JP2011501734A - Single fixed infusion dose of ocrelizumab (2H7) - Google Patents

Abstract

The present invention relates to the use of a CD20 antagonist in the manufacture of a medicament for the treatment of an autoimmune disease, which medicament is administered to a human patient in a single therapeutically effective intravenous infusion (iv). Is to do. The CD20 antagonist is preferably a CD20 antibody and the autoimmune disease is preferably rheumatoid arthritis.

Description

The present invention relates to the treatment of autoimmune diseases, including rheumatoid arthritis, by the administration of CD20 antibodies using a treatment regimen that includes a single full effective dose of intravenous infusion at the start of treatment.

Background of the Invention Lymphocytes are one of several populations belonging to white blood cells, which specifically recognize and respond to foreign antigens. The three main categories of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK) cells. B lymphocytes are cells involved in antibody production and provide humoral immunity. B cells mature in the bone marrow, exit the bone marrow, and express antigen-binding antibodies on the cell surface. When a naive B cell first encounters an antigen specific for its membrane-bound antibody, it rapidly divides and differentiates its progeny into memory B cells and effector cells called “plasma cells”. Memory B cells have a longer life span and continue to express membrane-bound antibodies with the same specificity as the original parental cells. Instead of producing membrane-bound antibodies, plasma cells produce secreted antibodies. Secreted antibodies are the main effector molecules of humoral immunity.

  The CD20 antigen (also called human B lymphocyte restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al., J. Biol. Chem). 264 (19): 11282-11287 (1989); and Einfeld et al., EMBO J. 7 (3): 711-717 (1988)). This antigen is also expressed on more than 90% of B-cell non-Hodgkin lymphoma (NHL) (Anderson et al., Blood 63 (6): 1424-1433 (1984)), but hematopoietic stem cells, pro-B cells, normal Found on normal plasma cells, or other normal tissues (Tedder et al., J. Immunol. 135 (2): 973-979 (1985)). CD20 is believed to control the initial steps in activation methods for cell cycle initiation and differentiation (Tedder et al., Supra) and possibly functions as a calcium ion channel (Tedder et al., J. Cell. Biochem. 14D: 195 (1990)).

  When CD20 is expressed in B cell lymphomas, this antigen is a useful therapeutic target for treating such lymphomas. In the United States, more than 300,000 people suffer from B-cell NHL, and 56,000 new cases are diagnosed each year. CD20 is also a useful target antigen for treating autoimmune diseases.

  Rituximab (RITUXAN®, a Mabthera® in Europe) antibody (Genentech, Inc., South San Francisco, Calif., USA), a genetically engineered chimeric mouse / human monoclonal antibody directed against the human CD20 antigen. Has been used to treat patients with relapsed or refractory, low-grade or follicular, CD20 positive, B-cell non-Hodgkin lymphoma. Rituximab is an antibody also referred to as “C2B8” in US Pat. No. 5,736,137 (Anderson et al.) And US Pat. No. 5,776,456, issued April 7, 1998.

  Rituximab has been studied in a variety of non-malignant autoimmune diseases where B cells and autoantibodies appear to be involved in the pathophysiology of the disease. Edwards et al., Biochem Soc. Trans. 30: 824-828 (2002). Rituximab is, for example, rheumatoid arthritis (RA) (Leandro et al., Ann. Rheum. Dis. 61: 883-888 (2002); Edwards et al., Arthritis Rheum., 46 (Suppl. 9): S46 (2002); Stahl et al. Ann. Rheum. Dis., 62 (Suppl. 1): OP004 (2003); Emery et al., Arthritis Rheum. 48 (9): S439 (2003)), Lupus (Eisenberg, Arthritis. Res. Ther. 5: 157 -159 (2003); Leandro et al., Arthritis Rheum. 46: 2673-2677 (2002); Gorman et al., Lupus, 13: 312-316 (2004)), immune thrombocytopenic purpura (D 'Arena et al., Leuk) Lymphoma 44: 561-562 (2003); Stasi et al., Blood, 98: 952-957 (2001); Saleh et al., Semin. Oncol., 27 (Supp 12): 99-103 (2000); Zaja et al., Haematolgica , 87: 189-195 (2002); Ratanatharathorn et al., Ann. Int. Med., 133: 275-279 (2000)), erythroblast wax (Auner et al., Br. J. Haematol., 116: 725-728 (2002)), autoimmune anemia (Zaja et al., Haematologica 87: 189-195 (2002) (Typographical error in Haematologica 87: 336 (2002))), cold agglutinin disease (Layios et al., Leukemia, 15: 187- 8 (2001); Berentsen Blood, 103: 2925-2928 (2004); Berentsen et al., Br. J. Haematol., 115: 79-83 (2001); Bauduer, Br. J. Haematol., 112: 1083-1090 (2001); Zaja et al., Br. J. Haematol., 115: 232-233 (2001)), severe insulin resistance type B syndrome (Coll et al., N. Engl. J. Med., 350: 310-311 (2004)), Mixed cryoglobulinemia (DeVita et al., Arthritis Rheum. 46 Suppl. 9: S206 / S469 (2002)), myasthenia gravis (Zaja et al., Neurology, 55: 1062-63 (2000); Wylam et al. Pediatr., 143: 674-677 (2003)), Wegener's granulomatosis (Specks et al., Arthritis & Rheumatism 44: 2836-2840 (2001)), refractory pemphigus vulgaris (Dupuy et al., Arch Dermatol., 140: 91-96 (2004)), dermatomyositis (Levine, Arthritis Rheum., 46 (Suppl. 9): S1299 (2002)), Sjogren's syndrome (Somer et al., Arthritis & Rheumatism, 49: 394-398 (2003)), Acute type II mixed cryoglobulinemia (Zaja et al., Blood, 101: 3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch. Dermato l., 140: 91-95 (2004)), autoimmune neuropathy (Pestronk et al., J. Neurol. Neurosurg. Psychiatry 74: 485-489 (2003)), paraneoplastic ocular clonus-myoclonus syndrome (Pranzatelli et al. Neurology 60 (Suppl. 1) PO5.128: A395 (2003)), and is reported to strongly relieve the signs and symptoms of relapsing-remitting multiple sclerosis (RRMS). Cross et al. (Abstract) "Preliminary Results from a Phase II Trial of Rituximab in MS" Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis, 20-21 (2003).

  A phase II study (WA16291) was conducted in patients with rheumatoid arthritis (RA) and provided 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 randomly assigned equally to four treatment groups: methotrexate, rituximab only, rituximab + methotrexate, and rituximab + cyclophosphamide (CTX). The treatment regimen for rituximab was 1 g administered intravenously on days 1 and 15. In most patients with RA, rituximab infusion was well tolerated by most patients, and 36% of patients had at least one adverse event during the first infusion (as opposed to 30% of patients receiving placebo). Overall, most adverse events appeared to be mild to moderate in severity and were unbiased in all treatment groups. There were a total of 19 serious adverse events in the 4 treatment groups over 48 weeks, with a slightly higher frequency in the rituximab / CTX group. The incidence of infection was unbiased across all groups. The average prevalence of serious infections in this RA patient population is 4.66 per 100 patients per year, which is necessary for hospitalization in RA patients reported in community-based epidemiological studies. Lower than infection rate (9.57 per 100 patients per year). Doran et al., Arthritis Rheum. 46: 2287-2293 (2002).

  Rituximab reported in a small number of 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) The safety profile of was similar to that reported in oncology or RA. In an ongoing investigator-initiated trial (IST) of rituximab in combination with interferon-β (INF-β) or glatiramer acetate in patients with RRMS, 1 in 10 patients treated was the first of rituximab After having had a moderate fever and chills after the first infusion, he was hospitalized for overnight observation, but the other nine patients completed four infusion regimens without any reported adverse events.

Patents and patent publications relating to CD20 antibodies, CD20 binding molecules, and autoantigen vaccines include US Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399, 061, and 6,682,734, as well as U.S. Published Patent No. 2002/0197255, U.S. Published Patent No. 2003/0021781, U.S. Published Patent No. 2003/0082172, U.S. Published Patent No. 2003/0095963, US Publication No. 2003/0147885, US Publication No. 2005/0186205 and International Publication No. 1994/11026 (Anderson et al.);
US Pat. No. 6,455,043, US Publication No. 2003/0026804, US Publication No. 2003/0206903, and International Publication No. 2000/09160 (Grillo-Lopez, A.), International Publication No. 2000/27428 (Grillo-Lopez and Leonard), US Published Patent No. 2004/0213784, and International Publication No. 2000/27433 (Grillo-Lopez and Leonard), International Publication No. 2000/44788 (Braslawsky et al.). International Publication No. 2001/10462 (Rastetter, W.), International Publication No. 2001/10461 (Rastetter and White); International Publication No. 2001/10460 (White and Grillo-Lopez), US Publication No. 2001/0018041, U.S. Published Patent No. 2003/0180292, U.S. Published Patent 2002/0028. 78 No., WO 2001/34194, and WO 2002/22212 (Hanna and Hariharan), U.S. Publication No. 2002/0006406 and WO 2002/04021 (Hanna and Hariharan);
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Scientific publications on treatment with rituximab include Perotta and Abuel, "Response of chronic relapsing ITP of 10 years duration to rituximab" Abstract # 3360 Blood 10 (1) (part 1-2): p. 88B (1998 Perotta et al., "Rituxan in the treatment of chronic idiopathic thrombocytopaenic purpura (ITP)", Blood, 94: 49 (abstract) (1999); Matthews, R., "Medical Heretics", New Scientist (April 7, 2001) Leandro et al., “Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion”, Ann Rheum Dis, supra; Leandro et al., “Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficiency and dose response”, Arthritis and Rheumatism 44 (9): S370 (2001);
Leandro et al., “An open study of B lymphocyte depletion in systemic lupus erythematosus”, Arthritis and Rheumatism, 46: 2673-2677 (2002), where each patient received two 500 mg infusions of rituximab, Two 750 mg infusions of cyclophosphamide and high oral corticosteroids were given, 2 of the treated patients relapsed after 7 and 8 months respectively, and retreated with different protocols "Successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy" Weide et al., Lupus, 12: 779-782 (2003), where patients were treated with rituximab (375 mg once a week) / m ² × are treated with 4 iterations), and subjected to further rituximab applications every 5-6 months and then subjected to maintenance therapy with rituximab in 375 mg / m ² every three months, and refractory A second patient with LE is successfully treated with rituximab, and, upon receiving maintenance therapy to every three months, where both patients responding well against rituximab therapy;
Edwards and Cambridge, "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes" Rheumatology 40: 205-211 (2001); Cambridge et al., "B lymphocyte depletion in patients with rheumatoid arthritis: serial studies of immunological parameters" Arthritis Rheum ., 46 (Suppl. 9): S1350 (2002); Cambridge et al., "Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis" Arthritis Rheum., 48: 2146-2154 (2003); Edwards et al., "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders "Biochem Soc.Trans., supra; Edwards et al.," Efficacy and safety of rituximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis.Arthritis and Rheumatism 46 (9): S197 (2002);
Edwards et al., “Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoide arithritis” N Engl. J. Med. 350: 2572-2582 (2004); Pavelka et al., Ann. Rheum. Dis. 63: (S1) : 289-290 (2004); Emery et al., Arthritis Rheum. 50 (S9): S659 (2004); Levine and Pestronk, "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using rituximab" Neurology 52: 1701-1704 ( 1999); Uchida et al., "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J. Exp. Med. 199: 1659-1669 (2004); Gong et al., "Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy "J. Immunol. 174: 817-826 (2005); Hamaguchi et al.," The peritoneal cavity provides a protective niche for B1 and conventional B lymphocytes during anti-CD20 immunotherapy in mice "J. Immunol .174: 4389-4399 (2005); Cragg et al., "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun. 8: 140-174 (2005); Eise nberg, "Mechanisms of autoimmunity" Immunol. Res. 27: 203-218 (2003);
DeVita et al., "Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis" Arthritis & Rheum 46: 2029-2033 (2002); Higashida et al, "Treatment of DMARD-refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology; Oct24-29; (Abstract # LB11), New Orleans, LA2002; Tuscano, J. "Successful treatment of infliximab-refractory rheumatoid arthritis with rituximab" Annual Scientific Meeting of the American College of Rheumatology; Oct24 -29; published in New Orleans, LA 2002, and published in Tuscano, Arthritis Rheum. 46: 3420 (2002); "Pathogenic roles of B cells in human autoimmunity; insights from the clinic" Martin and Chan, Immunity20 : 517-527 (2004);
Silverman and Weisman, "Rituximab therapy and autoimmune disorders, prospects for anti-B cell therapy", Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz and Isenberg, "Anti B cell therapy (rituximab) in the treatment of autoimmune diseases ", Current opinion in pharmacology, 4: 398-402 (2004); Virgolini and Vanda," Rituximab in autoimmune diseases ", Biomedicine & pharmacotherapy, 58: 299-309 (2004); Klemmer et al.," Treatment of antibody mediated autoimmune disorders with a AntiCD20 monoclonal antibody Rituximab ", Arthritis And Rheumatism, 48: (9) 9, S (SEP), page: S624-S624 (2003); Kneitz et al.," Effective B cell depletion with rituximab in the treatment of autoimmune diseases "Immunobiology, 206: 519-527 (2002); Arzoo et al.," Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab) "Annals of the Rheumatic Diseases, 61 (10), p922-924 ( 2002) Comment in Ann Rheum Dis. 61: 863-866 (2002);
"Future strategies in immunotherapy", published by Lake and Dionne, in Burger's Medicinal Chemistry and Drug Discovery (2003, John Wiley & Sons, Inc.), published online January 15, 2003 (Chapter2 "Antibody-Directed Immunotherapy"); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target, published online article entitled “CD20”: January 15, 2002; entitled “Monoclonal Antibodies to Human Cell Surface Antigens” Appendix 4A, edited by Stockinger et al .: Coligan et al. In Current Protocols in Immunology (2003 John Wiley & Sons, Inc.), posted online: May 2003; printed publication date: February 2003; Penichet and Morrison, "CD Antibodies / molecules: Definition; Antibody Engineering" in the Wiley Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies; posted online on January 15, 2002 It is.

Furthermore, Looney "B cells as a therapeutic target in autoimmune diseases other than rheumatoid arthritis" Rheumatology, 44 Suppl2: ii13-ii17 (2005); Chambers and Isenberg, "Anti-B cell therapy (rituximab) in the treatment of autoimmune diseases" Lupus 14 (3): 210-214 (2005); Looney et al., “B-cell depletion as a novel treatment for systemic lupus erythematosus: a phase I / II dose-escalating trial of rituximab” Arthritis Rheum. 50: 2580-2589 ( 2004); Looney, "Treating human autoimmune disease by depleting B cells" Ann Rheum.Dis.61: 863-866 (2002); Edelbauer et al., "Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression Case report" Pediatr.Nephrol. 20 (6): 811-813 (2005); D'Cruz and Hughes, "The treatment of lupus nephritis" BMJ 330 (7488): 377-378 (2005);
Looney, "B cell-targeted therapy in diseases other than rheumatoid arthritis" J. Rheumatol.Suppl. 73: 25-28; discussion29-30 (2005); Sfikakis et al., "Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand: an open-label trial "Arthritis Rheum.52 (2): 501-513 (2005); Rastetter et al.," Rituximab: expanding role in therapy for lymphomas and autoimmune diseases " Annu.Rev.Med.55: 477-503 (2004); Silverman, "Anti-CD20 therapy in systemic lupus erythematosus: a step closer to the clinic" Arthritis Rheum.52 (2): 371-7 (2005), Arthritis Rheum. 52 (4): 1342 (2005) typo; Ahn et al., "Long-term remission from life-threatening hypercoagulable state associated with lupus anticoagulant (LA) following rituximab therapy" Am. J. Hematol. 78 (2) : 127-129 (2005);
Tahir et al., "Humanized anti-CD20 monoclonal antibody in the treatment of severe resistant systemic lupus erythematosus in a patient with antibodies against rituximab" Rheumatology, 44 (4): 561-562 (2005), published online 11 January 2005 Looney et al., "Treatment of SLE with anti-CD20 monoclonal antibody" Curr. Dir. Autoimmun. 8: 193-205 (2005); Cragg et al, "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun. 8: 140-174 (2005); Gottenberg et al., "Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases" Ann. Rheum. Dis. 64 (6): 913-920 (2005) online Published November 18, 2004; Tokunaga et al., "Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab" Rheumatology 44 (2): 176-182 (2005) See online publication date 19 October 2004. See also Leandro et al., “B cell repopulation occurs mainly from naive B cells in patient with rheumatoid arthritis and systemic lupus erythematosus” Arthritis Rheum., 48 (Suppl9): S1160 (2003).

Specks et al., "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism 44 (12): In 2836-2840 (2001), the treatment of Wegener's granulomatosis is, four of rituximab 375mg / m ² Have been successfully used with iv drip infusions and high doses of glucocorticoids. This therapy was performed again 11 months after cANCA relapses, but the therapy did not use glucocorticoids. Eight months after the second course of rituximab, the patient's illness remained in complete remission. Furthermore, in another study, rituximab was used at a dose of 375 mg / m ² × 4 with oral prednisone 1 mg / kg / day (decreased to 40 mg / day by week 4 and then discontinued for 16 weeks). When found, rituximab was found to be well tolerated and an effective remission inducer for severe ANCA-related vasculitis. Four patients were retreated with rituximab alone for ANCA recurrence / ANCA titer increase. Apart from glucocorticoids, no further immunosuppressive agents appear to be necessary for induction of remission and maintenance of sustained remission (over 6 months). In Keogh et al., Kidney Blood Press. Res. 26: 293 (2003), 11 patients with refractory ANCA-related vasculitis were administered four times 375 mg / m ² rituximab and high dose once a week. It was reported that treatment with glucocorticoids resulted in remission.

