HK1128004B

HK1128004B - Method for treating joint damage

Info

Publication number: HK1128004B
Application number: HK09107218.2A
Authority: HK
Inventors: Mark Totoritis; Timothy Mark Shaw; Sunil Agarwal; David Yocum; Ariella Kelman
Original assignee: 霍夫曼 - 拉罗奇有限公司; 生物基因公司; 健泰科生物技术公司
Priority date: 2005-11-15
Filing date: 2006-11-14
Publication date: 2014-01-17

Description

Method for treating joint damage

Technical Field

The present invention concerns a method for treating joint damage in a subject suffering from joint damage.

Background

Destruction and injury of joints

Inflammatory arthritis is a prominent clinical manifestation of a variety of autoimmune disorders including Rheumatoid Arthritis (RA), psoriatic arthritis (PsA), Systemic Lupus Erythematosus (SLE), sjogren's syndrome, and polymyositis. Most of these patients exhibit joint deformities in somatic examinations, but typically only RA and PsA patients exhibit bone erosion in 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 in a somewhat lesser proportion in other parts of the world (Alamonosa and dross, autoimmun. rev., 4: 130-. It is a systemic inflammatory disease characterized by chronic inflammation in the synovium of the affected joint, ultimately leading to loss of daily function due to chronic pain and fatigue. Most patients also experience progressive deterioration of cartilage and bone in the affected joint, possibly ultimately leading to permanent disability. The long-term prognosis of RA is poor, with approximately 50% of patients experiencing major functional disability within 10 years of diagnosis (Keystone, Rheumatology, 44 (suppl.2): ii8-ii12 (2005)). Life expectancy decreases by an average of 3-10 years (Alamanos and Rosos, supra). Patients with high titers of Rheumatoid Factor (RF) (approximately 80% of patients) have more aggressive disease (Bukhari et al, Arthritis Rheum.46: 906-.

The pathogenesis of chronic inflammatory bone diseases such as RA has not yet been fully elucidated. Such diseases are accompanied by bone loss around the affected joint due to increased bone-breaking resorption. This process is primarily mediated by increased local production of proinflammatory cytokines (Teitelbaum Science 289: 1504-. These cytokines may act directly on cells in the osteoclast lineage, or indirectly by affecting osteoblast/stromal cell production of key osteoclast differentiation factors, receptor activator of NF < B ligand (RANKL) I and/or its soluble rusty erbium receptor, Osteoprotegerin (OPG) (Hossbauer et al j. bone miner. res.15 (1): 2-12 (2000)). TNF-alpha is a major mediator of inflammation and its importance in the pathogenesis of various forms of bone loss is supported by several experimental and clinical evidences (Feldmann et al. Cell85 (3): 307-. However, TNF- α is not required for osteoclastogenesis (osteoplastic genetics) (Douni et al J. Inflamm.47: 27-38(1996)), erosive arthritis (Campbell et al J. Clin. invest.107 (12): 1519-.

In RA, in particular, the immune response is thought to be initiated/sustained by one or more antigens presented in the synovial compartment, causing the influx of acute inflammatory cells and lymphocytes into the joint. The continuous inflammatory wave results in the formation of invasive and erosive tissue called pannus (pannus). This involves proliferating fibroblast-like synoviocytes and macrophages, which produce proinflammatory cytokines such as tumor necrosis factor-alpha (TNF- α) and interleukin-1 (IL-1). Local release of proteolytic enzymes, various mediators of inflammation, and osteoclast activation contribute to most tissue damage. There is loss of articular cartilage and the formation of bone erosion. The surrounding tendons and bursa may be affected by the inflammatory process. Eventually, the integrity of the joint structure is compromised, causing disability.

The exact contribution of B cells to the immunopathogenesis of RA has not yet been fully characterized. However, there are several possible mechanisms by which B cells may be involved in the disease process (Silverman and Carson, Arthritis Res. ther., 5 suppl.4: S1-6 (2003)).

Historically, B cells have been thought to promote disease processes in RA, primarily by acting as precursors to autoantibody-producing cells. A variety of autoantibody specificities have been identified, including antibodies to type II collagen, and proteoglycans, as well as rheumatoid factors. The production of large amounts of antibodies results in the formation of immune complexes and activation of the complement cascade. This in turn amplifies the immune response and may end up with local cytolysis. Increased RF synthesis and complement consumption have been associated with disease viability. 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); river et al, int.Immunol., 13: 1583-1593(2001)) shows that B cells are highly potent Antigen Presenting Cells (APC). RF positive B cells may be particularly effective APCs because their surface immunoglobulin will readily capture any immune complexes regardless of the antigen present therein. Many antigens can be so processed for presentation to T cells. In addition, it has recently been suggested that this may also allow RF-positive B cells to self-continue (Edwards et al, Immunology, 97: 188-.

To activate T cells, two signals need to be transmitted to the cells; one via the T Cell Receptor (TCR), which recognizes the processed peptide in the presence of Major Histocompatibility Complex (MHC) antigens, and the other via the costimulatory molecule. Upon activation, B cells express co-stimulatory molecules on their surface, and may thus provide a second signal for activation of T cells and production of effector cells.

B cells can promote their own function as well as that of other cells by producing cytokines (Harris et al, nat. immunol., 1: 475-. Among the several cytokines that B cells may produce in the synovium of RA are TNF- α and IL-1, lymphotoxin- α, IL-6, and IL-10.

Although T cell activation is thought to be a key factor in the pathogenesis of RA, recent work with human synovial explants in Severe Combined Immunodeficiency Disease (SCID) mice demonstrated that T cell activation and retention in the joints is extremely dependent on the presence of B cells (Takemura et al, J.Immunol., 167: 4710-4718 (2001)). The exact role of B cells in this process is unclear, as other APCs do not appear to have the same effect on T cells.

Structural damage to the joint is an important consequence of chronic synovial inflammation. Between 60% and 95% of patients with Rheumatoid Arthritis (RA) develop at least one radiographic erosion within 3-8 years of disease onset (Paulus et al, J. Rheumatol., 23: 801-. In early RA, the correlation between radiographic damage score and functional capacity is weak, but after 8 years of disease, the correlation coefficient can reach as high as 0.68(Scott et al, Rheumatology, 39: 122-. A significant correlation between the rate of progression of the Larsen radiographic damage score (Larsen et al, Acta Radiol. Diagn.18: 481-491(1977)), increased socially guaranteed disability status, and decreased household income was found in Wolfe et al (Arthritis Rheum, 43 suppl.9: S403(2000)) among 1,007 patients with RA for at least four years of age less than 60 years.

Prevention or delay of radiographic damage is one of the targets for RA treatment (Edmonds et al, Arthritis Rheum. 36: 336- & 340 (1993)). A controlled clinical trial lasting 6 or 12 months has demonstrated that the development of the radiographic damage score in the placebo group is more rapid than in the group receiving Methotrexate (MTX) (Sharp et al, Arthritis Rheum.43: 495-plus 505(2000)), leflunomide (Sharp et al, supra), sulfasalazine (SSZ) (Sharp et al, supra), prednisolone (Kirwan et al, N.Engl. J. Med., 333: 142-plus 146 (1995); Wassenburg et al, Arthritis Rheum, 42: Suppl 9: S243(1999)), interleukin-1 receptor antagonist (Bressihan et al, Arthritis Rheum, 41: 2196-plus 2204(1998)), or Ingliximab + MTX combination (Lipsy et al, N.Eng.1604: 1594 (2000)), furthermore, radiography after etanercept treatment does not progress as rapidly as MTX treatment (Bathon et al, N.Engl. J.Med., 343: 1586-. Other studies have evaluated the use of corticosteroids (Joint Committee of the Medical research and Nuclear Foundation, Ann Rheum. Dis. 19: 331-337 (1960); VanEverdingen et al, Ann. Intern. Med., 136: 1-12(2002)), cyclosporin A (Passer et al, J. Rheum. Dis., 24: 2113-2118 (1997); Forre, Arthritis Rheum. 37: 1506-1512(1994)), MTX vs. thiozopurine (Jeurisen et al, Ann. Intern. Med., 114: 999-1004(1991)), MTX vs. aurora (Weinbell et al, Arthritis Rheum. Phehanum., 36: 186613 (MTX. 619(1993)), and Chloroq et al (Rabbit et al, Arthritis et al., Arthia. Reye. J. 1993), and Alzhen. Thiela. Reye. J. Thieland. 10, 35, 1993), and combinations of Chloroq. Rhein et al, Phe. Rheum. Thielaid et al, ML. This. RTM. 10, 35, and L. Thielaid. Thielagic, Miq. RTM. A. RTM. A. 1, a combination of the aforementioned, L. RTM. A. RTM. A. RTM. RT, lancet, 350: 309-318 (1997); landewe et al, Arthritis rheum, 46: 347-: 1287-1291 (1996); mottonen et al, Lancet, 353: 1568-1573(1999)), cyclophosphamide + azathioprine + HCQ (Csuka et al, JAMA, 255: 2115-: s205(2002)) progress of radiography in the treated patient.

The FDA has now approved label claims (labeling claims) that certain drugs such as leflunomide, etanercept, and infliximab slow the progression of radiographic joint damage. These claims are based on the statistically significant differences in the rates of progression observed between the randomly assigned treatment groups and the control group. However, there was a considerable degree of overlap in the rate of progression of individuals in the treatment group and the control group; thus, despite significant differences between treatment groups, these data cannot be used to assess the probability that a patient starting treatment will obtain a favorable outcome in terms of radiographic damage progression. Various methods have been proposed to classify paired radiographs from individual patients as not progressing, e.g., with zero lesion score, no increase in lesion score, no newly eroded joints, and no change in score beyond a minimum detectable difference (i.e., 95% confidence interval of difference between repeat readings of the same radiograph) at both time points (Lassere et al, j. rheumatol, 26: 731-.

It is difficult to determine whether a patient individual has had increased structural damage during the interval of paired radiographs obtained at the beginning and end of a 6 or 12 month clinical trial for several reasons. Radiographic damage rates are not consistent among RA patient populations; a few patients may have rapidly progressing lesions, but many may have little or no progression, especially if the intervals are relatively short. Methods for scoring radiographic lesions, such as Sharp (Sharp et al, Arthritis Rheum., 14: 706- & 720 (1971); Sharp et al, Arthritis Rheum., 28: 1326- & 1335(1985)), Larsen (Larsen et al, Acta RadioI. Diagn., 18: 481- & 491(1977)), and improvements in these methods (Van der Heijde, J.Rheumatol., 27: 261- & 263(2000)), depend on the judgment and interpretation of the reader, i.e., whether an apparent disruption of the subchondral outer plate is true, or whether shortening of the distance between the articular cortex is true or due to slight changes in the position of the joint relative to the film and radiographic bundle, changes in radiographic exposure, or some other technical factor.

Thus, the recorded scores are an approximation of the true lesion, and for many subjects, the smallest detectable difference between the repeat scores of the same radiograph is greater than the actual change that occurred during the interval between the baseline and final radiographs. If the reader is unaware of the chronological order of the films, these unavoidable scoring errors can be in either direction, resulting in an apparent "cure" as the score decreases or a rapid progression as the reading error increases the difference between the films. When the study involved a sufficiently large population of patients that had been randomly assigned to receive effective treatment versus placebo, the positive and negative reading errors offset each other and subtle but real differences could be detected between treatment groups.

The inaccuracy of clinical measurements used to quantify RA disease activity has caused similar problems; statistically significant differences between some measurements from clinical trials cannot be used to assess the probability of improvement in individuals who begin treatment (Paulus et al, Arthritis Rheum., 33: 477-484 (1990)). The assignment of individual improvement became feasible with the generation of a 20% composite criteria for improvement by the American society for rheumatology (ACR) (ACR20) which specifies that a patient is improved if there is a 20% improvement in tender and swollen joint count and a 20% improvement in at least 3 of the 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-. All of these measurements have large values of minimal detectable difference, but by requiring simultaneous improvement in 5 of 7 aspects of the same process (disease activity), the randomness of these 7 measurement errors is constrained and it is easier to attribute true improvements to individuals.

In RA, joint damage is a prominent feature. The radiological parameters of joint destruction are considered as a key outcome measure describing the outcome of the disease. On the recent general act (clinical trial outcome measure of rheumatology), radiology was chosen as part of the core set of outcome measures for longitudinal observation studies (Wolfe et al, Arthritis rheum, 41 Supp 9: S204(1998) abstrate). Radiology is also part of the core set of measurements for long-term clinical trials required by WHO/ILAR (world health organization/international federation of rheumatology societies) (Tugwell and Boers, j. rhematol., 20: 528-.

Available data on the outcome of the radiological lesions in RA was obtained in both short and long term studies. In a short-term study of recently-ill RA patients, radiographs taken every 6 months showed a decrease in the rate of progression of radiological lesions in the hands and feet after 2-3 years after initial rapid progression (Van der Heijdeet et al, Arthritis Rheum., 35: 26-34 (1992); Fex et al, Br. J. Rheumatol., 35: 1106-1055 (1996)). In long-term studies of radiographs taken at lower frequencies, progression at constant rates with asymptotic worsening of lesions was found to last for 25 years (Wolfe and Sharp, Arthritis Rheum., 41: 1571-. It is not clear whether these differences in the pattern of radiography progression are due to differences in the scoring method.

The scoring systems used differ in the number of joints scored, the presence of independent scores for Erosion (ERO) and Joint Space Narrowing (JSN), the maximum score for each joint, and the weighting of radiological abnormalities. So far, no agreement has been reached on the preferences of the scoring method. During the first 3 years of follow-up in a cohort study of early arthritic patients, JSN was found to contribute differently to the progression of radiological lesions detected in the hands and feet by ERO (Van der Heijde et al, Arthritis Rheum., 35: 26-34 (1992)). Furthermore, methods that score ERO and JSN independently, such as Sharp and Kellgren scores, were found to be more sensitive to changes in early RA than methods that use overall measurements, such as Larsen scores (Plant et al, J.Rheumatol., 21: 1808-. Sharp scores are a very laborious method (Van der Heijde, Baillieres Clin Rheumatol., 10: 435-. In late or destructive RA, the Sharp and Larsen methods were found to provide similar information. However, the sensitivity of various scoring methods to late-stage changes in disease has not been investigated, and there is controversy over the useful information provided by scoring methods that measure ERO and JSN independently (Pincus et al, J.Rheumatol., 24: 2106-. See also, Drossaers-Bakker et al, artritisrheum, 43: 1465-1472(2000), which compares these three radiology scoring systems for long-term assessment of RA.

Paulus et al, Arthritis rheum, 50: 1083-. It appears that in the routine clinical management of RA patients, there should be an interval change of at least five Sharp radiographic damage score units between a pair of radiographs before the structural change is deemed to be real and used as the basis for a treatment decision.

Over the last 10 years, there has been significant progress in the treatment of RA. The use of existing disease modifying antirheumatic drugs (DMARDs) in combination with new biological agents provides a higher level of efficacy in a larger proportion of patients, while improving the early diagnosis and treatment of the disease.

Etanercept is a fully human fusion protein that inhibits Tumor Necrosis Factor (TNF) and subsequent inflammatory cytokine cascades. Etanercept has proven safe and effective in rapidly reducing disease activity and maintaining the improvement in adults with RA (Bathon et al, N.Eng.J.Med., 343: 1586-. It is also effective in children with polyarticular juvenile RA (Lovell et al, n.engl.j.med., 342: 763-. Etanercept is approved as a monotherapy for the treatment of RA, as well as a combination therapy with MTX.

Loss of function and radiographic changes occur early in the disease process. These changes can be delayed or prevented with the aid of certain DMARDs. While 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-.

Recent studies examined radiographic progression in patients with advanced RA who take leflunomide, MTX, or placebo (Strandet al., Arch. Intern.Med., 159: 2542-. In the first year of the Enbrel ERA (early RA) trial, etanercept proved 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)) reported the results of the second year of the study, concluding that etanercept is safe as monotherapy and superior to MTX in reducing disease activity, arresting structural damage, and alleviating disability for 2 years in early invasive RA patients.

In addition, a reduction in the progression of hand and foot radiography was observed in patients with early stage rheumatoid arthritis who received infliximab in combination with methotrexate (Van der Heijde et al, Annals Rheomatic diseases 64: 418-419 (2005)). Clinically meaningful and sustained improvements in somatic function were achieved in early rheumatoid arthritis patients after treatment with infliximab (Smolen et al, annals pharmaceutical Diseases 64: 418 (2005)). Van der Heijde et al, Annals rhematic diseases 64: 417(2005) reported the effects of infliximab and methotrexate on radiographic progression in patients with early stage rheumatoid arthritis. Infliximab treatment of ankylosing spondylitis patients results in changes in markers of inflammation and bone turnover that are associated with clinical efficacy (Visvanathan et al, Annals rhematic Diseases 64: 319 (2005)).

Van der Heijde et al, Annals Rheomatic Diseases 64: 319(2005) the effect of infliximab therapy on bone mineral density in Ankylosing Spondylitis (AS) patients was reported from a randomized, placebo-controlled trial named ASSERT. Infliximab was found to improve fatigue and pain in AS patients in outcome of ASSERT (Van der Heijde et al, Annals rhematic Diseases 64: 318-. In addition, van der Heijde et al, Arthritis rheum.5: 582-. In the 24-cycle study, the authors concluded that infliximab was well tolerated and effective in a large cohort of AS patients. In addition, the effect of infliximab therapy on spinal inflammation was evaluated by magnetic resonance imaging in a randomized, placebo-controlled trial of 279 AS patients (Van der Heijde et al, Annals Rheomatic Diseases 64: 317 (2005)). van der Heijde et al, Arthritis rheum.52: 1979-1985(2005) proposed a way to measure the therapeutic effect of spinal radiography progression in patients with AS.

Antoni et al, Annals rhematic Diseases 64: 107(2005) the results of a radiographic analysis of infliximab multinational psoriatic arthritis one year after the control trial (IMPACT) were reported. Smolen et al, artritis rheum.52: 1020-1030(2005) reported evidence of the radiographic benefit of infliximab plus MTX treatment in rheumatoid arthritis patients without clinical improvement, with a detailed sub-analysis of data from the anti-tumor necrosis factor test and concomitant therapy studies in rheumatoid arthritis. The radiographic progression, measured by mean change in modified Sharp/van der Heijde scores, was much greater in patients receiving MTX plus placebo than in patients receiving infliximab plus MTX. The authors concluded that even in patients without clinical improvement, treatment with infliximab plus MTX provided significant benefit in terms of a destructive process, suggesting that these 2 measures of disease are dissociated in such patients. Breedveld et al, Annals Rheomatic Diseases 64: 52-55(2005) described the association between baseline radiographic damage and improved body function following treatment with infliximab in rheumatoid arthritis patients. The van der Heijde modified Sharp score was used to assess structural damage. The authors concluded that greater joint damage at baseline was associated with poorer physical function at baseline and less improvement in physical function after treatment, underscoring the importance of early intervention to slow the progression of joint destruction.

CD20 antibodies and therapies using CD20 antibodies

Lymphocytes are one of the many types of white blood cells that are produced in the bone marrow during the hematopoietic process. There are two major lymphocyte populations: b lymphocytes (B cells) and T lymphocytes (T cells). Lymphocytes of particular interest herein are B cells.

B cells mature within the bone marrow and then leave the bone marrow and express antigen-binding antibodies on their cell surface. When a naive B cell first encounters an 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 lifespan and continue to express membrane-bound antibodies with the same specificity as the original parent cells. Plasma cells do not produce membrane bound antibodies, but instead produce antibody in a secreted form. Secreted antibodies are the main effector molecules of humoral immunity.

The CD20 antigen (also known as human B lymphocyte restricted differentiation antigen, Bp35, or B1) is a tetraspanin, glycosyl group with a molecular weight of about 35kD located on pre-B lymphocytes and mature B lymphocytesIntegral membrane proteins (Valentine et al, J.biol. chem.264 (19): 11282-. The antigen is also expressed on more than 90% of B-cell non-Hodgkin's lymphomas (NHLs) (Anderson et al, Blood 63 (6): 1424-. CD20 regulates early steps in the activation process of cell cycle initiation and differentiation (Tedder et al, supra) and may function as a calcium channel (Tedder et al, j.cell. biochem.14d: 195 (1990)). CD20 is phosphorylated 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 can be found on mature and memory B cells, but not on 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 play a role in B cell development. Unclear in vitro peripheral CD20 ⁺The link between lysis of B cells and rituximab activity in vivo. rituximab exhibits antibody-dependent cellular cytotoxicity (ADCC) in vitro (Reff et al, Blood 83: 435-445 (1994)). Potent complement-dependent cytotoxic (CDC) activity of rituximab has also been observed on lymphoma cells and cell lines (Reff et al, supra, 1994) and in certain mouse xenograft models (DiGaetano et al, J.Immunol.171: 1581-1587 (2003)). Several anti-CD 20 antibodies, including rituximab, have been shown to induce apoptosis in vitro when crosslinked by a second antibody or by other means (Ghetie et al, Proc. Natl. Acad. Sci.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. Essentially, such targeting can be summarized as follows: an antibody specific for the CD20 surface antigen of B cells is administered to the patient. These anti-CD 20 antibodies specifically bind to CD20 antigen (on the surface) of both normal and malignant B cells; binding of antibodies to CD20 surface antigen can lead to the destruction and depletion of neoplastic B cells. In addition, chemical agents or radioactive labels with the potential to destroy tumors can be conjugated to anti-CD 20 antibodies, allowing the agent to be specifically "delivered" to neoplastic B cells. Regardless of the method, the primary goal is to destroy the tumor; the particular method may be determined by the particular anti-CD 20 antibody utilized, and as such, the available methods for targeting the CD20 antigen may vary considerably.

rituximab (rituximab) (RITUXAN)) The antibodies are genetically engineered chimeric murine/human monoclonal antibodies to the CD20 antigen. rituximab is an antibody called "C2B 8" in U.S. Pat. No.5,736,137(Anderson et al), issued on 7.4.1998. 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 studies of the mechanism of action indicate that rituximab binds to human complement and lyses lymphoid B cell lines by CDC (Reff et al, Blood 83 (2): 435-. Furthermore, it has significant activity in ADCC assays. Recently, rituximab was shown to have an anti-proliferative effect and directly induce apoptosis in tritiated thymidine incorporation assays, whereas other anti-CD 19 and anti-CD 20 antibodies did not (Maloney et al, Blood88 (10): 637a (1996)). Synergy between rituximab and chemotherapy and toxins has also been observed in experiments. 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 chemotherapeutics)&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, possibly through complement and cell-mediated processes (Reff et al, Blood 83: 435-445 (1994)).

Rituximab was approved for the treatment of patients with relapsed or refractory low-grade or follicular CD20 in the United states at 11 months 1997⁺B cell NHL patients, once a week, were dosed at 375mg/m²Four doses in total. In month 4 2001, the Food and Drug Administration (FDA) approved other requirements for the treatment of low-grade NHL: re-treatment (once weekly, four doses total) and additional dosing regimens (once weekly, eight doses total). Over 300,000 patients are exposed to rituximab either as monotherapy or in combination with immunosuppressive or chemotherapeutic agents. Patients have also been treated as maintenance therapy with rituximab for up to 2 years (Hainsworth et al, J.Clin.Oncol.21: 1746-. Also, rituximab has been used to treat malignant and non-malignant plasma cell disorders (Treon and Anderson, Semin. Oncol.27: 79-85 (2000)).

Rituximab (Edwards et al, Biochem Soc. Trans.30: 824-828(2002)) has also been studied in a variety of non-malignant autoimmune disorders in which B cells and autoantibodies appear to play a role in the pathophysiology of the disease. Rituximab has been reported to potentially reduce, for example, Rheumatoid Arthritis (RA) (Leandro et al, Ann. Rheum. Dis.61: 883-888 (2002); Edwards et al, Arthritis Rheum.46 (Supl.9): S46 (2002); Stahl et al, Ann. Rheum. Dis.62 (Supl.1): OP004 (2003); Emery et al, Arthritis Rheum.48 (9): S439(2003)), Lupus (Eisenberg, Arthritis. Res.5: Thera 159 (2003); Leandr et al, Arthritis Rheum. 46: 2673-2677 (2002); Gorman et al, Lupus 13: 312-316 (2004); immune thrombocytopenic purpura (D '20', Australia: 78-99; Biond et al) (Biotin et al) (2000); Biotin et 99; Biotin et Biotin) (Biotin et 11, Biotin) (H.15, Biotin) (2003), Biotin et Biotin) (H.15, Biotin) (H.11, H.);) disorder) (Biotin) (H.15, H.);.) A) (Biotin) (H.15, H, br.j. haematol.116: 725-728(2002)), autoimmune anemia (Zaja et al, haematologic 87: 189-195(2002) (prospecting see Haematologica 87: 336(2002)), cold agglutinin disease (Layioset 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); Baudeer, Br. J. Haematol.112: 1083-1090 (2001); Damiani et al, Br. J. Haematol.114: 229-234(2001)), severe insulin resistance B syndrome (Coll et al, N.Engl. J. Med.2001.310: 311(2004)), mixed cryoglobulinemia (Deja Vitula et al, Arthritis sodium 46 (206/469); S289, J. Med.2001: 310-2000), and Espergyria et al, Rhynchus-6755, Rhynchus-11, Sjorgualin J. Skyi et al, J. Skyo et al, 6755, J. Skyo et al, J. Skyo et 35, J. 10, J. RTM. A, J. A, J. A. Arch.dermaltol.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-: 91-95(2004), autoimmune neuropathy (pesstronk et al, j.neurol.neurosurg.psychiatry 74: 485- "and 489(2003)), extratumoral ocular clonus-myoclonus syndrome (Pranzatelli et al, Neurology 60(suppl.1) PO 5.128: a395(2003)), and relapsing-remitting Multiple Sclerosis (RRMS) (Cross et al, (Abstract) "preferably Results from a Phase II Trial of Rituximab in MS", irradiation Annual Meeting of the America Committee for research and Treatment in Multiple Sclerosis (American Council for Multiple Sclerosis research and Treatment), 20-21 (2003)).

Phase II studies (WA16291) have been performed in Rheumatoid Arthritis (RA) patients, providing 48-week follow-up data on the 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 randomized to four treatment groups: methotrexate, rituximab alone, rituximab plus methotrexate, and rituximab plus Cyclophosphamide (CTX). The therapeutic regimen for rituximab was 1 gram administered intravenously on days 1 and 15. Most RA patients were well-tolerated for rituximab infusion, with 36% of patients experiencing at least one adverse event during their first infusion (compared to 30% of patients receiving placebo). In summary, most adverse events were considered mild to moderate in severity and well balanced across all treatment groups. There were 19 severe adverse events in the four groups over 48 weeks, with slightly more rituximab/CTX groups. The incidence of infection was well balanced among all groups. The average rate of severe infection in this RA patient population was 4.66 per 100 patients-year, which is lower than the rate of infection in RA patients requiring hospitalization (9.57 per 100 patients-year) reported in social-based epidemiological studies (Doran et al, Arthritis Rheum.46: 2287-.

The reported safety profile of rituximab in a few patients with neurological disorders, including autoimmune neuropathy (Pestronk et al, supra), ocular clonus-myoclonus syndrome (Pranzatelli et al, supra), and RRMS (Cross et al, supra), is similar to that reported in oncology or RA. In a investigator-initiated trial (IST) of rituximab in combination with interferon-beta (IFN- β) or glatiramer acetate in RRMS patients (Cross et al, supra), 1 of 10 treated patients experienced moderate fever and chills after the first rituximab infusion, after which they were admitted to the hospital for observation overnight, while the remaining 9 patients completed a four-infusion protocol without reporting any adverse events.

Patents and patent publications focusing on CD20 antibodies and CD20 binding molecules include U.S. Pat. nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963, US2003/0147885(Anderson et al); U.S. Pat. No.6,455,043 and WO 2000/09160(Grillo-Lopez, A.); WO 2000/27428(Grillo-Lopez and White); WO 2000/27433(Grillo-Lopez and Leonard); WO 2000/44788(Braslawsky et al.); WO2001/10462(Rastetter, W.); WO 2001/10461(Rastetter and White); WO2001/10460(White and Grillo-Lopez); US 2001/0018041, US 2003/0180292, WO 2001/34194(Hanna and Hariharan); US 2002/0006404 and WO 2002/04021(Hanna and Hariharan); US 2002/0012665, WO 2001/74388 and 6,896,885B5(Hanna, N.); US 2002/0058029(Hanna, N.); US 2003/0103971(Hariharan and hanna); US 2005/0123540(Hanna et al); US 2002/0009444 and WO 2001/80884(Grillo-Lopez, a.); WO 2001/97858; US 2005/0112060, and U.S. patent No.6,846,476(White, C.); US 2002/0128488 and WO 2002/34790(Reff, M.); WO2002/060955(Braslawsky et al); WO 2002/096948(Braslawsky et al.); WO2002/079255(Reff and Davies); U.S. Pat. No.6,171,586 and WO 1998/56418(Lam et al); WO 1998/58964(Raju, S.); WO 1999/22764(Raju, S.); WO1999/51642, U.S. Pat. No.6,194,551, U.S. Pat. No.6,242,195, U.S. Pat. No.6,528,624 and U.S. Pat. No.6,538,124 (Idusogene et al); WO 2000/42072(Presta, L.); WO 2000/67796(Curd et al.); WO 2001/03734(Grillo-Lopez et al); US2002/0004587 and WO 2001/77342(Miller and Presta); US 2002/0197256(Grewal, I.); US 2003/0157108(Presta, L.); U.S. Pat. Nos. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721(Kaminski et al.); U.S. Pat. Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852, 6,893,625(Robinson et al); U.S. Pat. No.6,410,391(Raubitschek et al); U.S. Pat. No.6,224,866 and WO 00/20864(Barbera-Guillem, E.); WO 2001/13945(Barbera-Guillem, E.); WO 2000/67795(golden berg); US 2003/0133930; WO 2000/74718 and US2005/0191300A1(Goldenberg and Hansen); US2003/0219433 and WO2003/68821(Hansen et al); WO 2004/058298(golden berg and Hansen); WO2000/76542(Golay et al.); WO 2001/72333(Wolin and Rosenblatt); U.S. Pat. No.6,368,596(Ghetie et al); U.S. Pat. Nos. 6,306,393 and 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); WO2003/061694(Sing and Siegas); US 2003/0219818(Bohen et al); US2003/0219433 and WO 2003/068821(Hansen et al); US 2003/0219818(Bohen et.); US 2002/0136719(Shenoy et al); WO 2004/032828 and US 2005/0180972(Wahl et al); and WO 2002/56910 (Hayden-Ledbetter). See also U.S. Pat. No.5,849,898 and EP 330,191(Seed et al); EP332,865A2(Meyer and Weiss); U.S. Pat. No.4,861,579(Meyer et al); US 2001/0056066(Bugelski et al.); WO1995/03770(Bhat et al.); US2003/0219433 a1(Hansen et al); WO2004/035607(Teeling et al.); WO 2005/103081(Teeling et al); WO2004/056312(Lowman et al.); US 2004/0093621(Shitara et al.); WO2004/103404(Watkins et al.); WO 2005/000901(Tedder et al.); US2005/0025764(Watkins et al); WO 2005/016969(Carr et al.); US 2005/0069545(Carr et al); WO 2005/014618(Chang et al.); US 2005/0079174(Barbera-Guillem and Nelson); US 2005/0106108(Leung and Hansen); WO2005/044859 and US 2005/0123546(Umana et al); WO 2005/070963(Allan et al.); US 2005/0186216(Ledbetter and Hayden-Ledbetter); US 2005/0202534(Hayden-Ledbetter and Ledbetter); US 2005/0202028(Hayden-Ledbetter and Ledbetter); US 2005/0202023(Hayden-Ledbetter and Ledbetter); U.S. Pat. No.6,183,744(golden berg); and U.S. Pat. No.6,897,044(Braslawski et al).

