MXPA06011850A - Treatment of disorders - Google Patents

Abstract

The present invention concerns treatment of polychondritis or mononeuritis multiplex in a mammal with an effective amount of an antibody that binds to CD20, optionally also with another agent that treats such disorders in an effective amount.

Description

TREATMENT OF DISORDERS Related Requests This application claims the priority of the Provisional Requests of E.U. Nos .: 60 / 563,227 filed on April 16, 2004 and 60 / 565,098 filed on April 22, 2004, to which Provisional Requests this application claims priority under 35 U.S.C. §119, the contents of which are incorporated herein by reference. Field of the Invention The present invention relates to the treatment of disorders with antagonists that bind to surface markers of B cells, such as CD19 or CD20, e. g. the antibodies that bind to CD20. Background of the Invention Lymphocytes are one of many types of white blood cells produced in the bone marrow during the process of hematopoiesis. There are two main populations of lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells). Lymphocytes of particular interest herein are B cells. B cells mature within the bone marrow and leave the marrow expressing an antibody that binds to the antigen on its cell surface. When a simple B cell first encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into memory B cells and effector cells called "plasma cells." Memory B cells have a longer lifespan and continue to express the antibody bound to the membrane with the same specificity as the original progenitor cell. The plasma cells do not produce antibodies bound to the membrane but instead produce the antibody in a form that can be secreted. The secreted antibodies are the main effector molecules of humoral immunity. The CD20 antigen (also called B-cell restricted differentiation antigen Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located in a pre-B and mature B lymphocyte (Valentine et al., J. Biol. Chem. 264 (19): 11282-11287 (1989); and Einfeld et al., EMBO J. 1 (3) .711-717 (1988)). The antigen is also expressed in more than 90% of non B-cell Hodgkin lymphomas (NHL) (Anderson et al., Blood 63 (6): 1424-1433 (1984)), but it is not found in hematopoietic germ cells, pro-B cells, normal plasma cells or other normal tissues (Tedder et al., J. Immunol., 135 (2): 973-979 (1985)). CD20 regulates one (s) early stage (s) in the activation process for the initiation and differentiation of the cell cycle (Tedder et al., Supra) and possibly functions as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D: 195 (1990)).

Given the expression of CD20 in B-cell lymphomas, this antigen can serve as a candidate to "be the target" of such lymphomas. In essence, such an objective can be generalized as follows: antibodies specific for the CD20 B cell surface antigen are administered to a patient. These anti-CD20 antibodies bind specifically to the CD20 antigen of (ostensibly) both normal and malignant B cells; the binding of the antibody to the CD20 surface antigen can lead to the destruction and suppression of neoplastic B cells. Additionally, chemical agents or radioactive labels that have the potential to kill the tumor can be conjugated to the anti-CD20 antibody so that the agent is specifically "delivered" to the neoplastic B cells. Regardless of the procedure, a primary goal is to destroy the tumor; the specific procedure can be determined by the particular anti-CD20 antibody that is used and, thus, the methods available to direct the CD20 antigen can vary considerably. CD19 is another antigen that is expressed on the surface of cells of lineage B. Similarly to CD20, CD19 is found on cells during differentiation of the germ cell lineage to a point just before terminal differentiation towards plasma cells (Nadler, L. Lymphocyte Typing II 2: 3-37 and Appendix, Renling et al. eds. (1986) by Springer Verlag). However, unlike CD20, the antibody that binds to CD19 causes internalization of the CD19 antigen. The CD19 Antigen is identified by the CD19-HD237 Antibody (also called the "AB4" antibody) (Kiesel et al., Leukemia Research II, 12: 1119 (1987)), among others. The CD19 antigen is present in 4-8% of peripheral blood mononuclear cells and over 90% of B cells isolated from peripheral blood, spleen, lymph node or tonsils. CD19 is not detected in T cells of peripheral blood, monocytes, or granulocytes. Virtually all acute non-T-cell lymphoblastic leukemias (ALL), chronic B-cell lymphocytic leukemias (CLL) and B-cell lymphomas express CD19 detectable by antibody B4 (Nadler et al., J. Immunol., 131: 244 (1983 ); and Nadler et ai. in Progress in Hematology Vol. XII pp. 187-206, Brown, E. ed. (1981) by Gruñe & Stratton, Inc.). Additional antibodies have been identified that recognize the specific antigens of the differentiation stage expressed by cells of the B cell lineage. Among these is the B2 antibody directed against the .CD21 antigen; the B3 antibody directed against the CD22 antigen; and the J5 antibody directed against the CD10 antigen (also called CALLA). I will see . g. , Patent of E.U. No. 5,595,721 issued January 21, 1997 (Kaminski et al.). The rituximab antibody (RITUXAN®) is a genetically engineered murine chimeric / monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody called "AC2B8" in the U.S. Patent. No. 5,736,137 issued April 7, 1998 (Anderson et al.). RITUXAN® is indicated for the treatment of recurrent or refractory patients in low or follicular grade, non B-cell Hodgkin lymphoma, CD20 positive, (Maloney et al., Blood 82 (Suppl 1): 445a (1993); Maloney et al. al. Proc Am Soc Clin Oncol 13: 993 (1994)). In studies of in vitro mechanisms of action it has been shown that RITUXAN® binds to human complement and smooth lymphoid B cell lines through complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83 (2): 435 -445 (1994)). Additionally, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). More recently, RITUXAN® has been shown to have anti-proliferative effects in tritiated thymidine incorporation assays and to induce apoptosis directly, although other anti-CD19 and CD20 antibodies are not (Maloney et al., Blood 88 (10): 637a (1996)). The synergy between RITUXAN® and chemotherapies and toxins has also been observed experimentally. In particular, RITUXAN® sensitizes drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and ricin (Demidem et al., Cancer Chemotherapy &Radiopharmaceuticals 12 (3 ): 177-186 (1997); Demidem A et al. FASEB J 9: A206 (1995)). Pre-clinical studies in vivo have shown that RITUXAN® reduces B cells from peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, presumably through complement and cell-mediated processes (Reff et al., • supra). Rituximab has also been studied in a variety of non-malignant autoimmune disorders, in which B cells and autoantibodies seem to play a role in the pathophysiology of the disease. Ed ards et al., Biochem Soc. Trans. 30: 824-828 (2002). Rituximab has been reported to potentially alleviate signs and symptoms of, for example, rheumatoid arthritis (RA) (Leandro et al., Ann. Rheum, Dis. 61: 883-888 (2002); Edwards et al., Arthritis Rheum., 46 (Suppl 9): S46 (2002), Stahl et al., Ann. Rheum. Dis., 62 (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: 561-56 (2003), Stasi et al., Blood, 98: 952 -957 (2001.); Saleh et al., Semin. Oncol., 27 (Sup.12) .- 99-103 (2000); Zaia et al., Haematolgica, 87: 189-195 (2002); Ratanatharathorn; et al., Ann. Int. Med., 133: 275-279 (2000)), pure red cell aplasia (Auner et al., Br. J. Haematol., 116: 725-728 (2002)); autoimmune anemia (Zaja et al., Haematologica 87: 189-195 (2002) (appears errata in Haematologica 87: 336 (2002)), cold agglutinin disease (Layios et al., Leukemia, 15: 187-8 (2001); Berentsen et al., Blood, 103: 2925-2928 (2004), Berentsen et al., Br. J. Haematol., 115: 79-83 (2001), Bauduer, Br. J. Haematol., 112: 1083-1090. (2001), Damiani et al., Br. J. Haematol., 114: 229-234 (2001)), severe type B insulin resistance syndrome (Coll et al., N. Engl. J. Med., 350: 310-311 (2004), mixed cytoloubulinemia (DeVita et al., Arthritis Rheum 46 Suppl 9: S2 06 / S469 (2002)), myasthenia gravis (Zaja et al., Neurology, 55: 1062-63 (2000); Ylam et al., J. Pediatr., 143: 674-677 (2003)), Wegener's granulomatosis (Specks et al., Arthritis &Rheumatism 44: 2836-2840 (2001)), refractory pemphigus vulgaris (Dupuy et al. ., Arch Der atol., 140: 91-96 (2004)), dermatomyositis (Levine, Arthritis Rheum., 46 (Suppl. 9): S1299 (2002)), Sjögren's syndrome (So er et al., Arthritis & Rheumatism, 49: 394-398 (2003)), active type-II mixed cyoglobulinemia (Zaja et al., Blood, 101: 3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch. Dermatol., 140 : 91-95 (2004)), autoimmune neuropathy (Pestronk et al., J. Neurol. Neurosurg, Psychiatry 74: 485-489 (2003).}, Opioclonus paraneoplastic-myoclonus syndrome (Pranzatelli et al., Neurology 60 Supl. 1) P05.128: A395 (2003)), and relapsing-remitting multiple sclerosis (RRMS) Cross et al. (Summary) "Preliminary Results from a Phase II Trial of Rituximab in MS" Eighth Annual Meeting of Committees of America for Research and Treatment in Multiple Sclerosis, 20-21 (2003) .A Phase II study (WA16291) was conducted in patients with rheumatoid arthritis (RA), provided 48 weeks after increasing data on safety and efficiency of Rituximab Emery et al., Arthritis Rheum 48 (9): S439 (2003), Szczepanski et al., Arthritis Rheum 48 (9): S121 (2003). randomized for four treatment sections: methotrexate, rituximab alone, rituximab plus methotrexate, and rituximab plus cyclophosphamide (CTX). The treatment regimen of rituximab was one gram administered intravenously on days 1 and 15. The infusions of rituximab in the majority of patients with R? they were well tolerated by the majority of patients, with 36% of patients experiencing at least one adverse event during their first infusion (compared to 30% of patients who received placebo). All, most adverse cases were considered to be benign to moderate in severity and were well balanced across all treatment groups. There were a total of 19 severe adverse events through the four sections during the 48 weeks, which were slightly more frequent in the rituxima / CTX group. The incidence of infections was well balanced across the groups. The mean rate of serious infection in this population of patients with RA was 4.66 per 100 patients per year, which is lower than the rate of infections that require hospital admission in patients with RA (9.57 per 100 patients per year) reported in a study. of epidemiology based in the community. Doran et al. , Arthritis Rheum. 46: 2287-2293 (2002). The reported safety profile of rituximab in a small number of patients with neurological disorders, including autoimmune neuropathy (Pestronk et al., Supra), opsoclonus-myoclonus syndrome (Pranzatelli et al., Supra), and RRMS (Cross et al. , supra), was similar to that reported in oncology or RA. In an ongoing trial (IST) of rituximab sponsored by the researcher in combination with beta interferon (IFN-3.) Or glatiramer acetate in patients with RRMS (Cross et al., Supra), from 1 to 10 treated patients were admitted in the hospital for overnight observation after experiencing moderate fever and rigors after the first infusion of rituximab, although the other 9 patients completed all four infusion regimens without any reported adverse events. Patents and patent publications concerning molecules that bind to CD20 and CD20 antibodies include the U.S. Patents. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US 2002/0197255, US 2003/0021781, US 2003/0082172, US 2003/0095963, US 2003/0147885 (Anderson et al.); the Patent of E.U. No. 6,455,043 and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); WO 2000/27433 (Grillo-Lopez and Leonard); WO 2000/44788 (Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO01 / 10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez.); US 2001/0018041, US 2003/0180292, WO 2001/34194 (Hanna and Hariharan), US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); 2002/0012665 and WO 2001/74388 (Hanna, N.), US 2002/0058029 (Hanna, N.), US 2003/0103971 (Hariharan and Hanna), US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858 (White, C.), \ US 2002/0128488 and WO 2002/34790 (Reff, M.); WO 2002/060955 (Braslawsky et al.); WO 2002/096948 (Braslawsky et al.); WO 2002/079255 (Reff and Davies); Patent of E.U. No. 6,171,586 and WO 1998/56418 (Lam et al.); WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, Patent of E.U. No. 6,194,551, U.S. Patent. No. 6,242,195, U.S. Patent. No. 6,528,624 and Patent of E.ü. No. 6,538,124 (Idusogie et al.); WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO 2001/03734 (Grillo-Lopez et al.); US 2002/0004587 and WO 2001/77342 (Miller and Presta); US 2002/0197256 (Grewal, I.); US 2003/0157108 (Presta, L.); Patent of E.U. Nos. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721, (Kaminski et al.); Patent of E.U. Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, and 6,652,852 (Robinson et al.); Pat. From Eü or. 6,410,391 (Raubitschek et al.); Patent of E.U. Do not. 6,224,866 and WO00 / 20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); US 2003/0133930 and WO 2000/74718 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 (Hansen et al.); WO2004 / 058298 (Goldenberg and Hansen); WO 2000/76542 (Golay et al.); WO 2001/72333 (Wolin and Rosenblatt); Patent of E.ü. No. 6,368,596 (Ghetie et al.); Patent of E.U. No. 6,306,393 and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and Hart ann); WO 2002/102312 (Engleman, E.); US 2003/0068664 (Albitar et al.); WO 2003/002607 (Leung, S.); WO 2003/049694, US 2002/0009427, and US 2003/0185796 (Wolin et al.); WO 2003/061694 (Sing and Siegall); US 2003/0219818 (Bohen et al.); US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.); US2002 / 0136719 (Shenoy et al.); WO 2004/032828 (Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter).

See also the U.S. Patent. No. 5,849,898 and EP 330,191 (Verd et al.); EP332,865A2 (Meyer and Weiss); Patent of E.U. Do not. 4,861,579 (Meyer et al.); US2001 / 0056066 (Bugelski et al.); WO 1995/03770 (Bhat et al.); US 2003/0219433 Al (Hansen et al.); WO 2004/035607 (Teeling et al.); WO 2004/056312 (Lowman et al.); US 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); US 2005/0025764 (Watkins et al.); WO 2005/016969 (Carr et al.); and US 2005/0069545 (Carr et al.). WO 20O4 / 032828 mentions of recurrent polychondritis as one of a list of immune disorders to be treated with anti-CD20 antibodies. Publications concerning rituximab therapy include: Perotta and Abuel, "Chronic recurrent ITP response lasting 10 years to rituximab" Abstract # 3360 Blood 10 (1) (part 1-2): p. 88B (1998); Perotta et al. , "Rituxan in the treatment of chronic idiopathic thrombocytopenic purpura (ITP)", Blood, 94: 49 (abstract) (1999); Matthews, R., "Medical Heretics" New Scientist (April 1, 2001); Leandro et al. , "Clinical consequences in 22 patients with rheumatoid arthritis treated with decreased B lymphocytes" Ann Rheum Dis, supra; Leandro et al. , "Suppression of lymphocytes 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 suppression of B lymphocytes in systemic lupus erythematosus", Arthritis and Rheumatism, 46: 2673-2677 (2002), in the present for a period of 2 weeks, each patient received two infusions of 500 mg of rituximab, two infusions of 750 mg of cyclophosphamide, and oral corticosteroids in high doses, and where two of the treated patients relapsed at 7 and 8 months, respectively, and having been re-treated, although with different protocols; "Successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy" Weide et al. , Lupus, 12: 779-782 (2003), where one patient was treated with rituximab (375 mg / m2 x 4, repeated at weekly intervals) and also applications of rituximab were given every 5-6 months and after therapy of maintenance received rituximab 375 mg / m2 every three months, and a second patient with refractory SLE was treated successfully with rituximab and maintenance therapy was received every three months, both patients responding well to rituximab therapy; Edwards and Cambridge, "I am sustained in rheumatoid arthritis after a protocol designed to reduce B lymphocytes" Rheumatology 40: 205-211 (2001); Cambridge et al. , "Suppression of B lymphocytes in patients with rheumatoid arthritis: serial studies of immunological parameters"? Rt-ir? Tis Rheum., 46 (Suppl 9): S1350 (2002); Edwards et al., "B lymphocyte suppression therapy in rheumatoid arthritis and other autoimmune disorders" Biochem Soc. Trans. , supra; Edwards et al. , "Efficiency and safety of rituximab, a chimeric monoclonal antibody directed to B cells: A randomized controlled follow-up of placebo in patients with rheumatoid arthritis." Rheumatism 46 (9): S197 (2002); Edwards et al., "Therapy directed to the efficiency of B cells with rituximab in patients with rheumatoid arthritis" N Engl. J. Med. 350: 2572-82 (2004); Pavelka et al., Ann. Rheum. Dis. 63: (SI): 289-90 (2004), Emery et al., Arthritis Rheum 50 (S9): S659 (2004), Levine and Pestronk, "Polyneuropathies related to IgM antibody: B-cell suppression chemotherapy using rituximab" Neurology 52: 1701- 1704 (1999); DeVita et al. , "Efficiency of selective blocking of B cells in the treatment of rheumatoid arthritis" Arthritis & Rheum 46: 2029-2033 (2002); Hidashida et al. "Treatment of rheumatoid arthritis refractory to DMARD with rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002; Tuscano, J. "Successful treatment of rheumatoid arthritis refractory to infliximab with rituximab" Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002; "Pathological roles of B cells in human autoimmunity; clinical insights" Martin and Chan, Immunity 20: 517-527 (2004); Silverman and Weisman, "Rituximab Therapy and Autoimmune Disorders, Prospects for Anti-B Cell Therapy", Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz and Isenberg, "Anti B-cell therapy (rituximab) in the treatment of autoimmune diseases", Current opinion in pharmacology, 4: 398-402 (2004); Virgolini and Vanda, "Rituximab in autoimmune diseases", Biomedicine & pharmaco therapy, 58: 299-309 (2004); Klemmer et al. , "Treatment of autoimmune disorders mediated by antibody with a monoclonal antibody AntiCD20 Rituximab", Arthritis And Rheumatism, 48: (9) 9, S (SEP), page: S624-S624 (2003); Kneitz et al. , "Effective suppression of B cells with rituximab in the treatment of autoimmune diseases", Immunobiology, 206: 519-527 (2002); Arzoo et al. , "Treatment of autoimmune disorders mediated by refractory antibody with an anti-CD20 monoclonal antibody (rituximab)" Annals of the Rheumatic Diseases, 61 (10), p922-4 (2002) Comment in Ann Rheum Dís. 61: 863-866 (2002); "Future Strategies in Immunotherapy" by Lake and Dionne, in Burgerr s Medicinal Chemistry and Medicament Discovery (2003 by John Wiley &Sons, Inc.) Online article registration date: January 15, 2003 (Chapter 2"Targeted Immunotherapy to the Antibody"); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target, online article registration date: January 15, 2002 entitled "CD20"; Appendix 4A entitled "Monoclonal Antibodies for Antigens Surfaces of Human Cells "by Stockinger et al., Eds: Coligan et al. , in Current Protocols in Immunology (2003 John Wiley &Sons, Inc) Online Registration date: May, 2003; Publication Printing Date: February, 2003; Penichet and Morrison., "Antibodies CD / molecules: Definition; Design of Antibodies" in the Wiley Section Encyclopedia of Molecular Medicine: Human, Humanized and Chimeric Antibodies; put online January 15, 2002; Specks et al. "Wegener's granulomatosis response to chimeric anti-CD20 monoclonal antibody therapy" Arthritis & Rheumatism 44: 2836-2840 (2001); abstract presentation and online invitation by Koegh et al. "Rituximab for Remission Induction in ANCA-associated vasculitis Severa: Prospective Pilot Monitoring Report Open in 10 patients Tagged" American College of Rheumatology, Session Number: 28-100, Session Title: Vasculitis, Session Type: ACR Session concurrently, Primary Category: 28 vasculitis, Session 18/10/2004 (< www abstractsonline com / viewer / SearchResults asp >...) Eriksson, "score and short-term safety in 5 patients with ANCA positive vasculitis treated with rituximab ", Kidney and Blood Pressure Research, 26: 294 (2003); Jayne et al. , "Suppression of B cells with rituximab for refractory vasculitis" Kidney and Blood Pressure Research, 26: 294 (2003); Jayne, poster 88 (11th International Vasculitis Study Group and ANCA), 2003 American Society of Nephrology; Stone and Specks, "Rituximab Therapy for the Induction of Remission and Tolerance in Vasculitis Associated with ANCA", in the Summary of Clinical Follow-up Investigation of the Immune Tolerance Network 2002-2003 < www immunetolerance. org / research / autoim une / trails / stone .html > See also Leandro et al. , "Repopulation of B cells occurs mainly from simple B cells in patients with rheumatoid arthritis and systemic lupus erythematosus" Arthritis Rheum. , 48 (Suppl 9): S1160 (2003). Sarwal et al. N. Eng. J. Med. 349 (2): 125-138 (July 10, 2003) reported the molecular heterogeneity in rejection of acute renal graft identified by profiling of DNA microarray.