Administration of rituximab to patients with refractory ANCA-associated vasculitis with immunosuppressive drugs (eg, intravenous cyclophosphamide, mycophenolate mofetil, azathioprine, or leflunomide) was clearly effective . Eriksson, “Short-term outcome and safety in 5 patients with ANCA-positive vasculitis treated with rituximab”, Kidney and Blood Pressure Research, 26: 294 (2003) (once every week for 4 weeks, 375 mg / m ² rituximab 5 patients with ANCA-related vasculitis treated with) responded to that treatment; Jayne et al., “B-cell depletion with rituximab for refractory vasculitis” Kidney and Blood Pressure Research, 26: 294-295 (2003) (Six patients with refractory vasculitis who received background immunosuppression and infusion of 375 mg / m ² rituximab with prednisolone and cyclophosphamide once a week, 4 times a week had high vasculitis activity. Decreased). A further report using four 375 mg / m ² / dose rituximab with intravenous cyclophosphamide for administration to patients with refractory systemic vasculitis is Smith and Jayne, A prospective, open label trial of B-cell depletion with rituximab in refractory systemic vasculitis, poster ^{998 (11 th International Vasculitis and ANCA} workshop), American Society of Nephrology, J. Am Soc Nephrol, 14:... are provided in p.755A (2003) Yes. Eriksson, J. Internal Med., 257: 540-548 (2005) for 9 patients with ANCA positive vasculitis successfully treated with 2 or 4 rituximab doses of 500 mg / week, and B lymphocytes in 11 patients with refractory ANCA-related vasculitis treated or retreated 4 times with a 375 mg / m ² / week dose of rituximab from January 2000 to September 2002 See also Keogh et al., Arthritis and Rheumatism, 52: 262-268 (2005), which reports that remission was induced by depletion.

  Regarding the activity of the humanized anti-CD20 antibody, see, for example, Vugmeyster et al., “Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca fascicularis” J. Immunother. 28: 212-219 (2005). For a discussion on human monoclonal antibodies, see Baker et al., “Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator” Arthritis Rheum. 48: 3253-3265 (2003). Furthermore, the MINT trial with rituximab to treat high-grade non-Hodgkin's lymphoma in a young patient was successful (Lancet Oncology, April 5, 2006).

  Trubion Pharmaceuticals Inc. has recently announced that TRU- The safety and efficacy of an anti-CD20 antibody fragment type molecule designated 015 was evaluated. These patients were randomly and equally divided into five groups that received placebo, 200 mg, 400 mg, 800 mg, or 1600 mg of TRU-015 by a single intravenous infusion. TRU-015 was generally reported to be well tolerated.

  Drugs are treated in such a way that they are effective, safe, well tolerated by patients with little or no side effects, and can be carried out advantageously in daily medical care so as to be as inconvenient to the patient as possible. It is beneficial to be able to administer As will be apparent from the detailed description below, the present invention satisfies this need by providing a new protocol for the administration of CD20 antibodies in the treatment of autoimmune diseases.

SUMMARY OF THE INVENTION In one aspect, the present invention relates to the treatment of human patients with autoimmune disease comprising administering to a patient a complete therapeutically effective amount of a CD20 antagonist as a single intravenous infusion (iv). It relates to a method for depleting cells. Therefore, the present invention relates to the use of a CD20 antagonist in the manufacture of a medicament for treating an autoimmune disease, wherein the medicament comprises a single therapeutically effective intravenous infusion (iv. ) For administration to human patients.

  In one embodiment of the use according to the invention, 4 to 6 months after the first administration, a second intravenous infusion of a full therapeutically effective amount of the CD20 antagonist takes place after said administration. The second dose is usually given when the treated patient responded to the first treatment but showed signs of recurrence. Accordingly, in another embodiment of the use according to the invention, the administration of the second intravenous infusion is for administration to a patient who has responded but has relapsed after the first administration. In another embodiment of the use according to the invention, the therapeutically effective dose in the first and second intravenous infusion is essentially the same.

  In another embodiment of the use according to the invention, the CD20 antagonist is a CD20 monoclonal antibody. In a preferred embodiment of the use according to the invention, the CD20 monoclonal antibody is chimeric, humanized or human. More preferably, the CD20 antibody is a humanized 2H7 antibody.

  In another aspect of the use according to the invention, the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Crohn's disease, Sjogren's syndrome, optic neuromyelitis (NMO), ANCA-related vasculitis Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM Selected from the group consisting of polyneuritis, myasthenia gravis, diabetes, Raynaud's syndrome, and glomerulonephritis. In a preferred embodiment, the autoimmune disease is rheumatoid arthritis. In an even more preferred embodiment, the autoimmune disease is active rheumatoid arthritis. In the most preferred embodiment, the active rheumatoid arthritis is moderate to severe rheumatoid arthritis.

  In another aspect of the use according to the invention, the treated patient is treated with previously administered methotrexate, a TNF antagonist, and / or other anti-CD20 antagonists (including CD20 antibodies other than those administered according to the invention). An inappropriate response was shown.

  The therapeutically effective amount for use in accordance with the present invention is typically 10 mg to 2000 mg, preferably about 400 mg to 1500 mg. In another embodiment, the therapeutically effective amount is 400 mg, 1000 mg, and 1500 mg.

  In a preferred embodiment, the autoimmune disease is rheumatoid arthritis and the CD20 antibody is a humanized 2H7 antibody.

  In various embodiments, a humanized CD20 antibody comprises (1) a 2H7 variant A, B comprising a full length L chain set forth in SEQ ID NO: 6 and a full length heavy chain set forth in SEQ ID NOs: 7, 8, and 15, respectively. And 2 or a fragment thereof, or (2) a 2H7 variant A comprising a full length L chain represented by SEQ ID NO: 6 and a full length H chain represented by SEQ ID NO: 7 Or a fragment thereof, or (3) 2H7 variant A comprising the full-length light chain represented by SEQ ID NO: 6 and the full-length heavy chain represented by SEQ ID NO: 7, or an antibody that binds to essentially the same epitope as the fragment thereof Or (4) 2H7 variants C, D, F comprising the full length L chain shown in SEQ ID NO: 9 and the full length H chains shown in SEQ ID NOs: 10, 11, 12, 13, and 14, respectively. , G, and H Or (5) a 2H7 variant B comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 8, or a fragment thereof, or (6) 2H7 variant C, or a fragment thereof, comprising the full-length L chain represented by SEQ ID NO: 9 and the full-length H chain represented by SEQ ID NO: 10.

  In one particular embodiment, the antibody is the chimeric antibody rituximab.

  In another embodiment, the CD20 antibody is the human antibody HUMAX-CD20 ™.

  In a further embodiment of the use according to the invention, the CD20 antibody comprises non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticosteroids, cyclophosphamide, adalimumab, leflunomide, infliximab, etanercept, ofatumumab, tocilizumab, For administration in combination with a drug selected from the group consisting of AME-133, Immu-106, and COX-2 inhibitors.

  In still further embodiments, the use is for administration comprising a second therapeutic agent, which may be, for example, an immunosuppressive agent. Preferably, the use comprises methotrexate. Most preferably, methotrexate is at a dose of 10-25 mg / week.

  In another embodiment, the use comprises administration of 1-6 DMARDs prior to administration of the CD20 antibody.

  In yet another embodiment, the use does not include any steroid treatment prior to administration of the CD20 antibody.

  In another aspect, the invention relates to the use of a CD20 antibody in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA), wherein the medicament comprises a dose of 400 mg to 1500 mg of the antibody as a single therapeutic. Intravenous infusion (iv) effective for administration to human patients. More preferably, the dose is selected from the group consisting of 400 mg, 1000 mg, or 1500 mg.

  As before, the antibody is a chimeric, humanized, or human monoclonal antibody and can be, for example, any of the CD20 antibodies listed above.

  In another aspect, the invention includes a 2H7 comprising a full length L chain shown in SEQ ID NO: 6 and a full length H chain shown in SEQ ID NO: 7 in the manufacture of a medicament for treatment of active rheumatoid arthritis (RA). With respect to the use of variant A, or a fragment thereof, the medicament herein is for administration of a 400 mg dose of the antibody in a single therapeutically effective intravenous infusion (iv).

  In yet another aspect, the invention comprises a full length light chain represented by SEQ ID NO: 6 and a full length heavy chain represented by SEQ ID NO: 7 in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA). With respect to the use of 2H7 variant A, or a fragment thereof, the medicament here is for administering a 1000 mg dose of the antibody in a single therapeutically effective intravenous infusion (iv). .

  In another aspect, the invention includes a 2H7 comprising a full length L chain shown in SEQ ID NO: 6 and a full length H chain shown in SEQ ID NO: 7 in the manufacture of a medicament for treatment of active rheumatoid arthritis (RA). With respect to the use of variant A, or a fragment thereof, the medicament herein is for administration of a 1500 mg dose of the antibody in a single therapeutically effective intravenous infusion (iv).

  In a particular embodiment of the use according to the invention, the CD20 antibody is for administration without a second administration of CD20 antibody for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months. It is.

  In a further aspect, the invention comprises administering to a 400 mg patient 2H7 variant A, comprising a full length L chain set forth in SEQ ID NO: 6 and a full length H chain set forth in SEQ ID NO: 7, or a fragment thereof. , For the treatment of active rheumatoid arthritis (RA) in human patients.

  In yet a further aspect, the present invention administers CD20 antibody 2H7 variant A, or a fragment thereof, comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 7 to a 1000 mg patient. This invention relates to a method for treating active rheumatoid arthritis (RA) in human patients.

  In certain embodiments, a second dose of CD20 antibody is not administered for at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months after the first dose.

  For all uses herein, the initial infusion rate is within the range of normal standard infusion rates, for example, 50 mg / h to 100 mg / h. The infusion rate need not be the same for all dosing times, but rather may be accelerated, eg, as described in Example 1.

  In a different aspect, the present invention provides a package insert comprising (a) a container containing a CD20 antagonist, and (b) instructions for treating an autoimmune disease in a human subject (the instructions herein include: Has been shown to be administered a full therapeutically effective amount of a CD20 antagonist as a single intravenous infusion). Preferably, the CD20 antagonist is a chimeric, humanized, or human CD20 monoclonal antibody. Also preferably, the autoimmune disease is rheumatoid arthritis.

  In a more preferred embodiment, the CD20 antagonist is CD20 antibody 2H7 variant A, or a fragment thereof, comprising the full length L chain shown in SEQ ID NO: 6 and the full length H chain shown in SEQ ID NO: 7. In another preferred embodiment, the instructions indicate a complete therapeutically effective amount between 400 mg and 1500 mg. More preferably, the product includes: (a) a container containing the CD20 antibody; and (b) a package insert containing instructions for treating rheumatoid arthritis in a human subject (the instructions herein include the subject A complete therapeutically effective amount of CD20 antibody from 400 mg to 1500 mg is shown to be administered as a single intravenous infusion).

  More preferably, in the product according to the invention, the CD20 antibody is a humanized 2H7 variant A, or a fragment thereof, comprising a full length L chain as shown in SEQ ID NO: 6 and a full length H chain as shown in SEQ ID NO: 7. . Most preferably, the humanized 2H7 variant A will be 20 mg / ml antibody at pH 5.8 in 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20, and sterile water for injection. To be formulated.

  In a further aspect, the present invention relates to a pharmaceutical formulation comprising a fully effective amount of a CD20 antibody in a form suitable for one-time intravenous administration. Preferably, the pharmaceutical preparation according to the present invention comprises a humanized 2H7 variant A comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 7, or a fragment of CD20 antibody. Including. More preferably, the pharmaceutical formulation comprises an effective amount of 400 mg to 1500 mg. Even more preferably, this amount is selected from the group of 400 mg, 1000 mg, or 1500 mg. Most preferably, the humanized 2H7 variant A will be 20 mg / ml antibody at pH 5.8 in 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20, and sterile water for injection. To be formulated.

Detailed Description of Preferred Embodiments Definitions “B cells” are lymphocytes that mature in the bone marrow and include naive B cells, memory B cells, or effector B cells (plasma cells) at all developmental stages. As used herein, B cells are normal or non-malignant B cells.

  “B cell depletion” as used herein refers to a decrease in the level of B cells in an animal or human after treatment with a drug or antibody as compared to the level prior to treatment. B cell levels can be measured by performing a complete blood count, by FACS analysis staining for known B cell markers, and using well known assays such as other methods well known in the art. In one embodiment, the depletion of CD20 expressing B cells is at least 25%. In patients receiving B cell depleting drugs, B cells are generally depleted from the time the drug is circulating in the patient's body to the time for B cell recovery.

  A “B cell surface marker” or “B cell surface antigen” or “B cell antigen” is an antigen that is expressed on the surface of a B cell and can be targeted with an antagonist that binds thereto. 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 details, see The Leukocyte Antigen Facts Book, 2nd edition, 1997, edited by Barclay et al. Academic Press, Harcourt Brace & Co., New York. See) 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. Certain B cell surface markers of interest are preferentially expressed on B cells compared to other non-B cell tissues in mammals, and are expressed on both progenitor and mature B cells. There may be. Preferred B cell surface markers are CD20 and CD22 herein.

  The “CD20” antigen or “CD20” is an approximately 35 kDa non-glycosylated phosphorylated protein found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on both normal B cells and malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include “B lymphocyte restricted antigen” and “Bp35”. The CD20 antigen is described, for example, in Clark et al., Proc. Natl. Acad. Sci (USA) 82: 1766 (1985).

  A “CD20 antagonist” destroys or depletes B cells in a mammal upon binding to CD20 on B cells and / or reduces, for example, the humoral response elicited by B cells or A molecule that interferes with the function of one or more B cells by interfering with it. A CD20 antagonist is preferably capable of depleting B cells (ie, reducing circulating B cell levels) in a mammal, eg, a human subject, treated therewith. Such depletion can be achieved through various mechanisms such as ADCC and / or CDC, inhibition of B cell proliferation, and / or induction of B cell death (eg, via apoptosis). Antagonists included within the scope of the present invention include antibodies, other CD20 targeting (binding) proteins, synthetic or native sequence binding to CD20 and optionally conjugated or fused to another molecule. Peptides, immunoadhesins, and small molecule antagonists. Preferred antagonists consist of antibodies, consist essentially of antibodies, or include antibodies.

  An “antibody antagonist” as used herein refers to a fluid that destroys or depletes B cells in a mammal upon binding to a B cell surface marker on B cells and / or is e.g. induced by B cells An antibody that interferes with one or more B cell functions by reducing or preventing a sexual response. The antibody antagonist is preferably capable of depleting B cells (ie, reducing circulating B cell levels) in a mammal treated therewith. Such depletion can be achieved through various mechanisms such as ADCC and / or CDC, inhibition of B cell proliferation, and / or induction of B cell death (eg, via apoptosis). Thus, a “CD20 antibody antagonist” or “antagonist CD20 antibody” destroys or depletes B cells in a mammal, eg, a human subject, and / or, for example, B, upon binding to CD20 on B cells. An antibody that interferes with one or more B cell functions by reducing or preventing the humoral response elicited by the cell. CD20 antagonist antibodies include rituximab (Genentech), TRU-015 (Trubion, Wyeth), ofatumumab (HuMax-CD20; Genmab); AME-133 (Eli Lilly), Immu-106 (Immunomedics), all of which Referenced in the background chapter.

  The term “inappropriate response to a CD20 antagonist” refers to an inappropriate response to previous and current treatment with a CD20 antagonist due to toxicity and / or inappropriate efficacy. Inappropriate responses can be evaluated by clinicians with skills in treating the disease in question. Mammals (eg, humans) that have become “toxic” by previous or current treatment with a CD20 antagonist may have one or more negative side effects associated therewith, eg, heart, lung, kidney side effects, particularly severe Serious side effects are caused, such as acute respiratory distress syndrome, myocardial infarction, ventricular fibrillation, cardiogenic shock, hypoxia, pulmonary infiltration, acute renal failure, mococutaneous reactions, or infections such as viral infections. Mammals in which “inappropriate efficacy” is caused continue to have active disease after previous or current treatment with a CD20 antagonist. For example, a patient may have active disease activity after one or three months of treatment with a CD20 antagonist.