Publications concerning treatment with rituximab include: perotta and Abuel, "Response of cyclic playback ITP of 10 layers duration to rituximab", Abstract #3360 Blood10(1) (part 1-2): p.88B (1998); "Rituxan in the treatment of cyclic thiophosphoric purpura (ITP)", Blood 94: 49 (abstrat) (1999); matthews, R., "Medical genetics", New Scientist (7April, 2001); "Clinical out of 22 copies with rhematous incomplete deletion" Ann Rheum Dis, supra; leandro et al, "lymphocytedization in rheumatoid arthritis: early evidence for safety, efficacy and doseresponse ", Arthritis and Rheumatism 44 (9): s370 (2001); leandro et al, "open study of B lymphocyte depletionin system lupus erythromatosus ", Arthritis and Rheumatism 46: 2673-2677(2002), wherein each patient received two 500mg rituximab infusions, two 750mg cyclophosphamide infusions, and a high dose oral corticosteroid during a 2 week period, wherein the two treated patients relapsed at 7 months and 8 months respectively and had been re-treated with different regimens; "Successful Long-term transaction of system throughput with throughput mail throughput" by glide et al, "throughput 12: 779- ²X 4, repeated at weekly intervals) and deliver more rituximab applications every 5-6 months, then receive 375mg/m of rituximab every three months²The second patient with refractory SLE was successfully treated with rituximab and received maintenance therapy every three months, with both patients responding better to rituximab therapy; edwards and Cambridge, "contained improvement in rhematoid reaction from protocol designed to delete B lymphocytes", Rheumatology 40: 205-211 (2001); cambridge et al, "B lymphocyte depletion in tissues with rhematoid arthritis: serial studios of immunological parameters ", Arthritis Rheum.46 (suppl.9): s1350 (2002); cambridge et al, "roller changes foaming B lymphocyte depletion for rhematoid Arthritis" Arthritis Rheum, 48: 2146 and 2154 (2003); edwards et al, "B-lymphocyte depletion therapy in rheumatoid and autoimmune disorders" Biochem Soc. Transs, supra; edwards et al, "effectiveness and safety of rituximab, a B-cell targeted textual monoclonalantitis: a randomised, placebo controlled tertiary in tissues with rhematous Arthritis ", Arthritis and Rheumatism 46 (9): s197 (2002); edwards et al, "effectiveness of B-cell-targeted therapy with rituximab in tissues with rheumatoid characterization" NEngl.J. Med.350: 2572, 2582 (2004); pavelka et al, ann.rheum.dis.63 (S1): 289-90 (2004); emery et al, Arthritis rheum.50 (S9): s659 (20) 04)；Levine and Pestronk，“IgM antibody-related polyneuropathies：B-cell depletionchemotherapy using rituximab”，Neurology 52：1701-1704(1999)；Uchida et al.，″The innate mononuclear phagocyte network depletes B lymphocytes through Fcreceptor-dependent mechanisms during anti-CD20antibody immunotherapy″J.Exp.Med.199：1659-1669(2004)；Gong et al.，″Importance of cellularmicroenvironment and circulatory dynamics in B cell immunotherapy″J.Immunol.174：817-826(2005)；Hamaguchi et al.，″The peritoneal cavityprovides a protective niche for B1 and conventional B lymphocytes duringanti-CD20immunotherapy in mice″J.Immunol.174：4389-4399(2005)；Cragget 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 cellblockade in the treatment of rheumatoid arthritis”，Arthritis&Rheum 46: 2029-2033 (2002); "Treatment of DMARD-retroreflective adhesive with rituximab", shown in Annual Scientific Meeting of the American College of Rheumatology, Oct 24-29, New Oreans, LA 2002; tuscano, J., "Successful stream of infliximab-regenerative rheumoid arthritis with rituximab", shown in the annual scientific Meeting of the American College of Rheumatology, Oct 24-29, New Orleanans, LA 2002 and published Tuscano, Arthritis Rheum.46: 3420 (2002); martin and Chan, "Pathological roles of B cells in human autoimmunity; inertia from the clinical ", Immunity 20: 517-527 (2004); silvermanand Weisman, "Rituximab therapy and autoimmune disorders, physicians for anti-B cell therapy", Arthritis and Rheumatism 48: 1484 + 1492 (2003); kazkaz and Senberg, "Anti B cell therapy (rituximab) in thetreatment of autoimmunediseases”，Current opinion in pharmacology 4：398-402(2004)；Virgolini andVanda，“Rituximab in autoimmune diseases”，Biomedicine&pharmacotherapy 58: 299-309 (2004); klemmer et al, "Treatment of antibody media disorders with a AntiCD20monoclonal antibody Rituximab", Arthrit And Rheumatous 48: (9)9, S (sep) pp.s624-S624 (2003); kneitz et al, "Effective B cell deletion with rituximab in the treatment of autoimmune diseases", immunology 206: 519 (2002); arzo et al, "Treatment of regenerative inorganic media with an anti-CD20monoclonal antibody (rituximab)", Annals of the radioactive Diseases61 (10): 922-; comment in Ann Rheum Dis.61: 863-866 (2002); lakeand Dione, "Future Strategies in Immunotherapy" in Burger's medicinal chemistry and Drug Discovery (2003 by John Wiley &Sons, Inc.) paper on-line date of posting: 15/1/2003 (Chapter 2, "Antibody-Directed Immunotherapy"); liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 asan immunotherpy Target, paper online paste date: 1/15 in 2002 entitled "CD 20"; appendix 4A entitled "Monoclonal Antibodies to Human Cell Surface Antibodies" byStockinger et al, eds: coligan et al, Current Protocols in Immunology (2003John Wiley)&Sons, Inc) online pasting date: 5 months in 2003; printing and publishing date: month 2 in 2003; penichet and Morrison, "CD Antibodies/molecules: definition; antibody engineering ", by Wiley Encyclopedia of Molecular Medicine, Section: chimeric, Humanized and Human Antibodies; and (3) online pasting date: 1, 15 days in 2002.

In addition, see Looney "B cells as a thermal target in autoimmability disease thermal and in a rheumoid assay," Rheumatology, 44Suppl 2: ii13-ii17 (2005); chambers and Isenberg, "Anti-B cell therapy (rit)uximab) in the treatment of animal diseases "Lupus 14 (3): 210, 214 (2005); looney et al, "B-cell displacement as a novel treatment for system luma regenerative: a phase I/IIdose-evaluating tertiary of rituximab "Arthritis Rheum.50: 2580, 2589 (2004); looney, "Treating human autoimmune disease by eliminating B cells" an Rheum. Dis.61: 863-866 (2002); edelbauer et al, "Rituximab in children system great lipids recovery to conservation immunological delivery 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-target dtherapy in diseases other than the renal tissue characterization" J.Rheumatol.Suppl.73: 25-28; discission 29-30 (2005); sfikakis et al, "recommendation of productive lupulin following B cell deletion therapy is predicted by down-regulation of T cell synergy expression CD40 ligand: an open-latex three "ArthritisRheum.52 (2): 501-513 (2005); rasetter et al, "Rituximab: expanding roll understanding for lymphamas and autoimmune diseases "Annu. Rev. Med. 55: 477-503 (2004); silverman, "Anti-CD 20 therapy in system unpus erythromatosus: a stepclock to the clinic "Arthritis Rheum.52 (2): 371-7(2005), the wrong finding: artritisrheum.52 (4): 1342 (2005); ahn et al, "Long-term reliability from a Long-lasting refractory state associated with a Long porous aggregate (LA) flowing and finishing, thermal approach" am.j.hematol.78 (2): 127-129 (2005); tahir et al, "manipulated anti-CD20monoclonal antibody in the treatment of polypeptide resistant system in a patient with antibodies against viral infection, 44 (4): 561-; looney et, Treatment of SLE with anti-CD20monoclonal antibody "current, dir. 193- > 205 (2005); cragg et al, "The biology of CD20and itspoten the total 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 Patientswitch system autoimmune diseases" Ann. Rheum. Dis.64 (6): 913-920(2005) electronic publication at 11/18 of 2004; tokunaga et al, "Down-registration of CD40 and DC D80on B cells in tissues with lithium-treating system filtration flux treatment with a puncturing flux" Rheumatology 44 (2): 176- "2005", published electronically on 10/19/2004. See also Leandro et al, "B cell repopulatoccurs mail fromB cells in patient with rheumatoid arthritis andsystemic lupus erythematosus″Arthritis Rheum.，48(Suppl 9)：S1160(2003)。

Specks et al.“Response of Wegener’s granulomatosis to anti-CD20chimericmonoclonal antibody therapy”Arthritis&Rheumatic 44 (12): 2836 Annune 2840(2001) discloses a four-infusion of 375mg/m²rituximab and high-dose glucocorticoids were successfully used to treat wegener's granulomatosis. Treatment was repeated when cANCA recurred after 11 months, but did not include glucocorticoids. The patient's disease remained completely resolved at 8 months after the second rituximab course of treatment. In addition, in another study, rituximab was found to be a well-tolerated agent effective in inducing regression of severe ANCA-associated vasculitis at a dose of 375mg/m ²X 4 with oral prednisone 1 mg/kg/day, decreased to 40 mg/day at week 4 and to complete withdrawal for the following 16 weeks. Four patients were retreated with relapsed/rising ANCA titers with rituximab alone. With the exception of glucocorticoids, no other immunosuppressant appears to be necessary to induce regression and maintain sustained regression (6 months or longer). See online summary submission and invitation Keogh et al, "Rituximab for recommendation indication in SevereANCA-Associated Vasculitis: report of a productive Open-Label Pilot Trial in10 Panels″，American College of Rheumatology，Session Number：28-100，Session Title：Vasculitis，Session Type：ACR Concurrent Session，PrimaryCategory：28 Vasculitis，Session 10/18/2004(<www.abstractsonline.com/viewer/SearchResults.asp>). See also, Keogh et al, Kidney Blood press. res.26: 293(2003), in which 11 patients with refractory ANCA-associated vasculitis were reported at a four week dose of 375mg/m²The regression was achieved with rituximab and high dose glucocorticoid treatment.

The administration of rituximab and immunosuppressive drugs such as intravenous cyclophosphamide, mycophenolate mofetil, azathioprine or leflunomide to patients with refractory ANCA-associated vasculitis has significant efficacy. Eriksson, "Short-term out monomer and safety in 5 patents with ANCA-positional tissue engineered with rituximab", Kidneyand Blood Pressure Research, 26: 294(2003) (5 patients with ANCA-associated vasculitis who used rituximab 375mg/m once a week ²Treatment for 4 weeks, they responded to treatment); jayne et al, "B-cell deletion with rituximab for reactivity variation and Blood Pressure Research," Kidney and Blood Pressure Research, 26: 294-²Cyclophosphamide plus background immunosuppression and prednisolone experienced a significant decrease in vasculitic activity). Administration of rituximab and intravenous cyclophosphamide per 375mg/m dose to a patient suffering from refractory systemic vasculitis²Another report of 4 doses is provided in Jayne, poster 88 (11)^thInternational Vasculitis and dANCA works hop), 2003American Society of neuroprology. See also Stone and Specks, "Rituximab Therapy for the indication of recommendation and toll in ANCA-associated vasculitides" in Clinical Trial Research study of the2002-2003 Immune Tolerance Network, http:// www.immunetolerance.org/Research/autoimmune/trials/store. html, where an 18 month total length Rituximab test is proposed for ANCA-related Vasculitis. See also Eriksson, J.Internal med., 257: 540-: 262- ²rituximab, treated or retreated, induced regression by B lymphocyte depletion, a study conducted between month 1 of 2000 and month 9 of 2002.

For the activity of humanized anti-CD20 antibodies see, e.g., Vugmeyster et al, "simplistic B cells by a humanized anti-CD20antibody PRO70769 in macavacaciciculatis" j. immunother.28: 212-219(2005). For a discussion of human monoclonal antibodies, see Baker et al, "Generation and characterization of Lymphostat-B, a human monoclonal antibody of the biological antibodies" antibodies of B lymphomatosis Rheum.48: 3253-3265(2003).

Study WA17043, phase IIb, randomized, double blind, dose-determination study in rheumatoid arthritis patients who responded inappropriately to DMARDs (including anti-TNF agents) (Emery et al, european league against rhe (EULAR) (June 2005) OP 0008; Van Vollenhoven et al, EULAR (June 2005) SAT0072) found that rituximab in combination with MTX WAs associated with clinically and statistically significant improvement in disease symptoms. This study determined that the dosage of rituximab in combination with MTX required further investigation and confirmation in the setting of phase III clinical studies. See also world pharmaceutical News, www.scrippharma.com, script paper, date 2005, 6.13.6.2005, entitled "Rituximab a future challenge for anti-TNFs? It is described in the EULAR study and it was thought whether x-ray data from the phase III REFLEX study would show whether rituximab could slow joint damage. In addition, WO 2004/091657 published on day 28/10/2004 discloses that treatment with a CD20antibody in a rheumatoid arthritis patient who exhibits an inappropriate response to TNF α inhibitor therapy, wherein the patient may have radiographic evidence that at least one joint has definite erosion attributable to rheumatoid arthritis, as determined by central reading sites (any joint of the hand, wrist or foot, except for the DIP joint of the hand). Potential secondary endpoints include changes in the modified Sharp radiography total score, the erosion score, and the joint space narrowing score, which may be analyzed using continuous or categorical methodologies as appropriate. Exploratory endpoints and analyses may involve radiographic analyses, including the proportion of patients with no erosive progression, which may be assessed at week 24 and later. See also US2005/00001862 published on 25.8.2005, corresponding to WO 2005/060999 published on 7.7.2005, for treatment of rheumatoid arthritis patients with rituximab, where potential secondary endpoints and exploratory endpoints and analyses include those in WO 2004/091657.

Despite some advances in the treatment of arthropathy, a significant number of patients are inapplicable, intolerant, or inadequately responsive to current treatments. Therefore, new treatment options are needed, particularly those that can target different aspects of the disease pathology and provide similar or better levels of clinical efficacy.

Summary of The Invention

The present invention involves the administration of a CD20 antagonist that provides a safe and effective treatment regimen for a subject with joint damage, including the selection of an effective dosing regimen and scheduled or unscheduled re-treatment (re-treatment). This antagonist is effective both in initial treatment and in the management of refractory diseases.

Accordingly, the invention is as claimed. In a first aspect, the invention concerns a method for treating joint damage in a subject, comprising administering to the subject a CD20 antibody, and at least about one month after the administration, subjecting the subject to a radiographic test to measure a reduction in joint damage compared to baseline prior to the administration, wherein the amount of CD20 antibody administered is effective in achieving a reduction in joint damage.

In another aspect, the invention relates to a method of monitoring treatment for joint damage in a subject comprising administering to the subject an effective amount of a CD20 antibody, and measuring by radiography after at least about one month from the administration whether the joint damage is reduced relative to baseline prior to the administration, wherein a reduction from baseline in the subject after treatment indicates that the CD20 antibody is having an effect on the joint damage. In a preferred embodiment, the degree of reduction from baseline is again measured after administration of the CD20 antibody.

In yet another aspect, the invention provides a method of determining whether to continue administering a CD20 antibody to a subject with joint damage, comprising measuring by radiography reduction in joint damage after a first administration of CD20 antibody by the subject, measuring by radiography reduction in joint damage after a second administration of CD20 antibody by the subject, comparing the radiography scores of the subject at the first and second times, and if the score at the second time is less than the first time, continuing administration of the CD20 antibody.

In yet another aspect, the present invention is directed to an article comprising:

(a) a container containing the CD20 antibody; and

(b) a package insert printed with instructions for treating joint damage in a subject, wherein the instructions instruct administering a CD20 antibody to the subject and then, at least about one month after the administration, performing a radiographic examination to measure a reduction in the joint damage as compared to baseline prior to the administration, wherein the amount of CD20 antibody administered is effective in achieving a reduction in joint damage.

In a preferred aspect, the article of manufacture further comprises a container comprising a second medicament, wherein the CD20 antibody is the first medicament, further comprising instructions printed on the package insert for treating the subject with an effective amount of the second medicament.

In another embodiment of the invention, there is provided a method for treating joint damage in a subject comprising administering to the subject a CD20 antibody and, at least about 52 weeks after the administration, giving the subject a radiographic test to measure a reduction in joint damage as compared to baseline prior to the administration, wherein the amount of CD20 antibody administered is effective in achieving a reduction in joint damage.

In yet another embodiment, the invention provides a method of monitoring treatment for joint damage in a subject, comprising administering to the subject an effective amount of a CD20 antibody, and measuring by radiography after at least about 52 weeks from the administration whether the joint damage is reduced relative to baseline prior to the administration, wherein a reduction from baseline in the subject after treatment indicates that the CD20 antibody is having an effect on the joint damage.

Still further, the present invention provides an article comprising:

(a) a container containing the CD20 antibody; and

(b) a package insert printed with instructions for treating joint damage in a subject, wherein the instructions instruct administering a CD20 antibody to the subject and then at least about 52 weeks after the administration, performing a radiographic test to measure a reduction in the joint damage as compared to baseline prior to the administration, wherein the amount of CD20 antibody administered is effective in achieving a reduction in joint damage.

In yet another aspect, the present invention provides a method for treating joint damage in a subject, wherein

(a) The subject exhibits an inadequate response to one or more anti-Tumor Necrosis Factor (TNF) inhibitors;

(b) the subject receiving at least one prior course of treatment with the CD20 antibody; and (c) the treatment comprises administering at least one more course of treatment with the CD20 antibody.

These and other aspects will be apparent from the remainder of the disclosure, including the examples and the appended claims.

Brief Description of Drawings

Figure 1 shows a study design for treatment of RA patients with either control (placebo plus MTX) or rituximab (1000mg x 2) plus MTX (example 1 herein).

Figure 2 shows ACR response in RA patients treated for six months with either control or rituximab (1000mg x 2) plus MTX.

Figure 3 shows ACR20 responses over six months in RA patients treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 4 shows the change in 328DS for six months in RA patients treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 5 shows EULAR response when RA patients were treated for six months with control or with rituximab (1000mg x 2) plus MTX.

Figure 6 shows EULAR regression or low disease at six months of RA patient treatment with either control or rituximab (1000mg x 2) plus MTX.

Figure 7 shows the median C-reactive protein (CRP) for six months in RA patients treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 8 shows the proportion of RA patients with clinically relevant functional improvement at six months, where the patients were treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 9 shows the percent change in ACR score at six months for RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

FIG. 10 shows the change in FACIT-F at six months in RA patients treated with either control or rituximab (1000mg X2) plus MTX.

Figure 11 shows the change in SF-36 grade (mental and physical health) at six months in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 12 shows total rheumatoid factor at six months in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 13 shows the mean change in Sharp-gent total score for RA patients treated with control or with rituximab (1000mg x 2) plus MTX for six months.

Figure 14 shows the mean change in Sharp-gent erosion score for six months in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 15 shows the mean change in Sharp-gent articular cavity narrowing (JSN) score for six months in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 16 shows the proportion of RA patients with no change in erosion score at six months, where patients were treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 17 shows the change in radiographic end-point at 24 weeks (exploratory end-point) for RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 18 shows the mean percent change in ACR core set parameters at 24 weeks in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 19 shows median CD19 at six months for RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 20 shows the most commonly reported adverse events in a study of RA patients over six months, where patients were treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 21 shows adverse events leading to withdrawal in a six month study of RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 22 shows events occurring during/within 24 hours of infusion in a six month study in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 23 shows acute infusion response in a six month study of RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 24 shows severe adverse events occurring during/within 24 hours of infusion in a six month study in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 25 shows systemic organ organoid-infection and invasion in a six month study in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

Figure 26 shows severe infection in a study with RA patients over six months, where patients were treated with either control or rituximab (1000mg x 2) plus MTX.

Figure 27 shows the infection rate in a study of RA patients over six months, wherein patients were treated with control or with rituximab (1000mg x 2) plus MTX.

Fig. 28 shows HACA in a study of RA patients over six months, where patients were treated with either control or rituximab (1000mg × 2) plus MTX.

Figure 29 shows IgG levels in RA patients treated with control or with rituximab (1000mg × 2) plus MTX over six months.

Fig. 30 shows IgA levels over six months in RA patients treated with control or with rituximab (1000mg × 2) plus MTX.

Figure 31 shows IgM levels over six months in RA patients treated with control or with rituximab (1000mg x 2) plus MTX.

FIG. 32A is a sequence alignment comparing the light chain variable domains (V) of murine 2H7(SEQ ID NO: 1), humanized 2H7.V16 variant (SEQ ID NO: 2), and human kappa light chain subgroup I (SEQ ID NO: 3)_L) The amino acid sequence of (a). V of 2H7 and hu2H7.V16_LThe CDRs of (A) are as follows: CDR1(SEQ ID NO: 4), CDR2(SEQ ID N)O: 5) and CDR3(SEQ ID NO: 6).

FIG. 32B is a sequence alignment comparing the heavy chain variable domains (V) of murine 2H7(SEQ ID NO: 7), humanized 2H7.V16 variant (SEQ ID NO: 8), and human heavy chain subgroup III consensus sequence (SEQ ID NO: 9)_H) The amino acid sequence of (a). V of 2H7 and hu2H7.V16_HThe CDRs of (A) are as follows: CDR1(SEQ ID NO: 10), CDR2(SEQ ID NO: 11), and CDR3(SEQ ID NO: 12).

In FIGS. 32A and 32B, CDR1, CDR2, and CDR3 in each chain are included within parentheses and flanked by the framework regions FR1-FR4, as indicated. 2H7 refers to murine 2H7 antibody. The asterisks between the two rows of sequences indicate the different positions between the two sequences. Residue numbering is according to Kabat et al, sequence of Immunological Interest, 5th Ed.public Health Service, National Institutes of Health, Bethesda, Md. (1991), where insertions are shown as a, b, c, d, and e.

FIG. 33 shows the amino acid sequence of mature 2H7.v16 light chain (SEQ ID NO: 13).

FIG. 34 shows the amino acid sequence of the mature 2H7.v16 heavy chain (SEQ ID NO: 14).

FIG. 35 shows the amino acid sequence of the mature 2H7.v31 heavy chain (SEQ ID NO: 15). The light chain of 2h7.v31 is identical to 2h7.v 16.

FIG. 36 is a sequence alignment comparing the light chain amino acid sequences of the humanized 2H7.v16 variant (SEQ ID NO: 2) and the humanized 2H7.v138 variant (SEQ ID NO: 28).

FIG. 37 is a sequence alignment comparing the heavy chain amino acid sequences of the humanized 2H7.v16 variant (SEQ ID NO: 8) and the humanized 2H7.v138 variant (SEQ ID NO: 29).

FIG. 38 shows an alignment of mature 2H7.v16 and 2H7.v511 light chains (SEQ ID NOS: 13 and 30, respectively) using Kabat variable domain residue numbering and Eu constant domain residue numbering.

FIG. 39 shows an alignment of mature 2H7.v16 and 2H7.v511 heavy chains (SEQ ID NOS: 14 and 31, respectively) using Kabat variable domain residue numbering and Eu constant domain residue numbering.

FIG. 40A shows the sequence of the humanized 2H7.v114 light chain variable domain (SEQ ID NO: 32); FIG. 40B shows the sequence of the humanized 2H7.v114 heavy chain variable domain (SEQ ID NO: 33); and FIG. 40C shows the sequence of the humanized 2H7.v114 full length heavy chain (SEQ ID NO: 34) using Kabat variable domain residue numbering and Eu constant domain residue numbering.

Figure 41 illustrates patient profiles for the REFLEX clinical trial at week 56, including ongoing treatment of a subgroup of patients selected from the treatment group and placebo group of the phase III REFLEX clinical trial.

Fig. 42 shows the change in the end point of radiography at week 56.

FIG. 43 is the mean change in Sharp-Genant total score over time.

FIG. 44 is a cumulative distribution of Sharp-Genant total score changes.

FIG. 45 is a sensitivity analysis: change in Sharp-Genant Total score.

Figure 46 is a patient with no radiographic changes at week 56.

Detailed description of the preferred embodiments

I. Definition of

"B cells" are lymphocytes that mature within the bone marrow, including naive B cells, memory B cells, or effector B cells (plasma cells). A B cell herein is a normal or non-malignant B cell.

"B cell surface marker" or "B cell surface antigen" as used herein refers to an antigen expressed on the surface of a B cell that can be targeted to it with an antagonist that binds to it. Exemplary B cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79B, CD80, CD81, CD82, CD83, CDw84, CD85, and CD86 Leukocyte surface markers (for description see The Leukocyte antibodies books, 2 nd edition, 1997, Barclay al. Other B cell surface markers include RP105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287. B cell surface markers of particular interest are preferentially expressed on B cells compared to other non-B cell tissues of mammals, and may be expressed on both precursor B cells and mature B cells. Preferred B cell surface markers herein are CD20 and CD 22.

The "CD 20" antigen or "CD 20" is an approximately 35kDa non-glycosylated phosphoprotein found on the surface of B cells from peripheral blood or lymphoid organs in more than 90%. CD20 is present on both normal B cells as well as malignant B cells, but is not expressed on stem cells. Other names of CD20 in the literature include "B lymphocyte restriction antigen" and "Bp 35". CD20 antigen is described, for example, in Clark et al, proc.natl.acad.sci. (USA) 82: 1766(1985).

The "CD 22" antigen or "CD 22", also known as BL-CAM or Lyb8, is a type 1 integral membrane glycoprotein with a molecular weight of about 130 (shortened) to 140kD (unshortened). It is expressed in both the cytoplasm and the cell membrane of B lymphocytes. The CD22 antigen appears early in the differentiation of B cell lymphocytes, at approximately the same stage as the CD19 antigen. Unlike other B cell markers, CD22 membrane expression is restricted to the late stage of differentiation involved between mature B cells (CD22+) and plasma cells (CD 22-). 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 "antagonist" refers to a molecule that destroys or depletes B cells in a mammal upon binding CD20 on the B cells and/or interferes with one or more B cell functions, for example by reducing or preventing the humoral response elicited by the B cells. The antagonist is preferably capable of depleting B cells in a mammal treated therewith (i.e., reducing the level of B cells in the circulation). Such depletion may be achieved by a variety of mechanisms, such as ADCC and/or CDC, inhibition of B-cell proliferation, and/or induction of B-cell death (e.g., by apoptosis). Antagonists included within the scope of the present invention include antibodies that bind CD20, synthetic or naturally-occurring sequence peptides, immunoadhesins, and small molecule antagonists, optionally coupled or fused to another molecule. Preferred antagonists include antibodies.

An "antibody antagonist" as used herein refers to an antibody that destroys or depletes B cells in a mammal upon binding to a B cell surface marker on the B cell and/or interferes with one or more B cell functions, e.g., by reducing or preventing the humoral response elicited by the B cell. Preferably, the antibody antagonist is capable of depleting B cells (i.e., reducing the level of B cells in circulation) in a mammal treated therewith. Such depletion may be achieved by a variety of mechanisms, such as ADCC and/or CDC, inhibition of B-cell proliferation, and/or induction of B-cell death (e.g., by apoptosis).

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; diabodies (diabodies); a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

For purposes herein, a "whole antibody" refers to an antibody comprising heavy and light chain variable domains and an Fc region.

An "antibody that binds to a B cell surface marker" refers to 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 B cells. Preferably, the antibody is capable of depleting B cells (i.e., reducing the level of B cells in circulation) in a mammal treated therewith. Such depletion may be achieved by a variety of mechanisms, such as ADCC and/or CDC, inhibition of B-cell proliferation, and/or induction of B-cell death (e.g., by apoptosis). In a preferred embodiment, the B cell surface marker is CD20 or CD22, and thus the antibody that binds to the B cell surface marker is an antibody that binds to CD20 or CD22, respectively, or is a "CD 20 antibody" or "CD 22 antibody", respectively. Examples of CD22 antibodies include those disclosed in EP 1,476,120(Tedder andduscano), EP 1,485,130(Tedder), and EP 1,504,035(Popplewell et al), as well as those disclosed in US 2004/0258682(Leung et al). In an even more preferred embodiment, the antibody is a CD20 antibody. A particularly preferred embodiment is a CD20 or CD22 antibody, preferably a CD20 antibody.

Examples of CD20 antibodies include: "C2B 8", now called "rituximab" ("RITUXAN") /MABTHERA"(U.S. Pat. No.5,736,137); yttrium [90 ]]Labeled 2B8 murine antibody, designated "Y2B 8" or "Ibritumomab Tiuxetan" (ZEVALIN)) Commercially available from BiogenIdec corporation (e.g., U.S. patent nos. 5,736,137; 2B8 deposited with the ATCC at month 6 and 22 of 1993, accession No. HB 11388); murine IgG2a "B1", also known as "Tositumomab", optionally with¹³¹I labelling to produce "¹³¹1I-B1 "or" iodine 131tositumomab "(BEXXAR)^TM) Available from Corixa (see also, e.g., U.S. Pat. No.5,595,721); murine monoclonal antibody "1F 5" (e.g., pressure et al, Blood, 69 (2): 584-; murine 2H7 and chimeric 2H7 antibodies (e.g., U.S. patent No.5,677,180); humanized 2H7 (e.g., WO2004/056312(Lowman et al.) and listed below); HUMAX-CD20^TMIs completely human,High affinity antibodies that target CD20 molecules in the cell membrane of B cells (Genmab, Denmark; see, e.g., Glenniean van de Winkel, Drug Discovery Today, 8: 503-; human monoclonal antibodies listed in WO 2004/035607 and WO 2005/103081(Teelinget al, GenMab/Medarex); antibodies with complex N-glycoside linked sugar chains bound to the Fc region as described in US 2004/0093621(Shitara et.); monoclonal antibodies and antigen-binding fragments that bind CD20 (e.g., WO 2005/000901, Tedder et al.) such as HB20-3, HB20-4, HB20-25, and MB 20-11; single chain proteins that bind CD20 (e.g., US2005/0186216(Ledbetter and Hayden-Ledbetter); US 2005/0202534(Hayden-Ledbetter and Ledbetter); US 2005/0202028(Hayden-Ledbetter and Ledbetter); US 2005/0202023(Hayden-Ledbetter and Ledbetter) -trubinpharm Inc.); CD20 binding molecules, such as antibodies of the AME series, e.g. AME-33 ^TMAntibodies, such as those listed in WO 2004/103404 and US 2005/0025764(Watkins et al, Applied Molecular Evolution, Inc.), and CD20 antibodies with Fc mutations, such as those listed in WO 2005/070963 (alan 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, such as those listed in WO 2005/014618(Chang et al); humanized LL2 monoclonal antibodies, such as those listed in US 2005/0106108(Leung and Hansen; immunolodics); chimeric or humanized B-Ly1 antibodies to CD20, such as those described in WO 2005/044859 and US 2005/0123546(Umana et al; GlycArt Biotechnology AG); a20 antibody or variants thereof, such as chimeric or humanized a20 antibodies (cA 20, hA20, respectively) and IMMUN-106 (e.g. US2003/0219433, immunology); and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2, available from the International Leukocyte Classification research group (International Leucocyte TypingWorkshop) (e.g., Valentine et al, Leucocyte Typing III, McMichael eds., p.440, Oxford University Press, 1987). Preferred CD20 antibodies herein are chimeric, humanized, or human CD20 antibodies, more preferably rituximab, humanized 2H7, chimeric Synthetic or humanized A20 antibodies (Immunomedics), HUMAX-CD20^TMHuman CD20 antibody (Genmab), and immunoglobulin/protein binding to CD20 (trubium Pharm Inc.).