Recurrent polychondritis is a rare, chronic, cartilage disorder characterized by recurrent episodes of cartilage inflammation in various tissues of the body. Tissues that contain cartilage that can become inflamed include the ears, nose, joints, spine, and trachea (trachea). The ears, heart, and blood vessels, which have a biochemical composition similar to that of cartilage, can also be affected. The cause of recurrent polychondritis is unknown. It is suspected that this condition is caused by a disorder of the immune system (autoimmunity) in which the immune system of the body (which normally fight the invaders of the body, particularly infections) is wrong. This results in inflammation that targets various tissues of the body. Relief can be found through anti-inflammatory agents and various steroids. Mononeuritis multiplex is an asynchronous, asymmetric, sensory and motor peripheral neuropathy that involves isolated damage to at least two separate nerve areas. Multiple nerves can be affected in random areas of the body. As the condition worsens, it becomes less multifocal and more symmetric, resembling polyneuropathy. Syndromes of multiple mononeuropathies can be distributed bilaterally, distally, and proximally throughout the body. Damage to the nerves involves the destruction of the axon (i.e., the part of the nerve cell that is analogous to the copper part of a cable), thus interfering with the nerve conduction at the location of the damage. Common causes include diabetes and multiple nerve compressions, as well as lack of oxygen caused by decreased blood flow or inflammation of blood vessels. The cause is not identified for approximately one third of the cases. Specific multiple disorders are not associated with multiple mononeuritis, including (but not limited to) blood vessel diseases such as polyarteritis nodosa and other vascular diseases, diabetes, and connective tissue diseases such as rheumatoid arthritis or systemic lupus erythematosus. Connective tissue disease is the most common cause in children. Less common causes include the following: Sjögren's syndrome, Wegener's granulomatosis, hypersensitivity (allergic reactions) that cause blood vessel inflammation, leprosy, sarcoidosis, amyloidosis, multifocal forms of neuropathy • Diabetic, and blood disorders (such as hypereosinophilia and cryoglobulinemia). See, for example, Hattori et al. Brain 122 (3): 427-439 (1999) in which the clinicopathological characteristics of 28 patients with peripheral neuropathy associated with Churg-Strauss syndrome, and sensory and motor complication showing mainly a multiple mononeuritis pattern in the initial phase are evaluated, progressing in asymmetric polyneuropathy, restricted to limbs. CD20-positive B lymphocytes were seen only occasionally. Treatment for neuropathy depends on its causes, and many neuropathies can be treated by addressing the underlying cause (such as vitamin deficiency). Others can be prevented from occurring. For example, controlling diabetes can prevent diabetic neuropathy. In cases where the cause is a broken tumor or disc, therapy may involve surgery to remove the tumor or repair the ruptured disc. In entrapment or compression neuropathy the treatment may consist of splinting or surgical decompression of the ulnar or middle nerves. Neuropathies of peroneal and radial compression may need to avoid pressure. Physical therapy and / or splints can be helpful in preventing contractures (a condition in which the muscles shortened around the joints cause abnormal positioning and sometimes joint pain). The neuropathies that are associated with immune diseases can be improved with treatment directed at the abnormal characteristics of the immune system. Such treatments include intravenous immunoglobulin, plasma exchange and immunosuppressive therapy (Cook et al., Neurology 40: 212-214 (1990); Dyck et al. N. Engl. J. Med 325: 1482-1486 (1991); Ernerudh et al. J. Neurol. Neurosurg. Psychiatry 55: 930-934 (1992); Blume et al. Neurology 45: 1577-1580 (1995); Pestronk et al. Neurology 44: 2027-2031 (1994)). This can produce minimal functional improvement. In addition, the treatment can be expensive and time consuming. The literature on the treatment directed against the antibody against membrane markers of the B cell surface is extremely limited. Levine and Pestronk describe five patients with neuropathy and immunoglobulin M antibodies for GM1 or MAG who were treated with rituximab. Within the 3-6 months of treatment all five had improved function and reduced serum antibody titre (Levine and Pestornk Am. J. Neurol, 52: 1701-1704 (1999)). If a specific treatment is not available, the pain of the neuropathy can usually be controlled, such as with the use of analgesics, pain medication, tricyclic antidepressants, anti-seizure medications, or a nerve blocker. Sutton and Winer Current Opinion in Pharmacology 2/3: 291-295 (June 1, 2002) state that plasma exchange, intravenous immunoglobulin and corticosteroids continue to be the mainstay of treatment for inflammatory neuropathies. Recent evidence shows that combining these therapies is not significantly more effective than the treatment of a single agent. The usefulness of new immunotherapies and cytotoxic agents is difficult to find out due to the treatment of small numbers of patients in open studies. Lee et al. Bone marrow Transplantation 30/1: 53-56 (2002) proposes that high-dose chemotherapy and transplantation of autologous peripheral blood germ cells (.PBSC) may have a role 'in the treatment of peripheral neuropathy secondary to severe, progressive and monoclonal gammopathy resistant to treatment of unknown significance (MGUS). Latov et al. Neurology 52: A551 (1999) describes that RITUXAN® appears to be a safe and effective treatment in two patients with neuropathy-associated IgM monoclonal gammopathy and anti-MAG antibody activity. Canavan et ai. Neurology 58/7 (Suppl 3): A233 (April 2002) describes that RITUXAN® was associated with sustained clinical improvement in the majority of treated patients who exhibited polyneuropathy associated with IgM antibody. With respect to the treatment of mixed cytoplasmic antibody monoclonal antibody resistant to interferon alpha with an anti-CD20 antibody, Sansonno et al. Blood 101 (10): 3818-3826 (May 15, 2003) describes the treatment of peripheral neuropathy with RITUXAN®. Hattori et al. Brain 122/3: 427-439 (1999) assesses the clinicopathological characteristics of patients with peripheral neuropathy associated with Churg-Strauss syndrome, establishing that CD20-positive B lymphocytes were seen only occasionally.

Zaja et al. Blood 101 (10): 3827-3834 (May 15, 2003) describes that RITUXAN® may represent a safe and effective alternative for standard immunosuppression in mixed cytologlobulinemia (CM) type II. RITUXAN® proved to be effective on manifestations of vasculitis on the skin (ulcers, purpura, or urticaria), subjective symptoms of peripheral neuropathy, mild B-cell lymphadenopathy, arthralgias, and fever. Zaidi et al. Leukemia and Lymphoma 45/4: 777-780 (2004) describes a case of limfomatoid granulomatosis (LYG), a rare lymphoproliferative disorder with a mortality rate of approximately 60% in the first year, with the pulmonary, hepatic, nervous systems Central and peripheral involved, it was successfully treated with RITUXAN®. Still, Trojan et al. Annals of Oncology 13/5: 802-805 (2002) describes that RITUXAN® does not seem to be effective for a patient suffering from peripheral neuropathy due to neurolymphomatosis. The representation of fused PET-CT images, made on an online PET-CT system, showed multiple small nodular lesions that extend along the peripheral nerves corresponding to an early recurrence of a non-Hodgkin lymphoma of transformed B cells. Binstadt et al. Journal of Pediatrics 143/5: 598-604 (November 2003) concluded that RITUXAN® was safe and effective in four pediatric patients with multisystem autoimmune diseases refractory to conventional immunosuppressive drugs, each with the central nervous system (CNS) involved. A patient with autoimmune cytopenias and autoimmune CNS disease and peripheral nervous system had resolution of cytopenias and marked improvement in neurological symptoms; he reports that he is currently receiving non-immunosuppressive medications. Two half brothers with lymphoplasmacytic colitis, pulmonary nodules, and CNS disease had improvement of their symptoms. A fourth patient with chorea and antiphospholipid antibody syndrome of seizures secondary to primary had improvement in fine and coarse motor functions and reduced the frequency of seizures. Saito et al. Lupus 12/10: 798-800 (2003) describes that RITUXAN® was useful to treat a patient with refractory lupus nephritis and CNS complications of systemic lupus erythematosus (SLE) associated with highly active B lymphocytes. There is a need in the art for additional drugs to treat various indications such as polychondritis and mononeuritis multiplex. SUMMARY OF THE INVENTION Accordingly, the invention is as claimed.

Specifically, the present invention provides, in a first aspect, a method for treating polychondritis or multiple mononeuritis in a mammal comprising administering to the mammal an effective amount of an antibody that binds CD20.

In one embodiment of this method, the antibody is not conjugated with another molecule. In another embodiment, the antibody is conjugated to another molecule, for example, a cytotoxic agent such as a radioactive compound, e. g. , Y2B8 or 131I-B1. In another embodiment, the antibody comprises humanized rituximab or 2H7. The humanized 2H7 in one embodiment comprises the variable domain sequences in SEQ ID Nos. 2 and 8. In another embodiment, the humanized 2H7 comprises a heavy chain variable domain with the alteration (s) N100A or D56A, N100A in SEQ ID NO: 8 and a light chain variable domain with the alteration (s) M32L, S92A, or M32L, S92A in SEQ ID NO: 2. In a further embodiment, the humanized 2H7 comprises the region sequence light chain variable (VL) of SEQ ID NO: 30 and the heavy chain variable sequence (VH) of SEQ ID NO: 8, wherein the antibody further contains a substitution of amino acids D56A in VH-CDR2, and N100 at VH-CDR3 is substituted with Y or W, and more preferably the antibody comprises the light chain sequence v511 of SEQ ID NO: 44 and the heavy chain sequence v511 of SEQ ID NO: 45. The antibody is preferably administered at a dose of about 20 mg / m2 to about 250 mg / m2 of the antibody to the mammal, more preferably, from about 50 mg / m2 to about 200 mg / m2. In another preferred embodiment, the method comprises administering an initial dose of the antibody followed by a subsequent dose, wherein the dose in mg / m2 of the antibody in the subsequent dose exceeds the mg / m2 dose of the antibody in the initial dose. In yet another preferred embodiment, the mammal is human. The antibody is preferably administered intravenously or subcutaneously. In a preferred embodiment, the method consists essentially of administering an effective amount of the antibody to the mammal. In another preferred aspect, the method further comprises administering to the mammal an effective amount of an immunosuppressive agent, anti-pain agent, or chemotherapeutic agent. In still further embodiments, if polychondritis is treated, the method further comprises administering to the mammal an effective amount of a non-spheroidal anti-inflammatory drug, steroid, or immunosuppressant such as methotrexate, cyclophosphamide, dapsone, azathioprine, penicillamine, or cyclosporin. If treating multiple mononeuritis, the method further comprises administering to the mammal an effective amount of an anti-pain agent, steroid, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulin, cyclosporin, or mycophenolate mofetil. In a further aspect, the present invention pertains to a manufacturing article comprising a container and a composition contained therein, wherein the composition comprises an antibody that binds CD20, and further comprises an insert package that instructs the user of the composition for treating polychondritis or mononeuritis multiplex in a mammal. In a preferred embodiment, the article further comprises a container comprising an agent other than the antibody for treatment and further comprising instructions for treating the mammal with such an agent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a sequence alignment that compares the amino acid sequences of the light chain variable domain (VL) of each of murine 2H7 (SEQ ID NO: l), 2H7.vl6 humanized variant (SEQ ID NO: 2), and subgroup I human kappa light chain (SEQ ID NO: 3). The VL CDRs of 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO:), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6). Figure IB is a sequence alignment that compares the amino acid sequences of the heavy chain variable domain (VH) of each of murine 2H7 (SEQ ID NO: 7), 2H7.vl6 humanized variant (SEQ ID NO: 8) , and the human consensus sequence of heavy chain subgroup III (SEQ ID NO: 9). The VH CDRs of 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: ll), and CDR3 (SEQ ID NO: 12). In Figure IA and Figure IB, the CDR1, CDR2 and CDR3 in each chain are enclosed within parentheses, flanked by the structure regions, FR1-FR4, as indicated. 2H7 refers to the murine 2H7 antibody. The asterisks between two lines of the sequences indicate the positions that are different between the two sequences. The numbering of the residue is according to Kabat et al. Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions shown as, b, c, d, and e. Figure 2 shows the nucleotide sequence of phagemid pVX4 (SEQ ID NO: 13. {Sequence 5 '.} And SEQ ID NO: 14. {Complementary sequence 3'.}.) Used for the construction of plasmids 2H7 Fab (see Example 1) as well as the amino acid sequences of the L chain (SEQ ID NO: 15) and H chain (SEQ ID NO: 16) of the Fab for the humanized anti-IFN-a CDR-grafted antibody. Figure 3 shows the nucleotide sequence of the expression plasmid encoding 2H7.v6.8 Chimeric Fab (SEQ ID NO: 17 { Sequence 5 '.}. And SEQ ID NO: 18 { Complementary sequence 3'.}.). The amino acid sequences of the L chain (SEQ ID NO: 19) and H chain (SEQ ID NO: 20) are shown .. Figure 4 shows the nucleotide sequence of the plasmid pDRl (SEQ ID NO: 21; 5391 bp) for the expression of the immunoglobulin light chains as described in Example 1. pDR1 contains the sequences encoding an irrelevant antibody, the light chain of a humanized anti-CD3 antibody (Shalaby et al., J. Exp. Med. 175: 217-225 (1992)), start and stop codons which are indicated in bold and underlined Figure 5 shows the nucleotide sequence of plasmid pDR2 (SEQ ID NO: 22; 6135 bp) for the expression of heavy chains of immunoglobulin as described in Example 1. pDR2 contains sequences encoding an irrelevant antibody, the heavy chain of a humanized anti-CD3 antibody (Shalaby et al., supra), the start and stop codons which are indicated in bold and underlined, Figures 6A and 6B show the amino acid sequences of the L chain 2H7.vl6, with Figure 6A showing the complete L chain containing the first 19 amino acids before DIQ which are the secretory signal sequence not present in the mature polypeptide chain (SEQ ID NO. : 23), and Figure 6B showing the mature polypeptide L chain (SEQ ID NO: 24) Figures 7A and 7B show the amino acid sequences of the 2H7.vlß H chain, with Figure 7A showing the H chain complete containing the first 19 amino acids before EVQ which are the sequence of the secretory signal not present in the chain of mature polypeptides (SEQ ID NO: 25), and Figure 7B showing the H chain of mature polypeptide (SEQ ID NO : 26). The alignment of the VH sequence in Figure IB (SEQ ID NO: 8) with the complete H chain sequence, the human? L constant region forms amino acid position 114-471 in SEQ ID NO: 25. Figures 8A and 8B show the amino acid sequences of the 2H7.v31 H chain, with Figure 8A showing the complete H chain containing the first 19 amino acids before EVQ which are the secretory signal sequence not present in the mature polypeptide chain (SEQ. ID NO: 27), and Figure 8B showing the H chain of mature polypeptides (SEQ ID NO: 28). The string L is the same as 2H7.vl6 (see Figure 6). Figure 9 is a flow chart that adds the amino acid changes from murine 2H7 to a subset of humanized versions up to v75. Figure 10 is a sequence alignment comparing the light chain amino acid sequence of the humanized variant 2H7.vl6 (SEQ ID NO: 2) and humanized variant 2H7.vl38 (SEQ ID NO: 29). Figure 11 is a sequence alignment that compares the heavy chain amino acid sequences of the humanized 2H7.vl6 variant (SEQ ID NO: 8) and the humanized 2H7.vl38 variant (SEQ ID NO: 30). DETAILED DESCRIPTION OF THE PREFERRED MODALITIES I. Definitions A "B cell surface marker" or "B cell surface antigen" herein is an antigen expressed on the surface of a B cell that can be targeted with an antagonist that binds thereto. Exemplary B cell surface markers include the leukocyte surface markers CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD74, CD74, CD74, CD77, CD78, CD79, CD79b, CD80, CD81, CD82, CD83, CD84, CD85 and CD86. (For descriptions, see The Leukocyte Antigen Facts Book, 2nd Edition, 1997, ed. Barclay et al., Academic Press, Harcourt Brace &Co., New York). Other surface markers of B cells include RP105, FcRH2, CD79A, C79B, B cell CR2, CCR6, CD72, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRHl, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287_at. The B cell surface marker of particular interest is preferably expressed on B cells compared to other non-B cell tissues of a mammal and can be expressed on both precursor B cells and mature B cells. The B cell surface markers preferred herein are CD20 and CD22. The antigen "CD20", or "CD20," is a non-glycosylated phosphoprotein of approximately 35-kDa, found on the surface of more than 90% of B cells of peripheral blood or lymphoid organs. CD20 is present in both normal B cells as well as malignant B cells, but is not expressed on germ cells. Other names for CD20 in the literature include "antigen restricted to B lymphocyte" and "Bp35". The CD20 antigen is described, for example, in Clark et al. Proc. Nati Acad. Sci. (USA) 82: 1766 (1985) ,. The "CD22" antigen, or "CD22," also known as BL-CAM or Lyb8, is a type 1 integral membrane glycoprotein with a molecular weight of about 130 (reduced) to 140 kD (not reduced). It expresses both the cytoplasm and the cell membrane of B lymphocytes. The CD22 antigen appears early in B cell lymphocyte differentiation at approximately the same stage as the CD19 antigen. Unlike other B-cell markers, membrane expression of CD22 is limited to the post-differentiation stages comprised between mature B cells (CD22 +) and plasma cells CD22-). The CD22 antigen is described, for example, in Wilson et al. J. Exp. Med. 173: 137 (1991) and Wilson et al. J. Immunol. 150: 5013 (1993). A "non-malignant disorder" in the present is polychondritis or mononeuritis multiplex, preferably mononeuritis multiplex. Additionally, it can be spino-optic multiple sclerosis; vulgar pemphigus; vasculitis or Churg-Strauss syndrome (CSS); lupus cerebritis; lupus nephritis; cutaneous systemic erythematosus lupus (SLE); IgE-mediated diseases other than asthma, specifically, allergic rhinitis, anaphylaxis, or atopic dermatitis; chronic neuropathy; opsoclonus-ioclonus syndrome; pulmonary alveolar proteinosis; scleritis; microscopic polyangiitis; paraneoplastic syndrome, which is a remote effectro produced by a tumor, such as hypercalcemia, but does not include Lambert-Eaton, anemia, or hypoglycemia; Rasmussen encephalitis; vasculitis of the central nervous system (CNS); channelopathies, which are diseases with various properties associated with ion channel dysfunction such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and CNS channelopathies, but not including inflammatory CNS disorders; autism; or neuropathic, myopathic, or CNS sarcoidosis. "Polychondritis" as used herein means any polychondritis, including recurrent polychondritis, von Meyenburg disease, Meyenburg's disease or syndrome, Meyerburg Altherr Uehlinger syndrome, polychondropathy, Askenazy syndrome, Jaksch Wartenhorst, Meyenburg, or Von Jaksch Wartenhorst, perichondritis which is chondromalcic arthritis or pancondritis, chondroitic, diffuse or recurrent. "Mononeuritis multiplex" as used herein describes a condition characterized by inflammation caused by various nerves in unrelated body positions, i.e., nerve damage involves isolated damage to at least two separate nerve areas. As it gets worse, it becomes more diffuse and less focused on particular areas, resembling polyneuropathy. Symptoms of a disease of this kind can include numbness, weakness, burning pain (especially at night), and loss of reflexes. The pain can be severe and disabling. An "antagonist" is a molecule that, when bound to a surface marker of B cells, destroys or reduces B cells in a mammal and / or interferes with one or more functions of B cells, e.g. by reducing or preventing a humoral response produced by the B cell. The antagonist is preferably able to consume the B cells (i.e. reduces the levels of B cells in circulation) in a mammal treated therewith. Such suppression can be achieved through various mechanisms such as inhibition of ADCC and / or CDC, proliferation of B cells, and / or induction of B cell death (e.g. through apoptosis). Antagonists included within the scope of the present invention include antibodies, native or synthetic sequence peptides, and small molecule antagonists that bind to the B cell marker, optionally conjugated or fused to a cytotoxic agent. The preferred antagonist comprises an antibody, ie, an antibody that binds to B cell surface marker. "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to the cell-mediated reaction in which non-cytotoxic cells Specific receptors that express Fe (FcRs) receptors (eg Natural Killer (NK) cells, neutrophils, and macrophages) recognize the bound antibody on a target cell and subsequently cause lysis of the target cell. Primary cells to mediate ADCC, NK cells, express only FcyRIII, while monocytes express Fc? RI, FcyRII and Fc? RIII. The expression of FcR on hematopoietic cells is shown in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Inmunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in the U.S. Patent, can be performed. No. 5,500,362 or 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.

Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, e. g. , in an animal model such as that described in Clynes et al. PNAS (USA) 95: 652-656 (1998). "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform effector functions of ADCC. Examples of human leukocytes that mediate ADCC include PBMC, NK cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being preferred. The terms "Fe receptor" or "FcR" are used to describe a receptor that binds to the Fe region of an antibody. The preferred FcR is a human FcR of natural sequence. In addition, a preferred FcR is one that binds to the IgG antibody (a gamma receptor) and includes receptors of the Fc? RI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc? RII receptors include FcyRIIA (an "activation receptor") and FcyRlIB (an "inhibition receptor"), which have similar amino acid sequences that differ mainly in the cytoplasmic domains of these. The activation receptor Fc? RIIA contains an activation motif based on a tyrosine immunoreceptor (ITAM) in its cytoplasmic domain. The Fc? RIIB inhibition receptor contains a motif of inhibition based on an immunoreceptor tyrosine (ITIM) in its cytoplasmic domain. (See Daéron, Annu, Rev. Inmunol., 15: 203-234 (1997)). The FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Inmunol 9: 457-92 (1991); Capel et al. Immunomods 4: 25-34 (1994); and de Haas et al. J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those that identify themselves in the future, are comprised by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 ( 1994)). "Complement-dependent cytotoxicity" or "CDC" will refer to the ability of a molecule to lyse an objective in the presence of complement. The complement activation path is initiated by joining the first components of the coplement system (Clq) to a molecule (eg, an antibody) that forms a complex with an analogous antigen. To assess complement activation, a CDC assay can be performed, e. g. as described in Gazzano-Santoro et al. J. Immunol. Methods 202: 163 (1996) ,. "Growth inhibitory" antagonists are those that prevent or reduce the proliferation of a cell that expresses an antigen to which the antagonist binds. For example, the antagonist can prevent or reduce the proliferation of B cells in vitro and / or in vivo. Antagonists that "induce apoptosis" are those that induce programmed cell death, e. g. of a B cell, as determined by standard apoptosis assays, such as annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum dilatation, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies) . The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies) formed from at least two intact antibodies, and antibody fragments while exhibiting the desired biological activity. "Antibody fragments" comprise a portion of an intact antibody, which preferably comprises binding to the antigen or variable region thereof. Examples of antibody fragments include the Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; antibody molecules of a chain; and multispecific antibodies formed from antibody fragments. For the purposes of the present, an "intact antibody" is one that comprises heavy- and light chain variable domains as well as an Fe region. "Native antibodies" are typically heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two light chains (L ) identical and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes.

Each heavy and light chain also have interspersed disulfide bridges regularly separated. Each heavy chain has at one end a variable domain (VH) followed by a number of variable domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domains of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. The residues of particular amino acids are considered to form an interface between the variable domains of light chain and heavy chain. The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among the antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not uniformly distributed across the variable domains of the antibodies. It is concentrated in three segments called hypervariable regions of the variable domains of both light chain and heavy chain. The most highly conserved portions of the variable domains are called structure regions (FRs). The variable domains of the light and heavy native chains each comprise four FRs, which widely adopt a β-sheet configuration, connected by three hypervariable regions, which form cycle connections, and in some cases are part of the structure of hoha ß. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The variable domains do not directly involve the binding to an antibody to an antigen, but exhibit several effector functions, such as participation of the antibody in ADCC. The papain digestion of the antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fe" fragment, whose name reflects its ability to crystallize easily. The pepsin treatment produces an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-linking the antigen. "Fv" is the minimum antibody fragment that contains a complete recognition of the antigen and the antigen binding site. This region consists of a dimer of a variable domain of a heavy chain and a light chain in close association, non-covalent. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity for the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind to the antigen, albeit at a lower affinity than the entire binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain domain including one or more cysteines from the antibody articulation region. Fab '-SH is the designation herein for Fab' in which the cystine residue (s) of the constant domains carry at least one free thio group. The F (ab ') 2 antibody fragments are originally produced as pairs of Fab' fragments having articulacipon cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chain" antibodies (immunoglobulins) of any vertebrate species can be assigned to one of the two clearly distinct types, called kappa (K) and lambda (?), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned for different diseases. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can also be expressed in subclasses. (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain variable domains corresponding to different classes of antibodies are called a, d, e,?, And μ, respectively. The structures of subunits and the three-dimensional configurations of different classes of immunoglobulins are well known. The "single chain Fv" or "scFv" antibody fragments comprise the VH and VL antibody domains, wherein these domains are present in a single chain of polypeptides. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allow the scFv to form the desired structure for the antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small fragments of antibody with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). When using a linker that is too short to allow pairs between the two domains in the same chain, the domains are forced to be pairs with the complementary domains of another chain and create two antigen-binding sites. The diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be precarious. in smaller quantities. Monoclonal antibodies are highly specific, targeting a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody that is being obtained from a substantially homogeneous antibody population, and is not constructed as required for the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be made by the hybridoma method first described by Kohler et al. Nature, 256: 495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature, 352: 624-628 (1991) and Marks et al. J. Mol. Biol. , 222: 581-597 (1991), for example. Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of particular antibody, while the rest of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, while exhibiting the activity desired biological (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nati, Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (eg, Old World Monkey)., such as baboon, rhesus or cynomolgus monkeys) and human constant region sequences (US Pat No. 5,693,780). The "Humanized" forms of non-human antibodies (e.g., murine) are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (antibody receptors) in which the residues of a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate that have the specificity, affinity, and desired capacity. In some cases, the residues of the structure region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are also made to perform refined antibodies. In general, the humanized antibody will substantially comprise all of at least one, and typically two, variable domain, in which all or substantially all of the hypervariable cycles correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For additional details, see Jones et al. Nature 321: 522-525 (1986); Riechmann et al. Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for binding to the antigen. The hypervariable region comprises amino acid residues from a "region of complementarity determination" or "CDR" (eg residues 24-34 (Ll), 50-56 (L2) and 89-97 (L3) in the variable domain of light chain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or those residues of a "hypervariable cycle" (eg residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) in the variable domain of light chain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J.

Mol. Biol. 196: 901-917 (1987)). "Structure" waste or "FR" are the variable domain residues different from the hypervariable region residues as defined herein. An antagonist "that binds" to an antigen of interest, e. g. a B cell surface marker is one capable of binding that antigen with sufficient affinity and / or avidity such that the antagonist is useful as a therapeutic agent to target a cell that expresses the antigen. Examples of antibodies that bind to the CD19 antigen include the anti-CD19 antibodies in Hekman et al. Cancer Immunol. Inmunother. 32: 364-372 (1991) and Vlasveld et al. Cancer Immunol. Inmunother. 40: 37-47 (1995); and the B4 antibody in Kiesel et al. Leukemia Research II, 12: 1119 (1987). An "antibody that binds CD20" refers to an antibody that binds to the CD20 antigen with sufficient affinity and / or avidity such that the antibody is useful as a therapeutic agent to target a cell that expresses or overexpresses the antigen. CD20. Examples of such antibodies include: "C2B8" which is now called "rituximab" ("RITUXAN®") (U.S. Patent No. 5,736,137); the murine antibody 2B8 tagged with yttrium- [90] designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN® (Patent of US Pat. No. 5,736,137); Murine IgG2a "Bl," also called "Tositumomab," optionally labeled with 131I to generate the "131I-B1" antibody (1131 iodine tositumomab, BEXXAR ™) (U.S. Patent No. 5,595,721); murine monoclonal antibody "1F5" (Press et al. Blood 69 (2): 584-591 (1987) and the "patched structure" or humanized 1F5 (WO03 / 002607, Leung, S.); deposit in ATCC HB-96450); murine 2H7 antibody and chimeric 2H7 (U.S. Patent No. 5,677,180); a humanized 2H7; huMax-CD20 (Genmab, Denmark); AME-133 (Applied Molecular Evaluation); and monoclonal antibodies L27, G28-2, 93-1B3, B-Cl or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987). The terms "rituximab" and "RITUXAN®" herein refer to the genetically engineered chimeric / human murine monoclonal antibody directed against the CD20 antigen and designated "C2B8" in U.S. Patent No. 5,736,137, including fragments thereof retaining the ability to bind to CD20. Only for purposes herein, "humanized 2H7" refers to a humanized antibody that binds to human CD20, or a fragment that binds to the antigen thereof, wherein the antibody is effective to consume primate B cells in vivo, the antibody comprising in the variable region of the H chain (VH) at least one CDR3 sequence of SEQ ID NO: 12 (FIG. IB) of an anti-human CD20 antibody and substantially the waste of the human consensus structure (FR) of human heavy chain subgroup III (VHIII). In a preferred embodiment, this antibody further comprises the CDR1 sequence of the H chain of SEQ ID NO: 10 and the CDR2 sequence of SEQ ID NO: 11, and more preferably further comprises the CDR1 sequence of L chain of SEQ ID NO: 4, the CDR2 sequence of SEQ ID NO: 5, CDR3 sequence of SEQ ID NO: 6 and substantially the human consensus structure (FR) residues of subgroup I of human K light chain (VKI), where the VH region can be linked to a human IgG chain constant region, wherein the region can be, for example, IgGl or IgG3.

In a preferred embodiment, such an antibody comprises the sequence VH of SEQ ID NO: 8 (vld, as shown in Figure IB), optionally also comprises the sequence VL of SEQ ID NO: 2 (vl6, as shown in FIG. Figure 1A), which may have the amino acid substitutions of D56A and N100A in the H chain and S92A in the L chain (v.96). A more preferred antibody is 2H7.vl6 having the light and heavy chain amino acid sequences of SEQ ID NOS: 24 and 26, respectively, as shown in Figures 6B and 7B. Another preferred embodiment is wherein the antibody is 2H7.v31 having the light and heavy chain amino acid sequences of SEQ ID NOS: 24 and 28, respectively, as shown in Figures 6B and 8B. The antibody herein may further comprise at least one amino acid substitution in the Fe region that enhances ADCC and / or CDC activity, such as one wherein the amino acid substitutions are S298A / E333A / K334A, more preferably 2H7.v31 quye has the heavy chain amino acid sequence of SEQ ID NO: 28 (as shown in Figure 8B). Any of these antibodies may further comprise at least one amino acid substitution in the Fe region that decreases CDC activity, for example, comprising at least the K322A substitution. Such antibodies are preferably 2H7.vll4 or 2H7.vll5 having at least ADCC activity improved 10-fold in comparison to RITUXAN®. A preferred humanized 2H7 is an intact antibody or antibody fragment comprising the variable light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID N0: 2); and the variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQK FKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 8). Wherein the antibody is humanized 2H7 an intact antibody, preferably comprising the amino acid sequence of light chain: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVADNLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 24); and the amino acid sequence of heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQK FKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 26) or the amino acid sequence of heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGDTSYNQK FKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSNSYWYFDVWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHY TQKSLSLSPGK (SEQ ID NO: 28).

An "isolated" antagonist is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are materials that could interfere with the diagnostic or therapeutic use for the antagonist, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antagonist will be purified (1) more than 95% by weight of the antagonist as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a sufficient degree to obtain at least 15 residues. of the N-terminal or internal amino acid sequence by use of a rotary vessel sequencer, or (3) for homogenity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver tincture. The isolated antagonist includes the in situ antagonist within the recombinant cells since at least one component of the natural environment of the antagonist will not be present. However, ordinarily, the isolated antagonist will be prepared by at least one purification step. "Mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo animals, sports, or pets, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. "Treatment" refers to both therapeutic and prophylactic treatment or prevention measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented. Accordingly, the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease or disorder. The term "an effective amount" refers to an amount of the antagonist that is effective to prevent, diminish or treat the autoimmune disease in question. The term "immunosuppressive agent" as used herein for auxiliary therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, sub-regulate or suppress self-expression of the antigen, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see US Pat. No. 4,665,077); mycophenolate mofetil such as CELLCEPT®; azathioprine (IMURAN®, AZASAN® / 6-mercaptopurine, bromocriptine, danazol, dapsone, glutaraldeido (which masks the MHC antigens, as described in US Patent No. 4,120,649), anti-idiotypic antibodies to MHC antigens and MHC fragments cyclosporin A; steroids such as corticosteroids and glucocorticosteroids, eg, prednisone, prednisolone such as PEDIAPRED® (sodium prednisolone phosphate) or ORAPRED® (prednisolone sodium phosphate oral solution), methylprednisolone, and dexamethasone; methotrexate (oral or subcutaneous) ( RHEUMATREX®, TREXALL ™), hydroxychlorocin / chlorocin, sulfasalazine, leflunomide, cytokine or cytokine receptor antagonists including anti-interferon- ?, -β, or -a antibodies, anti-tumor necrosis factor-a antibodies (infliximab or adalimumab), anti-TNFa immunoadhesin (ENBREL® etanercept), anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; -LFA-1, including anti-CDlla and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; polyclonal or pan-T antibodies, or anti-CD3 or anti-CD4 / CD4a monoclonal antibodies; soluble peptide containing an LFA-3 binding domain (WO 1990/08187 published 7/26/90); streptokinase; TGF-β; streptodornase; RNA or host DNA; FK506; RS-61443; deoxyspergualine; rapamycin; T-cell receptor (Cohen et al., U.S. Pat. No. 5,114,721); fragments of T cell receptor (Offner et al., Science, 251: 430-432 (1991), WO 1990/11294, Ianeway, Nature, 341: 482 (1989), and WO 1991/01133); T cell receptor antibodies (EP 340,109) such as T10B9; cyclophosphamide (CYTOXAN®); dapsone; penicillamine (CUPRIMINE®); plasma exchange; or intravenous immunoglobulin (IVIG). These can be used alone or in combination with each other, particularly combinations of steroid and other immunosuppressive agent or such combinations followed by a maintenance dose with a non-steroidal agent to reduce the need for steroids. "Anti-pain agent" refers to a medicament acts to inhibit or suppress pain, such as an analgesic or prescription pain medication for controlling neuralgia, such as non-spheroidal anti-inflammatory drugs (NSAIDs) including ibuprofen (MOTRIN) ®), naproxen (NAPROSYN®), as well as several other medications used to reduce the acute pain that may occur, including anticonvulsants (gabapentin, phenytoin, carbamazepine) or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenytoin (DILANTIN®), gabapentin (NEURONTIN®), (E) -N-Vanityl-8-methyl-6-noneamide (CAPSAICIN® ), or a nerve blocker. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes the destruction of cells. The term is intended to include radioactive isotopes (eg At21M I13M I12M Y90, Re185, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial origin, fungal, vegetable or animal, or fragments thereof. A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as cyclophosphamide thiotepa and CYTOXAN®; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylamelamines including altretamine, triethylene methamine, triethylene phosphoramide, triethylene-thiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including synthetic analog topotecan); Bryostatin; Callistatin; CC-1065 (including its synthetic adozelesina, carzelesina and bizelesina analog); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarcinol (including synthetic analogues, KW-2189 and CBl-TMl); eleuterobin; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the antibiotics enediin (eg, calicheamicin, especially calicheamicin ga mall and omegall calicheamicin (see, eg, Agnew, Chem Intl. Ed. Engl., .33: 183-186 (1994)), dinemicin, including dynemycin A; bisphosphonates, such as clodronate, a esperamycin, as well as neocarzinostatin chromophore and the related chromoproteins enediin chronophores antibiotic), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, -diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, esububicin, idarubicin, marcellomicin, mitomycin such as mitomycin C, ichophenolic acid, nogalamicin, olivomycins, peplomicin, potfiromicin, puromicin, chelamicin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinost atin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal such as aminoglutethimide, mitotane, trilostane; suppliers of folic acid such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornitin; eliptinium acetate; an epothilone; etoglucide; nitrate gallium; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; Pentostatin; fenamet; pirarubicin; losoxantrone; podofílinic acid; 2-ethylhydrazide; procarbazine; PSK® complex polysaccharide (Products Natural JHS, Eugene, OR); razoxana; rizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziq-uono; 2, 2 ', 2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine), urethane, vindesine, dacarbazine, manomustine, mitobronitol, mitolactol, pipobroman, gacitosin, arabinoside (" Ara-C " ), cyclophosphamide, thiotepa, taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), CREAMOPORO-free ABRAXANE ™, formulation of designed nanoparticles of paclitaxel albumin (American Pharmaceutical Partners, Schaumberg, Illinois) ), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France), chloranbucil, GEMZAR® gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16),; ifosfamide, mitoxantrone, vincristine, NAVELBINE® vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as innoic, capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormonal action in tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, ceoxifene, LY117018, onapristone, and FARESTON-toremifene; aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogens in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestania, fadrozole, RIVISOR® vorozol, FEMARA® letrozole, and ARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in the signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as an inhibitor of VEGF expression (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® r RH; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. The term "cytokine" is a generic term for proteins released by a cell population that acts on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocins, interleukins (ILs) such as IL-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL-11, IL-12, IL-15, a tumor necrosis factor such as TNF-α or TNF-β, and other polypeptide factors including LIF and ligand equipment (KL). As used herein, the term "cytokine" includes naturally occurring or recombinant cell culture proteins and biologically active equivalents of the native sequence cytokines.

The term "hormone" refers to polypeptide hormones, which are generally secreted by glandular organs with ducts. Included among the hormones are, for example, growth hormone such as human growth hormone, N-ethionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); prolactin; placental lactogen; peptide associated with mouse gonadotropin; inhibin; activin; - Mullerian inhibitor substance; and thrombopoietin. The term "growth factor" refers to proteins that promote growth, and include, for example, liver growth factor; fibroblast growth factor; Vascular endothelial growth factor; nerve growth factor such as NGF-β; platelet-derived growth factor; transformation growth factor (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -ß- Y ~ Y 'Y colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF ( G-CSF).

