MX2007003857A - Method for treating vasculitis - Google Patents

Abstract

A method of treating anti-neutrophil cytoplasmic antibodies-associated vasculitis (ANCA-associated vasculitis) in a patient eligible for treatment is provided involving administering an antagonist that binds to a B-cell surface marker, such as CD20 antibody, to the patient in a dose of about 400 mg to 1.3 grams at a frequency of one to three doses within a period of about one month. Another method of treating ANCA-associated vasculitis in a subject eligible for treatment is provided involving administering an effective amount of an antibody that binds to a B-cell surface marker to the subject to provide an initial exposure and a subsequent exposure to the antibody within certain dosing regimens. Further provided are articles of manufacture useful for such methods.

Description

METHOD OF TREATING VASCULITIS Related Requests This application is a non-provisional application filed under 37 CFR 1.53 (b) (1), which claims priority under 35 USC 119 (e) of the US Provisional Patent application. Serial Number 60 \ 616,104 filed on October 5, 2004, the contents of which are hereby incorporated by reference. Field of the Invention The present invention relates to methods for treating vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA · = Anti-Neutrophil Cytoplasmic Antibodies) in a subject and equipment with instructions for such uses. BACKGROUND OF INVENTION Vasculitis Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, vasculitis and lupus, among others, remain clinically important diseases in humans. As the name implies, auto immune diseases wreak havoc through the body's own immune system. While the pathological mechanisms differ between individual types of autoimmune diseases, a general mechanism involves the binding of certain antibodies (here referred to as auto-reactive antibodies or auto-antibodies) present. Vasculitis is defined by inflammation of the blood vessel wall and forms the pathological basis of a diverse group of entities of individual diseases. Vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA), which is a common primary systemic vasculitis, includes microscopic polyangiitis, egener's granulomatosis, Churg-Strauss syndrome, renal limit vasculitis (idiopathic crescentic necrotizing glomerulonephritis) (Falk et al. Engl. J. Med., 318: 1651-1657 (1988)), and certain types of drug-induced vasculitis, Jennette et al. Arthritis Rheum 37: 187-92 (1994); Jennette and Falk, N. Engl. J. Med. 337: 1512-1523 (1997). "The aforementioned diseases affect people of all ages but are more common in older adults in their 50s and 60s and can affect men and women equally." Pettersson et al, Clin. Nephrol., 43: 141-149 (1995), Falk et al., Ann.Inter.Med. 113: 656-663 (1990). ANCA are antigen-specific antibodies in cytoplasmic granules of neutrophils and monolith lysosomes, first reported in 1982, Niles et al., Arch. Intern. Med., 156: 440-445 (1996) ANCA was originally detected by indirect immunofluorescence in ethanol-fixed neutrophils Wiik, "Delineation of a standard procedure for indirect immunofluorescence detection of ANCA" APMIS Suppl 6: 12 -13 (1989) At least three different patterns of fluorescence have been distinguished: a cytoplasmic / classical pattern (cANCA) with attenuation of fluorescence intensity in the area with the nuclear lobes, a perinuclear pattern (pANCA) and a pattern of cytoplasmic staining more diffuse mica (ANCA atypical). Approximately 90% of the serum that produces a cANCA pattern reacts with proteinase 3 (PR3), a serine protease. of the azurophilic granules of myeloid cells. Jennette and Falk, N Engl. J. Med., Supra. In patients with primary systemic vasculitis that predominantly affects medium and small blood vessels, approximately 75% of the serum that produces the perinuclear pattern (pANCA) reacts with myeloperoxidase (MPO), a myeloid lysosomal enzyme. Cohen Tervaert et al., Am. J. Med. 91: 59-66 (1991). In ANCA-positive patients with other non-vasculitic diseases, other antigenic specificities are recognized. The diagnostic potential of PR3-ANCA and MPO-ANCA is now quite well established. In a patient with signs and symptoms of vasculitis, ANCA with specificity for PR3 (PR3-ANCA) suggests a diagnosis of Wegener's granulomatosis, while ANCA with a specificity for MPO (MPO-ANCA) is highly sensitive for microscopic polyangiitis, crescentic glomerulonephritis idiopathic necrotizing or active Churg-S'trauss syndrome. Cohen Tervaert et al., Sarcoidosis Vasc. Diffuse Lung Dis. 13: 241-245 (1996). See also Xiao et al., J. Clin. Invest., 110: 955-963 (2002), which describes an animal model that offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis, and Popa et al., J. Allergy Clin. Immunol., 103: 885-894 (1999) shows that Wegener's granulomatosis, B cell activation is related to active disease, while T cell activation persists during remission of the disease, which signals a disordered immune system intrinsic in this disease. See also Cupps et al., J. Immunol., 128: 2453-2457 (1982) regarding the role of cyclophosphamide in suppressing the function of human B lymphocytes. Within the spectrum of primary vasculitic syndromes, the syndromes related to ANCA form a distinct group with superimposed characteristics. Most patients have a prodromal flu-like onset consisting of malaise, myalgia, arthralgia, fever and weight loss. This influenza-like onset appears within days or weeks before the onset of manifest nephritic or vasculitic disease. Wegener's granulomatosis differs from the others by the presence of granulomatous inflammation of the upper and lower respiratory tract that is usually accompanied by vasculitis and glomerulonephritis of small necrotizing systemic vessels. The Churg-Strauss syndrome is differentiated by the presence of a (history of) asthma, allergic rhinitis, systemic eosinophilia, as well as systemic vasculitis with or without glomerulonephritis. Microscopic polyangiitis is characterized by necrotizing and / or crescentic glomerulonephritis and a vasculitis of multiple systems involving small vessels. Microscopic polyangiitis shares many characteristics with Wegener's granulomatosis and Churg-Strauss syndrome, but lacks necrotizing granulomatous inflammation of the respiratory tract and asthma. Jennette et al., Arthritis Rheum. , supra. In crescentic glomerulonephritis and / or idiopathic necrotizing, the vasculitic process is limited to the kidneys. Due to the treatment of patients with microscopic polyangiitis or Wegener's granulomatosis, it is essentially the same when there is major organ injury, it is unnecessary to distinguish conclusively between these closely related variants of ANCA-associated vasculitis before starting treatment. Jennette et al. Arthritis Rheum., Supra.

Before the treatment became available, patients with generalized Wegener's granulomatosis had a median survival of five months. In the early 1970s, Fauci and Olff introduced a regimen that combined daily cyclophosphamide therapy delivered for one year after remission was achieved with prednisone therapy initiated at a dose of 1 mg per kilogram of body weight per day and reduced in an alternate day program. This treatment has been found in reproducible form that induces remission in 80 to 100% of patients and may result in long-term survival. In fact, prolonged immunosuppressive therapy (longer than one year) with cyclophosphamide and steroids is effective in inducing remission of the disease and avoiding early relapses in most vasculitic disorders. Balow et al., "Vasculitic diseases of the kidney, polyarteritis, Wegener's granulomatosis, necrotizing and crescentic glomerulonephritis, and other disorders". In: Schrier and Gottschalk (eds): Diseases of the kidney, 5th edition, (Little, Brown and Company, Boston, 1993), pp. 2095-2117; Jayne et al., N. Engl. J. Med., 349: 36-44 (2003); Gaskin et al, "Systemic vasculitis". In: Cameon et al. (eds): Oxford textbook of clinical nephrology. (Oxford University Press, Oxford, 1992), pp. 612-636; Fauci et al, Ann. Intern. Med. 89: 660-676 (1978); Fauci et al., Ann. Intern. Med., 98: 76-85 (1983); Hoffman et al. , Ann. Int. Med., 116: 488-498 (1992); and Andrassy et al., Clin. Nephrol., 35: 139-147 (1991). However, when therapy is reduced and interrupted, relapses are common. In a study in which patients with Wegener's granulomatosis were followed up for an average of eight years, relapse occurred in 50% of the patients. Furthermore, continuous use of cyclophosphamide to sustain remission is not recommended, since this treatment regimen is associated with severe and potentially lethal adverse effects such as the occurrence of opportunistic infections and the development of malignancies. For example, repeated uses of cyclophosphamide are associated with bone marrow suppression, infection, cystitis, infertility, myelodysplasia, and transitional cell carcinoma of the bladder. In some instances, these toxic effects prevent greater use of cyclophosphamide. Stillwell et al., Arthritis Rheum., 31: 465-470 (1988); Radis et al., Arthritis Rheum. 38: 1120-1127 (1995). Therefore, cyclophosphamide is reduced or stopped and replaced by azathioprine once remission is achieved to avoid adverse effects, a proven policy in a rigorous multi-center trial that proved to be equally effective at the 18-month follow-up. Gaskin et al, supra, 1992; Jayne, Rheumatology 39: 585-595 (2000). Azathioprine is considered less effective at inducing remission than cyclophosphamide, but its long-term toxicity is much less. Bouroncle et al., Am. J. Med., 42: 314-318 (1967); Norton et al., Arch. Interin. Med., 121: 554-560 (1968). Other alternative maintenance treatment regimens include methotrexate ((de Groot et al, Arthritis Rheum., 39: 2052-2061 (1996)), cyclosporin A (Haubitz et al., Nephrol. Dial. Transplant, 13: 2074-2076 ( 1998)), mycophenolate (No ack et al., J. Am. Soc. Nephrol., 10: 1965-1971 (1999)), or trimethoprim-sulfamethoxazole (Stegeman et al., N. Engl. J. Med., .335: 16-20 (1996).) See also Sanders, et al., N. Engl. J. Med. 349: 2072-2073 (2003), since, however, relapses are frequently observed in vasculitis associated with ANCA. , treatment in these cases has to be intensified or restored Hoffman et al, supra, Gordon et al, Q J. Med., 86: 779-789 (1993), Nachman et al., J. Am. Soc. Nephrol., 7: 33-39 (1996), Guillevin et al., Medicine 78: 26-37 (1999), Reinhold-Keller et al., Arthritis Rheum 43: 1021-1032 (2000), Langford, New Eng. Med., 349: 3-4 (July 2003) Blocking tumor necrosis factor alpha (TNF-alpha) with infliximab is a potential therapy for vasculitis associated with ANCA, both for the initial therapy and in the administration of refractory disease. Infliximab was effective to induce remission in 88% of patients with vasculitis associated with ANCA and allowed reduction in steroid doses. Booth et al., J. Am. Soc. Nephrol. 15: 717-721 (2004). In addition, Stone et al., Arthritis and Rheumatism, 44: 1149-1154 (2001) describes that the TNF-alpha inhibitor etanercept (ENBREL®), given 25 mg subcutaneously twice a week in combination with standard treatment for granulomatosis of Wegener was well tolerated in patients with few adverse events, but an intermittently active (occasionally severe) disease was common. Patients with Churg-Strauss syndrome usually respond to high-dose corticosteroid therapy alone, although some cases may require the addition of cytotoxic drugs. Jayne and Rasmussen, Mayo Clin. Proc. 72: 737-47 (1997). Co-morbid conditions that accelerate vascular damage, for example hypertension, diabetes, hypercholesterolemia and smoking, should be appropriately controlled. In drug-induced vasculitis, the offending agent should be stopped. Antihistamines and non-spheroidal anti-inflammatory drugs help to alleviate discomfort in the skin and reduce associated allergies and myalgias. Severe cutaneous disease can guarantee oral corticosteroid therapy. Jennette et al., Arthritis Rheum, supra. The persistence or reappearance of ANCA is a risk factor for the development of a relapse of disease activity, suggesting a physiological role in vivo for these autoantibodies. Stegeman et al., Ann. Tntern. Med., 120: 12-17 (1994); De'Oliviera et al, Am. J. Kidney Dis., 25: 380-389 (1995); Jayne et al., Q. J. Med., 88: 127-133 (1995). Wegener granulomatosis relapses are often preceded by increases in the title of cANCA as. it is detected by indirect immunofluorescence (Cohen Tervaert et al., Arch. Intern. Med., 149: 2461-2465 (1989)), and can be avoided by treatment with immunosuppressants based on the increase in cANCA (Cohen Tervaert et al., Lancet, 336: 706-711 (1990)). For a general discussion on vasculitis associated with ANCA, see Lhote and Guillevin, Rheum. Dis. Clin. North Am. 21: 911-947 (1995); "ANCA-associated vasculitis: occurrence, prediction, prevention, and outcome of relapses" by Maarten Boomsma, PhD Thesis, Thesis University Groningen, ISBN 90-367-1451-6 (M.M.

Boomsma, Groningen, 2001) Kamesh et al. , J. Am. Soc. Nephrol. 13: 1953-1960 (2002); and Jayne, Kidney & Blood Pressure Research 26: 231-239 (2003). CD20 antibodies and their therapy Lymphocytes are one of the 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 here are B cells. B cells mature within the bone marrow and leave the marrow to express an antigen binding antibody on its cell surface. When an untreated 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 B "plasma cells". Memory B cells have a longer life span and continue to express membrane bound antibody with the same specificity as the original precursor cell. Plasma cells do not produce antibody bound to membrane, but instead produce the antibody in a form that can be secreted. Secreted antibodies are the main effector molecules of humoral immunity. The CD20 antigen (also termed the human B cell-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located in mature pre-B and B lymphocytes. Valentine et al., J. Biol. Chem. 264 (19): 11282-11287 (1989) and Einfeld et al., EMBO J. 7 (3): 711-717 (1988). The antigen is also expressed in more than 90% of non-Hodgkin's B-cell lymphomas (NHL = Non-Hodgkin's Lymphoma) (Anderson et al., Blood 63 (6): 1424-1433 (1984)), but no found in hematopoietic stem cells, pro-beta cells, normal plasma cells or other normal tissues (Tedder et al., J. Immunol., 135 (2): 973-979 (1985)). CD20 regulates the previous stages in the activation process for cell cycle initiation and differentiation (Tedder et al., Supra), and possibly functions as 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 "target" these lymphomas. In essence, this target can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are administered to a patient. These anti-CD20 antibodies bind specifically to the CD20 antigen of (ostensibly) both normal and malignant B cells, the antibody bound to the CD20 surface antigen can lead to the destruction and depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels that have the potential to destroy the tumor can be conjugated to the anti-CD20 antibody so that the agent is "delivered" specifically to the neoplastic B cells. Regardless of the approach, a primary goal is to destroy the tumor; the specific approach can be determined by the particular anti-CD20 antibody that is used and thus the approaches available to target the CD20 antigen can vary considerably. The rituximab antibody (RITUXAN®) is a genetically engineered chimeric human / mouse monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody termed "C2B8" in U.S. Pat. No. 5,736,137 issued April 7, 1998 (Anderson et al.). Rituximab is indicated for the treatment of patients with relapse or low refractory or follicular grade of non-Hodgkin lymphoma of B cell positive to CD20. In vitro mechanism-of-action studies have shown that rituximab binds human complement and smooth lymphoid B-cell lines through complement-dependent cytotoxicity (CDC). Reff et al., Blood 83 (2): 35-445 (1994). Additionally, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC = Antibody-Dependent Cellular Cytotoxicity). More recently, rituximab has been shown to have anti-proliferative effects in tritiated thymidine incorporation assays and to induce apoptosis directly while other anti-CD19 and anti-CD20 antibodies do not. Maloney et al. Blood 88 (10): 637a (1996). The synergy between rituximab and chemotherapies and toxins has also been observed experimentally. In particular, rituximab sensitizes drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria and ricin toxin (Demidem et al., Cancer Chemotherapy &; Radiopharmaceuticals 12 (3): 177-186 (1997)). In vivo preclinical studies have shown that rituximab depletes B cells from the peripheral blood, lymph nodes and bone marrow of macaque monkeys, presumably through complement-mediated and cell-mediated processes. Reff et al., Blood 83: 435-445 (1994). Rituximab was approved in the United States of America in November 1997 for the treatment of patients with follicular or refractory low-grade or relapse B-cell NHL at a dose of 375 mg / m2 weekly for four doses. In April 2001, the Food and Drug Administration (FDA) approved additional applications for the treatment of low-grade NHL: re-treatment (weekly for four doses) and an additional dose regimen (weekly for eight doses) ). There have been more than three hundred thousand patient exposures to Rituximab either as monotherapy or in combination with immunosuppressive or chemotherapeutic drugs. Patients with Rituximab have also been treated as maintenance therapy for up to two years. Hainsworth et. al., J. Clin. Oncol. 21: 1746-1751 (2? 03 '); Hainsworth et al., J. Clin. Oncol. 20: 4261-4267 (2002). Also, Rituximab has been used in the treatment of malignant and non-malignant plasma cell disorders. Treon and Anderson, Semin. Oncol. 27: 79-85 (2000). Rituximab has also been studied in a variety of non-malignant autoimmune disorders, where B cells and autoantibodies seem to play a role in the pathophysiology of the disease. Edwards et al., Biochem Soc. Trans. 30: 824-828 (2002). Rituximab has been reported to potentially alleviate signs and symptoms for example of rheumatoid arthritis (RA = Rheumatoid Arthritis) (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); Shaw et al., Ann. Rheum. Dis. 62 Suppl 2 : ii55-ii59 (2003), Weyand and Goronzy, Ann., NY Acad. Sci. 987: 140-149 (2003), Emery et al., Arthritis Rheum 48 (9): S439 (2003)), lupus (Eisenberg , Arthritis, Res. Ther.5: 157-159 (2003), Anolik et al., Arthritis Rheum 48: 455-459 (2003), Leandro et al., Arthritis Rheum 46: 2673-2677 (2002), Gorman. et al., Lupus, 13: 312-316 (2004), Tomietto et al., Thromb. Raemost., 92: 1150-1153 (2004)), purple immune thrombocyte pneumonia (D 'Arena et al., Leuk. : 561-562 (2003), Stas.i et al., Blood, 98: 952-957 (2001), Saleh et al., Semin. Oncol., 27 (Supp 12): 99-103 (2000); et al., Haematologica, 87: 189-195 (2002); Zaja et al., Haemat Hologram 88: 538-546 (2003); Cooper et al., Br. J. Haematol. 125: 232-239 (2004); Ratanatharathorn et al., Ann. Int. Med., 133: 275-279 (2000)), flattening of pure red blood cells (Auner et al., Br. J. Haematol., 116: 725-728 (2002)); autoimmune anemia (Zaja et al., Haematologica 87: 189-195 (2002) (erratum appears in Haematologica 87: 336 (2002); Raj et al., J. Pediatr Hematol Oncol. 26: 312-314 (2004), Zecca et al., Blood 101: 3857-3861 (2003), Quartier et al., Lancet 358: 1511-1513 (2001)), autoimmune cytopenias (Robak, Eur. J. Haematol. 72: 79 -88 (2004)), cold aglyutilin 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), Lee and Kueck, Blood 92: 3490-3491 (1998)), type B syndrome of severe insulin resistance (Coll et al., N. Engl. J. Med., 350: 310-311 (.2004 ), mixed cryoglobulinemia (DeVita et al., Arthritis Rheum 46 Suppl 9: S206 / S469 (2002), Zaja et al., Haematologica 84: 1157-1158 (1999)), myasthenia gravis (Zaja et al., Neurology, 55: 1062-63 (2000); Wylam et al., J. Pediatr., 143: 674-677 (2003)), granulomatosis of Wegener Specks et al., Arthritis & Rheumatism 44: 2836-2840 (2001)), pemphigus vulgaris refractory (Dupuy et al., Arch Dermatol., 140: 91-96 (2004)), dermatomyositis (Levine, Arthritis Rheum., 46 (Suppl 9): S1299 (2002)), Sjogren's syndrome (Somer et al., Arthritis & amp;; Rheumatism, 49: 394-398 (2003)), active type II mixed cryoglobulinemia (Zaja et al., Blood, 101: 3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch. Dermatol., 140 : 91-95 (2004)), autoimmune neuropathy (Pestronk et al., J. Neurol. Neurosurg, Psychiatry 74: 485-489 (2003); Nobile-Orazio, Curr. Opin. Neurol. 17: 599-605 ( 2004), Rojas-Garcia et al., Neurology 61: 1814-1816 (2003), Renaud et al. Muscle Nerve 27: 611-615 (2003)), paranooplastic obsoclonus-myoclonus syndrome (Pranzatelli et al., Neurology 60 (Suppl. 1) P05.128: A395 (2003)), inhibitors of acquired factor VIII (iestner et al., Blood 100: 3426-3428 (2002); and multiple sclerosis of remission-relapse (RRMS = Relapsing Remitting Multiple Sclerosis) Cross et (abstract) "Preliminary Results from a Phase II Trial of Rituximab in S" Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis, 20-21 (2003) A Phase II Study (WA16291) has been conducted in patients with arthritis Rheumatoid (RA) that provides 48-week follow-up data on safety and efficacy of Rituximab. Emery et al. Arthritis Rheum 48 (9): S439 (2003); Szczepanski et al. Arthritis Rheum 48 (9): S121 (2003). A total of 161 patients were evenly distributed randomly into four treatment branches: methotrexate, rituximab alone, rituximab plus methotrexate and rituximab plus cyclophosphamide (CTX). The treatment regimen rituximab was one gram administered intravenously on days 1 and 15. Infusions of rituximab in the majority of patients with RA 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 receiving placebo). In total, most adverse events were considered mild to moderate in severity and well balanced across all treatment groups. There were a total of 19 serious adverse events across the four branches during the 48 weeks that was slightly more frequent in the rituximab / CTX group. The incidence of infections was well balanced across all groups. The average rate of serious infection in this population of RA patients was copied per 100 patient-years, which is less than the rate of infections requiring hospital admission in RA patients (9.57 per 100 patient-years) reported in epidemiological studies of community base. 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), obsoclonus-myoclonus syndrome (Pranzatelli et al., Supra), and RRMS (Cross et al., supra), was similar to that reported in oncology or RA. In a trial sponsored by a researcher in process (IST = Investigator Sponsored Trial) of rituximab in combination with glatinamer acetate or interferon-beta (IFN-?) In patients with RRMS (Cross et al., Supra), one of ten patients treated He was admitted to the hospital for observation during the night after experiencing moderate fever and chills after the first infusion of rituximab, while the other 9 patients completed the four-infusion regimen without any reported adverse events. Patents and patent publications relating to the CD20 antibody and CD20 binding molecules include U.S. Patents. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US patent application. Number 2002/0197255, 2003/0021781, 2003/0082172, 2003/0095963, 2003/0147885 (Anderson et al.); Patent of the U.S.A. Number 6,455,043 and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); WO 2000/27433 (Grillo-Lopez and Leonard); WO 2000/44788 (Braslawsky et al.); WO 2001/10462 (Rastetter, W.); WO 2001/10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); 2001/0018041, 2003/0180292, 2001/34194 (Hanna and Hariharan); 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); 2002/0012665, WO 2001/74388 and 6,896,885B5 (Hanna, N.); 2002/0058029 (Hanna, N.); 2003/0103971 (Hariharan and Hanna); 2005/0123540 (Hanna et al.); 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858; 2005/0112060, and U.S. Pat. Number 6, 846, 476 (White, C); 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 No. 6,171,586 and WO 1998/56418 (Lam et al.); WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, U.S. Pat. Number 6,194,551, U.S. patent. Number 6,242,195, U.S. patent. Number 6,528,624 and U.S. Patent No. Number 6,538,124 (Idusogie et al.); WO 2000/42072 (Presta, L.); WO 2000/67796 (Curd et al.); WO 2001/03734 (Grillo-Lopez et al.); 2002/0004587 and WO 2001/77342 (Miller and Presta); 2002/0197256 (Grewal, I.); 2003/0157108 (Presta, L.); U.S. Patent Numbers 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721, (Kaminski et al.); U.S. Patent Numbers 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852, 6,893,625 (Robinson et al.); U.S. Patent Number 6,410,391 (Raubitschek et al.); U.S. Patent Number 6,224,866 and WO00 / 20864 (Barbera-Guil lem, E.); WO 2001/13945 (Barbera-Guillem, E.); WO 2000/67795 (Goldenberg); 2003/0133930 and WO 2000/74718 (Goldenberg and Hansen); 2003/0219433 and WO 2003/68821 (Hansen et al.); WO 2004/058298 (Goldenberg and Hansen); WO 2000/76542 (Golay et al.); WO 2001/72333 (Wolin and Rosenblatt); U.S. Patent Number 6,368,596 (Ghetie et al.); U.S. Patent Number 6,306,393 and US 2002/0041847 (Goldenberg, D.); 2003/0026801 (einer and Hartmann); WO 2002/102312 (Engleman, E.); 2003/0068664 (Albitar et al.); WO 2003/002607 (Leung, S.); WO 2003/049694, 2002/0009427, and 2003/0185796 (Wolin et al.); WO 2003/061694 (Sing and Siegall); 2003/0219818 (Bohen et al.); 2003/0219433 and WO 2003/068821 (Hansen et al.); 2003/0219818 (Bohen et al.); 2002/0136719 (Shenoy et al.); WO 2004/032828 and 2005/0180972 (Wahl et al.); and WO 2002/56910 (Hayden-Ledbetter). See also US patent. Number 5,849,898 and EP 330,191 (Seed et al.); EP332,865A2 (Meyer and Weiss); U.S. Patent Number 4,861,579 (Meyer et al.); 2001/0056066 (Bugelski et al.); WO 1995/03770 (Bhat et al.); 2003/0219433 Al (Hansen et al.); WO 2004/035607 (Teeling et al.); WO 2004/056312 (Lowman et al.); 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); 2005/0025764 (Watkins et al.); WO 2005/016969 (Carr et al.); 2005/0069545 (Carr et al.); WO 2005/014618 (Chang et al.); 2005/0079174 (Barbera-Guil lem and Nelson); 2005/0106108 (Leung and Hansen); WO2005 / 044859 and 2005/0123546 (Umana et al.); WO 2005/070963 (Alian et al.); 2005/0186216 (Ledbetter and Hayden-Ledbetter); and US patent. Number 6,897,044 (Braslawski et al.).