The term "rituximab" (rituximab) or "RITUXAN"genetically engineered chimeric murine/human monoclonal antibodies directed to the CD20 antigen, referred to herein as" C2B8 "in U.S. Pat. No.5,736,137, include fragments thereof that retain the ability to bind CD 20.

Purely for the purposes herein and unless otherwise indicated, "humanized 2H 7" refers to a humanized CD20 antibody or antigen-binding fragment thereof that comprises one, two, three, four, five or six of the following CDR sequences:

CDR L1 sequence, RASSSSVSYXH, wherein X is M or L (SEQ ID NO: 35), e.g. SEQ ID

NO: 4 (fig. 32A);

CDR L2 sequence, SEQ ID NO: 5 (fig. 32A);

CDR L3 sequence, QQWXFNNPPT, wherein X is S or A (SEQ ID NO: 36), e.g. SEQ ID

NO: 6 (FIG. 32A);

CDRH1 sequence, SEQ ID NO: 10 (fig. 32B);

CDR H2 sequence, AIYPGNGXTSYNQKFKG, wherein X is D or A (SEQ ID NO: 37),

for example, SEQ ID NO: 11 (fig. 32B); and

CDR H3 sequence, VVVYYSXXYWYFDV, wherein X at position 6 is N, A, Y, W or D and X at position 7 is S or R (SEQ ID NO: 38), e.g. SEQ ID NO: 12 (fig. 32B).

Humanized 2H7 antibodies herein include those in which the heavy chain amino acid sequence comprises a C-terminal lysine and those in which it does not. The above CDR sequences are typically present in the human light chainAnd heavy chain variable region framework sequences, such as substantially human light chain kappa subgroup I (V)_LKappa I) and substantially human heavy chain subgroup III (V)_HIII) human consensus FR residues. See also WO 2004/056312(Lowman et al.).

The heavy chain variable region may be linked to a human IgG chain constant region, wherein the region may be, for example, IgG1 or IgG3, including both native and non-native sequence constant regions.

In a preferred embodiment, such antibodies comprise SEQ ID NO: 8 (v16, as shown in figure 32B), optionally further comprising the heavy chain variable domain sequence of SEQ ID NO: 2 (v16, as shown in figure 32A), optionally comprising one or more amino acid substitutions at positions 56, 100 and/or 100a in the heavy chain variable domain, e.g. D56A, N100A or N100Y, and/or S100aR and one or more amino acid substitutions at positions 32 and/or 92 in the light chain variable domain, e.g. M32L and/or S92A. Preferably, the antibody is a whole antibody comprising SEQ ID NO: 13 or 30 and the light chain amino acid sequence of SEQ id no: 14. 15, 29, 31, 34 or 39, the amino acid sequence of the heavy chain of SEQ ID NO: 39, or a sequence of the sequence of (1).

A preferred humanized 2H7 antibody is ocrelizumab (Genentech, Inc.).

The antibodies herein may also comprise at least one amino acid substitution in the Fc region that improves ADCC activity, such as amino acid substitutions at positions 298, 333, and 334, preferably S298A, E333A, and K334A, using EU heavy chain residue numbering. See also U.S. Pat. No.6,737,056, L.Presta.

Any of these antibodies may comprise at least one substitution in the Fc region that improves FcRn binding or serum half-life, for example a substitution at position 434 of the heavy chain, such as N434W. See also U.S. Pat. No.6,737,056, L.Presta.

Any of these antibodies may further comprise at least one amino acid substitution in the Fc region that increases CDC activity, for example comprising at least a substitution at position 326, preferably K326A or K326W. See also U.S. Pat. No.6,528,624 (Idusogene et al.).

Some preferred humanized 2H7 variants are those comprising SEQ ID NO: 2 and the light chain variable domain of SEQ ID NO: 8, including those with or without substitutions in the Fc region (if any), and those comprising a heavy chain variable domain of SEQ ID NO: 8 has a change of N100A; or D56A and N100A; or the heavy chain variable domains of D56A, N100Y and S100 aR and the amino acid sequence set forth in SEQ ID NO: 2 with change M32L; or S92A; or the light chain variable domains of M32L and S92A.

M34 in the 2h7.v16 heavy chain variable domain has been identified as a potential source of antibody stability and as another potential candidate for substitution.

In a summary of some of the various preferred embodiments of the invention, the variable region of the 2h7.v 16-based variant comprises the amino acid sequence of v16, except for the positions of the amino acid substitutions shown in the table below. Unless otherwise indicated, the 2H7 variants will have the same light chain as v 16.

Exemplary humanized 2H7 antibody variants

2H7 type	Heavy chain (V)_H) Variations in	Light chain (V)_L) Variations in	Fc changes
				16 for reference			-
31	-	-	S298A，E333A，K334A
				73	N100A	M32L
75	N100A	M32L	S298A，E333A，K334A
				96	D56A，N100A	S92A
114	D56A，N100A	M32L，S92A	S298A，E333A，K334A
				115	D56A，N100A	M32L，S92A	S298A，E333A，K334A，E356D，M358L
116	D56A，N100A	M32L，S92A	S298A，K322A，K334A
				138	D56A，N100A	M32L，S92A	S298A，K326A，E333A，K334A
477	D56A，N100A	M32L，S92A	S298A，K326A，E333A，K334A，N434W
				375	-	-	K334L
588	-	-	S298A，K326A，E333A，K334A
				511	D56A，N100Y，S100aR	M32L，S92A	S298A，K326A，E333A，K334A

One preferred humanized 2H7 comprises the 2H7.v16 light chain variable domain sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKR(SEQ ID NO：2)；

and 2h7.v16 heavy chain variable domain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSS(SEQ ID NO：8)。

if the humanized 2h7.v16 antibody is an intact antibody, it may comprise the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC(SEQ ID NO：13)；

and heavy chain amino acid sequence SEQ ID NO: 14 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPG (SEQ ID NO：15)。

another preferred humanized 2H7 antibody comprises the 2H7.v511 light chain variable domain sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKR(SEQ IDNO：39)；

and 2h7.v511 heavy chain variable domain sequence:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSS(SEQ ID NO：40)。

if the humanized 2h7.v511 antibody is an intact antibody, it may comprise the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC(SEQ ID NO：30)；

and heavy chain amino acid sequence SEQ ID NO: 31 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG(SEQ ID NO：41)。

see fig. 38 and 39, which align the mature light and heavy chains of humanized 2h7.v511 with humanized 2h7.v16, respectively, the heavy chain using C-terminal lysine sequences.

If the humanized 2h7.v31 antibody is an intact antibody, it may comprise the light chain amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC(SEQ ID NO：13)；

and heavy chain amino acid sequence SEQ ID NO: 15 or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPG(SEQ ID NO：42)

or:

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPS SKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG(SEQ ID NO：43)。

in a preferred embodiment herein, the antibody is a monoclonal antibody comprising SEQ ID NO: 2 and 8 (version 16). In another preferred embodiment herein, the antibody is a monoclonal antibody comprising SEQ ID NO: 39 and 40 variable domain sequences (version 511). Further preferred antibodies are antibodies comprising SEQ ID NO: 32 and 33 (see fig. 40 version 114), such as a humanized 2H7 comprising a light chain variable domain SEQ ID NO: 32 and the heavy chain variable domain SEQ ID NO: 34. Further preferred antibodies are those comprised in SEQ ID NO: 8 has a change of N100A; or D56A and N100A; or D56A, N100Y, and S100aR and the light chain variable domain of SEQ ID NO: change M32L in 2; or S92A; or humanized 2H7 of the light chain variable domain of M32L and S92A.

"antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell, followed by causing lysis of the target cell. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravech and Kinet, annu.rev.immunol.9: 457-492(1991) page 464, page 3 summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. patent No.5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, pnas (usa) 95: 652-.

"human effector cells" refer to leukocytes which express one or more fcrs and which exert effector function. Preferably, the cells express at least Fc γ RIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), Natural Killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, preferably PBMC and NK cells.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see e.g. seqAnnu, rev, immunol.15: 203-234(1997)). For a review of fcrs see ravech and Kinet, annu. 457-492 (1991); capel et al, immunolmethods 4: 25-34 (1994); de Haas et al, j.lab.clin.med.126: 330-341(1995). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117: 587 (1976); Kim et al, J.Immunol.24: 249 (1994)).

"complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to solubilize a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) that complexes with a cognate antigen. To assess complement activation, CDC assays can be performed, for example, as described in Gazzano-Santoro et al, j.immunol.methods 202: 163 (1996).

"growth inhibitory" antibodies are those antibodies that prevent or reduce the proliferation of cells expressing the antigen to which the antibody binds. For example, the antibody can prevent or reduce B cell proliferation in vitro and/or in vivo.

Antibodies that "induce apoptosis" refer to those that induce programmed cell death (of e.g., B cells) according to the assays of standard apoptosis assays, such as annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell rupture, and/or membrane vesicle (referred to as apoptotic bodies) formation.

"native antibody" refers to a heterotetrameric glycoprotein of about 150,000 daltons, typically 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, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end _H) Followed by a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And the other end is a constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form the interface between the light and heavy chain variable domains.

The term "variable" refers to the fact that certain portions of the variable domains differ widely between antibody sequences and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β -sheet conformation, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and together with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of immunological interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in ADCC.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having an antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced an F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to form V_H-V_LAn antigen binding site is defined on the surface of the dimer. The six hypervariable regions together confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

The Fab fragment also comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant domain carry at least one free thiol group. F (ab') ₂Antibody fragments were originally generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), depending on the amino acid sequence of their constant domains.

Antibodies can be classified into different classes according to the amino acid sequence of their heavy chain constant domains. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"Single chain Fv" or "scFv" antibody fragments comprise the V of an antibody_HAnd V_LDomains, wherein the domains are present on a single polypeptide chain. Preferably, the Fv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains to enable the scFv to form the desired structure for binding to an antigen. For reviews on scFv see Pl ü ckthun, in: the Pharmacology of Monoclonal Antibodies, vol.113, eds., Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments are in the same polypeptide chain (V)_H-V_L) Comprising a linked heavy chain variable domain (V)_H) And a light chain variable domain (V)_L). By using linkers that are too short to allow pairing between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; hollinger et al, proc.natl.acad.sci.usa 90: 6444-6448(1993).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, with the exception of possible variants that may arise during the course of production of the monoclonal antibody, which variants are typically present in minimal amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the invention may be produced by monoclonal antibodies originally produced by Kohler et al, Nature 256: 495(1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. patent No.4,816,567). "monoclonal antibodies" can also be used, for example, as described in Clackson et al, Nature 352: 624-: the technique described in 581-597(1991) was isolated from phage antibody libraries.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, such as baboon, rhesus Monkey, or macaque) and human constant region sequences (U.S. patent No.5,693,780).

"humanized" forms of non-human (e.g., murine) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for the FR substitutions described above. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. See Jones et al, Nature 321: 522-525 (1986); riechmann et al, Nature 332: 323-329 (1988); presta, curr, op, struct, biol.2: 593-596(1992).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g.residues 24-34(L1), 50-56(L2) and 89-97(L3) in the light chain variable domain and residues 31-35(H1), 50-65(H2) and 95-102(H3) in the heavy chain variable domain; Kabat et al, Sequences of Proteins of immunological interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md 1991)) and/or those from the "hypervariable loops" (e.g.residues 26-32(L1), 50-52(L2) and 91-96(L3) in the light chain variable domain and residues 26-32(H1), 53-55(H2) and 96-96 (H3J 917J.; Biooth 901: Biooth: 901) in the heavy chain variable domain). "framework region" or "FR" residues refer to those residues in the variable domain other than the hypervariable region residues defined herein.

"naked antibody" or "naked antibody" refers to an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety, a polymer, or a radiolabel.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions and staining with Coomassie blue or preferably silver. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, an isolated antibody will typically be prepared by at least one purification step.

"joint damage" is used in the broadest sense and refers to damage or partial or complete destruction of any portion of one or more joints, including connective tissue and cartilage, where damage includes structural and/or functional damage of any cause and may or may not cause joint pain/arthralgia. It includes, but is not limited to, joint damage associated with or caused by inflammatory as well as non-inflammatory joint diseases. The injury may be caused by any condition, such as autoimmune diseases, such as lupus (e.g., systemic lupus erythematosus), arthritis (e.g., acute and chronic arthritis, rheumatoid arthritis including juvenile rheumatoid arthritis, Juvenile Idiopathic Arthritis (JIA), or juvenile ra (jra), and stages such as rheumatoid synovitis, gout or gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, collagen II-induced arthritis, infectious arthritis, septic arthritis, lyme arthritis, proliferative arthritis, psoriatic arthritis, stigmatic disease, spondyloarthritis, osteoarthritis, chronic progressive arthritis (arthritis progrientendosis), osteoarthritis (arthritis deformans), chronic primary polyarthritis (polyarthritis chronomania), reactive arthritis, menopausal arthritis, and menopausal arthritis, Estrogen-wasting arthritis, and ankylosing spondylitis/rheumatoid spondylitis), rheumatic autoimmune diseases other than RA, major systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis or Fisher's syndrome), Sjogren's syndrome, specific secondary syndromes, secondary restrictive cutaneous vasculitis with RA, seronegative spondyloarthropathies, Lyme disease, inflammatory bowel disease, scleroderma, inflammatory myopathy, mixed connective tissue disease, any overlapping syndrome, bursitis, tendonitis, osteomyelitis, infectious diseases including influenza, measles (rubella), rheumatic fever, Epstein-Barr disease virus syndrome, hepatitis, parotitis, rubella (German measles), and chicken pox (fowl pox), patellar chondromalacia, collagenosis, collagen colitis, autoimmune disorders related to collagen disease, inflammatory diseases including rheumatoid arthritis, rheumatoid arthritis, Joint inflammation, abnormal exertion or overuse such as sprains or strains, injuries including bone fractures, gout, especially found in the big toe, and caused by neurological disorders, hemophilia disorders (e.g., hemophiliac arthropathy), muscle disorders, progressive disorders, bone disorders, cartilage disorders, and vascular disorders. For purposes herein, the points of contact between the various components of the bones of the joint fingers (vertebrate, such as an animal) and the parts that surround and support it include, but are not limited to, for example, the hip, the joints between the vertebrae of the spine, the joints between the spine and the pelvis (sacroiliac joints), the joints where tendons and ligaments attach to bones, the joints between ribs and the spine, the shoulders, knees, feet, elbows, hands, fingers, ankles and toes, particularly the joints in the hands and feet.

"subject" herein refers to a human subject, including a patient suitable for receiving treatment, who is experiencing or has experienced one or more signs, symptoms, or other indicators of joint damage, has been diagnosed with joint damage, whether newly diagnosed or previously diagnosed, for example, and is now experiencing a reoccurrence or recurrence, or is at risk of developing joint damage, regardless of the cause. The subject may or may not have been previously treated with the CD20 antibody. Subjects eligible to receive treatment for joint damage can optionally be identified as subjects who have been screened for elevated levels of infiltrating CD20 cells, like in his blood, or screened using an assay that detects autoantibodies, wherein the production of autoantibodies is assessed qualitatively and preferably quantitatively. The subject may have not been treated with, for example, an immunosuppressive agent such as methotrexate at "baseline" (i.e., a set point prior to administration of the first dose of CD20 antibody in the treatment methods herein, such as the day of screening the subject prior to initiation of treatment), i.e., the subject has not been previously treated with, for example, an immunosuppressive agent such as methotrexate. Such subjects are generally considered candidates for treatment with the second drug.

"treatment" of a subject refers herein to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with joint damage as well as those in need of prevention of joint damage or progression of joint damage. Thus, the subject may have been diagnosed as having joint damage or may be predisposed or susceptible to joint damage, or may have limited joint damage that is likely to progress without treatment. The treatment is successful if the joint damage is alleviated or cured, or the progression of the joint or structure damage is halted or slowed as compared to before the administration. Successful treatment also includes complete or partial prevention of the occurrence/development of joint damage. For purposes herein, slowing or reducing joint damage or the progression of joint damage is equivalent to blocking, reducing, or reversing joint damage.

"clinical improvement" refers to preventing further progression of joint damage or any improvement in joint damage due to treatment, as determined by methods other than radiographic examination. As such, clinical improvement may be determined, for example, by assessing the number of tender or swollen joints, psoriasis assessment severity index, subject global clinical assessment, assessing erythrocyte sedimentation rate, or assessing the amount of C-reactive protein levels.

For purposes herein, a subject is in "remission" if he/she has no symptoms of active joint damage, such as those detectable by the methods disclosed herein, and does not progress according to an assessment of joint damage at baseline or at some point during treatment. Those not in remission include, for example, those subjects experiencing worsening or progression of joint damage. Subjects experiencing a recurrence of symptoms, including active joint injury, refer to those with "relapse" or "re-onset".

"symptoms" of joint damage refers to any pathological phenomena or deviation from normal in the structure, function, or sensation experienced by the subject, which is indicative of joint damage, such as those listed above, including tender or swollen joints.

The expression "effective amount" refers to an amount of an antibody or antagonist effective to treat joint damage, including an amount effective to achieve a reduction in joint damage as determined by a radiographic examination as compared to baseline prior to administration of the amount. An effective amount of an additional agent, such as a second agent, refers to an amount of the agent effective to treat joint damage or other adverse effects, including side effects or symptoms or other conditions that accompany joint damage, including underlying diseases or disorders.

"total modified Sharp score" refers to the use of Genant, am.J.Med., 30: 35-47(1983) improved evaluation of the scores obtained from radiographs according to Sharp. The primary assessment was the change in Sharp-gent total scores from the screen. The Sharp-gent score combines the hand and foot erosion score and the joint space narrowing score. Joint damage was measured in these tests and scored by a score at which the mean change was less than baseline (the time the patient was screened or tested prior to the first administration of the CD20 antagonist).

As used herein, "rheumatoid arthritis" refers to a recognized disease state that can be diagnosed according to the 2000 revised american rheumatoid association standards for the classification of rheumatoid arthritis or any similar standard. Physiological indicators of RA include symmetric joint swelling, which is characteristic, although not constant, in rheumatoid arthritis. Spindle-shaped swelling of the Proximal Interphalangeal (PIP) joints of the hand, as well as the Metacarpophalangeal (MCP), wrist, elbow, knee, ankle, and Metatarsophalangeal (MTP) joints, is often diseased and readily detectable. Pain during passive exercise is the most sensitive test for joint inflammation, and inflammation and structural deformation often limit the range of motion of the affected joint. Typical visible changes include ulnar deviation of the fingers at the MCP joint, hyperextension or hyper-flexion of the MCP and PIP joints, flexion contracture of the elbow, and subluxation of the carpal bones and toes. Rheumatoid arthritis subjects may tolerate DMARDs, i.e., DMARDs are not or not fully effective in treating symptoms. In addition, candidates for therapy according to the present invention include those subjects who have experienced an inadequate response to previous or current therapy with TNF inhibitors such as etanercept, infliximab, and/or adalimumab due to toxicity or inadequate therapeutic efficacy (e.g., etanercept for 3 months, 25 mg, twice weekly, or infliximab infused at 3mg/kg for at least 4 times). Patients with "active rheumatoid arthritis" refer to patients with active and non-latent symptoms of rheumatoid arthritis. Subjects with "early active rheumatoid arthritis" refer to those with active rheumatoid arthritis confirmed diagnosis for at least 8 weeks but not longer than 4 years according to the 1987ACR criteria for correction of RA classification.

Psoriatic arthritis (PsA) refers to an inflammatory joint disease characterized by extensive bone resorption. It is also disclosed herein that blood samples from PsA patients, particularly those with bone erosion on plain radiographs, exhibit a significant increase in osteoclast precursor (OCP) compared to healthy controls.

"antibody exposure" refers to contact or exposure to one or more doses of an antibody herein administered over a period of about 1 day to about 5 weeks. The dose may be administered once, or at fixed or irregular intervals over the exposure period, such as, for example, one dose per week for four weeks or two doses separated by a period of about 13-17 days. As detailed herein, the primary and subsequent antibody exposures are temporally spaced from each other.

Not applying or providing an exposure until "from the initial exposure" or a period of time from any prior exposure means measuring the time of a second or subsequent exposure from the time any dose of a prior exposure was applied if more than one dose was applied in the exposure. For example, if two doses are administered in a first exposure, then a second exposure is not administered until at least about 16-54 weeks from the time of the first or second dose administered within the prior exposure. Similarly, if three doses are administered, a second exposure may be measured at the time of the first, second, or third dose within the first exposure. Preferably, the time from the first dose is measured "from initial exposure" or from any prior exposure.

The term "immunosuppressive agent" as used herein in connection with adjuvant therapy refers to a substance that acts to suppress or mask the immune system of the mammal being treated herein. This would include substances that inhibit cytokine production, down-regulate or inhibit autoantigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No.4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir (ganciclovir), tacrolimus (tacrolimus), glucocorticoids such as cortisol (cortisol) or aldosterone (aldosterone), anti-inflammatory agents such as cyclooxygenase inhibitors, 5-lipoxygenase inhibitors or leukotriene receptor antagonists; purine antagonists, such as azathioprine (azathioprine) or Mycophenolate Mofetil (MMF); alkylating agents such as cyclophosphamide (cyclophosphamide), bromocriptine (bromocriptine), danazol (danazol), dapsone (dapsone), glutaraldehyde (which masks MHC antigens, as described in U.S. patent No.4,120,649); anti-idiotypic antibodies to MHC antigens and MHC fragments; cyclosporine A; steroids, such as corticosteroids or glucocorticosteroids or glucocorticoid analogues, e.g. prednisone (prednisone), methylprednisolone (methylprednisone) including SOLU-MEDROL Methylprednisolone sodium succinate, and dexamethasone (dexamethasone); dihydrofolate reductase inhibitors, such as methotrexate (oral or subcutaneous); antimalarial agents such as chloroquine (chloroquine) and hydroxychloroquine (hydroxychloroquine); sulfasalazine (sulfasalazine); leflunomide (leflunomide); cytokine antagonists such as cytokine antibodies or cytokine receptor antibodies, including anti-interferon-alpha, -beta or-gamma antibodies, anti-Tumor Necrosis Factor (TNF) -alpha antibody (infliximab) (REMICADE)) Or ADamascenan antibody (adalimumab)), anti-TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-2 (IL-2) antibodies and anti-IL-2 receptor antibodies, and anti-interleukin-6 (IL-6) receptor antibodies and antagonists; anti-LFA-1 antibodies, including anti-CD 11a and anti-CD 18 antibodies; anti-L3T 4 antibody; heterologous anti-lymphocyte globulin; pan (pan) T antibodies, preferably anti-CD 3 or anti-CD 4/CD4a antibodies; a soluble peptide comprising the LFA-3 binding domain (WO 90/08187, published at 90 years, 7 months, 26 days); a streptokinase; transforming growth factor-beta (TGF-beta); a streptococcal enzyme; RNA or DNA from a host; FK 506; RS-61443; chlorambucil (chlorambucil); deoxyspergualin (deoxyspergualin); rapamycin (rapamycin); t cell receptors (Cohen et al, U.S. patent No.5,114,721); t cell receptor fragments (Offner et al, Science 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); WO 91/01133); BAFF antagonists, such as BAFF antibodies and BR3 antibodies and zTNF4 antagonists (for review see Mackay and Mackay, Trends immunol.23: 113-5 (2002)); biologics that interfere with T helper cell signaling, such as anti-CD 40 receptor or anti-CD 40 ligand (CD154), including blocking antibodies to CD40-CD40 ligand (e.g., Durie et al, Science, 261: 1328-30 (1993); Mohan et al, J.Immunol., 154: 1470-80(1995)) and CTLA4-Ig (Finck et al, Science, 265: 1225-7 (1994)); and T cell receptor antibodies (EP 340,109), such as T10B 9. Some immunosuppressive agents herein are also DMARDs, such as methotrexate. Examples of preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

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 (IL), such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, including PROLEUKINrIL-2; tumor necrosis factor, such as TNF-alpha 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 natural sequence cytokines, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof. "cytokine antagonist" refers to a molecule that inhibits or antagonizes such cytokines by any mechanism, including, for example, antibodies to cytokines, antibodies to cytokine receptors, and immunoadhesins.

The term "hormone" refers to a polypeptide hormone, which is typically secreted by glandular organs with ducts. Hormones include, for example, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; (ii) a relaxin; estradiol; hormone replacement therapy; androgens such as carotinoid (calusterone), dromostanolonenepropionate (dromostanolone), epitioandrostane (mepitiostane), or testolactone (testolactone); a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); prolactin, placental lactogen, mouse gonadotropin-related peptides, gonadotropin-releasing hormone; a statin; an activin; mullerian (Mullerian) inhibitory substances; and thrombopoietin. As used herein, the term hormone includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence hormones, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

The term "growth factor" refers to a protein that promotes growth, including, for example, liver growth factor; fibroblast growth factor; vascular endothelial growth factor; nerve growth factors such as NGF-beta; platelet-derived growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); osteoinductive factor (osteoinductive factor); interferons such as interferon- α, - β, and- γ; and Colony Stimulating Factors (CSFs), such as macrophage CSFs (M-CSF), granulocyte-macrophage CSFs (GM-CSF), and granulocyte CSFs (G-CSF). As used herein, the term growth factor includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence growth factors, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

The term "integrin" refers to a receptor protein that allows cells to bind and respond to the extracellular matrix and is involved in a variety of cellular functions such as wound healing, cell differentiation, tumor cell homing, and apoptosis. They are part of a large family of cell adhesion receptors involved in cell-extracellular matrix and cell-cell interactions. Functional integrins consist of two transmembrane glycoprotein subunits, called α and β, that are non-covalently associated. The alpha subunits share some homology with each other, as do the beta subunits. Receptors always contain one alpha chain and one beta chain. Examples include α 6 β 1, α 3 β 1, α 7 β 1, LFA-1, and the like. As used herein, the term "integrin" includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the natural sequence integrins, including small molecule entities produced by artificial synthesis, and pharmaceutically acceptable derivatives and salts thereof.

For the purposes of the present invention, "tumor necrosis factor-alpha (TNF-alpha)" is intended to include Pennica et al, Nature 312: 721(1984) or Aggarwal et al, JBC 260: 2345(1985) human TNF- α molecule of the amino acid sequence set forth in (i) 5. "TNF- α inhibitor" as used herein refers to an agent that inhibits to some extent the biological function of TNF- α, typically by binding to TNF- α and neutralizing its activity. An example of a TNF inhibitor specifically contemplated herein is etanercept (ENBREL)) Infliximab (REMICADE), infliximab (REMICADE)) And Adalimumab (HUMIRA)^TM)。

Examples of "disease modifying antirheumatic drugs" or "DMARDs" include hydroxychloroquine (hydroxychloroquine), sulfasalazine (sulfasalazine), methotrexate (methotrexate), leflunomide (leflunomide), etanercept (etanercept), infliximab (infliximab) (plus oral and subcutaneous methotrexate), azathioprine (azathioprine), D-penicillamine, gold salts (oral), gold salts (intramuscular), minocycline (minocycline), cyclosporine (cyclosporine) including cyclosporine a and topical cyclosporine, staphylococcal protein a (Goodyear and silverer, j.exp.med.197 (9): 1125-39(2003), including salts and derivatives thereof, and the like. The preferred DMARD herein is methotrexate.

Examples of "non-steroidal anti-inflammatory drugs" or "NSAIDs" include aspirin (aspirin), acetylsalicylic acid (acetylsalicylic acid), ibuprofen (ibuprofen), flurbiprofen (flurbiprofen), naproxen (naproxen), indomethacin (indomethacin), sulindac (sulindac), tolmetin (tolmetin), phenylbutazone (phenylbutazone), diclofenac (diclofenac), ketoprofen (ketoprofen), benorilate (benorilate), mefenamic acid (mefenamic acid), methotrexate (methotrexate), fenbufen (fenbufen), azapropazone (axazopapazone), COX-2 inhibitors such as celecoxib (celecoxib)(ii) a 4- (5- (4-tolyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl) benzenesulfonamide, valdecoxib (BEXTRA)) Meloxicam (meloxicam) (MOBIC)) GR 253035(Glaxo Wellcome), and MK966(Merck Sharp)&Dohme), including salts and derivatives thereof, and the like. Preferably, they are aspirin, naproxen, ibuprofen, indomethacin or tolmetin.

In this textExamples of a catenin antagonist or antibody "include LFA-1 antibodies, such as efalizumab (RAPTIVA) available from Genentech) Or alpha 4 integrin antibodies, such as natelizumab (ANTEGREN) available from Biogen ) Or diazacyclophenylalanine derivatives (WO2003/89410), phenylalanine derivatives (WO 2003/70709, WO 2002/28830, WO2002/16329 and WO 2003/53926), phenylpropionic acid derivatives (WO 2003/10135), enamine derivatives (WO 2001/79173), propionic acid derivatives (WO 2000/37444), alkanoic acid derivatives (WO2000/32575), substituted phenyl derivatives (U.S. Pat. Nos. 6,677,339 and 6,348,463), aromatic amine derivatives (U.S. Pat. No.6,369,229), ADAM disintegrin domain polypeptides (U.S. Pat. No. 2002/0042368), antibodies to α v β 3 integrin (EP 633945), nitrogen bridged bicyclic amino acid derivatives (WO 2002/02556), and the like.

"corticosteroid" refers to any of several synthetic or naturally occurring substances that have the general chemical structure of steroids, mimicking or enhancing the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone (prednisone), prednisolone (prednisone) (including methylprednisolone), such as SOLU-MEDROLMethylprednisolone sodium succinate), dexamethasone (dexamethasone) or dexamethasone triamcinolone (dexamethasone triamcinolone), hydrocortisone (hydrocortisone) and betamethasone (betamethasone). Preferred corticosteroids herein are prednisone, methylprednisolone, hydrocortisone or dexamethasone.

The terms "BAFF," BAFF polypeptide, "" TALL-1 "or" TALL-1 polypeptide, "and" BLyS "as used herein encompass" native sequence BAFF polypeptides "and" BAFF variants. "BAFF" is used to refer to those polypeptides having any one of the amino acid sequences shown below, and homologs and fragments and variants thereof that have the biological activity of native BAFF:

human BAFF sequence (SEQ ID NO: 16):

1 MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALLSCC

61 LTVVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEPPAP

121 GEGNSSQNSRNKRAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEE

181 KENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETL

241 PNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL

mouse BAFF sequence (SEQ ID NO: 17):

1 MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLAATLLLALLSS

61 SFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPELTAGVKLLTPAAPRPHNSSR

121 GHRNRRAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNIIQDCLQLIADSDTP

181 TIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFIYSQVLYTDPIFAMGHVIQRKK

241 VHVFGDELSLVTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRNGDD

301 TFFGALKLL

the biological activity of BAFF may be selected from the group consisting of promoting B cell survival, promoting B cell maturation, and binding to BR 3. BAFF variants preferably have at least 80% or any consecutive integer up to 100% of the native sequence of a BAFF polypeptide, including more preferably at least 90%, even more preferably at least 95% amino acid sequence identity.

"native sequence" BAFF polypeptides include polypeptides having the same amino acid sequence as a corresponding BAFF polypeptide derived from nature. For example, BAFF exists in a soluble form after cleavage from the cell surface with a furin-type protease. Such native sequence BAFF polypeptides may be isolated from nature, or may be produced by recombinant and/or synthetic means.