The term "integrin" refers to a receptor protein that allows cells to both bind and respond to the extracellular matrix and become involved in a variety of cellular functions such as wound healing, cell differentiation, tumor cell guiding and apoptosis They are part of a large family of cell adhesion receptors that are involved in the cell's extracellular matrix and cell-cell interactions. Functional integrins consist of two subunits of transmembrane glycoprotein, called alpha and beta, that do not covalently bind. The alpha subunits all share some homology with each other, as do the beta subunits.

The receptors always contain an alpha chain and a beta chain. Examples include Alphadbetal, Alpha3betal and Alpha7betal. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the original drug and is capable of being enzymatically activated to become the most active original form. . I will see. g. , Wilman, "Promedicamentos in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al. "Promedications: A Chemical Approach to Targeted Medication Delivery," Directed Medication Delivery, Borchardt et al. (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, modified prodrugs by D-amino acid, glycosylated prodrugs, p-containing prodrugs. lactam, prodrugs containing optionally substituted phenoxyacetamide or prodrugs containing optionally substituted phenylacetamide, 5-fluorocytosine and other prodrugs of 5-fluorouridine which may become the most active free cytotoxic drug. Examples of cytotoxic drugs that can be derived in a form of prodrug for use in this invention include, but are not limited to, the chemotherapeutic agents described above. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and / or surfactants that are useful for the delivery of a medicament (such as the antagonists described herein and, optionally, a chemotherapeutic agent) to a mammal. The components of the liposome are commonly arranged in a bistratified formation, similar to the lipid arrangement of biological membranes. The term "package insert" is used to refer to instructions for the client included in the commercial package of therapeutic products, which contain information about the indications, use, dosage, administration, contraindications and / or warnings related to the use of such products. therapeutic products. II. Production of Antagonists The methods and articles of manufacture of the present invention utilize, or incorporate, an antagonist that binds to the B cell surface marker. Accordingly, methods for generating such antagonists will be described herein. The B cell surface marker to be used for the production of, or detection of, antagonist (s) may be, e. g. , a soluble form of the antigen, or a portion thereof, that contains the desired epitope. Alternatively, or additionally, cells expressing the surface marker of B cells on their cell surface can be used to generate, or detect, antagonist (s). Other forms of the B cell surface marker useful for the generation of antagonists will be apparent to those of skill in the art. Preferably, the B cell surface marker is the CD19 or CD20 antigen. Although the preferred antagonist is an antibody, different antagonists to the antibodies are contemplated herein. For example, the antagonist may comprise a small molecule antagonist optionally fused, or conjugated with, a cytotoxic agent (such as those described herein). Small molecule libraries can be detected against the B cell surface marker of interest herein in order to identify a small molecule that binds to that antigen. The small molecule can also be selected for its antagonistic and / or conjugated properties with a cytotoxic agent. The antagonist can also be a peptide generated by rational design or by phage display (see, e.g., WO 1998/35036 published August 13, 1998). In one embodiment, the selection molecule can be "CDR mimic" or analogous antibody designed based on the CDRs of an antibody. Although such peptides can be antagonists themselves, the peptide can optionally be fused to a cytotoxic agent thereby adding or improved antagonistic properties of the peptide. A description below as the exemplary techniques used for the production of the antibody antagonists according to the present invention. (i) Polyclonal Antibodies Polyclonal antibodies originate preferentially in animals by multiple subcutaneous (se) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e. g. , keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a derivative or bifunctional agent, eg, sulfosuccinimide maleimidobenzoyl ester (conjugation through cysteine residues), N-hydroxysuccinimide (a through lysine residues), glutaraldehyde, succinic anhydride, S0C12, or RXN = C = NR, wherein R and R1 are different alkyl groups. The animals were immunized against the antigen, immunogenic conjugates, or derivatives by combining, eg, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's Complete Adjuvant and injecting the solution in a manner intradermal in multiple sites. One month later the animals were boosted with 1/5 to 1/10 of the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals were bled and the serum was analyzed for the antibody titer.

The animals were reinforced to the flat title. Preferably, the animal was boosted with the conjugate of the same antigen, but was conjugated to a different protein and / or through a different cross-reagent. The conjugates can also be made in recombinant cell culture as protein fusions. Also, aggregation agents such as alum are suitably used to improve the immune response. (ii) Monoclonal Antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the "monoclonal" modifier indicates the character of the antibody, not being a mixture of separated antibodies. For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al. Na ture, 256: 495 (1975), or can be made by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to produce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization . Alternatively, the lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). The hybridoma cell thus prepared is seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the original unfused myeloma cells. For example, if the original myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), the substances of which prevent growth of the cells. cells deficient in HGPRT. Preferred myeloma cells are those that melt efficiently, support the production of high stable level of antibody by the cells that produce the selected antibody, and are sensitive to a medium such as the HAT medium. Among these, preferred myeloma cell lines are urine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells or X63-Ag8-653 available from the American Type Culture Collection, Manassas, Virginia USA. Mouse-human heteromyeloma cell lines and human myeloma have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, p. 51-63 (Marcel Dekker, Inc., New York, 1987)). The culture medium in which the hybridoma cell grows was analyzed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cell is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al. Anal Biochem. , 107: 220 (1980). After the hybridomas cell is identified as producing antibodies of the specificity, affinity, and / or desired activity, the clones can be subcloned by limiting the dilution and growth procedures by standard methods (Goding, Monoclonal Antibodies: Principies and Practice, pp.59-103 (Academic Press, 1986)). The culture medium suitable for this purpose includes, for example, D-MEM medium or RPMI-1640. In addition, the hybridoma cells can grow in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by standard immunoglobulin purification methods such as, for example, A-SEPHAROSE ™ protein agarose chromatography, hydroxylapatite chromatography, gel electrophoresis. , dialysis, or affinity chromatography. Monoclonal antibodies can also be produced recombinantly. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (eg, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cell serves as a preferred source of such DNA. Once isolated, the DNA can be placed in expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster's ovary (CHO) cells, or myeloma cells that otherwise they do not produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The review of the articles on recombinant expression in bacteria of DNA encoding the antibody includes Skerra et al. Curr. Opinion ín Immunol. , 5: 256-262 (1993) and Plückthun, Immunol. Revs. , 130: 151-188 (1992). In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al. Nature, 348: 552-554 (1990). Clackson et al. Nature, 352: 624-628 (1991) and Marks et al. J. Mol. Biol. , 222: 581-597 (1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM range) upon chain recombination (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for build very large phage libraries (Waterhouse et al., Nuc Acids, Res., 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives for traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain variable domains in place of murine homologous sequences (U.S. Patent No. 4,816,567; Morrison, et al. Proc. Nati Acad. Sci. USA, 81: 6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, each non-immunoglobulin non-polypeptide is replaced by the variable domains of an antibody, or replaced by the variable domains of a combination site with the antigen of an antibody to create a bivalent chimeric antibody comprising a combination site to the antigen having specificity for an antigen and another antigen combining site that has specificity for a different antigen. Humanized Antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "imported" residues, which are typically taken from an "imported" variable domain. Humanization can be performed essentially following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986), Riech ann et al., Nature, 332: 323-327 (1988), Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting the hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residue and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies. The selection of human variable domains, both light and heavy, to be used to make humanized antibodies is very important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is detected against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the region of human structure (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al. Mol. Biol., 196: 901 (1987)). Another method uses a region of particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same structure can be used for several different humanized antibodies (Cárter et al., Proc. Nati, Acad. Sci. USA, 89: 4285 (1992), Presta et al., J. Immunol., 151: 2623 (1993)). It is also important that the antibodies are humanized with high affinity retention for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the original sequences and several conceptual humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those with experience in the field. computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. The inspection of these deployments allows the analysis of the probable role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, the FR residues can be selected and combined from the receptor and import sequences so as to achieve the desired characteristics of the antibody, such as increased affinity for the target antigen (s). In general, hypervariable region residues are directly and more substantially involved in the binding of the influencing antigen. Human Antibodies As an alternative for humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of the production of endogenous immunoglobulin. For example, it has been described that homozygous deletion of the gene from the binding region (JH) of the heavy chain of the germline or chimeric antibody mutates the results of mice in complete inhibition of the production of endogenous antibodies. The transfer of the human cell line immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies by antigen challenge. I will see. g. , Jakobovits et al. Proc. Nati Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al. Nature, 362: 255-258 (1993); Bruggermann et al. Year in Immuno. , 7:33 (1993); and US Patents Nos. 5,591,669, 5,589,369 and 5,545,807. Alternatively, the phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce antibodies and human antibody fragments in vitro, from repertoires of immunoglobulin variable domain (V) genes. from non-immunized donors. According to this technique, the antibody domain V genes are cloned into either a coating protein gene greater or less than a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the antibody. phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on functional properties of the antibody also result in the selection of the gene encoding the antibody that exhibits these properties. Thus, the phage mimics some of the properties of the B cell. The display can be made in a variety of formats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J., 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) isolating a diverse array of anti-oxazolone antibodies from a small randomized combinational library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including autoantigens) can be essentially isolated following the techniques described by Marks et al. J. Mol. Biol. 222: 581-597 (1991), or Griffith et al. EMBO J. 12: 725-734 (1993). See, also, US Patent. Nos. 5,565,332 and 5,573,905. Human antibodies can also be generated by B cells activated in vitro (see U.S. Patents 5,567,610 and 5,229,275). (v) Antibody fragments Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived through proteolytic digestion of intact antibodies (see, eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). ). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragment can be isolated from the above-treated antibody phage libraries. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al. Bio / Technology 10: 163-167 (1992)). According to another procedure, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to practitioners of experience. In other embodiments, the selection antibody is a single chain Fv fragment (scFv). See WO 1993/16185; Patent of E.U. No. 5,571,894; and US Patent. No. 5,587,458. The antibody fragment can also be a "linear antibody," e. g. , as described for example, in the U.S. Patent. 5,641,870 ,. Such linear antibody fragments may be onospecific 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 B cell surface marker. Other antibodies can bind to a first B cell surface marker and further bind to a second B cell surface marker. Alternatively, an anti-label of B cell surface that binds to the arm can be combined with an arm that binds to the activation molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fe receptors for IgG (Fc? R), such as Fc? RI (CD64), FcyRII (CD32), and FcyRIII (CD16), in order to focus the cellular defense mechanisms towards the B cell. Bispecific antibodies can also be used to localize cytotoxic agents for the B cell. These antibodies have a B-cell surface marker binding arm and an arm that binds to the cytotoxic agent (eg saporin, anti-interferon-a, vinca alkaloid, A-chain ric ina, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, bispecific antibodies F (ab ') 2). Methods for making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two heavy chain-immunoglobulin light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (198 -3)). Due to the randomization of the light and heavy immunoglobulin chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather difficult, and the yield of the product is low. Similar procedures are described in WO 1993/08829, and in Traunecker et al. EMBO J., 10: 3655-3659 (1991). According to a different procedure, the variable domains of antibody with the desired specificities (antibody-antigen combination sites) are fused to the immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the joint regions, CH2, and CH3. It is preferred to have the first heavy chain constant region (CH1), which contains the site necessary for light chain binding, present in at least one of the fusions. The DNAs encoding the immunoglobulin heavy chain fusions, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. This provides great flexibility for adjusting the mutual proportions of the three polypeptide fragments in modalities when the unequal ratios of the three polypeptide chains used in the construction provide the optimum performance. However, it is possible to insert the coding sequence for two or all three polypeptide chains into an expression vector when the expression of at least two polypeptide chains in equal proportions results in high yields or when the proportions are of one meaning particular. In a preferred embodiment of this method, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in arm one, and a heavy chain-hybrid immunoglobulin light chain (providing a second binding specificity in the other arm.) This asymmetric structure was found to facilitate separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, such as the presence of an immunoglobulin light chain in only half of the bispecific molecule provides a form of separation path.

This method is described in WO 1994/04690. For further details to generate bispecific antibodies, see, for example, Suresh et al. Methods in Enzymology, 121: 210 (1986). According to another procedure described in the Patent of E.U. No. 5,731,168, the interference between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are recovered from the recombinant cell culture. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain.

In this method, one or more side chains of small amino acids from the interface of the first antibody molecules are replaced with large side chains (eg tyrosine or tryptophan). Compensatory "cavities" of similar or identical size to that of the large lateral chain (s) are created at the interface of the second antibody molecule by replacing the side chains of large amino acids with small (eg, alanine or threonine). This provides a mechanism for increasing the performance of the heterodimer over other undesired end products such as homodimers. Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have been, for example, proposed to direct the cells of the immune system to unwanted cells (US Patent No. 4,676,980), and for the treatment of HIV infection (WO 1991/00360, WO 1992/200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking methods. Cross-linking agents are well known in the art, and are described, for example, in the U.S. Patent. No. 4,676,980, together with a number of cross-linking techniques. Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al. Science, 229: 81 (1985) describes a method wherein the intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol sodium arsenite coupling agent to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted to thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to the thiol Fab '-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al. J. Exp. Med., 175: 217-225 (1992) describes the production of an F (ab ') 2 molecule of fully humanised bispecific antibody. Each Fab 'fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against the human breast tumor objectified.

Several techniques for making and isolating the bispecific antibody fragments directly from the recombinant cell culture have also been described. For example, -bispecific antibodies have been produced using leucine zippers. Kostelny et al. J. Immunol. , 148 (5): 1547-1553 (1992).

The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by genetic fusion. The antibody homodimers were reduced in the region of articulation to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The technology of. "diabody" described by Hollinger et al. Proc. Nati Acad. Sci.

USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments.

The fragments comprise a variable domain of heavy chain (VH) connected to a light chain variable domain (Vj.) By a linker that is too short to allow pairs between the two domains in the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy has also been reported for making bispecific antibody fragments by the use of single chain Fv (sFv) dimers. See Gruber et al. J.

Immunol. , 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991). III. Conjugates and Other Modifications of the Antagonist The antagonist used in the methods or included in the articles of manufacture herein is optionally conjugated to a cytotoxic agent. Chemotherapeutic agents useful in the generation of such cytotoxic antagonist conjugates have been described above. The conjugates of an antagonist and one or more small molecule toxins, such as a calicheamicin, to maytansin (U.S. Patent No. 5,208,020), a trichothene, and CC1065, are also contemplated herein. In one embodiment of the invention, the antagonist is conjugated to one or more maytansine molecules (eg, about 1 to about 10 molecules of maytansine per antagonist molecule). Maytansine can, for example, be converted to May-SS-Me, which can be reduced to May-SH3 and reacted with a modified antagonist (Chari et al, Cancer Research 52: 127-131 (1992)) to generate a maytansinoid-antagonist conjugate. . Alternatively, the antagonist is conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA fractures at sub-picomolar concentrations. Structural calicheamicin analogs that can be used include, but are not limited to,? A or 21, oA, N-acetyl-? I1, PSAG and? 1! (Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)). Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, unbound diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecina chain A , sarcin alfa, proteins of Aleurites fordii, proteins diantina, proteins of Phytolaca americana (PAPI, PAPII, and PAP-S), inhibitor of momordica charantia, curcin, crotin, inhibitor of - sapaonaria officinalis, gelonin, mitogellin, restrictocin, fenomicin, enomicin and trichothecenes. See, for example, WO 1993/21232 published October 28, 1993. The present invention further contemplates antagonist conjugated to a compound with nucleolytic activity (eg, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease, DNase). A variety of radioactive isotopes are available for the production of radioconjugated antagonists. Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu. The conjugates of the antagonist and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1- carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehydo), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine derivatives) ), bis-diazonium (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as tolieno 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, an immunotoxin ricin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid labeled with Carbon-14 (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotide to the antagonist. See WO 1994/11026. The linker can be a "cleavage linker" facilitating the release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or linker containing disulfide can be used (Chari et al, Cancer Research 52: 127-131 (1992)). Alternatively, a fusion protein comprising the antagonist and cytotoxic agent can be made, e. g. , by recombinant techniques or peptide synthesis. In yet another embodiment, the antagonist can be conjugated to a "receptor" (such as streptavidin) for use in pre-targeting to a tumor wherein the conjugate of? antagonist receptor is administered to the patient, followed by removal of the unbound conjugate from the circulation using a cleansing agent and then administration of a "ligand" (eg avidin) which is conjugated to a cytotoxic agent (e.g. to radionucleotide). The antagonists of the present invention can also be conjugated to an enzyme that activates the prodrug that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO 1981/01145) to an active anti-cancer drug. See, for example, WO 1988/07378 and U.S. Patent No. 4,975,278. The enzyme component of such a conjugate includes any enzyme capable of acting on a prodrug in such a way that it converts it into its most active cytotoxic form,. Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing drugs into free medicines; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as protease serratia, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), which are useful for converting peptide containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs containing D-amino acid substituents; enzymes that unfold the carbohydrate such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; ß-lactamase useful for converting drugs derived with ß-lactams into free medicines; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derived in their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active medicaments (see, e.g., Massey, Nature 328: 457-458 (1987)). The antagonist-abzyme conjugates can be prepared as described herein to deliver the abzyme to a tumor cell population. The enzymes of this invention can be covalently linked to the antagonist by techniques well known in the art such as the use of heterobifunctional cross-linking reagents discussed above. Alternatively, fusion proteins comprising at least the binding to the antigen region of an antagonist of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art ( see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)). Other modifications of the antagonist are contemplated herein. For example, the antagonist can be linked to one of a variety of nonprotein polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Antagonists described herein can also be formulated as liposomes. The liposomes containing the antagonist are prepared by methods known in the art, such as those described in Epstein et al. Proc. Nati Acad. Sci. USA, 82: 3688 (1985); Hwang et al. Proc. Na ti. Acad. Sci. USA, 77: 4030 (1980); Pat. of E.ü. you. 4,485,045 and 4,544,545; and WO 1997/38731 published October 23, 1997. Liposomes with improved circulation time are described in the U.S. Patent. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and derived PEG-phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter. The Fab 'fragments of an antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) through a disulfide exchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19): 1484 (1989). Modifications (s) of the amino acid sequence of protein or peptide antagonists described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antagonist. Variants of the amino acid sequence of the antagonist are prepared by introducing appropriate nucleotide changes into the nucleic acid of the antagonist, or by peptide synthesis. Such modifications include, for example, deletions of, and / or insertions in and / or substitutions of, residues within the amino acid sequences of the antagonist. Any combination of suppression, insertion, and substitution is made to arrive at the final construction, as long as the final construction possesses the desired characteristics. The amino acid changes can also alter the post-translational processes of the antagonists, such as changing the number or position of the glycosylation sites. A useful method for the identification of certain residues or regions of the antagonist that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residue or group of target residues (eg, changed residues such as arg, asp, his, lys, and glu) is identified and replaced by a negatively changed or neutral amino acid (more preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. These locations of amino acids that demonstrate functional sensitivity for the substitutions are then retined by introducing additional or other variants into, or for, the substitution sites. Thus, while the site is predetermined to introduce a sequence variation amino acids, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, wing scanning or random mutagenesis is conducted at the codon or target region and variants of the expressed antagonist are screened for the desired activity. The amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from a residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antagonist with an N-terminal methionyl residue or the antagonist fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include fusion of the N- or C-terminal antagonist of an enzyme, or a polypeptide that increases the serum half-life of the antagonist. Another type of variant is a substitution of amino acid variant. These variants have at least one amino acid residue in the antagonist molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis of antibody antagonists include hypervariable regions, but alterations in FR are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantive changes, termed "exemplary substitutions" in Table 1, or as further described below with reference to the amino acid classes, the products can be introduced and selected.

