19 Yeda Research And Development Co. Ltd. Preparation of t-cell and t-cell membrane for use in prevention and treatment of autoimmune diseases
WO1992020373A1 (en) 1991-05-14 1992-11-26 Repligen Corporation Heteroconjugate antibodies for treatment of hiv infection
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
WO1993025673A1 (en) 1992-06-04 1993-12-23 The Regents Of The University Of California In vivo gene therapy with intron-free sequence of interest
US5283187A (en) 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
WO1994004690A1 (en) 1992-08-17 1994-03-03 Genentech, Inc. Bispecific immunoadhesins
WO1994011026A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma
WO1994011523A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Fully impaired consensus kozac sequences for mammalian expression
EP0633945A1 (en) 1992-04-03 1995-01-18 Genentech, Inc. ANTIBODIES TO ALPHAvBETA3 INTEGRIN
WO1995003770A1 (en) 1993-08-02 1995-02-09 The Board Of Trustees Of The Leland Stanford Junior University Methods for b-cell population control
US5445934A (en) 1989-06-07 1995-08-29 Affymax Technologies N.V. Array of oligonucleotides on a solid substrate
US5484892A (en) 1993-05-21 1996-01-16 Dana-Farber Cancer Institute, Inc. Monoclonal antibodies that block ligand binding to the CD22 receptor in mature B cells
WO1996007321A1 (en) 1994-09-06 1996-03-14 The Uab Research Foundation Methods for modulating protein function in cells using intracellular antibody homologues
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5595721A (en) 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
WO1997038731A1 (en) 1996-04-18 1997-10-23 The Regents Of The University Of California Immunoliposomes that optimize internalization into target cells
US5700637A (en) 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
WO1998008943A1 (en) 1996-08-30 1998-03-05 Mayo Foundation For Medical Education And Research Hla-drb1 peptides with specific binding affinity for hla-dq molecules: prevention and treatment of rheumatoid arthritis
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5733779A (en) 1992-11-13 1998-03-31 Idec Pharmaceuticals Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
WO1998018921A1 (en) 1996-10-25 1998-05-07 Human Genome Sciences, Inc. NEUTROKINE $g(a)
WO1998024893A2 (en) 1996-12-03 1998-06-11 Abgenix, Inc. TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
WO1998027114A2 (en) 1996-12-17 1998-06-25 Schering Corporation Mammalian cell surface antigens; related reagents
WO1998035036A1 (en) 1997-02-10 1998-08-13 Genentech, Inc. Heregulin variants
US5807522A (en) 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
EP0869180A1 (en) 1997-04-02 1998-10-07 Smithkline Beecham Corporation A TNF homologue, TL5
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5846818A (en) 1985-11-01 1998-12-08 Xoma Corporation Pectate lyase signal sequence
US5849898A (en) 1988-02-25 1998-12-15 The General Hospital Corporation CD40 coding sequences
WO1998056418A1 (en) 1997-06-13 1998-12-17 Genentech, Inc. Stabilized antibody formulation
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
WO1999012964A2 (en) 1997-09-12 1999-03-18 Biogen, Inc. Kay - a novel immune system protein
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
WO1999033980A2 (en) 1997-12-30 1999-07-08 Chiron Corporation Members of tnf and tnfr families
WO1999036548A1 (en) 1998-01-16 1999-07-22 Hsc Research And Development Limited Partnership Human lymphoid protein tyrosine phosphatases
US5965787A (en) 1995-08-31 1999-10-12 Mayo Foundation For Medical Education And Research HLA-DRBI peptides with specific binding affinity for HLA-DQ molecules: prevention and treatment of rheumatoid arthritis
WO1999051642A1 (en) 1998-04-02 1999-10-14 Genentech, Inc. Antibody variants and fragments thereof
WO2000009160A1 (en) 1998-08-11 2000-02-24 Idec Pharmaceuticals Corporation Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody
WO2000020864A1 (en) 1998-10-07 2000-04-13 Biocrystal Limited Immunotherapy of b cell involvement in progression of solid, nonlymphoid tumors
WO2000027428A1 (en) 1998-11-09 2000-05-18 Idec Pharmaceuticals Corporation Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody
WO2000027433A1 (en) 1998-11-09 2000-05-18 Idec Pharmaceuticals Corporation Chimeric anti-cd20 antibody treatment of patients receiving bmt or pbsc transplants
WO2000032575A1 (en) 1998-11-30 2000-06-08 Celltech Therapeutics Limited β-ALANINE DERIVATIVES AS α4 INTEGRIN INHIBITORS
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000037444A1 (en) 1998-12-18 2000-06-29 Glaxo Group Limited Compounds useful in the treatment of inflammatory diseases
WO2000040716A2 (en) 1999-01-07 2000-07-13 Zymogenetics, Inc. Soluble receptor br43x2 and methods of using them for therapy
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
WO2000044788A1 (en) 1999-01-28 2000-08-03 Idec Pharmaceuticals Corporation Production of tetravalent antibodies
WO2000058522A1 (en) 1999-03-30 2000-10-05 Nanogen, Inc. Single nucleotide polymorphic discrimination by electronic dot blot assay on semiconductor microchips
WO2000061739A1 (en) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
WO2000067795A1 (en) 1999-05-10 2000-11-16 Immunomedics, Inc. Immunotherapy of b-cell malignancies using anti-cd22 antibodies
WO2000067796A1 (en) 1999-05-07 2000-11-16 Genentech, Inc. Treatment of autoimmune diseases with antagonists which bind to b cell surface markers
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000074718A1 (en) 1999-06-09 2000-12-14 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target b-cells
WO2000076542A1 (en) 1999-06-11 2000-12-21 Consiglio Nazionale Delle Ricerche Use of antibodies against cd20 for the treatment of the graft versus host disease
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
WO2001003734A1 (en) 1999-07-12 2001-01-18 Genentech, Inc. Blocking immune response to a foreign antigen using an antagonist which binds to cd20
US6183744B1 (en) 1997-03-24 2001-02-06 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
WO2001010462A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation Treatment of patients having non-hodgkins lymphoma with bone marrow involvement with anti-cd20 antibodies
WO2001010460A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation Treatment of intermediate- and high-grade non-hodgkins lymphoma with anti-cd20 antibody
WO2001010461A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation New clinical parameters for determining hematologic toxicity prior to radioimmunotherapy
WO2001012812A2 (en) 1999-08-17 2001-02-22 Biogen, Inc. Baff receptor (bcma), an immunoregulatory agent
WO2001012848A2 (en) 1999-08-14 2001-02-22 Univ Leeds Genetic marker for rheumatoid arthritis
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO2001013945A1 (en) 1999-08-23 2001-03-01 Biocrystal Ltd. Methods and compositions for immunotherapy of b cell involvement in promotion of a disease condition comprising multiple sclerosis
WO2001018240A2 (en) 1999-09-08 2001-03-15 The University Of British Columbia Diagnostic and therapeutic methods in autoimmune disease
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
WO2001029246A1 (en) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Process for producing polypeptide
WO2001034194A1 (en) 1999-11-08 2001-05-17 Idec Pharmaceuticals Corporation Treatment of b cell malignancies using anti-cd40l antibodies in combination with anti-cd20 antibodies and/or chemotherapeutics and radiotherapy
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6283761B1 (en) 1992-09-08 2001-09-04 Raymond Anthony Joao Apparatus and method for processing and/or for providing healthcare information and/or healthcare-related information
WO2001072333A1 (en) 2000-03-24 2001-10-04 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma using a combination_of an antibody to cd20 and interleuking-2
WO2001075166A2 (en) 2000-03-31 2001-10-11 Genentech, Inc. Compositions and methods for detecting and quantifying gene expression