Publications concerning treatment with rituximab include: Perotta and Abuel, "Response of chronic relapse ITP of 10 years duration to rituximab" Abstract # 3360 Blood 10 (1) (part 1-2): p. 88B (1998); Perotta et al., "Rituxan in the treatment of chronic idiopathic thrombocytopaenic purpura (ITP)", Blood, 94: 49 (abstract) (1999); Matthews, R., "Medical Heretics" New Scientist (April 7, 2001); Leandro et al., "Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion" Ann Rheum Dis, supra; Leandro et al., "Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficacy and dose response" Arthritis and Rheumatism 44 (9): S370 (2001); Leandro et al., "An open study of B lymphocyte depletion in systemic lupus erythematosus", Arthritis and Rheumatism, 46: 2673-2677 (2002), where a period of two weeks, each patient received two infusions of 500 mg of rituximab , two infusions of 750 mg of cyclophosphamide and oral corticosteroids of high dose and where two of the treated patients had relapse at 7 and 8 months, respectively and have been treated again 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 intervals weekly) and greater applications of rituximab were given every 5 to 6 months and then maintenance therapy was received with rituximab 375 mg / m2 every three months, and a second patient with refractory SLE was successfully treated with rituximab and receives maintenance therapy every three months. months, with both patients responding well to rituximab therapy; Edwards and Cambridge, "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes" Rheumatology 40: 205-211 (2001); Cambridge et al., "B lymphocyte depletion in patients · with rheumatoid arthritis: serial studies of immunological parameters" Arthritis Rheum. , 46 (Suppl 9): S1350 (2002); Cambridge et al., "Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis" Arthritis Rheum., 48: 2146-2154 (2003); Edwards et al., "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders" Biochem Soc. Trans. , supra; Edwards et al., "Efficacy and safety of rituximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis, Arthritis and Rheumatism 46 (9): S197 (2002); Edwards et al. , "Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis" N Engl.

J. Med. 350: 2572-2582 (2004); Pavelka et al., Ann. Rheum. Dis. 63: (Sl): 289-290 (2004); Emery et al. , Arthritis Rheum. 50 (S9): S659 (2004); Levine and Pestronk, "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using rituximab" Neurology 52: 1701-1704 (1999); Uchida et al. , "The innate mononuclear phagocyte network depletes B lymphocytes through Fc receptor-dependent mechanisms during anti-CD20 antibody immunotherapy" J. Exp. Med. 199: 1659-1669 (2004); Gong et al., "Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy" J. Immunol. 174: 817-826 (2005); Hamaguchi et al., "The peritoneal cavity provides a protective niche for Bl and conventional B lymphocytes during anti-CD20 immunotherapy in mice" J. Immunol. 174: 4389-4399 (2005); Cragg et al. "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun. 8: 140-174 (2005); Eisenberg, "Mechanisms of autoimmunity" Immunol. Res. 27: 203-218 (2003); DeVita et al. , "Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis" Arthritis & Rheum 46: 2029-2033 (2002); Hidashida et al. "Treatment of DMARD-refractory rheumatoid arthritis with rituximab". Presented at the annual scientific meeting of the American College of Rheumatology (Annual Scientific Meeting of the American College of Rheumatology); Oct 24-29; New Orleans, LA 2002; Tuscano, J. "Successful treatment of infliximab-refractory rheumatoid arthritis with rituximab" Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002 and published in Tuscano, Arthritis Rheum. 46: 3420 (2002); "Pathogenic roles of B cells in human autoimmunity; insights from the clinic" Martin and Chan, Immunity 20: 517-527 (2004); Silverman and Weisman, "Rituximab therapy and autoimmune disorders, prospects for anti-B cell therapy," Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz and Isenberg, "Anti B cell therapy (rituximab) in the treatment of autoimmune diseases", Current opinion in pharmacology, 4: 398.-402 (2004); Virgolini and Vanda, "Rituximab in autoimmune diseases", Biomedicine & pharmacotherapy, 58: 299-309 (2004); Klemmer et al., "Treatment of antibody mediated autoimmune disorders with a AntiCD20 monoclonal antibody Rituximab", Arthritis And Rheumatism, 48: (9) 9, S (SEP), page: S624-S624 (2003); Kneitz et al., "Effective B cell depletion with rituximab in the treatment of autoimmune diseases", Immunobiology, 206: 519-527 (2002); Arzoo et al., "Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab)" Annals of the Rheumatic Diseases, 61 (10), p 922-924 (2002) Comment in Ann Rheum Dis. 61: 863-866 (2002); "Future strategies in immunotherapy" by Lake and Dionne, in Burger's Medicinal Chemistry and Drug Discovery (2003 by John Wiley &Sons, Inc.) Article Online Posting Date: January 15, 2003 (Chapter 2"Antibody-Directed Immunotherapy"); Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, section: CD20 as an Immunotherapy Target, online article dated January 15, 2002 with the title "CD20"; appendix 4A with the title "Monoclonal Antibodies to Human Cell Surface Antigens" by Stockinger et al., eds: |Coligan et al., in Current Protocols in Immunology (2003 John Wiley &Sons, Inc) Online Posting Date: May, 2003; |Print Publication Date: February, 2003; Penichet and Morri.son, "CD Antibodies / molecules: Definition; Antibody Engineering" in Wiley Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies; posted online January 15, 2002.

In addition, see Looney "B cells as a therapeutic target in autoimmune diseases other than rheumatoid arthritis" Rheumatology, 44 Suppl 2: Ü13-iil7 (2005); Chambers and Isenberg, "Anti-B cell therapy (rituximab) in the treatment of autoimmune diseases" Lupus 14 (3): 210-214 (2005); Looney et al., "B-cell depletion as a novel treatment for systemic lupus erythematosus: a phase I / II dose-escalating trial of rituximab" Arthritis Rheum. 50: 2580-2589 (2004); Looney, "Treating human autoimmune disease by depleting B cells" Ann Rheum. Dis. 61: 863-866 (2002); Edelbauer et al., "Rituximab in childhood systemic lupus erythematosus refractory to conventional immunosuppression Case report" Pediatr. Nephrol. 20 (6): 811-813 (2005); D'Cruz and Hughes, "The treatment of lupus nephritis" BMJ 330 (7488): 377-378 (2005); Looney, "B cell-targeted therapy. In diseases other than rheumatoid arthritis" J. Rheumatol. Suppl. 73: 25-28; discussion 29-30 (2005); Sfikakis et al., "Remission of proliferative lupus nephritis following B cell depletion therapy is preceded by down-regulation of the T cell costimulatory molecule CD40 ligand: an open-label trial" Arthritis Rheum. 52 (2): 501-513 (2005); Rastetter et al., "Rituximab: expanding role in therapy for lymphomas and autoimmune diseases" Annu. Rev. Med. 55: 477-503 (2004); Silverman, "Anti-CD20 therapy in systemic lupus erythematosus: a step closer to the clinic" Arthritis Rheum. 52 (2): 371-7 (2005), Erratum in: Arthritis Rheum. 52 (4): 1342 (2005); Ahn et al., "Long-term remission from life-threatening hypercoagulable state associated with lupus anticoagulant (LA) following rituximab therapy" Am. J. Hematol. 78 (2): 127-129 (2005); Tahir et al., "Humanized anti-CD20 monoclonal antibody in the treatment of severe resistant systemic lupus erythematosus in a patient with antibodies against rituximab" Rheumatology, 44 (4): 561-562 (2005), Epub January 11 2005; Looney et al., "Treatment of SLE with anti-CD20 monoclonal antibody" Curr. Dir. Autoimmun. 8: 193-205 (2005); Cragg et al. , "The biology of CD20 and its potential as a target for mAb therapy" Curr. Dir. Autoimmun. 8: 140-174 (2005); Gottenberg et al., "Tolerance and short term efficacy of rituximab in 43 patients with systemic autoimmune diseases" Ann. Rheum. Dis. 64 (6): 913-920 (2005) Epub 2004 November 18; Tokunaga et al., "Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab" Rheumatology 44 (2): 176-182 (2005), Epub 2004 Oct 19. See also Leandro et al., "B cell repopulation occurs mainly from naive B cells in patient with rheumatoid arthritis and systemic lupus erythematosus" Arthritis Rheum., 48 (Suppl 9): S1160 (2003).