Table 1 Substantial modifications in the biological properties of the antagonist are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, eg, as a sheet or helical conformation, (b) the change or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. The residues that occur naturally are divided into groups based on common properties of the side chain: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) hydrophilic neutral: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the orientation of the chain: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will substitute exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the antagonist can also be substituted, generally with serine, to improve the oxidative stability of the molecule and avoid aberrant cross-linking. Conversely, the cysteine linkage (s) can be added to the antagonist to improve its stability (particularly where the antagonist is an antibody fragment such as an Fv fragment). A particularly preferred type of substitutional variant includes substituting one or more hypervariable region residues of an original antibody. Generally, the resulting variant (s) selected for further development will have improved biological properties relative to the original antibody from which they were generated. A convenient way to generate such substitutional variants is affinity maturation using phage display. In summary, several hypervariable region sites (e.g., sites 6-7) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are deployed in a monovalent form from phage filament particles as fusions for the gene III product of the M13 packaged within each particle. The phage display variants are then selected for their biological activity (e.g., binding affinity) as described herein. In order to identify the hypervariable sites of the candidate region for modification, the mutagenesis of alanine scanning can be performed to identify the hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the points of contact between the antibody and the antigen. Such contact residues and surrounding residues are candidates for substitution according to the techniques elaborated herein. Once such variants have been generated, the panel of variants is screened as described herein and antibodies with superior properties in one or more relevant assays can be selected for further development. Another type of amino acid variant of the antagonist alters the original glycoside pattern of the antagonist. By altering means suppressing one or more carbohydrate residues found in the antagonist, and / or adding one or more glycosylamine sites that are not present in the antagonist. The glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate residues to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences for enzyme assembly of the carbohydrate residue to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the annexation of one of the sugars N-acetylgalactosamine, galactose, or xylosa to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The addition of glycosylated sites to the antagonist is done covalently by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for the N-linked glycosylation sites). The alteration may also be made by the addition, or substitution by, one or more serine or threonine residues to the original antagonist sequence (for the O-linked glycosylation site). The nucleic acid molecules encoding the variant amino acid sequences of the antagonist are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis genes, mutated PCR genesis , and mutates the cassette genesis of a previously prepared variant or a non-variant version of the antagonist. It may be desirable to modify the antagonist of the invention with respect to effector function, e.g. in order to improve ADCC and / or CDC of the antagonist. This can be achieved by introducing one or more amino acid substitutions in an Fe region of an antibody antagonist. Alternatively or additionally, the cysteine residue (s) is introduced into the Fe region, thereby allowing the formation of interchain disulfide bonding in this region. The homodimeric antibody thus generated may have improved internalization capacity and / or cell destruction mediated by increased complement and ADCC. See Carón et al. J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinks as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be designed to have dual Fes region and can thereby have improved complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Medicamento Design 3: 219-230 (1989). To increase the serum half-life of the antagonist, a binding epitope to the receptor secured in the antagonist (especially an antibody frat) can be incorporated as described in the U.S. Patent. 5,739,277, for example. As used herein, the term "assured receptor binding epitope" refers to an epitope of the Fe region of an IgG molecule (eg, IgG ?, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half life in vivo of the IgG molecule. IV. Pharmaceutical Formulations Therapeutic formulations of the antagonists used in accordance with the present invention are prepared in stages by mixing an antagonist having the desired degree of purity with optional pharmaceutically acceptable excipients or stabilizers, (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable vehicles, excipients, or stabilizers are not toxic to recipients at spliced doses and concentrations, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Exemplary anti-CD20 antibody formulations are described in WO 1998/56418. This publication describes a multi-dose liquid formulation comprising 40 mg / ml of rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, and 0.02% POLISORBATE ™ 20 (sorbitan polyoxyethylene monooleate) at pH 5.0 which has a life minimum average of two years in storage at 2-8 ° C. Other anti-CD20 formulation of interest comprises 10mg / mL rituximab in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7mg / mL POLISORBATE ™ 80 (sorbitan polyoxyethylene monooleate), and Sterile Water by Injection, pH 6.5. Lyophilized formulations adapted for subcutaneous administration are described in WO 1997/04801. Such lyophilized formulations can be reconstituted with a suitable diluent at a high protein concentration and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein. The formulation herein may also contain more than one active compound as necessary for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide a cytotoxic agent, chemotherapeutic agent, cytokine, or immunosuppressant agent (eg one that acts on T cells, such as cyclosporin or an antibody that binds to T cells, eg, one that is links to LFA-1). The effective amount of such other agents depends on the amount of antagonists present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same doses and administration routes as used up to now or approximately from 1 to 99% of the doses used up to now. The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly- (methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. , liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980). Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained release matrices include polyester, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and? ethyl-L-glutamate ethylene vinyl acetate, non-degradable, lactic acid-degradable glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) - 3-hydroxybutyric acid. The formulations to be used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile V filtration membranes. Treatment with the antagonist The composition comprising an antagonist that binds to a B cell surface antigen will be formulated, dosed, and administered in a manner consistent with good medical practice Factors for consideration in this context include the particular disease or condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of agent delivery, the method of administration, administration scheduling, and other factors known to medical practitioners. The therapeutically effective amount of the antagonist to be administered will be governed by such considerations. As a general proposition, the therapeutically effective amount of the antagonist administered parenterally per dose will be in the range of about 0.1 to 20 mg / kg of the patient's body weight per day, with the typical initial range of the antagonist used being in the range of about 2 to 10 mg / kg. The preferred antagonist is an antibody, e.g. an antibody such as RITUXAN®, which is not conjugated to a cytotoxic agent. Suitable doses for a conjugated antibody are, for example, in the range of about 20 mg / m2 to about 1000 mg / m2. In one embodiment, the dose of the antibody differs from that currently recommended for RITUXAN®.

For example, one or more doses of substantially less than 375mg / m2 of the antibody may be administered to the patient, e. g. wherein the dose is in the range of about 20mg / m2 to about 250mg / m2, for example, from about 50mg / m2 to about 200mg / m2. In addition, one or more initial dose (s) of the antibody may be administered followed by one or more subsequent dose (s), wherein the dose of mg / m2 of the antibody in the subsequent dose (s) exceeds the dose of mg / m2 of the antibody in the initial dose (s). For example, the initial dose may be in the range of about 20mg / m2 to about 250mg / m2 (eg, from about 50mg / m2 to about 200mg / m2) and the subsequent dose may be in the range of about 250mg / m2 to approximately 1000mg / m2. However, as noted above, these amounts of the suggested antagonist are subject to a large amount of therapeutic discretion. The key factor in selecting an appropriate dose and schedule is the result obtained, as indicated above. For example, relatively higher doses may be necessary initially for the treatment of ongoing and acute diseases. To obtain the most effective results, depending on the disease or disorder, the antagonist is administered as close to the first signs, diagnosis, onset, or occurrence of the disease or disorder as possible or during remissions of the disease or disorder. The antagonist is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, inhalation, intra-thecal, intra-articular, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In addition, the antagonist can be suitably administered by pulse infusion, e.g., with decreasing doses of the antagonist. Preferably the dosage is given by injections, more preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. One or more other compounds can be administered, such as cytotoxic agents, chemotherapeutic agents, immunosuppressive agents, anti-pain agents, hormones, integrins, growth factors, and / or cytokines with the antagonists herein, or to apply various other therapies known to those skilled in the art. Preferably, depending, for example, on the type of indication, the degree or severity of the indication, and the type of the antagonist, the other compound administered is an immunosuppressive agent, an anti-pain agent, or a chemotherapeutic agent. If polychondritis such as relapsing polychondritis is treated, preferably the other compound, if the symptoms are not severe, is a non-spheroidal anti-inflammatory drug (NSAID), including ibuprofen (MOTRIN®), naproxen (NAPROSYN®), or sulindac (CLINORIL®), to control inflammation. However, normally, medications related to cortisone are required, e. g. , steroids such as prednisone and prednisolone. Frequently, high doses of steroids are initially necessary, especially when the eyes or respiratory airways are involved. In addition, most patients require steroids for long-term use. Another preferred compound that can be used in combination with the antagonist for the treatment of polychondritis is methotrexate (RHEÜMATREX®, TREXALL ™), which has shown promise as a treatment for recurrent polychondritis in combination with steroids as well as maintenance treatment. Studies have shown that methotrexate can help reduce steroid requirements. Other preferred compounds include cyclophosphamide (CYTOXAN®), dapsone, azathioprine (IMURAN®), AZASAN®), penicillamine (CUPRIMINE®), cyclosporine (NEORAL®, SANDIMMUNE®), and combinations of these medications with steroids. Regarding the treatment of multiple mononeuritis with another agent, if a specific treatment is not available, pain of neuropathy can usually be controlled. The simplest treatment is an over-the-counter pain reliever, such as acetaminophen (TYLENOL®), an NSAID such as ibuprofen as noted above, or aspirin, followed by a prescription medication for pain. Tricyclic antidepressants such as amitriptyline (ELAVIL®) and anti-seizure medications, such as carbamazepine (TEGRETOL®), phenyl- toine (DILANTIN®), or gabapentin (NEURONTIN®), have been used to relieve the pain of neuropathy. CAPSAICIN® ((E) -N-Vanilil-8-methyl-6-noneamid), the chemical responsible for hot pepper, is used as a cream to help relieve the pain of peripheral neuropathy. Additionally, a nerve blocker may be effective in relieving pain. Other preferred compounds for the treatment of peripheral neuropathies include PBSC anticancer transplantation, steroids such as corticosteroids including pulse therapy thereof and prednisone, prednisolone and methyl prednisolone including pulse therapy thereof, methotrexate, cyclophosphamide (eg, CYTOXAN ®) including intravenous cyclophosphamide pulse therapy, plasma exchange or plasmapheresis, intravenous immunoglobulin, cyclosporins such as cyclosporin A, mycophenolate mofetil (eg, CELLCEPT®) or - chemotherapeutic agents (including high doses thereof) including those of lower IgM concentrations, such as FLUDARA® (fludarabine phosphate) or LEUKERAN® (chlorambucil). Other particularly preferred compounds for this indication are anti-pain agents, steroids, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulin, cyclosporine or mycophenolate mofetil. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation and consecutive administration in any order, where preferably there is a period of time while both (or all) of the active agents simultaneously exert their biological activities. In addition to the administration of protein antagonists to the patient, the present application contemplates the administration of antagonists by genetic therapy. Such administration of nucleic acid encoding the antagonist is encompassed by the term "administering an effective amount of an antagonist." See, for example, WO 1996/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies. There are two main methods for introducing the nucleic acid (optionally contained in a vector) into the cells of the patient: in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antagonist is required. For the ex vivo treatment, the cells of the patient are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or for example, encapsulated within porous membranes that are implanted in the patient (see , eg US Patent Nos. 4,892,538 and 5,283,187). There are a variety of techniques available to introduce nucleic acids into viable cells. The techniques are dependent on whether the nucleic acid is transferred into cells grown in vitro or in vivo in the cells of the proposed host. Suitable techniques for nucleic acid transfer in mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the method of calcium phosphate precipitation, etc. A vector commonly used for the ex vivo delivery of the gene is a retrovirus. Current preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex virus, or adeno-associated virus) and lipid-based systems (lipids useful for lipid-mediated transfer of the gene they are for example DOTMA, DOPE and DC-Chol). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor in the target cell, etc. Where liposomes are employed, proteins that bind to a cell surface membrane associated with endocytosis can be used to target and / or facilitate absorption, e. g. capsid proteins or fragments thereof, typical for a particular cell type, antibodies for proteins that undergo internalization in cycling and proteins that target intracellular localization and improve intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al. J. Biol. Chem. 262: 4429-4432 (1987); and Wagner et al. Proc. Nati Acad. Sci. USA 87: 3410-3414 (1990). For the review of currently known gene labeling and gene therapy protocols, see Anderson et al. Science 256: 808-813 (1992). See also WO 1993/25673 and references cited therein. Articles of Manufacture In another embodiment of the invention, there is provided a manufacturing article containing materials useful for the treatment of diseases or disorders described above. The article of manufacture comprises a container and a package label or insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers can be formed from a variety of materials such as glass or plastic. The container carries or contains a composition that is effective in treating the selection disease or disorder and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a plunger pierced by a needle of hypodermic injection). At least one active agent in the composition is the antagonist that binds to the B cell surface marker. The label or package insert indicates that the composition is used to treat a patient who has or is predisposed to an autoimmune disease, such as those listed here. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may also include other desirable materials from a commercial and user point of sale, including other buffers, diluents, filters, needles, and syringes. The additional details of the invention are illustrated by the following non-limiting examples. The exposition of all citations in the specification are expressly incorporated herein by reference. EXAMPLE 1 Humanization of Monoclonal Antibody 2H7 anti-CD20 Murine Humanization of the murine anti-human antibody CD20, 2H7 (also referred to herein as m2H7, m for murine), was carried out in a series of site-directed steps. Variable region sequences of murine 2H7 antibody and chimeric 2H7 have been described with V for mouse and C for human; see, e.g., US Patent. 5,846,818 and 6,204,023. The CDR residues of 2H7 were identified by comparing the amino acid sequence of the variable domains 2H7murinos (described in US Patent No. 5,846,818) with the known antibody sequences (Kabat et al., Sequences of proteins of immunological interest, Ed. 5. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The CDR regions for light and heavy chains were defined based on sequence hypervariability (Kabat et al., Supra) and are shown in Figure IA and Figure IB, respectively. Using synthetic oligonucleotides (Table 2), site-directed mutagenesis (Kunkel, Proc. Nati. Acad. Sci. 82: 488-492 (1985)) was used to introduce all six of the murine CDR 2H7 regions into a Fab structure. complete human corresponding to a consensus sequence VKI, VHIH (V kappa subgroup I, VH subgroup III) contained in the plasmid pVX4 (Figure 2). The phagemid pVX4 (Figure 2) was used by mutagenesis as well as by expression of F (ab) s in E. coli. Based on phagemid pb0720, a derivative of pB0475 (Cunningham et al., Science 243: 1330-1336 (1989)), pVX4 contains a DNA fragment encoding a consensus humanized K-subgroup I light chain (VL? I-CL ) and a humanized consensus antibody subgroup III heavy chain (VHIII-CH1) anti-IFN-a (interferon-a). PVX4 also has an alkaline phosphatase promoter and Shine-Dalgarno sequence both split from others previously described based on plasmid pUC119, pAK2 (Carter et al., Proc. Nati, Acad. Sci. USA 89: 4285 (1992)). A single Spel restriction site was introduced between the DNA encoding the light and heavy chains of F (ab). The first 23 amino acids in both heavy and light anti-IFN-oc chains are the STII secretion signal sequence (Chang et al., Gene 55: 189-196 (1987)). To construct the CDR-swap version of 2H7 (2H7.v2), the site-directed mutagenesis was performed using a model containing deoxiuridine from pVX4; all six CDRs of the anti-IFN-a were loaded onto the murine 2H7 CDRs. The resulting molecule is referred to as humanized 2H7 version 2 (2H7.v2), or the "CDR-swap version" of 2H7; has the CDR m2H7 residues with the human consensus residues FR shown in Figures 1A and IB. Humanized 2H7.v2 using additional humanization. Table 2 shows the sequence of oligonucleotides used to create each of the murine CDRs 2H7 (m2H7) in the H and L chain. For example, the oligonucleotide CDR-H1 was used to recreate the H chain m2H7 of CDR1. CDR-HI, CDR-H2 and CDR-H3 refers to the H chain CDR1, CDR2 and CDR3, respectively; similarly, CDR-L1, CDR-L2 and CDR-L3 refer to each of L-chain CDRs. The substitutions in CDR-H2 were made in two stages with two oligonucleotides, CDR-H2A and CDR-H2B. Table 2 Sequence of oligonucleotides used for the construction of murine CDR-swap 2H7 CDRs in a human structure in pVX4. The residues exchanged for each oligonucleotide are underlined.

Substitution Sequence of oligonucleotides CDR-H1 C TAC ACC TTC ACG AGC TAT AAC ATG CAC TGG GTC CG (SEQ ID N0: 31) CDR-H2A G ATT AAT CCT GAC AAC GGC GAC ACG AGC TAT AAC CAG AAG TTC AAG GGC CG (SEQ ID NO: 32) CDR-H2B GAA TGG GTT GCA GCG ATC TAT CCT GGC AAC GGC GAC AC (SEQ ID NO: 33) CDR-H3 AT TAT TGT GCT CGA GTG GTC TAC TAT AGC AAC AGC TAC TGG TAC TTC GAC GTC TGG GGT CAA GGA (SEQ ID NO: 34) CDR-L1 C TGC ACA GCC AGC TCT TCT GTC AGC TAT ATG CAT TG (SEQ ID NO: 35) CDR-L2 AA CTA CTG ATT TAC GCT CCA TCG AAC CTC GCG TCT GGA GTC C (SEQ ID NO: 36) CDR-L3 TAT TAC TGT CAA CAG TGG AGC TTC AAT CCG CCC ACA TTT GGA CAG (SEQ ID NO: 37) For comparison with humanized constructs, a plasmid expressing a 2H7Chimeric Fab (containing VL and VH domains, and human CL and CHi murine) site-directed mutagenesis was constructed (Kunkel, supra) using synthetic oligonucleotides to introduce the murine structure residues in 2H7.v2. The sequence of the resulting plasmid construct by expression of the chimeric Fab known as 2H7.v6.8, is shown in Figure 3. Each encoded strand of the Fab has a 23 amino acid STII secretion signal sequence as described by pVX4 ( Figure 2) above. Based on a sequence of comparison of murine 2H7 structure residues with the human consensus structure VKI, VHIII (Figures 1A and IB) and previously humanized antibodies (Cárter et al., Proc. Nati. Acad. Sci. USA 89: 4285 -4289 (1992)), several structure mutations were introduced into the 2H7.v2 Fab construct by site-directed mutagenesis. These mutations result in a change of certain consensus residues of human structure to those found in the murine 2H7 structure, at sites that could affect the CDR conformations or antigen contacts. Version 3 contained in VH (R71V, N73K), version 4 contained in VH (R71V), version 5 contained in VH (R71V, N73K) and VL (L46P), and version 6 contained in VH (R71V, N73K) and VL ( L46P, L47W). The humanized and chimeric Fab versions of the antibody m2H7 were expressed in E. coli and purified as follows. The plasmids were transformed into E. coli strain XL-1 Blue (Stratagene, San Diego, CA) for the preparation of double and single strand DNA. For each variant, both light and heavy chains were completely sequenced using the dideoxynucleotide method (SEQUENASE ® labeled first cycle sequencing, U.S. Biochemical Corp.). The plasmids were transformed into E. coli strain 16C9, a derivative of MM294, plated on LB plates containing 5 μg / ml carbenicillin, and a single colony was selected for protein expression. The only colony grew in 5 ml LB-100 μg / ml carbenicillin for 5-8 h at 37 ° C. The 5 ml culture was added 500 ml AP5-100 μg / ml carbenicillin and allowed to develop for 16 h in a 4-L shake flask with a screen at 37 ° C. The AP5 medium consists of: 1.5 g glucose, 11.0 g HYCASE SF ™ (hydrolysed casein), 0.6 g yeast extract (certified), 0.19 g MgSO4 anhydride, 1.07 g NH4C1, 3.73 g KCl, 1.2 g NaCl, 120 ml 1 M triethanolamine, pH 7.4, to 1 1 of water and then sterile filtered through a SEACLEEN® 0.1 μm biocide filter. The cells were harvested by centrifugation in a 1-L centrifuge bottle (Nalgene) at 3000xg and the supernatant was removed. After freezing for 1 h, the pellet was resuspended in 25 ml cold 10 mM MES-10 mM EDTA, pH 5.0 (buffer A). 250 μl of 0.1M phenylmethylsulfonyl fluoride (PMSF) (Sigma) was added to inhibit proteolysis and 3.5 ml of stock 10 mg / ml white egg chicken lysozyme (Sigma) was added to aid in the lysis of the cell wall of the bacterium. After gentle stirring on ice for 1 h, the sample was centrifuged at 40,000xg for 15 min. The supernatant was taken to 50 ml of buffer A and loaded onto a column equilibrated with A 2-mi DEAE buffer. The flow-through was then applied to a CL-4B ™ agarose chromatography column (Pharmacia) of protein G-SEPHAROSE (0.5-ml bed volume) equilibrated with buffer A. The column was washed with 10 ml of buffer A and was eluted with 3 ml of 0.3 M glycine, pH 3.0, in 1.25 ml of 1 M TRIS, pH 8.0. The F (ab) buffer was then exchanged in phosphate buffered saline (PBS) using a centrifugal filter device CENTRICON-30 ® (Amicon) and concentrated to a final volume of 0.5 ml. The SDS-PAGE gels of all F (ab) s were run to ensure purity, and the molecular weight of each variant was verified by electro-spray mass spectrometry. In the cell-based ELISA binding assay (described below), the binding of Fabs, including chimeric Fab 2H7, to CD20 was difficult to detect. Therefore, the Fab 2H7 versions were reformatted as full length IgGl antibodies for further assays and mutagenesis. Plasmids for IgG 's full-length expression were constructed by subcloning the VL and VH domains of Fab (v6.8) chimeric 2H7 as well as humanized Fab versions 2 through 6 into previously described pRK vectors for mammalian cell expression (Gorman et al. DNA Prot. Eng. Tech. 2: 3-10 (1990)). Briefly, each Fab construct was digested with EcoRV and Blpl to excise the VL fragment, which was cloned into the EcoRV / Blpl sites of plasmid pDRl (Figure 4) for the expression of the complete light chain (V ^ -CL domains). Additionally, each Fab construct was digested with PvuII and ApaI to excise a VH fragment, which was cloned into the PvuII / Apal sites of the pDR2 plasmid (Figure 5) for the expression of the complete heavy chain (VH-CHi-joint-domains). CH2-CH3). For each IgG variant, transient transfections were performed by cotransfecting a light chain expressing the plasmid and a heavy chain expressing the plasmid in a human embryonic kidney cell line transformed with adenovirus, 293 (Graham et al., J. Gen. Virol 36: 59-74 (1977)). Briefly, 293 cells were divided on the day before transfection, and plated in medium containing serum. On the next day, the double-stranded DNA prepared as a precipitate of calcium phosphate was added, followed by PADVANTAGE ™ DNA (Promega, Madison, Wl), and the cells were incubated overnight at 37 ° C. The cells were cultured in serum-free medium and harvested after 4 days. The antibodies were purified from the culture supernatants using agarose chromatography protein A-SEPHAROSE CL-4B ™, then the buffer was exchanged in 10 mM sodium succinate, 140 mM NaCl, pH 6.0, and sew was concentrated using a filter device CENTRICON-10® centrifuge (Amicon). The protein concentration was determined by quantitative amino acid analysis. To measure the relative binding affinities for the CD20 antigen, a cell-based ELISA was developed. Human lymphoblastoid B-WIL2-S cells (ATCC CRL 8885, American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 20 mM HEPES, pH 7.2 and 10% inactivated fetal bovine serum. hot in a humidified 5% C02 incubator. The cells were washed with PBS containing 1% fetal bovine serum (FBS) (assay buffer) and seeded at 250-300,000 cells / well in 96-well round bottom plates (Nunc, Roskilde, Denmark). Two (15.6-1000 ng / ml of 2H7 v6.8 chimeric IgG) standard serially diluted twice and samples (2.7-2000 ng / ml) diluted serially three times in assay buffer. The plates were buried in ice and incubated for 45 min. To remove unbound antibodies, 0.1 ml of assay buffer was added to the wells.