WO2001074388A1 (en) 2000-03-31 2001-10-11 Idec Pharmaceuticals Corporation Combined use of anti-cytokine antibodies or antagonists and anti-cd20 for the treatment of b cell lymphoma
WO2001077342A1 (en) 2000-04-11 2001-10-18 Genentech, Inc. Multivalent antibodies and uses therefor
WO2001079173A2 (en) 2000-04-17 2001-10-25 Celltech R & D Limited Enamine derivatives as cell adhesion molecules
WO2001080884A1 (en) 2000-04-25 2001-11-01 Idec Pharmaceuticals Corporation Intrathecal administration of rituximab for treatment of central nervous system lymphomas
WO2001087979A2 (en) 2000-05-12 2001-11-22 Amgen Inc. Methods and compositions of matter concerning april/g70, bcma, blys/agp-3, and taci
US20010056066A1 (en) 1996-07-26 2001-12-27 Smithkline Beecham Corporation Method of treating immune cell mediated systemic diseases
WO2001097858A2 (en) 2000-06-20 2001-12-27 Idec Pharmaceuticals Corporation Cold anti-cd20 antibody/radiolabeled anti-cd22 antibody combination
WO2002002641A1 (en) 2000-06-16 2002-01-10 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
WO2002002556A2 (en) 2000-06-30 2002-01-10 Ortho Mcneil Pharmaceutical, Inc. AZA-BRIDGED-BICYCLIC AMINO ACID DERIVATIVES AS α4 INTEGRIN ANTAGONISTS
WO2002004021A1 (en) 2000-07-12 2002-01-17 Idec Pharmaceuticals Corporation Treatment of b cell malignancies using combination of b cell depleting antibody and immune modulating antibody related applications
US20020006404A1 (en) 1999-11-08 2002-01-17 Idec Pharmaceuticals Corporation Treatment of cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US6348463B1 (en) 1998-09-28 2002-02-19 Celltech Therapeutics Limited Phenylalanine derivatives
WO2002016312A2 (en) 2000-08-23 2002-02-28 Morphochem Ag Novel compounds inhibiting factor xa activity
WO2002016412A2 (en) 2000-08-18 2002-02-28 Dyax Corp. Binding polypeptides for b lymphocyte stimulator protein (blys)
WO2002016329A1 (en) 2000-08-18 2002-02-28 Ajinomoto Co., Inc. Novel phenylalanine derivatives
US20020028178A1 (en) 2000-07-12 2002-03-07 Nabil Hanna Treatment of B cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
WO2002022212A2 (en) 2000-09-18 2002-03-21 Idec Pharmaceuticals Corporation Combination therapy for treatment of autoimmune diseases using b cell depleting/immunoregulatory antibody combination
WO2002024909A2 (en) 2000-09-18 2002-03-28 Biogen, Inc. Receptor nucleic acids and polypeptides
US6368596B1 (en) 1997-07-08 2002-04-09 Board Of Regents, The University Of Texas System Compositions and methods for homoconjugates of antibodies which induce growth arrest or apoptosis of tumor cells
US6369229B1 (en) 1998-06-03 2002-04-09 Celltech Therapeutics, Limited Pyridylalanine derivatives
US20020042368A1 (en) 2000-02-25 2002-04-11 Fanslow William C. Integrin antagonists
WO2002028830A1 (en) 2000-09-29 2002-04-11 Ajinomoto Co.,Inc. Novel phenylalanine derivatives
US20020041847A1 (en) 1998-03-12 2002-04-11 Goldenberg David M. Immunotherapy of malignant and autoimmune disorders in domestic animals using naked antibodies, immunoconjugates and fusion proteins
WO2002034790A1 (en) 2000-10-20 2002-05-02 Idec Pharmaceuticals Corporation Variant igg3 rituxan r and therapeutic use thereof
WO2002038766A2 (en) 2000-11-07 2002-05-16 Zymogenetics, Inc. Human tumor necrosis factor receptor
US6410391B1 (en) 1999-07-02 2002-06-25 Infineon Technologies Ag Method for producing an EEPROM memory cell with a trench capacitor
WO2002056910A1 (en) 2001-01-17 2002-07-25 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2002060955A2 (en) 2001-01-29 2002-08-08 Idec Pharmaceuticals Corporation Modified antibodies and methods of use
WO2002066516A2 (en) 2001-02-20 2002-08-29 Zymogenetics, Inc. Antibodies that bind both bcma and taci
US20020136719A1 (en) 2000-12-28 2002-09-26 Bhami Shenoy Crystals of whole antibodies and fragments thereof and methods for making and using them
WO2002078766A2 (en) 2001-04-02 2002-10-10 Genentech, Inc. Combination therapy
WO2002079255A1 (en) 2001-04-02 2002-10-10 Idec Pharmaceuticals Corporation RECOMBINANT ANTIBODIES COEXPRESSED WITH GnTIII
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
WO2002092620A2 (en) 2001-05-11 2002-11-21 Amgen, Inc. Peptides and related molecules that bind to tall-1
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
WO2002102312A2 (en) 2001-06-14 2002-12-27 Intermune, Inc. Combination therapy of gamma-interferon and b cell specific antibodies
WO2003002607A1 (en) 2001-06-27 2003-01-09 Shawn Shui-On Leung Reducing immunogenicities of immunoglobulins by framework-patching
US20030026801A1 (en) 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
WO2003010135A1 (en) 2001-07-26 2003-02-06 Ajinomoto Co., Inc. Novel phenylpropionic acid derivatives
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003014294A2 (en) 2001-08-03 2003-02-20 Genentech, Inc. Tacis and br3 polypeptides and uses thereof
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
WO2003024991A2 (en) 2001-09-21 2003-03-27 Amgen Inc. Tall-1 receptor molecules and uses thereof
US20030068664A1 (en) 2001-09-20 2003-04-10 Board Of Regents, The University Of Texas System Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using elisa assays
WO2003035846A2 (en) 2001-10-24 2003-05-01 National Jewish Medical And Research Center Structure of tall-1 and its cognate receptor
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
CN1420129A (en) 2001-11-16 2003-05-28 上海中信国健药业有限公司 Humanized anti-CD 20 monoclonal antibody
US20030099943A1 (en) 2000-07-10 2003-05-29 Conaris Research Institute Gmbh Diagnostic use of polymorphisms in the gene coding for the TNF receptor II and method for detecting non-responders to anti-TNF therapy
US20030103971A1 (en) 2001-11-09 2003-06-05 Kandasamy Hariharan Immunoregulatory antibodies and uses thereof
WO2003049694A2 (en) 2001-12-07 2003-06-19 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
WO2003050542A2 (en) 2001-12-11 2003-06-19 Stichting Voor De Technische Wetenschappen Method of detecting autoantibodies from patients suffering from rheumatoid arthritis, a peptide and an assaykit
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20030118592A1 (en) 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
WO2003053926A1 (en) 2001-12-13 2003-07-03 Ajinomoto Co.,Inc. Novel phenylalanine derivative
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
WO2003061694A1 (en) 2001-05-10 2003-07-31 Seattle Genetics, Inc. Immunosuppression of the humoral immune response by anti-cd20 antibodies
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US20030147885A1 (en) 1992-11-13 2003-08-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
WO2003068821A2 (en) 2002-02-14 2003-08-21 Immunomedics, Inc. Anti-cd20 antibodies and fusion proteins thereof and methods of use
WO2003068822A2 (en) 2002-02-13 2003-08-21 Micromet Ag De-immunized (poly)peptide constructs
WO2003070709A1 (en) 2002-02-20 2003-08-28 Ajinomoto Co.,Inc. Novel phenylalanine derivative
US20030180292A1 (en) 2002-03-14 2003-09-25 Idec Pharmaceuticals Treatment of B cell malignancies using anti-CD40L antibodies in combination with anti-CD20 antibodies and/or chemotherapeutics and radiotherapy
US20030185796A1 (en) 2000-03-24 2003-10-02 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
WO2003084570A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM
WO2003085119A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa
WO2003089410A1 (en) 2002-04-19 2003-10-30 Kyowa Hakko Kogyo Co., Ltd. Phenylalanine derivative
US6652852B1 (en) 1986-10-27 2003-11-25 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US20030219818A1 (en) 2002-05-10 2003-11-27 Bohen Sean P. Methods and compositions for determining neoplastic disease responsiveness to antibody therapy
US20030232055A1 (en) 2000-07-31 2003-12-18 Ruslan Medzhitov Innate immune system-directed vaccines
US20040018590A1 (en) 2000-06-28 2004-01-29 Gerngross Tillman U. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
WO2004032828A2 (en) 2002-07-31 2004-04-22 Seattle Genetics, Inc. Anti-cd20 antibody-drug conjugates for the treatment of cancer and immune disorders
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2004056312A2 (en) 2002-12-16 2004-07-08 Genentech, Inc. Immunoglobulin variants and uses thereof