The term "native-sequence BAFF polypeptide" or "native BAFF" specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide. The term "BAFF" includes those polypeptides described in the following documents: shu et al, j.leukocyte biol., 65: 680 (1999); GenBank accession No. AF 136293; WO 1998/18921, published on 5/7/1998; EP 869,180, published in 1998 at 10 months and 7 days; WO 1998/27114, published at 25.6.1998; WO 1999/12964, published on 18.3.1999; WO 1999/33980, published on 7/8/1999; moore et al, Science, 285: 260-263 (1999); schneider et al, j.exp.med., 189: 1747 — 1756 (1999); mukhopadhyay et al, j.biol.chem., 274: 15978-15981(1999).

The term "BAFF antagonist" is used in the broadest sense as used herein and includes any of the molecules described below: (1) binds to a native-sequence BAFF polypeptide or to native-sequence BR3 thereby partially or completely blocking the interaction of BR3 with a BAFF polypeptide, and (2) partially or completely blocking, inhibiting or neutralizing the activity of native-sequence BAFF. In a preferred embodiment, the BAFF receptor to be blocked is the BR3 receptor. Native BAFF activity promotes B cell survival and/or B cell maturation, among others. In one embodiment, inhibition, blocking, or neutralization of BAFF activity results in a reduction in B cell number. BAFF antagonists according to the invention will partially or completely block, inhibit or neutralize one or more biological activities of a BAFF polypeptide in vitro and/or in vivo. In one embodiment, biologically active BAFF potentiates any one or combination of the following events in vitro and/or in vivo: increased B cell survival, increased levels of IgG and/or IgM, increased plasma cell numbers, and processing of NF-. kappa.b 2/100 to p52 NF-. kappa.b in splenic B cells (e.g., Batten et al, J.Exp. Med.192: 1453. sup. 1465 (2000); Moore et al, Science 285: 260. sup. 263 (1999); Kayagaki et al, Immunity 17: 515. sup. 524 (2002)).

As described above, BAFF antagonists may act in a direct or indirect manner to partially or completely block, inhibit or neutralize BAFF signaling in vitro or in vivo. For example, a BAFF antagonist can bind BAFF directly. For example, such BAFF antibodies are contemplated: they bind to a region of human BAFF comprising residues 162-275 and/or residues adjacent to residues selected from residues 162, 163, 206, 211, 231, 233, 264 and 265 of human BAFF such that the antibody sterically blocks BAFF binding to BR3, wherein the residue numbering is according to SEQ ID NO: 16. in another example, a direct binder is a polypeptide comprising any BAFF-binding portion of a BAFF receptor, such as the extracellular domain of a BAFF receptor, or fragments and variants thereof that bind native BAFF. In another example, BAFF antagonists include polypeptides having a sequence comprising a polypeptide having a sequence represented by formula I:

X₁-C-X₃-D-X₅-L-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-C-X₁₄-X₁₅-X₁₆-X₁₇

(formula I) (SEQ ID NO: 18)

Wherein X₁、X₃、X₅、X₇、X₈、X₉、X₁₀、X₁₁、X₁₂、X₁₄、X₁₅And X₁₇Is any amino acid other than cysteine; and

wherein X₁₆Is an amino acid selected from L, F, I and V; and is

Wherein the polypeptide does not contain a cysteine within 7 amino acid residues from the N-terminal side of the N-most cysteine C and within 7 amino acid residues from the C-terminal side of the C-most cysteine C of formula I.

In one embodiment, the polypeptide comprising a sequence of formula I has a disulfide bondTwo connected C; x₅LX₇X₈Conformation forming a type I beta turn structure in which the turn is centered at L and X₇To (c) to (d); and X₈Dihedral angle of(phi) has a positive value. In one embodiment, X₁₀Selected from W, F, V, L, I, Y, M and a non-polar amino acid. In another embodiment, X₁₀Is W. In another embodiment, X₃Is an amino acid selected from M, V, L, I, Y, F, W and a non-polar amino acid. In another embodiment, X₅Selected from V, L, P, S, I, A and R. In another embodiment, X₇Selected from V, T, I and L. In another embodiment, X₈Selected from R, K, G, N, H and D-amino acids. In another embodiment, X₉Selected from H, K, A, R and Q. In another embodiment, X₁₁Is I or V. In another embodiment, X₁₂Selected from P, A, D, E and S. In another embodiment, X₁₆Is L. In a particular embodiment, the sequence of formula I is a sequence selected from ECFDLLVRAWVPCSVLK (SEQ ID NO: 19), ECFDLLVRHWVPCGLLR (SEQ ID NO: 20), ECFDLLVRRWVPCEMLG (SEQ ID NO: 21), ECFDLLVRSWVPCHMLR (SEQ ID NO: 22), ECFDLLVRHWVACGLLR (SEQ ID NO: 23) and QCFDRLNAWVPCSVLK (SEQ ID NO: 24). In a preferred embodiment, the BAFF antagonist comprises a sequence selected from SEQ ID NO: 19. 20, 21, 22 and 23.

In another example, BAFF antagonists include polypeptides having a sequence comprising a polypeptide having a sequence of formula II:

X₁-C-X₃-D-X₅-L-V-X₈-X₉-W-V-P-C-X₁₄-X₁₅-L-X₁₇

(formula II) (SEQ ID NO: 25)

Wherein X₁、X₃、X₅、X₈、X₉、X₁₄、X₁₅And X₁₇Is any amino acid other than cysteine; and is

Wherein the polypeptide does not contain a cysteine within 7 amino acid residues from the N-terminal side of the N-most cysteine C and within 7 amino acid residues from the C-terminal side of the C-most cysteine C of formula II.

In one embodiment, the polypeptide comprising the sequence of formula II has a disulfide bond between two cs; having X₅LX₇X₈Conformation forming a type I beta turn structure in which the turn is centered at L and X₇To (c) to (d); and X₈Dihedral angle ofHas a positive value. In another embodiment of formula II, X₃Is an amino acid selected from M, A, V, L, I, Y, F, W and a non-polar amino acid. In another embodiment of formula II, X₅Selected from V, L, P, S, I, A and R. In another embodiment of formula II, X₈Selected from R, K, G, N, H and D-amino acids. In another embodiment of formula II, X₉Selected from H, K, A, R and Q.

In yet another embodiment, the BAFF receptor from which the extracellular domain or BAFF-binding fragment or BAFF-binding variant thereof is derived is TACI, BR3, or BCMA. Alternatively, a BAFF antagonist may bind to the extracellular domain of native sequence BR3 at its BAFF binding region, thereby partially or completely blocking, inhibiting, or neutralizing BAFF binding to BR3 in vitro, in situ, or in vivo. For example, such indirect antagonists are anti-BR 3 antibodies as described below: it binds to the region of BR3 comprising residues 23-38 of human BR3(SEQ ID NO: 26) as defined below, or to the vicinity of these residues, thereby sterically hindering the binding of human BR3 to BAFF.

In some embodiments, BAFF antagonists according to the invention include BAFF antibodies and immunoadhesins comprising BAFF receptor extracellular domains or fragments and variants thereof that bind native BAFF. In yet another embodiment, the BAFF receptor from which the extracellular domain or BAFF-binding fragment or BAFF-binding variant thereof is derived is TACI, BR3, or BCMA. In another embodiment, the immunoadhesin comprises an amino acid sequence of formula I or formula II as described above, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19. 20, 21, 22, 23 and 24.

According to one embodiment, the BAFF antagonist binds to a BAFF polypeptide or BR3 polypeptide with a binding affinity of 100nM or less. According to another embodiment, the BAFF antagonist binds to a BAFF polypeptide or BR3 polypeptide with a binding affinity of 10nM or less. According to another embodiment, the BAFF antagonist binds to a BAFF polypeptide or BR3 polypeptide with a binding affinity of 1nM or less.

The terms "BR 3", "BR 3 polypeptide" or "BR 3 receptor", as used herein, encompass "native sequence BR3 polypeptide" and "BR 3 variants" (which are further defined herein). "BR 3" is used to refer to those polypeptides comprising the amino acid sequence shown below and homologs thereof, and variants or fragments thereof that bind native BAFF:

Human BR3 sequence (SEQ ID NO: 26):

1 MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQ

61 ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD

121 KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAG

181 PEQQ

the BR3 polypeptides of the invention may be isolated from a variety of sources, for example from a human tissue type or from another source, or prepared by recombinant and/or synthetic methods. The term BR3 includes the BR3 polypeptides described in WO 2002/24909 and WO 2003/14294.

"native sequence" BR3 polypeptide or "native BR 3" includes polypeptides having the same amino acid sequence as the corresponding BR3 polypeptide derived from nature. Such native sequence BR3 polypeptides may be isolated from nature or may 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., extracellular domain sequences), naturally occurring variant forms (e.g., alternatively spliced forms), and naturally occurring allelic variants of the polypeptide. The BR3 polypeptides of the invention include BR3 polypeptides: which comprises or consists of the contiguous sequence of amino acid residues 1 to 184 of human BR3(SEQ ID NO: 26).

BR3 "extracellular domain" or "ECD" refers to a form of BR3 polypeptide that is substantially free of transmembrane and cytoplasmic domains. The ECD form of BR3 includes polypeptides comprising any of the amino acid sequences selected from amino acids 1-77, 2-62, 2-71, 1-61, 7-71, 23-38 and 2-63 of human BR 3. The present invention contemplates such BAFF antagonists: they are polypeptides comprising any of the above ECD forms of human BR3 and variants and fragments thereof that bind native BAFF.

Mini (Mini) BR3 refers to the 26 residue core region of the BAFF binding domain of BR3, i.e. the amino acid sequence: TPCVPAECFD LLVRHCVACG LLRTPR (SEQ ID NO: 27).

By "BR 3 variant" is meant a BR3 polypeptide having at least about 80% amino acid sequence identity to the native sequence full length BR3 or BR3ECD and which binds to the native sequence BAFF polypeptide. Optionally, the BR3 variant comprises a single cysteine-rich domain. Such BR3 variant polypeptides include, for example, BR3 polypeptides in which one or more amino acid residues are added or deleted at the N-terminus and/or C-terminus and in one or more internal domains of the full-length amino acid sequence. Fragments of BR3ECD that bind to native sequence BAFF polypeptides are also contemplated. According to one embodiment, the BR3 variant polypeptide has 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 to a human BR3 polypeptide or a specific fragment thereof (e.g., ECD), 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. BR3 variant polypeptides do not encompass native BR3 polypeptide sequences. According to another embodiment, a BR3 variant polypeptide is 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.

In a preferred embodiment, the BAFF antagonist herein is an immunoadhesin comprising BR3, TACI or BCMA or a variant thereof that binds to BAFF. In other embodiments, the BAFF antagonist is a BAFF antibody. A "BAFF antibody" is an antibody that binds to BAFF, preferably in the region of human BAFF comprising residues 162-275 of the human BAFF sequence disclosed herein under the definition of "BAFF" (SEQ ID NO: 16). In another embodiment, the BAFF antagonist is a BR3 antibody. A "BR 3 antibody" is an antibody that binds BR3, preferably an antibody that binds BR3 in the region of human BR3 comprising residues 23-38 of the human BR3 sequence disclosed herein under the definition of "BR 3" (SEQ ID NO: 26). In general, the amino acid positions of human BAFF and human BR3 described herein are according to the sequence numbering of human BAFF and human BR3 (i.e., SEQ ID NOs: 16 and 26, respectively) as disclosed herein under the definition of "BAFF" and "BR 3".

Other examples of BAFF binding polypeptides or BAFF antibodies can be found in, for example, WO 2002/092620; WO 2003/014294; gordon et al, Biochemistry 42 (20): 5977-5983 (2003); kelleyeet al, j.biol.chem.279 (16): 16727-; WO 1998/18921; WO 2001/12812; WO 2000/68378; WO 2000/40716.

"package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding indications, usage, dosage, administration, contraindications, other therapeutic products associated with the packaged product, and/or warnings relating to the use of such therapeutic products.

"drug" or "agent" refers to an active agent that treats joint damage or symptoms or side effects thereof.

Treatment of

In one aspect, the invention provides a method of treating joint damage in a subject, such as a patient, comprising administering to the subject an antagonist, preferably an antibody, more preferably a CD20 antibody, that binds to a B cell surface marker and assessing whether the subject exhibits reduced joint damage after treatment by radiographic x-ray.

Thus, the invention contemplates a method for treating joint damage in a subject comprising administering to the subject an antagonist that binds a B cell surface marker and, at least about one month after the administration, giving the subject a radiographic test to measure a reduction in joint damage as compared to baseline prior to the administration, wherein the amount of antagonist administered is effective in achieving a reduction in joint damage, indicating that the subject has been successfully treated for joint damage.

The invention also contemplates a method for treating joint damage in a subject comprising administering to the subject an antibody that binds a B cell surface marker and, at least about one month after the administration, giving the subject a radiographic test to measure a reduction in joint damage as compared to baseline prior to the administration, wherein the amount of antibody administered is effective in achieving a reduction in joint damage, indicating that the subject has been successfully treated for joint damage.

The invention further contemplates a method for treating joint damage in a subject comprising administering to the subject a CD20 antibody and, at least about one month after the administration, giving the subject a radiographic test to measure a reduction in joint damage as compared to baseline prior to the administration, wherein the amount of CD20 antibody administered is effective in achieving a reduction in joint damage, indicating that the subject was successfully treated for joint damage.

In a preferred embodiment, radiographic testing after administration of the antagonist or antibody, such as the CD20 antibody, occurs at least about two months, more preferably at least about 10 weeks, still more preferably at least about three months, further preferably at least about four months, still more preferably at least about five months, further more preferably at least about 24 weeks, or at least about six months, and most preferably at least about 52 weeks after administration of the antagonist or antibody. In another preferred embodiment, the test measures the corrected Sharp total score.

In another preferred embodiment, the subject is retreated in that the method further comprises administering to the subject an antagonist or antibody, such as a CD20 antibody, in an amount effective to achieve a sustained or sustained reduction in joint damage as compared to the effect of prior administration of the antagonist or antibody, such as a CD20 antibody. Thus, a second dose of antagonist or antibody can be administered to the subject and evaluated by radiographic examination at least about one month (preferably more than about two months, more preferably about 24 weeks or about 6 months) after the second dose to determine whether the second dose is effective (i.e., administered an effective amount of antagonist or antibody) to maintain the effect of the first dose or to promote a reduction in joint damage as compared to the effect of the first dose. This re-treatment regimen may be repeated as long as needed or necessary to achieve or maintain a reduction in joint damage, which is indicative of successful treatment of the joint damage.

In another embodiment, an antagonist or antibody, such as the CD20 antibody, is additionally (continuously) administered to the subject even if the subject has no clinical improvement upon a radiographic examination following a prior administration, such as a first administration of the antagonist or antibody. In the latter embodiment, it is preferred that the clinical improvement is determined by assessing the number of tender or swollen joints, psoriasis assessment severity index, overall clinical assessment of the subject, assessing erythrocyte sedimentation rate, or assessing the amount of C-reactive protein levels.

For purposes of the present invention, a second antagonist or antibody exposure for re-treatment refers to the next treatment of the subject with an antagonist or, e.g., a CD20 antibody after the initial antibody exposure, with no intervening antagonist or, e.g., a CD20 antibody treatment or exposure between the initial exposure and the second exposure. Such re-treatment may be scheduled or unscheduled, but is preferably scheduled for re-administration, particularly for the protection of organs such as the kidney from injury. If an antibody, particularly a CD20 antibody, is used, it is preferred that the second antibody exposure be about 0.5 to 4 grams, more preferably about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5 grams, and that the second exposure not be provided until about 20 to 35 weeks (preferably about 23 to 30 weeks, more preferably about 23 to 28 weeks) from the initial exposure.

The method contemplates administering to the subject an effective amount of an antagonist or, for example, a CD20 antibody to provide a third antagonist or antibody exposure (more preferably a CD20 antibody, if an antibody is present), preferably about 0.5 to 4 grams, more preferably about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5 grams, until about 46 to 60 weeks (preferably about 46 to 55 weeks, more preferably about 46 to 52 weeks) from the initial exposure does not provide the third exposure. Preferably, no further antagonist or antibody exposure is provided until at least about 70-75 weeks from the initial exposure, and still more preferably, no further antagonist or antibody exposure is provided until about 74 to 80 weeks from the initial exposure.

Where antibodies are employed, any one or more antibody exposures herein can be provided to a subject in the form of a single dose of antibody or multiple doses of antibody, e.g., 1-4 doses of antibody (e.g., consisting of first and second, or first, second, and third, or first, second, third, and fourth, etc.). The specific number of doses (whether one, two, three, or more) employed per antibody exposure depends, for example, on the type of joint damage being treated, the type of antibody employed, whether the second drug described below is employed, what type of second drug described below is employed, and how much of the second drug described below is employed, and the method and frequency of administration. In the case of multiple doses, the subsequent dose (e.g., second or third dose) is preferably administered from about 1 to 20 days, more preferably from about 6 to 16 days, and most preferably from about 14 to 16 days from the time the previous dose is administered. The multiple doses are preferably administered over a total period of between about 1 day and 4 weeks, more preferably between about 1 and 20 days (e.g., over a period of 6-18 days). In one such aspect, the multiple doses are administered about once a week, the second dose is administered about one week from the first dose, and any third or subsequent dose is administered about one week from the second dose. Preferably, the multiple doses are about 0.5 to 1.5 grams, more preferably about 0.75 to 1.3 grams, of antibody per dose.

In a most preferred embodiment, a method of treating joint damage in a subject is provided comprising administering to the subject an effective amount of an antibody that binds to a B cell surface marker (e.g., a CD20 antibody) to provide an initial antibody exposure followed by a second antibody exposure, wherein the second exposure is not provided until about 16 to 54 weeks from the initial exposure and each antibody exposure is provided to the subject as a single dose or as two or three doses of antibody. Preferably, in such a method, the antibody exposure is about 0.5 to 4 grams per time, most preferably in the amounts given above.

In one embodiment, the subject is provided with at least about three antibody exposures, for example, about 3 to 60 exposures, more specifically about 3 to 40 exposures, and most specifically about 3 to 20 exposures. Preferably, such exposure is administered at intervals of 24 weeks. In one embodiment, each antibody exposure is provided as a single dose of antibody. In an alternative embodiment, each antibody exposure is provided as multiple doses of antibody. However, not every antibody exposure need be provided in a single dose or in multiple doses.

In a preferred embodiment, about 2-3 grams of the CD20 antibody is administered as an initial exposure. If about 3 grams is administered, about 1 gram of the CD20 antibody is administered once a week for about three weeks as an initial exposure. If about 2 grams of CD20 antibody is administered as an initial exposure, then about 1 gram of CD20 antibody is administered, followed by another about 1 gram of antibody about two weeks later as an initial exposure. In a preferred aspect, the second exposure is about 24 weeks or six months from the initial exposure and an amount of about 2 grams is administered. In another preferred aspect, the second exposure is about 24 weeks or six months from the initial exposure and about 1 gram of CD20 antibody is administered followed by another about 1 gram of antibody about two weeks later.

In a preferred embodiment of the multiple exposure method herein, the subject is in remission after initial or any later antagonist or antibody exposure. More preferably, the multiple exposure methods herein involve a re-administration or re-treatment that is scheduled such that the subject is in remission when a second, and preferably all, antagonist or antibody exposure is provided. Such re-administration is arranged to prevent any recurrence, re-onset or organ damage, rather than to treat it therapeutically. Most preferably, the subject is in remission for at least about 24 weeks or six months, still most preferably at least about nine months, and even still most preferably at least about 52 weeks or a year from the last antagonist or antibody exposure used in the method of treatment.

In yet another embodiment, the subject is treated with at least two antagonist or antibody exposures, preferably each antagonist or antibody exposure with the same antagonist or antibody, such as a CD20 antibody. Thus, the initial and second antagonist or antibody exposures preferably use the same antagonist or antibody, more preferably all antagonist or antibody exposures use the same antagonist or antibody, i.e., the first two exposures, preferably all exposures are treated with one type of antagonist or antibody that binds to a B cell surface marker, such as a CD20 antibody, e.g., both rituximab or both the same humanized 2h 7.

In all of the methods listed herein, the antagonist (e.g., CD20 or B cell surface marker antibody) may be unconjugated, such as a naked antibody, or may be conjugated with another molecule for increased effectiveness, such as, for example, to increase half-life. Preferred CD20 antibodies herein are chimeric, humanized, or humanCD20 antibody, more preferably rituximab, humanized 2H7 (e.g.comprising the variable domain sequences in SEQ ID NOS: 2 and 8, or comprising the variable domain sequences in SEQ ID NOS: 39 and 40, or comprising the variable domain sequences in SEQ ID NOS: 32 and 33, or comprising the heavy chain variable domain having the alterations N100A, or D56A + N100A, or D56A + N100Y + S100aR in SEQ ID NO: 8 and the light chain variable domain having the alterations M32L, or S92A, or M32L + S92A in SEQ ID NO: 2), chimeric or humanized A20 antibodies (Immunomedics), HUMAX-CD20^TMHuman CD20 antibody (Genmab), or a single chain protein that binds to CD20 (trubiun Pharm Inc.). More preferred is rituximab or humanized 2H 7.

In another embodiment of all of the methods herein, the subject has never been previously treated with one or more drugs, such as with an anti-TNF- α inhibitor, e.g., an anti-TNF- α or anti-TNF- α receptor antibody, for example, to treat arthritis, or with an immunosuppressive agent to treat joint damage or underlying cause such as an autoimmune disorder, and/or has never been previously treated with an antagonist (e.g., an antibody) against a B cell surface marker (e.g., has never been previously treated with a CD20 antibody). In such an embodiment, the subject has never been previously treated with: anti- α 4 integrin antibodies or co-stimulatory modulators, biological agents, DMARDs other than MTX, in addition to azathioprine and/or leflunomide, cell-depleting therapies including investigational agents (e.g., camp, anti-CD 4, anti-CD 5, anti-CD 3, anti-CD 19, anti-CD 11a, anti-CD 22, or BLys/BAFF), live/attenuated vaccines (within 28 days prior to baseline), or intra-articular or parenteral glucocorticoids (within 4 weeks prior to baseline).

In yet another aspect, the subject may have had a relapse of joint injury or suffered an organ injury such as a kidney injury prior to receiving treatment by any of the methods described above, including after initial or later antagonist or antibody exposure. However, it is preferred that the subject does not have a recurrence of the joint injury, and more preferably, such a recurrence is not present at least prior to the initial treatment.

In another embodiment, the antagonist (e.g., CD20 antibody) is the only drug administered to the subject to treat joint damage. In another embodiment, the antagonist (e.g., CD20 antibody) is one of the drugs used to treat joint damage. In yet another embodiment, the subject is free of malignancies, including solid tumors, hematologic malignancies, or carcinoma in situ (except for basal cell and squamous cell carcinoma of the skin that has been excised and healed). In yet another embodiment, the subject is free of Rheumatoid Arthritis (RA). In another aspect, the subject does not have a rheumatic autoimmune disease other than RA, or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis, or fischer-tropsch syndrome). In another embodiment, the subject has secondary sjogren's syndrome or secondary restrictive cutaneous vasculitis. In another embodiment, the subject does not have a functional level IV as defined by the ACR classification of RA functional state. In yet another embodiment, the subject is free of inflammatory joint diseases other than RA (including but not limited to gout, reactive arthritis, psoriatic arthritis, seronegative spondyloarthropathies, lyme disease), or other systemic autoimmune disorders (including but not limited to systemic lupus erythematosus, inflammatory bowel disease, scleroderma, inflammatory myopathy, mixed connective tissue disease, or any overlapping syndrome). In another embodiment, the subject does not have Juvenile Idiopathic Arthritis (JIA) or juvenile RA (jra) and/or RA by 16 years of age. In another embodiment, the subject is free of significant and/or uncontrolled cardiac or pulmonary diseases (including obstructive pulmonary disease), or significant concomitant diseases, including but not limited to neurological, renal, hepatic, endocrine, or gastrointestinal disorders, and is also free of primary or secondary immunodeficiency (historical or currently active), including a known history of HIV infection. In another aspect, the subject is free of any neurological (congenital or acquired), vascular or systemic disorder, particularly joint pain and swelling (e.g., parkinson's disease, cerebral palsy, diabetic neuropathy) that may affect any assessment of efficacy. In yet another embodiment, the subject is not multiple sclerosis. In yet another embodiment, the subject does not have lupus or sjogren's syndrome. In yet another embodiment, the subject does not have an autoimmune disease. In yet another aspect of the invention, the joint damage is not associated with or at risk of developing an autoimmune disease other than arthritis. For the purposes of this last statement, an "autoimmune disease" herein refers to a disease or disorder or co-segregation (co-segregate) or manifestation thereof or condition resulting therefrom that originates from and is directed against an individual's own tissues or organs. Without being bound to any one theory, B cells exhibit pathogenic effects in human autoimmune diseases through a number of mechanistic pathways, including autoantibody production, immune complex formation, dendritic cell and T cell activation, cytokine synthesis, direct chemokine release, and provision of nests (nidus) for ectopic neo-lymphogenesis. Each of these pathways is involved in the pathology of autoimmune diseases to varying degrees.

In a preferred embodiment, the joint damage is caused by: arthritis, sterile joint loosening of orthopedic implants, nonunion of fractures, spondyloarthropathies, psoriasis, or crohn's disease. More preferably, the joint damage is caused by arthritis, and more preferably rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, or psoriatic arthritis.

In a further embodiment, if the antagonist is an antibody, then the antibody is administered intravenously or subcutaneously.

In a further preferred aspect, if the antagonist is an antibody, the antibody is administered in a dose of about 0.4 to 4 grams, more preferably about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5 grams. In another aspect, the antibody is preferably administered in a dose of about 0.4 to 1.3 grams at a frequency of one to four doses over a period of about one month, more preferably about 500 mg to 1.2 grams, still more preferably about 500 mg or about 750 mg to about 1.1 grams, and more preferably the antibody is administered in two to three doses. Still more preferably, the antibody is administered over a period of about 2 to 3 weeks.

In another preferred aspect, the subject is rheumatoid factor negative. In another aspect, the subject is rheumatoid factor positive.

In another preferred aspect of the methods described above, methotrexate is administered to the subject prior to baseline or prior to initiation of treatment. More preferably, methotrexate is administered in one dose of about 10-25 mg/week. Also, it is preferred that methotrexate is administered at least about 12 weeks prior to baseline, and still more preferred that a stable dose of methotrexate is administered for the last 4 weeks prior to baseline. In other embodiments, methotrexate is administered orally or parenterally.

In particularly preferred embodiments of the methods described above, the joint damage is caused by rheumatoid arthritis and the subject exhibits inadequate response to one or more anti-Tumor Necrosis Factor (TNF) inhibitors, and/or methotrexate is administered to the subject with the antagonist, e.g., CD20 antibody, and/or the antagonist is CD20 antibody administered intravenously at a dose of approximately 1000 milligrams x 2 on days 1 and 15 of the start of treatment.

In yet another embodiment, the invention provides a method of monitoring treatment for joint damage in a subject comprising administering to the subject an effective amount of a B cell surface marker antagonist (such as an antibody, including a CD20 antibody), and measuring by radiography after at least about one month from the administration whether joint damage is reduced relative to baseline prior to the administration, wherein a reduction from baseline in the subject after treatment indicates that the antagonist or antibody, such as a CD20 antibody, is having an effect on joint damage. Preferably, the degree of reduction from baseline is measured again after administration of an antagonist or antibody, such as a CD20 antibody. And, preferably, at least about 24 weeks after the administration.

The invention also includes a method of monitoring treatment of joint damage in a subject comprising administering to the subject an effective amount of a B cell surface marker antagonist (such as an antibody, including a CD20 antibody), and measuring by radiography after at least about 52 weeks from the administration whether joint damage is reduced relative to baseline prior to the administration, wherein a reduction from baseline in the subject after treatment indicates that the antagonist or antibody, such as CD20 antibody, is having an effect on joint damage. Preferably, the degree of reduction from baseline is measured again after administration of an antagonist or antibody, such as a CD20 antibody.

In yet another aspect, the invention provides a method of determining whether to continue administering a B cell surface marker antagonist (such as an antibody, including a CD20 antibody) to a subject having joint damage, comprising measuring by radiography a reduction in joint damage in the subject after a first administration of the antagonist, such as a CD20 antibody, measuring by radiography a reduction in joint damage in the subject after a second administration of the antagonist, such as a CD20 antibody, comparing the radiographic scores of the subject at the first and second times, and continuing administration of the antagonist or antibody, such as a CD20 antibody, if the second time score is less than the first time.

In yet another embodiment, the method of treatment comprises the step of testing the subject's response to treatment after the administering step for determining whether the level of response is effective for treating joint damage. For example, a step of testing the radiography score after administration and comparing it with a baseline radiography score obtained before administration is included for determining whether the treatment is effective by measuring whether and how much the change occurred. This test may be repeated at various scheduled or unscheduled intervals following administration for the purpose of determining the maintenance of any partial or complete regression. Alternatively, the methods herein comprise the step of testing the subject prior to administration for the presence or absence of one or more biomarkers or symptoms of joint damage, as listed above. In another method, a step of examining the medical history of the subject prior to administering the antibody or antagonist to the subject can be included, as detailed above, e.g., for the exclusion of infection or malignancy as a cause, e.g., a leading cause, of a condition in the subject. Preferably, the joint injury is primary (e.g., primary disease) and not secondary, e.g., secondary to an infection or malignancy, whether a solid or liquid tumor.

In one embodiment of all of the methods herein, no other drug is administered to the subject in addition to the antagonist, such as the CD20 antibody, to treat joint damage.

In any of the methods herein, it is preferred that an effective amount of a second drug (wherein the antagonist or antibody that binds a B cell surface marker (e.g., CD20 antibody) is the first drug) can be administered to the subject along with the antagonist or antibody that binds a B cell surface marker. The second agent may be one or more agents including, for example, an immunosuppressive agent, a cytokine antagonist such as a cytokine antibody, a growth factor, a hormone, an integrin antagonist or antibody, or any combination thereof. The type of such second drug depends on a variety of factors, including the type of joint injury, the severity of the joint injury, the condition and age of the subject, the type and dose of the first drug employed, and the like.