Specks et al. "Response of Wegener 's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism 44 (12): 2836-2840 (2001) describes successful use of four infusions of 375mg / m2 of rituximab and high-dose glucocorticoid to treat Wegener's granulomatosis. The therapy was repeated after 11 months when cANCA recurred, but the therapy was without glucocorticoids. At 8 months after the second course rituximab, the patient's illness remained in complete remission. In addition, in another study, rituximab was found to be an effective remission induction agent, well tolerated for severe vasculitis associated with ANCA when used at a dose of 375 mg / m2 for 4 together with oral predmison at 1 mg / kg / day that is reduced to week 4 to 40 mg / day and to complete interruption during the next 16 weeks. Four patients with rituximab alone were treated again. ascending / recurrent ANCA titles. Apart from glucocorticoids, additional immunosuppressive agents did not appear to be necessary for induction of remission and maintenance of sustained remission (6 months or more). See online abstract presentation and invitation from Keogh et al., "Rituximab for Remission Induction in Severe ANCA-Associated Vasculitis: Report of a Prospective Open-Label Pilot Trial in 10 Patients," American College of Rheumatology, Session Number: 28- 100, Session Title: Vasculitis, Session Type: ACR Concurrent Session, Primary Category: 28 Vasculitis, Session 10/18/2004 (< w w. abstractsonline. com / viewer / SearchResults. asp >). See also Keogh et al., Kidney Blood Press. Beef. (2003), where it is reported that 11 patients with refractory ANCA-associated vasculitis were treated with 4 weekly doses of 375 mg / m2 of rituximab and high-dose glucocorticoids resulting in remission. Patients with vasculitis associated with refractory ANCA were administered rituximab together with immunosuppressive drugs such as intravenous cyclophosphamide, mycophenolate mofetil, azathioprine, or leflunomide, with apparent efficacy. Eriksson, "Short-term outcome and safety in 5 patients with ANCA-positive vasculitis treated with rituximab", Kidney and Blood Pressure Research, 26: 294 (2003) (five patients with vasculitis associated with ANCA treated with rituximab 375 mg / m2 a once a week for 4 weeks they responded to the treatment); Jayne et al., "B-cell depletion with rituximab for refractory vasculitis" Kidney and Blood Pressure Research, 26: 294-295 (2003) (six patients with refractory vasculitis received 4 weekly infusions of rituximab at 375 mg / m2 with cyclophosphamide together with background immunosuppression and predmisolone experienced major falls in vasculitic activity). An additional report of using rituximab together with intravenous cyclophosphamide at 375 mg / m2 per dose in 4 doses to be administered to patients with refractory systemic vasculitis is provided by Jayne, poster 88 (IIth International Vasculitis and ANCA workshop), 2003 American Society of Nephrology. See also Stone and Specks, "Rituximab Therapy for the Induction of Remission and Tolerance in ANCA-associated Vasculitis," in the Clinical Trial Research Summary of the 2002-2003 Immune Tolerance Network, http: // www. irninunetolerance. crg / research / autoinraune / tria 's / s tone. html, where a test of rituximab in vasculitis associated with ANCA is proposed for a total duration of 18 months. See also Eriksson, J. Internal Med., 257: 540-548 (2005) regarding 9 patients with ANCA-positive vasculitis who were successfully treated with 2 or 4 weekly doses of 500 mg of rituximab, as well as Keogh et al., Arthritis and Rheumatism, 52: 262-268 (2005), who reports that in 11 patients with vasculitis associated with refractory ANCA, treatment or re-treatment with 4 weekly doses of 375 mg / m2 of rituximab induced remission due to depletion of cell lymphocyte B, the study was conducted between January 2000 and September 2002. Regarding the humanized CD20 antibody activity see for example Vugmeyster et al., "Depletion of B cells by a humanized anti-CD20 antibody PRO70769 in Macaca fascicularis" J. Immunother. 28: 212-219 (2005). For a discussion of a human monoclonal antibody, see Baker et al., "Generation and characterization of LymphoStat-B, a human monoclonal antibody that antagonizes the bioactivities of B lymphocyte stimulator" Arthritis Rheum. 48: 3253-3265 (2003) There is a need for approaches to treat that reduce the frequency of active drug infusions within a month. In addition, there is a need to reduce the risk of toxic effects of currently used drugs such as steroids and chemotherapeutic agents and reduce the risk of sudden recurrence or worsening of disease symptoms, relapses and recurrences in patients with vasculitis associated with ANCA, and for ' sustain remission and maintain remission sustained by. a long period of time. SUMMARY OF THE INVENTION The present invention involves administration of a CD20 antibody that provides a safe and active treatment regimen in subjects with vasculitis associated with ANCA, including selection of an effective dose regimen and scheduled or unscheduled re-treatment. This antagonism is as effective for initial therapy as in the management of refractory disease. According to this, the invention is as claimed. In a first aspect, the present invention relates to treating vasculitis associated with ANCA in a patient comprising administering a CD20 antibody to the patient in a dose of about 400 mg to 1.3 grams at a frequency of one to three doses within a period of approximately one month. In a further aspect, the invention provides an article of manufacture comprising: a container comprising a CD20 antibody and a packing graft with instructions for treating vasculitis associated with ANCA in a patient where the instructions indicate that a dose of CD20 antibody of approximately 400 mg to 1.3 grams at a frequency of one to three doses is administered to the patient within a period of about one month .... In preferred embodiments of the above inventive aspects, the vasculitis is Wegener's granulomatosis or microscopic polyangiitis and / or a second medicament is administered in an effective amount to the patient, wherein the CD20 antibody is a first drug. This medication can be one or more medications. More preferably, this second medicament is a chemotherapeutic agent, an immunosuppressive agent, an antirheumatic drug modifying disease (DMARD = Disease-Modifying Anti-Rheumatic Drug), a cytotoxic agent, an integrin antagonist, a non-steroidal anti-inflammatory drug (NSAID = Non-Steroidal Anti-Inflammatory Drug), a cytokine or cytokine antagonist, a hormone or a combination thereof. In still further aspects, the present invention relates to a method for treating ANCA associated vasculitis in a subject which comprises administering an effective amount of a CD20 antibody to the subject to provide an initial exposure of antibody followed by a second antibody challenge, wherein the second exposure is not provided until approximately 16 to 54 weeks from an initial exposure. In a preferred embodiment of this more recent method involving multiple antibody exposures, the present invention relates to a method for treating vasculitis associated with ANCA in a subject, which comprises administering to the subject an effective amount of a CD20 antibody to provide a initial exposure of antibody of approximately 0.5 to 4 grams followed by a second antibody exposure of approximately 0.5 to 4 grams, where the second exposure is not provided until approximately 16 to 54 weeks of the initial exposure and each of the exposures of antibody is provided to the subject as approximately 1 to 4 doses of antibody, more preferably as a single dose or as two or three separate doses of antibody. A specific preferred embodiment herein is a method for treating vasculitis associated with ANCA in a subject, which comprises administering an effective amount of a CD20 antibody to the subject to provide an initial antibody challenge followed by a second antibody challenge, wherein the second exposure it is not provided until approximately 16 to 54 weeks from the initial exposure and. each of the antibody exposures is provided to the subject as a single dose or as two or three separate doses of antibody. Preferably, in this method, antibody exposures are approximately 0.5 to 4 grams each. In another preferred embodiment of the latter methods, a second medicament is administered with the initial exposure and / or subsequent exposures, wherein the antibody is a first medicament. In a preferred embodiment, the second medicament is one or more of those previously established as preferred. In a more preferred embodiment, the second medicament is a steroid and / or an immunosuppressive agent. In an even more preferred embodiment, a steroid is administered with the first exposure, but not with the second exposure, or administered in smaller amounts than those used with the initial exposure. In yet another preferred embodiment of these more recent aspects, the subject has never previously been treated with a CD20 antibody, and / or another drug other than the CD20 antibody is administered to the subject to treat vasculitis. In another preferred embodiment, the initial and second antibody exposures are with the same antibody, and more preferably all antibody exposures are with the same antibody. In another preferred embodiment, the subject is in remission after the initial or subsequent antibody exposures, preferably when provided with the second antibody exposure. More preferably, the subject is in remission when all antibody exposures are provided. More preferably, this subject is in remission at least about six months after the last antibody challenge is provided. In yet another preferred embodiment of the most recent aspects, the subject has a high level of anti-nuclear antibodies (ANA), anti-rheumatoid factor (RF) antibodies, creatinine, blood urea nitrogen, anti-endothelial antibodies, antibodies anti-neutrophil cytoplasmic (ANCA), or a combination of two or more thereof. Additionally, in major aspects, the invention provides an article of manufacture comprising: (a) a container comprising an antibody CD20; and (b) a packaging insert with instructions for treating ANCA-associated vasculitis in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial antibody challenge followed by a second exposure of the antibody. antibody, wherein the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure. Preferably, this packing insert is provided with instructions for treating vasculitis associated with ANCA in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject, which is effective to provide an initial antibody exposure of about 0.5 to 4 grams followed by a second antibody exposure of approximately 0.5 to 4 grams, wherein the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure and each of the antibody exposures is provided to the subject as about one to four doses, preferably as a single dose or as two or three separate doses of antibody. In a specific aspect, a manufacturing article is provided comprising: (a) a container comprising an antibody GD20; and (b) a packaging insert with instructions for treating ANCA-associated vasculitis in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial antibody exposure followed by a second exposure. of antibody wherein the second exposure is not provided until about 16 to 54 weeks from the initial exposure, and each of the antibody exposures is provided to the subject as a single dose or as two or three separate doses of antibody. The treatments herein preferably reduce, minimize or eliminate the need for co-, pre- or post-administration of excessive amounts of second medications such as immunosuppressive agents and / or chemotherapeutic agents which are the standard standard treatment for these subjects, to avoid as much as possible the side effects of this standard treatment, as well as reducing costs and increasing convenience or comfort to the subject, such as convenience in time and frequency of administration. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A is a sequence alignment comparing the amino acid sequences of the light chain variable domain (VL) of each of the murine 2H7 subgroups (SEQ ID N0: 1), humanized 2H7.vl6 variant. (SEQ ID NO: 2), and the human kappa light chain sub-group I (SEQ ID NO: 3). The VL CDRs of 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO: 4), 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 murine 2H7 variant (SEQ ID N0: 7), humanized 2H7.vl6 variant (SEQ ID N0: 8), and the human consensus sequence of heavy chain sub-group 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 1A and Figure IB, the chain CDR1, CDR2 and CDR3 are circumscribed within claudontists, flanked by framework regions, FR1-FR4, as indicated. 2H7 refers to the murine 2H7 antibody. The asterisks between two rows of sequences indicate the positions that are different between the two sequences. Numbering of waste is according to Kabat et al. Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions shown as a, b, c, d, and e. Figure 2 shows the amino acid sequence of the mature 2H7.vl6 L chain (SEQ ID NO: 13) Figure 3 shows the amino acid sequence of the mature 2H7.vl6 H chain (SEQ ID NO: 14). Figure 4 shows the amino acid sequence of the mature 2H7.v31 H chain (SEQ ID NO: 15). The string L of 2H7.v31 'is the same as for 2H7.V16. Figure 5 is a sequence alignment comparing the light chain amino acid sequences of the humanized variant 2H7.vl6 (SEQ ID NO: 2) and the humanized variant 2H7.vl38 (SEQ ID NO: 28). Figure 6 is a sequence alignment comparing 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: 29). Figure 7 shows an alignment of the mature light chains 2H7.vl6 and 2H7.v511 (SEQ ID NOS: 13 and 30, respectively), with Kabat variable domain residue numbering and Eu domain constant residue numbering. Figure 8 shows an alignment of the heavy chains 2H7.vl6 and 2H7.v511 mature (SEQ ID NOS: 14 and 31, respectively), with Kabat variable domain residue numbering and constant domain residue numbering Eu. Figure 9A shows the sequence of the variable light chain domain 2H7.vll4 (SEQ ID NO: 32); Figure 9B shows the sequence of the humanized variable heavy chain domain 2H7.vll4 (SEQ ID NO: 33); and Figure 9C shows the sequence of the humanized integrated length heavy chain 2H7.vll4 (SEQ ID NO: 34), with Kabat variable domain residue numbering and constant domain numbering Eu. Detailed Description of the Preferred Modalities I. Definitions A "B cell" is a lymphocyte that matures within the bone marrow, and includes a pre-treated B cell, memory B cell, or effector B cell (plasma cells). The B cell here can be a normal or non-malignant B cell.

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 it. Exemplary cell surface B markers include the leukocyte surface markers CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD74, CD74, CD74, CD77, CD78, CD79a, 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 B cell surface markers include RP105, FcRH2, B-cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, .NAG14, SLGC16270, FcRHl, IRTA2, AT D578, FcRH3, IRTA1, FcRH6 , BCMA, and 239287. The B cell surface marker of particular interest is preferably expressed in B cells compared to other tissues that are not B-cells from a mammal and can be expressed in both precursor B cells and mature B cells. The preferred B cell surface markers here are CD20 and CD22. The "CD20" or "CD20" antigen is a non-glycosylated 35-kDa phosphoprotein, which is found on the surface of more than 90% B cells of the peripheral blood or lymphoid organs. CD20 is present in both normal B cells as well as malignant B cells, but is not expressed in stem cells. Other names for CD20 in the literature include "antigen restricted to B lymphocyte" and "Bp35". The CD20 antigen is described in Clark et al., Proc. Nati Acad. Sci. (USA) 82: 1766 (1985), for example. The "CD22" or "CD22" antigen, 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 (unreduced). It is expressed both in the cytoplasm and in the cell membrane of B lymphocytes. The CD22 antigen appears early in the B cell lymphocyte differentiation at approximately the same stage as the CD19 antigen. The. Unlike other B cell markers, CD22 membrane expression is limited to the late differentiation stages comprised between mature B cells (CD22 +) and plasma cells (CD22-). The CD22 antigen is described, for example in Wilson et al., J. Exp. Med. 173: 137 (1991) and Wilson et al., J. Immunol. 150: 5013 (1993). An "antagonist" is a molecule that, by binding to CD20 on B cells, destroys or depletes B cells in a mammal and / or interferes with one or more B cell functions, for example by reducing or preventing a humoral response produced by the B cell. The antagonist is preferably capable of depleting B cells (i.e. reducing circulating B cell levels) in a treated mammal. This depletion can be achieved by various mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC = = antibody-dependent cell-mediated cytotoxicity) and / or complement-dependent cytotoxicity (CDC = complement dependent cytotoxicity), inhibition of B cell proliferation and / or induction of B cell death [for example by apoptosis). Antagonists included within the scope of the present invention include antibodies, native or synthetic sequence peptides, immunoadhesins, and small molecule antagonists. which bind, to CD20, optionally conjugated with or fused to a cytotoxic agent. The preferred antagonist comprises an antibody. An "antibody antagonist" herein is an antibody that, by binding to a B cell surface marker in B cells, destroys or depletes B cells in a mammal and / or interferes with one or more B cell functions, e.g. , by reducing or preventing a humoral response produced by the B cell. The antibody antagonist is preferably capable of depleting B cells (i.e., reducing circulating B cell levels) in a treated mammal. This depletion can be achieved by various mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), inhibition of B-cell proliferation and / or induction of B-cell death (e.g. apoptosis). The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), formed of at least two intact antibodies, and antibody fragments provided they exhibit the activity desired biological "Antibody fragments" comprise a portion of the intact antibody. what. preferably it comprises its antigen binding region. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. For the present purposes, an "intact antibody" is one that comprises heavy and light variable domains as well as a Fe region. An "antibody that binds to a B cell surface marker" is a molecule that, by binding to a label of B cell surface, destroys or depletes B cells in a mammal and / or interferes with one or more B cell functions, for example by reducing or preventing a humoral response produced by the B cell. The antibody is preferably able to deplete cells B (i.e. reducing levels of B cells in circulation) in a mammal treated therewith. This depletion can be achieved by various mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and / or induction of B cell death (e.g. by apoptosis). ). In a preferred embodiment, the antibody-induces a greater clinical response. In another preferred embodiment, the cell surface marker B is CD20, such that the antibody that binds to a cell surface marker B is an antibody that binds CD20 'or an "antibody CD20". A particularly preferred embodiment is a CD20 antibody that induces a greater clinical response. For the present purposes, a "major clinical response" is defined as achieving an answer 70 according to the American College of Rheumatology (ACR 70 = American College of Rheumatology 70 response) for six consecutive months. ACR Response Ratings are characterized as ACR 20, ACR 50 and ACR 70, with ACR 70 which is the highest level of symptoms and signs control in this evaluation system. The ACR response scores measure improvement in rheumatoid arthritis disease activity, including joint inflammation and softness, pain, disability level, and total evaluation of the patient and physician. An example of a different type of antibody that induces a greater clinical response as recognized by the FDA and as defined herein is etanercept (ENBREL®). Examples of CD20 antibodies include: "C2B8", which is now called "rituximab" ( "RITUXAN®") (U.S. Patent No. 5,736,137); the murine antibody labeled 2B8 with yttrium- [90] "Y2B8" or "Ibritumomab Tiuxetan" (ZEVALIN®) commercially available from IDEC Pharmaceuticals, Inc. (U.S. Patent No. 5,736,137; 2B8 filed with ATCC under accession number HB11388 on June 22, 1993); Murine IgG2a "Bl", also referred to as "Tositumomab", optionally labeled with 131I to generate the antibody "131I-B1" or "iodine 1131 tositumomab" (BEXXAR ™) commercially available from Corixa (see also U.S. Patent No. 5,595,721); murine monoclonal antibody "1F5" (Press et al., Blood 69 (2): 584-591 (1987) and its variants, including humanized IF5 or "patching in frame" (WO 2003/002607, Leung, S .; ATCC deposit HB -96450), murine 2H7 and chimeric 2H7 antibody (U.S. Patent No. 5,677,180), a humanized 2H7 (WO 2004/056312 (Lo man et al.) And as set forth below), fully human high affinity antibody HUMAX- CD20MR, in the CD20 molecule in the cell membrane of B cells (Genmab, Denmark, see for example, Glennie and van de Winkel, Drug Discovery Today 8: 503-510 (2003) and Cragg et al., Blood 101: 1045- 1052 (2003)); the human monoclonal antibodies established in WO04 / 035607 (Teeling et al.); AME-133MR antibodies (Applied Molecular Evolution); antibody A20 or its variants such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Patent 2003/0219433, Immunomedics); and Monoclonal Antibodies L27, G28-2, 93-1B3,. B-Cl or NU-B2 available from International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., P.400, Oxford University Press (1987).) CD20 antibodies preferred here are CD20 antibodies. humanized or human chimeric, more preferably rituximab, a humanized 2H7 antibody, chimeric or humanized A20 (Immunomedics), and human CD20 antibody HUMAX-CD20MR (Genmab) The terms "rituximab" or "RITUXAN®" herein refer to the genetically engineered chimeric murine / human monoclonal antibody directed against the CD20 antigen and designated "C2B8" in US Pat. No. 5,736,137, including its fragments that retain the ability to bind CD20. For the present purposes only and unless indicated otherwise, a "humanized 2H7" refers to a humanized CD20 antibody or its antigen binding fragment, wherein the antibody is effective to deplete primate B cells in vivo, the antibody comprises in its H chain variable region (VH), at least one CDR H3 sequence of SEQ ID NO: 12 (FIG. IB) of an anti-human CD20 antibody and substantially the human consensus framework (FR) residues of the subgroup of human heavy chain III (VHIII). In a preferred embodiment, the antibody further comprises the H chain CDR Hl sequence of SEQ ID NO: 10 and the CDR H2 sequence of SEQ ID NO: 11, and more preferably further comprises the L chain CDR sequence of SEQ ID NO. : 4; the CDR sequence L2 of SEQ ID NO: 5; the CDR L3 sequence of SEQ ID NO: 6 and substantially the human consensus framework residues (FR) of the human light chain subgroup I (VI), wherein the VH region can be linked to a human IgG chain constant region, wherein the region can be for example IgGl or IgG3. See also WO 2004/056312 (Lowman et al.). In a preferred embodiment, this antibody comprises the VH sequence of SEQ ID NO: 8 (vl6, as illustrated in FIG. IB), optionally also comprises the sequence VL of SEQ ID NO: 2 (vl6, as shown in FIG. Fig. 1A), which may have the amino acid substitutions of D56A and N100A in the H chain and S92A in the L chain (v96). Preferably, the antibody is an intact antibody comprising the light and heavy chain amino acid sequences of SEQ ID NOS: 13 and 14, respectively, as shown in Figs. 2 and 3. Another preferred embodiment is when the antibody is 2H7.v31 comprising the light and heavy chain amino acid sequences of SEQ ID NOS: 13 and 15, respectively, as shown in Figs. 2 and 4. 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 that wherein the amino acid substitutions are S298A / E333A / K334A, more preferably 2H7.v31 having the heavy chain amino acid sequence of SEQ ID NO: 15 (as shown in Fig. 4). Another preferred embodiment is when the antibody is 2H7.vl38 comprising the light chain and heavy chain amino acid sequences of SEQ ID NOS: 28 and 29, respectively, as shown in Figs. 5 and 6, which are alignments of these sequences with the corresponding light chain and heavy chain amino acid sequences of 2H7.vl6. Alternately, this preferred intact humanized 2H7 antibody is 2H7.v477, which has light and heavy chain sequences of 2H7.vl38 except for the amino acid substitution of N434. 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 substituent K322A. See U.S. Pat. No. 6,528,624B1 (Idusogie et al.). . The most preferred humanized 2H7 variants are those having the variable light chain domain of SEQ ID NO: 2 and the variable heavy chain domain of SEQ ID NO: 8,. ie those with or without substitutions in the Fe region and those having a variable heavy chain domain with alteration of N100A or D56A and N100A in SEQ ID NO: 8 and a variable light chain domain with 32L alteration, or S92A, or M32L and S92A in SEQ ID NO: 2, ie those with or without substitutions in the Fe region. If substitutions are made in the Fe region, they are preferably those established in the following table. In a summary of various preferred embodiments of the invention, the V region of variants based on version 2H7 will have the amino acid sequences of vl6 except at the positions of amino acid substitutions that are indicated in the following table. Unless indicated otherwise, the 2H7 variants will have the same L chain as the vl6 one.

A particularly preferred humanized 2H7 is an intact antibody or antibody fragment comprising the variable light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 2); and the variable heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKFKGRFTI SVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYS SYWYFDVWGQ GTLVTVSS (SEQ ID NO: 8). Where the humanized 2H7 antibody is an intact antibody, preferably comprising the amino acid sequence of light chain: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ KVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13); and the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYS SYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTL ISRTPEVTCVVVDVSHEDPEVKFN YVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD LNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE ESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) or the heavy chain amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT.CVVVDVSHEDPEVKFN YVD GVEVHNAKTKPREEQYNATYRWSVLTVLHQD LNGKEYKCKVSNKALPAPIAATIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE ESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15). In another preferred embodiment, the intact humanized 2H7 antibody comprises the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGV PSR FSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSVFIF PPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28) and the sequence of heavy chain amino acids: EVQLVESGGGLVQPGGSLRLSCAASG YTFTSYNMHWVRQAPGKGLEWVGAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQM NSL RAEDTAVYYCARVVYYSASYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAA LGCLVKDYFPEPVTVS NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTC VVVDVSHEDPEVKFN YVDGVEVHNAKTKPREEQYNATYRVVSVLT.VLHQDWLNGKE YKC. KVSNAALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ SL SLSPGK (SEQ ID NO: 29). "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refers to a cell-mediated reaction where non-specific cytotoxic cells expressing Fe (FcRs) receptors (e.g., natural killer (NK) cells, neutrophils and macrophages (recognize antibody bound to a target cell and subsequently cause lysis of the target cell.The primary cells to mediate ADCC, NK cells, which express FcyRIII only, while the monoliths express FcyRI, FcRII and FcRIII.The expression FcR in hematopoietic cells is solved in Table 3 on page 464 of Ravetch and Kinet, Annu, Rev. Immunol., 9: 457-492 (1991) To estimate the ADCC activity of a molecule of interest, an in vitro ADCC assay such as that described in U.S. Patent Nos. 5,500,362 or 5,821,337 can be made.Effective effector cells for these assays include peripheral blood mononuclear cells (PBMC = Pperipheral Blood Mononuclea) r Cells) and natural killer cells (NK = Natural Killer.) .. Alternatively, or additionally, the ADCC activity of the molecule of interest can be estimated in vivo, for example 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 FcyRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells that are 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 native sequence. Still further, a preferred FcR is that which binds an IgG antibody (a gamma receptor) and includes receptors of the subclasses Fc ^ RI, Fc ^ RII, and Fc ^ RUI, including allelic variants and altered forms of these receptors, Fc RII receptors include Fc RIIA (an "activation receptor") and Fc ^ RIIB (an "inhibition receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The Fc ^ RIIA activation receptor contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The Fc / RIIB inhibition receptor contains a motif of inhibition based on tyrosine immunoreceptor (ITIM) in its cytoplasmic domain. (See Daéron, Annu, Rev. Immuno1, 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-341 (1995). Other FcRs, including those that will be identified in the future, are covered by the term "FcR" here. Temrino also includes the neonatal receptor FcRn, which is co-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" refers to the ability of a molecule to lyse a target in the presence of complements. The complement activation pathway is initiated by linking the first component of the complement system (Clq) to a molecule (eg, an antibody) complexed with a connate antigen. To estimate complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J. Immunol :. Methods 202: 163 (.1996), can be performed. . "Growth inhibitory" antibodies are those that prevent or reduce proliferation of a cell that expresses an antigen to which the antibody binds. For example, the antibody can prevent or reduce B cell proliferation in vitro and / or in vivo. Antibodies that "induce apoptosis" are those that induce programmed cell death, for example from a B cell, as determined by standard apoptosis assays, such as annexin V binding, DNA fragmentation, cellular shrinkage, endoplasmic reticulum dilatation, cellular fragmentation, and / or formation of membrane vesicles (called apoptotic bodies). "Native antibodies" are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains (L) and two identical heavy (H) chains. 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 has intra-chain disuLfuro bridges regularly spaced. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are considered to form an interface between the variable domain 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 antibodies, and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the variable domains of light chain and heavy chain. The most highly conserved portions of variable domains are called frame regions (FRs) :. The variable domains of heavy chains, and light native ones each comprise a four FRs, substantially adopting a ß sheet configuration, connected by three hypervariable regions, which form loops that. they connect, and in some cases they are part of the sheet structure ß. The regions . hypervariables in each chain are held together in immediate proximity by the FRs and with the hypervariable regions of the other chain, the formation of the antibody antigen binding site contributes (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions such as participation of the antibody in antibody-dependent cellular cytoxicity (ADCC).