WO2004058298A1 (en) 2002-12-31 2004-07-15 Immunomedics, Inc. Immunotherapy of b cell malignancies and autoimmune disease using unconjugated antibodies and conjugated antibodies, antibody combinations and fusion proteins
WO2004060052A2 (en) 2002-12-16 2004-07-22 Genentech, Inc. Transgenic mice expressing human cd20 and/or cd16
US20040191256A1 (en) 1997-06-24 2004-09-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US20040202658A1 (en) 2003-04-09 2004-10-14 Genentech, Inc. Therapy of autoimmune disease in a patient with an inadequate response to TNF-alpha inhibitor
WO2004092219A2 (en) 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
EP1476120A2 (en) 2002-02-21 2004-11-17 Duke University Treatment methods using anti-cd22 antibodies
WO2004103404A1 (en) 2003-05-20 2004-12-02 Applied Molecular Evolution, Inc. Cd20 binding molecules
US20040258682A1 (en) 1996-03-20 2004-12-23 Immunomedics, Inc Gylocosylated humanized B-cell specific antibodies
WO2005000901A2 (en) 2003-05-09 2005-01-06 Duke University Cd20-specific antibodies and methods of employing same
WO2005000351A2 (en) 2003-06-05 2005-01-06 Genentech, Inc. Combination therapy for b cell disorders
EP1504035A2 (en) 2002-05-02 2005-02-09 Celltech R &amp; D Limited Antibodies specific for human cd22 and their therapeutic and diagnostic uses
US20050032130A1 (en) 2003-07-29 2005-02-10 Genentech, Inc. Neutralizing antibody assay and uses therefor
WO2005011428A1 (en) 2003-07-25 2005-02-10 Bally Gaming International, Inc. Uniquely identifiable casino gaming chips
WO2005014618A2 (en) 2003-08-08 2005-02-17 Immunomedics, Inc. Bispecific antibodies for inducing apoptosis of tumor and diseased cells
WO2005014622A2 (en) 2003-08-07 2005-02-17 F. Hoffmann-La Roche Ag Ra antigenic peptides
WO2005016969A2 (en) 2003-08-14 2005-02-24 Merck Patent Gmbh Cd20-binding polypeptide compositions
WO2005017148A1 (en) 2003-07-26 2005-02-24 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
US20050053602A1 (en) 2003-08-29 2005-03-10 Genentech, Inc. Therapy of ocular disorders
WO2005029091A2 (en) 2003-09-15 2005-03-31 Oklahoma Medical Research Foundation Method of using cytokine assays to diagnose, treat, and evaluate inflammatory and autoimmune diseases
WO2005035778A1 (en) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. PROCESS FOR PRODUCING ANTIBODY COMPOSITION BY USING RNA INHIBITING THE FUNCTION OF α1,6-FUCOSYLTRANSFERASE
WO2005035586A1 (en) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Fused protein composition
WO2005044859A2 (en) 2003-11-05 2005-05-19 Glycart Biotechnology Ag Cd20 antibodies with increased fc receptor binding affinity and effector function
US20050106108A1 (en) 1994-08-12 2005-05-19 Immunomedics, Inc. Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
WO2005053742A1 (en) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicine containing antibody composition
US20050136044A1 (en) 2003-12-04 2005-06-23 Watkins Jeffry D. Butyrylcholinesterase variants that alter the activity of chemotherapeutic agents
WO2005061542A2 (en) 2003-12-19 2005-07-07 Genentech, Inc. Detection of cd20 in transplant rejection
WO2005060999A2 (en) 2003-12-19 2005-07-07 Genentech, Inc. Detection of cd20 in therapy of autoimmune diseases
WO2005064307A2 (en) 2003-12-23 2005-07-14 Roche Diagnostics Gmbh Method of assessing rheumatoid arthritis by measuring anti-ccp and interleukin 6
US20050163775A1 (en) 2003-06-05 2005-07-28 Genentech, Inc. Combination therapy for B cell disorders
WO2005070963A1 (en) 2004-01-12 2005-08-04 Applied Molecular Evolution, Inc Fc region variants
WO2005085858A1 (en) 2004-02-27 2005-09-15 Roche Diagnostics Gmbh Method of assessing rheumatoid arthritis by measuring anti-ccp and serum amyloid a
WO2005086872A2 (en) 2004-03-10 2005-09-22 Celera, An Applera Corporation Business Ptpn22 polymorphisms in diagnosis and therapy
US6953665B1 (en) 1998-09-01 2005-10-11 Mayo Foundation For Medical Education And Research Methods and materials for evaluating rheumatoid arthritis
WO2005103081A2 (en) 2004-04-20 2005-11-03 Genmab A/S Human monoclonal antibodies against cd20
WO2005108989A2 (en) 2004-04-16 2005-11-17 Genentech, Inc. Assay for antibodies
US20050255527A1 (en) 2004-05-15 2005-11-17 Genentech, Inc. Cross-screening system and methods for detecting a molecule having binding affinity for a target molecule
US20050266410A1 (en) 2004-05-19 2005-12-01 Emily Walsh Methods of Human Leukocyte Antigen typing by neighboring single nucleotide polymorphism haplotypes
WO2005113003A2 (en) 2004-04-16 2005-12-01 Genentech, Inc. Method for augmenting b cell depletion
WO2005115453A2 (en) 2004-04-16 2005-12-08 Genentech, Inc. Treatment of polychondritis and mononeuritis multiplex with anti-cd20 antibodies
US20050271658A1 (en) 2004-05-05 2005-12-08 Genentech, Inc. Preventing autoimmune disease
WO2005117978A2 (en) 2004-06-04 2005-12-15 Genentech, Inc. Method for treating multiple sclerosis
WO2005120437A2 (en) 2004-06-04 2005-12-22 Genentech, Inc. Method for treating lupus
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
CN1718587A (en) 2005-07-11 2006-01-11 中国人民解放军军事医学科学院生物工程研究所 Ant CD20 chimeric antibody
WO2006005477A1 (en) 2004-07-09 2006-01-19 Schering Ag Combination therapywith radiolabeled anti-cd20 antibody in the treatment of b-cell lymphoma
WO2006010146A2 (en) 2004-07-09 2006-01-26 The Burnham Institute Functional variant of lymphoid tyrosine phosphatase is associated with autoimmune disorders
WO2006008183A1 (en) 2004-07-23 2006-01-26 Novartis Ag Biomarkers for rheumatoid arthritis (ra)
US20060018900A1 (en) 1999-09-24 2006-01-26 Mccormick Alison A Self antigen vaccines for treating B-cell lymphomas and other cancers
US6991790B1 (en) 1997-06-13 2006-01-31 Genentech, Inc. Antibody formulation
WO2006012508A2 (en) 2004-07-22 2006-02-02 Genentech, Inc. Method of treating sjögren's syndrome
US20060024304A1 (en) 2000-06-28 2006-02-02 Gerngross Tillman U Immunoglobulins comprising predominantly a Man5GlcNAc2 glycoform
WO2006014725A1 (en) 2004-07-21 2006-02-09 Glycofi, Inc. IMMUNOGLOBULINS COMPRISING PREDOMINANTLY A GlcNAcMAN5GLCNAC2 GLYCOFORM
WO2006014679A1 (en) 2004-07-21 2006-02-09 Glycofi, Inc. Immunoglobulins comprising predominantly a glcnac2man3glcnac2 glycoform
US20060029604A1 (en) 2000-06-28 2006-02-09 Gerngross Tillman U Immunoglobulins comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform
WO2006014726A2 (en) 2004-07-21 2006-02-09 Glycofi, Inc. Immunoglobulins comprising predominantly a man5glcnac2 glycoform
WO2006014685A1 (en) 2004-07-21 2006-02-09 Glycofi, Inc. Immunoglobulins comprising predominantly a man3glcnac2 glycoform
WO2006014683A2 (en) 2004-07-21 2006-02-09 Glycofi, Inc. Immunoglobulins comprising predominantly a gal2glcnac2man3glcnac2 glycoform
US20060034830A1 (en) 2000-06-28 2006-02-16 Gerngross Tillman U Immunoglobulins comprising predominantly a GalGlcNAcMan5GLcNAc2 glycoform
US20060034828A1 (en) 2000-06-28 2006-02-16 Gerngross Tillman U Immunoglobulins comprising predominantly a GlcNAcMAN5GLCNAC2 glycoform
WO2006029224A2 (en) 2004-09-08 2006-03-16 Genentech, Inc. Methods of using death receptor ligands and cd20 antibodies
WO2006031370A2 (en) 2004-08-19 2006-03-23 Genentech, Inc. Polypeptide variants with altered effector function
US20060062859A1 (en) 2004-08-05 2006-03-23 Kenneth Blum Composition and method to optimize and customize nutritional supplement formulations by measuring genetic and metabolomic contributing factors to disease diagnosis, stratification, prognosis, metabolism, and therapeutic outcomes
US7029872B2 (en) 2000-06-28 2006-04-18 Glycofi, Inc Methods for producing modified glycoproteins
WO2006042240A2 (en) 2004-10-08 2006-04-20 Wyeth Immunotherapy of autoimmune disorders
WO2006041680A2 (en) 2004-10-05 2006-04-20 Genentech, Inc. Method for treating vasculitis
US20060088523A1 (en) 2004-10-20 2006-04-27 Genentech, Inc. Antibody formulations
US20060093601A1 (en) 2004-09-03 2006-05-04 Genentech, Inc. Humanized anti-beta7 antagonists and uses therefor