Examples of such additional drugs include interferon-based drugs such as interferon-alpha (e.g., from Amarillo Biosciences, Inc.), IFN-beta-1 a (REBIF)And AVONEX) Or IFN-beta-1 b (Betaseron)) Oligopeptides such as glatiramer acetate (COPAXONE)) Agents that block the CD40-CD40 ligand, immunosuppressive agents (e.g., mitoxantrone (NOVANTRONE) ) Methotrexate, cyclophosphamide, chlorambucil, leflunomide, and azathioprine), intravenous immunoglobulin (gamma globulin), gonorrheaBasocyte-depleting therapies (e.g. mitoxantrone, cyclophosphamide, CAMPATH)^TMAntibodies, anti-CD 4, cladribine, polypeptide constructs having at least two domains comprising de-immunized (de-immunized) autoreactive antigens or fragments thereof specifically recognized by an Ig receptor of an autoreactive B cell (WO 2003/68822), whole body irradiation, bone marrow transplantation), integrin antagonists or antibodies (e.g., LFA-1 antibodies such as efalizumab/RAPTIVA available from GenentechOr alpha 4 integrin antibodies such as natalizumab/ANTEGREN available from BiogenOr other antibodies described above), agents for treating secondary or related symptoms of joint injury such as those described herein, steroids such as corticosteroids (e.g., prednisolone, methylprednisolone such as SOLU-MEDROL)^TMMethylprednisolone sodium succinate, prednisone such as low-dose prednisone, dexamethasone, or glucocorticoids for injection, e.g., via joint injection, including systemic corticosteroid therapy, non-lymphocyte-depleting immunosuppressive therapy (e.g., MMF or cyclosporine), "statin/statin" (statin) cholesterol-lowering drugs (including cerivastatin) (BAYCOL) ^TM) Fluvastatin (LESCOL)^TM) Atorvastatin (LIPITOR)^TM) Lovastatin (MEVACOR)^TM) Pravastatin (pravastatin) (pravacachol)^TM) And simvastatin (simvastatin) (ZOCOR)^TM) Estradiol, testosterone (optionally elevated dose) (Stuve et al neurology 8: 290 (2002)), androgens, hormone replacement therapy, TNF inhibitors such as antibodies to TNF- α, DMARDs, NSAIDs, plasmapheresis or plasma exchange, trimethoprim-sulfamethoxazole (BACTRIM)^TM，SEPTRA^TM) Mycophenolate mofetil, H2-blockers or proton pump inhibitors (during immunosuppressive therapy with potential ulcerogenicity), levothyroxine, cyclosporin A (e.g. Sandomone)) A somatostatin analogue, DMARD or NSAID, cytokine antagonist such as an antibody, antimetabolite, immunosuppressant, reconstructive surgery, radioiodine, thyroidectomy, BAFF antagonist such as BAFF or BR3 antibody or immunoadhesin, anti-CD 40 receptor or anti-CD 40 ligand (CD154), anti-IL-6 receptor antagonist/antibody, another B cell surface antagonist or antibody such as humanized 2H7 or other humanized or human CD20 antibody with rituximab; IL-1 blockers, such as rHUIL-1Ra (Amgen-Synergen) and tiaprofenic acid I-1B inhibitors (Hoechst); and co-stimulatory modifiers, such as ORENCIA (abatacept) (Bristol-MyersSquibb); enromomab (enlimomab) (anti-ICAM-1 monoclonal antibody); CDO-855 (humanized antibody, which specifically binds to a region of MHC class II complex, Celltech); CH-3298 (Chiroscience); acemetacin (Merck); GW353430 (anti-CD 23 monoclonal antibody, Glaxo Wellcome); GR 252025(COX02 inhibitor, Glaxo Wellcome); 4162W94 (anti-CD 4 humanized antibody; Glaxo Wellcome); azathioprine (DMARD, glaxowelome); penicillamine and fenoprofen (fenoprofen) (Eli Lilly); and so on.

Preferred such drugs are antibiotics, anti-integrins, gamma globulin, pain control agents, integrin antagonists, anti-CD 4, cladribine, trimethoprim-sulfamethoxazole, H2 blockers, proton pump inhibitors, cyclosporines, statin/statin cholesterol-lowering drugs, estradiol, testosterone, androgens, hormone replacement drugs, TNF inhibitors such as TNF-alpha inhibitors, DMARDs, NSAIDs (for treating symptoms such as muscle bone), levothyroxine, cyclosporin A, somatostatin (somatastatin) analogs, cytokine antagonists (including cytokine receptor antagonists), antimetabolites, BAFF antagonists such as BAFF antibodies or BR3 antibodies (especially BAFF antibodies), immunosuppressive agents such as methotrexate or corticosteroids, bisphosphonates (bisphosphates), hormones, and another B cell surface marker antibody, a cytotoxic agent, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, a pharmaceutically acceptable, Such as rituximab in combination with humanized 2H7 or other humanized CD20 antibodies.

More preferred such drugs are antibiotics, immunosuppressants such as methotrexate or corticosteroids, DMARDs, pain control agents, integrin antagonists, NSAIDs, cytokine antagonists, bisphosphonate compounds, or hormones, or combinations thereof.

In a particularly preferred embodiment, the second drug is a DMARD, which is preferably selected from the group consisting of auranofin, chloroquine, D-penicillamine, injectable gold, oral gold, hydroxychloroquine, sulfasalazine, thiomycorisin, and methotrexate.

In another such embodiment, the second drug is an NSAID, which is preferably selected from the group consisting of bedsteadin, mesalazine, amsaceae, codeine phosphate, benorilate, fenbufen, naproxen, diclofenac, etodolac and indomethacin, aspirin and ibuprofen.

In another such embodiment, the second drug is a pain control agent, preferably selected from acetaminophen and dextropropoxyphene.

In yet another such embodiment, the second drug is an immunosuppressive agent, preferably selected from the group consisting of etanercept, infliximab, adalimumab, leflunomide, anakinra, azathioprine, methotrexate, and cyclophosphamide.

In other preferred embodiments, the second agent is selected from the group consisting of OPG, etanercept, infliximab, etanercept, adalimumab, kinaret, raptiva, Osteoprotegerin (OPG), RANKFc, anti-RANKL, pamidronate, alendronate, actone, zoledronate, clodronate, methotrexate, azulfidine, hydroxychloroquine, doxycycline, leflunomide, sulfasalazine (SSZ), prednisolone, interleukin-1 receptor antagonist, prednisone, and methylprednisolone.

In a further preferred embodiment, the second drug is selected from the group consisting of infliximab, infliximab + Methotrexate (MTX) combination, etanercept, corticosteroid, cyclosporin a, azathioprine, auranofin, Hydroxychloroquine (HCQ), prednisolone + MTX + SSZ combination, MTX + SSZ + HCQ combination, cyclophosphamide + azathioprine + HCQ combination, adalimumab + MTX combination. If the second drug is a corticosteroid, it is preferred that it is prednisone, prednisolone, methylprednisolone, hydrocortisone, or dexamethasone. Also, it is preferred that the amount of corticosteroid administered is lower than the amount in the absence of administration of the CD20 antibody to a subject treated with a corticosteroid. Most preferably, the second drug is methotrexate.

All of these second drugs may be used in combination with each other or alone with the first drug, and thus the expression "second drug" as used herein does not mean that it is the only drug other than the first drug. Thus, the second medicament need not be one medicament, but may comprise more than one such agent.

These second drugs listed herein are typically used at the same dosages and routes of administration as used above or at about 1-99% of the dosages used so far. If such second drugs are indeed used, they are preferably used in lower amounts than in the absence of the first drug, especially in subsequent administrations after the initial administration of the first drug, in order to eliminate or reduce the side effects caused thereby.

For the method of retreatment described herein, wherein the second agent is administered in an effective amount with an antagonist or antibody exposure, it can be administered with any exposure, e.g., with only 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 yet another embodiment, the second medicament is administered with all exposures. Preferably, the amount of such second drug is reduced or eliminated after the initial exposure (such as to a steroid) to reduce the subject's exposure to agents having side effects such as prednisone, prednisolone, methylprednisolone, and cyclophosphamide.

The combined administration of the second medicament includes co-administration (simultaneous administration) using separate formulations or a single pharmaceutical formulation, as well as sequential administration in either order, wherein preferably there is a period of time during which both (or more) active agents (medicaments) exert their biological activity simultaneously.

The antibody or antagonist herein is administered by any suitable means, including parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration. Parenteral infusion includes intramuscular, intravenous (i.v.), intraarterial, intraperitoneal or subcutaneous administration. Membrane/intrathecal administration is also envisaged (see e.g. US2002/0009444, Grillo-Lopez, A cosmetic intravenous delivery of a CD20 antibody). In addition, the antibody or antagonist may suitably be administered by pulsed infusion, for example with decreasing doses of the antibody or antagonist. Intravenous or subcutaneous administration is preferred, and intravenous infusion is more preferred.

If multiple antibody exposures are provided, each exposure may be provided using the same or different means of administration. In one embodiment, each exposure is administered intravenously. In another embodiment, each exposure is given by subcutaneous administration. In yet another embodiment, the exposure is given by both intravenous and subcutaneous administration.

In one embodiment, the CD20 antibody is administered as a slow intravenous infusion rather than as an intravenous bolus or as a rapid injection. For example, a steroid such as prednisolone or methylprednisolone (e.g., about 80-120 mg intravenously, more preferably about 100 mg intravenously) is administered about 30 minutes prior to any infusion of the CD20 antibody. The CD20 antibody is infused, for example, by a dedicated line.

Such infusions are preferably initiated at a rate of about 50 mg/hour for the initial dose of multiple doses of CD20 antibody exposure, or for a single dose where the exposure comprises only one dose. This may be increased in steps, for example, at a rate of about 50 mg/hour every about 30 minutes up to a maximum of about 400 mg/hour. However, if the subject is experiencing an infusion-related reaction, the infusion rate is preferably reduced, for example, to half the current rate, for example from 100 mg/hour to 50 mg/hour. Preferably, infusion of such agents (e.g., about 1000 mg total dose) of the CD20 antibody is completed in about 255 minutes (4 hours and 15 minutes). Preferably, the subject receives prophylactic treatment orally of acetaminophen/acetaminophen (e.g., about 1 gram) and diphenhydramine HCl (e.g., about 50 mg or an equivalent dose of a similar agent) about 30 to 60 minutes prior to initiation of infusion.

If more than one CD20 antibody infusion is given to complete the entire exposure, then preferably in this infusion embodiment a second or subsequent CD20 antibody infusion is initiated at a higher rate than the initial infusion, e.g., about 100 mg/hour. This rate may be increased stepwise, for example at a rate of about 100 mg/hour every about 30 minutes up to a maximum of about 400 mg/hour. Subjects who have an infusion-related response preferably reduce the infusion rate to half that rate, for example from 100 mg/hour to 50 mg/hour. Preferably, infusion of such second or subsequent doses of CD20 antibody (e.g., about 1000 mg total dose) is completed over about 195 minutes (3 hours 15 minutes).

In another embodiment, a method of treating joint damage in a subject is provided comprising administering to the subject a B cell surface marker antagonist such as an antibody, e.g., a CD20 antibody, and, at least about 52 weeks after the administration, giving the subject a radiographic test to measure a reduction in joint damage compared to baseline prior to the administration, wherein the amount of antagonist or antibody, e.g., CD20 antibody, administered is effective in achieving a reduction in joint damage, indicating that the subject was successfully treated.

In this method, preferably, the test measures the total modified Sharp score. In another preferred embodiment, the antagonist is a CD20 antibody. More preferably, the CD20 antibody is rituximab, or a polypeptide comprising SEQ ID NO: 2 and 8, or a humanized 2H7 comprising the variable domain sequence of SEQ ID NO: 39 and 40 or a humanized 2H7 comprising the variable domain sequence of seq id NO: 32 and 33, or a humanized 2H7 comprising the variable domain sequence in SEQ ID NO: 8, and a light chain variable domain having changes N100A, or D56A + N100A, or D56A + N100Y + S100aR and the amino acid sequence set forth in SEQ ID NO: 2, humanized 2H7 with a light chain variable domain that alters M32L, or S92A, or M32L + S92A.

In another preferred embodiment, the joint damage is caused by arthritis, preferably RA, more preferably early active RA. In another preferred embodiment, the subject has never been previously treated with an immunosuppressive agent prior to administering the first dose of antagonist or antibody, such as the CD20 antibody, in this method of treatment. In a preferred embodiment, the antagonist or antibody is administered in one dose of about 0.4 to 4 grams, more preferably, the antagonist or antibody is administered in one dose of about 0.4 to 1.3 grams, at a frequency of one to four doses over a period of about one month. Still more preferably, the agent is from about 500 mg to 1.2 g, and in other embodiments, from about 750 mg to 1.1 g. In such aspects, the antagonist or antibody is preferably administered in two to three doses, and/or over a period of about 2 to 3 weeks.

In another aspect, such methods further comprise re-treating the subject by providing to the subject an additional administration of an antagonist, such as a CD20 antibody, in an amount effective to achieve a sustained or sustained reduction in joint damage as compared to the effect of a prior administration of the antagonist or antibody, such as a CD20 antibody. In one aspect of this embodiment, the antagonist or antibody, such as the CD20 antibody, is additionally administered to the subject even if the subject has no clinical improvement upon radiographic testing following prior dosing. The re-treatment may begin at least about 24 weeks after the first administration of the antagonist, such as the CD20 antibody, and optionally begin one or more further re-treatments. In another embodiment, the further re-treatment begins at least about 24 weeks after the second administration of the antagonist, such as the CD20 antibody. In yet another preferred aspect, the joint damage after the re-treatment is reduced as compared to the degree of joint damage after the first radiographic evaluation.

Preferably, in this method, which is evaluated for about 52 weeks, an effective amount of a second drug is administered, wherein an antagonist or antibody, such as a CD20 antibody, is the first drug. In one aspect, the second medicament is more than one medicament. In another aspect, the second drug is an antibiotic, an immunosuppressant, a disease modifying anti-rheumatic drug (DMARD), a pain control agent, an integrin antagonist, a non-steroidal anti-inflammatory drug (NSAID), a cytokine antagonist, a bisphosphonate, or a hormone, or a combination thereof, most preferably methotrexate. The subject may be rheumatoid factor negative or positive. Also, it is preferred that antagonists such as CD20 antibodies are administered intravenously or subcutaneously, most preferably intravenously.

Methods of generating, modifying, and formulating such antibodies are discussed below.

Production of antibodies

The methods and articles of the invention use or comprise antibodies that bind to B cell surface markers, particularly antibodies that bind to CD 20. Accordingly, methods for generating such antibodies will be described herein.

The CD20 antigen to be used for generating or screening antibodies may be, for example, CD20 in soluble form or a portion thereof comprising the desired epitope. Alternatively, or in addition, cells expressing CD20 on their cell surface may be used to generate or screen antibodies. Other forms of CD20 that may be used to generate antibodies will be apparent to those skilled in the art.

The following description exemplifies a method for producing the antibody used according to the present invention.

(i) Polyclonal antibodies

Polyclonal antibodies are preferably generated in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional or derivatizing reagents, e.g. maleimidobenzoyl sulphosuccinimidyl ester (coupled via cysteine residues), NHydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR (where R and R are¹Are different hydrocarbon groups) it may be useful to couple 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.

Animals are immunized against an antigen, immunogenic conjugate or derivative by mixing, for example, 100 or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. After 1 month, animals were boosted with an initial amount of 1/5-1/10 of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals and the antibody titer of the serum was determined. Animals were boosted until the titer reached a plateau (plateau). Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and/or via a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Also, a coagulant such as alum is suitably used to enhance the immune response.

(ii) 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, with such variants generally being present in minor amounts, except for possible variants that arise during the course of production of the monoclonal antibody. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.

For example, monoclonal antibodies may be used as originally produced by Kohler et al, Nature 256: 495(1975), or can be prepared by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above 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. The lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent HGPRT-deficient cells from growing.

Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma Cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center (San Diego, California, USA) and SP-2 or X63-Ag8-653 cells available from the American Type culture Collection (American Type culture Collection, Rockville, Maryland, USA). Human myeloma and mouse-human heteromyeloma cell lines have also 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., NewYork, 1987).

The culture broth in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies to 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 immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Munson et al, anal. biochem.107: 2201980) was analyzed by Scatchard.

After identification of hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and cultured by standard methods (Goding, monoclonal antibodies: Principles and Practice, pp.59-103, academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors.

Can be purified by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose^TMHydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by the subclones being suitably separated from the culture medium, ascites fluid or serum.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are preferred sources of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as e.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, to obtain synthesis of monoclonal antibodies in the recombinant host cells. A review article on recombinant expression of DNA encoding an antibody in bacteria includes Skerra et al, curr. 256-charge 262(1993) and Pl ü ckthun, Immunol. Revs.130: 151-188(1992).

In yet another embodiment, the method may be selected from the group consisting of using McCafferty et al, Nature 348: 552 (1990) and isolating antibodies or antibody fragments from phage antibody libraries. Clackson et al, Nature 352: 624-: 581-597(1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology 10: 779-. As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequences for the constant domains of the human heavy and light chains (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851(1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

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-binding site of an antibody, to produce a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

In addition, antibodies comprising a variant Fc region with high affinity for Fc γ R may be useful in the treatment of diseases where enhanced efficacy of effector cell function is desired, such as autoimmune diseases, for example as exemplified in US 2005/0037000 and WO 2004/63351 (macrogenetics, inc.

(iii) Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced from a non-human source. 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 performed essentially following the method of Winter and co-workers (Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536(1988)), by replacing the corresponding sequences of human antibodies with hypervariable region sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than the entire human variable domain is replaced with 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 replaced by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, used to construct humanized antibodies is important for reducing antigenicity. The entire library of known human variable domain sequences is screened with the variable domain sequences of rodent antibodies according to the so-called "best-fit" method. The human sequence closest to the rodent sequence is then selected as the human Framework Region (FR) of the humanized antibody (Sims et al, J.Immunol.151: 2296 (1993); Chothia et al, J.mol.biol.196: 901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework can 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)).

More importantly, the antibodies retain high affinity for the antigen and other favorable biological properties after humanization. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to obtain desired antibody characteristics, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.

(iv) Human antibodies

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, the region of the antibody heavy chain junction (J) has been described in chimeric and germline mutant mice_H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of large numbers of human germline immunoglobulin genes in such germline 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, Yeast in immune.7: 33 (1993); U.S. patent nos. 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 generate human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) domain genes from an unimmunized donor. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous phages such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous phage particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying 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 a review see, e.g., Johnson et al, Current Opinion in structural biology 3: 564-571(1993). Several sources of V gene segments are available for phage display. Clackson et al, Nature 352: 624-628(1991) a large number of different anti-oxazolone antibodies were isolated from a random combinatorial library of small V genes derived from the spleen of immunized mice. May essentially follow Marks et, j.mol.biol.222: 581-597(1991) or Griffith et al, EMBO J.12: 725-734(1993) by constructing a V gene repertoire from non-immunized human donors and isolating antibodies against a number of different antigens, including self-antigens. See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies can also be produced by in vitro activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

(v) Antibody fragments

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al, Science 229: 81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from phage antibody libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragment (Carter et al, Bio/Technology 10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures₂And (3) fragment. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent nos. 5,571,894; U.S. Pat. No.5,587,458. Antibody fragments may also be "linear antibodies," for example as described in U.S. Pat. No.5,641,870. Such linear antibody fragments may be monospecific or bispecific.

(vi) Bispecific antibodies

Bispecific antibodies refer to antibodies having binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the CD20 antigen. Other such antibodies may bind CD20 and further bind a second B cell surface marker. Alternatively, an anti-CD 20 binding arm can 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 an Fc receptor (Fc γ R) of IgG, such as Fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD16), such that the cellular defense mechanisms focus on the B cell. Bispecific antibodies can also be used to localize certain agents to B cells. These antibodies have a CD20 binding arm and an arm that binds the agent (e.g., methotrexate). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods for constructing bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature 305: 537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. WO 93/08829 and Traunecker et al, EMBO j.10: 3655-3659(1991) similar procedures are disclosed.

According to a different approach, antibody variable domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. The fusion preferably uses an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, a first constant region of the heavy chain (CH1) is present in at least one of the fusions that includes the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide optimal yields, this provides great flexibility in adjusting the ratios of the three polypeptide fragments to each other. However, where expression of at least two polypeptide chains in the same ratio results in high yields or where the ratio is of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains into the same expression vector.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient way of isolation, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex from the unwanted immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology 121: 210(1986).

According to another approach described in U.S. Pat. No.5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise a C of at least part of the antibody constant domain_H3 domain. In this method, one or more small amino acid side chains of the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be conjugated to avidin and the other to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treating HIV infection (WO 91/00360; WO 92/200373; EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229: 81(1985) describes the proteolytic cleavage of intact antibodies to F (ab')₂Protocol for fragmentation. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Various techniques for the preparation and isolation of bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, leucine zippers have been used to generate bispecific antibodies (Kostelny et al, J.Immunol.148 (5): 1547-. Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Prepared from Hollinger et al, proc.natl.acad.sci.usa 90: 6444-. The fragment comprises heavy chain variable domains (V) connected by a linker _H) And a light chain variable domain (V)_L) The linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single chain fv (sFv) dimers has also been reported. See Gruber et al, j.immunol.152: 5368(1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, j.immunol.147: 60(1991).

Conjugates and other modifications of antibodies

Modifications of the antibodies are contemplated herein. Thus, in one embodiment, the antibody may be conjugated to another molecule, for example to increase half-life or stability or otherwise improve the pharmacokinetics of the antibody. For example, the antibody can be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol. Antibody fragments (such as Fab') linked to one or more PEG molecules are a particularly preferred embodiment of the invention.

The antibodies disclosed herein can also be formulated as liposomes. Antibody-containing liposomes can be prepared by methods known in the art, such as Epstein et al, proc.natl.acad.sci.usa 82: 3688 (1985); hwang et al, proc.natl.acad.sci.usa 77: 4030 (1980); U.S. patent nos. 4,485,045 and 4,544,545; WO 97/38731, published on 23/10/1997. Liposomes with extended circulation times are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be formed by reverse phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a defined pore size to produce liposomes having a desired diameter. Fab' fragments of the antibodies of the invention can be coupled to liposomes via a disulfide exchange reaction, such as Martin et al, j.biol.chem.257: 286 and288 (1982).

Amino acid sequence modifications of the protein or peptide antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody 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 sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, so long as the final construct possesses the desired properties. Amino acid changes can also alter post-translational processing of the antibody, such as changing the number or position of glycosylation sites.

One method that can be used to identify certain residues or regions of an antibody that are preferred mutagenesis positions is referred to as "alanine scanning mutagenesis," e.g., Cunningham and Wells, Science 244: 1081-. Here, a residue or set of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid positions exhibiting functional sensitivity to substitution are then refined by introducing more or other variants at or for the substitution site. Thus, although the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the consequences of a mutation at a given site, alanine scanning mutagenesis or random mutagenesis is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino-and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a polypeptide or polymer. Other insertional variants of the antibody molecule include fusion to the N-or C-terminus of the antibody or polypeptides that extend the serum half-life of the antibody.

Another class of variants are amino acid substitution variants. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. Sites of most interest in antibodies for substitutional mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in the table below under the heading "preferred substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to in the following table as "exemplary substitutions," or as described further below with respect to amino acid species, can be introduced and the products screened.

Original residues	Example alternatives	Preferred alternatives
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Asp；Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
			Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
			Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine	Leu
			Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr

Thr(T)	Val；Ser	Ser
			Trp(W)	TYr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile; leu; met; phe; ala; norleucine	Leu

Substantial modification of antibody biological properties is accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone of the surrogate region, e.g., as a folded sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to their similarity in side chain properties (A.L. Lehninger, Biochemistry, 2 nd edition, pp.73-75, Worth Publishers, New York, (1975)):

(1) Non-polar: ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) Uncharged, polar: gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) Acidic: asp (D), Glu (E)

(4) Basic: lys (K), Arg (R), His (H)

Alternatively, based on common side chain properties, naturally occurring residues can be grouped as follows:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

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

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will entail replacing one member of one of these classes for another.

Any cysteine residue not involved in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred class of substitutional variants involves substituting one or more hypervariable region residues of a parent antibody. In general, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. One convenient method for generating such surrogate variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development.

Another class of amino acid variants of antibodies alters the original glycosylation pattern of the antibody. Such alterations include deletion of one or more carbohydrate moieties found in the antibody, and/or addition of one or more glycosylation sites not present in the antibody.

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 recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by adding or replacing one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. For example, U.S. patent application US 2003/0157108(Presta, L.) describes antibodies with a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody. See also US 2004/0093621(Kyowa Hakko kogyo co., Ltd.). Antibodies having an aliquot of N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are mentioned in WO 03/011878(Jean-Mairet et al) and U.S. Pat. No.6,602,684(Umanaet al). Antibodies having at least one galactose residue in an oligosaccharide attached to the Fc region of an antibody are reported in WO 97/30087(Patel et al). For antibodies with altered carbohydrate attachment to their Fc region see also WO 1998/58964(Raju, S.) and WO 1999/22764(Raju, S.). See also US 2005/0123546(Umana et al) for antigen binding molecules with improved glycosylation.

Preferred glycosylation variants herein comprise an Fc region, wherein the 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 that further improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (Eu residue numbering). Examples of publications relating to "defucose" 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; WO 2005/053742; okazakiet, j.mol.biol.336: 1239-1249 (2004); yamane-ohniki et al, biotech.bioeng.87: 614(2004). Examples of cell lines producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, Arch. biochem. Biophys.249: 533-545 (1986); U.S. patent application No. US 2003/0157108A1, Presta, L; WO 2004/056312A1, Adams et al, especially example 11) and knock-out cell lines such as α -1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (Yamane-Ohnuki et al, Biotech. Bioeng.87: 614 (2004)).

Nucleic acid molecules encoding antibody amino acid sequence variants can be 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 non-variant version of the antibody.

It may be desirable to modify an antibody of the invention with respect to effector function, e.g., to enhance ADCC and/or CDC of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, cysteine residues may be introduced into the Fc region to allow for interchain disulfide bond formation in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and ADCC. See Caron et al, j.exp.med.176: 1191-1195(1992) and shop, J.Immunol.148: 2918-2922(1992). Homodimeric antibodies may also be used as described in Wolff et, Cancer Research 53: 2560, 2565 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, and thus can have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer drug design 3: 219-230(1989).

WO 00/42072(Presta, L.) describes antibodies with improved ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fc region. Preferably, antibodies with improved ADCC comprise substitutions at positions 298, 333 and/or 334 of the Fc region. Preferably, the altered Fc region is a human IgG1Fc region comprising, replacing, or consisting of at one, two, or three of these positions.

Antibodies with altered Clq binding and/or CDC are described in WO 99/51642, U.S. patent No.6,194,551B 1, U.S. patent No.6,242,195B 1, U.S. patent No.6,528,624B 1, and U.S. patent No.6,538,124(Idusogie et al). The antibody comprises an amino acid substitution at one or more of amino acids 270, 322, 326, 327, 329, 313, 333 and/or 334 of its Fc region.

To extend the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment), as described, for example, in U.S. Pat. No.5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., IgG)₁、IgG₂、IgG₃Or IgG₄) Is responsible for extending the serum half-life of the IgG molecule in vivo. WO 00/42072(Presta, L.) describes antibodies with substitutions in their Fc region and increased serum half-life.

Engineered antibodies having three or more (preferably four) functional antigen binding sites are also contemplated (U.S. patent application No. US 2002/0004587a1, Miller et al).

V. pharmaceutical formulation

Therapeutic formulations of antibodies for use in accordance with the present invention are prepared for storage as lyophilized formulations or as aqueous solutions by mixing the antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's pharmaceutical sciences, 16 th edition, Osol, a. eds. (1980)). Acceptable carriers, excipients or stabilizersThe dosing agent is non-toxic to recipients at the dosages and concentrations employed, and includes buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol 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 counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Exemplary anti-CD 20 antibody formulations are described in WO 98/56418. This publication describes a liquid multi-dose formulation comprising 40mg/mL rituximab, 25mM acetate, 150mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20ph5.0, with a minimum shelf life of two years at 2-8 ℃. Another anti-CD 20 formulation of interest comprises 10mg/mL rituximab in 9.0mg/mL sodium chloride, 7.35mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80, and sterile water for injection pH 6.5.

Lyophilized formulations suitable for subcutaneous administration are described in U.S. Pat. No.6,267,958(Andya et al.). Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein.

Crystalline forms of the antibody are also contemplated. See, for example, US 2002/0136719a1(Shenoy et al).

The formulations herein may also contain more than one active compound (the second drug described above) as necessary, preferably those whose activities are complementary and do not adversely affect each other. The type and effective amount of such drugs depends, for example, on the amount of antibody present in the formulation and the clinical parameters of the subject. Preferred such agents are those described above.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, 16 th edition, Osol, A. eds (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid with gamma ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.

VI. product

In another embodiment of the present invention, an article of manufacture is provided that contains materials useful for treating joint damage as described above. Specifically, the present invention provides an article comprising: (a) a container containing an antagonist such as an antibody (e.g., a CD20 antibody) that binds to a B cell surface marker (preferably, the container contains the antibody and a pharmaceutically acceptable carrier or diluent within the container); and (b) a package insert printed with instructions for treating joint damage in a subject, wherein the instructions indicate that an antagonist or antibody (e.g., a CD20 antibody) is administered to the subject followed by a radiographic test at least about 1 month after administration to measure a reduction in joint damage as compared to baseline prior to administration, wherein the amount of antagonist or antibody, such as CD20 antibody, administered is effective to achieve a reduction in joint damage, indicating that the subject has been successfully treated.

In a preferred embodiment of this aspect of the invention, the article of manufacture of the invention further comprises a container containing a second medicament, wherein the antagonist or antibody is the first medicament, and the article of manufacture further comprises instructions on the package insert for treating the subject with an effective amount of the second medicament. The second agent can be any of those listed above, with exemplary second agents being those listed above and including antibiotics, immunosuppressants, disease modifying antirheumatic drugs (DMARDs), pain control agents, integrin antagonists, nonsteroidal anti-inflammatory drugs (NSAIDs), cytokine antagonists, bisphosphonates, or hormones, or combinations thereof, more preferably DMARDs, NSAIDs, pain control agents, or immunosuppressants. Most preferably, the second drug is methotrexate.

In this aspect, the package insert is on or accompanying the container. Suitable containers include, for example, bottles (bottles), vials (vitamins), syringes (syringees), and the like. The container may be made of a variety of materials such as glass or plastic. The container contains or contains a composition effective for treating joint damage and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antagonist or an antibody. The label or package insert indicates that the composition is useful for treating joint damage in a subject suitable for treatment, as well as specific instructions regarding the amount and interval of administration of the provided antagonist or antibody and any other drugs. The article of manufacture may further comprise another container containing a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and/or dextrose solution. The article of manufacture may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

In a more specific embodiment, an article comprises: (a) a container containing an antagonist such as an antibody (e.g., a CD20 antibody) that binds to a B cell surface marker (preferably, the container contains the antibody and a pharmaceutically acceptable carrier or diluent within the container); and (b) a package insert printed with instructions for treating joint damage in a subject, wherein the instructions indicate that an antagonist or antibody (e.g., a CD20 antibody) is administered to the subject followed by a radiographic test at least about 52 weeks after administration to measure a reduction in joint damage as compared to baseline prior to administration, wherein the amount of CD20 antibody administered is effective to achieve a reduction in joint damage, indicating that the subject was successfully treated. In a preferred embodiment, the article of manufacture comprises a container containing a second medicament, wherein the antagonist or antibody (such as a CD20 antibody) is the first medicament, and further comprising instructions on the package insert for treating the subject with an effective amount of the second medicament. Preferably, the second drug is methotrexate.

The following non-limiting examples illustrate further details of the invention. The disclosures of all citations in this specification are expressly incorporated herein by reference.

Examples

Example 1: efficacy and safety of rituximab in patients who respond poorly or lack tolerance to previous anti-TNF therapy

This is a phase III randomized, double-blind, parallel-group, multicenter clinical trial, termed: randomized assessment of long-term efficacy of rituximab in RA (REFLEX). The study design is shown in figure 1. The objectives of this study were:

● to determine the efficacy and safety of rituximab in combination with Methotrexate (MTX) for 517 patients with active rheumatoid arthritis who responded poorly to one or more anti-TNF therapies.

● the pharmacokinetics and pharmacodynamics (e.g. length of duration of B-cell depletion and effect on immunoglobulins and rheumatoid factors) of rituximab in this patient population were studied.