The papain digestion of 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 easily crystallize. The treatment with pepsin produces an F (ab ') 2 fragment that has two antigen binding sites and is still capable of cross-linking antigen. "Fv" is the minimum antibody fragment that contains an antigen binding site and complete antigen recognition. This region consists of a heavy chain dimer and a light chain variable domain in a closed, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer specificity of antigen binding to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit at 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. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation here for Fab1 wherein the cysteine residue or cysteine residues contain at least one free thiol group. F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The "light chains" of antibodies v (immunoglobulins) of any vertebrate species can be assigned to one of two clearly distinct types, called kappa (?) And lambda. { ?), based on the amino acid sequences of their constant domains. Depending on the amino acid sequences of the constant domain of their heavy chains, the antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can further be divided into subclasses (isotypes), eg, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The constant heavy chain domains corresponding to the different classes of antibodies are called? A, d, e,? , and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. "Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody wherein these domains are present in a single polypeptide chain. 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 antigen binding. For a review of scFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,.; Rosenburg and Moore eds. , SprAnger-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments with two antigen binding sites, these 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 pairing between the two domains in the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully for example in 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 that is obtained from a substantially homogeneous antibody population, ie, the individual antibodies comprising the population are identical and / or link the same epitope, except for possible variants which may arise during production of the monoclonal antibody, these variants are generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants .. (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous since they are not contaminated by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody that is obtained from a substantially homogeneous population of antibodies, and is not considered to require 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 they can be made by recombinant DNA methods (see for example , 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. Blol., 222: 581-597 (1991), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) wherein a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or that per.tene.ee to a class or subclass of particular antibody while the rest of the chain (s) is or are identical to or with homologs to corresponding sequences in antibodies derived from another species or belonging to another class or sub-class of antibody as well as fragments of these antibodies, provided they exhibit the desired biological activity (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, resus or monkey macaque), and human constant region sequences (U.S. Patent No. 5, 693, 780). "Humanized" forms of non-human antibodies (eg, murine) are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues of a hypervariable region of the container are replaced by residues of a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, primate not human, that has the desired specificity, affinity and capacity. In some cases, framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all or all of the FRs are those in the sequence human immunoglobulin except for the FR substitution (s) previously noted. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin. For more 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 amino acid residues of an antibody that is responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity termination region" 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 loop" (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 variable domain of heavy chain; Chothia and Lesk J. Mol. Biol. 196: 901-917 ( 1987)). "Frame" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein. A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule such as a cytotoxic portion or radiolabel. An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Pollutant components of its natural environment are materials that will interfere with therapeutic diagnostic uses for the antibody and may include enzymes, hormones: and others, proteinaceous or non-proteinaceous solutes. In preferred methods, the antibody will be purified, (.1) to more than 95% by weight of the antibody as determined by the Lowry method, and more preferably more than 99% by weight, (2) to a sufficient to obtain at least 15 internal or N-terminal amino acid sequence residues by the use of a centrifuge cup sequencer or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably staining with silver. Isolated antibody includes an antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. "Vasculitis associated with ANCA" or "vasculitis associated with anti-neutrophil cytoplasmic antibody" "AAV" as used herein is an autoimmune disease or disorder that involves systemic vasculitis (or inflammation of the walls of blood vessels) where cytoplasmic antibodies Anti-neutrophils in circulation (ANCA) are normally present in the subject's blood, or other clinical manifestations are present that define vasculitis, as noted below. The term "Vasculitis associated with ANCA "as used herein, it applies to the vasculitis associated with ANCA no matter what type and stage or severity it does not matter what symptoms are evident, provided the diagnosis is made.

ANCA include microscopic polyangiitis, Wegener's granulomatosis, Churg-Strauss syndrome, limited renal vasculitis, (idiopathic crescentic necritizing glorulonephritis) and certain types of drug-induced vasculitis. Diagnoses for vasculitis associated with ANCA and its various manifestations include those established below. Several diagnostic tests are commonly used in people suspected of having vasculitis associated with ANCA. Characteristics that may help in defining the specific type of vasculitic disorder include the type of organ involvement, presence and type of ANCA (ANCA-myeloperoxidase or ANCA-proteinase 3), the presence of serum cryoglobulins, and the presence of evidence for granulomatous inflammation. . Exemplary autoantibodies associated with vasculitis associated with ANCA include elevated level of anti-nuclear antibodies (ANA), anti-rheumatoid factor (RF) antibodies, creatinine, blood urea nitrogen, anti-endothelial antibodies, anti- cytoplasmic antibodies, neutrophil (ANCA),. such as auto antibodies directed against proteinase 3 (PR3) or against myeloperoxidase (MPO), or a combination thereof. ANCA antibodies can be detected using antigen-specific immunochemical assay to characterize PR3-ANCA and MPO-ANCA. Niles et al., Supra. Since an ELISA test for ANCA is associated with a substantially higher positive predictive value and likelihood ratios for vasculitis associated with ANCA, ELISA tests are performed only on samples that are positive for ANCA by immunofluorescence. Stone et al., Arthritis Care and Research, 13: 424-34 (2000); Comment on Arthritis Care Res. 13: 341-342 (2000); Russell et al., Clin. Immunol., 103: 196-203 (2002). Approximately 10% of patients with microscopic polyangiitis (the most common type of vasculitis associated with ANCA) and egener granulomatosis have negative tests for ANCA; however, this finding does not completely rule out these diseases and the ANCA titers do not always correlate with the activity of the disease. Jennette and Falk,? 7. Engl J Med., Supra. On the other hand, an ANCA positive test result is only a diagnosis of vasculitis associated with ANCA. Table 1 summarizes the potential clinical manifestations of vasculitis associated with. ANCA, which should be suspected in any patient who presents a disease of multiple systems not caused by an infectious or malignant process (for example kidney dysfunction, skin irritation, pulmonary manifestations, or neurological manifestation). Constitutional symptoms are common. The frequency and combination of various system shares varies between entities of individual diseases. See also Guillevin et al. Arthritis Rheum. 42: 421-430 (1999); Pettersson et al., Clin. Nephrol. 43: 141-149 (1995); Savage et al., Lancet 349: 553-558 (1997); Guillevin et al., Br. J. Rheumatol 35: 958-964 (1996).

Table 1 Clinical Manifestations of Vasculitis Associated with ANCA The most common skin lesion is purple, a slightly elevated, non-bleaching rash that usually begins in the lower extremities. Occasionally, the rash or rash is vesicular or slightly ulcerated. Urticaria may also be a manifestation of vasculitis associated with ANCA. Unlike non-vasculitic allergic urticaria, vasculitic urticaria lasts more than a day and can develop into purple lesions. The presence of hypocomplementemia may indicate that the vasculitis is nicked by immune complex instead of vasculitis associated with ANCA. Renal involvement in vasculitis may progress in renal failure. Kidney biopsy results commonly reveal, glomerulonephritis. Focal necrosis, crecéntica formation and the absence or insufficiency of immunoglobulin deposits characterize glomerolunefritis in patients with vasculitis associated with ANCA. Pettersson et al., Clin. Nephrol. 43: 141-149 (1995). Lung involvement is in the range from ephemeral focal infiltrates or iaterstisial disease to massive pulmonary hemorrhagic alveolar capillaritis. The latter is the most life-threatening feature of small vessel vasculitis. It is important, however, to differentiate vasculitis associated with ANCA from other diseases that result in manifestations of multiple systems.

Diseases with extensive embolism to different organs (for example arterioembolic disease, endocarditis, antiphospholipid syndrome, and atrial myxoma) can produce similar clinical presentations. Kelley, "Vasculitis and related disorders" In: Textbook of rheumatology. 5th ed. (Philadelphia: Saunders, 1997), pp.1079-1101. People with sepsis can also have participation from multiple systems. It is also important to realize that the vasculitis associated with ANCA may be secondary to infection or malignancy. Some viral, bacterial and fungal infections can be complicated by vasculitis, which is predominantly a dermal vasculitis. Diagnosis is suggested by clinical history. Malignancy, such as lymphomas, leukemia, myeloproliferative, and myelodysplastic syndromes, may be associated with vasculitis associated with ANCA; however, solid tumors are less commonly associated with this vasculitis. Infection of underlying or malignant cases should be fully evaluated before the diagnosis of vasculitis associated with ANCA is made - even if the ANCA test result is positive. Table 2 illustrates some of the clinical features that can help in the diagnosis of the specific type of vasculitis. Laboratory evaluation should include a routine chemistry profile and whole blood cell count urinalysis, faecal occult blood test and chest x-ray. There may be normocytic anemia, bromocytosis, high sediment sedimentation rate, increased liver function, or evidence of renal involvement. ANCA serum levels should also be measured. Other laboratory tests that must be performed to exclude vasculitis associated with ANCA include antinuclear antibody, rheumatoid factor, cryoglobulins, complement, antibodies to hepatitis B and C, and human immunodeficiency virus (HIV) test. Computed tomographic scans of The breasts and chest can also be performed, if appropriate. Pathological examination of the tissue involved (eg skin, nerves, lung or kidney) can help in documenting the type of vasculitis associated with ANCA. The biopsy should be obtained from symptomatic and accessible sites. Biopsies of asymptomatic sites have low yield of positive results. Table 2 Clinical characteristics that favor the diagnosis of a specific type of vasculitis.

The information in table 2 is from Jennette and Falk, N. Engl. J. Med., Supra, and Kelley, supra. Wegener's granulomatosis is characterized by necrotizing granulomas of the upper and lower respiratory tract along with glomerulonephritis and systemic vasculitis, which usually involves medium-sized vessels, with formation of granulomas and parenchyma necrosis. Kelley, supra. Signs and symptoms of the upper respiratory tract include sinusitis, nasal ulcers, otitis media or hearing loss. Signs and symptoms of the upper respiratory tract are seen in 70% of the infiltrated patients or pulmonary nodules that can cavitate develop in 85% of patients. Kelley, supra. Antiprotease 3 serum-ANCA (c-ANCA) is positive in 75 to 90%, although 20% may have positive p-ANCA. Open lung biopsy is the most definitive diagnostic test. Breast biopsy is diagnosed in only 30% of cases because inflammatory findings are often non-specific and kidney biopsy is also relatively non-specific. Radiographic findings are opacities of middle and lower areas, which are diffuse and alveolar and intersitial. Nodules, which can cavitate, are rare in children. CT scan may show poorly defined | diffuse perivascular opacities. W.egener granulomatosis can affect patients of any age, with the highest incidence during the fourth decade of life and is slightly more common in men. Duna et al., Rheum. Dis. Clin. North Am. 21: 949-986 (1995). The most definitive way to diagnose Wegener's granulomatosis is to perform a biopsy of an involved organ site (usually the breasts), lung or kidney) to confirm the presence of vasculitis and granulomas, which together are diagnostic of the disease. Microscopic polyangiitis is characterized by the presence of ANCA and few or no immune deposits in the vessels involved. Savage et al., Lancet 349: 553-558 (1997). The kidneys are the most commonly affected organs in 90% of patients who have this type of vasculitis. Kelley, supra. Patients present with variable combinations of renal manifestations, palpable purpura, abdominal pain, cough and hemotisis. The majority of patients have positive MPO-ANCA (p-ANCA), although PR3-ANCA (c-ANCA) may also be present in 40% of patients. The most common age of onset is 40 to 60 years and more common is the male sex. Churg-Strauss syndrome is a rare disease and has three phases: allergic rhinitis and asthma, an infiltrating eosinophilic disease that resembles pneumonia and small vessel vasculitis with granulomatous inflammation. Guillevin et al., Br. J. Rheumatol., 35: 958-964 (1996). The vasculitic phase usually develops three years after the onset of asthma. Almost all patients have more than 10% eosinophils in their blood. Coronary arthritis and myocarditis are the main causes of morbidity and mortality. The age of onset varies from 15 to 70 years and is more common in men. Drug-induced vasculitis usually develops in 7 to 21 days after it starts with the drug and can be confined to the skin. Jennette and Falk, N. Engl. J. Med., Supra. Skin lesions are identical to those seen in systemic small vessel vasculitis. The drugs cause approximately 10% of vasculitic lesions on the skin. Drugs that have been implicated include penicillin, aminopenicillins, sulfonamides, allopurinol, thiazides, quinolones, hydrantoins and propylthiouracil. Some drugs such as propylthioauracil and hydralazine (APRESOLINEMR) seem to cause vasculitis by inducing ANCA. Another way to test active disease and determine which patients / subject are eligible for treatment, is to determine the Wegener granulomatosis value / Birmingham vasculitis activity score (BVAS / WG = Birmingham Vasculitis Activity Score / Wegener's granulomatosis.) For the patient, whether older or younger. This rating is an index of vasculitis activity and is designed to document clinical features that are directly due to active Wegener's granulomatosis. It has been found to be a specific marker in the valid and reliable disease for Wegener's granulomatosis. Stone et al., Arthritis &; Rheumatism, 44: 912-920 (2001). It can also be used for other diseases of vasculitis associated with ANCA. The instrument separates characteristics that represent new or worse disease activity from those that represent persistent activity. Typically, the patient's BVAS / WG rating is 3 or higher (or has been 3 or higher within 28 days of treatment). Each major item in the BVAS / WG evaluation form is scored with 3 points. Each minor item is rated with 1 point. However, another distinction used is that of acute disease, either first presentation or relapse, shows a BVAS / WG of at least 10, while persistent disease shows a BVAS / WG of at least 4. Lymphopenia can also be a good marker for Wegener's granulomatosis. Izzedine et al., Nephron 92: 466-471 (2002). A "subject" here, is a human subject, including a. patient, susceptible to treatment of vasculitis associated with ..- ANCA, · who experiences or has experienced one. or more signs, symptoms or other indicators of vasculitis associated with ANCA, would be diagnosed with vasculitis associated with ANCA, whether for example recently diagnosed or previously diagnosed and now experiencing a recurrence or relapse, or is at risk of developing vasculitis associated with ANCA . The subject may have been previously treated with CD20 antibodies or not treated. A susceptible subject for treatment of vasculitis associated with ANCA can optionally be identified as one who has been monitored such as in the blood, by high levels of infiltrating CD20 cells or is monitored using an assay to detect autoantibodies, where the production of auto-antibodies is estimated qualitatively and preferably in quantitative form. A "patient" here is a susceptible human subject for treatment of ANCA-associated vasculitis who experiences or has experienced one or more signs, symptoms or other indicators of vasculitis associated with ANCA, whether for example recently diagnosed or previously diagnosed and now experiences a recurrence or relapse. The patient may have been previously treated with antibody. CD2.0 or not having been treated. A susceptible patient. for treatment of vasculitis associated with ANCA - it can be optionally identified as the one to be monitored using an assay to detect autoantibodies, such as those previously noted, where the production of autoantibodies is estimated qualitatively and preferably quantitatively. "Treatment" of a subject here refers to both therapeutic treatment and prophylactic or preventive measures. Those that require treatment include those that already have vasculitis associated with ANCA as well as those in which the vasculitis associated with ANCA is to be avoided. Therefore, the subject may have been diagnosed as having vasculitis associated with ANCA, or may be predisposed or susceptible to vasculitis associated with ANCA. Treatment of a subject includes treatment of a patient. "Treatment" of a patient here refers to therapeutic treatment. Those patients who require treatment are those diagnosed with vasculitis associated with ANCA. For the present purposes, a patient or subject is in "remission", if he / she has not had symptoms of vasculitis disease associated with..ANCA, such as those detected by the methods described herein, and has not had recurrence of ANCA or increasing ANCA titers that coincide with or after B cell reconstitution, since sustained or recurrent ANCA levels have been found as predictors of relapses in patients in clinical remissions of Wegener's granulomatosis. Boomsma et al., Arthritis Rheum., 43: 2025-2033 (2000). Those who are not in remission include, for example, those who experience a sudden recurrence or worsening of disease symptoms after reconstitution of B cells, those who suffer from organ damage, such as kidney damage, or those who are asymptomatic but who have had a recurrence of ANCA recurrence title or an ANCA titration coincident with or after reconstitution of B cells. These subjects and Patients who experience a return of symptoms, including active disease and / or organ damage, or exhibit recurrent or increasing ANCA titles are those who have had "relapse" or have had a "recurrence." A "symptom" of vasculitis associated with ANCA is any morbid phenomenon or separation from normal in structure function or sensation, experienced by the subject or patient and indicative of. disease, such as those previously noted. The term "effective amount" refers to an amount of the antibody or antagonist that is effective to treat vasculitis associated with ANCA. "Antibody exposure" refers to contact with or exposure to the antibody herein in one or more doses that are administered for a period of time from about 1 day to about 5 weeks. The doses may be delivered at a fixed or irregular time or intervals over this period of exposure, such as for example a weekly dose for four weeks or two doses separated by a time interval of approximately 13 to 17 days. Initial and subsequent antibody exposures are separated in time from each other as described in detail here. An exposure that is not administered or provided under a certain time "from the initial exposure" or from any previous exposure, means the time for the second or subsequent exposure is measured from the time that any of the doses of the previous exposure is measured. administer, if more than one dose is administered in that exposure. For example, when two doses are administered at an initial exposure, the second exposure is not delivered until at least approximately 16 to 54 weeks as measured from the time the first or second dose was administered within that prior exposure. ' Similarly, when three doses are administered, the second exposure can be measured from the time of the first second or third dose within the previous exposure. Preferably, "from the initial exposure" or from any previous description is measured from the time of the first dose. 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 will include substances that suppress cytokine production, reduce or suppress self-antigen expression or mask MHC antigens.