The plates were centrifuged and the supernatants were removed.

The cells were washed twice with 0.2 ml of assay buffer. Antibodies bound to the plates were detected by adding peroxidase-conjugated goat anti-human Fe antibody (Jackson ImmunoResearch, West Grove, PA) to the plates. After a 45-min incubation, the cells were washed as described below. The TMB substrate (3, 3 ', 5,5'-tetramethyl benzidine; Kirkegaard &Perry Laboratories, Gaithersburg, MD) was added to the plates. The reaction was stopped by adding 1 M phosphoric acid. The titration curves were adjusted with a regression curve adjustment program of four non-linear parameters (KALEIDAGRAPH ™, Synergy software, Reading, PA). The absorbance at the midpoint of the titration curve (mid-OD) and its corresponding concentrations of the standard were determined. Then the concentration of each variant in this mid-OD was determined, and the concentration of the standard was divided between that of each variant. Therefore, the values are a proportion of the union of each variant with respect to the standard. The standard deviations in relative affinity (equivalent concentration) was generally +/- 10% between experiments. As shown in Table 3, the binding of the CDR-swap variant (v.2) was extremely reduced compared to the 2H7chimeric (v.6.8). However, versions 3 to 6 show improved binding. To determine the minimum number of mutations that might be required to re-establish the binding affinity for the chimeric 2H7, mutations and additional combinations of mutations were constructed by site-directed mutagenesis to produce variants 7 to 17 as indicated in Table 4. In particular , these include VH mutations A49G, F67A, I69L, N73K, and L78A; and mutations VL M4L, M33I, and F71Y. Versions 16 and 17 show the best affinities, within 2-fold of that of the chimeric version, without significant difference (s.d. = +/- 10%) between the two. To minimize the number of mutations, version 16, having only 4 mutations of human structure residues to residues of murine structure (Table 4), they were therefore selected as the humanized form for further characterization.

Table 3 The relative binding affinity of humanized 2H7 IgG variants to CD20 compared to chimeric 2H7 using cell-based ELISA. The relative binding is expressed as the concentration of the chimeric 2H7 on the concentration of the variant required for equivalent binding; consequently a proportion < 1 indicates weak affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%. The structure substitutions in the variable domains are in relation to the CDR-swap version according to the Kabat numerical system (Kabat et al., Supra).

Table 4 Sequence of oligonucleotides used for the construction of VH mutations (A49G, R71V, N73K) and VL (L46P.) In humanized version 2H7 (2H7.vl6) .The underlined codons code for the indicated amino acid substitutions. (R71V, N73K) and VL (L46P), the oligonucleotides are shown as the sense string since these were used for mutagenesis in the Fab pattern, whereas for VH (A49G), the oligonucleotide is shown as the anti-sense strand, since it was used with the pRK (heavy chain IgG) standard, the protein sequence of version 16 is shown in Figure 6 and Figure 7.

Example 2 Determinants of Antigen binding (paratopes) of 2H7 The alanine substitutions (Cunningham &Wells, Science 244: 1081-1085 (1989)) were prepared in 2H7.vl6 or 2H7.vl7 in order to test the contributions of individual side chains of the antibody in binding to CD20. The variant IgGs were expressed in 293 vector cells pDRl and pDR2, purified and tested by relative binding affinity as described above. Several alanine substitutions resulted in a significant decrease in binding relative to CD20 in WIL-2S cells (Table 5).

Table 5 Effects of alanine substitutions on CDR regions of humanized 2H7.vl6 measured using cell-based ELISA (WIL2-S cells). Relative binding is expressed as the concentration of 2H7.vl6 of origin over the concentration of • the variant required for the equivalent binding; therefore a proportion < 1 indicates a weaker affinity for the variant; a proportion > 1 indicates a greater affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%. The structure substitutions in the variable domains are relative to 2H7.vl6 according to the Kabat numbering system (Kabat et al., Supra). NBD means no detectable linkage. The two numbers for version 45 are from separate experiments.

EXAMPLE 3 Additional Mutations Within 2H7 CDR Regions Residual substitutions and additional combinations of substitutions at CDR positions were also tested which were identified as important by Ala scanning. Various combination variants, particularly v.96, seemed to bind more closely than v.16.

Table 6 Effects of mutagen combinations and non-alanine substitutions on the CDR regions of humanized 2H7.V16 measured using cell-based ELISA (WIL2-S cells). Relative binding is expressed as the concentration of the 2H7.vl6 of origin over the concentration of the variant required for the equivalent binding; therefore a proportion < 1 indicates a weaker affinity for the variant; a proportion > 1 indicates a greater affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%. The structure substitutions in the variable domains are relative to 2H7.vl6 according to the Kabat numbering system (Kabat et al., Supra).

Example 4 Mutations in sites of structure humanization substitutions Substitutions of additional residues in structure positions that changed during humanization were also tested in the background 2H7.vl6. In particular, alternative structure substitutions that were found neither in the murine 2H7 of origin nor in the human consensus structure were performed in VL (P46) and VH (G49, V71, and K73). These substitutions generally led to a small change in the relative union (Table 7), indicating that there is some flexibility in the structure residues in these positions. Table 7 Relative binding in a cell-based assay (WIL2-S) of structure substitutions. The variant IgGs are shown with mutations with respect to the background 2H7.vl6. Relative binding is expressed as the concentration of 2H7.v6.8 chimera over the concentration of the variant required for the equivalent union; therefore, a proportion < 1 indicates a weaker affinity for the variant; a proportion > 1 indicates a greater affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%. The structure substitutions in the variable domains are relative to 2H7.vl6 according to the Kabat numbering system (Kabat et al., Supra).

(*) Variants that were analyzed with 2H7.vl6 as a standard comparator; the relative values are normalized with that of the chimera.

Example 5 Humanized 2H7 Variants with Enhanced Effector Functions Because 2H7 can mediate lysis of B cells through CDC and ADCC, variants of humanized 2H7.vl6 with enhanced CDC and ADCC activity are sought. Mutations of certain residues within the Fes region of other antibodies have been described (Idusogie et al., J. Immunol., 166: 2571-2575 (2001)) to improve CDC through increased binding to the complement component Clq. Mutations have also been described (Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Presta et al., Biochem. Soc. Trans. 30: 487-490 (2002)) to improve ADCC through of an increase in the binding of IgG to Fc activation receptors? and reduction of IgG binding to Fc? inhibitory receptors. In particular, three mutations have been identified to improve the activity of CDC and ADCC: S298A / E333A / K334A (also referred to herein as triple mutant or variant Ala; the numbering in the Fe region is in accordance with the EU numbering system; Kabat et al. , supra), as described (Idusogie et al., supra (2001); Shields et al., supra). In order to improve the CDC and ADCC activity of 2H7, a triple mutant Ala of 2H7 Fe was constructed. A humanized variant of anti-HER2 antibody 4D5 with mutations S298A / E333A / K334A has been produced and is known as 4D5FcllO (ie, anti -p185HER2 IgGl (S298A / E333A / K334A); Shields et al., supra).

A plasmid, p4D5FcllO that codes for the antibody 4D5FcllO (Shields et al., Supra) was digested with Apal and HindlH, and the Fe fragment (containing mutations S298A / E333A / K334A) was ligated into the Apal / ífincilII sites of the heavy chain vector 2H7 pDR2-vl6, to produce pDR2-v31. The amino acid sequence of the complete chain H version 31 is shown in Figure 8. The chain L is the same as that of vl6. Although the variable domains of the Fe region of IgGl antibodies are relatively conserved within a given species, there are allelic variations (reviewed by Lefranc and Lefranc, in The Human IgG Subclasses: molecular analysis of structure, function, and regulation, pp. 43 -78, F. Shakib (ed.), Pergamon Press, Oxford (1990)).

Table 8 Effects of substitutions in the Fe region in the binding of CD20. The relative binding to CD20 was measured in a cell-based assay (WIL2-S) of the structure substitutions. The Fe mutations (*) are indicated by the EU numbering (Kabat, supra) and are relative to the 2H7.vl6 of origin. The combination of three Ala changes in the Fe region of v.31 is described as "FcllO." The variant IgGs are shown with mutations with respect to the background 2H7.vl6. Relative binding is expressed as the concentration of 2H7.v6.8 chimera over the concentration of the variant required for the equivalent union; therefore, a proportion < 1 indicates a weaker affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%.

EXAMPLE 6 Humanized 2H7 Variants with Improved Stability For development as therapeutic proteins, it is desirable to select variants that remain stable with respect to oxidation, deamidation, or other processes that may affect the quality of the product, in a suitable formulation buffer. In 2H7.vl6, several residues were identified as possible sources of instability: VL (M32) and VH (M34, N100). Consequently, mutations were introduced at these sites for comparison with vl6. Table 9 Relative binding of 2H7 variants, designed for improved stability and / or effector function, to CD20 in a cell-based assay (WIL2-S). The variant IgGs are shown with mutations with respect to the background 2H7.vl6. Relative binding is expressed as the concentration of 2H7.v6.8 chimera over the concentration of the variant required for the equivalent union; therefore, a proportion < 1 indicates a weaker affinity for the variant. The standard deviation in the determination of relative affinity averaged +/- 10%. The structure substitutions in the variable domains are relative to 2H7.vl6 according to the Kabat numbering system and the Fe (*) mutations are indicated by the EU numbering (Kabat et al., Supra).

(**) Variants that were measured with 2H7 .vl6 as a comparator; The relative binding values are normalized to that of the chimera. Additional Fe mutations were combined with mutations that improve stability or affinity to alter or improve effector functions on the basis of previously reported mutations (Idusogie et al., J. Immunol 164: 4178-4184 (2000); Idusogie et al. Immunol., 166: 2571-2575 (2001); Shields et al., J. Biol. Chem. 276: 6591-6604 (2001)). These changes include S298, E333A, K334A as described in Example 5; K322A to reduce CDC activity; D265A to reduce ADCC activity; K326A or K326W to improve CDC activity; and E356D / M358L to test the effects of allotypic changes in the Fe region. None of these mutations caused significant differences in CD20 binding affinity. To test the effects of stability mutations on the proportion of protein degradation, 2H7.vl6 and 2H7.v73 were formulated at 12-14 mg / mL in 10 mM histidine, 6% sucrose, 0.02% POLISORBATE 20 ™ emulsifier, pH 5.8 and incubated at 40 ° C for 16 days. The incubated samples were then analyzed for changes in charge variants by ion exchange chromatography, aggregation, and fragmentation by size exclusion chromatography, and relative binding by testing in a cell-based assay (WIL2-S). The results show that 2H7 v.73 has greater stability compared to 2H7 v.16 with respect to the losses in the main peak fraction by ion exchange chromatography under accelerated stability conditions. No significant differences were observed with respect to aggregation, fragmentation, or binding affinity. Example 7 Scatchard Analysis of CD20 Antibody Binding in WIL2-S Cells Equilibrium dissociation constants (Kd) were determined for the binding of 2H7 variant IgGs to WIL2-S cells using radiolabeled 2H7 IgG. The variant IgGs were produced in CHO cells. RITUXAN® (the source for all experiments is Genentech, S. San Francisco, CA) and murine 2H7 (BD PharMingen, San Diego, CA) was used for comparison with humanized variants. The murine 2H7 antibody is also available from other sources, e. g. , Bioscience, and Calbiochem (both of San Diego, CA), Accurate Chemical &; Scientific Corp., (Westbury, NY), Ancell (Bayport, MN), and Vinci-Biochem (Vinci, Italy). All dilutions were carried out in binding assay buffer (DMEM medium containing 1% bovine serum albumin, 25 mM HEPES pH 7.2, and 0.01% sodium azide). Aliquots (0.025 mL) of 125 I-2H7.vl6 (iodinated with lactoperoxidase) were dispensed at a concentration of 0.8 nM into wells of a 96-well bottom V microassay plate, and serial dilutions (0.05) were added and mixed. mL) of antibody. WIL2-S cells (60,000 cells in 0.025 mL) were then added. The plate was sealed and incubated at room temperature for 24 hours, then centrifuged for 15 min at 3,500 RPM. The supernatant was then aspirated and the cell pill was washed and centrifuged. The supernatant was aspirated again, and the pills were dissolved in IN NaOH and transferred to tubes for gamma counting. The data were used for Scatchard analysis (Munson and Rodbard Anal. Biochem. 107: 220-239 (1980)) using the program binder (McPherson Comput. Programs Biomed. 17: 107-114 (1983)). The results, shown in Table 10, indicate that humanized 2H7 variants had a similar binding affinity of CD20 compared to murine 2H7, and similar binding affinity withRITUXAN®. It is expected that 2H7.v31 will have a Kd very similar to v.16 based on the union shown in Table 8 above. Table 10 Equilibrium binding affinity of 2H7 variants from the Scatchard analysis Example 8 Complement-Dependent Cytotoxicity (CDC) Assays 2H7 variant IgGs were analyzed for their ability to mediate complement-dependent lysis of WIL2-S cells, a CD20 expressing the B-cell lymphoblastoid line, essentially as described (Idusogie et al. J. Immunol. 164: 4178-4184 (2000); Idusogie et al. J. Immunol. 166: 2571-2575 (2001)). Antibodies were serially diluted 1: 3 from a stock solution of 0.1 mg / mL. 0.05 mL of aliquot of each dilution was added to a 96-well tissue culture plate containing 0.05 mL of a normal human complement solution (Quidel, San Diego, CA). To this mixture, 50,000 WIL2-S cells were added in a volume of 0.05 mL. After incubation for 2 hours at 37 ° C, 0.05 L of a solution of ALAMAR BLUE ™ resazurin (Accumed International, Westlake, OH) was added, and the incubation was continued for an additional 18 hours at 37 ° C. the covers were then removed from the plates, and shaken for 15 min at room temperature in an orbital shaker. The relative fluorescent units (RFU) were read using a 530 nm excitation filter and a 590 nm emission filter. An EC50 was calculated by adjusting the RFU as a concentration function for each antibody using KALEIDAGRAPH ™ software. The results (Table 11) show a surprising improvement in CDC by humanized 2H7 antibodies, with relative potency similar to RITUXAN® for v.73, 3-times more potent than RITUXAN® for v.75, and 3-fold weaker than RITUXAN® for v.16. Table 11 CDC activity of 2H7 antibodies compared to RITUXAN®. The numbers > 1 indicate less potent CDC activity than RITUXAN® and the numbers < 1 indicate more potent activity than RITUXAN®. The antibodies were produced from stable CHO lines, except that those indicated by (*) occurred transiently.

EXAMPLE 9 Antibody-dependent Cell Cytotoxicity Assays (ADCC) The 2H7 IgGs variants were analyzed for their ability-to mediate NK cell lysis of WIL2-S cells, a CD20 expressing the B-cell lymphoblastoid line, essentially as described above. describes (Shields et al., J. Biol. Chem. 276: 6591-6604 (2001)) using a lactate dehydrogenase (LDH) reading.

NK cells were prepared from 100 L of heparinized blood, diluted with 100 mL of PBS, obtained from normal human donors that were isotyped for FcγRIII, also known as CD16 (Koene et al., Blood 90: 1109-1114 (1997 )).