US20060121032A1 (en) 2003-03-03 2006-06-08 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
WO2006066086A1 (en) 2004-12-17 2006-06-22 Genentech, Inc. Antiangiogenesis therapy of autoimmune disease in patients who have failed prior therapy
WO2006064121A2 (en) 2004-12-15 2006-06-22 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Cytotoxic antibody directed against type b lymphoid hematopoietic proliferations
WO2006069403A2 (en) 2004-12-22 2006-06-29 Genentech, Inc. Methods for producing soluble multi-membrane-spanning proteins
CN1796997A (en) 2004-12-22 2006-07-05 上海富纯中南生物技术有限公司 Detection kit for diagnosing RA, preparating kit, and method for completing standard of quality detection
US20060153838A1 (en) 2002-02-20 2006-07-13 Watkins Jeffry D Fc region variants
WO2006076651A2 (en) 2005-01-13 2006-07-20 Genentech, Inc. Treatment method
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
WO2006084264A2 (en) 2005-02-07 2006-08-10 Genentech, Inc. Cd20 antibody variants and uses thereof
WO2006093923A2 (en) 2005-02-28 2006-09-08 Genentech, Inc. Treatment of bone disorders
WO2006106959A1 (en) 2005-03-31 2006-10-12 Biomedics Inc. Anti-cd-20 monoclonal antibody
US20060263349A1 (en) 2005-05-20 2006-11-23 Genentech, Inc. Pretreatment of a biological sample from an autoimmune disease subject
US20060263783A1 (en) 2003-07-03 2006-11-23 Podhajcer Osvaldo L Methods and systems for diagnosis of non-central nervous system (cns) diseases in cns samples
WO2006126069A2 (en) 2005-05-24 2006-11-30 Avestha Gengraine Technologies Pvt Ltd. A method for the production of a monoclonal antibody to cd20 for the treatment of b-cell lymphoma
WO2006130458A2 (en) 2005-06-02 2006-12-07 Astrazeneca Ab Antibodies directed to cd20 and uses thereof
WO2007000169A2 (en) 2005-06-29 2007-01-04 Genmab A/S Non-human mammalian arthritis model featuring human antibodies against citrullinated proteins
WO2007014238A2 (en) 2005-07-25 2007-02-01 Trubion Pharmaceuticals, Inc. Single dose use of cd20-specific binding molecules
US7175985B1 (en) 1999-11-08 2007-02-13 Eiken Kagaku Kabushiki Kaisha Method of detecting variation or polymorphism
EP1753878A2 (en) 2004-05-12 2007-02-21 The Board of Trustees of The Leland Stanford Junior University Dna profiling and snp detection utilizing microarrays
US20070048300A1 (en) 2003-08-22 2007-03-01 Biogen Idec Ma Inc. Antibodies having altered effector function and methods for making the same
US20070059306A1 (en) 2005-07-25 2007-03-15 Trubion Pharmaceuticals, Inc. B-cell reduction using CD37-specific and CD20-specific binding molecules
US20070072237A1 (en) 2004-02-27 2007-03-29 Norbert Wild Method of assessing rheumatoid arthritis by measuring rheumatoid factor and interleukin-6
US20070071747A1 (en) 2005-05-16 2007-03-29 Hoffman Rebecca S Use of TNFalpha inhibitor for treatment of erosive polyarthritis
US20070071760A1 (en) 2005-08-09 2007-03-29 Herve Broly Methods for treating B-cell malignancies using a TACI-Ig fusion molecule
WO2007039280A1 (en) 2005-10-06 2007-04-12 Roche Diagnostics Gmbh Anti-ccp and antinuclear antibodies in diagnosis of rheumatoid arthritis
WO2007059188A1 (en) 2005-11-15 2007-05-24 F. Hoffman - La Roche Ag Method for treating joint damage
US20070128626A1 (en) 2005-08-18 2007-06-07 The University Of Iowa Research Foundation Assessing response to anti-CD20 therapy by genotyping C1q components
US7244571B2 (en) 2001-09-06 2007-07-17 Decode Genetics Ehf. Methods for producing Ex vivo models for inflammatory disease and uses thereof
US20070196835A1 (en) 2005-09-27 2007-08-23 Danute Bankaitis-Davis Gene expression profiling for identification monitoring and treatment of rheumatoid arthritis
US20070224189A1 (en) 2002-03-01 2007-09-27 Xencor, Inc. CD20 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
WO2008003319A1 (en) 2006-07-04 2008-01-10 Genmab A/S Cd20 binding molecules for the treatment of copd

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2137325A1 (en) * 2007-04-02 2009-12-30 Genentech, Inc. Biological markers predictive of rheumatoid arthritis response to b-cell antagonists

Patent Citations (415)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
US4018653A (en) 1971-10-29 1977-04-19 U.S. Packaging Corporation Instrument for the detection of Neisseria gonorrhoeae without culture
US4120649A (en) 1975-04-10 1978-10-17 Israel Schechter Transplants
US4016043A (en) 1975-09-04 1977-04-05 Akzona Incorporated Enzymatic immunological method for the determination of antigens and antibodies
EP0003089A1 (en) 1978-01-06 1979-07-25 Bernard David Drier for silkscreen printed sheets
US4665077A (en) 1979-03-19 1987-05-12 The Upjohn Company Method for treating rejection of organ or skin grafts with 6-aryl pyrimidine compounds
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4424279A (en) 1982-08-12 1984-01-03 Quidel Rapid plunger immunoassay method and apparatus
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5846818A (en) 1985-11-01 1998-12-08 Xoma Corporation Pectate lyase signal sequence
US6204023B1 (en) 1985-11-01 2001-03-20 Xoma Ltd. Modular assembly of antibody genes, antibodies prepared thereby and use
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US5567610A (en) 1986-09-04 1996-10-22 Bioinvent International Ab Method of producing human monoclonal antibodies and kit therefor
US6893625B1 (en) 1986-10-27 2005-05-17 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US6652852B1 (en) 1986-10-27 2003-11-25 Royalty Pharma Finance Trust Chimeric antibody with specificity to human B cell surface antigen
US6120767A (en) 1986-10-27 2000-09-19 Pharmaceutical Royalties, L.L.C. Chimeric antibody with specificity to human B cell surface antigen
US5677180A (en) 1987-01-08 1997-10-14 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5500362A (en) 1987-01-08 1996-03-19 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
US5721108A (en) 1987-01-08 1998-02-24 Xoma Corporation Chimeric antibody with specificity to human B cell surface antigen
WO1988004936A1 (en) 1987-01-08 1988-07-14 International Genetic Engineering, Inc. Chimeric antibody with specificity to human b cell surface antigen
US4892538A (en) 1987-11-17 1990-01-09 Brown University Research Foundation In vivo delivery of neurotransmitters by implanted, encapsulated cells
US5283187A (en) 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
US5849898A (en) 1988-02-25 1998-12-15 The General Hospital Corporation CD40 coding sequences
EP0330191A2 (en) 1988-02-25 1989-08-30 The General Hospital Corporation DNA encoding CD40
US5114721A (en) 1988-03-15 1992-05-19 Yeda Research And Development Co. Ltd. Preparation of t-cell and t-cell membrane for use in prevention and treatment of autoimmune diseases
US4861579A (en) 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
EP0332865A2 (en) 1988-03-17 1989-09-20 American Cyanamid Company Suppression of B-cells in mammals
EP0340109A2 (en) 1988-04-28 1989-11-02 The Board Of Trustees Of The Leland Stanford Junior University Anti-T-cell receptor determinants as autoimmune disease treatment
US5700637A (en) 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
WO1990008187A1 (en) 1989-01-19 1990-07-26 Dana Farber Cancer Institute Soluble two domain cd2 protein
WO1990011294A1 (en) 1989-03-21 1990-10-04 The Immune Response Corporation Vaccination and methods against diseases resulting from pathogenic responses by specific t cell populations
US5445934A (en) 1989-06-07 1995-08-29 Affymax Technologies N.V. Array of oligonucleotides on a solid substrate
EP0404097A2 (en) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Bispecific and oligospecific, mono- and oligovalent receptors, production and applications thereof
WO1991000360A1 (en) 1989-06-29 1991-01-10 Medarex, Inc. Bispecific reagents for aids therapy
WO1991001133A1 (en) 1989-07-19 1991-02-07 Arthur Allen Vandenbark T cell receptor peptides as therapeutics for autoimmune and malignant disease