The terms “autoimmune disease” and “autoimmune disease” are used synonymously and are diseases or disorders resulting from or directed to an individual's own tissue or co-segregate, or It refers to the sign or condition resulting from it. An “autoimmune disease” is an organ-specific disease (ie, the immune response is endocrine, hematopoietic, skin, cardiopulmonary, gastrointestinal and liver, kidney, thyroid, eye, neuromuscular, central nervous system, etc. Or systemic diseases (eg, systemic lupus erythematosus (SLE), rheumatoid arthritis, polymyositis) that can affect multiple organ systems).
Examples of autoimmune diseases or disorders include autoimmune rheumatic diseases such as arthritis (rheumatoid arthritis such as acute arthritis, rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, osteoarthritis, infectious Arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis ),
Inflammatory hyperproliferative skin diseases, psoriasis (eg psoriasis vulgaris, psoriasis vulgaris, pustular psoriasis, and nail psoriasis), atopy (including atopic diseases such as hay fever and Job syndrome), dermatitis (contactability) Dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpes dermatitis, and atopic dermatitis), x-linked high IgM syndrome, urticaria (eg, chronic allergic) Urticaria and chronic idiopathic urticaria, eg chronic autoimmune urticaria), polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrosis, scleroderma (including systemic scleroderma), sclerosis (Eg, systemic sclerosis, multiple sclerosis (MS), eg spino-optical MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic Sclerosis, atherosclerosis Arteriosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis), inflammatory bowel disease (IBD) (eg, Crohn's disease, autoimmune-mediated gastrointestinal disease, colitis, eg, ulcerative colitis, Colon ulcer, microscopic colitis, collagen colitis, colon polyp, necrotizing enterocolitis and transmural colitis, and autoimmune inflammatory bowel disease), pyoderma gangrenosum, erythema nodosum, primary Sclerosing cholangitis, episclerosis, respiratory distress syndrome (including adult or acute respiratory distress syndrome (ARDS)),
Meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disease, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases (eg anaphylaxis and allergic and atopic) Rhinitis), encephalitis (eg, Rasmussen encephalopathy, and limbic and / or brainstem encephalitis), uveitis (eg, anterior uveitis, acute pre- uveitis, granulomatous uveitis, non-granular uveitis) Meningitis, lens antigenic uveitis, posterior uveitis, or autoimmune uveitis), glomerulonephritis (GN) with or without nephrotic syndrome (eg, chronic or acute glomerulonephritis, eg Primary GN, immune GN, membranous GN (membrane nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrane or membranous proliferative GN (MPGN) (including type I and type II) ,as well as Rapidly progressive GN, allergic condition and response, allergic reaction, rash (including allergic and atopic rash), asthma (eg, asthmatic bronchitis, bronchial asthma, and autoimmune asthma), T cell infiltration and chronic inflammation Condition with response, immune response to foreign antigens such as fetal ABO blood group during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency,
Systemic lupus erythematosus (systemic lupus erythematosus) or systemic lupus erythematosus (eg cutaneous SLE), subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus lupus erythematosus, lupus (lupus nephritis, lupus encephalitis) , Childhood lupus, non-renal lupus, extrarenal lupus, discoid lupus, alopecia lupus), juvenile-onset (type I) diabetes (including childhood insulin-dependent diabetes mellitus (IDDM)), adult-onset diabetes (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis (lymphoma-like granuloma) Disease, including Wegener's granulomatosis), agranulocytosis, vasculitides (vasculitides (va sculitis) (including macrovascular vasculitis (including rheumatoid polymyalgia and giant cell (takayasu) arteritis)), medium vasculitis (including Kawasaki disease and nodular polyarteritis / nodal periarteritis), Microscopic polyangiitis, CNS vasculitis, necrotizing vasculitis, cutaneous vasculitis, or hypersensitivity vasculitis, systemic necrotizing vasculitis, ANCA-related vasculitis (AAV) (eg Churg Strauss vasculitis or syndrome) (Including CSS)), including ANCA-negative vasculitis], including temporal arteritis),
Aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, diamond blackfan anemia, hemolytic anemia, or immune hemolytic anemia (including autoimmune hemolytic anemia (AIHA)), malignant anemia ( Anemia malignant fever), Addison's disease, erythroblastic anemia or erythroblastic fistula (PRCA), factor VIII deficiency, hemophilia A, autoimmune anti-neutropenia, cytopenia, leukopenia , Diseases with leukocyte extravasation, CNS inflammatory diseases, multiple organ injury syndromes (eg secondary to sepsis, trauma, or hemorrhage), diseases mediated by antigen-antibody complexes, anti-glomerular basement membrane Disease, antiphospholipid syndrome, allergic neuritis, Bechet disease or Behcet disease, Castleman syndrome, Goodpasture syndrome, Raynaud's syndrome, Sjogren Symptom groups, Steven Johnson syndrome, pemphigoid (eg, blistering pemphigoid and pemphigoid skin), pemphigus (pemphigus vulgaris, deciduous pemphigus, pemphigus mucosa, erythematous pemphigus) ), Autoimmune multiglandular endocrine disorders, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, optic neuritis (NMO; also known as Devic syndrome), polyneuropathy, chronic neuropathy ( For example, IgM polyneuropathy or IgM-mediated neuropathy),
Thrombocytopenia (eg, occurring in patients with myocardial infarction) (thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia, and autoimmune or immune-mediated thrombocytopenia Diseases, including idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP), testicular and ovarian autoimmune diseases (including autoimmune orchiditis and ovitis), primary hypothyroidism , Hypoparathyroidism, autoimmune endocrine disease [thyroiditis (eg, autoimmune thyroiditis, Hashimoto disease, chronic thyroiditis (Hashimoto thyroiditis), or subacute thyroiditis), autoimmune thyroid disease, idiopathic Hypothyroidism, Grave's disease, multi-glandular syndromes (eg autoimmune multi-gland syndrome (or multi-gland endocrine disorder syndrome)), paraneoplastic syndromes (neurogenic paraneoplastic syndromes, Lambert Eaton myasthenia syndrome or Eaton Lambert syndrome, stiff man or stiff person syndrome), encephalomyelitis (eg allergic encephalomyelitis or encephalomyelitic allergy and experimental allergic encephalomyelitis (EAE)) , Myasthenia gravis (eg thymoma-related severe myasthenia gravis), cerebellar degeneration, neuromuscular angina, clonal ocular or clonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor nerves Disability, sheehan syndrome,
Autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia (LIP), obstructive bronchiolitis (non-transplant) NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA skin disease, primary biliary cirrhosis, pulmonary fibrosis, autoimmune bowel disease syndrome, celiac disease, koreaic disease, Celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease (eg autoimmunity) Inner ear disease (AIED)), autoimmune hearing loss, ocular clonus-myoclonus syndrome (OMS), polychondritis (eg refractory or relapsing polychondritis), alveolar tachycardia Parkinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, monoclonal B-cell lymphocytosis (eg, benign monoclonal immunoglobulin and unknown monoclonal immunoglobulin, MGUS) Primary lymphocytosis, peripheral neuropathy, paraneoplastic syndrome, channel disease such as epilepsy, migraine, arrhythmia, muscle disease, hearing loss, blindness, periodic paralysis, and CNS channel disease,
Autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine eye disorder, uveoretinitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine insufficiency , Schmidt syndrome, adrenalitis, gastric atony, presenile dementia, demyelinating diseases (eg autoimmune demyelinating disease and chronic inflammatory demyelinating polyneuropathy), diabetic nephropathy, dresser syndrome, circular hair loss , CREST syndrome (calcification, Raynaud's phenomenon, decreased canine peristalsis, sclerotia, and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage , Farmer lung, erythema multiforme, post-opening syndrome, Cushing syndrome, avian herd, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis (eg allergic alveolitis and fibrosis) lung Inflammation), interstitial pneumonia, blood transfusion reaction, leprosy, malaria, leishmaniasis, kypanosomiasis, schistosomiasis, roundworm, aspergillosis, Sumpter syndrome, Kaplan syndrome, dengue fever, endocarditis, endocardium Myocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, permanent erythema erythema, fetal erythroblastosis , Eosinophilic fasciitis, Charman syndrome, Ferti syndrome, flariasis, ciliary inflammation (eg chronic ciliitis, metachronous ciliitis, iris ciliitis (acute or chronic) ), Or Fuchs ciliary inflammation), Henoch-Schönlein purpura,
Human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmunity Gonadal dysfunction, sydenom chorea, streptococcal post-nephritis, obstructive thromboangiitis, thyrotoxicosis, spinal cord fistula, choroiditis, giant cell polymyalgia, endocrine eye disorder, chronic hypersensitivity pneumonia, dry horn Conjunctivitis, epidemic keratoconjunctivitis, idiopathic nephritis syndrome, smile-changing nephrosis, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, pneumoconiosis, after, aphthous Stomatitis, arteriosclerotic disease, aspermiogenese, autoimmune hemolysis, Beck disease, cryoglobulinemia Dupuytren's contracture, lens anaphylaxis endophthalmia phacoanaphylactica, allergic enteritis, erythema nodosum erythema, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hanman-rich disease, sensorineural hearing loss, paroxysmal Hemoglobinuria, hypogonadism, localized ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis,
Sympathetic ophthalmitis, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Kelvan thyroiditis, acquired spleen atrophy, infertility due to anti-sperm antibody, nonmalignant thymoma, vitiligo, Diseases related to SCID and Epstein-Barr virus, acquired immune deficiency syndrome (AIDS), parasitic diseases (eg Leishmania), toxic shock syndrome, food poisoning, condition with T cell infiltration, leukocyte adhesion deficiency, cytokine And immune responses associated with acute and delayed hypersensitivity mediated by T lymphocytes, diseases with leukocyte extravasation, multi-organ injury syndrome, diseases mediated by antigen-antibody complexes, anti-glomerular basement membrane disease , Allergic neuritis, autoimmune multiglandular endocrine disorder, ovitis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmitis, rheumatic disease, mixed connective tissue disease, nef -Seed syndrome, pancreatic insulitis, multigland endocrine insufficiency, peripheral neuropathy, multigland autoimmune syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), total head alopecia, dilated cardiomyopathy, Acquired epidermolysis bullosa (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinus Inflammation, maxillary sinusitis, or sphenoid sinusitis, diseases related to eosinophils (eg eosinophilia, pulmonary infiltrating eosinophilia, hypereosinophilic myalgia syndrome, Lefler syndrome, chronic Eosinophilic pneumonia, localized pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or granulomas including eosinophils), anaphylaxis,
Seronegative spondyloarthritis, polyadenoendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Breton syndrome, infancy transient hypogammaglobulinemia, Wiscott Aldrich syndrome, telangiectasia ataxia, autoimmune disease related to collagen disease, rheumatism, neurological disease, lymphadenitis, ischemia-reperfusion disease, decreased blood pressure response, vascular dysfunction, vasodilatation, tissue damage , Cardiovascular ischemia, hyperalgesia, cerebral ischemia, and angiogenic diseases, allergic hypersensitivity disease, glomerulonephritis, reperfusion injury, reperfusion injury of myocardium or other tissues, skin with acute inflammatory components , Acute purulent meningitis or other central nervous system inflammatory disease, eye and orbital inflammatory disease, granulocyte transfusion related syndrome, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelonephritis, pulmonary fibrosis ,sugar Disease retinopathy, diabetic macrovascular disease, arterial hyperplasia, peptic ulcer, valvular disease, as well as include endometriosis but are not limited thereto.

  The term “rheumatoid arthritis” or “RA” is used in its broadest sense herein and is recognized, which can be diagnosed according to the 2000 revised American Rheumatoid Association criteria for rheumatoid arthritis classification or any similar criteria. It refers to the disease state. Physiological indicators of RA include symmetric joint swelling that is characteristic but not unchanged in rheumatoid arthritis. It usually develops spindle-shaped swelling of the proximal interphalangeal joint (PIP) as well as the metacarpophalangeal joint (MCP), wrist, elbow, knee, ankle and metatarsophalangeal (MTP) joints, Easily detected. 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 finger ulnar displacement at the MCP joint, hyperextension or hyperflexion of the MCP and PIP joints, elbow flexion contracture, and carpal and toe subluxation. . A subject with rheumatoid arthritis can be resistant to DMARDs in that DMARDs are not effective or are not fully effective in treating symptoms. Rheumatoid arthritis can be described as mild, moderate, or severe. Mild RA is characterized by the development of at least 3 joints (simultaneous) with no extra-articular lesions. Rheumatoid factor (RF) is typically negative and there is no erosion on the radiograph. Moderate illness shows active inflammation of 6-20 joints, usually changes in radiographs, is positive for RF, and typically has no extra-articular signs. For moderate illnesses, it is suggested that both NSAID and DMARDS are required, not just NSAID therapy. Severe RA is characterized by the development of more than 20 joints, RF (usually high titer) is positive, includes extra-articular lesions, and typically further includes hypoalbuminemia and chronic disease. Causes other systemic effects, including anemia. In severe forms of RA, functional capacity is rapidly decreasing. As used herein, the term “rheumatoid arthritis” or “RA” refers to active rheumatoid arthritis, early and late RA based on how long a patient has suffered from RA, and moderate to severe rheumatoid arthritis. Includes all forms and stages of RA, including but not limited to.

  Rheumatoid factor (RF) is an immunoglobulin directed against the Fc portion of another immunoglobulin commonly used as a blood test for the diagnosis of rheumatoid arthritis. It self-aggregates in the joint space into a lattice-like form on which inflammatory cells can adhere and act. Rheumatoid arthritis patients who showed high titers of RF (approximately 80% of patients) had worse long-term outcomes and more aggressive disease and higher mortality than RF negative patients .

  A patient having “active rheumatoid arthritis” refers to a patient having rheumatoid arthritis symptoms that are active and not latent. For the purposes of the present invention, “active rheumatoid arthritis” is defined by the presence of at least 4 swollen joints.

  A subject with “early active rheumatoid arthritis” is a subject with active rheumatoid arthritis who has been diagnosed according to the ACR criteria revised in 1987 for the classification of RA in at least 8 weeks, but not more than 4 years Is the body.

  “Treatment” or “treatment” or “remission” means therapeutic treatment and preventive or preventive measures, the purpose of which is to prevent or delay (reduce) the targeted pathological condition or disease. is there. Following administration of a therapeutically effective amount of a CD20 antagonist, such as a CD20 binding antibody of the invention, according to the method of the invention, the subject is observable and / or measurable for one or more signs and symptoms of a particular autoimmune disease Has been successfully “treated” for an autoimmune disease. For example, if the target disease is rheumatoid arthritis, the subject in need of treatment includes a subject already suffering from the disease and a disease that includes various characteristics of the disease such as joint damage or progression of joint damage. Subjects who wish to prevent the onset of Thus, the subject may have been diagnosed as having rheumatoid arthritis, or may be predisposed to or susceptible to rheumatoid arthritis, or may progress without treatment. You may have mild or moderate rheumatoid arthritis. Treatment is successful if the disease and / or various features or symptoms of the disease have been ameliorated or cured, or the progression of the disease has stopped or slowed compared to before administration. Successful treatment further includes complete or partial prevention of the onset of the disease. For purposes herein, slowing or reducing the disease or symptom of the disease or progression of the symptom of the disease is the same as cessation, reduction or recovery of the symptom. Symptoms of rheumatoid arthritis include any symptom listed in the American Rheumatoid Association criteria revised in 2000 for the classification of rheumatoid arthritis, such as joint swelling, joint damage, bone deformity, bone erosion, pain, inflammation, and Systemic effects associated with more advanced forms of rheumatoid arthritis, including but not limited to hypoalbuminemia and chronic disease anemia.

  The term “effective amount” as used herein refers to a therapeutically and prophylactically effective amount. Thus, in the methods of the present invention, an “effective amount” refers to the amount of CD20 antibody or another CD20 antagonist that is effective in treating (including preventing) rheumatoid arthritis. As a result of administering an “effective amount”, the target disease and / or various features or symptoms of the disease are ameliorated or cured, or the progression of the disease is stopped or slowed compared to before administration. Successful treatment further includes complete or partial prevention of the onset of the disease. For purposes herein, slowing or reducing the progression of disease or disease symptoms or disease symptoms is the same as stopping, reducing, or ameliorating symptoms. Symptoms of rheumatoid arthritis include any symptom listed in the American Rheumatoid Association criteria revised in 2000 for the classification of rheumatoid arthritis, such as joint swelling, joint damage, bone deformity, bone erosion, pain, inflammation, and Systemic effects associated with more advanced forms of rheumatoid arthritis, including but not limited to hypoalbuminemia and chronic disease anemia.

  The term “fully therapeutically effective amount” administered as “single intravenous infusion (iv)” as used herein refers to CD20 antibodies and the like that are effective for therapeutic treatment of target diseases such as rheumatoid arthritis. Intravenous infusion of the total amount of CD20 antagonist at two or more divided doses (less than the therapeutically effective amount) sequentially, eg, in separate infusions over several days or weeks To be administered as

  The “CD20” antigen is a non-glycosylated transmembrane phosphorylated protein with a molecular weight of about 35 kDa found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until it differentiates into a plasma cell and is not found on human stem cells, lymphocyte progenitor cells, or normal plasma cells. CD20 is present on both normal B cells as well as malignant B cells. Other names for CD20 in the literature include “B lymphocyte restricted differentiation antigen” and “Bp35”. The CD20 antigen is described, for example, in Clark and Ledbetter, Adv. Can. Res. 52: 81-149 (1989) and Valentine et al., J. Biol. Chem. 264 (19): 11282-11287 (1989). .

  The term “antibody” is used in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (eg, bispecific antibodies), single chain antibodies and antibody fragments (desired organisms). Cover as long as they exhibit a biological activity or function).

Biological activity of a CD20 binding antibody of the invention includes binding of the antibody to human CD20, more preferably binding of the antibody to human and other primate CD20 (including cynomolgus monkeys, rhesus monkeys, chimpanzees, baboons). It is. Antibody, 1 × ¹⁰ lower than ^-8 _{K d} values, preferably when bound to CD20 with a _{K d} values from 1 × ^{10 -9,} compared such not treated with antibody and appropriate negative control B cells can be killed or depleted, preferably at least 20% in vivo. B cell depletion may be the result of one or more of ADCC, CDC, apoptosis, or other mechanisms. In certain aspects of disease treatment herein, certain effector functions or mechanisms are desired over others, and certain variants of humanized 2H7 or certain human CD20 binding antibodies may have biological functions such as ADCC. Preferred to achieve.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecificity formed from antibody fragments Sex antibodies are included. “Fv” is the minimum antibody fragment which contains a complete antigen recognition and binding site. This fragment consists of a dimer in which one heavy chain and one light chain variable region domain are closely associated non-covalently. From the folding of these two domains, six hypervariable loops (each three loops derived from the heavy and light chains) result in amino acid residues for antigen binding, making the antibody antigen specific Sex is imparted. However, even a single variable domain (or half of the Fv containing only three CDRs specific for the antigen, half of the Fv) has lower affinity than the whole binding site, but recognizes and binds to the antigen. Have the ability to

As used herein, the term “monoclonal antibody” refers to an antibody derived from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are the same and / or bind to the same epitope, except for variants that may generally be present in small amounts that may occur during the production of monoclonal antibodies. Such monoclonal antibodies typically comprise an antibody comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence comprises a single target binding polypeptide sequence from a plurality of polypeptide sequences. Obtained by a method comprising selecting. For example, the selection method can be to select unique clones from multiple clones (eg, a hybridoma clone, a phage clone, or a pool of recombinant DNA clones). The selected target binding sequence may be immunized in vivo to improve its production in cell culture, for example, to improve the affinity for the target, to humanize the target binding sequence, It should be understood that antibodies can be further modified, such as to create multispecific antibodies, to reduce originality, and antibodies that include modified target binding sequences are also monoclonal antibodies of the invention. . In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation has a single determinant on the antigen. Directed against. In addition to its specificity, monoclonal antibody preparations are advantageous in that they are typically free of other immunoglobulins.
The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should be construed as requiring that the antibody be produced by any specific method is not. For example, monoclonal antibodies used in accordance with the present invention can be produced, for example, by hybridoma methods (eg, Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition). Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier, NY, 1981)), recombinant DNA methods (see, eg, US Pat. No. 4,816,567), phage display technology ( For example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991); Sidhu et al., J. Mol. Biol. 338 (2): 299 -310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101 (34): 12467-12472 (2004); And Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004)),
In addition, a technique for producing a human or human-like antibody in an animal having a human immunoglobulin locus encoding a human immunoglobulin sequence or a part or all of a gene (for example, International Publication No. 1998/24893; International Publication No. 1996) International Publication No. 1996/33735; International Publication No. 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255. -258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); US Patent Nos. 5,545,806; 5,569,825; 5,591,669 (all GenPharm) No. 5,545,807; WO 1997/17852; US Pat. No. 5,545,807; 5,545,806; 5,569,825; No. 5,633,425; and 5,661,016; Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison , Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).