Examples of these agents include substituted 2-amino-6-aryl-5-primidines (see U.S. Patent No. 4,665,077); non-spheroidal anti-inflammatory drugs (NSAIDs); ganciclovir, tacrolimus, glucocorticoids such as cortisol or aldosterone, anti-inflammatory agents such as cyclooxygenase inhibitor, a 5-lipoxygenase inhibitor, or a leukotriene receptor antagonist; purine antagonist such as azathioprine or mycophenolate mofetil (MMF); alkylating agents such as cyclophosphamide, bromocriptine, danazol, dapsone, glutaraldehyde (which masks the MHC antigens as described in US Patent Number 4, 120, 649), anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogues, for example prednisone, methyl prednisolone including methyl prednisolone sodium succinate SOLU-MEDROL® methylprednisolone and dexamethasone; inhibitors of dihydrofolate reductase and methotrexate (oral or subcutaneous); antimalarial agents such as chloroquine and hydroxychloroquine, sulfasalazine, leflunomide, cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta or -gamma antibodies, anti-tumor necrosis factor (TNF) -alpha antibodies (infliximab (REMICADE®) or adalimumab), anti- TNF-alpha immunoadhesin (etanercept), anti-TNF-beta antibodies, anti-interleukin-antibodies 2 (IL-2) and anti-IL-2 receptor antibodies and antibodies and anti-interleukin-6 (IL-6) receptor antagonists; anti-LFA-1 antibodies, including anti-CDlla and anti-CD18 antibodies; anti-L374 antibodies; anti-lymphocyte heterologous globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; soluble peptide containing an LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; transforming growth factor-beta (TGF-beta); streptodornase; RNA or host DNA; FK506; RS-61443; chlorambucil; . deoxyspergualine; rapamycin; T-cell receptor (Cohen et al., U.S. Patent No. 5,114,721); T-cell receptor fragments (Offner et al., Science, 251: 430-432 (1991), WO 90/11294, Ianeway, Nature, 341: 482 (1989), and WO 91/01133); BAFF antagonists such as BAFF antibodies and BR3 antibodies and zTNF4 antagonists (for review see Mackay and Mackay, Trends Im unol., 23: 113-5 (2002) and also the following definition); biological agents that interfere with T cell helper signals such as anti-CD40 receptor or anti-CD40 ligand (CD154), including CD40-CD40 ligand blocking agents (eg, Durie et al., Science, 261: 1328-30 ( 1993), Mohán et al., J. Immunol., 154: 1470-80 (1995)) and CTLA4-Ig (Finck et al., Science, 265: 1225-7 (1994)); and T cell receptor antibodies (EP 340,109) such as T10B9. Some preferred immunosuppressive agents include cyclophosphamide, chlorambucil, azathioprine, leflunomide, MMF or methotrexate. The term "cytotoxic agent" as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term is intended to include radioactive isotopes (eg At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes, Lu), chemotherapeutic agents and toxins such as small molecule toxins or enzymatically toxins active bacteria, funga! or of plants or animals or their fragments. A "chemotherapeutic agent" is a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylamelamines including altretamine, triethylmenemelamine, triethylene-phosphoramide, triethylethylene-phosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachona; lapachol; Colchicines; betulinic acid; and camptothecin (including the synthetic analog topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); Bryostatin; Callistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-T 1); eleutherobin; pancratistatin; a sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediin antibiotics (eg calicheamicin, especially gammall calicheamicin and omegall calicheamicin (see for example Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)), dinemicin, including dynemycin A; esperamycin, as well as neocarzinostatin chromophore and chromophores antibiotics of chromoprotein enediin), aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including ADRIAMICINA®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, injection of liposomes doxorubicin HC1 (DOXIL®) and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, acid mycophenolic, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamicin, rodorubicin, streptonigr ina, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, tiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocythabin, floxuridine; anti-adrenal such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornitin; eliptinium acetate; ethoglucid gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; fenamet; . pirarubicin; losoxantrone; 2-ethylhydrazide; . procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxana; rhizoxin; sizofirano; spirogermanium; tenuazonic acid; triaziquone; 2, 2, ', 2"-trichlorotriethylamine, trichothenes (especially toxin, T-2, verracurin A, roridin A and ariguidine)"; urethane; vindesina (ELDISINE®, FILDESIN®); Dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitoquina; arabinoside ("Ara-C"); thiotepa; taxoids, for example paclitaxel (TAXOL®), paclitaxel albumin engineered nanoparticle formulation (ABRAXANEMR), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); Oxaliplatin; leucovovina; vinorelbine (NAVELBINE®); novantrone; edatrexate; Daunomycin; aminopterin; ibandronate; Topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINMR) combined with 5-FU and leucovovina.

Also included in this definition are anti-hormonal agents that 'act' to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, and are often in the form of systemic or whole-body treatment. . They can be the hormones themselves. Examples include anti-estrogens and selective estrogen sector modulators (SERMs), including for example tamoxifen (including tamoxifen NOLVADEX®), raloxifen (EVISTA®), droloxifen, 4-hydroxy tamoxifen, trioxifen, keoxifen, LY117018, onapristone, and toremifen ( FARESTON®); anti-progesterone; descending estrogen receptor regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); agents that function to suppress or interrupt the operation of the ovaries, for example leutinizing hormone releasing hormone (LHRH = leutinizing hormone-releasing hormone) such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the aromatase enzyme, which. regulates the production of estrogen in the adenoid glands such as for example 4 (5) -imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMAS IN®), formestani, fadrozole, vorozole (RIVISOR®).,. . l.etrozole (FEMARA®), and anastrozole (ARIMIDEX®). In addition, .. this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); as well as troxacitabine (an analogue of 1,3-dioxolan nucleoside cytokine); anti-sense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in proliferation of aberrant cells, such as for example PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine and gene therapy vaccines, for example the ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; Topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (a small molecule inhibitor of dual tyrosine sinase ErbB-2 and EGFR also known as GW572016); and the 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 in another cell as intercellular mediators. Examples of these cytokines are lihphocins, monocycins; interleukins (ILs) such as IL-1, IL-? , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, including PROLEUKIN® rIL- 2; a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and natural ligand of the kit receptor (KL). As used herein, the term cytokine includes proteins from natural or recombinant cell culture sources and biologically active equivalents of the native sequence cytokines, including synthetically produced small molecule entities and pharmaceutically acceptable derivatives and their salts. The term "hormone" refers to polypeptide hormones that are generally secreted by glandular organs with ducts. The hormones include, for example, growth hormone such as human growth hormone, human growth hormone N-methionyl and bovine growth hormone; parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, estradiol, hormone replacement therapy, androgens such as calusterone, dromostanolone propionate, episthostanol, mepitiostane, or testolactone; prorelaxin; , glycoprotein hormones such as follicle stimulating hormone or follicle stimulants. (FSH = follicle stimulating hormone), thyroid stimulating hormone (TSH = thyroid stimulating hormone), and luteinizing hormone (LH = luteinizing hormone); prolactin, placental lactogen, mouse gonadotropin-associated peptide, gonadotropin-releasing hormone, inhibin; actibin; substance of mulerian inhibition and thrombopoietin. As used herein, the term "hormone" includes proteins from natural or recombinant cell culture sources and biologically active equivalents of the native sequence hormone, including synthetically produced small molecule entities and their pharmaceutically acceptable salts. 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 factors such as NGF-? platelet-derived growth factor; transformation growth factors (TGFs = transforming growth factors) such as TGF-a and TGF-.fi; insulin-like growth factor -I and -II; erythropoietin (EPO); Osteoinductive factors; interferons such as interferon-a, -ß, and -y; and colony stimulus factors; (CSFs = colony stimulating factors) such as .CSF-macrophage (M-CSF = macrophage-CSF); CSF-macrophage-granulocyte (GM-CSF = granulocyte-macrophage-CSF); and CSF- granulocyte (G-CSF = granulocyte-CSF). As used herein, the term "growth factor" includes proteins from natural or recombinant cell culture sources and biologically active equivalents of the native sequence growth factor, including synthetically produced small molecule entities and their derivatives and pharmaceutically acceptable salts. The term "integrin" refers to a receptor protein that allows both cells to bind to and respond to the extracellular matrix and is involved in a variety of cellular functions such as wound healing, cell differentiation, tumor cell settlement and apoptosis. They are part of a large family of cell adhesion receptors that are involved in cell-cell and extracellular-cell matrix interactions. Functional integrins consist of two subunits of transmembrane glycoproteins, called alpha and beta, which do not bind, cooperatively. 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 alpha 6 beta 1, alpha 3 beta 1, alpha 7 beta 1, LFA-1, etc. As used herein, the term "integrin" includes proteins, from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence integrin, including synthetically produced small molecule entities and their pharmaceutically acceptable salts and derivatives. For the present purposes"tumor necrosis factor alpha (TNF-alpha)" refers to a human TNF-alpha molecule comprising the amino acid sequence as described in Pennica et al., Nature, 312: 721 (1984) or Aggarwal et al. ., JBC, 260: 2345 (1985). A "TNF-alpha inhibitor" here is an agent that inhibits, to some extent, a biological function of TNF-alpha, generally through binding to TNF-alpha and neutralizing its activity. Examples of TNF inhibitors specifically contemplated here are ENBREL®), infliximab (REMICADE®), and adalimumab (HUMIRA ™). Examples of "disease-modifying antirheumatic drugs" or "DMARDs" include hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab (plus oral and subcutaneous methotrexate), azathioprine, D-penicillamine, gold salts (oral), gold salts (intramuscular), minocycline, cyclosporine including cycles.por.ina A and topical cyclosporin, staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197, (9) ·, pll25- 39 (2003)), including its salts and derivatives, etc. Examples of "non-spheroidal anti-inflammatory drugs" or "NSAIDs" include aspirin, acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, tolmetin, COX-2 inhibitors such as celecoxib (CELEBREX®; 4- (5- (4-methylphenyl) - 3- (trifluoromethyl) -lH-pyrazole-l-yl) benzenesulfonamide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®), including their salts and derivatives, etc. Preferably they are, aspirin, naproxen, ibuprofen, indomethacin, or tolmetin.

Examples of "integrin antagonists or antibodies" herein include an LFA-1 antibody, such as (RAPTIVA®) commercially available from Genentech, or an alpha 4 integrin antibody such as (ANTEGREN®) available from Biogen, or diazacycline phenylalanine derivatives (WO 2003/89410), phenylalanine derivatives (WO 2003/70709, WO 2002/28830, WO 2002/16329 and WO 2003/53926), phenylpropionic acid derivatives (WO • 2003/10135), enamine derivatives (WO 2001 / 79173), propionic acid derivatives (WO 2000/37444), derivatives of; alkanoic acid (WO 2000/32575), substituted phenyl derivatives (U.S. Patent No. 6,677,339 and 6, 348 i, 463), aromatic amine derivatives (U.S. Patent No. 6,369,229) ,. ADA disintegrin domain polypeptides (US2002 / 0042368), alpha v beta 3 integrin antibodies (EP 633945), bicyclic amino acid or aza bridge derivatives (WO 2002/02556). "Corticosteroid" refers to any one of several substances of natural or synthetic origin with the general chemical structure of spheroids that mimic or enhance the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include prednisone, prednisolone (including methylprednisolone, such as sodium methylprednisolone succinate SOLU-MEDROL®) dexamethasone or dexamethasone triamcinolone, hydrocortisone, and betamethasone. Preferred corticosteroids here are prednisone, methylprednisolone, hydrocortisone, or dexamethasone. The terms "BAFF," "BAFF polypeptide," "TALL-1" or "TALL-1 polypeptide," and "BLyS" when used herein, encompass "native sequence BAFF polypeptides" and "BAFF variants." "BAFF" is a designation given to those polypeptides having any of the amino acid sequences shown below:; Human BAFF sequence (SEQ ID NO: 16): 1 DDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAATLLLALL SCC. 61 LT.VVSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKAGLEEAPAVTAGLKIFEP PAP 121 181 LEE GEGNSSQNSRNKRAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSA KENKILVKETGYFFIYGQVLYTDKTYA GHLIQRKKVHVFGDELSLVTLFRCIQNMP ETL 241 PNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL BAFF mouse sequence (SEQ ID NO: 17): 1 MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGA FGICRDGRLLAATLLLAL LSS 61 SFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPELTAGVKLLTPAAPRPHN SSR 121 GHRNRRAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNI IQDCLQLIADS DTP 181 TIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFIYSQVLYTDPI FAMGHVIQ RKK '241; VHVFGDELSLVTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRN GDD 301 TFFGALKLL and homologs and fragments and their variants, which have the biological activity of the native BAFF. A biological activity BAFF can be selected from the group consisting of promoting B cell survival, promoting B cell maturation and binding to BR3. BAFF variants preferably have at least 80% of any successive integer up to 100% including, more preferably, at least 90%, and even more preferably at least 95% amino acid sequence identity with a native sequence of a BAFF polypeptide.

A "native sequence" BAFF polypeptide comprises a polypeptide having the same amino acid sequence as the corresponding BAFF polypeptide derived from nature. For example, BAFF exists in a soluble form after excision of the cell surface by furin-type proteases. These BAFF polypeptides of native sequence can be isolated from nature and can be produced by recombinant and / or synthetic means. The term "native sequence BAFF polypeptide" or "native BAFF" specifically encompasses secreted or truncated forms of natural origin (e.g., a sequence of extracellular domain), variant forms of natural origin (for example, alternatively combined forms) and allelic variants of natural origin of the polypeptide. The term "BAFF" includes those polypeptides described in Shu et al., J. Leukocyte Biol., 65: 680 (1999); access number GenBank AF136293; WO 1998/18921 published May 7, 1998; EP 869,180 published October 7, 1998; WO 1998/27114 published June 25, 1998; WO 1999/12964 published March 18, 1999; WO 1999/33980 published July 8, 1999; Moore et al., Science, 285: 260-263 (1999); Schneider et al., J. Exp. Med., 189: 1747-1756 (1999) and Mukhopadhyay et al., J. Biol. Chem., 274: 15978-15981 (1999).

The term "BAFF antagonist" as used herein, is used in the broadest sense and includes any molecule that (1) ligates a native sequence BAFF polypeptide or ligates a native BR3 sequence to partially or completely block BR3 interaction with the BAFF polypeptide and (2) blocks, inhibits or partially or completely neutralizes native sequence BAFF activity. In a preferred embodiment, the BAFF receptor to be blocked is the BR3 receptor. Native BAFF activity promotes, among other things, B cell survival and / or B cell maturation. In one embodiment, the inhibition, blocking or neutralization of BAFF activity results in a reduction, in the number of B cells. A BAFF antagonist according to this invention partially or completely blocks, inhibits or neutralizes one or more of the biological activities of a BAFF polypeptide, in vitro and / or in vivo. In one embodiment, a biologically active BAFF potentiates any or a combination of the following events in vitro and / or in vivo: increased survival of B cells, an increased level of IgG and / or IgM, an increased number of plasma cells, and processing NF- / rb2 / 100 to p52 NF-Arb in splenic B cells. { e.g., Batten et al. , J. Exp. Med. 192: 1453-1465 (2000); Moore et al., Science 285: 260-263 (1999); Kayagaki et al. Immunity 17: 515-524 (2002)). As mentioned above, a BAFF antagonist can function in a direct or indirect way to partially or completely block, inhibit or neutralize BAFF signaling, in vitro and / or in vivo. For example, the BAFF antagonist can directly bind BAFF. For example, BAFF antibodies that bind within a region of human BAFF comprising residues 162-27.5 and / or a neighboring residue of a residue selected from the group consisting of 162, 163, 206, 211, 231, 233, 264 and 265 of human BAFF such that the antibody spherically prevents BAFF from binding with BR3 is contemplated, where, these residue numbers are. refer. to SEQ ID NO: 16. In another example, -, a. "direct linker" is a polypeptide comprising any portion of a BAFF receptor that binds BAFF such as an extracellular domain of a BAFF receptor, or its fragments and variants that bind native BAFF. In another example, BAFF antagonists include polypeptides having a sequence of a polypeptide comprising the sequence of Formula I: Xi-C-Xa-D-Xs-LX-Xs-g-io-n-iz-CX ^ - Xis-Xie- (Formula I) (SEQ ID NO: 18) Where Xi, X3, X5, X7 / Xs X91 ??? Xii / Xi2 »X14, X15 and 17 are any amino acid except cysteine; and wherein Xi6 is an amino acid selected from the group consisting of L, F, I and V; and wherein the polypeptide does not comprise a cysteine within seven amino acid residues N-terminal to cysteine C plus N-terminal and C-terminal to cistern C plus C-terminal formule I.

In one embodiment, a polypeptide comprising the sequence of Formula I has two Cs joined by disulfide bonding: XsLX7X8 which form the conformation of a beta-type spin structure with the center of rotation between L and X7; and has a positive value for the dihedral angle fi of X8. In one embodiment, Xio is selected from the group 'consisting of W, F,' V, L, I, Y, M and a non-polar amino acid. In another embodiment Xi0 is W. In another embodiment, X3 is an amino acid selected from the group consisting of M, V, L, I, Y, F, and a non-polar amino acid. In another embodiment, X5 is selected from the group consisting of V, L, P, S, I, A and R. In another embodiment, X7 is selected from the group consisting of V, T, I and L. In another embodiment, Xe is selected from the group consisting of R, K, G, N, H and a D-amino acid. In another embodiment, Xg is selected from the group consisting of H, K, A, R and Q. In another embodiment X is I or V. In another embodiment, X12 is selected from the group consisting of P, A, D, E and S. In another embodiment Xi6 is L. In a specific embodiment, the sequence of Formula I is a sequence selected from the group consisting of ECFDLLVRAWVPCSVLK (SEQ ID NO: 19), ECFDLLVRHWVPCGLLR (SEQ ID NO: 20), ECFDLLVRRWVPCEMLG ( SEQ ID NO: 21), ECFDLLVRS VPCHMLR (SEQ ID NO: 22), ECFDLLVRHWVACGLLR (SEQ ID NO: 23), and QCFDRLNA VPCSVLK (SEQ ID NO: 24). In a preferred embodiment, the BAFF antagonist comprises any of the amino acid sequences selected from the group consisting of SEQ ID NO: 19, 20, 21, 22, and 23.

In still another example, BAFF antagonists include polypeptides having a sequence of a polypeptide comprising the sequence of Formula II: Xi-C-X3-D-X5-LV-X8-X9-VPC-Xi4-Xi5-L -Xi7 (Formula II) (SEQ ID NO: 25) Where Xx, X3, X5, Xs / Xi-w X15 and X1 are any amino acid except cysteine; and wherein the polypeptide does not comprise a cysteine within seven amino acid residues N-terminal to C-plus C-terminal and C-terminal to C-terminal plus C-terminal of Formula II. In one embodiment, a polypeptide comprising the sequence of Formula II has a disulfide bond between the two Cs and has the conformation of X5LX7X8 which forms a type I beta rotation structure with the center of rotation between L and X7; and has a positive value for the dihedral angle fi of X8. In another embodiment of Formula II, X3 is an amino acid selected from the group consisting of M, A, V, L, I, Y, F, W and a non-polar amino acid. In another embodiment of Formula II, X5 is selected from the group consisting of V, L, P, S, I, A and R. In another embodiment of Formula II, X8 is selected from the group consisting of R, K, G, N, H and D-amino acid. In another embodiment of Formula II, Xg is selected from the group consisting of H, K, A, R and Q. In a further embodiment, the BAFF receptor of which the extracellular domain or fragment of. link BAFF or its link variant BAFF is derived is TACI, BR3 or BCMA. Alternatively, the BAFF antagonist can bind an extracellular domain of a native BR3 sequence in its BAFF binding region to block, inhibit or partially or completely neutralize BAFF with BR3 in vitro, in situ, or in vivo. For example, this indirect antagonist is an anti-BR3 antibody that binds in a region of BR3 comprising residues 23-38 of human BR3 as defined below (SEQ ID NO: 26) or a neighboring region of such residues such that the binding of human BR3 to BAFF is prevented sterically. In some embodiments, a BAFF antagonist according to this invention includes BAFF antibodies and immuniadesins comprising an extracellular domain of a BAFF receptor or its fragments and variants that bind native BAFF. In a further embodiment, the BAFF receptor from which the extracellular domain or BAFF binding fragment or its BAFF link variant is derived is TACI, BR3 or BCMA. In yet another embodiment, the immunoadesin comprising an amino acid sequence of that of Formula I or Formula II as set forth above, including an amino acid sequence selected from any of the group consisting of ID NOS: 19, 20, 21, 22, 23, and 24.: |: According to one modality, the antagonist BAFF .. binds to a BAFF polypeptide or a BR3 polypeptide with a binding affinity of ????? or less .. According to another embodiment, the BAFF antagonist binds with a BAFF polypeptide or a BR3 polypeptide with a binding affinity of ???? or less. According to yet another embodiment, the BAFF antagonist ligates with a BAFF polypeptide or a BR3 polypeptide with a binding affinity of InM or less. The terms "BR3", "BR3 polypeptide" or "BR3 receptor" when used herein encompass "native sequence BR3 polypeptides" and "BR3 variants" (which are also defined herein). "BR3" is a designation given to those polypeptides comprising the following amino acid sequence and their homologs, and variants or fragments thereof that bind native BAFFs: Human BR3 sequence (SEQ ID NO: 26): 1 MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTAL QPQ 61 ESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAP DGD 121 KDAPEPLDKVI ILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTK TAG. 181 PEQQ. The BR3s polypeptides of the invention can be isolated from a variety of sources such as from human-type tissues or from another source, or prepared by recombinant and / or synthetic methods. The term BR3 includes the BR3s polypeptides described in WO 2002/24909 and O 2003/14294. A BR3 polypeptide of "native sequence" or "native BR3" comprises a polypeptide having the same amino acid sequence as the corresponding BR3 polypeptide derived from nature. These native sequence BR3s polypeptides can be isolated from nature or can be produced by recombinant and / or synthetic means. The term "native sequence BR3 polypeptide" specifically encompasses soluble or secreted truncated forms of natural origin (e.g., a sequence of extracellular domain), variant forms of natural origin (for example, combined forms in alternating form) and allelic variants of natural origin of the polypeptide. The BR3s polypeptides of the invention include the BR3 polypeptide comprising or consisting of the contiguous sequence of amino acid residues 1 to 184 of a human BR3 (SEQ ID NO: 26). An "extracellular domain" BR3 or "ECD" refers to a form of the BR3 polypeptide that is essentially free of the transmembrane and cytoplasmic domains. ECD forms of BR3 include a polypeptide comprising any of the amino acid sequences selected from the group consisting of the amino acids 1-77, 2-62, 2-71, 1-61, 7-71, 23-38 and 2 -63 of human BR3. The invention contemplates antagonists BAFF which are polypeptides comprising any of the aforementioned ECD forms of human BR3 and variants and fragments thereof which bind to native BAFF. Mini-BR3 is a core region of 26 residues of the BAFF binding domain of BR3, ie, the amino acid sequence: TPCVPAECFD LLVRHCVACG LLRTPR (SEQ ID NO: 27) "Variant BR3" means a BR3 polypeptide having at least approximately 80% amino acid sequence identity with the amino acid sequence of a full-length, native sequence BR3 or BR3 ECD and ligated to a native sequence BAFF polypeptide. Optionally, the BR3 variant includes a single domain rich in cysteine. This variant BR3 polypeptide includes for example BR3s polypeptides wherein one or more amino acid residues are added, or deleted, at the N- and / or C-terminus, as well as within: one or. more internal domains of the amino acid sequence. of integral length. Fragments of the BR3 ECD that bind to a BAFF polypeptide of native sequence as well. they are contemplated According to one embodiment, a variant BR3 polypeptide will have at least about 80% amino acid sequence identity, at least about 81% amino acid sequence identity, at least about 82% amino acid sequence identity, at least about 83% amino acid sequence identity, at least about 84% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 86% amino sequence identity acids, at least about 87% amino acid sequence identity, at least about 88% amino acid sequence identity, at least about 89% amino acid sequence identity, at least about 90% sequence identity of amino acids, at least about 91% amino acid sequence identity, at least about 92% sequence identity of amino acids, at least about 93% amino acid sequence identity, at least about 94% sequence identity of. amino acids, at least about 95% amino acid sequence identity, at least about 96% amino acid sequence identity, at least about 97% amino acid sequence identity, at least about 98% identity of amino acid sequence, at least about 99% amino acid sequence identity with a human BR3 polypeptide or its specified fragment (e.g., ECD).