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1991010741A1 (en) 1990-01-12 1991-07-25 Cell Genesys, Inc. Generation of xenogeneic antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5229275A (en) 1990-04-26 1993-07-20 Akzo N.V. In-vitro method for producing antigen-specific human monoclonal antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5569825A (en) 1990-08-29 1996-10-29 Genpharm International Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5571894A (en) 1991-02-05 1996-11-05 Ciba-Geigy Corporation Recombinant antibodies specific for a growth factor receptor
WO1992020373A1 (en) 1991-05-14 1992-11-26 Repligen Corporation Heteroconjugate antibodies for treatment of hiv infection
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO1993001161A1 (en) 1991-07-11 1993-01-21 Pfizer Limited Process for preparing sertraline intermediates
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5587458A (en) 1991-10-07 1996-12-24 Aronex Pharmaceuticals, Inc. Anti-erbB-2 antibodies, combinations thereof, and therapeutic and diagnostic uses thereof
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993016185A2 (en) 1992-02-06 1993-08-19 Creative Biomolecules, Inc. Biosynthetic binding protein for cancer marker
US5589369A (en) 1992-02-11 1996-12-31 Cell Genesys Inc. Cells homozygous for disrupted target loci
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
EP0633945A1 (en) 1992-04-03 1995-01-18 Genentech, Inc. ANTIBODIES TO ALPHAvBETA3 INTEGRIN
WO1993025673A1 (en) 1992-06-04 1993-12-23 The Regents Of The University Of California In vivo gene therapy with intron-free sequence of interest
WO1994004690A1 (en) 1992-08-17 1994-03-03 Genentech, Inc. Bispecific immunoadhesins
US6283761B1 (en) 1992-09-08 2001-09-04 Raymond Anthony Joao Apparatus and method for processing and/or for providing healthcare information and/or healthcare-related information
US20030147885A1 (en) 1992-11-13 2003-08-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US20030095963A1 (en) 1992-11-13 2003-05-22 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restriced differentiation antigen for treatment of B cell lymphoma
US6159730A (en) 1992-11-13 2000-12-12 Idec Pharmaceutical Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
US5733779A (en) 1992-11-13 1998-03-31 Idec Pharmaceuticals Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US20030082172A1 (en) 1992-11-13 2003-05-01 Idec Pharmaceuticals Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US20030021781A1 (en) 1992-11-13 2003-01-30 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabelled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5648267A (en) 1992-11-13 1997-07-15 Idec Pharmaceuticals Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
US20050186205A1 (en) 1992-11-13 2005-08-25 Anderson Darrell R. Therapeutic application of chimeric and radiolabeled antibodies to human B Lymphocyte restricted differentiation antigen for treatment of B cell Lymphoma
US5776456A (en) 1992-11-13 1998-07-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
WO1994011026A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma
US6017733A (en) 1992-11-13 2000-01-25 Idec Pharmaceuticals Corporation Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same
US6682734B1 (en) 1992-11-13 2004-01-27 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US6399061B1 (en) 1992-11-13 2002-06-04 Idec Pharmaceutical Corporation Chimeric and radiolabelled antibodies specific to human CD20 antigen and use thereof for treatment of B-cell lymphoma
US5843439A (en) 1992-11-13 1998-12-01 Anderson; Darrell R. Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
WO1994011523A2 (en) 1992-11-13 1994-05-26 Idec Pharmaceuticals Corporation Fully impaired consensus kozac sequences for mammalian expression
US20020197255A1 (en) 1992-11-13 2002-12-26 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabelled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5484892A (en) 1993-05-21 1996-01-16 Dana-Farber Cancer Institute, Inc. Monoclonal antibodies that block ligand binding to the CD22 receptor in mature B cells
WO1995003770A1 (en) 1993-08-02 1995-02-09 The Board Of Trustees Of The Leland Stanford Junior University Methods for b-cell population control
US6287537B1 (en) 1993-09-16 2001-09-11 The Regents Of The University Of Michigan Radioimmunotherapy of lymphoma using anti-CD20 antibodies
US5595721A (en) 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
US6565827B1 (en) 1993-09-16 2003-05-20 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20 antibodies
US5843398A (en) 1993-09-16 1998-12-01 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20 antibodies
US6015542A (en) 1993-09-16 2000-01-18 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20 antibodies
US6090365A (en) 1993-09-16 2000-07-18 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20 antibodies
US5807522A (en) 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
US20050106108A1 (en) 1994-08-12 2005-05-19 Immunomedics, Inc. Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells
WO1996007321A1 (en) 1994-09-06 1996-03-14 The Uab Research Foundation Methods for modulating protein function in cells using intracellular antibody homologues
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5739277A (en) 1995-04-14 1998-04-14 Genentech Inc. Altered polypeptides with increased half-life
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO1996033735A1 (en) 1995-04-27 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6267958B1 (en) 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US5965787A (en) 1995-08-31 1999-10-12 Mayo Foundation For Medical Education And Research HLA-DRBI peptides with specific binding affinity for HLA-DQ molecules: prevention and treatment of rheumatoid arthritis
WO1997030087A1 (en) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation of glycosylated antibodies
US20040258682A1 (en) 1996-03-20 2004-12-23 Immunomedics, Inc Gylocosylated humanized B-cell specific antibodies
WO1997038731A1 (en) 1996-04-18 1997-10-23 The Regents Of The University Of California Immunoliposomes that optimize internalization into target cells
US20010056066A1 (en) 1996-07-26 2001-12-27 Smithkline Beecham Corporation Method of treating immune cell mediated systemic diseases
WO1998008943A1 (en) 1996-08-30 1998-03-05 Mayo Foundation For Medical Education And Research Hla-drb1 peptides with specific binding affinity for hla-dq molecules: prevention and treatment of rheumatoid arthritis
WO1998018921A1 (en) 1996-10-25 1998-05-07 Human Genome Sciences, Inc. NEUTROKINE $g(a)
WO1998024893A2 (en) 1996-12-03 1998-06-11 Abgenix, Inc. TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
WO1998027114A2 (en) 1996-12-17 1998-06-25 Schering Corporation Mammalian cell surface antigens; related reagents
WO1998035036A1 (en) 1997-02-10 1998-08-13 Genentech, Inc. Heregulin variants
US6306393B1 (en) 1997-03-24 2001-10-23 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US6183744B1 (en) 1997-03-24 2001-02-06 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
EP0869180A1 (en) 1997-04-02 1998-10-07 Smithkline Beecham Corporation A TNF homologue, TL5
WO1998056418A1 (en) 1997-06-13 1998-12-17 Genentech, Inc. Stabilized antibody formulation
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
US6991790B1 (en) 1997-06-13 2006-01-31 Genentech, Inc. Antibody formulation
WO1998058964A1 (en) 1997-06-24 1998-12-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US20040191256A1 (en) 1997-06-24 2004-09-30 Genentech, Inc. Methods and compositions for galactosylated glycoproteins
US6368596B1 (en) 1997-07-08 2002-04-09 Board Of Regents, The University Of Texas System Compositions and methods for homoconjugates of antibodies which induce growth arrest or apoptosis of tumor cells
WO1999012964A2 (en) 1997-09-12 1999-03-18 Biogen, Inc. Kay - a novel immune system protein
WO1999022764A1 (en) 1997-10-31 1999-05-14 Genentech, Inc. Methods and compositions comprising glycoprotein glycoforms
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
WO1999033980A2 (en) 1997-12-30 1999-07-08 Chiron Corporation Members of tnf and tnfr families
WO1999036548A1 (en) 1998-01-16 1999-07-22 Hsc Research And Development Limited Partnership Human lymphoid protein tyrosine phosphatases