In this experiment, the NK cells were from human donors heterozygous for CD16 (F158 / V158). The diluted blood was placed on 15 mL of lymphocyte separation medium (ICN Biochemical, Aurora, Ohio) and centrifuged for 20 min at 2000 RPM. The white cells at the interface between layers were dispensed to 4 clean 50-mL tubes, which were filled with RPMI medium containing 15% fetal calf serum. The tubes were centrifuged for 5 min at 1400 RPM and the supernatant was discarded. The pills were resuspended in MACS Shock Absorber (0.5% BSA, 2mM EDTA), and NK cells were purified using beads (NK Cells Isolation Kit, 130-046-502) according to the manufacturer's protocol (Miltenyi Biotech.). The NK cells were diluted in MACS buffer at 2xl06 cells / mL. Serial dilutions of antibody (0.05 mL) were added in assay medium (F12 / DMEM 50:50 without glycine, 1 mM buffer HEPES pH 7.2, Penicillin / Streptomycin (100 units / mL, Gibco), glutamine, and 1% serum bovine fetal inactivated with heat) to a 96-well round bottom tissue culture plate. The WIL2-S cells were diluted in assay buffer at a concentration of 4 x 105 / mL. WIL2-S cells (0.05 mL per well) were mixed with antibody diluted in the 96-well plate and incubated for 30 min at room temperature to allow binding of the antibody to CD20 (opsonization). The ADCC reaction was initiated by adding 0.1 mL of NK cells to each well. In control wells, 2% TRITON® X-100 alkylaryl polyether alcohol was added. The plate was then incubated for 4 hours at 37 ° C. Released LDH levels were measured using a cytotoxicity detection (LDH) kit (Kit # 1644793, Roche Diagnostics, Indianapolis, Indiana) following the manufacturer's instructions. 0.1 mL of LDH developer was added to each well, followed by mixing for 10 seconds. The plate was then covered with aluminum foil and incubated in the dark at room temperature for 15 min. The optical density was then read at 490 nm and used to calculate the% lysis by dividing the total LDH measured in the control wells. Lysis was illustrated as a function of antibody concentration, and a 4-parameter curve fitting (KALEIDAGRAPH ™ software) was used to determine EC50 concentrations. The results showed that humanized 2H7 antibodies were active in ADCC, with relative power 20-fold higher than RITUXAN® for v.31 and v.75, 5-times more potent than RITUXAN® for v.16, and almost 4 -Times higher than RITUXAN® for v.73. Table 12 ADCC activity of 2H7 antibodies in WIL2-S cells compared to 2H7.vl6, based on n experiments. (the values >; 1 indicate less power than 2H7.vl6, and the values < 1 indicate more power) Additional ADCC assays were carried out to compare the combination variants of 2H7 with RITUXAN®. The results of these tests indicated that 2H7.vll4 and 2H7.V115 have > 10-fold enhanced ADCC power compared to RITUXAN® (Table 13). Table 13 ADCC activity of 2H7 antibodies in WIL2-S cells compared to RITUXAN®, based on n experiments (Values> 1 indicate lower potency than RITUXAN®, and values <1 indicate greater potency).

EXAMPLE 10 In Vivo Effects of 2H7 Variants in a Pilot Study in Cynomolgus Monkeys The 2H7 variants, produced by transient transfection of CHO cells, were tested in normal male cynomolgus monkeys (Macaca fascicularis) in order to evaluate their activities in vivo. Other anti-CD20 antibodies, such as C2B8 (RITUXAN®), have demonstrated an ability to consume B cells in normal primates (Reff et al., Blood 83: 435-445 (1994)).

In one study, humanized 2H7 variants were compared. In a parallel study, RITUXAN® was also tested in cynomolgus monkeys. Four monkeys were used in each group of five doses: (1) vehicle, (2) 0.05 mg / kg hu2H7.vl6, (3) 10 mg / kg hu2H7.vl6, (4) 0.05 mg / kg hu2H7.v31, and (5) 10 mg / kg hu2H7.v31. The antibodies were administered intravenously at a concentration of 0, 0.2, or 20 mg / mL, for a total of two doses, one on day 1 of the study, and another on day 8. The first day of dosing is designated day 1 and the previous day is designated day -1; the first day of recovery (for 2 animals in each group) designated as day 11. Blood samples were collected on days -19, -12, 1 (before dosing), and at 6 hours, 24 hours, and 72 hours after the first dose. Additional samples were taken on day 8 (before dosing), day 10 (prior to slaughter of 2 animals / group), and on days 36 and 67 (for recovery animals). The concentrations of peripheral B cells were determined by a FACS method which counted CD3- / CD40 + cells. The percentage of CD3-CD40 + B cells of the total lymphocytes in monkey samples was obtained by the following strategy. The lymphocyte population was marked in the forward scatter / lateral scatter scatter diagram to define region 1 (Rl). Using the events in Rl, fuorescence intensity point diagrams were displayed for CD40 and CD3 markers. Isotype controls marked with fluorescence were used to determine the respective cut-off points for CD40 and CD3 positivity. The results indicated that both 2H7.vl6 and 2H7.v31 were able to produce total suppression of peripheral B cells in the dose of 10 mg / kg and partial suppression of peripheral B cells at a dose of 0.05 mg / kg. The time course and extent of B cell suppression measured during the first 72 hours of dosing was similar for the two antibodies. Subsequent analyzes of the recovery animals indicated that the animals treated with 2H7.v31 showed a prolonged suppression of B cells compared to those dosed with 2H7.vl6. In particular, for recovery animals treated with 10 mg / kg 2H7.vl6, B cells showed substantial recovery of B cells sometime between sampling on day 10 and day 36. However, for recovery animals treated with 10 mg / kg 2H7.v31, B cells show no recovery until sometime between day 36 and day 67. This suggests a longer duration of total suppression for approximately one month for 2H7.v31 compared to 2H7.vl6. No toxicity was observed in the study in monkeys at low or high doses and the general pathology was normal. In other studies, vl6 was well tolerated up to the highest dose evaluated (100mg / kgx2 = 1200 mg / m2 x2) following i.v. of 2 doses given 2 additional weeks in these monkeys. Data on cynomolgus monkeys with 2H7.vl6 versus RITUXAN® suggest that a 5-fold reduction in the CDC activity does not adversely affect the potency. An antibody with potent ADCC activity but with reduced CDC activity may have a more favorable safety profile with respect to the first reactions to infusion reactions than one with greater CDC activity. EXAMPLE 11 2H7 Antibodies Fucose Deficient Variant with Enhanced Effector Function Normal CHO and HEK293 cells add fucose to IgG oligosaccharide at a high degree (97-98%). Serum IgG is also highly fucosylated. DP12, a CHO cell line of dihydrofolate-reductase-minus (DHFR) competent to fucosylation, and Lecl3, a cell line deficient in protein fucosylation, were used to produce antibodies for this study. The CHO cell line, Pro-Lecl3.6a (Lecl3), was obtained from Professor Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University. The lines of origin are Pro- (auxotrophic proline) and Gat- (glycine, adenosine, auxotrophic thymidine). The CHO-DP12 cell line is a derivative of the CHO-Kl cell line (ATCC # CCL-61), which is deficient in dihydrofolate reductase, and has a reduced insulin requirement. Cell lines were transfected with cDNA using the SUPERFECT ™ transfection reagent method (Qiagen, Valencia, CA). The selection of Lecl3 cells expressing transfected antibodies was carried out using puromycin dihydrochloride (Calbiochem, San Diego, CA) at 10 μg / ml in growth medium containing: MEM MEM medium with L-glutamine, ribonucleosides and deoxyribonucleosides (GIBCO- BRL, Gaithersburg, MD), supplemented with 10% inactivated FBS (Gibco), 10 mM HEPES, and IX penicillin / streptomycin (Gibco). CHO cells were similarly selected in growth medium containing Ham's F12 without GHT: low glycine DMEM without glycine with NaHCO 3 supplemented with 5% FBS (Gibco), 10 mM HEPES, 2 mM L-glutamine, IX GHT ( glycine, hypoxanthine, thymidine), and IX penicillin / streptomycin. The colonies formed within two to three weeks and were deposited for protein expansion and expression. Cell deposits were initially seeded at 3 x 10 6 cells / 10 cm plate for expression by small batches of protein. The cells were converted to serum free medium once they grew to a confluence of 90-95%, and after 3-5 days the cell supernatants were collected and tested in an IgG-IgG and intact IgG-ELISA for estimate the levels of protein expression. Lecl3 and CHO cells were seeded at approximately 8 x 10 6 cells / 15-cm plate one day before converting to PS24 production medium, supplemented with 10 mg / L of recombinant human insulin and 1 mg / L of trace elements. Lecl3 cells and DP12 cells remained in serum free production medium for 3-5 days. Supernatants were collected and clarified by centrifugation in 150-ml conical tubes to remove cells and powder. Protease inhibitors PMSF and aprotinin (Sigma, St. Louis, MO) were added and the supernatants were concentrated 5-fold in stirred cells using MWCO30 ™ filters (Amicon, Beverly, MA) prior to immediate purification using G protein chromatography ( Amersham Pharmacia Biotech, Piscataway, NJ)). All proteins were exchanged with buffer to PBS using CENTRIPREP-30 ™ concentrators (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using A280 absorbance values and verified using amino acid composition analysis. CHO cells were transfected with vectors expressing humanized 2H7vl6, 2H7v.31 and selected as described. The 2H7v.l6 antibody retains the wild type Fe region, while v.31 (see Example 5, Table 8 above) has a Fe region where 3 amino acid changes were made (S298A, E333A, K334A), which gave as resulting in a higher affinity for the Fc? RIIIa receptor (Shields et al., J.

Biol. Chem. 276 (9): 6591-6604 (2001)). After transfection and selection, individual cell colonies were isolated and evaluated for level of protein expression, and the highest producers were subjected to selection of methotrexate to select cells that had amplified the number of plasmid copies and that, consequently, they produced higher levels of antibody. The cells were cultured and transferred to serum-free medium for a period of 7 days, then the medium was collected and loaded onto a protein A column and the antibody was eluted using standard techniques. The final concentration of the antibody was determined using an ELISA which measures the intact antibody. All proteins were exchanged with buffer to PBS using CENTRIPREP-30 ™ concentrators (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Analysis of Spectral Mass of Desorption Flight Time / Liaser Assisted Ionization by Matrix (MALDI-TOF) of Oligosaccharides United to Asparagine. N-binding oligosaccharides of recombinant glycoproteins were released using the method of Papac et al. Glycobiology 8: 445-454 (1998). Briefly, the wells of a microtitre plate coated with 96-well polyvinylidine difluoride (PVDF) (Millipore, Bedford, MA) were conditioned with 100 μl methanol which was extracted through PVDF membranes by applying vacuum to the Millipore MULTISCREEN ™ vacuum apparatus. . The conditioned PVDF membranes were washed with 3 X 250 μl water. Between all the washing steps, the wells were drained completely by applying a gentle vacuum to the appliances. The membranes were washed with reduction buffer and carboxymethylation (RCM) consisting of 6 M guanidine hydrochloride, 360 mM TRIS, 2 mM EDTA, pH 8.6. Samples of glycoprotein (50 μg) were applied to the individual wells, again extracted through the PVDF membranes by gentle vacuum and the wells were washed with 2 X 50 μl of RCM buffer. The immobilized samples were reduced by adding 50 μl of a 0.1 M solution of dithiothreitol (DTT) to each well and incubating the microtitre plate at 37 ° C for 1 hour. The DTT was removed by vacuum and the wells were washed with 4 x 250 μl water. The cysteine residues were carboxylmethylated by the addition of 50 μl of a 0.1 M solution of iodoacetic acid (IAA) which was freshly prepared in 1 M NaOH and diluted to 0.1 M with RCM buffer. Carboxymethylation was achieved by incubation for 30 min in the dark at room temperature. Vacuum was applied to the plate to remove the IAA solution and the wells were washed with 4 x 250 μl of purified water. The PVDF membranes were blocked by the addition of 100 μl of 1% PVP-360 solution (polyvinylpyrrolidine 360,000 MW) (Sigma) and incubation for 1 hour at room temperature. The PVP-360 solution was removed by gentle vacuum and the wells were washed with 4 x 250 μl of water. The PNGASE F ™ amidase digestion solution (New England Biolabs, Beverly, MA), 25 μl of a 25 unit / ml solution in 10 mM TRIS acetate, pH 8.4, was added to each well and the digestion proceeded for 3 hr at 37 ° C. After digestion, samples were transferred to 500 μl Eppendorf tubes and 2.5 μL of a 1.5 M solution of acetic acid was added to each sample. The acidified samples were incubated for 3 hr at room temperature to convert the glycosylamino oligosaccharides to the hydroxyl form. Prior to the MALDI-TOF spectral mass analysis, the released oligosaccharides were desalted using a 0.7-ml bed of cation exchange resin (AG50W-X8 ™ resin in the hydrogen form) (Bio-Rad, Hercules, CA) packed in mixing in compact reaction tubes (US Biochemistry, Cleveland, OH). For the MALDI-TOF spectral mass analysis of the samples in the positive mode, the desalted oligosaccharides (0.5 μl aliquots) were applied to the stainless target with 0.5 μl of the acid matrix 2, 5-dihydroxybenzoic acid (sDHB) which was prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid with 0.1 mg of 5-methoxyscyclic acid in 1 ml of ethanol / 10 mM sodium chloride 1: 1 (v / v). The sample / matrix mixture was dried by vacuum. For the negative mode analysis, the N desalted oligosaccharides (0.5 μl aliquots) were applied to the stainless target together with a 0.5 μl matrix of 2 ', 4', 6 '-trihydroxyacetophenone (THAP) prepared in 1: 3 buffer (v / v) acetonitrile / 13.3 mM ammonium citrate. The sample / matrix mixture was dried under vacuum and then allowed to absorb atmospheric moisture prior to analysis. The released oligosaccharides were analyzed by MALDI-TOF on a PERSEPTIVE BIOSYSTEMS ™ VOYAGER-DE ™ mass spectrometer. The mass spectrometer was operated at 20 kV in the positive or negative mode with the linear configuration and using delayed extraction. The data was acquired using a laser energy of 1300 and in the data sum mode (240 scan) to improve the signal-to-noise ratio. The instrument was calibrated with a mixture of standard oligosaccharides and the data was smoothed using a 19-point Savitsky-Golay algorithm before allocating the masses. The integration of the spectral mass data was achieved using the CAESAR 7.0 ™ analysis data software equipment (SciBridge Software). NK cell ADCCs. The ADCC assays were carried out as described in Example 9. The target ratio of NK-to-cell (WIL2-S) was 4 to 1, the tests were carried out for 4 hours, and the toxicity was measured as before. using the lactose dehydrogenase assay. Target cells twere opsonized with antibody concentrations were indicated for 30 min prior to the addition of NK cells. The RITUXAN® antibody used was from Genentech (S. San Francisco, CA).

The results show that low-fucosylation antibodies mediate the destruction of the target NK cell more efficiently than antibodies with a total complement of fucose. The low-fucosylation antibody, 2H7v.31, is more efficient in mediating the destruction of the target cell. This antibody is effective at lower concentrations and is capable of mediating the destruction of a higher percentage of target cells at higher concentrations than the other antibodies. The activity of the antibodies is as follows: Lecl3-derivative 2H7 v31 > Read 13 derivative 2H7vl6 > Dpl2 derivative 2H7v31 > Dpl2 derivative 2H7vl6 > o = a RITUXAN®. Alterations of protein and carbohydrate are additional. The comparison of the carbohydrate found in natural IgG from the Lecl3-produced and CHO-produced IgG did not show appreciable differences in the degree of galactosylation, and therefore the results can be attributed only in the presence / absence of fucose. Example 12 Cloning of CD20 from Mono cinomolgo and Antibody binding The CD20 DNA sequence for cynomolgus monkey (Macaca fascicularis) was determined by isolating the cDNA encoding CD20 from a cinomolgus spleen cDNA library. A SUPERSCRIPT ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat # 18248-013, Invitrogen, Carlsbad, CA) was used with slight modifications to build the library. The cDNA library was ligated into a pRK5E vector using Xhol and Notl restriction sites. The mRNA was isolated from spleen tissue (California Regional Research Primate Center, Davis, Calif.) Initiators were designated to amplify the cDNA encoding CD20 based on human CD20 non-coding sequences. N 5 '-AGTTTTGAGAGCAAAATG-3' (SEQ ID NO: 41) and C 5 'termination region initiator -AAGCTATGAACACTAATG-3' (SEQ ID NO: 42) to clone by polymerase chain reaction (PCR) the coding cDNA for CD20 of cynomolgus monkey The PCR reaction was carried out using the PLATINUM TAQ DNA POLIMERASE HIGH FIDELITY ™ system according to the manufacturer's recommendation (Gibco, Rockville, MD) The PCR product was subcloned into the PCR®2.1- vector TOPO® (Invitrogen) and transformed into XL-1 blue E. coli (Stratagene, La Jolla, CA) The DNA plasmid containing PCR-linked products was isolated from individual clones and sequenced. CD20 of cynomolgus monkey mu in Figure 19. Figure 20 shows a comparison of cynomolgus and human CD20. The CD20 of cynomolgus monkey is 97.3% similar to human CD20 with 8 differences. The extracellular domain contains a change in V157A, while the remaining 7 residues can be found in the cytoplasmic or transmembrane regions. Antibodies directed against human CD20 were analyzed for the ability to bind and displace the binding of murine FITC-conjugated 2H7 to cynomolgus monkey cells expressing CD20. Twenty milliliters of blood were extracted from 2 cynomolgus monkeys (California Regional Research Primate Center, Davis, CA) in sodium heparin and sent directly to Genentech, Inc. On the same day, the blood samples were deposited and did 1: 1. by adding 40 ml of PBS. 20 ml of did blood was placed in 4 x 20 ml of FICOLL-PAQUE ™ PLUS (Amersham Biosciences, üppsala, Sweden) in 50 ml conical tubes (Cat # 352098, Falcon, Franklin Lakes, NJ) and centrifuged at 1300 rpm for 30 minutes at room temperature in a SORVAL ™ 7 centrifuge (Dupont, Newtown, CT). The PBMC layer was isolated and washed in PBS. The red blood cells were used in a 0.2% soon of NaCl, restored to isotonicity with an equivalent volume of a 1.6% soon of NaCl, and centrifuged for 10 minutes at 1000 RPM. The PBMC pill was resuspended in RPMI 1640 (Gibco, Rockville, MD) containing 5% FBS and dispensed in a 10-cm tissue culture dish for 1 hour at 37 ° C. the non-adherent populations of cell B and T were removed by aspiration, they were centrifuged, and counted. A total of 2.4 x 107 cells was recovered. The resuspended PBMC was distributed in twenty 12 x 75-mm culture tubes (Cat # 352053, Falcon), with each tube containing 1 x 10 6 cells in a volume of 0.25 ml. The tubes were divided into four sets of five tubes. To each set was added either medium (RPMI16 0.5% FBS), titrated amounts of control human IgGx antibody, RITUXAN®, 2H7.vl6, or 2H7.v31.