The results of this study were:

● Primary Endpoint (Primary Endpoint)

● at week 24, the proportion of patients with ACR20 response.

● minor end point (Secondary Endpoints)

● proportion of patients with ACR50 and ACR70 responses at week 24.

● change in DAS28 from baseline to week 24.

● EULAR response at week 24.

● ACR core gathers changes from baseline.

● SF-36 change from baseline.

● change in Genant modified Sharp radiographic total score at week 56.

● change in Genant modified Sharp radiographic score at week 24 (exploratory).

● erosion score and articular cavity narrowing score.

The key selection criteria (inclusion criterion) for this study were:

● experienced inadequate response to previous or current etanercept, infliximab, or adalimumab treatments due to toxicity or lack of efficacy

● etanercept 25mg twice a week for 3 months or more

● infliximab is not less than 3mg/kg, and is infused for at least 4 times

● adalimumab is 40mg, once every other week, for more than 3 months.

● must have received a dose of 10-25 mg/week of MTX (oral, p.o.) or parenteral) for at least 12 weeks and the dose stabilized for the last 4 weeks prior to screening.

● all other DMARD/biological response modifiers were discontinued for at least 4 weeks prior to randomization (8 weeks for infliximab, leflunomide, and adalimumab).

● prednisone equivalent is less than or equal to 10 mg/day.

● swelling joint number (SJC) is more than or equal to 8(66 joint numbers), and tender joint number (TJC) is more than or equal to 8(68 joint numbers).

● CRP is more than or equal to 1.5mg/dL (15mg/L) or ESR is more than or equal to 28 mm/h.

● at least one joint has radiographic evidence of exact erosion attributable to rheumatoid arthritis.

The study treatments for this study were:

● group A:

● at day 1 and day 15, two i.v. infusions of 1000mg rituximab.

● group B:

● placebo i.v. infusion on days 1 and 15.

● two groups of the Chinese herbal medicine are provided with

● 100mg i.v. methylprednisolone before each rituximab/placebo infusion.

● day 2-7 at 60mg/d and day 8-14 at 30 mg/d.

And 6-month results:

patient population:

patient number placebo rituximab

Registered 209308

ITT 201 298

Vial Breaks 43

Audited Site 14

Treatment before randomization (Treated priority Randomisation) 33

Zoned ITT (ITT by region) 201298

US 116(58％) 172(58％)

non-US 85 (42%) 126 (42%)

ITT (ITT By RF) 201298

RF+ve 160(80％) 234(79％)

RF-ve 41(20％) 64(21％)

Compliance protocol 161259

Exclusion 48 (24%) 49 (16%)

Security 209308

●ITT

● randomized

● receiving partial infusion

● analysis as randomization

● compliance protocol

● are as above, but comply with the protocol

● security

● randomized

● receiving partial infusion

● analysis as accepted

Patient demographics:

placebo rituximab

2×1000mg

(n＝201)(n＝298)

Sex

82% of women

Male 18% to 19%

Age (mean, year) 5352

Methotrexate dose (mg/wk) 1515

Early DMARD (mean) 2.52.6

Number of previous anti-TNF treatments 1.51.5

Patients receiving concurrent corticosteroids^* 78％ 74％

Patients receiving NSAID or COX2^* 75％ 67％

Baseline disease characteristics of patients:

placebo rituximab

2×1000mg

(n＝201) (n＝298)

Duration of disease (years) 11.712.2

SJC (mean) 2323

TJC (mean) 3334

RF positivity 80% 79%

RF (mean, IU/L) 320328

CRP (mean, mg/dL) 3.83.8

ESR (mean, mm/h) 4848

DAS28 6.8 6.9

Configuration of the patient:

placebo rituximab

2×1000mg

Randomized 209308

Exit 97 (46%) 54 (18%)

Adverse event 1 (< 1%) 8 (3%)

Death-

Response was less than 83 (40%) 36 (12%)

Treatment rejection 5 (2%)

Other 8 (4%) 5 (2%)

Complete 24 weeks 112 (54%) 254 (82%)

The efficacy results of this study are shown in figures 1-12.

The radiographic results of this study are shown in FIGS. 13-17.

The mean variation of the ACR kernel set parameters is shown in fig. 18.

The safety analysis of this study is shown in FIGS. 19-31.

The conclusion of the REFLEX study is:

● rituximab was associated with a significant increase in response rate to ACR20 relative to placebo (primary endpoint)

● all secondary and exploratory endpoints (DAS, EULAR, ACR core set) support the primary analysis

● the radiographic results show that the treatment group showed a lower Sharp-Genant total score at 24 weeks compared to the placebo group (FIG. 13), a lower Sharp-Genant erosion score at 24 weeks compared to the placebo group (FIG. 14), a lower Sharp-Genant JSN score at 24 weeks compared to the placebo group (FIG. 15), no change in erosion score at 24 weeks of treatment compared to the placebo group (FIG. 16), and a summary of the placebo and treatment group radiographic endpoints at 24 weeks is given in FIG. 17.

● rituximab is generally well tolerated.

● infusion-related events

● all infection rates were comparable to placebo

● slight increase in the rate/incidence of severe infection

● has no significant effect on immunoglobulins

● Low HACA

Results at 56 weeks:

number of patients (%)^*

Placebo rituximab

(n＝186) (n＝277)

Completion of 56 weeks 141(76) 202(73)

Exit 46(25) 75(27)

Withdrawal and receipt of TNF inhibitor therapy 12(6.5) 29(10.5)

^*Patients with radiographic data available at week 56.

Figure 41 further illustrates patient profiles for the REFLEX clinical trial at week 56, including ongoing treatment of a subgroup of patients selected from the phase III REFLEX clinical trial treatment group and the placebo group.

As shown in figure 42, mean changes in gent modified Sharp total score (gent, am.j.med., 30: 35-47(1983)), articular cavity narrowing (JSN), and erosion score at week 56 all showed statistically significant increases relative to placebo.

The efficacy of rituximab treatment is further illustrated by the mean change of the Sharp-gent total score over time. As shown in fig. 43, the increase continued from week 24 to week 56.

FIG. 44 shows the cumulative distribution of Sharp-Genant total score changes.

FIG. 45 shows the results of the sensitivity analysis expressed as changes in Sharp-Genant total score. The rituximab + MTX treated group was consistently superior to the placebo + MTX treated group.

Figure 46 shows the percentage of patients showing no radiographic changes in joint condition at observation point at week 56, as measured by erosion score and gent modified Sharp score, respectively.

In conclusion, the results of this clinical trial show that rituximab significantly inhibits radiographic progression in Rheumatoid Arthritis (RA) patients with inadequate or intolerant response to one or more TNF inhibitors. Furthermore, this study demonstrates for the first time that B cell targeted therapy can inhibit radiographic progression.

Example 2: rituximab in RA: in the phase III procedure

Important outcomes evaluated in RA include inhibition of the progression of structural joint damage and improvement of somatic function. This result is particularly important for patients recently diagnosed with RA, as there may be a higher long-term benefit to having these early effects. Genovese et al, Arthritis Rheum.46: 1443-1450(2002). Therefore, the early RA patient population is a suitable population to study these important outcomes.

For the appropriate control treatment of these patients, the current gold standard therapy for early RA is MTX. Thus, selecting a patient population that has not been exposed to MTX and comparing rituximab plus MTX to MTX alone provides a comparison of the novel treatment method to the current gold standard for these patients.

The study was a randomized phase III, controlled, double-blind (soluble-blid), parallel group, multicenter study to assess the safety and efficacy of rituximab in combination with MTX compared to MTX alone in active RA patients who were not exposed to MTX.

The main purpose is as follows:

1. the efficacy of rituximab in preventing the progression of structural joint damage was determined and the safety of rituximab in active RA patients on initiation of treatment with MTX was assessed.

2. The efficacy of rituximab in improving physical function and RA signs and symptoms in patients was evaluated.

3. The effect of Pharmacokinetics (PK) and covariates (covariates) of rituximab on PK parameters in the target RA patient population was studied by population analysis means.

4. The long-term efficacy and safety of more rituximab courses were studied.

First course of treatment-study design:

group A: rituximab 500mg i.v.x2 plus MTX (7.5mg escalation to 20mg p.o.)

Group B: rituximab 1000mg i.v.x 2 plus MTX (7.5mg escalation to 20mg p.o.)

Group C: placebo rituximab i.v.x 2 plus MTX (7.5mg escalation to 20mg p.o.)

For group C patients, from week 104 onwards, eligible patients may be retreated with a course of rituximab 500mg i.v.x 2 plus MTX.

The following countermeasures, consistent with the above protocol, apply:

the second course of treatment:

a second course of rituximab plus MTX or placebo plus MTX is administered as soon as possible when the following conditions are met:

1. a minimum of 24 weeks has elapsed since the first infusion of the last course of study medication.

2.DAS28-ESR＞2.6。

3. The final blood sample analysis satisfied absolute neutrophil counts (not less than 1.5X 10)³mu.L), IgG (not less than 5.0mg/mL) and IgM (not less than 0.40mg/mL) as qualified and selected (Eligibility) standards.

Furthermore, the patient must meet the following eligibility exclusion criteria 8, 10 and 11:

1. significant heart or lung diseases (including obstructive pulmonary disease).

2. Primary or secondary immunodeficiency (historical, or currently active), including a known history of HIV infection.

3. Any class of active infections (excluding fungal infections of the nail bed), or any major infectious attack, which requires hospitalization within 4 weeks of infection or treatment with i.v. anti-infective drugs or completion of oral anti-infective drugs within 2 weeks prior to infusion, are known.

Patients who did not meet the criteria for the second rituximab/placebo course of treatment at week 24 were followed every 4 to 8 weeks and then received the second course of treatment when they became eligible based on the criteria described above.

More treatment courses:

from week 48 the patient may become eligible to receive a third rituximab/placebo session. The third and subsequent courses of treatment may be administered only to patients who have met the criteria of the second course of treatment described above and to patients who the investigator believes have had an associated clinical response after the first or second course of rituximab. Patients who never experienced a clinical response should exit to enter safety tracking (SFU).

Patients considered to have developed clinical responses but currently not meeting more rituximab course criteria are followed every 4 weeks from week 48 to week 56, then every 8 weeks, and then receive more courses of treatment as they become eligible based on the above criteria.

All patients received a pre-medication of 100mg i.v. methylprednisolone prior to each infusion. All patients also received a stable dose of folic acid (folate) given as a single dose or divided doses once a week (at least 5 mg/week).

All patients should continue to receive a stable dose of any background corticosteroid (at least 10 mg/day of prednisone or equivalent) or oral non-steroidal anti-inflammatory drugs (NSAIDs). Randomization was grouped by region (US or ROW) and rheumatoid factor (positive or negative) to ensure balanced configuration of patients across regions and RF conditions among treatment groups.

Patients participated in the clinic every 4 weeks for the first 24 weeks, and every 8 weeks thereafter (except every 4 weeks at weeks 48-56) to assess efficacy, safety, immunology and quality of life. Radiographic assessments were performed at screening, week 24 and week 52. Radiographic assessments were also made 2 and 3 years after the first dose of study medication.

The primary endpoint (change in total modified Sharp score) was assessed at week 52. Secondary and exploratory endpoints included more radiographic endpoints and signs and symptoms, physical function and regression endpoints.

At any time after radiographic assessment at week 52, 20% of patients with no improvement in swelling and tender joint count (improvement) received rescue therapy with elevated doses of MTX or a non-biological DMARD.

All patients who were withdrawn from the study or at any time point or completed the entire treatment session should be returned for SFU assessment at weeks 4, 12, 24, 36 and 48 after withdrawal or completion. This effectively followed patients one year after they exited/completed the study. If the patient's peripheral B cell count (CD19) does not return to its baseline level or returns to within the normal range after one year, calculated as the lower of the two, then the safety follow-up should be continued at 12 week intervals until B cell saturation occurs.

CBC, differential (differential), platelet, quantitative Ig and CD19 counts should be determined within one week of the infectious adverse event becoming severe for all reported severe infectious adverse events.

Patients who exited into the SFU are strongly encouraged to return to all scheduled radiographic assessments (weeks 24, 52, 104 and 152), regardless of their exit from the study at any point in time. Radiographs were taken for these patients on the original assessment schedule relative to the original date of randomization (original day).

Approximately 852 patients were enrolled in the study and equally randomized into three treatment groups. Patients are grouped by region (US or elsewhere in the world (ROW)) and RF condition (positive RF at least 20IU/mL or negative RF less than 20 IU/mL). The overall proportion of RF-negative patients is limited to 20% of the total sample size. Recruitment was competitive with no more than 70% and no less than 30% additions in any area. The patient's treatment is discontinued for any reason and is no longer replaced by another patient.

Target population:

the target population for this study was patients with early active RA who had not been exposed to MTX.

Selection criteria (inclusion criterion):

The patients must meet the following criteria to meet the conditions for study participation:

1. can provide informed consent to comply with the requirements of the study protocol.

2. RA patients were diagnosed for at least 8 weeks but no more than 4 years according to the revised ACR criteria of 1987 for RA taxonomy.

3. The patient was not exposed to MTX and was considered a candidate for MTX therapy.

4. Swollen joint number (SJC) at screening and baseline was at least 8(66 joint numbers) and tender joint number (TJC) was at least 8(68 joint numbers).

5. CRP was at least 1.2mg/dL (12mg/L) at screening.

6. The age is 18-80 years.

7. If stable at least four weeks prior to baseline, a glucocorticoid of at least 10 mg/day prednisolone (prednisolone) or an equivalent is allowed.

8. NSAIDs are allowed if stable at least two weeks prior to baseline.

9. For fertile patients (both male and female), reliable contraceptive means (e.g. hormonal contraceptives, patches, intrauterine devices, physical barriers) are used throughout the study.

10. It must be acceptable to take oral folic acid.

11. For RF-negative patients only, at least one joint suffers from radiographic evidence attributable to the exact erosion of RA.

12. The patient is to receive or is currently receiving treatment based on the clinic.

Exclusion criteria:

exclusion associated with RA

1. non-RA rheumatic autoimmune diseases, or significant systemic involvement secondary to RA (including but not limited to vasculitis, pulmonary fibrosis, or Felty's syndrome). (ii) having secondary Sjogren's syndrome with RA: (syndrome) or secondary localized cutaneous vasculitis is permissible.

2. Functional class IV as defined by ACR taxonomy of functional status of RA.

3. Historical or current non-RA inflammatory joint diseases including, but not limited to, gout, reactive arthritis, psoriatic arthritis, seronegative spondyloarthropathy, lyme disease, or other systemic autoimmune disorders including, but not limited to, systemic lupus erythematosus, inflammatory bowel disease, scleroderma, inflammatory myopathy, mixed connective tissue disease, or any overlapping syndrome.

4. Juvenile Idiopathic Arthritis (JIA) or juvenile RA (jra) and/or RA before 16 years of age are diagnosed.

General health related exclusion

5. Any surgical procedure, including bone/joint surgery/synovectomy (including joint fusion or replacement) within 12 weeks prior to baseline or planned during the study.

6. Absence of peripheral venous access.

7. Pregnancy or lactation.

8. Significant and/or uncontrolled heart or lung disease (including obstructive pulmonary disease).

9. There are significant concomitant signs of disease including, but not limited to, neurological, renal, hepatic, endocrine, or gastrointestinal disorders that the patient would be excluded from participating in from the investigator's point of view.

10. Primary or secondary immunodeficiency (historical, or currently active), including a known history of HIV infection.

11. Any class of active infections (excluding fungal infections of the nail bed), or any major infectious attack, which requires hospitalization within 4 weeks of infection or treatment with i.v. anti-infective drugs or completion of oral anti-infective drugs within 2 weeks prior to infusion, are known.

12. History of deep space/tissue infection (e.g. fasciitis, abscess, osteomyelitis) within 52 weeks prior to baseline.

13. History of severe recurrent or chronic infections (for screening of breast infections, if not performed within 12 weeks prior to screening, a chest radiograph was taken at screening).

14. A history of cancer, including solid tumors, haematological malignancies and carcinoma in situ (in addition to skin-based and squamous cell carcinomas that have been resected and cured).

15. Any neurological (congenital or acquired), vascular or systemic disorder (e.g. parkinson's disease, cerebral palsy, diabetic neuropathy) that may affect any one of the efficacy assessments, particularly joint pain and swelling.

16. Current active alcohol or drug abuse or history of alcohol or drug abuse within 24 weeks prior to baseline.

Exclusion associated with drug treatment

17. A history of severe allergic or anaphylactic reactions to biological agents or a known hypersensitivity to any component of rituximab or to murine proteins.

18. Prior treatment with any approved or investigational biological agent for RA.

19. Previous treatments with anti- α 4 integrin antibodies or co-stimulatory modulators.

20. Concurrent treatment with any biological agent or non-MTX DMARD. Treatment must be discontinued 14 days prior to baseline, except for the following: azathioprine for at least 28 days; leflunomide for at least 8 weeks (or at least 14 days after 11 days of standard cholestyramine or activated carbon washing).

21. Previous treatments with any cell depleting therapy include investigational agents (e.g., CAMPATH, anti-CD 4, anti-CD 5, anti-CD 3, anti-CD 19, anti-CD 11a, anti-CD 22, BLys/BAFF, and anti-CD 20).

22. Treatment with any investigational agent within 28 days of baseline or within 5 half-lives (whichever is longer) of the investigational drug.

23. Live/attenuated vaccines were received within 28 days prior to baseline (it is recommended to carefully investigate the patient's vaccination record and need for immunization before receiving rituximab/placebo).

24. Intra-articular or parenteral glucocorticoids within the first 4 weeks of baseline.

25. Intolerance or contraindication to i.v. glucocorticoids (contin-indicia).

Exclusion associated with experimental findings

26. Positive serum human chorionic gonadotropin (hCG) measured before the first rituximab infusion.

27. Positive tests against hepatitis b surface antigen (HBsAg), hepatitis b core antibody (HBsAb) or hepatitis c serology.

28. The hemoglobin is less than 8.0 g/dL.

29. Serum IgG and/or IgM concentrations were below 5.0 and 0.40mg/mL, respectively.

30. Absolute Neutrophil Count (ANC) was less than 1.5 × 103/μ L.

AST or ALT greater than 2.5 times the upper limit of normal.

The end of treatment was defined as a 3 year time point followed by an additional SFU period of at least one year.

Rituximab (500mg or 1000mg) plus MTX or placebo plus MTX were administered by IV infusion on days 1 and 15. Patients may be eligible for re-treatment (two doses 14 days apart), with the highest frequency being once every 24 weeks of re-treatment. Patients who were initially randomized to receive rituximab plus MTX received the same dose of re-treatment throughout the study. From week 104, patients who were initially randomized to receive placebo plus MTX may be eligible to receive rituximab (500mg) plus MTX. A pre-medication of 100mg i.v. methylprednisolone was administered before each infusion.

Methotrexate tablets were administered orally (7.5 mg/week escalated to 20 mg/week) for all groups.

It is recommended that all patients should be pre-medicated with acetaminophen/acetaminophen (1gm p.o.) and diphenhydramine HCl (100mg i.v. or oral equivalent antihistamine) 30 to 60 minutes prior to infusion initiation to reduce the likelihood of infusion reactions.

The patient received folic acid or an equivalent (at least 5 mg/week) administered as a single dose or as divided doses once a week.

The patient may continue to receive any background glucocorticoid (at least 10 mg/day of prednisone or equivalent). Analgesics may be used to relieve pain if desired.

The primary endpoint was change in total modified Sharp score at week 52 relative to screening with modified intent-to-treat (ITT) populations.

The radiographic secondary endpoints were:

1. changes in total modified Sharp score at weeks 24 and 104

2. Change in modified Sharp erosion score at week 52

3. Change in the score of improved articular luminal narrowing at week 52

4. Proportion of patients with no radiographic progression at week 52 (defined as change in total modified Sharp score less than or equal to 0). In addition, the proportion of patients with no radiographic progression at weeks 24 and 104 was analyzed.

Radiographic exploratory endpoints were:

1. changes in the modified Sharp score are exhibited over time.

2. The proportion of patients with no radiographic progression was further presented in the following subcategories at weeks 24, 52 and 104:

a. proportion of patients with no change in modified Sharp erosion score relative to screening

b. Ratio of improved articular cavity narrowing score to screening of patients without change

c. The proportion of patients without newly eroded joints.

Radiographic evaluations were performed as follows: radiographs of each hand posterior-anterior (PA) and each foot Anteroposterior (AP) were taken according to the evaluation plan. At the screening visit, the readability and quality of the radiographs (which can be found in the manual of the hand, wrist and foot radiographic examination procedures) must be confirmed before the patient leaves the work site. Radiographs of RF-negative patients at the screening visit were examined for radiographic evidence that at least one joint is attributable to the exact erosion of RA according to the central reading site. All radiographs were measured using a gent, Am j.med., 30: 35-47(1983) evaluated by the modified Sharp method. The primary assessment was the change in total modified Sharp score at week 52 relative to screening. The total modified Sharp score combines the erosion score and joint space narrowing score for both hands and feet. The greatest total erosion in the hand and foot is scored as 100 and 42, and the greatest narrowing of the joint space in the hand and foot is scored as 100 and 48. The maximum total modified Sharp score achievable is 290. The change in the 52 nd week score was calculated as follows:

The filter score was subtracted from the score at week 52.

Radiographs of the hands and feet were taken to calculate the total modified Sharp score. All radiology departments attend training sessions to standardize radiographs prior to the start of the trial. This includes standardization of equipment, film, cassettes, procedures for confirmation of hand/foot position and procedures for obtaining consistent radiographs.

The change in total modified Sharp score is skewed and therefore not normally distributed. Therefore, the change in the total share score at week 52 was statistically examined between treatment groups using non-parametric tests grouped according to regional and RF conditions. However, if the data is shown to be approximately normally distributed, the data is analyzed using an analysis of variance (ANOVA) model, with the region, RF condition, and treatment group as test items in the model (experimental term).

The closing principle (closure principle) was used to regulate multiple comparisons in the primary endpoint. The first comparison was performed statistically using the Kruskal-Wallistest (Kruskal-Wallistest) between each of the three treatment groups.

If there is sufficient statistical evidence to negate the following null hypothesis:

H₀：μ₁＝μ₂＝μ₃that is, there was no overall Sharp in either treatment group

There is evidence of any difference in the change in score,

and accepts an alternative hypothesis (alternative hypothesis):

H₁：μ₁is not equal to mu₂I.e. improved in at least one pairwise therapeutic comparison

Or μ₁Is not equal to mu₃The change of the Sharp total score is different,

or μ₂Is not equal to mu₃

The three treatment groups will be considered different.

If the test results were statistically significant at the p-0.05 level, a difference in the change in total modified Sharp score from baseline at week 52 was inferred between treatment groups.

Subsequently, each rituximab group was compared to the placebo group at the p ═ 0.05 level, as described below. The main comparative consideration was the use of Van Elteren test statistics in the individual rituximab dose groups compared to the placebo group.

If there is sufficient statistical evidence to negate the following invalid assumption:

H₀：μ₁＝μ₂that is, the absence of modified Sharp in the rituximab group

Evidence of better overall score change than placebo,

and accepts an alternative hypothesis:

H₁：μ₁is not equal to mu₂That is, the total fraction of modified Sharp in the rituximab group

The change was superior to that of the placebo group,

then each rituximab-treated group was considered superior to the placebo group.

If the test results were statistically significant at the p-0.05 level, it was concluded that the rituximab group exhibited a change in the total modified Sharp score from baseline at week 52 that was better than the placebo group.

All efforts were made to ensure that the patient returned, had his radiographic visit, whenever possible, even if the study drug was discontinued. Missing week 52 data was entered using the following method:

if week 52 radiographic data is missing, then week 24 radiographic data is used to linearly extrapolate the patient's 52 week results. Those patients who exited earlier from the study by week 52 were included as part of the radiographic data analysis by week 52. Any patient without post-screening radiographic data was excluded from the modified ITT population and thus from the primary endpoint analysis.

The effect of potential imbalances in treatment configurations within a particular workplace is investigated. Sensitivity analysis was performed to assess the impact of heavily unbalanced workplaces on the primary analysis.

For the radiographic secondary endpoints, the change in total modified Sharp score at weeks 24 and 104 was analyzed in the same manner as specified for the primary endpoint. The change in modified Sharp erosion score at week 52 was analyzed in the same manner as specified for the primary endpoint. In addition, the change in modified Sharp erosion score at weeks 24 and 104 was analyzed. The change in improved articular cavity narrowing score at week 52 was analyzed in the same manner as specified for the primary endpoint. In addition, the change in the improved articular cavity narrowing score at weeks 24 and 104 was analyzed. The proportion of patients with no radiographic progression (defined as a change in total modified Sharp score of less than or equal to 0) at week 52 was assessed as follows: the difference in the ratio was statistically examined using the Cochran-mantelHaenszel (CMH) test grouped according to area and RF conditions. In addition, the proportion of patients with no radiographic progression at weeks 24 and 104 was analyzed.

For radiographic exploratory endpoints, the modified Sharp score is presented as a function of time. The proportion of patients with no radiographic progression at weeks 24, 52 and 104 is further presented in the following subclasses:

● ratio of modified Sharp erosion score to patient with no change in screening

● improving the ratio of articular cavity narrowing score relative to patients who did not change during screening

● proportion of patients who have no newly eroded joints.

Details of the joints and rating scales (grading scales) (gent, am.j.med., 30: 35-47(1983)) used for radiographic evaluation are as follows:

rating scale:

1. narrowing of joint cavity (JSN)

Level 0-normal.

Level 0.5-mild JSN or fuzzy discovery

Grade 1.0-mild JSN (focal or smaller)

Grade 1.5-mild to moderate JSN

Level 2.0-moderate JSN

Grade 2.5-moderate to severe JSN

3.0 level-severe JSN

Grade 3.5-Severe JSN approximating Joint rigidity

Grade 4.0 — exact ankylosis.

2. Erosion (discontinuous discontinuity in the surface of the skin)

Grade 0-Normal

Grade 0.5-mild loss of cortical continuity or ambiguous findings of bone erosion

Grade 1.0-mild. Erosion of the exact, but small, articular bone or bones is usually located in the exposed area that encompasses less than 25% of the articular surface.

Grade 1.5-mild to moderate. Small-medium erosions involving less than 25% of the articular surface of one or both articular bones.

Grade 2.0-medium. While involving medium-large erosions of 26-50% of the articular surface of both articular bones.

Grade 2.5-moderate to severe. Erosion of 51-75% of the articular surface.

Grade 3.0-severe. Erosion of 76-90% of the articular surface.

Grade 3.5-very severe. Erosion of 100% of the articular surface (total destruction of the articular surface). Joint assessment:

1. narrowing of the articular cavity of the hand (13 joints per hand)

a. All Proximal Interphalangeal (PIP) joints on the II-V fingers.

b. The interphalangeal joints on the ith finger.

c. All Metacarpophalangeal (MCP) joints.

d. The carpometacarpal (CMC) joint of the III-V finger as a single unit.

e. The pericapsular (joint of scaphoid-skull and lunate-skull) region.

f. The radial and wrist joints.

2. Hand erosion score (14 joints per hand)

a. All Proximal Interphalangeal (PIP) joints on the II-V fingers.

b. The interphalangeal joints on the ith finger.

c. All Metacarpophalangeal (MCP) joints.

d. Carpometacarpal (CMC) joint of the I-th finger.

e. Scaphoid bone.

f. The distal radius.

g. The distal ulna.

The scores are summed up separately (maximum x 2 for erosion 14 x 3.5, i.e. per joint, 98, and maximum x 2 for JSN 13 x 4, i.e. per joint). Each sum is normalized to a scale of 0-100. The two scores are added to obtain a total score (scale 0-200).

3. Narrowing and erosion of the articular cavity (6 joints per foot)

a. All Metatarsophalangeal (MTP) joints.

b. All interphalangeal joints on the ith digit.

The scores are summed up separately (6 × 3.5 for erosion, i.e. maximum per joint × 2 — 42, and 6 × 4 for JSN, i.e. maximum per joint × 2). The two scores are added to obtain a total score on a scale of 0-90.

The original radiographs are sent to a central reading site where their quality is controlled. If the original radiograph is of unacceptable quality, the center is advised of what correction is needed and informed to take the second radiograph.

All patients randomized to receive at least a partial rituximab dose in the study (including those patients who have exited to enter safety tracking) were radiographed of the hands/wrists and feet at weeks 24, 52, 104 and 152. Patients were strongly encouraged to return for all radiographic assessments regardless of their point of exit from the study. Radiographs of these patients were taken according to the original assessment schedule relative to the initial day of randomization.

Other secondary and exploratory endpoints have signs and symptoms, physical function, regression, and patient reported outcomes.

Therefore, the change in total modified Sharp score at week 52 between treatment groups was examined using non-parametric test statistics grouped according to region and RF conditions. However, if the data is shown to be approximately normally distributed, the data is analyzed using an analysis of variance (ANOVA) model, with the region, RF condition, and treatment group as test items in the model.

It is clearly desirable to maintain patients in a state of low disease activity by limiting the onset of disease and potentially limiting the progression of structural damage. In the DANCE study mentioned above (Emery et al, EULAR, supra and Van Vollenhoven et al, EULAR, supra), at week 24, approximately 90% of patients treated with rituximab had not achieved EULAR (DAS28) regression. In this study, the patients who were subjects of this example were eligible to receive their first rituximab re-treatment at week 24. DAS28-ESR based mandatory re-treatment provided objective information about a re-treatment paradigm based on disease activity. Based on the pharmacokinetics and pharmacodynamics of rituximab, a minimum interval period of 24 weeks between treatment courses is recommended. Without being bound by any one theory, it appears that the pharmacokinetics and pharmacodynamics of rituximab show that at this time (week 24) drug levels are below detection levels with recovery of peripheral CD19⁺Evidence of cells. After this time point, a significant increase in disease activity parallels this and therefore represents a reasonable time point at which more courses can be given.

Rituximab (or a humanized 2H7 antibody in place of rituximab) in combination with MTX was effective in meeting the primary endpoint of prevention of progression of structural joint damage set forth in this example, and the protocol of this study also met one or more secondary radiographic endpoints. Thus, administration of the first dose of rituximab or humanized 2H7(500mg x 2 or 100mg x 2) and methotrexate reduces joint damage relative to baseline (prior to the first administration of CD20 antibody) at least about one month from baseline or from the start of treatment, preferably at least about 24 weeks from baseline, more preferably at least about 52 weeks from baseline, and up to more time points of 104 weeks from baseline, as measured by the modified Sharp total score. Patients can also be effectively retreated at week 24 or 52 from baseline to maintain prevention of the progression of the joint injury.

Administration of rituximab or humanized 2H7 to a subject in a scheduled re-dosing regimen as set forth above was effective at preventing the progression of structural joint damage at or after week 52. These results are significantly better than the control results.

Administration of another dose of 1g or 2g of CD20 antibody (e.g., rituximab or humanized 2H7) at one time at about weeks 48-54 or dispersed over a period of about 14-16 days in an amount of 0.5 or 1 gram of CD20 antibody is also effective to treat joint damage throughout the second year, with or without one or more second agents such as immunosuppressive agents. Thus, successful administration of the CD20 antibody first over a period of about 2 weeks, followed by another treatment at about 4-8 months from the initial treatment (calculated from the administration of any dose), followed by another treatment at about one year from the initial treatment, followed by treatment at about two years from the initial treatment, together with about one-half gram or one gram x 2 "4 doses for each treatment, administered about once per week or about once every other week over a period of about two to four weeks, was successful. The results of this treatment were much better than the results of the control with placebo. The re-treatment regimen is successfully used for years with no or minimal side effects.