US20020041847A1 (en) 1998-03-12 2002-04-11 Goldenberg David M. Immunotherapy of malignant and autoimmune disorders in domestic animals using naked antibodies, immunoconjugates and fusion proteins
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
WO1999051642A1 (en) 1998-04-02 1999-10-14 Genentech, Inc. Antibody variants and fragments thereof
US6538124B1 (en) 1998-04-02 2003-03-25 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US20040072290A1 (en) 1998-04-20 2004-04-15 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US6369229B1 (en) 1998-06-03 2002-04-09 Celltech Therapeutics, Limited Pyridylalanine derivatives
US6455043B1 (en) 1998-08-11 2002-09-24 Idec Pharmaceuticals Corporation Combination therapies for B-cell lymphomas comprising administration of anti-CD20 antibody
WO2000009160A1 (en) 1998-08-11 2000-02-24 Idec Pharmaceuticals Corporation Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody
US20030026804A1 (en) 1998-08-11 2003-02-06 Idec Pharmaceuticals Corporation Combination therapies for B-cell lymphomas comprising administration of anti-CD20 antibody
US20030206903A1 (en) 1998-08-11 2003-11-06 Idec Pharmaceuticals Corporation Combination therapies for B-cell lynphomas comprising administration of anti-CD20 antibody
US6953665B1 (en) 1998-09-01 2005-10-11 Mayo Foundation For Medical Education And Research Methods and materials for evaluating rheumatoid arthritis
US6348463B1 (en) 1998-09-28 2002-02-19 Celltech Therapeutics Limited Phenylalanine derivatives
US6677339B2 (en) 1998-09-28 2004-01-13 Celltech R & D Limited Phenylalanine derivatives
WO2000020864A1 (en) 1998-10-07 2000-04-13 Biocrystal Limited Immunotherapy of b cell involvement in progression of solid, nonlymphoid tumors
US6224866B1 (en) 1998-10-07 2001-05-01 Biocrystal Ltd. Immunotherapy of B cell involvement in progression of solid, nonlymphoid tumors
WO2000027433A1 (en) 1998-11-09 2000-05-18 Idec Pharmaceuticals Corporation Chimeric anti-cd20 antibody treatment of patients receiving bmt or pbsc transplants
WO2000027428A1 (en) 1998-11-09 2000-05-18 Idec Pharmaceuticals Corporation Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody
US20040213784A1 (en) 1998-11-09 2004-10-28 Idec Pharmaceuticals Corporation Use of chimeric anti-CD20 antibody as in vitro or in vivo purging agent in patients receiving BMT or PBSC transplant
WO2000032575A1 (en) 1998-11-30 2000-06-08 Celltech Therapeutics Limited β-ALANINE DERIVATIVES AS α4 INTEGRIN INHIBITORS
WO2000037444A1 (en) 1998-12-18 2000-06-29 Glaxo Group Limited Compounds useful in the treatment of inflammatory diseases
WO2000040716A2 (en) 1999-01-07 2000-07-13 Zymogenetics, Inc. Soluble receptor br43x2 and methods of using them for therapy
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US20060194957A1 (en) 1999-01-15 2006-08-31 Genentech, Inc. Polypeptide variants with altered effector function
WO2000042072A2 (en) 1999-01-15 2000-07-20 Genentech, Inc. Polypeptide variants with altered effector function
US20060194291A1 (en) 1999-01-15 2006-08-31 Genentech, Inc. Polypeptide variants with altered effector function
US20050118174A1 (en) 1999-01-15 2005-06-02 Genentech, Inc. Polypeptide variants with altered effector function
US7122637B2 (en) 1999-01-15 2006-10-17 Genentech, Inc. Polypeptide variants with altered effector function
US20050233382A1 (en) 1999-01-15 2005-10-20 Genentech, Inc. Polypeptide variants with altered effector function
US20060194290A1 (en) 1999-01-15 2006-08-31 Genentech, Inc. Polypeptide variants with altered effector function
US6897044B1 (en) 1999-01-28 2005-05-24 Biogen Idec, Inc. Production of tetravalent antibodies
WO2000044788A1 (en) 1999-01-28 2000-08-03 Idec Pharmaceuticals Corporation Production of tetravalent antibodies
WO2000058522A1 (en) 1999-03-30 2000-10-05 Nanogen, Inc. Single nucleotide polymorphic discrimination by electronic dot blot assay on semiconductor microchips
WO2000061739A1 (en) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Method for controlling the activity of immunologically functional molecule
WO2000067796A1 (en) 1999-05-07 2000-11-16 Genentech, Inc. Treatment of autoimmune diseases with antagonists which bind to b cell surface markers
WO2000067795A1 (en) 1999-05-10 2000-11-16 Immunomedics, Inc. Immunotherapy of b-cell malignancies using anti-cd22 antibodies
US7074403B1 (en) 1999-06-09 2006-07-11 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20070020265A1 (en) 1999-06-09 2007-01-25 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target b-cells
US20050191300A1 (en) 1999-06-09 2005-09-01 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20060051349A1 (en) 1999-06-09 2006-03-09 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
WO2000074718A1 (en) 1999-06-09 2000-12-14 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target b-cells
US20030133930A1 (en) 1999-06-09 2003-07-17 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US20070014785A1 (en) 1999-06-11 2007-01-18 Consiglio Nazionale Delle Richerche Use of antibodies against CD20 for the treatment of the graft versus host disease
WO2000076542A1 (en) 1999-06-11 2000-12-21 Consiglio Nazionale Delle Ricerche Use of antibodies against cd20 for the treatment of the graft versus host disease
US6410391B1 (en) 1999-07-02 2002-06-25 Infineon Technologies Ag Method for producing an EEPROM memory cell with a trench capacitor
WO2001003734A1 (en) 1999-07-12 2001-01-18 Genentech, Inc. Blocking immune response to a foreign antigen using an antagonist which binds to cd20
WO2001010462A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation Treatment of patients having non-hodgkins lymphoma with bone marrow involvement with anti-cd20 antibodies
WO2001010461A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation New clinical parameters for determining hematologic toxicity prior to radioimmunotherapy
WO2001010460A1 (en) 1999-08-11 2001-02-15 Idec Pharmaceuticals Corporation Treatment of intermediate- and high-grade non-hodgkins lymphoma with anti-cd20 antibody
WO2001012848A2 (en) 1999-08-14 2001-02-22 Univ Leeds Genetic marker for rheumatoid arthritis
WO2001012812A2 (en) 1999-08-17 2001-02-22 Biogen, Inc. Baff receptor (bcma), an immunoregulatory agent
WO2001013945A1 (en) 1999-08-23 2001-03-01 Biocrystal Ltd. Methods and compositions for immunotherapy of b cell involvement in promotion of a disease condition comprising multiple sclerosis
US20050079174A1 (en) 1999-08-23 2005-04-14 Biocrystal, Ltd. Methods and compositions for immunotherapy of B cell involvement in promotion of a disease condition comprising multiple sclerosis
WO2001018240A2 (en) 1999-09-08 2001-03-15 The University Of British Columbia Diagnostic and therapeutic methods in autoimmune disease
US20060018900A1 (en) 1999-09-24 2006-01-26 Mccormick Alison A Self antigen vaccines for treating B-cell lymphomas and other cancers
WO2001029246A1 (en) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Process for producing polypeptide
WO2001034194A1 (en) 1999-11-08 2001-05-17 Idec Pharmaceuticals Corporation Treatment of b cell malignancies using anti-cd40l antibodies in combination with anti-cd20 antibodies and/or chemotherapeutics and radiotherapy
US20010018041A1 (en) 1999-11-08 2001-08-30 Idec Pharmaceuticals Corporation Treatment of B cell malignancies using anti-CD40L antibodies in combination with anti-CD20 antibodies and/or chemotherapeutics and radiotherapy
US20050123540A1 (en) 1999-11-08 2005-06-09 Biogen Idec Inc. Treatment of B cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US7175985B1 (en) 1999-11-08 2007-02-13 Eiken Kagaku Kabushiki Kaisha Method of detecting variation or polymorphism
US20020006404A1 (en) 1999-11-08 2002-01-17 Idec Pharmaceuticals Corporation Treatment of cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US20020042368A1 (en) 2000-02-25 2002-04-11 Fanslow William C. Integrin antagonists
US20030185796A1 (en) 2000-03-24 2003-10-02 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
US20020009427A1 (en) 2000-03-24 2002-01-24 Wolin Maurice J. Methods of therapy for non-hodgkin's lymphoma