  A “functional fragment” of a CD20 binding antibody of the invention retains binding to CD20 with substantially the same affinity as the intact full-length molecule from which it is derived, and in vitro as described herein. Or a fragment that exhibits biological activity including B cell depletion as measured by in vivo assays.

  The term “variable” refers to the fact that certain segments of a variable domain vary greatly in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for that particular antigen. However, variability is not evenly distributed over the 110 amino acid span of the variable domain. Instead, the V regions are separated by a highly variable short region called the “hypervariable region”, each 9-12 amino acids long, and is a relatively invariant called the 15-30 amino acid framework region (FR). Consists of stretch. The natural heavy and light chain variable domains each contain four FRs (mostly in β-sheet conformation) connected by three hypervariable regions, where the hypervariable regions are β Loops connecting the sheet three-dimensional structures are formed, and in some cases form part of the β sheet structure. The hypervariable regions in each chain are held together adjacent to the FR and together with the hypervariable region from the other chain contribute to the formation of the antigen binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, (See 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in antibody binding to antigen, but exhibit various effector functions such as antibody involvement in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region generally "complementarity determining region" or "CDR" derived amino acid residues (e.g., approximately 24-34 (L1 of _{V L), 50~56 (L2)} , and 89-97 (L3) residues, as well as approximately 31~35B of _{V H (H1), 50~65 (} H2) and residues of 95~102 (H3); Kabat et al, Sequences of Proteins of Immunological Interest, fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or residues from “hypervariable loops” (eg, _VL 26-32 (L1), 50-52 (L2) and 91 -96 (L3) and _VH 26-32 (H1), 52A-55 (H2), and 96-101 (H3); Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987) ))including.

  A “consensus sequence” or consensus V domain sequence referred to herein is an artificial sequence derived from a comparison of amino acid sequences of known human immunoglobulin variable region sequences. Based on these comparisons, a recombinant amino acid sequence encoding a V domain amino acid, a consensus sequence obtained from human kappa and human heavy chain subgroup IIIV domains, was prepared. The consensus V sequence does not have any known antibody binding specificity or affinity.

  A “chimeric” antibody (immunoglobulin) is one in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or antibody belonging to a particular antibody class or subclass. While the remainder of the chain is identical to the corresponding sequence in an antibody from another species or an antibody belonging to another antibody class or subclass, and fragments of such an antibody as long as it exhibits the desired biological activity. Or homologous (US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Humanized antibodies as used herein are a subset of chimeric antibodies.

  “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are derived from non-human species (eg, mouse, rat, rabbit, or non-human primate) in which the recipient's hypervariable region residues have the desired specificity, affinity, and ability. A human immunoglobulin (recipient or acceptor antibody) that is substituted with a hypervariable region residue (donor antibody). In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody comprises substantially all of at least one, typically two variable domains, wherein all or substantially all of its hypervariable loops are hypervariable of non-human immunoglobulin. Corresponds to the loop and all or substantially all of the FR region is an FR region of a human immunoglobulin sequence, but the FR region may contain one or more amino acid substitutions that improve binding affinity . The number of these amino acid substitutions in the FR is typically 6 or less for the H chain and 3 or less for the L chain. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); and Presta, Cur. Op. Struct. Biol. 2: 593-596 (1992). Refer to).

  Antibody “effector function” refers to a biological activity attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region), which varies with the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors) As well as B cell activation.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to Fc receptors (FcRs) that are present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils, and macrophages. A cytotoxic form in which these cytotoxic effector cells are able to specifically bind to antigen-bearing target cells and then kill the target cells with a cytotoxin by secreting Ig bound to Say. Antibodies “arm” cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay as described in US Pat. Nos. 5,500,362 and 5,821,337 can be performed. Effector molecules useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model as disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998). .

  “Fc receptor” or “FcR” refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors), including those of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of these receptors and alternatively spliced forms. Can be mentioned. FcγRII receptors include FcγRIIA ([Activating Receptor]) and FcγRIIB (“Inhibitory Receptor”), which have similar amino acid sequences, but differ mainly in their cytoplasmic domains. Yes. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See review by M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330. -41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes FcRn, a neonatal receptor involved in the transport of IgG from the mother to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994). )).

  International Publication No. WO 00/42072 (Presta) and International Publication No. 2004/056312 (Lowman et al.) Describe antibody variants with improved or reduced binding to FcR. The contents of these patent publications are specifically incorporated herein by reference. See also Shields et al., Journal of Biological Chemistry 276 (9): 6591-6604 (2001).

  “Complement dependent cytotoxicity” or “CDC” refers to the lysis of target cells in the presence of complement. The classical complement activation pathway is initiated by binding of the first component of the complement system (C1q) to an antibody (appropriate subclass antibody) that is bound to a cognate antigen. To assess complement activation, CDC assays such as Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed.

  Polypeptide variants in which the amino acid sequence of the Fc region is changed to improve or decrease the C1q binding ability are described in US Pat. No. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

  A “naked antibody” is an antibody (as defined herein) that is not conjugated to a heterologous molecule (eg, a cytotoxic moiety, polymer, or radiolabel).

  An “isolated” antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies, which can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified to (1) greater than 95%, most preferably greater than 99% by weight of the antibody as measured by the Lowry method, or (2) a spinning cup. ) Purified to a sufficient extent to obtain at least 15 residues of the N-terminal amino acid sequence or internal amino acid sequence by using a sequencer, or (3) using Coomassie blue or preferably silver staining And purified to homogeneity by SDS-PAGE under reducing or non-reducing conditions. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  “Joint damage” is used in the broadest sense and means damage or partial or complete destruction of any part of one or more joints (including connective tissue and cartilage), where damage May include structural and / or functional damage due to any cause and may or may not cause joint pain / joint pain. Joint damage specifically includes, but is not limited to, joint damage associated with or caused by inflammatory joint disease as well as non-inflammatory joint disease. This damage can be caused, for example, by autoimmune diseases such as lupus (eg systemic lupus erythematosus), arthritis (eg acute and chronic arthritis, rheumatoid arthritis (juvenile onset rheumatoid arthritis, juvenile idiopathic arthritis (JIA)), or juvenile Rheumatoid synovitis, gout or gout arthritis, acute immune arthritis, chronic inflammatory arthritis, osteoarthritis, type II collagen-induced arthritis, infectious arthritis, purulent arthritis, lime Arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis, chronic primary polyarthritis, reactive arthritis, menopausal arthritis, estrogen deficient arthritis, and ankylosing Spondylitis / rheumatic spondylitis), rheumatic autoimmune diseases other than RA, significant systemic onset secondary to RA (vasculitis) May include, but is not limited to, pulmonary fibrosis, or Ferti syndrome), Sjogren's syndrome, certain such secondary syndromes, and secondary limited cutaneous vasculitis due to RA.

The term “immunosuppressive agent” as used herein for adjunct therapy refers to a substance that acts to suppress or mask the immune system of the mammal being treated herein. This includes substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (US Pat. No. 4,665,077); nonsteroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids (Eg, cortisol), or aldosterone, anti-inflammatory agents (eg, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, or leukotriene receptor antagonists); purine antagonists such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclo Phosphamide; bromocriptine; danazol; dapsone; glutaraldehyde (which shields MHC antigens as described in US Pat. No. 4,120,649);
Anti-idiotype antibodies against MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs such as prednisone, methylprednisolone (SOLU-MEDROL® methyl succinate) Prednisolone sodium), and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); antimalarials such as chloroquine and hydroxychloroquine; sulfasalazine; leflunomide; cytokine antagonists such as cytokine antibodies or cytokine receptor antibodies (Anti-interferon-α, -β, or -γ antibody, anti-tumor necrosis factor (TNF) α antibody (infliximab (REMICADE )) Or adalimumab), anti-TNFα immunoadhesin (etanercept), anti-TNF-β antibody, anti-interleukin-2 (IL-2) antibody, anti-IL-2 receptor antibody, and anti-interleukin-6 (IL- 6) including receptor antibodies and antagonists); anti-LFA-1 antibodies (including anti-CD-11a and anti-CD18 antibodies); anti-L3T4 antibodies; heterologous anti-lymphocyte globulins; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibody;
A soluble peptide comprising an LFA-3 binding domain (WO 90/08187 published July 26, 1990); streptokinase; transforming growth factor-β (TGF-β); streptodolase; host RNA or DNA from; FK506; RS-61443; chlorambucil; deoxyspergualin; rapamycin; T cell receptor (Cohen et al., US Pat. No. 5,114,721); T cell receptor fragment (Offner et al., Science, 251) 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); BAFF antagonists such as BAFF and BR3 antibodies and xTNF4 Antagonists (for review see Mackay and Mackay, Trends Immunol., 23: 113-5 (2002)) Biological agents that interfere with T cell helper signals, such as anti-CD40 receptor or anti-CD40 ligand (CD154) (blocking antibodies against CD40-CD40 ligand (eg, 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)));
In addition, a T cell receptor antibody (European Patent No. 340,109), for example, T10B9 may be mentioned. Some immunosuppressive agents herein are also DMARDs such as methotrexate. Examples of preferred immunosuppressive agents herein include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF, or methotrexate.

  Examples of “disease modifying antirheumatic drugs” or “DMARDs” include hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (+ oral and subcutaneous methotrexate), azathioprine, D-penicillamine, gold salt (oral ), Gold salt (for intramuscular use), minocycline, cyclosporine (including cyclosporin A and topical cyclosporin), Staphylococcus protein A (Goodyear and Silverman, J. Exp. Med., 197, (9), p1125- 39 (2003)) (including salts and derivatives thereof). A preferred DMARD herein is methotrexate.

  Examples of “non-steroidal anti-inflammatory drugs” or “NSAIDs” include aspirin, acetylsalicylic acid, ibuproifen, flurbiprofen, naproxen, indomethacin, sulindac, tolmethine, phenylbutazone, diclofenac, ketoprofen, benolylate, mefenam Acids, methotrexate, fenbufen, azapropazone; COX-2 inhibitors such as celecoxib (CELEBREX®; 4- (5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl ) Benzenesulfonamide), valdecoxib (BEXTRA®), meloxicam (MOBIC®), GR253035 (Glaxo Wellcome); and MK966 (Merck Sharp & Dohme) (including salts and derivatives thereof) And the like. Preferably they are aspirin, naproxen, ibuprofen, indomethacin, or tolmetin.

  Examples of “integrin antagonists or antibodies” herein include LFA-1 antibody (eg, efalizumab (RAPTIVA®) commercially available from Genentech), or α4 integrin antibody (eg, natalizumab (available from Biogen) ANTEGREN (registered trademark)), or diazacyclic phenylalanine derivative (International Publication No. 2003/89410), phenylalanine derivative (International Publication No. 2003/70709, International Publication No. 2002/28830, International Publication) Publication No. 2002/16329 and International Publication No. 2003/53926), phenylpropionic acid derivatives (International Publication No. 2003/10135), enamine derivatives (International Publication No. 2001/79173), propanoic acid derivatives Body (International Publication No. 2000/37444), alkanoic acid derivatives (International Publication No. 2000/32575), substituted phenyl derivatives (US Pat. Nos. 6,677,339 and 6,348,463), aroma Group amine derivatives (US Pat. No. 6,369,229), ADAM disintegrin domain polypeptide (US 2002/0042368), antibodies to αvβ3 integrin (European Patent 633945), aza-bridged bicyclic amino acids Derivatives (International Publication No. 2002/02556) and the like.

  "Corticosteroid" refers to any one of several synthetic or naturally occurring substances having the general chemical structure of steroids that mimic or enhance the action of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone, eg, SOLU-MEDROL® methyl prednisolone sodium succinate), dexamethasone, or dexamethasone triamcinolone, hydrocortisone, and betamethasone. Preferred corticosteroids herein are prednisone, methylprednisolone, hydrocortisone, or dexamethasone.

  “TNF” drugs for the treatment of rheumatoid arthritis include molecules that target the TNFα ligand and molecules that target the receptors for TNF-TNFα, ie TNFR-1 and TNFR-2.

  For purposes of this specification, “tumor necrosis factor α (TNF-α)” is as described in Pennica et al., Nature, 312: 721 (1984) or Aggarwal et al., JBC, 260: 2345 (1985). It refers to a human TNFα molecule containing the complete amino acid sequence.

  As used herein, a “TNFα inhibitor” or “TNFα antagonist” is an agent that generally inhibits the biological function of TNFα to some extent by binding to TNFα or a TNFα receptor and neutralizing the activity of TNFα. . Examples of TNF inhibitors specifically contemplated herein are etanercept (ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA®).

  The term “inappropriate response to a TNFα inhibitor” refers to an inappropriate response to previous or current treatment with a TNFα inhibitor due to toxicity and / or inappropriate efficacy. Inappropriate responses can be evaluated by clinicians with skills in treating the disease in question. Mammals that have become “toxic” by previous or current treatment with TNFα inhibitors may have one or more negative side effects associated therewith, such as infection (particularly severe infection), congestive heart failure, prolapse. Spinal cord (leading to multiple sclerosis), hypersensitivity, neurological events, autoimmunity, non-Hodgkin lymphoma, tuberculosis (TB), autoantibodies, etc. are caused. Mammals in which “inappropriate efficacy” has been caused continue to have active disease after previous or current treatment with TNFα inhibitors. For example, a patient may have active disease activity after 1 month or 3 months of treatment with a TNFα inhibitor.

  “Packaging” refers to information about indications, usage, dosage, administration, contraindications, other therapeutic products used in conjunction with the packaged product, and / or warnings regarding the use of such therapeutic products, etc. Used to refer to instructions customarily included in commercial packages of therapeutic products.

  A “medicament” is an active drug for treating joint damage or symptoms or side effects thereof.

  The term “recurrence” is used herein to refer to the reversal of signs and symptoms of disease after a patient has been in remission. Thus, initially the target disease and / or various features or symptoms of the disease have been reduced or cured, or the progression of the disease has stopped or slowed and then one or more characteristics of the disease or disease have returned If so, the patient is said to have “relapsed”. Symptoms of rheumatoid arthritis include any symptom listed in the 2000 revised American Rheumatoid Association criteria for classification of rheumatoid arthritis, such as joint swelling, joint damage, bone deformity, bone erosion, pain, inflammation, and more Systemic effects associated with advanced forms of rheumatoid arthritis include but are not limited to hypoalbuminemia and chronic disease anemia. If one or more of these symptoms return after an initial successful treatment, the patient is considered to have relapsed.

II. Therapeutic Methods The present invention relates to a novel mode of administration of CD20 antagonists to human subjects for treating autoimmune diseases. Accordingly, in one aspect, the invention relates to a novel treatment protocol for administering CD20 antibodies to treat autoimmune diseases, such as chronic CD20-related autoimmune diseases, such as rheumatoid arthritis (RA) or lupus.

  When the CD20 antibody is Rituxan®, the current protocol includes two 1000 mg intravenous infusions separated by 2 weeks. In contrast, the treatment protocol of the present invention, as discussed above, initially comprises, consists essentially of, or consists of administration of a total effective amount in the form of intravenous infusion.

  According to the present invention, an effective amount of a CD20 antagonist (eg, a CD20 antibody) is administered in the form of an intravenous infusion (iv) at the beginning of treatment, in the form of an intravenous infusion (iv), optionally from the first dose. After about 3-7 months, typically 4-6 months, a second total effective dose (usually but not necessarily the same dose as the first) is given intravenously. If the autoimmune disease being treated is active RA, a second i. v. Administration is usually done 6 months after the initial treatment. In the case of lupus, the second i. v. Administration is usually scheduled 4 months or 16 weeks after the first treatment, but the timing of the second administration depends on the nature and stage of the autoimmune disease being treated and the patient's response to the first treatment (this is May vary depending on the individual). The second effective amount is typically the same as the initial dose, but may be more or less than the initial dose depending on the patient's response to the initial treatment and the resulting change in patient condition.

The CD20 antibody includes “C2B8” (US Pat. No. 5,736,137), currently called “rituximab” (“RITUXAN®”); “Y2B8” commercially available from IDEC Pharmaceuticals, Inc. Alternatively, an yttrium- [90] -labeled 2B8 mouse antibody referred to as “Ibritumomab Tiuxetane” (ZEVALIN®) (US Pat. No. 5,736,137; accession number HB11388 on June 22, 1993) 2B8) was deposited with the ATCC as; "tositumomab" and also mouse IgG2a "B1" called, optionally generated is labeled with ¹³¹ I, are commercially available from Corixa "131I-B1" or "iodine I131 tositumomab" antibody (BEXAR®) (US Pat. No. 5,595,721) See also); mouse monoclonal antibody "1F5" (Press et al., Blood 69 (2): 584-591 (1987)) and "framework patched" or humanized 1F5 (WO 2003/002607). Leung, S .; ATCC deposit HB-96450) mutants thereof; mouse 2H7 and chimeric 2H7 antibodies (US Pat. No. 5,677,180); humanized 2H7 (WO 2004/056312 (Lowman) And HUMAX-CD20 ™ fully human antibody (Genmab, Denmark; eg Glennie and van de Winkel, Drug Discovery Today 8: 503-510 (2003) and Cragg et al., Blood 101: 1045. -1052 (2003)); human monoclonal antibodies shown in WO 2004/035607 (Teeling et al.); US 2004/0093621 (Shitara et al.). An antibody having a complex of an N-glycoside-linked sugar chain bound to an Fc region, as described in WO 2004/103404 (Watkins et al., Applied Molecular Evolution). A CD20 binding molecule such as an AME series of antibodies such as ATM antibody; an A20 antibody or variant thereof, such as a chimeric or humanized A20 antibody (cA20, hA20, respectively, US 2003/0219433, Immunomedics); Monoclonal antibodies L27, G28-2, 93-1B3, B-C1, or NU-B2 available from International Leukocyte Typing Workshop (Valentine et al., Leukocyte Typing III (McMichael, Ed., P. 440, Oxford University Press (1987) ). Preferred CD20 antibodies herein are humanized, chimeric, or human CD20 antibodies, more preferably humanized 2H7 antibodies, rituximab, chimeric or humanized A20 antibodies (Immunomedics), and HUMAX-CD20 ™ human CD20. It is an antibody (Genmab).