The primary endpoint was also met with the same regimen at the same or higher dose and without a second agent such as MTX using rituximab or another CD20 antibody as monotherapy, and success was achieved with the same regimen at the same or higher dose as monotherapy.

Example 3: efficacy study of rituximab re-treatment in patients with rheumatoid arthritis

This example describes a phase III, randomized, double-blind, placebo-controlled, multicenter study of rituximab re-treatment in RA subjects receiving background methotrexate.

The primary objective of this study was to assess the efficacy of rituximab re-treatment in active RA subjects who are receiving MTX and are responding poorly to TNF inhibitors.

Secondary objectives of the study were as follows:

● the safety of rituximab re-treatment in active RA subjects who are receiving MTX and are responding poorly to TNF inhibitors.

● the safety of rituximab in active RA subjects who are receiving MTX and are responding poorly to TNF inhibitors is evaluated.

This is a phase III, randomized, double-blind, placebo-controlled, multicenter study that assesses the efficacy of rituximab re-treatment in active RA subjects who are receiving MTX. The study consisted of four parts: screening, treatment period (rituximab open first course label, double-blind, randomized re-treatment for eligible subjects), safety tracking (SFU), and B-cell tracking. The subject must be insufficiently responsive to treatment with one or more TNF inhibitors due to toxicity or insufficient efficacy. Approximately 555 subjects in the united states entered a treatment session at approximately 150 study sites. RF positive and RF negative subjects were enrolled and equally configured between treatment groups, with the total proportion of RF negative subjects limited to 20% of the total sample size.

Day 1 ago, subjects discontinued all DMARDs except MTX (8 weeks or more for leflunomide, adalimumab, and infliximab, and 4 weeks or more for etanercept). All subjects continued to receive MTX 10-25mg/wk at a stable dose during the study. Depending on clearance requirements, the screening visit may be made as early as 56 days prior to receiving the first dose of study treatment.

All subjects who met the eligibility criteria and were added to the trial received a first course of rituximab. A course of rituximab is defined as two 1000mg Intravenous (IV) doses given 14 days apart, with methylprednisolone 100mg IV pre-treatment prior to each rituximab dose. All subjects also received a stable dose of folic acid (. gtoreq.5 mg/wk). All subjects should continue to receive a stable dose of any background corticosteroid (< 10 mg/day prednisone or equivalent) or oral nonsteroidal anti-inflammatory drugs (NSAIDs).

The first dose of rituximab should be administered within 24 hours after baseline assessment. However, up to 72 hours between baseline assessment and first dose of study drug was allowed if necessary.

During weeks 24-40, subjects with active disease based on a disease activity score of 28 joints (DAS 28-erythrocyte sedimentation rate [ ESR ]) of ≧ 2.6 were considered eligible for re-treatment and randomized at a 2: 1 ratio for one additional rituximab (group A) or placebo (group B) course of re-treatment. Follow-up of safety and efficacy was continued for subjects who did not meet the second rituximab course criteria during the 24 th-40 th cycle. Subjects who met the criteria for re-treatment between cycles 24-40 and were denied re-treatment for any reason exit the treatment phase and enter the SFU phase.

Standard arthritis and safety assessments were performed. Pharmacodynamic measurements include CD19⁺B cells, immunoglobulins, and autoantibodies. The researchers calculated the DAS 28-ESR score at regular scheduled visits (Prevoo et al Arthritis Rheum 38: 44-48 (1995); DAS-score. nl2005 DAS score. nl 2005: home of the DAS. department of Rheumatology University medical center Nijmegan-the Netherlands [2005, 9, 1, referenced)]CanObtained as http:// www.das-score.nl/www.das-score.nl/index. html).

The treatment period was 72 weeks (day 1 to 72 weeks). All subjects who were withdrawn from the treatment period at any time or who received rituximab/placebo re-treatment between weeks 24-40 and completed the treatment period should be returned for SFU assessment at weeks 4, 12, 24, 36 and 48 SFU after withdrawal or completion. All subjects were followed for at least 48 weeks after their last dose of rituximab. All subjects who received only one course of rituximab therapy (i.e., subjects who did not meet the re-treatment conditions during the study) and completed the treatment period did not return to SFU.

Subjects whose peripheral B cell count has not been restored at the end of the treatment or SFU period continue to follow the laboratory assessments and the occurrence of severe adverse events every 12 weeks until B cells are restored. B cell recovery is defined as the return of peripheral B cell count to baseline or lower normal limit (LLN), whichever is lower.

At or after 16 weeks post-retreatment, subjects who have not achieved a 20% improvement in both Tender Joint Count (TJC) and Swollen Joint Count (SJC) compared to baseline can initiate rescue treatment with a non-biological DMARD, the choice of which is judged by their treating physician.

In a Rituximab re-treatment study in RA, subjects obtained sustained ACR (American College of Rheumatology)20, 50 and 70 responses (Pavelka et al, "Efficacy and safety following diseases of rituximab in patients with active rhematoid arthritis," abstract provided by EULAR 2005). However, because this study is a label-open study, there is no control data to assess the efficacy and safety of rituximab re-treatment in RA. The study was designed to evaluate the efficacy of re-treatment in a placebo-controlled trial in which active RA subjects received a single additional course of rituximab. Active disease subjects characterized by a DAS 28-ESR ≧ 2.6 are treated during weeks 24-40 with a course of the second study drug (rituximab or placebo).

The purpose of re-treatment with rituximab is to prevent onset, promote continued control of the disease, and potentially prevent disease progression. The retreatment standard of DAS 28-ESR more than or equal to 2.6 ensures that the subject with clinically significant disease activity is retreated. DAS 28-ESR was calculated using the swelling and tender joint count, ESR, and patient's overall disease activity assessment (visual analog scale [ VAS ] at 100 mm).

The primary endpoint of this study was the proportion of subjects retreated with ACR20 at week 48 relative to baseline (day 1) rather than relative to the time of retreatment. The baseline (day 1) was chosen without time for re-treatment in order to assess the overall benefit of re-treatment with rituximab in moderate to severe RA subjects. Without being bound by any one theory, it is hypothesized that retreatment with RTX resulted in the maintenance of slightly improved effects at week 48 relative to week 24 compared to exacerbations in placebo-treated subjects. Although there may be significant therapeutic benefit in the retreated subjects, the ACR20 response at week 48 relative to week 24 baseline may be meaningless for both groups. As such, the present study was not designed to evaluate improvement over treatment time.

Based on the data at week 24 in the phase III study (REFLEX, WA17042/U2646s/IDEC102-20), approximately 91% of subjects treated with rituximab met the criteria for re-treatment at week 24 of the regimen (DAS 28-ESR ≧ 2.6). DAS 28-ESR based mandatory re-treatment provides objective efficacy information using a re-treatment paradigm based on disease activity. The minimum period of 24 weeks is based on the pharmacokinetics and pharmacodynamics of rituximab. This parallels a significant increase in disease activity after this time point and therefore represents a reasonable time for re-treatment.

The primary outcome measure was the proportion of retreated subjects who had an ACR20 response at week 48 relative to baseline (day 1).

Secondary results were measured as follows:

● proportion of retreated subjects with ACR50 and ACR70 responses at week 48 relative to baseline (day 1).

● change in DAS 28-ESR compared to baseline (day 1) at week 48 for retreated subjects

● proportion of subjects receiving a combined European Rheumatism prevention and treatment (EULAR) response (fair or moderate) at week 48 relative to baseline (day 1)

● for the retreated subjects, changes in the ACR core set (SJC, TJC, Health Assessment Questionnaire [ Health Assessment questonnaire, HAQ ], global Assessment by patient and physician, patient pain Assessment, C-reactive protein [ CRP ], and ESR) at week 48 compared to baseline (day 1)

● Redo ACR-N at week 48 in subjects

● Recreatments of changes in subjects from baseline (day 1) to week 48 SF-36 subscale (subscale) and summary score (summary score)

● Change in function Assessment-Fatigue (Functional Assessment of Chronic Illness Therapy-Fatigue) (FACIT-F) Assessment of Subjects from Baseline (day 1) to week 48 of Chronic disease Therapy

● the proportional exploratory results of the ACR20, ACR50, and ACR70 responses at week 48 in all subjects were measured as:

● proportion of all subjects who obtained ACR20, ACR50 and ACR70 responses at week 72 compared to baseline

● proportion of subjects with DAS 28-ESR regression (DAS 28-ESR < 2.6) at week 48

● proportion of subjects with DAS 28-ESR Low disease (low disease) (DAS 28-ESR ≦ 3.2) at week 48

● proportion of subjects with DAS 28-ESR regression (DAS 28-ESR < 2.6) at week 72

● proportion of subjects with DAS 28-ESR Low disease (DAS 28-ESR. ltoreq.3.2) at week 72

Subjects selected for eligibility to have active RF positive (. gtoreq.20 IU/mL) or RF negative RA, receiving MTX, and previously or currently responding insufficiently to one or more TNF inhibitors were screened for participation in the study. RF negative subjects were limited to 20% of the total enrolled population.

The subjects must meet the following criteria as conditions for entry into the study:

● signing on informed consent forms

● are able and willing to comply with the requirements of the research protocol

● 18-80 years old

● was diagnosed as RA for at least 6 months according to the 1987 revised ACR criteria for the RA classification (Hochberg et al, Arthritis Rheum 35: 498-502 (1992)):

Class I

Full functional ability with the ability to engage in all ordinary duties without difficulty

Class II

Functional capability sufficient to perform normal activities despite inconvenient difficulties or limited mobility of one or more joints

Class III

Functional capacity sufficient only to perform a very few jobs or self-care of the usual occupation

Class IV

Substantially or completely incapacitated, bedridden or wheelchair-bound, hardly self-care subjects

● receiving RA treatment on an outpatient basis

● moderate to severe active RA activity recorded at screening was as follows:

TJC ≧ 8(68 joint counts), and

SJC ≧ 8(66 joint counts), and

abnormal CRP of 0.6mg/dL or more, or ESR of 28mm/hr or more

Insufficient response to previous or current treatment with one or more of the following drugs, either due to toxicity or inadequate efficacy, is documented: etanercept, infliximab, and/or adalimumab. Insufficient efficacy includes the following treatments: etanercept, 25mg twice a week or 50mg once a week for 3 months or more; infliximab for at least four infusions of greater than or equal to 3 mg/kg; or adalimumab, 40mg every other week, for more than or equal to 3 months.

● MTX 10-25mg/wk is used before day 1 for more than 12 weeks, and the stable dose is more than 4 weeks

● willing to accept folic acid orally

● if a background corticosteroid is administered (≦ 10 mg/day prednisone or equivalent), the corticosteroid must be administered at a stable dose during the 4 week period prior to day 1

● if the dosage is stable for 2 weeks or more before day 1, then NSAID use is allowed

● for male and female with fertility ≧ 30 days before day 1 and study period or subject peripheral CD19⁺In the period of B cell depletion, the older the period, reliable means of contraception are preferred

(e.g., hormonal contraceptives, intrauterine devices, physical barriers)

Subjects who met any of the following criteria were excluded from the study:

a. general (General)

● rheumatic autoimmune disease other than RA or major systemic spread secondary to RA (e.g., vasculitis, pulmonary fibrosis, or Fisher's syndrome)

Secondary sjogren's syndrome with RA is allowed.

● History or present inflammatory joint diseases other than RA (e.g., gout, reactive arthritis, psoriatic arthritis, seronegative spondyloarthropathy, or Lyme disease) or other systemic rheumatic diseases (e.g., systemic lupus erythematosus, inflammatory bowel disease, scleroderma, inflammatory myopathy, or overlap syndrome)

● functional class IV, as defined by the ACR classification of functional status in rheumatoid arthritis

● any surgical procedure, including bone/joint surgery/synovectomy (including joint fusion or replacement), within 12 weeks before day 1 or within 48 weeks after day 1

● known hypersensitivity to any component of humanized or murine monoclonal antibodies

● received live inoculations within 4 weeks before day 1

● major heart or lung diseases including obstructive pulmonary disease

● evidence of significant uncontrolled concomitant diseases such as, but not limited to, neurological, renal, hepatic, endocrine, or gastrointestinal disorders

● known active bacteria, viruses, fungi, mycobacteria or other infections (including tuberculosis or atypical mycobacteriosis but excluding fungal infection of the nail bed), or any significant outbreaks of infections requiring hospitalization within 4 weeks of day 1 or treatment with an IV antibiotic or oral antibiotic within 2 weeks of day 1

● history of severe recurrent or chronic infection (for screening of chest infections, if not performed 12 weeks before screening, chest radiographs are performed at screening time)

● History or present active primary or secondary immunodeficiency including HIV infection

● history of cancer, including solid tumors and haematological malignancies (with the exception of basal cell or squamous cell carcinoma of the skin that has been resected and cured)

● Severe cytopenia or history of other bone marrow disorders

● history of alcohol, drug or chemical abuse within 24 weeks before day 1

● pregnant or lactating

● neuropathy and neurovascular disorders that may interfere with pain assessment

● poor peripheral venous access (access)

● intolerance or contraindication to oral or IV corticosteroids

b. Laboratory exclusion criteria

● hemoglobin < 8.0g/dL

● Absolute neutrophil count < 1.5X 10³/μL

●IgM＜0.40mg/mL

●IgG＜5.0mg/mL

● aspartate Aminotransferase (AST) or alanine Aminotransferase (ALT) > 2.5 fold upper limit of normal

● positive hepatitis B surface antigen or hepatitis C antibody serology

● Positive serum pregnancy test at screening for fertility in women (including those with ligated oviducts)

● excluded prior or concurrent medication

● currently any DMARD not using MTX is used

● concurrent therapy with any biological agent

Treatment had to be discontinued at least 4 weeks prior to day 1, except: leflunomide for more than or equal to 8 weeks (or more than or equal to 14 days after 11 days of standard cholestyramine clearance); infliximab for not less than 8 weeks;

And adalimumab for not less than 8 weeks

● treatment with any study agent within a half-life of 4 weeks or 5 study drugs (whichever is longer) before day 1

● any previous treatment with rituximab or other cell depleting therapies, including CAMPATH, anti-CD 4, anti-CD 5, anti-CD 3, anti-CD 19, anti-CD 11a, anti-CD 22, BLys/BAFF, and other anti-CD 20 agents

● previously used anti-alpha 4 integrin agents, including natalizumab

● previously used IV gamma globulin or IV gamma globulin during a 6 month screening periodTreatment of columns

Once all screening evaluations and verifications are completed that the subject has met all selection criteria and exclusion criteria, personnel at the workplace contact an interactive voice response system (ivrs) to obtain the number of subjects and confirm subject enrollment for study drug inventory management (study drug inventory). All enrolled subjects received rituximab as their initial course of treatment.

Subjects eligible for re-treatment were randomized to group A (rituximab re-treatment) or group B (placebo; see Table 1) at a 2: 1 ratio during weeks 24-40. If the subject meets eligibility criteria for re-treatment during the 24 th-40 th cycle, the staff at the workplace contacts the IVRS to initiate randomization of the subject and obtain the study drug kit number.

Independent IVRS providers implement randomization and retain treatment assignment codes. Randomization was grouped by study center, baseline RF conditions (RF positive, RF negative), and improvement in tender and swollen joint numbers at week 24 (≧ 20% improvement or < 20% improvement). Non-blind operations (unblinding) require the IVRS provider to provide a treatment assignment code and kit treatment number. The transfer and a record of the data included in the transfer are saved on the archive.

TABLE 1

Study treatment group

Group a rituximab: for the first course of treatment, 1000mg IV on days 1 and 15 during weeks 24-40, for a course of re-treatment of eligible subjects (DAS 28-ESR ≧ 2.6 and meeting the re-treatment criteria herein), 1000mg IV corticosteroid on days 1 and 15: 100mg IV methylprednisolone MTX before each infusion: 10-25mg/wk folic acid: not less than 5mg/wk
	Group B rituximab: for the first course of treatment, 1000mg IV on days 1 and 15 during weeks 24-40, a course of re-treatment for eligible subjects (DAS 28-ESR ≧ 2.6 and meeting the re-treatment criteria herein), placebo IV corticosteroid on days 1 and 15: 100mg IV methylprednisolone MTX before each infusion: 10-25mg/wk folic acid: not less than 5mg/wk

During the re-treatment period, treatment group assignments were blinded to staff at the workplace and Genentech (clamped). To prevent potential non-blind operations (unblinding) due to efficacy or laboratory changes observed during retreatment, dual assessor methods were used to assess efficacy and safety.

Peripheral CD 19B-cell counts were blinded to the staff at the workplace and Genentech until the primary endpoint database was closed for all subjects enrolled in the trial at week 48. Thus, all subjects who had completed the treatment phase and SFU before the database was shut down were assumed to be peripherally depleted and B-cell tracking was maintained.

The efficacy assessor (or assignees) should be a rheumatologist or a skilled arthritis assessor. Efficacy assessors must not be the primary Investigator (Principal Investigator). Efficacy assessors are only entitled to exposure to efficacy data and are only responsible for completing the number of joints and the physician's assessment of overall disease activity VAS. To ensure consistency of joint assessment throughout the trial, subject individuals should be assessed by the same joint assessor for all study visits. During the study visit, all results reported by the subjects should be completed before all other assessments.

The safety assessor (or assignee) should be a rheumatologist and have access to both safety and efficacy data. The safety assessor may be the primary researcher. The security assessor has access to source files, laboratory results and Case Report Forms (CRF) and is responsible for calculating the DAS 28-ESR and making treatment decisions based on the subject's clinical response and laboratory parameters. The safety assessor cannot perform any efficacy assessment or record any efficacy assessment results on behalf of the efficacy assessor.

Rituximab used in this study was a sterile, clear, colorless, preservative-free liquid concentrate for IV administration. Rituximab is provided in a 500mg (50mL) single use vial at a concentration of 10 mg/mL. The product was formulated in 9.0mg/mL sodium chloride, 7.35mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80 and sterile water for injection for IV administration. The pH was adjusted to 6.5.

All enrolled subjects received a course of rituximab (1000 mg rituximab IV infusion on days 1 and 15) as their initial treatment. During weeks 24-40, subjects eligible for re-treatment were randomized to group a to receive an additional course of 1000mg IV rituximab (two doses) 14 days apart, or to group B to receive a course of 1000mg IV placebo (two doses) 14 days apart.

For all subjects, pre-medication with 1000mg oral acetaminophen and 50mg oral diphenhydramine is recommended and 100mg IV methylprednisolone needs to be completed within 30-60 minutes before each rituximab/placebo infusion.

All subjects continued to receive a stable dose of MTX of 10-25mg/wk and a stable dose of folic acid (. gtoreq.5 mg/week) given as a single dose or divided doses once a week.

Rituximab/placebo infusion should be administered to the subject under close supervision of the researcher or the assigned at the hospital or clinic where a full set of rescue (resuscitation) equipment is immediately available. Although rituximab may be administered on an outpatient basis, subjects may also be admitted for observation at the discretion of the investigator. Rituximab/placebo should be administered as a slow IV infusion. Not as an IV bolus (push or bolus). At the end of each infusion, the IV line should be left in place for at least 1 hour for IV administration of the drug if necessary. If no adverse events occur during this time, the IV line can be removed.

The necessary amount of rituximab/placebo is removed using appropriate aseptic techniques and diluted at a final concentration of 4mg/mL into infusion bags containing 0.9% sodium chloride, USP, or 5% dextrose in water, USP. The bag was gently inverted to mix the solution. Rituximab vials are biologically and chemically stable at 2 ℃ -8 ℃ (36 ° F-46 ° F). It is necessary to use the medicine bottle beyond the expiration date. Exposure of rituximab to direct sunlight should be avoided.

Once reconstituted in the IV bag, the rituximab solution for infusion can be stored at 2 ℃ -8 ℃ (36 ° F-46 ° F) for 24 hours. Rituximab solutions for infusion have been shown to be stable for storage at room temperature (23 ℃ or 73 ℃ F.) for an additional 24 hours. However, since rituximab solution does not contain preservatives, the diluted solution should be refrigerated (2 ℃ -8 ℃). No incompatibility between rituximab and polyvinyl chloride or polyethylene bags has been observed.

For retreatment, DAS 28-ESR was calculated using the number of joints from the current visit and the patient's overall disease activity (VAS) assessment and ESR from the current visit, only if ESR from the current visit was not available. Subjects meeting the following criteria at any visit between periods 24-40 met the criteria for receiving a re-treatment with study drug (rituximab or placebo):

●DAS 28-ESR≥2.6

● negative urinary pregnancy test in women with fertility, including those with ligated fallopian tubes

Furthermore, if current results are not available, subjects eligible for retreatment for DAS 28-ESR ≧ 2.6 during weeks 24-40 must also meet the following criteria in order to be retreated based on results from a previous visit:

● hemoglobin is greater than or equal to 8.0g/dL

● Absolute neutrophil count (1.5X 10)³/μL)

●IgM≥0.40mg/mL

●IgG≥5.0mg/mL

● No significant Heart or Lung disease (including obstructive Lung disease)

● lack of primary or secondary immunodeficiency, including a known history of HIV infection

● No evidence of significant uncontrolled concomitant diseases such as, but not limited to, neurological, renal, hepatic, endocrine, or gastrointestinal disorders

● have no active infection of any kind, (excluding fungal infections of the nail bed), or any significant episode of infection requiring hospitalization within 4 weeks of infusion or treatment with an IV antibiotic or completion of oral antibiotics within 2 weeks prior to infusion.

No alteration of rituximab doses was allowed during the study. The infusion rate may be adjusted if an infusion-related reaction occurs. If the subject develops an infusion-related reaction that requires discontinuation of the infusion and the investigator concludes that the infusion should not be restarted, the subject should exit from the study treatment session and enter the SFU.

All subjects were prescribed by their treating physicians and received 10-25mg/wk of MTX in parallel. Subjects must have been treated with MTX for > 12 weeks prior to study entry and must maintain a stable dose of MTX for > 4 weeks before day 1 and during the study unless changes are necessary due to toxicity.

Certain adverse events commonly associated with MTX treatment may occur. To minimize MTX toxicity, all subjects treated with MTX also received a stable dose of folic acid (≧ 5 mg/week) given in a weekly single dose or a daily divided dose. Dosing regimens are at the discretion of the investigator.

If the dose stabilizes for 4 weeks or more before day 1, treatment with 10mg prednisone or equivalent is allowed daily. The dose should remain stable throughout the study unless changes are required due to toxicity.

If the dose stabilizes for 2 weeks or more before day 1, one NSAID is allowed. The dose should remain stable throughout the study unless changes are required due to toxicity.

Furthermore, for cardiovascular prophylaxis, treatment with ≦ 325mg acetylsalicylic acid per day is allowed.

Additional analgesics may be used to relieve pain if desired. However, subjects should not take pain medication within the 12 hours prior to the visit, where efficacy assessments are performed at the time of the visit. The analgesic regimen can be adjusted, but the changes must be recorded in the appropriate CRF.

Intra-articular injection of corticosteroids is discouraged, particularly during the first 48 weeks; however, intra-articular injection of corticosteroids may be used on a limited basis to manage RA activity in subjects during the study. No more than one joint should be injected every 24 weeks, and no more than one injection should be injected for the same joint at any 48 weeks. The single injection should not exceed 40mg of triamcinolone (or equivalent), and the total corticosteroid dose should not exceed 80mg of triamcinolone (or equivalent) over any 48 week period.

During the study, subjects may continue to receive background corticosteroid at a dose of ≦ 10 mg/day prednisone (or equivalent). In the case of a seizure or non-RA disorder such as asthma that requires treatment with oral corticosteroids, the appropriate dose should be administered for up to 2 weeks and should decline to the previous level as quickly as medically possible.

Increasing the dose of corticosteroid is considered a worsening of the subject's condition from baseline and should be recorded on CRF as an adverse event.

IV or intramuscular corticosteroids were not allowed in the study except those prescribed in the treatment regimen.

Prior to day 1 and during the study, subjects must maintain a stable dose of MTX for 4 weeks or more, unless necessary to change due to toxicity.

At or after 16 weeks post-treatment, subjects who have not achieved a 20% improvement in both TJC and SJC compared to baseline can initiate rescue treatment with one non-biological DMARD, the choice of which is judged by their treating physician. Subjects receiving rescue were not withdrawn from the study.

Samples must be taken prior to the infusion of methylprednisolone IV and rituximab/placebo.

If a subject is difficult to write or unable to read during a visit, the data reported by the subject person (e.g., the patient's overall disease activity assessment, the patient's pain assessment) may only be recorded by the study nurse/researcher on behalf of the subject. This must be clearly recorded in the subject record.

To prevent potential non-blind operations (unblinding), a dual assessor (efficacy assessor and safety assessor) approach was used to assess efficacy and safety. The assessments completed by the subject and the efficacy assessor must be made prior to the assessments made by the safety assessor.

The subject should express his/her assessment of overall disease activity during the last 24 hours using a 100-mm level (horizontal) VAS in which the left-hand end of the line represents no disease activity (asymptomatic and without arthritic symptoms) and the right-hand end represents maximal disease activity (maximal arthritic disease activity).

The subject should express his/her assessment of pain level during the last 24 hours using a 100-mm level VAS in which the left-hand end of the line represents no pain and the right-hand end represents intolerable pain.

The stanford HAQ disability index is a subject report questionnaire specifically directed to RA. It includes 20 questions related to the eight component sets: clothing/finishing (dressing/finishing), living (claiming), eating (eating), walking (walking), hygiene (hydrene), stretching (reach), grasping (grip), and activity (activities). The questionnaires were scored according to the guidelines of the Stanford University Medical Center (Fries et al, Arthritis Rheum 23: 137-.

FACIT-F was used to assess fatigue. It is a questionnaire with 13 items, which subjects are asked to score each question on a scale of 0-4. This evaluation was originally developed for chronic disease and is now approved for RA subjects (Cella et al, JRheumatology 32 (5): 811-819 (2005)).

SF-36 is a general Health-related quality of life tool, its psychometric properties have been extensively tested (psychometric properties), and is widely used in clinical and epidemiological studies (Ware et al, How to Score Version Two of the SF-36Health surface. Lincoln, RI: Qualitymetric Incorporated, 2000). SF-36 (2 nd edition) is supplied by Medical Outcomes Trust (Boston, MA, USA).

Swelling of 66 joints and tenderness of 68 joints should be evaluated by rheumatologists or skilled joint evaluators. The joints were evaluated at the time of physical examination based on pressure and joint massage (pressure and joint massage) and classified as swollen or non-swollen and tender or non-tender. Joints that have been completely arthrodesis/prosthesis (prosthesis) or arthrodesis (arthirodesis) should not be evaluated; however, all other joints should be evaluated. The joints to be evaluated for swelling and tenderness are provided below:

Temporomandibular joint

Sternoclavicular joint

Acromioclavicular joint

Shoulder pad^a

Elbow^*

Wrist type^*

Interphalangeal joints on the 1 st finger^a

Distal interphalangeal joint on the 2 nd-5 th finger

Proximal interphalangeal joints on the 2 nd to 5 th fingers^a

Metacarpophalangeal joints on the 1 st to 5 th fingers^a

Buttocks (pain only)

Knee^a

Ankle joint

Metatarsus

Interphalangeal joints in the 1 st to 5 th digits

Metatarsophalangeal joints on the 1 st to 5 th toes

^a28 joints were included for calculating a Disease Activity Score (DAS) 28.

Joints that have undergone the following procedure should be evaluated as follows:

● surgery: any joint that has been replaced or fused at any time prior to or during the study should be recorded as unestimable (NE) during the study.

Any joint that has undergone synovectomy (including chemical and radiological synovectomy)

Non-progression (ND) should be recorded 24 weeks after synovectomy. The joint may thereafter be evaluated again.

● Intra-articular injection: any joint that has received intra-articular corticosteroid injections should be scored as ND for the next 12 weeks. The joint may thereafter be evaluated again.

● arthrocentesis: any synovial aspirated joint was not evaluated at the next scheduled visit and rated ND. The joint may thereafter be evaluated again.

The physician should express the assessment of disease activity in the subject over the last 24 hours with a 100-mm level VAS, the left hand end of the line in the 100-mm level VAS representing no disease activity (no symptoms and no arthritic symptoms) and the right hand end representing maximum disease activity. This should be done by a efficacy evaluator, which may or may not be a physician.

CRP was analyzed in a central laboratory. The ESR was determined in the local laboratory by the Westgren method.

DAS 28-ESR ≧ 2.6 has been chosen as the threshold for retreatment in this test. Subjects with DAS 28-ESR ≧ 2.6 during weeks 24-40 can be eligible to receive a second course of study medication (rituximab or placebo).

A full physical examination (including cardiovascular, respiratory, gastrointestinal and nervous systems) should be conducted at the times specified in the assessment schedule. Any persistent anomalies should be recorded each time a check is conducted. If appropriate, a diagnosis of a new abnormality should be recorded as an adverse event.

Vital signs (heart rate, systolic and diastolic blood pressure, and body temperature) were acquired at times specified in the assessment schedule. The evaluation should be performed after the subject has been held in a semi-supine position for at least 5 minutes.

An Electrocardiogram (ECG) of twelve leads should be conducted at the time specified in the evaluation schedule.

Screening should be done with posteroanterior and lateral chest radiographs and reviewed by the investigator or assignees. At screening, if a chest radiograph that has been taken over the past 12 weeks does not show clinically significant abnormalities and there are no signs or symptoms of pulmonary disease suggesting that the subject may be excluded from the trial, the chest radiograph need not be repeated.

Performing hematology, serology, chemistry, urinalysis, serum pregnancy test, flow cytometry, immunology and CRP analysis by a central laboratory; pharmacokinetic and HACA analyses were performed by Genentech; urine pregnancy tests and ESR assessments were performed locally at the study site. Instruction manuals and replenishment kits were provided for all laboratory evaluations, including pharmacokinetics and HACA replenishment. Laboratory evaluations included the following items:

● hematology/CBC: hemoglobin, hematocrit, Red Blood Cells (RBC), White Blood Cells (WBC) (differential), and platelet count.

● serology: hepatitis b surface antigen (HBsAg) and Hepatitis C Virus (HCV) antibodies.

● serum chemistry: AST/SGOT, ALT/SGPT, alkaline phosphatase, total protein, albumin, total bilirubin, Blood Urea Nitrogen (BUN), uric acid, creatinine, random glucose (randmglucose), potassium, sodium, chloride, calcium, and phosphorus.

● urinalysis: blood, protein and glucose (microscopic examination if abnormal and appropriate).

● pregnancy test: all women with fertility potential (including those who have undergone tubal ligation) were tested for serum pregnancy at screening. In addition, a periodic urinary pregnancy test was performed at all other visits. If the urine pregnancy test is positive, it must be confirmed by a serum pregnancy test.

● serum C3 and C4 complement levels.

● immunological evaluation: immunoglobulins (total Ig, IgG, IgA and IgM), RF (total and isotype concentrations) and anti-cyclic-citrullinated peptide (CCP) antibodies (IgG) were quantified.

● Expanded Fluorescence Activated Cell Sorter (FACS) analysis: the cell populations evaluated included monocytes (CD14 and CD 16); NK cells (CD 56); t cell subsets (CD3, CD4, CD8, CD45RO, and CD45 RA); and B cell subsets (CD19, CD27, CD38, and IgD). Activation markers (CD25, CD69, CD40L, and CD80) can also be evaluated.

● FACS analysis of B cells: absolute B cells only (CD 19).

● for all enrolled subjects, HACA response analysis was performed using ELISA.