BR3 variant polypeptides do not encompass the native BR3 polypeptide sequence. According to another embodiment, variant BR3 polypeptides are at least about 10 amino acids in length, at least about 20 amino acids in length, at least about 30 amino acids in length, at least about 40 amino acids in length, at least about 50 amino acids in length, at least about 60 amino acids in length, or at least about 70 amino acids in length. In a preferred embodiment, the BAFF antagonists present are immuno adhesins comprising a portion of BR3, TACI or BCMA that binds BAFF, or its variants that bind BAFF. In . other modalities, the BAFF antagonist is a BAFF antibody. A "BAFF antibody" is an antibody that binds BAFF, and preferably binds BAFF within a region of human BAFF comprising residues 162-275 of the human BAFF sequence described herein, under the definition "BAFF" (SEQ ID '). NO: 16) i In another embodiment, the BAFF antagonist is BR3 antibody. An "BR3 antibody" is an antibody that binds BAFF, and preferably that which binds BR3 within a region of human BAFF comprising residues 23-38 of the human BR3 sequence described herein, under the definition "BR3" (SEQ ID. NO: 26). In general, the amino acid positions of human BAFF and human BR3 referred to herein are in accordance with the sequence number under human BAFF and human BR3, SEQ ID NOS: 16 and 26, respectively, described herein, under the definitions "BAFF" and "BR3".

Other examples of BAFF binding polypeptides or BAFF antibodies can be found for example in WO 2002/092620, WO 2003/014294, Gordon et al., Biochemistry 42 (20): 5977-5983 (2003), Kelley et al., J. Biol. Chem., 279 (16) -.16727-16735 (2004), WO 1998/18921, WO 2001/12812, WO 2000/68378 and WO 2000/40716. A "package insert" is used to refer to instructions usually included in commercial packages of therapeutic products, which contain information regarding the indications use, dosage, administration, against indications other therapeutic products to be combined with the packaging product and / or warnings regarding the use of these therapeutic products, etc. A . "medication" is an active drug to treat vasculitis associated with ANCA or its symptoms or side effects. II. Therapy In one aspect, the present invention provides a method for treating vasculitis associated with ANCA in a patient comprising administering an antagonist, preferably an antibody, that binds a B cell surface marker (more preferably a CD20 antibody) to the patient. , in a dose of about 400 mg to 1.3 grams at a frequency of one to three doses within a period of about one month. Thus, the invention contemplates a method for treating vasculitis associated with ANCA in a patient, which comprises administering an antibody that binds a B cell surface marker with the patient in a dose of about 400 mg to 1.3 grams a a frequency of one to three doses within a period of approximately one month. The invention also contemplates a method for treating vasculitis associated with ANCA in a patient, which comprises administering an antagonist that binds to a surface marker: from B cell: to the patient in a dose of approximately 400 mg to 1.3 grams at a frequency of one to three doses within a period of approximately one month. The invention also contemplates a method for treating vasculitis associated with ANCA in a patient, characterized in that it comprises administering a CD20 antibody to the patient in a dose of about 400 mg to 1.3 grams at a frequency of one to three doses within a period of about month. In a preferred embodiment of each of these aspects, the dose is about 500 mg to 1.2 grams, more preferably 750 mg to 1.1 grams. In another preferred embodiment, the antibody is administered in two or three doses, more preferably in two doses, but alternately three doses. In an even more preferred embodiment, the antibody is administered within a period of about two to three weeks, more preferably about two weeks but alternately three weeks. In another embodiment, the present invention provides a method for treating vasculitis associated with ANCA in a subject susceptible to treatment, comprising administering an effective amount of an antibody that binds to a B cell surface marker, (preferably an antibody). CD20) to the subject to provide an initial exposure of antibody (or preferably about 0.5 to 4 grams, more preferably about 1.5 to 3.5 grams, and even more preferably about 1.5 to 2.5 grams) followed by a second exposure to antibody (preferably about 0.5 to 4 grams, more preferably about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5 grams), the second exposure is not delivered until about 16 to 54 weeks (preferably about 20 to 30 weeks, more preferably about 46 to 54 weeks) of the initial exposure.

For the purposes of this invention, the second antibody challenge is the next time the subject is treated with the CD20 antibody after the initial antibody exposure, with no treatment or exposure of intermediate CD20 antibody between the initial and second exposures. This re-treatment can be scheduled or unscheduled, but preferably it is a programmed re-dosing, particularly to protect against damage to organs such as kidneys. .. - The method of preference comprises administering to the subject an effective amount. of the CD20 antibody to provide a third antibody exposure (preferably about: - 0.5 to 4 grams, more preferably about 1.5 to 3.5 grams, still more preferably about 1.5 to 2.5 grams), the third exposure up to about 46 to 60 weeks (preferably approximately 46 to 55, more preferably approximately 46 to 52 weeks) of the initial exposure. Preferably, no more antibody challenge is provided until at least about 70-75 weeks of the initial exposure, and even more preferably no further antibody exposure is provided until about 74 to 80 weeks from the initial exposure.

Any one or more of the antibody exposures present can be provided to the subject as a single dose of antibody, or as separate doses, for example about 1-4 separate doses of the antibody (eg, which constitute a first and second dose, or a first, second and third dose or first, second, third and fourth doses, etc). The particular number of doses (whether one, two or three or more) used for each antibody exposure depends for example on the type of vasculitis associated with ANCA treated, the. type of antibody used, 'yes, what type and how much and how much of a second medicine is used as noted below, and the method. and frequency of administration. When administering separate doses, the last dose, for example. second or third dose (preferably if it is administered approximately 1 to 20 days more preferably approximately 6 to 16 days and more preferably approximately 14 to 16 days, from the time the previous dose is administered. they are administered within a total period of between about one day and 4 weeks, more preferably between about 1 and 20 days), for example within a period of 6 to 18 days). In that aspect, the separate doses are administered approximately weekly with the second dose which is administered approximately one week from the first dose and any third or subsequent dose is administered approximately one week after the second dose. Each separate dose of the antibody of preference is about 0.5 to 1.5 grams, more preferably about 0.75 to 1.3 grams. In a more preferred embodiment, a method for treating vasculitis associated with ANCA in a subject is provided comprising administering an effective amount of an antibody that binds to a cell surface marker. B (e.g., a CD20 antibody) to the subject to provide an initial exposure of the antibody followed. by a . second, antibody exposure, where the second:. Exposure is not provided until approximately 16 to 54 weeks from the initial exposure and each of the antibody exposures is provided to the subject as a single dose or as two or three separate doses of antibodies. Preferably in this method, the antibody exposures are approximately 0.5 to 4 grams each and more preferably the amounts given above. In one embodiment, the subject is provided at least about three exposures of the antibody, for example from about 3 to 60 exposures and more particularly 3 to 40 exposures, more particularly about 3 to 20 exposures. Preferably, these exposures are administered at intervals of 24 weeks each. In one embodiment, each antibody exposure is provided as a single dose of the antibody. In an alternate embodiment, each antibody exposure is provided as separate doses of the antibody. However, not all antibody exposure needs to be delivered as a single dose or as separate doses. In a preferred embodiment, approximately 2-3 grams of the CD20 antibody is administered as the initial exposure. If approximately 3 grams are administered, then approximately one gram of the CD20 antibody is administered weekly for approximately three weeks as initial exposure. If approximately two grams of the CD20 antibody is administered as the initial exposure, then approximately one gram of the CD20 antibody is administered followed by approximately two weeks by another approximate gram of the antibody as the initial exposure. In a preferred aspect, the second exposure is about 6 weeks from the initial exposure and is administered in an approximate amount of two grams. In an alternate preferred aspect, the second exposure is approximately six months from the initial exposure and is administered as approximately one gram of the antibody followed by approximately two weeks by another approximate gram of the antibody. In all the methods of the invention herein established, the B cell surface marker antibody or CD20 may be a naked antibody or may be conjugated to another molecule such as a cytotoxic agent, such as a radioactive compound. The preferred CD20 antibody present is a humanized or humanized chimeric CD20 antibody, more preferably rituximab, a humanized 2H7 (for example comprising the variable domain sequences in SEQ ID Nos. 2 and 8, or comprising a variable heavy chain domain with alteration N100A or D56A and N100A in SEQ ID? 0: · 8 and variable light chain domain with alteration M32L, or S92A, or M32L and S92A in SEQ ID N0: 2), chimeric or humanized A20 antibody (Immunomedics), or antibody Human CD20 HUMAX-CD20MR (Genmab). Even more preferred is humanized rituximab or 2H7. Also, while the vasculitis associated with ANCA in all methods can be any of said diseases, in a preferred embodiment, it is Wegener's granulomatosis or microscopic polyangiitis. In a further embodiment of all the present methods, the subject or patient has never been previously treated with the drug (s), such as immunosuppressant agent or people, to treat the vasculitis associated with ANCA and / or has never been previously treated with an antagonist (e.g. antibody) to a B cell surface marker (e.g. has never been previously treated with a CD20 antibody). In a still further aspect, the subject or patient may have had a relapse with the vasculitis associated with ANCA or suffered organ damage such as damage to the kidney before being treated with any of the above methods, including after exposure of the initial antibody or a later one. However, preferably the patient or subject has had no relapse with the vasculitis and more preferably has not had this relapse before at least the initial treatment. In another embodiment, the subject or patient has been previously treated with one or more drugs to treat vasculitis and / or has been previously treated with this antibody or antagonist. In another embodiment, the antagonist (e.g., CD20 antibody) is the only drug administered to the subject or patient to treat vasculitis. In another embodiment, the antagonist (for example CD20 antibody) is one of the drugs used to treat vasculitis. In a further embodiment, the subject or patient does not have a malignancy.

In a still further embodiment, the subject or patient does not have rheumatoid arthritis. In a still further embodiment, the subject or patient does not have multiple sclerosis. In a still further embodiment, the subject or patient does not have lupus or Sjögren's syndrome. In yet another embodiment, the subject or patient does not have an autoimmune disease other than vasculitis associated with ANCA. In yet another embodiment of the invention, the vasculitis associated with ANCA is not associated with a different autoimmune disease or with a risk of developing a different autoimmune disease. For purposes of the most recent statements, an "autoimmune disease" here is a disease or disorder that arises from and is directed against the individual's own tissues or organs or to a co-segregated or manifestation thereof of or a condition resulting therefrom. . Without being bound by any theory, B cells demonstrate a pathogenic effect in human autoimmune diseases through a multitude of mechanistic pathways, including production of autoantibodies, immune complex formation, activation of T cells and dendritic cells, synthesis of cytokine, direct chemokine release and provide a nest for ectopic neo-lymphogenesis. Each of these routes participates in different degrees in the pathology of autoimmune diseases.

In yet another embodiment, the subject or patient has a BVAS / WG score of less than 3, more preferably less than 2, still more preferable less than about 1 and more preferably from 0 (complete remission) to about 3 months, preferably about 6 months. months and more preferably about 1 year or more after administration of the antagonist or antibody. Specific modalities of this BVAS response achieve a score lower than 2 to three months after administration or less than 1 (for example 0.2 or 0.4) at 14 weeks or three months after administration, or less than 1 (for example 0.6) a 6 months after administration or more preferably, 0 to 3 or 6 months after administration. In another embodiment, the amount of steroids such as prednisone compared to the start of treatment is reduced without substantially affecting the reduced BVAS / WG rating. Thus, for example, a subject or patient at a set interval after treatment (such as 3 months or 6 months after treatment) preferably has a lower baseline BVAS / WG score and is given less than one dose of a baseline steroid (the baseline is at the start of administration). In a still further embodiment, a step in the treatment method is included to test the response of the subject or patient to treatment after the administration step to determine that the response level is effective to treat vasculitis. For example, a stage is included to test the BVAS / WG score after administration and compare it with a BVAS / WG baseline score that is obtained before administration to determine if the treatment is effective in measuring if and how much it has reduced . This test may be repeated at various scheduled or unscheduled time intervals after administration to determine maintenance of any partial or complete remission. Alternatively, the present methods comprise a step of testing the patient or subject, prior to administration to see if one or more bio-markers are present for vasculitis associated with ANCA, such as one or more auto-antibodies, a BVAS rating / WG or symptoms unique to vasculitis associated with ANCA, as stated above. In another method, a step can include verifying the clinical history of the patient or the subject, as detailed above, for example to rule out infections or malignancy as causes, for example, primary causes, of the patient or subject's condition, prior to administration of the antibody or antagonist to the subject or patient. Preferably, the vasculitis associated with ANCA is primary (that is, the main cause) and not secondary, such as secondary to infection or malignancy, whether solid or liquid tumors. In a preferred embodiment of the multiple exposure method present, the subject is in remission after initial antibody exposures or any subsequent exposures. More preferably, the method of multiple exposures here involves programming redosing or re-treatment such that the patient is in remission when the second and preferably all antibody exposures are provided. This re-dosing is programmed to avoid any relapse, recurrence or organ damage instead of treating it therapeutically. More preferably, the subject is in remission for at least about 6 months and even more preferably at least about 9 months, even more preferably at least about 1 year, since the last antibody exposure used in the re-treatment method . In yet another embodiment, the subject is treated with the same CD20 antibody for at least 2 antibody exposures and preferably for each antibody exposure. In this manner, the initial and second antibody exposures are preferably with the same antibody and more preferably all antibody exposures are with the same antibody, i.e., treatment for the first two exposures and preferably all exposures is with a type of antibody that binds to a surface marker of B cells, such as CD20 antibody, for example all with rituximab or all with the same humanized 2H7. In any of the present methods, the subject or patient may be administered together with the antagonist or antibody that binds cell surface marker B an effective amount of a second medicament (either the antagonist or antibody that binds to a cell surface marker). B (for example the CD20 antibody) is a first drug). The second medicament can be one or more medicaments and include for example a cytotoxic agent, chemo-therapeutic agent, immunosuppressive agent, cytokine, cytokine antagonist or antibody, growth factor, hormone, integrin, antagonist or integrin antibody or any combination of them. The type of this second medication depends on several factors including the type of vasculitis, the severity of the vasculitis, the condition and age of the patient, the type and dose of the first medication used, etc. Examples of these additional medicaments include a chemo-therapeutic agent, an interferon class drug such as interferon-alpha (eg, from Amarillo Biosciences, Inc.), IFN-beta-la (REBIF® and AVONEX®) or IFN-beta- lb (BETASERON®), an oligopeptide such as glatiramer acetate (COPAXONE®), an agent that blocks CD40-CD40 ligand, a cytotoxic or immunosuppressive agent (such as mitoxantrone (NOVANTRONE®), methotrexate, cyclophosphamide, chlorambucil, leflunomide, and azathioprine), intravenous immunoglobulin (gamma globulin), lymphocyte depletion therapy (eg mitoxantrone cyclophosphamide, CAMPATHMR antibodies, anti-CD4, cladribine, a polypeptide construct with at least two domains comprising a self-reactive de-immunized antigen or its fragment that is specifically known for its IG receptors of self-reactive B cells (WO 2003/68822), and total body radiation, bone marrow transplantation (antagonist or integrin antibody (po for example an LFA-1 antibody such as efalizumab / RAPTIVA® commercially available from Genentech, or an alpha 4 integrin antibody such as natalizumab / ANTEGREN® available from Biogen, or others as noted above), drugs that treat secondary or related symptoms to vasculitis associated with ANCA (eg, fungal infections and others) such as those noted herein, spheroids such as corticosteroids (eg prednisolone, methylprednisolone such as SOLU-MEDROL ™ methylprednisolone sodium succinate for injection, prednisone such as low dose prednisone, dexamethasone or glucocorticoid, for example by co-injection including systemic corticosteroid therapy), immunosuppressive therapy without lymphocyte depletion (eg, MMF or cyclosporin), drug for class cholesterol reduction " statin "(which includes cerivastatin (BAYCOLMR), fluvastatin (LESCOLMR), atorvastatin (LIPITORMR), lovastatin (MEVACORMR), pravastatin (PRAVACHOLMR), and simvastatin (ZOCORMR)), estradiol, testosterone (optionally at high doses; Stuve et al., Neurology 8: 290-301 (2002)), androgen, hormone replacement therapy, a TNF inhibitor such as a TNF-alpha antibody, DMARD, NSAID, plasmapheresis or plasma exchange , trimethoprim-sulfamethoxazole (BACTRIMMR, SEPTRAMR), mycophenolate mofetil, H2 blockers or proton pump inhibitors (during the use of potential ulcerogenic immunosuppressive therapy), levothyroxine, cyclosporin A (eg SANDIMMUNE®), somatastatin analogue, cytokine, antagonist or anti-cytokine antibody, antimetabolite, immunosuppressant agent, rehabilitative surgery, redio-iodine, thyroidectomy, BAFF antagonist such as BAFF or BR3 antibodies or immunoadhesins, anti-CD40 receptor or anti-CD40 ligand (CD154), anti-receptor antagonist / antibody -IL-6, another antagonist or B-cell surface antibody such as humanized 2H7 or other humanized or human CD20 antibody with rituximab, etc. Medications of these preferred are a chemo-therapeutic agent, a cytotoxic agent, anti-integrin, gamma globulin, anti CD4, cladribine, trimethoprimusulfamethoxazole, an H2 blocker, a proton pump inhibitor, a corticosteroid, cyclosporine, cholesterol-lowering drug of the class statin, estradiol, testosterone, androgen, drug for hormone replacement, a TNF inhibitor, DMARD, NSAID (to treat for example musculoskeletal symptoms), levothyroxine, cyclosporin A, somatostatin analogue, cytokine antagonist, or receptor antagonist of cytokine, antimetabolite, BAFF antagonist such as BAFF antibody or BR3 antibody, especially a BAFF antibody, immunosuppressive agent or other B cell surface marker antibody, such as a combination of rituximab and a humanized 2H7 or other humanized CD20 antibody . The most preferred of these medicaments are a chemo-therapeutic agent, an immunosuppressive agent including an antibody against TNF alpha, an antibody against ligand CD40-CD40, and a BAFF antagonist such as antibody BR3 or BAFF, a DMARD, a cytotoxic agent , an integrin antagonist, an NSAID, a cytokine antagonist, or a hormone or a combination thereof. Immuno-suppressors may be required, for example for a highly active disease with increased organ involvement, and include agents such as coclifosfamide (CYTOXAN®), chlorambucil, leflunomide, MMF, azathioprine (IMURAN®) and methotrexate. BAFF antagonists may be useful in combination with the first medication for efficacy. Still more preferred is a steroid, chemo-therapeutic agent, immunosuppressant agent, cytotoxic agent, integrin antagonist, cytokine antagonist, or a hormone, or combination thereof, more preferably a steroid and / or an immunogenic agent. suppressant, even more preferably, a corticosteroid and / or an immunosuppressive agent. In a particularly preferred embodiment, the second medicament is or comprises one or more steroids, for example a corticosteroid which is preferably prednisone, prednisolone, methylprednisolone, hydrocortisone or dexamethasone. This steroid is preferably administered in minor amounts which are employed if the first medicament a CD20 antibody is not administered to a patient treated with steroid. In one aspect, the steroid is not administered with any second exposure of antibody or is administered with the second exposure but in smaller amounts than those used with the initial antibody exposure. It is also preferred when the steroid is not administered with third or subsequent antibody exposures. In a further particularly preferred aspect, the second medicament is an immunosuppressive agent more preferably cyclophosphamide, MMF, chlorambucil, azathioprine, leflunomide, or methotrexate, and is preferably administered at least with the initial exposure of antibody. In one modality, acetioprine, methotrexate or MMF are preferably used in place of cyclophosphamide for maintenance of remission. In an even more preferred aspect, the second drug is a combination of one or more spheroids and immunosuppressive agent. Prophylactic treatment of vasculitis associated with ANCA with (DIFLUCANMR) orally for fungal infection can also be used, as well as trimetorpim-sulfamethoxasol (480 mg) three times weekly for prophylactic treatment of patients with pneumocystis carinii. Jayne and Rasmussen, supra. All these second drugs can be used in combination with each other or with the first drug, so that the term "second drug" as used here does not mean that it is the only medication apart from the first medicine, respectively. In this way, the second medication does not need to be a medication, but it can constitute and comprise more than one drug. These second drugs, as established herein, are generally used in the same doses and routes of administration as previously used or approximately 1 to 99% of the doses used to date. If these second medications are in fact used, they are preferably used in smaller quantities than if the first medication was not present, especially in subsequent doses beyond the initial dose with the first medication, to eliminate or reduce side effects caused in this way . For the re-treatment method herein, when a second medicament is administered in an effective amount with an antibody exposure, it can be administered with any exposure, for example only with one exposure or with more than one exposure. In one embodiment, the second medication is administered with the initial exposure. In another, modality, the second medication is administered with the initial and second exposures. In a still further embodiment, the second drug is administered with all exposures. It is preferred that after the initial exposure, such as spheroid, the amount of this second medicament be reduced or eliminated in order to reduce the subject's exposure to an agent with side effects such as prednisone, prednisolone, methylprednisolone and cyclophosphamide. As a specific example, treatment of patients with microscopic polyangiitis and egener granulomatosis has three phases: (1) induction of remission, (2) maintenance of remission, and (3) relapse treatment. Current induction therapy often consists of cyclophosphamide (CYTOXAN®) and short-steroids. This includes a high dose of intravenous methylprednisolone for several days (for example 1 to 5 days), plus reduced oral prednisone for a period of time, such as 3 to 5 months. For aggressive disease, the use of high-dose intravenous methylprednisolone for three days, as recommended, combined with intravenous or oral cyclophosphamide. The reduced doses of prednisone preferences continue along with maintenance of cyclophosphamide for 12 to 18 months. When the first medication is used, that amount and frequency of doses of preference are further reduced, since the lower dose of steroids that control the disease should be employed. The infection should be considered if the symptoms seem to be exacerbated. For patients in sustained remission at 12 months, it is preferred that the use of all these second medications be interrupted at a higher rate with the first medication administered here without it. Patients whose symptoms are in good control, however, should be closely followed at six-month intervals for signs and symptoms of relapse. During treatment with these agents, complete blood counts and liver function tests should be reviewed periodically.