  Humanized antibodies that also bind to human CD20 and preferably other primate CD20 as well have at least one, preferably two or all of the heavy chain CDRs of an anti-human CD20 antibody (donor antibody) of a non-human species. Contains substantially all of the heavy (H) chain and framework residues of the recipient antibody, the human consensus antibody. Donor antibodies may be derived from a variety of non-human species (including mice, rats, guinea pigs, goats, rabbits, horses, primates), but most often are rodent antibodies. “Substantially all” in this context means that the recipient FR region in the humanized antibody may contain one or more amino acid substitutions that are not present in the human consensus FR sequence. These FR changes may include residues not found in the recipient antibody or donor antibody.

In one embodiment, the donor antibody is a murine 2H7 antibody, and the V regions comprising the CDR and FR sequences of their heavy and light chains, respectively, are shown in FIGS. 1A and 1B. In a specific embodiment, the human Fab framework residues correspond to human Vκ subgroup I and _VH subgroup III consensus sequences, which are shown in FIGS. 1A and 1B, respectively. The humanized 2H7 antibody of the present invention has at least one CDR of a mouse donor antibody heavy chain. In one embodiment, the humanized 2H7 antibody that binds human CD20 comprises both the heavy and light chain CDRs of the donor antibody.

  For full-length antibodies, the humanized CD20 binding antibodies of the invention comprise a humanized V domain linked to the C domain of a human immunoglobulin. In a preferred embodiment, the H chain C region is derived from human IgG, preferably from IgG1 or IgG3. The light chain C domain is preferably derived from a human kappa chain.

  The numbering of the residues is according to Kabat et al., Sequences of Immunological Interest. 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The gap is indicated by a dash. In a CD20 binding antibody comprising an Fc region, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be engineered, for example, during antibody purification or by recombinant manipulation of the nucleic acid encoding the antibody polypeptide. May be removed. Thus, a CD20 binding antibody composition useful in the present invention may comprise an antibody having K447 residues, an antibody from which all K447 has been removed, or a mixture of antibodies having and without K447 residues.

  Generation and production of humanized 2H7 antibodies that bind to CD20 are described in WO 04/056312 and US 2006/0034835, the entire disclosures of which are hereby incorporated by reference.

  In a specific embodiment, the humanized 2H7 antibody is an antibody listed in Table 1.

及び、重鎖可変配列（Ｖ_Ｈ）：

を含む。 Each of the antibody variants A, B and I in Table 1 has a light chain variable sequence (V _L ):

And heavy chain variable sequence (V _H ):

including.

及び、重鎖可変配列（Ｖ_Ｈ）：

を含む。 Each of the antibody variants C, D, F, and G of Table 1 is a light chain variable sequence (V _L ):

And heavy chain variable sequence (V _H ):

including.

を含む。 The antibody variant H in Table 1 has a light chain variable sequence (V _L ) and a heavy chain variable sequence (V _H ) represented by SEQ ID NO: 3 (described above):

including.

を含む。 Each of the antibody variants A, B, and I of Table 1 is a full length light chain sequence:

including.

を含む。 Variant A in Table 1 is a full length heavy chain sequence:

including.

を含む。 Variant I in Table 1 shows the full length heavy chain sequence:

including.

を含む。 Variant B in Table 1 is a full length heavy chain sequence:

including.

を含む。 Each of the antibody variants C, D, F, G, and H of Table 1 is a full length light chain sequence:

including.

を含む。 Variant C in Table 1 is a full length heavy chain sequence:

including.

を含む。 Variant D in Table 1 is the full length heavy chain sequence:

including.

を含む。 Variant F in Table 1 is full length heavy chain sequence:

including.

を含む。 Variant G in Table 1 is a full length heavy chain sequence:

including.

を含む。 Variant H in Table 1 is a full length heavy chain sequence:

including.

  In certain embodiments, a humanized 2H7 antibody of the invention further comprises amino acid changes in IgG Fc, and the antibody has a binding affinity for human FcRn as compared to an antibody with wild type IgG Fc. An increase of at least 60 times, at least 70 times, at least 80 times, more preferably at least 100 times, preferably at least 125 times, even more preferably at least 150 times to about 170 times.

The N-glycosylation site in IgG is Asn297 of the C _H 2 domain. Humanized 2H7 antibody compositions of the invention include compositions of any of the preceding humanized 2H7 antibodies having an Fc region, wherein about 80-100% (preferably, preferably) of the antibodies in the composition About 90-99%) contain the mature core carbohydrate structure lacking fucose attached to the Fc region of the glycoprotein. Such a composition has been demonstrated herein to show a surprising improvement in binding to Fc (RIIIA (F158)), which is in the interaction with human IgG Fc (RIIIA ( V158)) is not as effective. Fc (RIIIA (F158)) is more common than Fc (RIIIA (V158)) in normal healthy African Americans and Caucasians. See Lehrnbecher et al., Blood 94: 4220 (1999). Historically, antibodies produced in Chinese hamster ovary cells (CHO), one of the most frequently used industrial hosts, account for about 2-6% of non-fucosylated antibodies in the population. Including. However, YB2 / 0 and Lec13 can produce antibodies with 78-98% non-fucosylated species. Shinkawa et al., J Bio. Chem. 278 (5), 3466-347 (2003) show that antibodies produced in YB2 / 0 and Lec13 cells have low FUT8 activity, which is a significantly increased ADCC activity in vitro. Reported to show. Production of antibodies with reduced fucose content is also described, for example, by Li et al. (GlycoFi) “Optimization of humanized IgGs in glycoengineered Pichia pastoris” Nature Biology online article published 22 January 2006; Niwa R. et al. Cancer Res. 64 (6): 2127-2133 (2004); US 2003/0157108 (Presta); US 6,602,684 and US 2003/0175884 (Glycart Biotechnology); US published patent No. 2004/0093621, US Published Patent No. 2004/0110704, US Published Patent No. 2004/0132140 (all Kyowa Hakko Kogyo).

  CD20 binding antibodies include bispecific CD20 binding antibodies, where one arm of the antibody includes the heavy (H) chain and light (L) chain of a CD20 binding antibody, eg, the humanized of the present invention. It has the H chain and L chain of the 2H7 antibody, and the other arm has a V region having binding specificity for the second antigen. In a specific embodiment, the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D, or other NL activating ligand.

A clinical study by Genentech and Biogen Idec evaluated the therapeutic efficacy for the treatment of autoimmune diseases using doses as low as 10 mg up to 1 g of anti-CD20 (humanized 2H7 variant and rituximab) (Rituximab study) In the Background chapter; and WO 04/056312, Example 16). In general, in these clinical studies, the antibody was administered in two doses spaced approximately two weeks apart. As an example of a regimen studied in clinical studies, humanized CD20 antibody 2H7 in rheumatoid arthritis is 2 × 10 mg (2 doses at 10 mg per dose; 70 kg, total amount of about 10.1 mg / m ² in a patient with a height of 67 inches). 2 × 50 mg (70 kg, in a patient with a height of 67, total amount 55 mg / m ² ), 2 × 200 mg (70 kg, in a patient with a height of 67, total amount 220 mg / m ² ), 2 × 500 mg (70 kg, height) 67 patients total about 550 mg / m ² ), and 2 × 1000 mg (70 kg, total height about 1100 mg / m ^{2 in} patients with height 67); Rituxan 2 × 500 mg (70 kg, total height about 550 mg / m ² for patients with height 67 m ² ), 2 × 1000 mg (70 kg, total amount of about 1100 mg / m ² in a patient having a height of 67).

  The therapeutic efficacy of the treatment of autoimmune diseases has been evaluated using doses of CD20 antibodies as low as 1 mg up to 2 g. As shown in the examples, a single infusion treatment showed similar results for all doses tested (400 mg, 1000 mg, 1500 mg, and 2000 mg), so the effective amount should be varied over a wide range Typically, an effective amount is about 0.1 mg to about 2000 mg, or about 1 mg to about 2000 mg, or about 10 mg to 2000 mg, or about 400 mg to 2000 mg of CD20 antibody. The effective amount will vary depending on the CD20 antibody used, the disease being treated (including the severity of the disease), the gender, weight, age, and general condition of the patient, and other factors normally considered by the clinician. The The determination of an effective amount in each particular situation is within the skill of the physician. Thus, in various embodiments, the CD20 antibody is 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300. , 350, 400, 500, 1000, 1500, or 2000 mg. Parameters for assessing the efficacy or success of treatment of an autoimmune or autoimmune related disease will be known by physicians with skill in the appropriate disease. In general, a skilled physician will expect a reduction in the signs and symptoms of a particular disease.

  In the treatment of autoimmune diseases, it may be desirable to modulate the extent of B cell depletion depending on the severity of the disease and / or condition in the individual patient by adjusting the dose of the CD20 binding antibody. Thus, B cell depletion can be complete, but need not be complete. Alternatively, total B cell depletion may be desirable in the initial treatment, but in subsequent treatments the dose may be adjusted to achieve only partial depletion. In one embodiment, B cell depletion is at least 20%, ie, 80% or less of CD20 positive B cells remain compared to baseline levels prior to treatment. In other embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70%, or more. Preferably, B cell depletion is sufficient to stop disease progression, more preferably sufficient to alleviate the signs and symptoms of a particular disease under treatment, and even more preferably cure the disease. Enough to do. If partial or short-term B cell depletion is the goal, or if the disease is not chronic or less severe, a lower dose of CD20, eg 20 mg, 10 mg or less may be used. In another embodiment, treatment with the low dose of the present invention is useful in maintenance therapy after successful treatment with the first higher dose administered according to the present invention. In certain embodiments, a fully effective amount is initially administered as a single intravenous infusion, followed by the same dose of intravenous infusion 4-6 months after the first administration, after which typically 4-6 Lower maintenance doses may be administered at intervals of months.

  Patients with autoimmune diseases where one or more current standard therapies are ineffective, poorly tolerated, or contraindicated can be treated using the dosage regimen of the present invention . For example, the present invention contemplates the treatment of the present invention for RA patients who have shown an inappropriate response to tumor necrosis factor (TNF) inhibitor therapy or disease modifying anti-rheumatic drug (DMARD) therapy.

  In one embodiment, the doses and dosing regimens of the invention are used to treat rheumatoid arthritis (RA), such as active RA, such as moderate to severe active RA.

  RA is a debilitating autoimmune disease that affects more than 2 million Americans and interferes with their daily activities. RA occurs when the body's own immune system improperly attacks joint tissue, causing chronic inflammation that destroys healthy tissue and damages the joint. Symptoms include joint inflammation, swelling, stiffness, and pain. Furthermore, since RA is a systemic disease, it can also affect other tissues (eg, lungs, eyes, and bone marrow).

  Currently there is no known cure for RA. Current treatments include various steroidal and non-steroidal anti-inflammatory drugs, immunosuppressants, disease modifying anti-rheumatic drugs (DMARDs), and biologicals. However, many patients still show inappropriate responses to such treatments.

  The CD20 antibody can be used as a first line therapy in patients with early stage RA (ie, methotrexate (MTX) naïve) and as a monotherapy or in combination with, for example, MTX or cyclophosphamide. Alternatively, the antibodies can be used as second line therapy for patients who are DMARD and / or MTX refractory and as monotherapy or in combination with other drug therapies (eg, MTX). Humanized CD20 binding antibodies are generally active RA (eg moderate to severe) to prevent and control joint damage in RA, to delay structural damage, to reduce inflammation-related pain. Useful for reducing signs and symptoms in RA). RA patients can be treated with a humanized CD20 antibody before, after, or in conjunction with other drugs used to treat RA (see combination therapy below). . In one embodiment, patients who have previously failed with a disease modifying anti-rheumatic drug and / or have shown an inappropriate response to methotrexate alone are treated with a humanized CD20 binding antibody herein. In one embodiment of this treatment, the patient is initially administered a humanized CD20 binding antibody alone, followed by CD20 antibody + cyclophosphamide, or CD20 antibody + methotrexate, according to the administration regimen of the present invention. The

One method of assessing treatment efficacy in RA is based on the American College of Rheumatology (ACR) criteria, which measures, among other things, the rate of improvement in tenderness and joint swelling. RA patients can be scored, for example, ACR20 compared to those not treated with any antibody (eg baseline before treatment) or treated with placebo (20% improvement). Other methods of assessing the efficacy of antibody treatment include X-ray scoring, such as the Sharp X-ray score used for scoring structural damage (such as bone erosion and joint space narrowing). Patients can be evaluated for the prevention or amelioration of disability based on the Health Assessment Questionnaire [HAQ] score, AIMS score, SF-36 during or after treatment. The ACR20 criteria may include a 20% improvement in tender (painful) joint number and joint swelling number, and at least three 20% improvements in the following five additional criteria:
1. Assessment of patient pain by visual analog scale (VAS);
2. Overall assessment (VAS) of the patient's disease activity,
3. Overall assessment of disease activity by doctors (VAS),
4). 4. Disability of self-assessment by patients as measured by health assessment questionnaire, and Acute phase reactant, CRP or ESR.

  ACRs 50 and 70 are defined similarly. Preferably, the patient has an amount of the invention effective to achieve a score of at least ACR 20, preferably at least ACR 30, more preferably at least ACR 50, even more preferably at least ACR 70, most preferably at least ACR 75, and higher. CD20 antibody is administered.

  Psoriatic arthritis has unique and distinct radiographic features. In psoriatic arthritis, joint erosion and joint space narrowing can also be assessed by the Sharp score. The humanized CD20 binding antibodies of the invention can be used to prevent joint damage and reduce signs and symptoms of disease.

  Yet another aspect of the invention is a method of treating lupus or SLE by administering to a patient suffering from SLE a therapeutically effective amount of a humanized CD20 antibody of the invention. SLE patients include patients with extrarenal manifestations as well as lupus nephritis. The SLEDAI score provides a numerical quantification of disease activity. SLEDAI is a weighted index of 24 clinical and experimental parameters known to correlate with disease activity and ranges from 0 to 103. See Bryan Gescuk & John Davis, “Novel therapeutic agent for systemic lupus erythematosus” Current Opinion in Rheumatology 2002, 14: 515-521. Antibodies against double-stranded DNA are thought to cause lupus renal redness and other symptoms. Patients undergoing antibody treatment can be monitored for time to renal redness, defined as a significant and reproducible increase in serum creatinine, urine protein, or urinary blood. Alternatively or additionally, patients can be monitored for levels of antinuclear antibodies and antibodies to double stranded DNA. Treatment of SLE includes high dose corticosteroids and / or cyclophosphamide (HDCC).

  Regarding vasculitis, approximately 75% of patients with systemic vasculitis have anti-neutrophil cytoplasmic antibodies and are clustered into one of three conditions involving small / medium sized blood vessels: Wegener's granulomatosis (WG), microscopic polyangiitis (MPA), and Churg-Strauss syndrome (CSS) (collectively known as ANCA-associated vasculitis (AAV)).

  Spondyloarthropathy is a type of joint disease, including ankylosing spondylitis, psoriatic arthritis, and Crohn's disease. The success of the treatment can be determined by an approved patient and physician general assessment measurement tool.

  Various drug therapies are used to treat psoriasis; treatment varies directly with the severity of the disease. Mild forms of psoriasis patients typically manage disease using topical treatments such as topical steroids, anthralin, calcipotriene, clobetasol, and tazarotene, whereas moderate to severe Patients with psoriasis appear to be more likely to use systemic (methotrexate, retinoid, cyclosporine, PUVA, and UVB) therapy. Tar is also used. These therapies have a combination of safety concerns, time consuming regimens, or inconvenient treatment methods. Furthermore, in some cases, expensive equipment and dedicated space are required in the examination room. Systemic medications can cause severe side effects, including hypertension, hyperlipidemia, myelosuppression, liver disease, kidney disease, and gastrointestinal upset. In addition, the use of phototherapy may increase the incidence of skin cancer. In addition to the inconvenience and discomfort associated with the use of local therapies, phototherapy and systemic treatment require patients to be placed in treatment and non-treatment cycles and to monitor lifetime exposure for their side effects.

  The efficacy of treatment for psoriasis is based on changes in the physician's overall assessment (PGA) compared to baseline conditions, as well as the Psoriasis Area and Severity Index (PASI) score, Psoriasis Symptom (Psoriasis Symptom) Assessment) (PSA) is assessed by monitoring changes in clinical signs and symptoms of the disease. Patients can be measured periodically over the entire duration of treatment on a visual analog scale used to indicate the degree of itch experienced at a particular time.

  Patients may cause an infusion reaction or symptoms associated with an infusion due to an initial infusion of therapeutic antibody. The severity of these symptoms varies and can generally be reversed by medical intervention. These symptoms include, but are not limited to, flu-like fever, chills / chills, nausea, urticaria, headache, bronchospasm, angioedema. It is desirable to minimize the drip response in the disease treatment methods of the present invention. To alleviate or minimize such adverse events, the patient may be administered an initial acclimatizing or tolerated dose of antibody followed by a therapeutically effective amount. The acclimation dose is lower than the therapeutically effective amount and is intended to acclimate the patient to a higher dose.