WO2001072333A1 (en) 2000-03-24 2001-10-04 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma using a combination_of an antibody to cd20 and interleuking-2
WO2001075166A2 (en) 2000-03-31 2001-10-11 Genentech, Inc. Compositions and methods for detecting and quantifying gene expression
US6896885B2 (en) 2000-03-31 2005-05-24 Biogen Idec Inc. Combined use of anti-cytokine antibodies or antagonists and anti-CD20 for treatment of B cell lymphoma
WO2001074388A1 (en) 2000-03-31 2001-10-11 Idec Pharmaceuticals Corporation Combined use of anti-cytokine antibodies or antagonists and anti-cd20 for the treatment of b cell lymphoma
US20020012665A1 (en) 2000-03-31 2002-01-31 Nabil Hanna Combined use of anti-cytokine antibodies or antagonists and anti-CD20 for treatment of B cell lymphoma
US20050180975A1 (en) 2000-03-31 2005-08-18 Biogen Idec Inc. Combined use of anti-cytokine antibodies or antagonists and anti-CD20 for treatment of B cell lymphoma
WO2001077342A1 (en) 2000-04-11 2001-10-18 Genentech, Inc. Multivalent antibodies and uses therefor
US20020004587A1 (en) 2000-04-11 2002-01-10 Genentech, Inc. Multivalent antibodies and uses therefor
US20060025576A1 (en) 2000-04-11 2006-02-02 Genentech, Inc. Multivalent antibodies and uses therefor
WO2001079173A2 (en) 2000-04-17 2001-10-25 Celltech R & D Limited Enamine derivatives as cell adhesion molecules
US20020009444A1 (en) 2000-04-25 2002-01-24 Idec Pharmaceuticals Corporation Intrathecal administration of rituximab for treatment of central nervous system lymphomas
WO2001080884A1 (en) 2000-04-25 2001-11-01 Idec Pharmaceuticals Corporation Intrathecal administration of rituximab for treatment of central nervous system lymphomas
WO2001087979A2 (en) 2000-05-12 2001-11-22 Amgen Inc. Methods and compositions of matter concerning april/g70, bcma, blys/agp-3, and taci
WO2002002641A1 (en) 2000-06-16 2002-01-10 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to blys
US20050112060A1 (en) 2000-06-20 2005-05-26 Idec Pharmaceuticals Corporation Treatment of B-cell associated diseases such as malignancies and autoimmune diseases using a cold anti-CD20 antibody/radiolabeled anti-CD22 antibody combination
US6846476B2 (en) 2000-06-20 2005-01-25 Idec Pharmaceuticals Corporation Treatment of B-cell associated diseases
WO2001097858A2 (en) 2000-06-20 2001-12-27 Idec Pharmaceuticals Corporation Cold anti-cd20 antibody/radiolabeled anti-cd22 antibody combination
US20020039557A1 (en) 2000-06-20 2002-04-04 Christine White Treatment of B-cell associated diseases such as malignances and autoimmune diseases using a cold anti-CD20 antibody/radiolabeled anti-CD22 antibody combination
US20030026801A1 (en) 2000-06-22 2003-02-06 George Weiner Methods for enhancing antibody-induced cell lysis and treating cancer
US20060024304A1 (en) 2000-06-28 2006-02-02 Gerngross Tillman U Immunoglobulins comprising predominantly a Man5GlcNAc2 glycoform
US20060034830A1 (en) 2000-06-28 2006-02-16 Gerngross Tillman U Immunoglobulins comprising predominantly a GalGlcNAcMan5GLcNAc2 glycoform
US20060029604A1 (en) 2000-06-28 2006-02-09 Gerngross Tillman U Immunoglobulins comprising predominantly a GlcNAc2Man3GlcNAc2 glycoform
US7029872B2 (en) 2000-06-28 2006-04-18 Glycofi, Inc Methods for producing modified glycoproteins
US20060034828A1 (en) 2000-06-28 2006-02-16 Gerngross Tillman U Immunoglobulins comprising predominantly a GlcNAcMAN5GLCNAC2 glycoform
US20040018590A1 (en) 2000-06-28 2004-01-29 Gerngross Tillman U. Combinatorial DNA library for producing modified N-glycans in lower eukaryotes
WO2002002556A2 (en) 2000-06-30 2002-01-10 Ortho Mcneil Pharmaceutical, Inc. AZA-BRIDGED-BICYCLIC AMINO ACID DERIVATIVES AS α4 INTEGRIN ANTAGONISTS
US20030099943A1 (en) 2000-07-10 2003-05-29 Conaris Research Institute Gmbh Diagnostic use of polymorphisms in the gene coding for the TNF receptor II and method for detecting non-responders to anti-TNF therapy
WO2002004021A1 (en) 2000-07-12 2002-01-17 Idec Pharmaceuticals Corporation Treatment of b cell malignancies using combination of b cell depleting antibody and immune modulating antibody related applications
US20020028178A1 (en) 2000-07-12 2002-03-07 Nabil Hanna Treatment of B cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US20030232055A1 (en) 2000-07-31 2003-12-18 Ruslan Medzhitov Innate immune system-directed vaccines
WO2002016412A2 (en) 2000-08-18 2002-02-28 Dyax Corp. Binding polypeptides for b lymphocyte stimulator protein (blys)
WO2002016329A1 (en) 2000-08-18 2002-02-28 Ajinomoto Co., Inc. Novel phenylalanine derivatives
WO2002016312A2 (en) 2000-08-23 2002-02-28 Morphochem Ag Novel compounds inhibiting factor xa activity
WO2002024909A2 (en) 2000-09-18 2002-03-28 Biogen, Inc. Receptor nucleic acids and polypeptides
US20020058029A1 (en) 2000-09-18 2002-05-16 Nabil Hanna Combination therapy for treatment of autoimmune diseases using B cell depleting/immunoregulatory antibody combination
WO2002022212A2 (en) 2000-09-18 2002-03-21 Idec Pharmaceuticals Corporation Combination therapy for treatment of autoimmune diseases using b cell depleting/immunoregulatory antibody combination
US20060275284A1 (en) 2000-09-18 2006-12-07 Biogen Idec Inc. Combination therapy for treatment of autoimmune diseases using b-cell depleting/immunoregulatory antibody combination
WO2002028830A1 (en) 2000-09-29 2002-04-11 Ajinomoto Co.,Inc. Novel phenylalanine derivatives
US20060078991A1 (en) 2000-10-06 2006-04-13 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
US20060078990A1 (en) 2000-10-06 2006-04-13 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US20060064781A1 (en) 2000-10-06 2006-03-23 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20060063254A1 (en) 2000-10-06 2006-03-23 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-producing cell
US20020128448A1 (en) 2000-10-20 2002-09-12 Idec Pharmaceuticals Corporation Variant IgG3 Rituxan and therapeutic use thereof
WO2002034790A1 (en) 2000-10-20 2002-05-02 Idec Pharmaceuticals Corporation Variant igg3 rituxan r and therapeutic use thereof
WO2002038766A2 (en) 2000-11-07 2002-05-16 Zymogenetics, Inc. Human tumor necrosis factor receptor
US20020136719A1 (en) 2000-12-28 2002-09-26 Bhami Shenoy Crystals of whole antibodies and fragments thereof and methods for making and using them
US20030133939A1 (en) 2001-01-17 2003-07-17 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US20050202012A1 (en) 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunogloubulin fusion proteins
US20050202023A1 (en) 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050202028A1 (en) 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050175614A1 (en) 2001-01-17 2005-08-11 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050136049A1 (en) 2001-01-17 2005-06-23 Ledbetter Jeffrey A. Binding constructs and methods for use thereof
WO2002056910A1 (en) 2001-01-17 2002-07-25 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2005037989A2 (en) 2001-01-17 2005-04-28 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050180970A1 (en) 2001-01-17 2005-08-18 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050186216A1 (en) 2001-01-17 2005-08-25 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20030118592A1 (en) 2001-01-17 2003-06-26 Genecraft, Inc. Binding domain-immunoglobulin fusion proteins
US20050202534A1 (en) 2001-01-17 2005-09-15 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
WO2002060955A2 (en) 2001-01-29 2002-08-08 Idec Pharmaceuticals Corporation Modified antibodies and methods of use
WO2002066516A2 (en) 2001-02-20 2002-08-29 Zymogenetics, Inc. Antibodies that bind both bcma and taci
WO2002078766A2 (en) 2001-04-02 2002-10-10 Genentech, Inc. Combination therapy
WO2002079255A1 (en) 2001-04-02 2002-10-10 Idec Pharmaceuticals Corporation RECOMBINANT ANTIBODIES COEXPRESSED WITH GnTIII
US20020197256A1 (en) 2001-04-02 2002-12-26 Genentech, Inc. Combination therapy
WO2003061694A1 (en) 2001-05-10 2003-07-31 Seattle Genetics, Inc. Immunosuppression of the humoral immune response by anti-cd20 antibodies
WO2002092620A2 (en) 2001-05-11 2002-11-21 Amgen, Inc. Peptides and related molecules that bind to tall-1