● pharmacokinetic testing: serum samples were taken at the visit specified in the SOA and at the same time point as HACA for pharmacokinetic testing. Serum rituximab concentrations are required to accurately interpret HACA results.

● optional biomarker sample:

for individually licensed subjects, optional study samples (whole blood, serum) were collected during the exploratory biomarker assessment study. Using PAXgene^TMWhole blood samples collected from RNA tubes were used to map gene expression profiles. Serum sample evaluation of markers involving RA or rituximab may include, but is not limited to, cytokine/chemokine measurements and quantification of bone and cartilage turnover (turnover) markers. To obtain the best data comparability, all samples were collected at the same time point.

The information obtained from this analysis is expected to facilitate and facilitate individualized healthcare (individulizedhealeath) by better understanding the mode of action of rituximab, the associated efficacy and safety, predictors of good response, and possibly the progression of RA (and other autoimmune) disease. These samples will be stored for up to 15 years after the database has expired.

All subjects must provide informed consent before any protocol or assessment specific to the study is performed, including changes to the subject's current treatment regimen. Screening assessments may be performed during the 56 days prior to the first rituximab infusion performed on day 1. The evaluation of the subject report should be conducted prior to other clinical evaluations.

If the subject at screening fails the laboratory exclusion criteria, the investigator may repeat the test up to two times during the screening period. If the subject fails the laboratory standard for the third time, they are considered as failing the screening. Blood samples or laboratory tests are not considered retests if they are re-sampled due to sample handling problems, breakage or sample integrity. Subjects who fail the screening can be screened again.

Subjects can be screened again if they do not meet all eligibility criteria within 56 days of the initial screening visit. Subjects who do this must repeat the entire screening process and re-approve before any procedure specific to the study is performed. Subjects can be screened only once again.

a. Screening visit (day-56)

● informed consent

● examination of selection and exclusion criteria

● contact IVRS to obtain assigned subject screening numbers

● demographic data (e.g., gender, age, race/ethnicity)

● complete medical history (including vaccination history)

● concurrent medications taken within the first 12 weeks of the screen, including vaccine, all previous DMARDs and biological agents

● Vital signs (Heart Rate, blood pressure and body temperature)

● comprehensive body examination including height and weight measurement

● Joint assessment

● 12 lead ECG

● chest X-ray

If chest X-rays have been administered within 12 weeks prior to screening and no clinically significant abnormalities are shown, chest X-rays need not be administered at the time of screening.

● Central laboratory evaluation

hematology/CBC

Hepatitis B surface antigen and hepatitis C antibody

Serum pregnancy test of women with fertility potential (including those with ligated oviducts)

Serum chemistry

Urine test

CRP

IgG and IgM

Total RF

● ESR (local laboratory; Westergren's method)

For each visit, all assessments during treatment should be made within a specified time window. The scheduled assessments and procedures performed on the day of rituximab administration should be completed prior to rituximab infusion unless otherwise indicated. For this study, day 1 was the day of the initial rituximab infusion. A day 1 visit should be conducted on a day that allows subsequent visits (e.g., day 15 visits) to be made without being delayed. The evaluation of the subject report should be conducted prior to other clinical evaluations. For a subject eligible for a re-treatment, the visit at which the subject meets the eligibility criteria for the re-treatment is considered a call to confirm eligibility.

a. Day 1

All assessments were performed 30 minutes prior to infusion, unless otherwise noted.

● examination of selection and exclusion criteria

● contact IVRS to obtain subject accession numbers and study drug lists

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● vital signs (heart rate, blood pressure and body temperature): pre-infusion, during infusion (every 15 minutes for 1 hour, then every 30 minutes until the end of infusion) and post-infusion (every 30 minutes for 1 hour post-infusion)

● body examination, including measurement of body weight

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● urinary pregnancy test of women with fertility potential, including those with ligated oviducts

● Central laboratory evaluation

hematology/CBC (pre-infusion and within 30 minutes post-infusion)

Serum chemistry

Urine test

Extended FACS (pre-infusion and within 30 minutes post-infusion)

CD19B cells (within 30 minutes before and after infusion)

CRP

Immunoglobulins

RF

anti-CCP antibodies

C3、C4

Pharmacokinetic samples (before and within 30 minutes after infusion)

HACA sample

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples)

● administration of Methylprednisolone

● administration of Rituximab

● adverse event

● concurrent medication (comitant mediations)

b. Day 15 (± 1 day)

● Central laboratory evaluation

hematology/CBC (Rituximab pre-infusion and within 30 minutes post-infusion)

Serum chemistry

Urine test

C3、C4

Pharmacokinetic samples (before and within 30 minutes after infusion)

● administration of Methylprednisolone

● administration of Rituximab

● adverse event

● concurrent medication

c. Weeks 4, 12 and 20 (days 28, 84 and 140; +. 3 days, respectively)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

● FACIT-F (week 12 only)

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS (week 12 only)

CD19B cells (week 4 and 20 only)

CRP

C3, C4 (week 4 only)

Pharmacokinetic sample

Immunoglobulins

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples) (week 12 only)

● adverse event

● concurrent medication

d. Week 24 (168 days. + -. 3 days)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● the DAS 28-ESR was calculated using the joint count from this visit and the patient's assessment of overall disease activity (VAS) and the ESR from the previous visit, if ESR results were not available.

The suitability (eligibility) of the subject for re-treatment was evaluated based on the calculated DAS 28-ESR and laboratory results from previous visits.

If the subject met the re-treatment criteria, day 1 of re-treatment was entered and the assessment was completed as indicated.

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS

CRP

Immunoglobulins

RF

anti-CCP antibodies

Pharmacokinetic sample

HACA sample

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples)

● adverse event

● concurrent medication

e. Week 28 (day 196. + -. 3)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

●This was done only when the subject was not treated again.DAS 28-ESR was calculated using the joint count from this visit and the patient's overall disease activity assessment (VAS) and ESR from the previous visit, if ESR results were not available.

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

CD 19B cells

CRP

Immunoglobulins

Pharmacokinetic sample

● ESR (local laboratory; Westergren's method)

● adverse event

● concurrent medication

f. Weeks 32, 40 and 44 (days 224, 280 and 308; day + -3, respectively)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

● FACIT-F (week 32 only)

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

●This was done only when the subject was not treated again.DAS 28-ESR was calculated using the joint count from this visit and the patient's overall disease activity assessment (VAS) and ESR from the previous visit, if ESR results were not available (only weeks 32 and 40).

The suitability (eligibility) of the subject for re-treatment was evaluated based on the calculated DAS 28-ESR and experimental results from previous visits.

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS (only when subjects were not treated again; only week 44)

CD19B cell

Immunoglobulins

CRP

Pharmacokinetic sample

● ESR (local laboratory; Westergren's method)

● adverse event

● concurrent medication

g. Week 48 (336 days. + -. 3 days)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS

CRP

Immunoglobulins

RF

anti-CCP antibodies

Pharmacokinetic sample

HACA sample

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples)

● adverse event

● concurrent medication

h. Week 60 (day 420. + -. 7)

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS

CRP

Immunoglobulins

Optional study biomarker samples (whole blood and serum samples)

● ESR (local laboratory; Westergren's method)

● adverse event

● concurrent medication

i. Week 72 (day 504. + -. 7)

Peripheral B cells (CD 19) at the completion of this visit⁺) Subjects who have not recovered are counted into the B cell follow-up phase. B cell recovery is defined as peripheral CD19 ⁺The count has returned to the baseline value or LLN whichever is lower.

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● Vital signs (Heart Rate, blood pressure and body temperature)

● physical examination including weight

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● 12 lead ECG

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

Extended FACS

CRP

Immunoglobulins

RF

anti-CCP antibodies

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples)

● adverse event

● concurrent medication

Subjects eligible for retreatment between cycles 24-40 were randomized to receive additional study drug sessions. Subjects should complete all follow-up day 1 assessments not performed during the qualifying visit (qualifying visit). The evaluation performed from the qualifying visit (qualifying visit) need not be repeated.

If an infusion cannot be given on the same day as the qualifying visit, the subject should return to give an infusion within 72 hours of the qualifying visit.

After a re-treatment session (days 1 and 15 of re-treatment), subjects should return from the point in time where they qualified for re-treatment to their next scheduled visit based on the assessment schedule. For example, if the subject met the re-treatment condition at week 32 visit, the subject's next scheduled visit should be week 40 visit after the re-treatment session (days 1 and 15 of re-treatment).

a. Second treatment day 1 (R1)

● randomization and distribution of study drugs by IVRS

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● body examination including body weight measurement

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● Central laboratory evaluation

hematology/CBC (pre-infusion and within 30 minutes post-infusion)

Serum chemistry

Urine test

CRP

Extended FACS (pre-infusion and within 30 minutes post-infusion)

CD19B cells (within 30 minutes before and after infusion)

Immunoglobulins

RF

anti-CCP antibodies

C3、C4

Pharmacokinetic samples (before and within 30 minutes after infusion)

HACA sample

● ESR (local laboratory; Westergren's method)

● optional study biomarker samples (Whole blood and serum samples)

● administration of Methylprednisolone

● administration of study drugs

● adverse event

● concurrent medication

b. Re-treatment day 15 (R15; +/-1 day)

● Central laboratory evaluation of urinary pregnancy tests for women with fertility potential, including those who have had fallopian tubes ligated

hematology/CBC (pre-infusion and within 30 minutes post-infusion)

Serum chemistry

Urine test

C3、C4

Pharmacokinetic samples (before and within 30 minutes after infusion)

● administration of Methylprednisolone

● administration of study drugs

● adverse event

● concurrent medication

Early treatment withdrawal (early treatment with a dry visit) was required for any reason to return from subjects who had withdrawn from the treatment period for an early treatment withdrawal visit (until 14 days after withdrawal), then returned on week 4 after withdrawal visit, and then returned every 12 weeks for at least 48 weeks from the time of subject withdrawal. The following assessments were made at the time of early treatment exit visit:

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

●FACIT-F

●SF-36

● Vital signs (Heart Rate, blood pressure and body temperature)

● physical examination including weight

● Joint assessment

● assessment of overall disease Activity by physicians (VAS)

● 12 lead ECG

● Central laboratory evaluation

hematology/CBC

Serum chemistry

Urine test

CRP

CD19B cell

Immunoglobulins

RF

anti-CCP antibodies

Pharmacokinetic sample

HACA sample

Optional study biomarker samples (whole blood and serum samples)

● ESR (local laboratory; Westergren's method)

● adverse event

● concurrent medication

Safety tracking (SFU) assessments were performed on weeks 4, 12, 24, 36, and 48 SFU following early withdrawal or completion of the determined treatment period for the following subjects:

● Subjects who were retreating between cycles 24-40 and completed the treatment period (i.e., visit at week 72)

● early withdrawal from treatment

The following evaluations were made at each SFU visit:

● Up to week 4 of all adverse events

● Severe adverse events and infectious adverse events after week 4

● concurrent medication for treating these events

● Central laboratory evaluation

hematology/CBC

Immunoglobulins

Pharmacokinetic sample

HACA sample

CD19B cell

Upon final SFU visit, peripheral B cells (CD 19)⁺) Subjects who have not recovered are counted into the B cell follow-up phase. B cell recovery is defined as peripheral CD19⁺The count has returned to the baseline value or LLN whichever is lower. For all severe infectious adverse events reported, CBC (with differential), quantitative Ig and CD19 counts should be determined within 1 week of onset.

Peripheral B cells (CD 19) at visit determined by last protocol (week 72 or end of SFU)⁺) Subjects who have not recovered are counted into the B cell follow-up phase and returned to study visits every 12 weeks since the last visit until B cells recover. B cell recovery is defined as peripheral CD19⁺The count has returned to the baseline value or LLN whichever is lower.

The following evaluations and procedures were performed at each B cell follow-up visit:

● Severe adverse events and infectious adverse events

● concurrent medication for treating these events

● Central laboratory evaluation

hematology/CBC

Immunoglobulins

Flow cytometry: b cell (CD 19)⁺) Counting

For all severe infectious adverse events reported, CBC should be determined within 1 week of onset

(with differential), quantitative Ig and CD19 counts.

Blood, serum and urine samples were taken at the indicated time points. Instructions for handling, storing, and transporting samples to Genentech and central clinical laboratories are provided in laboratory manuals. For hematology, serology, chemistry, serum β -hCG, flow cytometry, immunology and urinalysis, standard assays were used.

Rituximab pharmacokinetics ELISA measures rituximab levels in human serum samples. It uses affinity purified polyclonal goat anti-rituximab as a capture reagent, and uses goat anti-mouse IgG F (ab) conjugated with Horse Radish Peroxidase (HRP)₂The antibody is used as a detection reagent.

Anti-rituximab HACA ELISA is a bridging assay that uses rituximab as a capture reagent, biotinylated rituximab and streptavidin-HRP for detection. The assay uses a calibration curve prepared from affinity purified polyclonal goat anti-rituximab antibody and confirmed by immuno-subtraction using rituximab. The results of the test relative to the polyclonal antibody are reported in Relative Units (RU)/mL.

Subjects can withdraw or discontinue from the treatment session at any time, but should return to the study center within 14 days for early treatment withdrawal visits, SFU and potentially B-cell tracking. Subjects who have discontinued from this treatment session are tracked for safety assessment. Best efforts should be made to obtain these assessments in subjects who have aborted early. The primary reason for early termination must be recorded on the appropriate CRF page.

Reasons for subject discontinuation by the investigator include, but are not limited to, the following:

● authorized proactive exit

● any medical condition that may endanger the safety of a subject if he or she continues to participate in the study, at the discretion of the researcher

● investigator judges failure to meet maximum benefit of subject if continued

● Subjects with positive urine or serum pregnancy tests at any time during the study.

Early withdrawal subjects were no longer replaced.

Genentech has the right to terminate the study at any time. Reasons for terminating this study may include, but are not limited to, the following:

● the incidence or severity of adverse events in this or other studies indicates a potential health hazard to the subject.

● subject enrollment was not satisfactory.

● the data records are inaccurate or incomplete.

The study was designed to evaluate the efficacy of a course of treatment with rituximab or placebo for eligible subjects and to evaluate the safety of rituximab treatment in active RA subjects who are receiving MTX and have previously or currently inadequate response to one or more anti-TNF therapies. The eligibility of subjects undergoing re-treatment was based on DAS 28-ESR regression criteria (DAS 28-ESR ≧ 2.6). Approximately 555 subjects were added. The study was open label for the first course of treatment (with rituximab) and blinded for subjects eligible for a second course of treatment (study drug). Subjects meeting the re-treatment criteria were randomized to rituximab or placebo at a 2: 1 ratio during weeks 24-40.

Unless otherwise stated, all statistical tests were two-sided (two-sided) and were performed at a significance level of p ═ 0.05.

The primary endpoint was the proportion of subjects with ACR20 response at week 48 relative to baseline (day 1). Once week 48 assessments from all subjects entered the database and the database was cleared and frozen, analysis of non-blind data was initiated.

For the labeled open treatment session with rituximab (first 24 weeks of the study), the numbers of the added subjects are tabulated by the center. For subjects who discontinued from the labeled open treatment segment, the reason for discontinuation was summarized. The violation of selection criteria in key eligibility (virositions), other major protocol deviations (protocol definitions), and the number of subjects who completed each scheduled dose are summarized.

For the blinded treatment sessions with study drug, randomized subject numbers were tabulated by the center and treatment groups. The subject's configuration was summarized by the treatment group in terms of subject randomization, treatment, and completion of the study. Violation of key eligibility criteria, other major protocol deviations, and the number of subjects completing each scheduled dose are summarized by the treatment group. For subjects who had early discontinuations from the placebo-controlled, retreated segment, the reasons for discontinuation were summarized and listed by the treatment group.

The comparability of treatment group baseline was assessed in terms of demographic data (i.e., age, gender, race/ethnicity) and baseline characteristics (e.g., body weight, RA duration, baseline RF condition, baseline SJC/TJC, baseline DAS 28-ESR, and number of previous TNF therapies). The baseline value for any variable was defined as the last available value before the first rituximab administration (day 1).

Safety was assessed by a summary of adverse events, deaths, laboratory test results, vital signs and HACA. These summaries are provided as a comprehensive summary of the label open treatment segments by the treatment groups of placebo-controlled re-treatment segments. Safety analysis was based on subjects receiving any study drug dose. The subjects were analyzed according to the actual treatment received.

The following safety summary is included in the safety analysis.

Descriptions of treatment of emergency adverse events are written verbatim (mapped) to the MedDRA dictionary entry (the acurus term). Adverse events were tabulated according to system organ categories, treatment groups, and NCI CTCAE ratings. Adverse events were tabulated on a subject basis for severe adverse events, infection-related adverse events, rituximab-related infusion reactions, and adverse events leading to discontinuation of study medication, and tabulated after the first infusion of an initial course of rituximab and a second course of treatment.

In addition, severe adverse events were summarized as the incidence of each 100 patient-year following the initial rituximab course and the first infusion of the re-treatment course and throughout the observation period.

The subject's death and the leading causes of death are listed and/or summarized.

A narrative summary of laboratory values and changes from baseline throughout the study period is provided. The proportion of subjects experiencing treatment emergent laboratory abnormalities is summarized.

The treatment group provided a narrative summary of vital signs and changes from baseline throughout the study.

The proportion of subjects with measurable antibody responses to rituximab is summarized.

The intent-to-treat (ITT) population consisted of all subjects randomized into placebo-controlled re-treatment sessions and receiving any study drug. The ITT population is the primary analysis population for the primary and secondary endpoints. Subjects were analyzed for randomized treatment.

The primary endpoint was the proportion of retreating subjects with ACR20 response at week 48 compared to baseline (day 1).

To obtain ACR20, an improvement of 20% or more was required in both TJC and SJC compared to baseline, and similarly an improvement of 20% or more was required for three of the following five additional measurements:

● physician global disease activity assessment

● assessment of overall disease Activity of patients (VAS)

● evaluation of pain in patients (VAS)

●HAQ

● acute phase reactant (CRP or ESR)

CRP was used for the calculation of ACR 20. ESR is used if CRP is lost or not implemented.

Using the Cochran-Mantel Haenszel test statistics grouped by baseline RF conditions, a major analysis of the differences in ACR20response rates between placebo-treated and rituximab-treated groups was provided. The results are summarized narratively by treatment groups and expressed as a ratio, along with the corresponding adjusted 95% Confidence Intervals (CI) for the differences between response rates and p-values.

The ACR20response rates were also analyzed using logistic regression (logistic regression) and the correlation between ACR20 responses and treatment groups at Week 48 was examined while using logistic regression model to control baseline RF conditions (ACR20 responsiveness with a wide variety of sources for the association of logistic regression and treatment groups for the use of an association between ACR20 responses and treatment groups (ACR20 responsiveness with a wide variety of sources for the use of an association between ACR20 responsiveness at Week 48and a genetic control for the use of a logistic RF status). The parameter estimates from the model were examined by tabulating standard error, Wald statistics, and odds ratios (odds ratios) with corresponding 95% CI and p values.

Sensitivity analysis was also performed by adjusting possible explanatory items of the ACR20 response (e.g., baseline SJC).

The secondary endpoint was analyzed as follows:

● the proportion of subjects with ACR50 and ACR70 responses at week 48 was analyzed using the same approach as specified for the primary endpoint.

● DAS 28-ESR changes from baseline to week 48 were evaluated using an analysis of variance (ANOVA) model in which placebo/rituximab-retreated groups and baseline RF conditions were used as explanatory terms.

● analyzed the sorted (ordered) ACR response categories (ACR70 responders, ACR50-70 responders, ACR20-50 responders, and ACR20 non-responders) at week 48 using an additive logits model (logits model) grouped by baseline RF conditions. The parameter estimates from the model were examined by tabulating standard error, Wald statistics, and odds ratios with corresponding 95% CI and p values.

● the EULAR response rates (excellent or moderate) were analyzed in the same manner as specified for the primary endpoint.

● the ACR core set (SJC, TJC, HAQ, patient and physician global assessments, pain, CRP, and ESR) was analyzed for changes from baseline to week 48 in an ANOVA model in which placebo/rituximab-retreated groups and baseline RF status were used as instructions.

● the AUC of week 48 ACRn and week 48 ACRn was evaluated using an analysis of variance (ANOVA) model in which placebo/rituximab re-treatment groups and baseline RF conditions were used as an explanatory term.

● for eight regional (domain) scores and mental and somatic component scores, the SF-36 sub-scale (subscale) and summary scores were reported as a change from baseline to week 48 using the analysis in the ANOVA model in which the placebo/rituximab retreatment group and baseline RF conditions were used as instructions. In addition, mental and somatic component scores are further classified and analyzed.

● the change from baseline to week 48 was assessed using the analysis FACIT-F as analyzed in the ANOVA model in which placebo/rituximab re-treatment groups and baseline RF conditions were used as explanatory terms.

● the proportion of subjects who obtained DAS 28-ESR regression at week 48 (DAS 28-ESR < 2.6) was analyzed in the same manner as specified for the primary endpoint.

● the proportion of subjects at week 48 who had obtained a DAS 28-ESR low condition (DAS 28-ESR. ltoreq.3.2) was analyzed in the same manner as specified for the primary endpoint.

Pharmacokinetic (PK) parameters derived from rituximab serum concentrations were estimated for all subjects using a population PK model, including maximum serum concentration (C) _max) Time of maximum serum concentration (T)_max) Area under concentration-time curve (AUC), systemic Clearance (CL), volume of distribution (V) and half-life (t)_1/2). The distribution phase may not be well characterized due to the sparse sampling. PK data from this study were combined with data from other studies for population PK analysis.

For population PK analysis, the overall mean PK parameters were estimated across the study population, along with estimates of intra-and inter-subject variation and estimates of random error. Estimates of individual subject parameters were calculated using post hoc analysis procedures. A prospective analysis plan (prospective analysis plan) was prepared and population PK analyses were provided in separate reports from clinical study reports.

The population PK analysis includes an exploratory analysis to identify baseline covariates that affect the pharmacokinetics of rituximab in this patient population. The baseline covariates to be tested included demographics and other subject characteristics such as disease severity and selected laboratory measurements.

Data were summarized using descriptive statistics including mean, standard deviation, geometric mean, coefficient of variation, median, and range.

Potential pharmacodynamic markers from blood samples are descriptively summarized, including B cell counts, quantitative Ig levels, lymphocyte subtypes, and HACA concentrations. For these analyses, baseline values determined from day 1 pre-dose samples were used to calculate the change from baseline at each sampling time point.

Exploratory Analysis was performed to assess possible correlations between pharmacodynamic markers, PK measurements and clinical responses and detailed in the Statistical Analysis Plan (Statistical Analysis Plan).

Approximately 555 subjects were added. With a 25% withdrawal rate for the label open treatment zone, a 10% withdrawal of DAS 28-ESR was obtained at week 24 and subjects without re-treatment, and a 2: 1 randomization ratio, this sample size was 80% capable (power) in detecting a 16% difference in ACR20 response rate from baseline to week 48 between the rituximab re-treatment group (50% of ACR20 responders) and the placebo group (34% of ACR20 responders) using the Fisher's exact test.

Safety assessments include monitoring and recording protocol-determined Adverse Events (AEs) and Severe Adverse Events (SAE); protocol-specific laboratory (hematology, clinical chemistry, and urinalysis) variable measurements; protocol-specified vital sign measurements; and other protocol-specified tests deemed critical for safety assessment of research drugs.

To monitor infusion-related responses, vital signs were obtained every 15 minutes for the first hour of the infusion period, then every 30 minutes for the remainder of the infusion, and every 30 minutes for one hour after infusion, on the day of study drug administration, just prior to infusion. Other readings may be taken in the event of an infusion-related reaction (e.g., hypotension and/or fever).

An AE is any disliked and unwanted sign, symptom or disease, regardless of cause (attribute), temporarily associated with an intervention applied using a research (medical) product or other protocol.

This includes the following:

●, an AE in the subject not observed before the subject that occurred during the protocol-specified AE reporting period, including signs or symptoms associated with RA that did not occur before the AE reporting period

● complications that occur as a result of a regimen-mandatory intervention (e.g., an interventional procedure such as a biopsy)

● AE related to medication exclusion, no treatment run (no treatment run-in) or other protocol-mandatory intervention, occurring prior to dispensing study treatment, if applicable

● judged by investigators, pre-existing medical conditions (non-investigational conditions) that worsen in severity or frequency or change in trait during a protocol-specified AE reporting period should be classified as SAE if the AE meets the following criteria:

● it caused death (i.e., the AE actually triggered or caused death).

● it is life threatening (i.e., the AE, as seen by investigators, immediately places the subject at risk of death.

● it requires or prolongs patient admission therapy.

● it results in persistent or major disability/disability (i.e., the AE results in substantial disruption of the subject's ability to perform normal vital functions).

● it causes congenital anomalies/birth defects (birth defects) in the newborn/baby birth for mothers exposed to the investigational product.

● investigators consider it to be a significant medical event according to medical judgment (e.g., the subject may be compromised or medical/surgical intervention may be required to prevent one of the outcomes listed above).

All AEs that do not meet any severe criteria should be considered non-severe AEs.

The terms "severe (severe)" and "severe (serious)" are not synonymous. Severity (or intensity) refers to the grade of a particular AE, e.g., mild (grade 1), moderate (grade 2), or severe (grade 3) myocardial infarction. "severe" (Serious) is a regulatory concept based on subject or event outcomes or action criteria, which is generally associated with events that threaten the life or function of a subject. Severity (non-severity) is used as a guide to determine regulatory reporting obligations (regulatory reporting authorities) from the sponsor as applicable regulatory rights (applicable regulatory authorities).

Severity and severity should be assessed independently when AE and SAE are recorded on CRF.

Investigators assessed all subjects during the study for the occurrence of AEs and SAEs at time points. All AEs and SAEs, whether provided voluntarily by the subject, discovered by the investigator during interrogation, or detected by physical examination, laboratory testing, or other means, are recorded in the subject's medical record and on the appropriate AE or SAE CRF page.

Each AE or SAE recorded is described by its duration (i.e., start and end dates), severity (see table 2), regulatory severity criteria (if applicable), guessed relevance to the investigational product (see guidance below), and action taken.

The AE rating (severity) scale found in NCI CTCAE, V3.0 was used for AE reporting.

TABLE 2

Adverse event rating (severity) scale

Note: regardless of severity, some events may also meet regulatory severity criteria.

See SAE definition.

a use of observed or reported AEs when not in the NCI CTCAE List

Alternative definitions for level 1, 2, 3, and 4 events.

To ensure consistency in the causal evaluation of AE and SAE, the investigator should apply the following general guidelines:

● is

There is a plausible temporary correlation between the onset of an AE and the administration of an investigational product, and the AE cannot be easily explained by the clinical status of the subject, intercurrent disease, or concurrent treatment; and/or the AE follows a known pattern of response to the investigational product; and/or AE is reduced or eliminated upon discontinuation of the investigational product or dose reduction, and reappears upon re-challenge, if applicable.

● No

There is evidence that the AE has a non-investigational product etiology (e.g., preexisting medical condition, underlying disease, intercurrent disease, or concurrent medication); and/or a temporary association of the AE with the absence of plausibility of the investigational product (e.g., cancer diagnosed 2 days after initial dose of study drug).

Note: the investigator's assessment of the causality of an individual AE report is part of the study record process. Regardless of the causal assessments of "yes" or "no" reported for individual AEs, the sponsor rapidly assesses all SAEs reported for cumulative product experience to identify and rapidly communicate to investigators and appropriate regulatory authorities (regulatory issues) potential new safety findings.

An RA should be recorded as AE or SAE only if the investigator concludes at any time during the study that the severity and/or frequency has unexpectedly deteriorated or that the attributes have changed. When an unexpected worsening of rheumatoid arthritis is noted on the AE or SAE CRF pages, it is important that the expression of the condition by including an appropriate descriptor (e.g., "accelerated rheumatoid arthritis") has changed.

Retreatment according to the protocol herein (or with a different CD20 antibody) is effective to prevent or slow the progression of structural joint damage and erosion by RA. Monotherapy with the CD20 antibody, without MTX, yielded efficacious results.

Example 4: efficacy studies of rituximab in psoriatic arthritis patients

The protocol of example 2 was followed except that the patients were treated for joint damage caused by psoriatic arthritis. Similar results were observed for rheumatoid arthritis (using rituximab or humanized 2H7 antibody), i.e., prevention of progression of structural joint injury was achieved at more time points of at least about one month from baseline or start of treatment, preferably at least 24 weeks from baseline, more preferably at least 52 weeks from baseline, and up to 104 weeks from baseline, after the first dose of CD20 antibody (which may be appropriately adjusted depending on, e.g., the dose, with or without methotrexate).

These rituximab-based regimens challenge current standard of care, improve net clinical benefit, and primarily aim to prevent progression of joint damage. These results were significantly better than those of the control group.

Administration of rituximab or humanized 2H7 to the subject at the predetermined re-dosing schedule set forth in example 2 was effective at preventing the progression of structural joint damage at or after week 52. These results are significantly better than the results of the control.

Administration of another dose of 1g or 2g of CD20 antibody (e.g., rituximab or humanized 2H7) in one dose at about weeks 48-54 or dispersed over a period of about 14-16 days in an amount of 0.5 g or 1g of CD20 antibody is also effective to treat joint damage throughout the second year, with or without one or more second agents such as immunosuppressive agents. Thus, successful administration of the CD20 antibody first over a period of about 2 weeks, followed by another treatment at about 4-8 months from the initial treatment (calculated from the administration of any dose), followed by another treatment at about one year from the initial treatment, followed by treatment at about two years from the initial treatment, together with about one-half gram or one gram x 2 "4 doses for each treatment, administered about once per week or about once every other week over a period of about two to four weeks, was successful. The results of this treatment were much better than the results of the control with placebo. The re-treatment regimen is successfully used for years with no or minimal side effects.

Example 5: therapeutic study of Rituximab efficacy in osteoarthritis and Crohn's disease patients

The results of example 2 were successful in cases where the patient had osteoarthritis or joint damage due to crohn's disease, whether rituximab or another CD20 antibody such as humanized 2H7 was used, and whether an immunosuppressive agent was used or not, with adjustments in the dose being known to those skilled in the art. Also, if these patients were first treated with the CD20 antibody and then re-treated with this antibody about six months or about one year after the first treatment, they continued to experience prevention of the progression of joint damage for an extended period of time, otherwise using the same dosing and other regimens of example 2.

Rituximab or other CD20 antibodies are also at least as effective as conventional treatment regimens in inducing and maintaining regression of joint damage, providing substantial advantages over standard therapies by virtue of their superior side effect profile, e.g., less toxicity and better recovery tolerance than steroids.

Patients in the treatment group tolerated well rituximab infusion and their B-cell shortness (swiftly) was depleted. In addition to methotrexate, additional immunosuppressive agents are not necessary to induce regression and maintain sustained regression (6 months or longer) in CD20 antibody treated patients, if desired.

Claims

1. Use of rituximab in the manufacture of a medicament or article for preventing or reducing the rate of progression of structural joint damage caused by rheumatoid arthritis, by subjecting a subject to a radiographic examination at least one month after administration of said medicament or article to said subject, as determined in comparison to the radiographic results prior to said administration.

2. The use of claim 1, wherein the rheumatoid arthritis is active rheumatoid arthritis.

3. The use of claim 1, wherein the medicament or article of manufacture comprises rituximab and methotrexate.

4. The use of any of claims 1-3 wherein the results of the radiographic testing are expressed as a change in total modified Sharp score.