  One or more CD20s in combination with a second therapeutic agent, such as an immunosuppressant, in the treatment of an autoimmune disease, such as an autoimmune disease or condition associated with autoimmunity as described above, eg, in a multi-drug regimen. The binding antibody can be used to treat. The CD20 binding antibody may be administered simultaneously, sequentially or alternately with the immunosuppressive agent, or after non-response with other therapies. The immunosuppressive agent may be administered at the same dose as shown in the art or at a lower dose. Preferred adjuvant immunosuppressive agents depend on many factors, including the type of disease being treated as well as the patient's medical history.

  As used herein, “immunosuppressive agent” in adjuvant therapy refers to a substance that acts to suppress or shield the patient's immune system. Such agents include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask MHC antigens. Examples of such agents include steroids such as glucocorticosteroids such as prednisone, methylprednisolone, and dexamethasone; 2-amino-6-aryl-5-substituted pyrimidines (eg, US Pat. No. 4,665,077). ), Azathioprine (or cyclophosphamide, if there is an adverse reaction to azathioprine); bromocriptine; glutaraldehyde (masks MHC antigens as described in US Pat. No. 4,120,649) Anti-idiotype antibodies against MHC antigens and MHC fragments; cyclosporin A; cytokines or cytokine receptor antagonists (including anti-interferon-α, -β, or -γ antibodies); anti-tumor necrosis factor-α antibody; anti-tumor necrosis factor- β antibodies; anti-interleukin-2 antibodies and Anti-IL-2 receptor antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; pan-T antibody, preferably anti-CD3 or anti-CD4 / CD4a antibody; soluble peptide containing LFA-3 binding domain (July 1990) Streptokinase; TGF-β; Streptodornase; RNA or DNA derived from host; FK506; RS-61443; Deoxyspergualin; Rapamycin; T cell receptor (US Pat. No. 5,114,721); T cell receptor fragment (Offner et al., Science 251: 430-432 (1991); WO 90/11294; and WO 91/01133); And T cell receptor antibodies (European Patent No. 340,109), such as T10B9.

  In the treatment of rheumatoid arthritis, a patient can be treated with a CD20 binding antibody (eg, rituximab or ocrelizumab or a variant thereof) in combination with one or more of the following drugs: DMARDS (disease modifying anti-rheumatic drug) (E.g. methotrexate)), NSAI or NSAID (non-steroidal anti-inflammatory drugs), immunosuppressants (e.g. azathioprine; mycophenolate mofetil (CellCept (R); Roche)), analgesics, glucocorticosteroids, Cyclophosphamide, HUMIRA ™ (Adalimumab; Abbott Laboratories), ARAVA ™ (Leflunomide), REMICADE ™ (Infliximab; Centocor Inc., Malvern, Pa), ENBREL (Etanercept; Immunex, WA) , ACTEMRA (Toshirizuma Roch, Switzerland), COX-2 inhibitor. DMARDs commonly used in RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab, azathioprine, D-penicillamine, gold (oral), gold (intramuscular), minocycline, cyclosporine, staphylococcus Protein A immunoadsorption. Adalimumab is a human monoclonal antibody that binds to TNFα. Infliximab is a chimeric monoclonal antibody that binds to TNFα. Etanercept is an “immunoadhesin” fusion protein consisting of the extracellular ligand binding portion of human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. Actemra (tocilizumab) is a humanized anti-human interleukin-6 (IL-6) receptor. For conventional treatment of RA, see, for example, "Guidelines for the management of rheumatoid arthritis" Arthritis & Rheumatism 46 (2): 328-346 (February 2002). In a specific embodiment, RA patients are treated with a CD20 antibody of the invention in combination with methotrexate (MTX). An exemplary dose of MTX is about 7.5-25 mg / kg / week. MTX can be administered orally and subcutaneously.

  In the treatment of ankylosing spondylitis, psoriatic arthritis, and Crohn's disease, patients are used in combination with, for example, Remicade® (Infliximab; a product of Centocor Inc. Malvern, Pa.), ENBREL (Etanercept; Immunex, WA) Then, it can be treated with the CD20 binding antibody of the present invention.

  Treatment of SLE includes a combination of high dose corticosteroids and / or cyclophosphamide (HDCC) and CD20 antibody. Patients with SLE, AAV and NMO can be treated with a CD20 binding antibody of the invention in combination with any of the following: corticosteroids, NSAIDs, analgesics, COX-2 inhibitors, glucocorticosteroids, conventional DMARDS (eg, methotrexate, sulfasalazine, hydroxychloroquine, leflunomide), biological DMARDS, eg, anti-Blys (eg, belimumab), anti-IL6R, eg, tocilizumab; CTLA4-Ig (abatacept), anti-CD22 (eg, epratuzumab), immunosuppressant (Eg azathioprine; mycophenolate mofetil (CellCept®; Roche)), and cytotoxic agents (eg cyclophosphamide).

  In the treatment of psoriasis, patients are treated with a CD20 binding antibody in combination with topical treatments such as topical steroids, anthralin, calcipotriene, clobetasol, and tazarotene, or methotrexate, retinoids, cyclosporine, PUVA, and UVB therapy. Can be administered in combination. In one embodiment, psoriasis patients are treated sequentially or simultaneously with cyclosporine using a CD20 binding antibody.

  In order to minimize toxicity, traditional systemic therapy can be administered with a dose of a CD20 binding antibody composition of the invention, in a rotating, continuous, combinatorial or intermittent treatment regimen, or at a lower dose. May be administered in combination regimens.

  A therapeutic formulation of a CD20 binding antibody used in accordance with the present invention can produce an antibody having the desired purity by any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A (1980)) and is prepared for storage in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, including buffers such as phosphoric acid, citric acid, and other organic acids Antioxidants (including ascorbic acid and methionine); preservatives (eg octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or Propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer; For example Vinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin); chelating agents such as EDTA; A sugar, mannitol, trehalose, or sorbitol; a salt-forming counterion, such as sodium; a metal complex (eg, a Zn-protein complex); and / or a nonionic surfactant, such as TWEEN ™, PLURONICS ™, or Examples include polyethylene glycol (PEG).

III. Preparation of CD20 Antibodies The production methods and products of the present invention use or incorporate antibodies that bind to B cell surface markers, particularly antibodies that bind to CD20. Accordingly, methods for producing such antibodies are described herein.

  The CD20 antigen used for antibody production or for antibody screening can be, for example, a soluble form of CD20 or a portion thereof containing the desired epitope. Alternatively or additionally, cells expressing CD20 on their cell surface can be used to make or screen for antibodies. Other forms of CD20 useful for generating antibodies will be apparent to those skilled in the art.

  The following descriptions are examples of techniques for making antibodies used in accordance with the present invention.

(I) Polyclonal antibodies Polyclonal antibodies are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Bifunctional drugs or derivatization of related antigens into proteins that are immunogenic in the species to be immunized (eg, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) Agents such as maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residue), N-hydroxysuccinimide (via lysine residue), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N═C═ It may be useful to conjugate using NR where R and R ¹ are different alkyl groups.

  For example, by mixing 100 μg or 5 μg protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites, the antigen, immunogen Animals are immunized against sex conjugates or derivatives. One month later, the animals are boosted by subcutaneous injection at multiple sites of the original amount of 1/5 to 1/10 peptide or conjugate in Freund's complete adjuvant. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Preferably, the animal is boosted with a conjugate that is the same antigen but conjugated to a different protein and / or conjugated via a different cross-linking reagent. Conjugates may be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

(Ii) Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies included in the population are the same and / or bind to the same epitope, except such variants are generally small, except for variants that may occur during the production of monoclonal antibodies. Existing. Thus, the modifier “monoclonal” refers to a feature of an antibody that is not a mixture of separate antibodies or a polyclonal antibody.

  For example, monoclonal antibodies may be generated using the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (US Pat. No. 4,816,567). You may produce by.

  In hybridoma methods, mice or other suitable host animals (such as hamsters) are immunized as described hereinabove to produce antibodies that specifically bind to the protein used for immunization, or Trigger lymphocytes that can be produced. Alternatively, lymphocytes may be immunized in vitro. Hybridoma cells are then formed by fusing lymphocytes with myeloma cells using a suitable fusing agent such as polyethylene glycol (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

  The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium). ) (These substances prevent the growth of HGPRT-deficient cells).

  Preferred myeloma cells are cells that fuse efficiently, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, particularly preferred myeloma cell lines are mouse myeloma lines such as those derived from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and American Type Culture. SP-2 or X63-Ag8-653 cells available from Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines have also been reported in the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987)).

  Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

  The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

  After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones are subcloned by limiting dilution and standard methods (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEN or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in animals.

  Monoclonal antibodies secreted by the subclone can be purified by conventional immunoglobulin purification procedures (eg, protein A-SEPHAROSE ™, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography) in culture media, ascites, Or it is appropriately separated from serum.

  DNA encoding a monoclonal antibody can be easily obtained using conventional procedures (eg, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of a mouse antibody). Isolated and sequenced. Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is incorporated into an expression vector that is then transferred into host cells that do not separately produce immunoglobulin proteins (eg, E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells). , Or myeloma cells) can be used to synthesize monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs., 130: 151-188. (1992).

  In further embodiments, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) isolated mouse and human antibodies, respectively, using phage libraries. Is described. In subsequent publications, chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)) and combinatorial infection and recombination in vivo as strategies for constructing very large phage libraries. (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)) describes the production of high affinity (nM range) human antibodies. Thus, these techniques are a viable alternative to traditional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

  For example, by substituting coding sequences for human heavy and light chain constant domains in place of homologous mouse sequences (US Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA, 81 6851 (1984)), or the DNA may be modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the coding sequence of the immunoglobulin.

  Typically, such a non-immunoglobulin polypeptide has specificity for an antigen by substituting the constant domain of an antibody, or by substituting the variable domain of one antigen-binding site of an antibody. A chimeric bivalent antibody is made comprising one antigen binding site and another antigen binding site with specificity for a different antigen.

  In addition, antibodies comprising a variable Fc region with high affinity for FcγR include, for example, US Patent Publication No. 2005/0037000 and International Publication No. 2004/63351 (Macrogenics, Inc., STAVENHAGEN et al.) Useful for treating diseases where enhanced effector cell function is desired (eg, autoimmune diseases).

(Iii) Humanized antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is essentially the method of Winter and co-workers by replacing the hypervariable region sequences of the corresponding sequences of human antibodies (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al. Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)). Accordingly, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein substantially less than an intact human variable domain is derived from a non-human species. Has been replaced by a sequence. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. It is.

  The choice of human variable domains (both light and heavy chains) used in making humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the variable domain sequences of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then recognized 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 method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151 : 2623 (1993)).

  It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by methods of analysis of the parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and humanized sequence. The Three-dimensional immunoglobulin models are commercially available and are well known to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional conformational structure of selected immunoglobulin sequence candidates. By exploring these indications, it is possible to analyze the likely role of residues in the functioning of an immunoglobulin sequence candidate, i.e., to analyze the residues that affect the ability of an immunoglobulin candidate to bind its antigen. Become. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic (eg, high affinity for the target antigen). In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

(Iv) Human antibodies As an alternative to humanization, human antibodies may be generated. For example, it is now possible to produce transgenic animals (eg, mice) that can produce a full repertoire of human antibodies upon immunization without producing endogenous immunoglobulins. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (J _H ) gene in chimeric mice and germline mutant mice completely inhibits endogenous antibody production. By transferring a human germline immunoglobulin gene array to such germline mutant mice, human antibodies will be produced upon antigen challenge. For example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al. Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7: 33 (1993); And U.S. Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.

  Alternatively, human antibodies and antibodies in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors by using phage display technology (McCafferty et al., Nature 348: 552-553 (1990))) Fragments can be made. According to this technique, an antibody V domain gene is cloned in-frame into either the majority or minority coat protein gene of a filamentous bacteriophage (eg M13 or fd), and on the surface of the phage particle Presented as a functional antibody fragment. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also selects for genes encoding antibodies that exhibit those properties. In this way, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of ways; for a review, see, eg, Johnson et al., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. V gene repertoires from non-immunized human donors can be generated and antibodies against a diverse array of antigens (including self-antigens) can be obtained from Marks et al., J. Mol. Biol. 222: 581-597 (1991). Or essentially according to the technique described by Griffith et al., EMBO J. 12: 725-734 (1993). See also US Pat. Nos. 5,565,332 and 5,573,905.

  Human antibodies can also be generated by activated B cells in vitro (see US Pat. Nos. 5,567,610 and 5,229,275).

(V) Antibody fragments Various techniques for producing antibody fragments have been developed. Traditionally, these fragments have been obtained from proteolytic digestion of intact antibodies (eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229 : 81 (1985)). However, now these fragments can be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragment can be obtained from E. coli. F (ab ′) ₂ fragments can be formed by direct recovery from E. coli and chemically coupled (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ′) ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for making antibody fragments will be apparent to those skilled in the art. In other embodiments, the selected antibody is a single chain Fv fragment (scFv). See WO 93/16185; US Pat. No. 5,571,894; and US Pat. No. 5,587,458. The antibody fragment may also be, for example, a “chain antibody”, for example as described in US Pat. No. 5,641,870. Such chain antibody fragments can be monospecific or bispecific.

(Vi) Bispecific antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the CD20 antigen. Other such antibodies can bind to CD20 and can also bind to a second B cell surface marker. Alternatively, an anti-CD20 binding arm is an arm that binds to a trigger molecule on leukocytes, eg, a T cell receptor molecule (eg, CD2 or CD3) or an Fc receptor for IgG (FcγR), to focus the cellular defense mechanism on B cells. For example, FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). By using bispecific antibodies, certain drugs can be concentrated in B cells. These antibodies have a CD20 binding arm and an arm that binds to a drug (eg, methotrexate). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies).

  Methods for making bispecific antibodies are known in the art. The production of traditional full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, the two chains having different specificities (Millstein et al., Nature 305: 537-539 (1983)). Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) are a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Produce. Purification of the correct molecule is usually performed by an affinity chromatography step, which is cumbersome and the product yield is low. Similar procedures are disclosed in WO 93/08829 and Traunecker et al., EMBO J., 10: 3655-3659 (1991).

  According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. This fusion is preferably performed with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. Preferably it has a first heavy chain constant region (CH1) containing the site necessary for light chain binding, which is present in at least one of the fusions. The immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This greatly adjusts the ratio of the three polypeptide fragments to each other in an embodiment in which the ratio of the three polypeptide chains used in the construct results in optimal yield. It is done flexibly. However, when expression at an equal ratio of at least two polypeptide chains yields a high yield, or when the ratio is not particularly important, two or all three polypeptide chains in one expression vector It is possible to insert a coding sequence for.

  In a preferred embodiment of this approach, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (second) in the other arm. Of binding specificity). This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations. This is because the separation is facilitated by the presence of the immunoglobulin light chain in only one half of the bispecific molecule. This approach is disclosed in International Patent Publication No. 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in US Pat. No. 5,731,168, heterodimers recovered from recombinant cell culture can be engineered by genetically engineering the interface between a pair of antibody molecules. The ratio can be maximized. A preferred interface comprises at least a portion of the C _H 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (eg tyrosine or tryptophan). Replacing a large amino acid side chain with a smaller side chain (eg, alanine or threonine) creates a compensatory “cavity” of the same or similar size as the large side chain at the interface of the second antibody molecule. . This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.

  Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin and the other can be bound to biotin. Such antibodies can be used, for example, to target immune system cells to unwanted cells (US Pat. No. 4,676,980), for the treatment of HIV infection (WO 91/00360, International Publication No. 92 / 2303-3 and European Patent No. 03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980, along with a number of crosslinking techniques.

Techniques for making bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure for proteolytically cleaving intact antibodies to produce F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent diols and prevent intramolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of other Fab′-TNB derivatives for dual specificity. Antibodies are formed. This generated bispecific antibody can be used as an agent to selectively immobilize enzymes.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies are made using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab ′ portions of two different antibodies by gene fusion. Reduction of the antibody homodimer at the hinge region formed a monomer and then reoxidation to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provides an alternative mechanism for generating bispecific antibody fragments. It was done. The fragment contains a heavy chain variable domain (V _H ) connected to a light chain variable domain (V _L ) by a linker that is too short to pair between two domains on the same chain. Thus, the V _H and V _L domains of one fragment must pair with the complementary _VL and V _H domains of another fragment, thereby forming two antigen binding sites. Another strategy for generating 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 a valency greater than 2 are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

(Vii) Antibody Conjugates and Other Modifications Modifications of antibodies are contemplated herein. Thus, in one embodiment, an antibody may be conjugated to another molecule, eg, to increase the half-life or stability of the antibody, or to 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 (eg, Fab ′) linked to one or more PEG molecules are a particularly preferred embodiment of the present invention.

  The antibodies disclosed herein can also be formulated as liposomes. Liposomes containing antibodies are described in Epstein et al. Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); 485,045 and 4,544,545; and WO 97/38731 published Oct. 23, 1997, and prepared by methods known in the art. The Liposomes with extended circulation time are disclosed in US Pat. No. 5,013,556.

  Particularly useful liposomes can be made by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-GE). Liposomes are extruded through a filter with a defined pore size to obtain liposomes having a desired diameter. Fab 'fragments of the antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257: 286-288 (1982).

  Amino acid 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 the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletion from residues within the amino acid sequence of the antibody, and / or insertion into amino acid residues of the antibody, and / or substitution of amino acid residues of the antibody. Any combination of deletions, insertions and substitutions is made to arrive at the final construct, provided that the final construct has the desired characteristics. The amino acid change can also change the post-translational method of the antibody (eg, change the number or position of glycosylation sites).

  A useful method for identifying specific residues or regions of an antibody that are preferred positions for mutagenesis is described in "Alanine Scanning" as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). It is called “mutagenesis”. Here, a residue or group of residues of the target residue is identified (eg, charged residues such as arg, asp, his, lys, and glu) to affect the interaction of amino acids and antigens. To a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Those amino acids that are functionally sensitive to substitution are then narrowed down by introducing further or other mutations at or for that substitution site. In this way, the site for introducing an amino acid sequence variation is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutation is performed at the target codon or region and the expressed antibody variants are screened for the desired activity.