WO2002102312A2 (en) 2001-06-14 2002-12-27 Intermune, Inc. Combination therapy of gamma-interferon and b cell specific antibodies
WO2003002607A1 (en) 2001-06-27 2003-01-09 Shawn Shui-On Leung Reducing immunogenicities of immunoglobulins by framework-patching
WO2003010135A1 (en) 2001-07-26 2003-02-06 Ajinomoto Co., Inc. Novel phenylpropionic acid derivatives
US20030175884A1 (en) 2001-08-03 2003-09-18 Pablo Umana Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003011878A2 (en) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
WO2003014294A2 (en) 2001-08-03 2003-02-20 Genentech, Inc. Tacis and br3 polypeptides and uses thereof
US20050070689A1 (en) 2001-08-03 2005-03-31 Genentech, Inc. Taci and br3 polypeptides and uses thereof
US7244571B2 (en) 2001-09-06 2007-07-17 Decode Genetics Ehf. Methods for producing Ex vivo models for inflammatory disease and uses thereof
US20030068664A1 (en) 2001-09-20 2003-04-10 Board Of Regents, The University Of Texas System Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using elisa assays
WO2003024991A2 (en) 2001-09-21 2003-03-27 Amgen Inc. Tall-1 receptor molecules and uses thereof
WO2003035846A2 (en) 2001-10-24 2003-05-01 National Jewish Medical And Research Center Structure of tall-1 and its cognate receptor
WO2003035835A2 (en) 2001-10-25 2003-05-01 Genentech, Inc. Glycoprotein compositions
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20030103971A1 (en) 2001-11-09 2003-06-05 Kandasamy Hariharan Immunoregulatory antibodies and uses thereof
CN1420129A (en) 2001-11-16 2003-05-28 上海中信国健药业有限公司 Humanized anti-CD 20 monoclonal antibody
WO2003049694A2 (en) 2001-12-07 2003-06-19 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
WO2003050542A2 (en) 2001-12-11 2003-06-19 Stichting Voor De Technische Wetenschappen Method of detecting autoantibodies from patients suffering from rheumatoid arthritis, a peptide and an assaykit
WO2003053926A1 (en) 2001-12-13 2003-07-03 Ajinomoto Co.,Inc. Novel phenylalanine derivative
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
WO2003068822A2 (en) 2002-02-13 2003-08-21 Micromet Ag De-immunized (poly)peptide constructs
US20070020259A1 (en) 2002-02-14 2007-01-25 Immunomedics, Inc. Anti-cd20 antibodies and fusion proteins thereof and methods of use
US7151164B2 (en) 2002-02-14 2006-12-19 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
WO2003068821A2 (en) 2002-02-14 2003-08-21 Immunomedics, Inc. Anti-cd20 antibodies and fusion proteins thereof and methods of use
US20030219433A1 (en) 2002-02-14 2003-11-27 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US20060153838A1 (en) 2002-02-20 2006-07-13 Watkins Jeffry D Fc region variants
WO2003070709A1 (en) 2002-02-20 2003-08-28 Ajinomoto Co.,Inc. Novel phenylalanine derivative
EP1476120A2 (en) 2002-02-21 2004-11-17 Duke University Treatment methods using anti-cd22 antibodies
EP1485130A2 (en) 2002-02-21 2004-12-15 Duke University Reagents and treatment methods for autoimmune diseases
US20070224189A1 (en) 2002-03-01 2007-09-27 Xencor, Inc. CD20 OPTIMIZED Fc VARIANTS AND METHODS FOR THEIR GENERATION
US20030180292A1 (en) 2002-03-14 2003-09-25 Idec Pharmaceuticals Treatment of B cell malignancies using anti-CD40L antibodies in combination with anti-CD20 antibodies and/or chemotherapeutics and radiotherapy
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
WO2003085119A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. METHOD OF ENHANCING ACTIVITY OF ANTIBODY COMPOSITION OF BINDING TO FcϜ RECEPTOR IIIa
WO2003084570A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. DRUG CONTAINING ANTIBODY COMPOSITION APPROPRIATE FOR PATIENT SUFFERING FROM FcϜRIIIa POLYMORPHISM
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
WO2003089410A1 (en) 2002-04-19 2003-10-30 Kyowa Hakko Kogyo Co., Ltd. Phenylalanine derivative
EP1504035A2 (en) 2002-05-02 2005-02-09 Celltech R &amp; D Limited Antibodies specific for human cd22 and their therapeutic and diagnostic uses
US20030219818A1 (en) 2002-05-10 2003-11-27 Bohen Sean P. Methods and compositions for determining neoplastic disease responsiveness to antibody therapy
US20050180972A1 (en) 2002-07-31 2005-08-18 Wahl Alan F. Anti-CD20 antibody-drug conjugates for the treatment of cancer and immune disorders
WO2004032828A2 (en) 2002-07-31 2004-04-22 Seattle Genetics, Inc. Anti-cd20 antibody-drug conjugates for the treatment of cancer and immune disorders
WO2004035607A2 (en) 2002-10-17 2004-04-29 Genmab A/S Human monoclonal antibodies against cd20
US20040167319A1 (en) 2002-10-17 2004-08-26 Jessica Teeling Human monoclonal antibodies against CD20
US20060024300A1 (en) 2002-12-16 2006-02-02 Genentech, Inc. Immunoglobulin variants and uses thereof
WO2004056312A2 (en) 2002-12-16 2004-07-08 Genentech, Inc. Immunoglobulin variants and uses thereof
US20060034835A1 (en) 2002-12-16 2006-02-16 Genentech, Inc. Immunoglobulin variants and uses thereof
WO2004060052A2 (en) 2002-12-16 2004-07-22 Genentech, Inc. Transgenic mice expressing human cd20 and/or cd16
US20060179501A1 (en) 2002-12-16 2006-08-10 Chan Andrew C Transgenic mice expressing human cd20
WO2004060053A2 (en) 2002-12-16 2004-07-22 Genentech, Inc. Transgenic mice expressing human cd20
WO2004058298A1 (en) 2002-12-31 2004-07-15 Immunomedics, Inc. Immunotherapy of b cell malignancies and autoimmune disease using unconjugated antibodies and conjugated antibodies, antibody combinations and fusion proteins
US20060121032A1 (en) 2003-03-03 2006-06-08 Xencor, Inc. Optimized anti-CD20 monoclonal antibodies having Fc variants
US20040202658A1 (en) 2003-04-09 2004-10-14 Genentech, Inc. Therapy of autoimmune disease in a patient with an inadequate response to TNF-alpha inhibitor
WO2004091657A2 (en) 2003-04-09 2004-10-28 Genentech, Inc. Therapy of autoimmune disease in a patient with an inadequate response to a tnf-alpha inhibitor
WO2004092219A2 (en) 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
WO2005000901A2 (en) 2003-05-09 2005-01-06 Duke University Cd20-specific antibodies and methods of employing same
WO2004103404A1 (en) 2003-05-20 2004-12-02 Applied Molecular Evolution, Inc. Cd20 binding molecules
US20060251652A1 (en) 2003-05-20 2006-11-09 Applied Molecular Evolution, Inc., Cd20 binding molecules
US20050025764A1 (en) 2003-05-20 2005-02-03 Watkins Jeffry D. CD20 binding molecules
US20050163775A1 (en) 2003-06-05 2005-07-28 Genentech, Inc. Combination therapy for B cell disorders
US20050095243A1 (en) 2003-06-05 2005-05-05 Genentech, Inc. Combination therapy for B cell disorders
WO2005005462A2 (en) 2003-06-05 2005-01-20 Genentech, Inc. Blys antagonists and uses thereof
US20060135430A1 (en) 2003-06-05 2006-06-22 Genentech, Inc. BLy antagonists and uses thereof
WO2005000351A2 (en) 2003-06-05 2005-01-06 Genentech, Inc. Combination therapy for b cell disorders
US20060263783A1 (en) 2003-07-03 2006-11-23 Podhajcer Osvaldo L Methods and systems for diagnosis of non-central nervous system (cns) diseases in cns samples
WO2005011428A1 (en) 2003-07-25 2005-02-10 Bally Gaming International, Inc. Uniquely identifiable casino gaming chips
WO2005017148A1 (en) 2003-07-26 2005-02-24 Trubion Pharmaceuticals, Inc. Binding constructs and methods for use thereof
WO2005017529A1 (en) 2003-07-29 2005-02-24 Genentech, Inc. Assay for human anti cd20 antibodies and uses therefor
US20050032130A1 (en) 2003-07-29 2005-02-10 Genentech, Inc. Neutralizing antibody assay and uses therefor
WO2005014622A2 (en) 2003-08-07 2005-02-17 F. Hoffmann-La Roche Ag Ra antigenic peptides
WO2005014618A2 (en) 2003-08-08 2005-02-17 Immunomedics, Inc. Bispecific antibodies for inducing apoptosis of tumor and diseased cells
WO2005016969A2 (en) 2003-08-14 2005-02-24 Merck Patent Gmbh Cd20-binding polypeptide compositions
US20050069545A1 (en) 2003-08-14 2005-03-31 Carr Francis Joseph CD20-Binding polypeptide compositions and methods
US20070048300A1 (en) 2003-08-22 2007-03-01 Biogen Idec Ma Inc. Antibodies having altered effector function and methods for making the same
WO2005023302A2 (en) 2003-08-29 2005-03-17 Genentech, Inc. Anti-cd20 therapy of ocular disorders
US20050053602A1 (en) 2003-08-29