The combined administration of a second drug includes co-administration (concurrent 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 agents assets (medicine) simultaneously exercise their biological activities. The antibody or antagonist herein is administered by any convenient means, including topical parenterals, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and / or intralesional administration. Parenteral infusions include intramuscular, intravenous (i.v.), intraarterial, intraperitoneal, or subcutaneous administration.

Intrathecal administration is also contemplated (see for example US 2002/0009444, Grillo-Lopez, A concerning intrathecal delivery of a CD20 antibody). In addition, the antibody or antagonist can be conveniently administered by pulse infusion, for example with decreasing doses of the antibody or antagonist. Preferably, the dose is delivered intravenously or subcutaneously and more preferably by one or several intravenous infusions. If multiple antibody exposures are provided, each exposure can be provided using the same or different administration means. In one embodiment, each exposure is by intravenous administration. In another embodiment, each exposure is delivered by subcutaneous administration. In yet another embodiment, exposures are delivered by both intravenous and subcutaneous administration. In one embodiment, the CD20 antibody is administered as a slow intravenous infusion instead of an intravenous bolus or impulse. For example, a steroid such as prednisolone or methylprednisolone (e.g., about 80-120 mg i.v., more specifically about 100 mg i.v.) is administered approximately 30 minutes before any infusion of the CD20 antibody. The CD20 antibody for example is infused through a dedicated line. For the initial dose of exposure of multiple doses to CD20 antibody, or for the single dose if exposure involves only one dose, this infusion preferably starts at an expense of approximately 50 mg / hour. This can be increased in scale, for example at increments of about 50 mg / hour every 30 minutes to a maximum of about 400 mg / hour. However, if the subject experiences an infusion-related reaction, the infusion rate is preferably reduced, for example to half the current expense, for example from 100 mg / h to 50 mg / h. Preferably, the infusion of this dose of CD20 antibodies (for example to a total dose of approximately 1000 mg) is completed to approximately 255 minutes (4 hours 15 minutes). Optionally, the subjects receive a prophylactic treatment of acetaminophen / paracetamol (for example about 1 g) and diphenhydramine HC1 (for example about 50 mg or equivalent dose of similar agents) per mouth about 30 to 60 minutes before the start of the infusion. If more than one infusion (dose) of CD20 antibody is delivered to achieve full exposure, the second or subsequent infusions of CD20 antibody in this infusion mode are preferably initiated at a rate higher than the initial infusion, for example to approximately 100. mg / hour. This speed can be adjusted in scale, for example at an increase rate of about 100 mg / hour each about 30 minutes to a maximum of about 400 mg / hour. Subjects who experience an infusion-related reaction preferably have the infusion rate reduced to half that speed, for example from 100 mg / hr to 50 mg / hr. Preferably, the infusion of this second or subsequent dose of CD20 antibodies (e.g., a total dose of approximately 1000 mg) is completed for approximately 195 minutes (3 hours 15 minutes). A discussion of methods to produce, modify and formulate these antibodies follows. III. Production of Antibodies The methods and articles of manufacture of the present invention utilize, or incorporate an antibody that binds to a cell surface marker B, especially that which binds to CD20. Accordingly, methods for generating these antibodies will be described herein. CD20 antigen to be used for production of, or monitoring of antibody (s) can be for example a soluble form of CD20 or a portion thereof containing the desired epitope. Alternatively or additionally, cells expressing CD20 on their cell surface can be used to generate, or monitor antibody (s). Other forms of CD20 useful for generating antibodies will be apparent to those skilled in the art following the description as to exemplary techniques for production of the antibodies employed in accordance with the present invention. (i) Polyclonal Antibodies Polyclonal antibodies are preferably developed in animals by multiple subcutaneous (s.c.) or intraperitoneal (i.p) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, for example hemocyanin from sea urchin, serum albumin, bovine triroglobulin, or trypsin inhibitor from soy using a derivatizing or bifunctional agent, example maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, S0C12 or R1N = C = NR, wherein R and R1 are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates or derivatives by combining eg 100 μg or 5g of the protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites . One month later, the animals are boosted with 1/5 to 1/10 of the original amount of conjugated peptide in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. The animals are reinforced until the title reaches a plateau. Preferably, the animal is boosted with the conjugate of the same antigen but conjugated to a different protein and / or through a different reagent reagent. Conjugates can also be made in recombinant cell culture as protein fusions. Also, aggregation agents such as alum, are conveniently employed to improve the immune response. (ii) Monoclonal Antibodies Monoclonal antibodies are obtained from a substantially homogenous population of antibodies, ie the individual antibodies comprising the population are identical and / or ligate the same epitope except for possible variants that arise during production of the monoclonal antibody. These variants are generally present in smaller quantities. In this way, the "monoclonal" modifier indicates the character of the antibody that is not a mixture of discrete or polyclonal antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or they can be made by the recombinant DNA method (U.S. Patent No. 4,816,567). 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 specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a convenient 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 cells thus prepared are seeded and grown in a convenient culture medium which preferably contains one or more substances that inhibit the growth or survival of pre-fusion myeloma cells without fusion. For example, if the precursor 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), these substances prevent cell growth. deficient in HGPRT.

Preferred myeloma cells are those that are efficiently fused, support high stable level production of antibody by select antibody producing cells and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63- cells. Ag8-653 available from American Type Culture Collection, Rockville, Maryland USA. Human-mouse and human myeloma heteromyeloma cell lines have also been described for human monoclonal antibody reproduction (Kozbor, J. Immunol., 133: 3001 (1984)).; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which the hybridoma cells are grown is tested for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody for example can be determined by the Scatchard analysis of Munson et al., Anal. Biochem. , 107: 220 (1980). After hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity are identified, the cells can be subcloned by limiting dilution procedures and developed by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59 -103 (Academic Press, 1986)). Suitable culture medium for this purpose includes, for example, D-MEM or RPMI-1640 media. In addition, the hybridoma cells can develop in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the isolates are conveniently separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures, such as for example protein A-SEPHAROSEMR, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. . DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies) the hybridoma cells serve as a preferred source of this DNA. Once isolated, the DNA can be isolated. Once isolated, the DNA can be placed in expression vectors, which are then transected into host cells such as E. coli cells, simian COS cells, Chinese hamster 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. Review articles on recombinant expression in DNA bacteria encoding the antibody include Skerra et al., Curr. Opinion in 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) describe the isolation of murine and human antibodies respectively using phage libraries.

Subsequent publications describe the production of high affinity human antibodies (n-rank) by chain intermixing (Marks et al., Bio / 'Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy to build very large phage libraries (Waterhouse et al., Nuc Acids, Res., 21: 2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation for monoclonal antibodies. DNA can also be modified, for example by substituting the coding sequence for human light and heavy chain constant domains in place of homologous murine sequences (U.S. Patent Number 4,816,567); Morrison, et al., Proc. Nati Acad. Sci. USA, 81: 6851 U984)), or by covalent attachment to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically these non-immunoglobulin polypeptides are substituted by the constant domains of an antibody or substituted by the variable domains of an antigen combining site of an antibody to create a chimeric bivalent antibody comprising an antigen combining site having specificity for an antigen and another antigen combining site that has specificity for a different antigen. In addition, antibodies comprising a variant Fe region with high affinity for FcyR are useful for treating diseases wherein an improved efficacy of effector cell function is desired, such as autoimmune diseases, as set forth for example in US 2005 / 0037000 and WO 2004/63351 (Macrogenics, Inc. STAVENHAGEN et al.). (iii) 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 "import" residues, which are typically taken from a variable "import" domain. Humanization can be performed essentially following the method of Winter and co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al. ., Science, 239: 1534-1536 (1988)), by replacing the hypervariable region sequences with the corresponding sequences of a human antibody. Accordingly, these "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain is replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies wherein some hypervariable region residues and possibly some FR residues are substituted for residues of analogous sites in rodent antibodies. The selection of human variable domains, both light and heavy, to be used in producing humanized antibodies is very important in reducing antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is monitored against the entire library of known human variable domain sequences. The human sequence that is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993); Chothia et al., J Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework 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 precursor sequences and various conceptual humanized products using three-dimensional models of the precursor and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and exhibit probable tri-dimensional conformation structures of selected candidate immunoglobulin sequences. Inspection of these exhibits allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, ie the residue analysis that influences the ability of the candidate immunoglobulin to bind its antigen. In this way, RF residues can be selected and combined from the container and import sequences in such a manner that the desired antibody characteristic, such as increased affinity for the target or target antigen (s), is achieved. In general, these hypervariable region residues are directly and more substantially involved in influencing the antigen binding. (iv) Human antibodies As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies, in the absence of endogenous immunoglobulin production. For example, it has been reported that homozygous deletion of the antibody heavy chain binding region (JH) gene in germline and chimeric mice results in complete inhibition of endogenous antibody production. The transfer of the set of human germline immunoglobulin genes in this germline mutant mouse will result in the production of human antibodies upon antigen testing. See, for example, 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 U.S. Pat. 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 human antibodies and antibody fragments in vitro, from repertoires of variable domain immunoglobulin gene (V ) from non-immunized donors. According to this technique, the V antibody domain genes are cloned in-frame in either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and exhibit as functional antibody fragments in the surface of the phage particle. Because the filamentous particle contains a copy of single-stranded DNA from the phage genome, selections based on functional properties of the antibody also result in selection of the gene encoding the antibody that exhibits these properties. In this way, the phage mimic some of the properties of the B cell. The phage display can be made in a variety of formats; for review see, for example, Johnson et al., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolate a diverse set of anti-oxazolone antibodies from a small random V pool of genes derived from the vessels of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed and antibodies to a diverse set of antigens (including auto-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993). See, also the patents of the U.S. Nos. 5,565,332 and 5,573,905. Human antibodies can also be generated by B cells activated in vitro (see U.S. Patent 5,567,610 and 5,229,275). (v) Antibody fragments Various techniques have been developed for the production of antibody fragments.

Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, for example, 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, antibody fragments can be isolated from the antibody phage libraries discussed above. 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 approach, F (ab ') 2 fragments can be isolated directly from the culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to the person skilled in the art. In other embodiments, the selection antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and US patent. No. 5,587,458. The antibody fragment can also be a "linear antibody", for example, as described in U.S. Pat. 5,641,870 for example. These linear antibody fragments can be monospecific or bispecific. (vi) Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the CD20 antigen. Other of these antibodies can bind CD20 and in addition bind a second B cell surface marker. Alternatively, an anti-CD20 binding arm can be combined with an arm that binds an activation molecule on a leukocyte such as a receptor molecule. T cell (for example CD2 or CD3), or Fe receptors for IgG (Fc / R), such as Fc / RI (CD64), Fc RII (CD32) and Fc / RIII (CD16) in order to focus cellular defense mechanisms in cell B. Bispecific antibodies; - can also be used to localize cytotoxic agents to the B cell. These antibodies have a CD20 binding arm and an arm that binds the cytotoxic agent (eg, saporin, anti-interferon-a, vinca alvaloidr, ricin A chain, methotrexate or the radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., bispecific antibodies F (ab ') 2). Methods for producing bispecific antibodies are known in the art. Traditional production of integral length bispecific antibodies are based on the co-expression of two light chain-heavy chain immunoglobulin pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). ). Due to the random assortment of 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 problematic, and product yields are low. Similar procedures are described in WO 93/08829, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). According to a different approach, variable domains of antibody with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the necessary site for light chain linkage present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host organism. This allows greater flexibility to adjust the mutual proportions of the three polypeptide fragments in embodiments where different proportions of the three polypeptide chains used in the construction provide the optimum yields. Without . -but it is possible to insert ... the sequences, coding for two or all: the three polypeptide chains in an expression vector when the expression of at least two polypeptide chains in equal proportions results in high yields or when the proportions are not particular significance. In a preferred embodiment of this approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a light chain-heavy immunoglobulin heavy chain pair (which provides a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a form of easy separation. This approach is described in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the one inferred between a pair of antibody molecules can be engineered to maximize the "percent" of heterodimers that are recovered from recombinant cell culture.The preferred variant comprises at least a portion of the CH3 domain of a constant antibody domain In this method, one or more small amino acid side chains from the inferium of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). "Compensatory" cavities of identical size or Similar to the one or the large side chains are created in the inferium of the second antibody molecule by replacing large amino acid side chains with smaller ones [eg, alanine or threonine.] This provides a mechanism to increase the yield of the heterodimer over other undesired end products such as homodimers.

Bispecific antibodies include interlaced or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled with avidin, the other with biotin. These antibodies, for example, have been proposed to be targeted in cells of immune system to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (O 91/00360, O 92/200373, and EP 03089 ). Heteroconjugate antibodies can be made using any convenient crosslinking methods. Suitable crosslinking agents are well known in the art, and are described in US Pat. No. 4,676,980, together with a number of crosslinking techniques.

Techniques for generating bispecific antibodies to antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a method wherein intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the complex agent before dithiol sodium arsenite to stabilize vicinal dithiols and avoid intermolecular disulfide formation. The generated Fab 'fragments are then converted into thionitrobenzoate derivatives (TNB). One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixing with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by gene fusion. The antibody homodimers were reduced in the hinge region 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 "diabody" technology described by Hollinger et al., Proc. Nati Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for producing bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two sites. of antigen binding. Another strategy for producing bispecific antibody fragments by the use of single chain dimers Fv (sFv) has also been reported. See Gruber et al., J. Immunol., 152: 5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trichispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991). IV. Conjugates and Other Modifications of the Antibody The antibody used in the methods or included in the articles of manufacture herein is optionally conjugated to a cytotoxic agent. For example, the antibody (CD20) can be conjugated to a drug as described in WO2004 / 032828.