  Amino acid sequence insertions include fusions to amino and / or carboxyl termini ranging from one residue to a polypeptide (including 100 or more residues) and one or more amino acid residues. Includes insertion of the group into the sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus, or antibodies fused to a polypeptide or polymer. Other insertional variants of the antibody molecule include fusion of the enzyme to the N-terminus or C-terminus of the antibody, or to a polypeptide that increases the serum half-life of the antibody.

  Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The site of most interest for mutagenesis by antibody substitution includes the hypervariable region, but FR changes are also contemplated. Conservative substitutions are indicated in the table below under the heading “preferred substitutions”. If such a substitution results in a change in biological activity, it is referred to as an “exemplary substitution” in the table below, or as further described below with reference to the amino acid class. Substantial changes can be introduced and the product can be screened.

Substantial alterations in the biological properties of the antibody include: (a) the structure of the polypeptide backbone in the substitution region, eg the sheet or helix conformation, (b) the molecular charge or hydrophobicity at the target site, or (c) The effect on maintenance of side chain volume, is achieved by selecting substitutions that differ significantly. Amino acids can be classified by similarity in their side chain properties (AL Lehninger, in Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)):
(1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)
(2) Uncharged polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)
(3) Acidity: Asp (D), Glu (E)
(4) Basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be classified based on the consensus side chain properties:
(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;
(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;
(3) Acidity: Asp, Glu;
(4) Basic; His, Lys, Arg;
(5) Residues affecting chain orientation: Gly, Pro;
(6) Aromatic: Trp, Tyr, Phe.

  Non-conservative substitutions involve exchanging one member in these classes with a member of another class.

  Any cysteine residue that is not involved in maintaining the proper conformation of the antibody can also be generally replaced with serine to improve the oxidative stability of the molecule and to prevent abnormal cross-linking. Conversely, a cysteine bond may be added to the antibody to improve antibody stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

  A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. In general, variants selected for further development have improved biological properties compared to the parent antibody that produces them. A convenient method for generating such substitutional variants is affinity maturation using phage display. Briefly, all possible amino substitutions are made at each site by mutating several hypervariable region sites (eg, 6-7 sites). The antibody variants thus generated are presented in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (eg, binding affinity) as disclosed herein. To identify hypervariable region candidate sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex in order 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 mutants are made, the panel of mutants can be screened as described herein to further develop antibodies with superior properties in one or more related assays. Can be selected for.

  In another type of amino acid variant of the antibody, the original glycosylation pattern of the antibody is altered. Such changes include the deletion of one or more carbohydrate moieties found in the antibody and / or the addition of one or more glycosylation sites that are not present in the antibody.

  Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a 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 other than proline) are recognized for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Is an array. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a glycosylatable site. O-linked glycosylation refers to the sugar N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid (most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used). It refers to the attachment of one of them.

  Addition of glycosylation sites to the antibody is conveniently accomplished by changing the amino acid sequence to include one or more tripeptide sequences as described above (in the case of N-linked glycosylation sites). Changes may be made by adding or substituting one or more serine or threonine residues to or replacing the original antibody sequence (in the case of O-linked glycosylation sites).

  If the antibody contains an Fc region, the carbohydrate attached to it can be altered. For example, antibodies having a mature carbohydrate structure lacking fucose attached to the Fc region of the antibody are described in US Patent Application No. 2003/0157108 (Presta, L). See also US Published Patent No. 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Antibodies with branched N-acetylglucosamine (GlcNAc) in the carbohydrate attached to the Fc region of the antibody are described in WO 2003/011878, Jean-Mairet et al., And US Pat. No. 6,602,684, It is mentioned in Umana et al. An antibody having at least one galactose residue in the oligosaccharide attached to the Fc region of the antibody is reported in International Publication No. 1997/30087, Patel et al. See also International Publication No. 1998/58964 (Raju, S.) and International Publication No. 1999/22764 (Raju, S.) regarding antibodies in which the carbohydrate attached to the Fc region is altered. I want. See also US Patent Publication No. 2005/0123546 (Umana et al.) For antigen-binding molecules that have undergone modified glycosylation.

  Preferred glycosylation variants herein include an Fc region in which the carbohydrate structure attached to the Fc region lacks fucose. Such mutants have improved ADCC function. In some instances, the Fc region may further improve one or more amino acid substitutions in the Fc region that further improve ADCC (eg, positions 298, 333, and / or 334 of the Fc region (Eu numbering of residues)). including. Examples of publications relating to “defucosylated” or “fucose-deficient” antibodies include US 2003/0157108; WO 2000/61739, WO 2001/29246; U.S. Published Patent No. 2002/0164328; U.S. Published Patent No. 2004/0093621; U.S. Published Patent No. 2004/0132140; U.S. Published Patent No. 2004/0110704; U.S. Published Patent No. 2004/0110282; US Publication No. 2004/0109865; International Publication No. 2003/085119; International Publication No. 2003/084570; International Publication No. 2005/035586; International Publication No. 2005/035778; International Publication No. 2005. / 0 No. 3742; Okazaki et al., J Mol Biol 336:..... 1239-1249 (2004)); Yamane-Ohnuki et al, Biotech Bioeng 87: 614 (2004) and the like. Examples of cell lines that produce defucosylated antibodies include Lec13CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application 2003/0157108). A1, Presta, L; and WO 2004/056312 A1, Adams et al., In particular Example 11), and knockout cell lines such as α-1,6-fucosyltransferase gene, FUT8 knockout CHO cells (Yamane- Ohnuki et al., Biotech. Bioeng. 87: 614 (2004)).

  Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or oligonucleotide-mediated (or site-specificity) of previously prepared variant or non-mutant versions of antibodies. ) Preparation by, but not limited to, mutagenesis, PCR mutagenesis, and cassette mutagenesis.

  It may be desirable to modify the antibodies of the invention with respect to effector function, eg, to enhance the 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 additionally, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody produced in this way may have improved internalization capabilities and / or increased complement dependent cell killing and ADCC. See Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies may also be prepared using heterobifunctional crosslinkers as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, antibodies having two Fc regions and thereby enhanced complement lysis and ADCC capabilities can be generated by genetic engineering. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

  International Publication No. WO 00/42072 (Presta, L.) describes antibodies (including amino acid substitutions in their Fc region) having improved ADCC function in the presence of human effector cells. Preferably, the improved ADCC antibody comprises a substitution at positions 298, 333, and / or 334 of the Fc region. Preferably, the altered Fc region is a human IgG1 Fc region comprising or consisting of substitutions at one, two or three of these positions.

  Antibodies with altered C1q binding and / or CDC are disclosed in WO 99/51642, US Pat. No. 6,194,551B1, US Pat. No. 6,242,195B1, US Pat. No. 6,528,624B1. And US Pat. No. 6,538,124 (Idusogie et al.). The antibody comprises amino acid substitutions at one or more of the amino acids at positions 270, 322, 326, 327, 329, 313, 333, and / or 334 of the Fc region.

In order to increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an IgG molecule (eg, IgG ₁ , IgG ₂ , IgG ₃ , or IgG ₄ ) that is involved in increasing the serum half-life of an IgG molecule in vivo. The epitope of the Fc region. Antibodies with substitutions in their Fc region and increased serum half-life are also described in WO 00/42072 (Presta, L.).

  Genetically engineered antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (US Patent Application No. 2002 / 0004587A1, Miller et al.).

IV. Pharmaceutical Formulations Exemplary anti-CD20 antibody formulations are described in WO 98/56418, expressly incorporated herein by reference. Another formulation is 40 mg / mL anti-CD20 antibody, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 with a minimum shelf life of 2 years at 2-8 ° C. (PH 5.0) is a multi-dose liquid formulation. Another anti-CD20 formulation of interest includes 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and 10 mg / mL antibody in sterile water for injection. Contains (pH 6.5). Yet another aqueous pharmaceutical formulation has a surface activity of pH about 4.8 to about 5.5, preferably 10 to 30 mM sodium acetate, pH 5.5, in an amount of about 0.01 to 0.1% v / v. Polysorbate as an agent, trehalose in an amount of 2-10% w / v and benzyl alcohol as a preservative (US Pat. No. 6,171,586). A lyophilized formulation adapted for subcutaneous administration is described in WO 97/04801. Such a lyophilized formulation can be reconstituted to a high protein concentration using a suitable diluent, and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein.

  One formulation for the humanized 2H7 variant is 12-14 mg / mL antibody in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20 (pH 5.8).

  In a specific embodiment, the 2H7 variant and in particular variant A is formulated with 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20, and 20 mg / mL antibody in sterile water for injection. (PH 5.8).

  The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably those with ancillary activity that do not adversely affect each other. For example, cytotoxic agents, chemotherapeutic agents, cytokines or immunosuppressive agents (eg those acting on T cells, eg cyclosporine, or antibodies that bind T cells, eg antibodies that bind LFA-1) It may be desirable to provide. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used at the same doses and routes of administration as described herein, or at about 1-99% of the doses used so far.

  Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filter membrane.

V. Products In another aspect of the present invention, products comprising materials useful for the treatment of autoimmune diseases are provided. The invention particularly relates to (a) a container (preferably the container contains an antibody and a pharmaceutically acceptable carrier or diluent) containing an antagonist (eg an antibody that binds to a B cell surface marker, such as a CD20 antibody). And (b) a package insert containing instructions for treating an autoimmune disease (eg, rheumatoid arthritis) in a subject, wherein the instructions are The subject has been shown to receive a full therapeutically effective amount of an antagonist or antibody (eg, a CD20 antibody) as a single intravenous infusion.

  In certain embodiments, the product herein further comprises a container containing a second pharmaceutical product, wherein the antagonist or antibody is the first pharmaceutical product, and the product comprises an effective amount of the second pharmaceutical product. And further comprising instructions in a package insert for treating the subject. The second pharmaceutical agent can be any of those listed above, and exemplary second pharmaceutical agents are those listed above, which include antibiotics, immunosuppressants, disease modifying properties. Antirheumatic drugs (DMARD), pain control agents, integrin antagonists, non-steroidal anti-inflammatory drugs (NSAIDs), cytokine antagonists, bisphosphonates, or hormones, or combinations thereof, more preferably DMARDs, NSAIDs, pain control agents, Or it is an immunosuppressive agent. Most preferably, the second pharmaceutical agent is methotrexate.

  In this aspect, the package insert is present on or associated with the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition effective in treating the targeted autoimmune disease and may have a sterile outlet (eg, the container may be an intravenous solution) It can be a bag or a vial with a stopper that can be pierced by a needle for subcutaneous injection). At least one active agent in the composition is a CD20 antagonist, eg, a CD20 antibody. The product may further comprise a further container containing a pharmaceutically acceptable dilution buffer (eg bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and / or dextrose solution). . The product may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

  Further details of the invention will be apparent from the following non-limiting examples.

Reference Examples Humanized 2H7 antibody variants were prepared and biological functions (human) as described in WO 04/056312, which is hereby incorporated by reference in its entirety. Assayed for CD20 binding affinity, effector function, and B cell depletion). Chimeric 2H7 with mouse 2H7 antibody variable region sequences and mouse V and human C are described, for example, in US Pat. Nos. 5,846,818 and 6,204,023, which are incorporated herein by reference in their entirety. ).

Example 1
Phase I / II clinical trial to test the efficacy and safety of ocrelizumab administered as a single intravenous infusion to patients with active rheumatoid arthritis. Administered as a single intravenous infusion (iv). Stable doses (10-25 mg / week) of methotrexate after failure of 1-6 DMARDs (including biological agents) to demonstrate the efficacy of ocrelizumab (OCR), a humanized monoclonal antibody targeting CD20B cells Tested by ACR (American College of Rheumatology) response in patients with moderate to severe rheumatoid arthritis (RA) receiving concomitant administration of (MTX).

  In particular, a multicentre randomized placebo-controlled phase I / II study on the safety of increasing intravenous doses of rhuMAb2H7 (Ro496-4913, PRO70769) has received a combination of stable doses of methotrexate, It was performed in patients with moderate to severe rheumatoid arthritis with unsatisfactory clinical responses. The purpose of the trial was to evaluate the safety and tolerability of a escalating intravenous (IV) dose of rhuMAb2H7 in combination with methotrexate (MTX) in patients with moderate to severe rheumatoid arthritis (RA). It was.

  The trial was conducted using the following protocol:

Part I—Dose escalation Patients are administered with a single intravenous infusion of one dose of rhuMAb or placebo equivalent at the following dose levels:
First group: 400mg
Second group: 1000 mg
3rd group: 1500mg

  Dose escalation is performed after treating a minimum of 10 patients per dose level (8 active substances; 2 placebos). The incremental decision is based on an overview of the previous group of data in the study. The number of dose limiting toxicities can be assessed and based on this number, the study can proceed to the next dose level, or additional patients can be enrolled at the same dose level.

Part II After the dose escalation phase and interim analysis of the data, the three doses used in Part I (assumed to be 400, 1000, and 1500 mg) are tolerated during the dose escalation phase If demonstrated, an additional 120 patients (96 actives; 24 placebos) are randomly assigned to these three doses.

Number of patients At least 40 in Part I; 120 in Part II

Target population-adult patients (18 to 80 years)
-Moderate to severe RA
-Failed with one disease modifying anti-rheumatic drug (DMARD) or biological agent (ineffective or intolerable) but not more than five of these- Currently receiving MTX, but clinical response to treatment with MTX is unsatisfactory (ie, partial responders)

Duration of the study-one intravenous infusion; evaluated until week 24, followed by a further 48 weeks follow-up: total 72 weeks-patients discontinued at any time point or still have B cells at 72 weeks Patients who are depleted will be followed for 48 weeks for safety or until their B cells have recovered (long in any case).

Significant inclusion criteria with active RA:
-Diagnosed as RA for at least 6 months (according to ACR criteria revised in 1987)
-Serum rheumatoid factor is positive (more than 20 IU / L)
-C-reactive protein of 1.5 mg / dL or more at screening or erythrocyte sedimentation rate of 28 mm / h or more-8 or more joint swelling numbers (66 joints) and 8 or more tender joints at screening and at randomization Number (68 joints)

Previous treatment / background treatment:
-Current treatment on an outpatient basis-Current treatment of at least 12 weeks with 10-25 mg / week MTX, stable for the last 4 weeks prior to screening-All 4-8 weeks prior to randomization Of DMARDs (except MTX); Etanercept, Infliximab, Adalimumab, and Leflunomide discontinued 8 weeks prior (after drug wash with cholestyramine in patients receiving leflunomide)
-Corticosteroid dose does not exceed 10 mg / day prednisone or equivalent and is stable for the last 4 weeks before screening-When receiving NSAIDs, the dose is stable 2 weeks before screening -Willing to receive oral folic acid

Study Product Dose-400, 1000, 1500, and 2000 mg single dose rhuMAb2H7
Route-intravenous infusion (at the rate provided in Appendix 3 of the protocol)

Comparative “Drug” Dose / Route / Regiment—One Placebo Drip

Concomitant treatment Pre- and post-infusion treatment Prophylactic (recommended)-Prophylactic doses of acetaminophen and diphenhydramine (or other non-sedating antihistamine) recommended to be administered 30-60 minutes before infusion of rhuMAB2H7 Is done.

  Possible steroid protection during infusion-If significant infusion-related events are observed at a given dose level, and reducing the infusion rate does not reduce the number of infusion-related events, the sponsor It may be recommended to administer a premedication of intravenous corticosteroids to patients undergoing treatment.

  Methotrexate (required)-stable 10-25 mg / week dose (oral or parenteral); provided by Trial Center Pharmacy

  Corticosteroid (optional)-Continue oral doses of prednisone or prednisone equivalent up to 10 mg / day stable

  NSAIDs-continue at a stable dose

Re-treatment The onset of clinical response is expected to be slow. Therefore, if possible, background treatment should continue unchanged until week 24. From the 24th week onwards, eligible patients can undergo retreatment under another retreatment protocol.

Supply form rhuMAb2H7 and its corresponding placebo are supplied by the sponsor in 10 mL vials; rhuMAb2H7 is at a concentration of 20 mg / mL. rhuMAb2H7 must be diluted in 0.9% sodium chloride prior to infusion, thereby providing an appropriate dose in the 250-500 mL volume (dilution information is provided in Appendix 3 of the protocol).

  Other medicines will be prescribed by researchers and provided locally by Center Pharmacy.

The following evaluation:
-Safety Adverse Events-Adverse events (nature, intensity, severity, association, reversibility, and treatment) including infusion-related reactions and dose-limiting toxicities (see procedure section for definition); NCI CTCAEv3. Hematology, clinical chemistry, and urinalysis-occurrence of abnormal laboratory values immunology-generation of human anti-rhuMAB2H7 antibody-rheumatoid factor concentration-serum immunoglobulin concentration (IgG , IgM, IgA, IgE)
-Serum tetanus toxin antibody concentration-Peripheral blood B cell count; T cell count-Efficacy-Percentage of patients with clinical response of 20%, 50% and 70% according to ACR criteria and at day 1 as baseline Components of this result measured at 24 weeks using pre-infusion ACR core set values-Disease activity score at 24 weeks and the percentage of EULAR responders

Statistical analysis:
All analyzes are for investigation. Patients are classified according to the treatment they actually receive. All patients receiving any study drug (rhuMAb2H7 or placebo) are included for analytical purposes.

  Patients who require increased MTX dose, increased corticosteroid dose, introduction of additional interventions, or excluded drug therapy during the first 24 weeks of the study are considered non-responders in the efficacy analysis .

  Control (placebo) patients at various dose levels are combined into one control group. All results are presented separately for the control group and each active dose group: 400 mg, 1000 mg, 1500 mg, and 2000 mg.

  Two subgroup analyzes are performed: one is based on all participating patients and the other is based on patients randomly assigned to Part II of the study after completing Part I dose escalation.