The final concentration of each antibody was 30, 10, 3.3 and 1. 1 nM. Additionally, each tube also received 20 μl of human anti-CD20 conjugated with fluorescence isothiocyanate (FITC) (Cat # 555622, BD Biosciences, San Diego, CA). The cells were mixed gently, incubated for 1 hour on ice, and then washed twice in cold PBS. The cell surface coloration was analyzed on an EPIC XL-MCL ™ flow cytometer (Coulter, Miami, FL), and the geometric medium was derived and illustrated (KALEIDAGRAPH ™, Synergy Software, Reading, PA) versus the concentration of antibodies The data showed that 2H7 v.16 and 2H7 v.31 competitively displaced the binding of murine FITC-2H7 to cynomolgus monkey cells. In addition, RITUXAN® also displaced the binding of murine FITC-2H7, thus demonstrating that both 2H7 and RITUXAN® bind to an epitope superimposed on CD20. Additionally, the data shows that IC50 values for 2H7 v.16, 2H7 v.31 and RITUXAN® are similar and fall in the 4-6 nM range. Example 13 Phase I / II study of rhuMAb 2H7 (2H7.vl6) in moderate-to-severe rheumatoid arthritis Protocol overview A randomized, placebo-controlled, multicenter, blinded phase I / II study of the safety of staged doses of PRO70769 (rhuMAb 2H7) in subjects with moderate to severe rheumatoid arthritis who receive stable doses of concomitant methotrexate (MTX). Objectives The main objective of this study is to evaluate the safety and tolerance of stepped intravenous (IV) doses of PRO70769 (rhuMAb 2H7) in subjects with moderate to severe rheumatoid arthritis (RA). Study Design This is a randomized, placebo-controlled, multicenter, blinded phase I / II, investigator-and-subject-blinded trial of step-dose safety of PRO70769 in combination with MTX in subjects with moderate to severe RA. The study consists of a dose escalation phase and a second phase combining a greater number of subjects. The sponsor will remain without involvement in the treatment assignment. Will subjects be recruited with R? moderate to severe that have failed one to five medications or biological disease-modifying antirheumatic agents that currently have unsatisfactory clinical responses to treatment with MTX. Subjects will be required to receive MTX in the range of 10-25 mg weekly for at least 12 weeks prior to the start of the study to be in a stable dose for at least 4 weeks before receiving their initial dose of study medication (PRO70769 or placebo). Subjects can also receive stable doses of oral corticosteroids (up to 10 mg daily or equivalent prednisone) and stable doses of non-spheroidal anti-inflammatory drugs (NSAIDs). Subjects will receive two IV infusions of PRO70769 or placebo equivalent to the dose indicated on days 1 and 15 according to the following dose escalation plan. Dose escalation will occur according to specific criteria and then the safety of the data is reviewed through internal data security, committee review and acute toxicity establishment, 72 hours after the second infusion in the last subject treated in each cohort. After the dose escalation phase, 40 additional subjects (32 active and 8 placebos) will be randomized to each of the following dose levels: 2x50 mg, 2x200 mg, 2x500 mg, and 2x1000 mg, if dose levels have shown to be tolerable during the dose escalation phase, approximately 205 subjects will be recruited into the study. B cell counts will be obtained and recorded.

B cell counts will be assessed using flow cytometry in a period of 48 weeks beyond the 6-month efficacy evaluation. The suppression of B cells will not be considered a dose-limiting toxicity (DLC), but the expected pharmacodynamic result of the PRO70769 treatment.

In an optional sub-study, RNA analysis of blood and serum will be obtained, as well as urine samples, from subjects at various time points. These samples can be used to identify biomarkers that can be predictive of the response to the PRO70769 treatment in subjects with R? moderate to severe Outcome Measurements The measurement of the primary outcome for this study is the safety and tolerance of PRO70769 in subjects with moderate to severe RA. Study Treatment Cohorts of subjects will receive two IV infusions of PRO70769 or placebo equivalent to the indicated dose in the days 1 and 15 according to the following escalation plan: 10 mg PRO70769 or equivalent placebo: 4 subjects with active drug, 1 control 50 mg PRO70769 or equivalent placebo: 8 subjects with active drug, 2 control 200 mg PRO70769 or equivalent placebo: 8 subjects with active drug, 2 control 500 mg PRO70769 or equivalent placebo: 8 subjects with active drug, 2 control 1000 mg PRO70769 or equivalent placebo: 8 subjects with active drug, 2 control Efficacy The efficacy of PRO70769 will be measured by ACR responses. The percentage of subjects who achieved a response to ACR20, ACR50, and ACR70 will be summarized by treatment group and 95% confidence intervals for group layer will be generated. The components of these responses and their changes from the baseline will be summarized by treatment and visit. Conclusion of Examples 1-13 The above data demonstrated the success in the production of humanized CD20 binding antibodies, in particular, humanized 2H7 antibody variants, which maintained and even improved their biological properties. The humanized 2H7 antibody of the invention bound CD20 to affinities similar to the murine donor and chimeric 2H7 antibodies and was effective in the destruction of B cells in a primate, leading to the decrease of B cells. Certain variants showed ADCC increase on a chimeric anti-CD20 antibody currently used to treat non-Hodgkin's lymphoma (NHL), favoring the use of lower doses of therapeutic antibody in patients. Additionally, while it may be necessary for a chimeric antibody having murine FR residues to be administered in an effective dose to achieve complete suppression of B cells to obviate the antibody response against it, the present humanized antibodies can be administered in doses that they achieve complete or partial suppression of B cells, and for different durations of time, as described for the particular disease and patient. Additionally, these antibodies demonstrated stability in solution. These properties of humanized 2H7 antibodies make them ideal for use as immunotherapeutic agents in the treatment of autoimmune CD20-positive diseases; it is not expected that these antibodies are immunogenic or that they will be at least immunogenic than the fully murine ones or chimeric anti-CD20 antibodies in human patients. EXAMPLE 14 Preparation of Additional Humanized Antibodies The 2H7.v31 antibody comprising the light and heavy chain sequences of the amino acids of SEQ ID NOS: 24 and 28, respectively, may further comprise at least one amino acid substitution in the Fe region. improves ADCC and / or CDC activity, such as one wherein the amino acid substitutions are S298A / E333A / K334A, more preferably 2H7.v31 having the heavy chain amino acid sequence of SEQ ID NO: 28. The antibody may be 2H7.vl38 comprising the light and heavy chain amino acid sequences of SEQ ID NOS: 29 and 30, respectively, as shown in Figures 10 and 11, respectively, which are alignments of such sequences with the sequences of amino acids of light and heavy chain corresponding to 2H7.vl6. Alternatively, Such a preferred humanized 2H7 intact antibody is 2H7.v477, which has the light and heavy chain sequences of 2H7.vl38 except for the amino acid substitution of N434W. Any of these antibodies may further comprise at least one amino acid substitution in the Fe region that decreases CDC activity, for example, comprising at least the K322A substitution. See Patent of E.U. No. 6,528,624B1 (Idusogie et al.). Some preferred humanized 2H7 variants are those having the light chain variable domain of SEQ ID NO: 2 and the heavy chain variable domain of SEQ ID NO: 8, ie, those with or without substitutions in the Fe region, and those that have a heavy chain variable domain with alteration N100A or D56A and N100A in SEQ ID NO: 8 and a light chain variable domain with M32L alteration, or S92A , or M32L and S92A in SEQ ID NO: 2, ie, those with or without substitutions in the Fe region. If the substitutions are made in the Fe region, they are preferably one of those described in the table below. In a summary of several preferred embodiments of the invention, the V region of the variants based on version 16 of 2H7 will have the amino acid sequences of vl6 except at the positions of the amino acid substitutions indicated in the table below. Unless indicated otherwise, the 2H7 variants will have the same L chain as the vl6 one.

In addition to the above variants, the humanized 2H7 intact antibody may be version 138, which comprises the amino acid sequence of light chain: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVADNLQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29) and amino acid sequence of heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQ KFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSASYWYFDVWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRWSV LTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK (SEQ ID NO: 30). In another embodiment, the humanized 2H7 antibody can comprise the light chain variable region (VL) sequence of SEQ ID NO: 43 and the heavy chain variable region (VH) sequence of SEQ ID NO: 8, wherein the antibody further contains an amino acid substitution of D56A in VH-CDR2, and NlOO in VH-CDR3 is substituted with Y or W, wherein SEQ ID NO: 43 has the sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 43). In one embodiment of this latter humanized 2H7 antibody, NlOO is replaced by Y. In another embodiment, NlOO is replaced by W. In addition, in a further embodiment, the antibody comprises the SlOOaR substitution in VH-CDR3, preferably also comprising at least an amino acid substitution in the Fe region that improves ADCC and / or CDC activity, such as one comprising a Fe IgGl comprising the amino acid substitutions S298A, E333A, K334A, K326A. Alternatively, the antibody comprises the SlOOaR substitution in VH-CDR3, which preferably further comprises at least one amino acid substitution in the Fe region that enhances ADCC but decreases CDC activity, such as one comprising at least the K322A amino acid substitution, as well as one further comprising the amino acid substitutions S298A, E333A, K334A. In an especially preferred embodiment, the antibody is version 511 and comprises the 2H7.v511 light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVADNLQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44) and 2H7.v511 sequence heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSY NQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARWYYSYRYWYFDVWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 45). Example 15 Clinical study of Rituximab in polychondritis Patients diagnosed with polychondritis are treated with RITUXAN® antibody. The treated patient will not have B cell disease. RITUXAN® is administered intravenously (IV) to the patient according to any of the following dosing schedules: (A) 50mg / m2 IV day 1 150 mg / m2 IV in days 8, 15 & 22 (B) 150mg / m2 IV day 1 375 mg / m2 IV on days 8, 15 & 22 (C) 375 mg / m2 IV days 1, 8, 15 & Additional adjunct therapies (such as immunosuppressive agents as noted above) can be combined with RITUXAN® therapy, but preferably the patient is treated with RITUXAN® as the sole agent during the therapy course.

The proportion of total response is determined based on a reduction in inflammation of cartilaginous tissues as determined by standard chemical parameters. The administration of RITUXAN® will improve any or more of the symptoms of polychondritis in the patient treated as described above. Example 16 Clinical Study of Rituximab in Multiple Mononeuritis Patients with clinical diagnosis of mononeuritis multiplex as defined herein are treated with antibody rituximab (RITUXAN®), optionally in combination with steroid therapy. The treated patient will not have a B-cell disease. A detailed and complete medical history is vitally important in determining the possible underlying cause of the disorder. Pain often begins in the lower back or hip and extends to the thigh and knee on one side. The pain is usually characterized as deep and painful with superimposed stinging stitches that are more severe at night. Individuals with diabetes typically present with acute setting of unilateral severe hip pain followed quickly by weakness and atrophy of the anterior muscles of the hip and loss of knee reflex. Other possible symptoms that may be reported by the patient include: numbness, tingling, abnormal sensation, burning pain - dysesthesia, difficulty moving a part of the body - paralysis, lack of controlled movement of a part of the body. Loss of sensation and movement may be associated with specific nerve dysfunction. The examination reveals the preservation of reflexes and good resistance except in the most deeply affected regions. Some common discoveries of mononeuritis multiplex may include the following (not listed in order of frequency): siutic nerve dysfunction, femoral nerve dysfunction, common peroneal nerve dysfunction, auxiliary nerve dysfunction, radial nerve dysfunction, middle nerve dysfunction, ulnar nerve dysfunction, and autonomic dysfunction, ie, the part of the nervous system that controls involuntary bodily functions, such as the glands and the heart. A positive response to therapy is projected as an improvement in two of the four parameters listed below to count for this variation and also based on previous treatment studies in diabetic neuropathy (Jaradeh et al., Journal of Neurology, Neurosurgery and Psychiatry 67: 607-612 (1999)). Patients must have a measurable neuropathy as defined by the electrophysiological test. Patients with known diabetic or hereditary neuropathy are excluded. Patients must have adequate organ function measured by the following criteria (values must be obtained within 2 months prior to registration): Hepatic: AST < 3 x upper limit of normal lab and bilirubin < 2.0mg / dl. Kidney: Creatinine < 3. Omg / dl. Rituximab will be administered intravenously in an outpatient setting. An online filter is not required. The initial proportion is 50mg / hr for the first hours. If no toxicity is observed, the proportion can be scaled up gradually in increments of 50mg / hr at 30-minute intervals up to a maximum of 400mg / hr. If the first dose is well tolerated, the initial proportion for the subsequent dose is 100 mg / hr, incremented gradually in increments of 10-Omg / hr at 30-minute intervals, without exceeding 400 mg / hr. If the patient experiences fever and rigors, the antibody infusion is discontinued. The severity of side effects should be evaluated. If the symptoms improve, the infusion continues initially to the miyad of the previous proportion. After the infusion of antibody, the intravenous line should be maintained for medications as needed. If there are no complications after one hour of observation, the intravenous line may be discontinued. All patients registered in this study will receive weekly rituximab for 4 consecutive weeks. The dose is based on a real surface area.

The regimen of administration is rituximab: 375 mg / m2 weekly x 4 by IV infusion on day 1, 8, 15 and 22. All patients should be premedicated with 650 mg of TYLENOL® to relieve pain and 50 mg of allergic medication BENADRYL® given IV or PO to reduce adverse events 30-60 minutes before treatment. Medications for the treatment of hypersensitivity reactions, e.g. Epinephrine, antihistamines and corticosteroids should be available for immediate use in the case of a reaction during administration. Additionally, an anti-pain agent such as acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenylthin (DILANTIN®), gabapentin (NEURONTIN®), (E) -N-Vanityl-8-methyl-6-noneamide (CAPSAICIN®), or a nerve blocker can be used in conjunction with rituximab. Neuropathy will be evaluated by several different parameters: 1) EMG / NCS 2) Quantitative sensory test 3) Marking of neuropathic damage 4) Neuropathic symptoms and Change questionnaire. EMG / NCS: measurements of electromyography and nerve conduction velocity will be made at three, six and twelve months post-infusion of rituximab by the same electromyographer and technician. The summary of the data of each study will be used for comparison with the initial values including average potential of nervous sensory action (sural, mean and ulnar), average potential of nervous motor action (peroneal in tibial, tibial, ulnar and anterior media), and average driving speed of motor nerves. Average F-wave latencies and proximal-distal motor amplitude proportions will also be calculated. An objective response would require an improvement of > 10% from the baseline. Stable disease would indicate no significant change in neuropathy (+/-> 10). Progressive disease would indicate worsening neuropathy (> 10% from baseline). Quantitative sensory test: Quantitative sensory test with vibration detection threshold (VDT), cooling detection threshold (CDT), and heat pain threshold (HPT) on the back of the foot and hand in addition to the sudomotor axon reflex test (QSART) of the foot and distal hand, will be carried out at three, six and twelve months post-infusion of rituximab by the same technician. The abnormalities in these tests can be transformed into points based on the percentage mark in relation to the standard deviation. A change in two percentages of the pre-study measurements will be considered significant. Marking of neuropathic damage (NIS): This test measures reflexes, sensation and muscular strength. A functional determination of the lower limbs is performed walking in tips, hips and rising from a kneeling position. A marking will be performed at three, six and twelve months post-infusion of rituximab by the same neurologist during the study. Improvement will be defined as a decrease in NIS by 5 points or more (Dyck "Quantitative Severity of Neuropathy" In: Dyck et al., Eds. Peripheral Neuropathy, Philadelphia: WB Saunders, 686-697 (1993)). Neuropathic Symptoms and Change Questionnaire (NSC): This questionnaire consists of 38 concepts answered as true or false. This evaluates the presence or absence of neuropathic symptoms, their severity and change over time. It will be carried out by the same neurologist for each patient during the study. A change of 10% from the marking of the baseline will be considered significant. The primary measurement of study results is the patient's improvement. A patient is classified with improvement if he shows a significant improvement in 2 of the 4 parameters listed above, while not declining in any of the other measurements. Based on this response classification, accurate confidence intervals of 95% are computed for the response proportions based on a binomial calculation. With 14 patients the amplitude of this interval will be less than approximately 50% if the actual response rate is between 30-70%, approximately 40% if the ratio is between 70-90% or 10-30%, and approximately 30%. % if the proportion is >90% or < 10% Point estimates and 95% confidence intervals will be computed by the proportion of patients with a successful outcome in each parameter using exact binomial intervals. For each continuous or orifice measurement, accurate 95% confidence intervals will be computed for the change from the baseline using the Hodges-Lehmann statistic and the Tukey interval (See Hollender and Wolfe Nonparametric Statistical Methods 2nd Edition, Wiley, New York, 1999 p51-56). The calculations will be made using the statistical software equipment STATEXACT ™ (Cytel). The neuropathic damage marking test will provide a single mark of neuropathic deficits and subset markings related to cranial nerve function, muscle weakness, reflexes, and sensation. The deficits will be marked by the examiner when compared with the age and gender of the patients, considering the stature, weight and physical structure. Muscle weakness will be marked as 0 if it is normal, 1 if it is 25% weak, 2 if it is 50% weak, 3 if it is 75% weak, 3.25 if the muscle moves against gravity, 3.5 if there is movement by eliminating the severity , 3.75 when there is a spark without movement, and 4 if there is a total paralysis. This will apply to cranial nerves III, VI, VII, X and XII. The individual muscle groups tested for strength include respiratory, neck flexion, shoulder abduction, elbow flexion, brachial radius, elbow extension, flexion and extension waist, flexion and extension of fingers. Abduction of the thumb, hip flexion and extension, knee flexion and extension, ankle dorsiflexion, plantar flexion of the ankle, extensor and heel flexors for a total of 24 concepts. Each group will be tested on the right and left side. The reflections will be marked as 0 = normal, l = decreased, 2 = absent. Fiber tendon reflexes will be examined on each side including biceps, triceps, brachial radial, quadriceps, and triceps sulars. For patients 60 years of age or older, the decrease in ankle reflexes will graduate as 0 and their absence will graduate as 1. The sensory examination will be performed on the back of the finger and the tip. The touch pressure will be measured using a long cotton blanket. The needle puncture will be established with the use of straight needles. The sensation of vibration is tested with a 165 Hz tone fork, and the joint position will be tested by moving the terminal phalanx of the index finger and the middle finger. The examination will be carried out on each extremity and the marking will be 0 = normal, l = decreased and 2 = absent. It is expected that humanized rituximab or 2H7 will exhibit an improvement in the patient as defined above on a control (without such an antibody), and will therefore treat mononeuritis multiplex.

Claims (1)

CLAIMS 1. A method for treating polychondritis or mononeuritis multiplex in a mammal comprising administering to the mammal an effective amount of an antibody that binds CD20. 2. The method of claim 1 wherein the antibody is not conjugated with another molecule. 3. The method of claim 1 wherein the antibody is conjugated with another molecule. 4. The method of claim 3 wherein the other molecule is a cytotoxic agent. 5. The method of claim 4 wherein the cytotoxic agent is a radioactive compound. 6. The method of claim 4 or 5 wherein the cytotoxic agent comprises Y2B8 or 131I-B1. The method of any of claims 1-6 wherein the antibody comprises rituximab. 8. The method of any of claims 1-6 wherein the antibody comprises a humanized 2H7. 9. The method of any of the claims

1-8 comprising administering a dose of about 20 mg / m2 to about 250 mg / m2 of the antibody to the mammal. The method of claim 9 wherein the dose is from about 50 mg / m2 to about 200 mg / m2. 11. The method of any of claims 1-10 comprising administering an initial dose of the antibody followed by a subsequent dose, wherein the dose of mg / m2 of the antibody in the subsequent dose exceeds the mg / m2 dose of the antibody at the initial dose . 12. The method of any of claims 1-11 wherein the mammal is a human. The method of any of claims 1-12 wherein the antibody is administered intravenously. The method of any of claims 1-12 wherein the antibody is administered subcutaneously. 15. The method of any of claims 1-14 further comprising administering to the mammal an effective amount of an immunosuppressive agent, an anti-pain agent, or a chemotherapeutic agent. 16. The method of any of claims 1-15 wherein the polychondritis is treated. The method of claim 16 further comprising administering to the mammal an effective amount of a non-spheroidal anti-inflammatory drug, steroid, methotrexate, cyclophosphamide, dapsone, azathioprine, penicillamine, or cyclosporin. 18. The method of any of claims 1-15 wherein mononeuritis multiplex is treated. 19. The method of claim 18 further comprising administering to the mammal an effective amount of an anti-pain agent, steroid, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulin, cyclosporin, or mycophenolate mofetil. 20. A manufacturing article comprising a container and a composition contained therein, wherein the composition comprises an antibody that binds to CD20, and further comprises a package insert that instructs the user of the composition to treat polychondritis or multiple mononeuritis in a mammal. 21. The article of claim 20 further comprising a container comprising an agent other than the antibody for treatment and further comprising instructions for treating the mammal with such an agent.