Pharmacokinetics:
Serum concentrations of rhuMAb2H7 by patient and dose group and computational pharmacokinetic parameters are listed and summarized by description (mean, standard deviation,% coefficient of variation, minimum, maximum). Individual concentration and average concentration vs time plots are presented in both halves and logarithmic scales. Pharmacokinetic parameters are computed using a non-compartmental method.

  In all calculations, concentrations below the quantification limit of the assay are deleted or assigned a numerical value based on the reported lower limit of the assay.

Pharmacokinetics:
Each patient's B cell depletion profile is determined for each dose level and summarized by description. The pharmacokinetics of peripheral blood B cell depletion and sufficiency are characterized by the degree and duration of depletion at each dose level. In addition, to provide population pharmacokinetic modeling, a prospective analytical plan was developed as a pharmacokinetic / pharmacokinetic analysis of the relationship between pharmacokinetics of rhuMAb2H7 and peripheral blood B cell depletion and sufficiency pharmacokinetics. Be prepared.

Formulation:
The rhuMAb 2H7 drug product is manufactured by Genentech as a sterile, clear and colorless preservative-free liquid for dilution upon intravenous administration. The rhuMAb 2H7 drug product is supplied at a concentration of 20 mg / mL in a 10 cc single use vial containing nominally 10 mL (200 mg) rhuMAb 2H7 per vial. The rhuMAb 2H7 drug product is formulated in 10 mM histidine sulfate, 60 mg / mL sucrose, 0.2 mg / mL polysorbate 20, and sterile water for injection. The pH is adjusted to 5.8.

  The rhuMAb 2H7 drug product must be diluted prior to administration. A solution of rhuMAb2H7 for intravenous administration is prepared by diluting the drug product into an infusion bag containing 0.9% sodium chloride to a final drug concentration of 1.6-6 mg / mL. A placebo corresponding to rhuMAb 2H7 is also supplied in a 10 cc single use vial containing a nominal 10 mL placebo solution per vial. It is the same composition as the rhuMAb2H7 drug product, but does not contain rhuMAb2H7. A rhuMAb2H7 placebo solution for intravenous administration is prepared by diluting the rhuMAb2H7 placebo into an infusion bag containing 0.9% sodium chloride using the same procedure as the rhuMAb2H7 drug product.

Dosage, administration:
The test drug must be administered as a slow intravenous infusion at the indicated rate. The rate depends on the dose administered and does not exceed 250 mL / hour. It should not be administered as an IV push or IV bolus. Test drug infusions should be made through a dedicated line. An isotonic 0.9% sodium chloride solution should be used as the infusion medium. It is recommended that patients receive oral prophylactic treatment with acetaminophen (1 g) and diphenhydramine HCl (50 mg; or an equivalent dose of a similar drug) 30-60 minutes prior to the start of infusion. All doses other than 2000 mg are administered in a volume of 250 mL; 2000 mg (and the equivalent placebo) is administered in a volume of 500 mL.

Re-treatment feasibility Re-treatment of patients with recurrent symptoms has been treated with active substance, achieving at least ACR20 response 24 weeks after initial treatment and no significant adverse events during the study May be administered only after 24 weeks. Retreatment in these patients is at the same dose as the first regimen when symptoms recur (represented by more than 8 SJCs and more than 8 TJCs after 24 weeks).

Infusion schedule The rhuMAb 2H7 drug product is delivered as a liquid at a concentration of 20 mg / mL in a 10 cc, single use vial containing nominally 10 mL (200 mg) of rhuMAb 2H7 per vial. A corresponding placebo that is the same composition as the rhuMAb 2H7 drug product but does not contain rhuMAb 2H7 is also supplied as a liquid in a 10 cc, single use vial containing nominally 10 mL placebo solution per vial.

  Rituximab (MabThera) prescription information provides the following information regarding infusion:

  First infusion: The recommended initial rate for infusion is 50 mg / hour; after the first 30 minutes, it can be increased in increments of 50 mg / hour every 30 minutes to a maximum of 400 mg / hour. If hypersensitivity or an infusion-related event occurs, the infusion can be temporarily delayed or interrupted and continued at half the previous rate if symptoms improve. If the same adverse reaction occurs severely the second time, the decision to discontinue treatment should be considered. The infusion rate may be increased as symptoms improve.

  Subsequent infusion: Instill at an initial rate of 100 mg / hr, increase in increments of 100 mg / hr at 30 minute intervals, up to a maximum of 400 mg / hr.

  The ongoing ACT 2847g test uses the same standard infusion rate, with the highest dose being 1000 mg and increasing to 400 mg / hour, with an overall infusion time of about 4 hours and 15 minutes.

  To investigate faster infusion times, this protocol uses the following schedule, but with any indication that it is not tolerated for faster infusion rates in the lowest dose group in Part I: For example, in subsequent dose groups, the rate is reduced and the increase in infusion rate (mg / hour) is reduced to the standard rate of steps of 50 mg / hour.

  Active RA was defined by 8 or more swollen joints and 8 or more tender joints, and increased C-reactive protein (CRP) or increased erythrocyte sedimentation rate (ESR). Patients (n = 175) were randomly assigned to either a single intravenous infusion of OCR (400, 1000, 1500, or 2000 mg) or equivalent placebo (PLO). 40 patients (2 in each cohort, 2 placebos, 8 actives) and 135 randomly assigned to 3 lower doses or PLOs who participated in Part I of the 4 incremental doses in this study Other patients were included in the safety and ITT analysis. Before the test drug (OCR), glucocorticoid before and after infusion was not administered. Clinical evaluations were performed at 2 week intervals up to the 8th week and then at 4 week intervals up to the 24th week. The first endpoint was the proportion of patients who achieved an ACR20 response at 24 weeks.

Results 70% and 89% of the OCR and PLO groups were female, and at baseline the mean numbers of tender and swollen joints were 16 and 17, 28 and 28, ESR 45 and 49, CRP 25 and 21, RF + 92, respectively. % And 94%. B cell depletion (96% in OCR at 12 weeks) and clinical response were observed in all OCR dose groups. The ACR 20/50/70 responses at 24 weeks were 57%, 31% and 15% for the combined OCR group and 40%, 10% and 3% for the PLO group, respectively.

CONCLUSION Treatment with one infusion of humanized anti-CD20 ocrelizumab but no pre- and post-infusion steroids in RA patients receiving background MTX achieved B cell depletion at all doses, An increased ACR 20/50/70 response was seen compared to MTX alone. With only one drip, discontinuation due to non-tolerance was only 2 people, indicating good tolerability. The onset of infection and the type of infection were similar in the PLO and OCR groups.

  The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the specified construct. Because the specified embodiments are taken as an explanation of certain aspects of the invention, any construct that is functionally equivalent is within the scope of the invention. The material specification herein is not an admission that the description contained herein is inappropriate for the practice of any aspect of the present invention, including its best mode, and also claims Is not limited to the specific examples presented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and fall within the scope of the appended claims.

  From the description of the invention herein, it is manifest that various equivalents can be used to implement the concepts of the present invention without departing from its scope. Further, while the present invention has been described with particular reference to particular embodiments, those skilled in the art will recognize that changes can be made formally and in detail without departing from the spirit and scope of the present invention. You will recognize. The described embodiments are considered in all respects as illustrative and not restrictive. While the invention is not limited to the specific embodiments described herein, it should be understood that many equivalents, rearrangements, modifications, and substitutions can be made without departing from the scope of the invention. It is. Accordingly, further aspects are within the scope of the invention and are within the scope of the following claims.

  All US patents and patent applications; foreign patents and patent applications; scientific literature; books; and other publications mentioned in this specification, each in its entirety, although shown in its entirety (Including any drawings, figures and tables) are specifically incorporated herein by reference as if individually incorporated by reference.

Claims (62)

  Use of a CD20 antagonist in the manufacture of a medicament for the treatment of an autoimmune disease, wherein the medicament is for administering the antagonist to a human patient in a single therapeutically effective intravenous infusion (iv). The use.   Use according to claim 1, wherein a second complete therapeutically effective dose of CD20 antagonist is administered intravenously 4-6 months after the first administration.   Use according to claim 2, wherein the second intravenous infusion is for administration to a patient who has responded but has relapsed after the first administration.   4. Use according to any one of claims 1 to 3, wherein the CD20 antagonist is a CD20 monoclonal antibody.   Use according to any of claims 1 to 4, wherein the therapeutically effective amount for administration in the first and second intravenous infusion is essentially the same.   6. Use according to any one of claims 4 to 5, wherein the CD20 monoclonal antibody is chimeric, humanized or human.   Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, Crohn's disease, Sjogren's syndrome, optic neuromyelitis (NMO), ANCA-related vasculitis, Wegener's disease, inflammatory bowel disease, sudden onset Thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, Use according to any one of claims 1 to 6, selected from the group consisting of diabetes, Raynaud's syndrome, and glomerulonephritis.   The use according to any one of claims 1 to 7, wherein the autoimmune disease is rheumatoid arthritis.   9. Use according to any one of claims 1 to 8, wherein the patient has shown an inadequate response to previous treatment with methotrexate, a TNF antagonist or a different CD20 antagonist.   The use according to any one of claims 1 to 9, wherein the autoimmune disease is active rheumatoid arthritis.   11. Use according to claim 10, wherein the active rheumatoid arthritis is moderate to severe rheumatoid arthritis.   Use according to any one of claims 1 to 11, wherein the therapeutically effective amount is between 10 mg and 2000 mg.   Use according to any one of claims 1 to 12, wherein the CD20 antibody is a humanized 2H7 antibody.   14. Use according to any one of claims 1 to 13, wherein the therapeutically effective amount is between 400 mg and 1500 mg.   15. Use according to any one of claims 1 to 14, wherein the therapeutically effective amount is 400 mg.   16. Use according to any one of claims 1 to 15, wherein the therapeutically effective amount is 1000 mg.   Use according to any one of claims 1 to 16, wherein the therapeutically effective amount is 1500 mg.   The humanized CD20 antibody comprises 2H7 variants A, B, and I, or a fragment thereof, each comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NOs: 7, 8, and 15. 18. Use according to any one of claims 1 to 17, selected from the group consisting of:   The humanized CD20 antibody is 2H7 variant A comprising the full-length light chain represented by SEQ ID NO: 6 and the full-length heavy chain represented by SEQ ID NO: 7, or a fragment thereof. Use as described in section.   The humanized CD20 antibody binds to essentially the same epitope as 2H7 variant A comprising the full length L chain shown in SEQ ID NO: 6 and the full length H chain shown in SEQ ID NO: 7, or a fragment thereof. Use of any one of 1-17.   The humanized CD20 antibody comprises 2H7 variant C, D, comprising the full length L chain shown in SEQ ID NO: 9 and the full length H chain shown in SEQ ID NOs: 10, 11, 12, 13 and 14, respectively, or fragments thereof. 18. Use according to any one of claims 1 to 17, selected from the group consisting of F, F, G and H.   The humanized CD20 antibody is 2H7 variant B comprising the full-length light chain represented by SEQ ID NO: 6 and the full-length heavy chain represented by SEQ ID NO: 8, or a fragment thereof. Use as described in section.   The humanized CD20 antibody is 2H7 variant C comprising the full-length light chain represented by SEQ ID NO: 9 and the full-length heavy chain represented by SEQ ID NO: 10, or a fragment thereof. Or use according to claim 1.   18. Use according to any one of claims 1 to 17, wherein the antibody is the chimeric antibody rituximab.   18. Use according to any one of claims 1 to 17, wherein the CD20 antibody is the human antibody HUMAX-CD20 (TM).   CD20 antibodies are non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticosteroids, cyclophosphamide, adalimumab, leflunomide, infliximab, etanercept, ofatumumab, tocilizumab, AME-133, Immu-106, and COX 26. Use according to any one of claims 1 to 25, for administration in combination with a drug selected from the group consisting of -2 inhibitors.   27. Use according to any one of claims 1 to 26 for administration, comprising a second therapeutic agent.   28. Use according to any one of claims 1 to 27, wherein the second therapeutic agent is an immunosuppressant.   29. Use according to any one of claims 1 to 28, wherein the second therapeutic agent is methotrexate.   30. Use according to any one of claims 1 to 29, wherein the use of methotrexate is for administration at a dose of 10 to 25 mg / week.   31. Use according to any one of claims 1 to 30, comprising administration of 1 to 6 DMARDs prior to administration of the CD20 antibody.   32. Use according to any one of claims 1-31, comprising no steroid treatment prior to administration of the CD20 antibody.   Use of a CD20 antibody in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA), said medicament comprising a single therapeutically effective intravenous infusion (i. v.), wherein the use is for administration to a human patient.   34. Use according to claim 33, wherein the CD20 antibody is a chimeric, humanized or human monoclonal antibody.   The humanized CD20 antibody comprises 2H7 variants A, B, and I, each comprising a full length L chain represented by SEQ ID NO: 6 and a full length heavy chain represented by SEQ ID NOs: 7, 8, and 15, or a fragment thereof. 35. Use according to any one of claims 33 to 34, selected from the group consisting of:   36. The humanized CD20 antibody is a 2H7 variant A comprising the full length L chain represented by SEQ ID NO: 6 and the full length H chain represented by SEQ ID NO: 7, or a fragment thereof. Use as described in section.   The humanized CD20 antibody binds to essentially the same epitope as 2H7 variant A comprising the full length L chain shown in SEQ ID NO: 6 and the full length H chain shown in SEQ ID NO: 7, or a fragment thereof. Use of any one of 33-34.   The humanized CD20 antibody comprises 2H7 variant C comprising the full length L chain shown in SEQ ID NO: 9 and the full length H chain shown in SEQ ID NOs: 10, 11, 12, 13, and 14, respectively, or fragments thereof, 35. Use according to any one of claims 33 to 34, selected from the group consisting of D, F, G and H.   36. The humanized CD20 antibody is 2H7 variant B comprising the full length L chain represented by SEQ ID NO: 6 and the full length H chain represented by SEQ ID NO: 8, or a fragment thereof. Use as described in section.   40. The use of claim 38, wherein the humanized CD20 antibody is 2H7 variant C comprising the full length L chain shown in SEQ ID NO: 9 and the full length H chain shown in SEQ ID NO: 10, or a fragment thereof.   35. Use according to any one of claims 33 to 34, wherein the antibody is the chimeric antibody rituximab.   42. Use according to any one of claims 33 to 41, wherein the administration of the CD20 antibody for the second time is not performed for at least 4 months after the administration.   43. Use according to any one of claims 33 to 42, wherein a 400 mg dose of CD20 antibody is administered.   43. Use according to any one of claims 33 to 42, wherein a 1000 mg dose of CD20 antibody is administered.   43. Use according to any one of claims 33 to 42, wherein a 1500 mg dose of CD20 antibody is administered.   Use of 2H7 variant A comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 7, or a fragment thereof, in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA) The use, wherein the medicament is for administering a 400 mg dose of the antibody to a human patient in a single therapeutically effective intravenous infusion (iv).   Use of 2H7 variant A comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 7 or a fragment thereof in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA) Wherein the medicament is for administration of a 1000 mg dose of the antibody to a human patient in a single therapeutically effective intravenous infusion (iv).   Use of 2H7 variant A comprising a full-length light chain represented by SEQ ID NO: 6 and a full-length heavy chain represented by SEQ ID NO: 7 or a fragment thereof in the manufacture of a medicament for the treatment of active rheumatoid arthritis (RA) Wherein the medicament is for administering a 1500 mg dose of the antibody to a human patient in a single therapeutically effective intravenous infusion (iv).   49. Use according to any one of claims 46 to 48, wherein the administration of the CD20 antibody for the second time is not performed for at least 4 months after the administration.   A product comprising (a) a container containing a CD20 antagonist, and (b) a package insert containing instructions for treating an autoimmune disease in a human subject, wherein the instructions herein include: The product shown to be administered a full therapeutically effective amount of a CD20 antagonist as a single intravenous infusion.   52. The product of claim 51, wherein the CD20 antagonist is a chimeric, humanized, or human CD20 monoclonal antibody.   52. The product of any one of claims 50 to 51, wherein the autoimmune disease is rheumatoid arthritis.   53. The CD20 antagonist according to any one of claims 50 to 52, wherein the CD20 antagonist is a CD20 antibody 2H7 variant A comprising the full length L chain represented by SEQ ID NO: 6 and the full length H chain represented by SEQ ID NO: 7, or a fragment thereof. Product described in the section.   54. A product according to any one of claims 50 to 53, wherein the instructions indicate a fully therapeutically effective amount of 400 mg to 1500 mg.   A product comprising: (a) a container containing a CD20 antibody; and (b) a package insert containing instructions for treating rheumatoid arthritis in a human subject, wherein the instructions herein include: The product shown to be administered a complete therapeutically effective amount of CD20 antibody from 400 mg to 1500 mg as a single intravenous infusion.   57. The product of claim 56, wherein the CD20 antibody is a humanized 2H7 variant A comprising the full length L chain shown in SEQ ID NO: 6 and the full length H chain shown in SEQ ID NO: 7, or a fragment thereof.   Humanized 2H7 variant A is formulated to be 20 mg / ml antibody at pH 5.8 in 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20, and sterile water for injection. 57. A product according to any one of claims 55 to 56.   A pharmaceutical formulation comprising a fully effective amount of a CD20 antibody in a form suitable for one-time intravenous administration.   59. The pharmaceutical formulation of claim 58, wherein the CD20 antibody is a humanized 2H7 variant A comprising the full length L chain shown in SEQ ID NO: 6 and the full length H chain shown in SEQ ID NO: 7, or a fragment thereof.   60. The pharmaceutical formulation according to any one of claims 58 to 59, wherein the effective amount is from 400 mg to 1500 mg.   61. The CD20 antibody is a humanized 2H7 variant A comprising a full length L chain represented by SEQ ID NO: 6 and a full length H chain represented by SEQ ID NO: 7, or a fragment thereof. The pharmaceutical preparation according to Item.   Humanized 2H7 variant A is formulated to be 20 mg / ml antibody at pH 5.8 in 10 mM histidine sulfate, 60 mg / ml sucrose, 0.2 mg / ml polysorbate 20, and sterile water for injection. 62. A pharmaceutical formulation according to any one of claims 58 to 61.