Chemotherapeutic agents useful in the generation of these antibody-cytotoxic agent conjugates have been described above. Conjugates of an antibody and one or more small molecule toxins, such as calicheamicin, a maytansin (U.S. Patent No. 5,208,020), a trichotine, and CC1065 are also contemplated herein. In one embodiment of the invention, the antibody is conjugated to one or more maytansin molecules (eg, about 1 to about 10 molecules of maytansin per antibody molecule). Maytansin, for example, can be converted to May-SS-Me, which can be reduced in May-SH3 and reacted with a modified antibody (Chari et al., Cancer Research 52: 127-131 (1992)) to generate an antibody conjugate. maytansinoid Alternatively, the antibody is conjugated with one or more calicheamicin molecules. The family of calicheamicin antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogues of calicheamicin that may be employed include, but are not limited to,? , a2t, a3t, N-acetyl- ^ i1, PSAG and T1! (Hinman et al., Cancer Research 53: 3336-3342 (1993) and Lode et al., Cancer ROlesearch 58: 2925-2928 (1998)). 41 Entomatically active toxins and their fragments that can be used include diphtheria A chain, active fragments without diphtheria toxin binding, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii, diantine proteins, proteins of Phytolaca americana (PAPI, PAPII, and PAP-S), and inhibitor of momordica charantia, curcin, crotina, inhibitor of sapaonaria officinalis, gelonin, mitogeline, restrictocin, fenomycin, enomycin and trichortin. See, for example, WO 93/21232 published October 28, 1993. The present invention further contemplates antibody conjugated to a compound with nucleolytic activity (for example a ribonuclease or a DNA endonuclease such as deoxyribonuclease; DNase). A variety of radioactive isotopes are available for the production of conjugated radio antibodies. Examples include At211, I131, I125, Y90, Re186, Re1, Sm1, Bi, Pi, and radioactive isotopes of Lu. Antibody and cytotoxic agent conjugates 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 HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (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, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). L-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) labeled with carbon 14 is an exemplary chelating agent for conjugating radio nucleotide to the antibody. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, a labile acid linker, peptidase sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52: 127-131 (1992)) can be used. Alternatively, a fusion protein comprising the antibody and cytotoxic agent can be made, for example by recombinant techniques or peptide synthesis. In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pre-target, wherein the antibody-receptor conjugate is administered to the subject, followed by removal of unbound conjugate from the circulation using a release agent and then administering a "ligand" (e.g., avidin) that is conjugated with a cytotoxic agent (e.g., a radio nucleotide). The antibodies of the present invention can also be conjugated with a prodrug activation enzyme that converts a prodrug (eg, a peptide chemotherapeutic agent, see WO81 / 01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278. The enzyme component of these conjugates includes any enzyme capable of acting in a prodrug in a way that converts it into its more 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 prodrugs into free drugs; cytokine deaminase useful for converting non-toxic 5-fluorocytokine 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 cleave carbohydrates such as ß-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; /? - lactamase useful for converting drugs derivatized with /? - lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting derivatized drugs into their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity also known in the art as "aczymes, can be used to convert the prodrugs of the invention into free active drugs (see, for example, Massey, Nature 328: 457-458 (1987)). antibody-aczyme can be prepared as described herein for delivery of the enzyme to a population of tumor cells Enzymes of this invention can be covalently linked to the antibody by techniques well known in the art such as the use of crosslinking or crosslinking reagents In alternate form, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of the enzyme of the invention, can be constructed using recombinant DNA techniques either known in the art (see, for example, Neuberger et al., Nature, 312: 604-608 (1984).) Other modifications of the antibody herein are contemplated. For example, the antibody can be linked to one of a variety of non-protein polymers, for example, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Antibody fragments, such as Fab ', linked to one or more PEG molecules are an especially preferred embodiment of the invention. The antibodies described herein can also be formulated as liposomes. Liposomes containing the antibody are prepared by methods known in the art, as described in Epstein et al., Proc. Nati Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Nati Acad. Sci. USA, 77: 4030 (1980); US patents Nos. 4,485,045 and 4,544,545; and O 97/38731 published October 23, 1997. Liposomes with improved circulation time are described in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporative method with a lipid composition comprising phosphatidylcholine, cholesterol and phosphatidylethanolamine derivatized with PEG (PEG-PE). Liposomes are extruded through pore size filters to result in liposomes with the desired diameter. Fab 'fragments of an antibody of the present invention can be conjugated to liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) by a disulfide exchange reaction. A chemotherapeutic agent is optionally contained in the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989). Modification or modifications of amino acid sequence of peptide or protein antibodies described herein are contemplated. For example, it may be convenient to improve the binding affinity and / or other biological properties of the antibody. Sequences of the amino acid sequence of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. These modifications include for example, deletions of, and / or insertions in and / or substitutions of, residues within the amino acid sequences of the antibody. Any combination of elimination, insertion, and substitution is made to arrive at the final construction, provided that the final construction possesses the desired characteristics. Changes of amino acids can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. A useful method for identifying certain residues or regions of the antibody that are preferred locations for mutagenesis is termed "alanine scanning mutagenesis genes" as described by Cunningham and Wells, Science, 244: 1081-1085 (1989). Here, a residue or a group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a negatively or neutrally charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Those amino acid locations demonstrate functional sensitivity to substitutions that are refined by introducing additional or other variants into, or for, substitution sites. In this way, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, it leads random mutagenesis or wing scanning at the target region or codon and the expressed antibody variants are monitored for the desired activity. Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions in the length range of 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 antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusion of the N or C terminus of the antibody of an enzyme, or a polypeptide that increases the serum half-life of the antibody. Another type of variant is a variant in substitution of acidic amino acids. These variants have at least one amino acid residue in the antibody molecule replaced by different residue. The sites of greatest interest for antibody substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 3 under the heading of "preferred substitutions". If these substitutions result in a change in biological activity, then more substantial changes, termed "exemplary substitutions" in Table 3, or as described further below with reference to amino acid class, may be introduced and the products monitored. Table 3 • Substantial modifications in the biological properties of the antibody are achieved by selection of substitutions that differ significantly in their effect to maintain (a) the major structure of the polypeptide in the substitution area, eg, as a leaf or helical conformation, ( b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Amino acids can be grouped according to similarities in the properties of their side chains (in AL Lehninger, in Biochemistry, second ed., Pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M) (2) polar without charge: Gli (G), Ser (S), Thr (T), Cis (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp (D), Glu (E) (4) Basic: Lys ( K), Arg (R), His (H). Residues of natural origin in alternate form, can be divided into groups based on common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie; (2) neutral hydrophilites: Cis, Ser, Thr, Asn, Gln; : (3) Acidics: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gli, Pro; (6) aromatics: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. In contrast, one or more cysteine bonds can or can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment). A particularly preferred type of substitution variant involve replacing one or more hypervariable region residues of a precursor antibody. In general, the resulting variants or variants selected for further development will have improved biological properties with respect to the precursor antibody from which they are generated. A convenient way to generate these substitution variants is defined maturation using phage display. Briefly, several hypervariable region sites (for example S a l sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent form from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage display variants are then monitored for their biological activity (e.g. binding affinity) as described herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to the antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. These contact residues and neighboring residues are candidates for substitution according to the techniques elaborated here. Once these variants are generated, the panel of variants is subjected to supervision 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 antibody alters the original glycosylation pattern of the antibody. Said alteration includes removing one or more carbohydrate moieties that are in the antibody and / or adding one or more glycosylation sites that are not present in the antibody. The glycosylation of polypeptides is typically already N-linked or O-linked. N-linked refers to the connection of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-trionine, where X is an amino acid except proline, are the recognition sequences for enzymatic connection of the carbohydrate portion with the side chain asparagine. In this way, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the connection of one of the sugars N-acetyl galactosamine, galactose or xylose with a hydroxyamino acid, most commonly serine or trionine, although 5-hydroxyproline or 5-hydroxylysine may also be employed. The addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or trionine residues to the original antibody sequence (for 0-linked glycosylation sites.) When the antibody comprises an Fe region, the carbohydrate connected thereto can For example, antibodies with a mature carbohydrate structure lacking fucose connected to an antibody Fe region are described in U.S. Patent Application No. 2003/0157108 (Presta, L.). See also US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.) Antibodies with bisective N-acetylglucosamine (GlcNAc) in the carbohydrate connected to a Fe region of the antibody are reported in WO 2003/011878, Jean-Mairet et al. US 6,602,684, Umana et al Antibodies with at least one galactose residue in the oligo saccharide connected to an Fe region of the antibody are reported in WO 1997/30087, Patel et al., See also WO 1998/58964 (Raju, S. ) and WO 1999/22764 (Raju, S.) relating to antibodies with altered carbohydrate connected to their Fe region. See also US 2005/0123546 (Umana et al.) on antigen binding molecules with modified glycosylation .. The glycosylation variant preferred herein comprises an Fe region, wherein a carbohydrate structure is connected. the Fe region lacks fucose. These variants have improved ADCC function. Optionally, the Fe region further comprises one or more amino acid substitutions that further enhance ADCC, for example substitutions at positions 298, 333 and / or 334 of the Fe region (Eu numbering of residues). Examples of publications refer to "defucosylated" or "fucose deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; OR 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; O2005 / 053742; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech Bioeng. 87: 614 (2004). Examples of cell lines that produce desfucosylated antibodies include CHO Lecl3 cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys., 249: 533-545 (1986); U.S. Patent Application number 2003/0157108 Al, Presta. , L; and WO 2004/056312 Al, Adams et al., Especially in Example 11), and cell lines with genes inoperative with "knockout miles" such as the alpha-1,6-fucosyltransferase gene, to CHO cells with knockout genes FUT8, (Yamane-Ohnuki et al., Biotech, Bioeng 87: 614 (2004)). Acid molecules. nucleic acids encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include but are not limited to isolation from a natural source (in the case of amino acid sequence variants of natural origin) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a variant previously prepared or a non-variant version of the antibody. It may be convenient to modify the antibody of the invention with respect to effector function, for example to improve the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in an Fe region of an antibody. Alternatively or additionally, one or more cysteine residues can be introduced into the Fe region, thereby allowing interchain chain disulfide formation in this region. The homodimeric antibody thus generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) - .. See Caron et al., J. Exp Med. 176: 1191-1195 ( 1992) and'-Shopes, J. Im unol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody can be engineered having dual Fe regions and thus can have improved complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

WO 00/42072 (Presta, L.) discloses antibodies with enhanced ADCC function in the presence of human effector cells, wherein the antibodies comprise amino acid substitutions in their Fe region. Preferably, the antibody with enhanced ADCC comprises substitutions in the positions 298, 333 and / or 334 of the Fe region. Preferably, the altered Fe region is a human IgGl Fe region comprising or consisting of substitutions in one, two or three of these positions.

Antibodies with altered Clq bond and / or "complement dependent cytotoxicity (CDC)" are described in W099 / 51642, U.S. Patent No. 6,194,551B1, U.S. Patent No. 6,242,195B1, U.S. Patent No. 6,528 , 624B1 and U.S. Patent No. 6,538,124 (Idusdgie et al.) The antibodies comprise an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 313, 333 and / or 334 of its Fe region.

To increase the serum half-life of the antibody, a recovery receptor binding epitope can be incorporated into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term "recovery receptor binding epitope" refers to an epitope of the Fe region of an IgG molecule (eg, IgGi, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life. in vivo of the IgG molecule. Antibodies with substitutions in their Fe region and increased serum half-lives are also described in WO00 / 42072 (Presta, L.).

Engineering antibodies with three or more (preferably four) functional antigen binding sites are also contemplated (U.S. Patent Application number US2002 / 0004587 Al, Miller et al.). V. Pharmaceutical Formulations Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable stabilizing carriers and excipients. { Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Suitable excipient carriers or stabilizers are non-toxic to the containers at the doses and concentrations employed and include buffers such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol butyl or benzyl alcohol, alkyl parabens such as methyl propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as. polyvinyl pyrrolidone; amino acids such as glycine; glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). Exemplary anti-CD20 antibody formulations are described in W098 / 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, 20 to 0.02% polysorbate at pH 5.0 which has a shelf life of at least two years. storage at 2-8 degrees C. Another anti-CD20 formulation of interest comprises 10 mg / mL of rituximab in 9.0 mg / mL of sodium chloride, 7.35 mg / mL of sodium citrate dihydrate, 0.7 mg / mL of polysorbate 80, and sterile water for injection, pH 6.5.

Freeze-dried formulations adapted for subcutaneous administration are described in U.S. Pat. No. 6,267,958 (Andya et al.). These "lyophilized formulations can be reconstituted with a convenient diluent at a high protein concentration and the reconstituted formulation can be administered subcutaneously to the mammal to be treated here.

Crystallized forms of the antibody are also contemplated. See for example US 2002 / 0136719A1 (Shenoy et al.). The present formulation may also contain more than one active compound (a second medicament as noted above) as necessary, preferably those with complementary activities that do not adversely affect each other. The type and effective amounts of these drugs depend for example on the amount of antibody present in the formulation and clinical parameters of the subjects. The preferred drugs were noted above.

The active ingredients can also be entrapped in microcapsules prepared for example by preservation techniques or by interfacial polymerization, for example hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacrylate) microcapsules respectively, in colloidal drug delivery systems (e.g., liposomes, microspheres of albumin, microemulsions, nanoparticles and nano capsules) or in macroemulsions. These techniques are described in Remington's Pharmaceutical Sciences 16th editi.on, Osol, A. Ed. (1980) ..

Sustained-release preparations can be made. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, these matrices are in the form of shaped articles, for example films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), L-glutamic acid copolymers and? ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT (injectable microspheres composed of copolymer of lactic acid-glycolic acid and leuprolide acetate) and poly-D- (-) - acid 3-hydroxybutyric.

The formulations to be used for in vivo administration must be sterile. This is easily achieved by ration through sterile ration membranes. SAW . Articles of manufacture In another embodiment of the invention, articles of manufacture containing materials useful for the treatment of vasculitis associated with ANCA described above, are provided. In one aspect, the article of manufacture comprises (a) a container comprising an antagonist that binds to a B cell surface marker (e.g., an antibody that binds, including a CD20 antibody) (preferably the container comprises the antagonist or antibody and a pharmaceutically acceptable carrier or diluent within the container); and (b) a packing insert with instructions for treating vasculitis associated with ANCA in a patient, wherein the instructions indicate that a dose of the antagonist or antibody of about 400 mg to 1.3 grams at a frequency of one to three doses is administered at patient within a period of approximately one month. In this manner, the invention provides a manufacturing article comprising: a container comprising a CD20 antibody, or an antibody or antagonist that binds a B cell surface marker, and a packaging insert with instructions for treating vasculitis associated with ANCa in a patient, wherein the instructions indicate that a dose of the CD20 antibody, or the antibody or antagonist that binds to a B cell surface marker, of about 400 mg to 1.3 grams, at a frequency of one to three doses, is administers the patient within a period of approximately one month. In a preferred embodiment, the article of manufacture herein further comprises a container comprising a second medicament, wherein the antagonist or antibody is a first membrane. This article further comprises instructions on the package insert, to treat the patient with the second medication, in an effective amount. The second medicament can be any of those set forth above with a second exemplary medicament which is a chemotherapeutic agent, an immunosuppressive agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist or a hormone. The second preferred drugs are those preferred as set forth above and more preferred is a steroid or an immunosuppressive agent or both. In another aspect, the invention provides an article of manufacture comprising: (a) a container comprising an antibody that binds to a B cell surface marker (e.g., a CD20 antibody) (preferably the container comprises the antibody and a pharmaceutically acceptable carrier or diluent within the container); and (b) a packaging insert with instructions for treating vasculitis associated with ANCA in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial antibody challenge. Followed by a second antibody exposure, where the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure. Preferably, the package insert is provided with instructions for treating ANCA-associated vasculitis in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial antibody exposure of about 0.5 to 4. grams, followed by a second antibody exposure of approximately 0.5 to 4 grams, wherein the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure and each of the antibody exposures is given to the subject is approximately 1 to 4 doses, preferably as a single dose or as two or three separate doses of antibody. In a specific aspect, an article of manufacture is provided comprising: (a) a container comprising an antibody that binds to a B cell surface marker (e.g., a CD20 antibody) (preferably the container comprises the antibody and a pharmaceutically acceptable carrier or diluent within the container); and (b) a packaging insert with instructions for treating ANCA-associated vasculitis in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial exposure of antibodies followed by a second exposure of the patient. antibody, wherein the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure, and each antibody exposure is provided to the subject as a single dose or as two or three separate doses of antibody. Preferably, the antibody exposures are from about 0.5 to 4 grams. In a preferred embodiment of these inventive aspects, the article of manufacture further comprises a container comprising a second medicament, wherein the antibody is a first medicament and this article further comprises instructions in the packaging insert for treating the medicament. subject with the second medication, in an effective amount. The second medicament can be any of those established above with a second exemplary medicament which is a chemotherapeutic agent, an immuno suppressive agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist or a hormone, more preferably a steroid or an agent immunosuppressant or both. In all these aspects, the packing insert is in or associated with the container. Convenient containers include, for example, bottles, ampoules, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective to treat vasculitis associated with ANCA and may have a sterile access gate (e.g. the container may be an intravenous solution bag or a vial having a plug pierced by an injection needle. hypodermic). At least one active agent in the composition is the antagonist or antibody. The label or package insert indicates that the composition is used to treat vasculitis associated with ANCA in a patient or subject amenable to treatment with specific guidance regarding dosage amounts and ranges of antagonist or antibody and any other medication that is provided. The article of manufacture may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer such as bacteriostatic water for injection (B FI), buffered saline with phosphate, Ringer's solution, and / or dextrose solution. The article of manufacture can also include other suitable materials from a commercial and user point of view, including other shock absorbers, diluents, filters, needles and syringes. Further details of the invention are illustrated by the following non-limiting examples. The descriptions of all citations in the specification are expressly incorporated herein by reference.

Example 1 Efficacy Study of Rituximab in Patients with Wegener's Granulomatosis This study estimates the superiority of efficacy and safety of rituximab (MABTHERA® / RITUXAN®) in a certain dose regime compared to placebo for the treatment of signs and symptoms in patients with Wegener's granulomatosis that exhibit one or more symptoms of systemic disease. Rituximab (1000 mg i.v. x 2) is administered i.v. in two initial doses on days 1 and 15 with 1 mg / kg of oral prednisone, daily (which is reduced to 40 mg / day in week 4, and reduces utilizárido 'a standardized reduction regimen resulting in the complete interruption of prednisone during 'the' following 3 to 5 months). This experimental regimen is compared with the same regimen except using placebo rituximab instead of rituximab, with 1: 1 randomization between the two arms of the study, with approximately 48 patients per arm (total of 96 patients). Active disease is defined as Wegener's granulomatosis / Birmingham vasculitis activity score (BVAS / WG) greater than zero. For test inclusion, the patient's BVAS / WG rating must be 3 or higher (or has been 3 or higher within 28 days of randomization). Each main item in the form of evaluation 3 is scored 3 points. Each minor item is rated 1 point. To determine the degree of activity of the disease, the investigator will distinguish between active vasculitis (new / worse BVAS / G items as opposed to persistent items) and permanent organ damage (caused by previously active vasculitis). Severe sudden recurrence or worsening of symptoms is a new occurrence of one or more larger BVAS / WG items. (Major items have a * on the BVAS / WG rating sheet). In general, these sudden recurrences are treated by increases in the dose of prednisone and dose of cyclophosphamide. Severe Wegener's granulomatosis occurs in a patient whose disease is not classified as limited but defined below. A limited sudden recurrence is a new occurrence of one or more smaller BVAS / WG items. In general, these sudden occurrences are treated with increases in doses of prednisone or an increase in doses of methotrexate. Wegener's limited granulomatosis occurs in a patient who meets the modified criteria of the American College of Rheumatology (ACR) for a diagnosis of Wegener's granulomatosis but does not have the disease that poses an immediate threat to either a critical individual organ or to life of the patient. Specifically, this means that: The patient does not have red blood cells in the urine. • If hematuria is present (but there is no + RBC), serum creatinine should be less than or equal to 1.4 and there should be evidence that there is no increase in creatinine more than 25% above the baseline of the patient. • Lung involvement must be circumscribed, so that p02 of the ambient air is > . 70 mmHg or O2 saturation of ambient air by pulse oximetry is > 92% Pulmonary hemorrhage can be treated as a limited disease whenever there is no evidence of progress of the process. In the absence of advance data, pulmonary hemorrhage can be treated as a severe disease at the discretion of the physician. • There can be no disease within any other critical organ (for example, the gastrointestinal tract, eyes, central nervous system) that, without the immediate institution of maximum therapy (ie, methylprednisolone pulsed and daily oral cyclophosphamide), threatens function of that organ and / or the life of the patient.

A newly diagnosed patient is a patient in his first course of treatment of corticosteroids and / or a chemotherapeutic agent or immunosuppressant for Wegener's granulomatosis, without a history of increased immunosuppressive therapy before entering the study. For the purpose of rating BVAS / WG, persistent disease is defined as the presence of ongoing disease activity present in the previous test evaluation (ie, with no new or worse activity). A skin test derived from purified protein can be used. to detect latent tuberculosis infection. A refractory patient is a patient with a history of immunosuppressive therapy (corticosteroids and / or an immunosuppressive agent or chemotherapeutic agent) before the start of treatment for Wegener's granulomatosis activity that makes the patient susceptible to this study. Patients will be considered in referral when their BVAS / WG rating is 0. This regimen based on rituximab tests the current standard of care and is intended to limit the patient's exposure to steroids and their known toxicities, and to demonstrate improved net clinical benefit . Patients were monitored for disease activity, use of additional immune suppressors, use of spheroids and safety events on the length of the one-year trial, with the primary efficacy endpoint of the test measured at three months, followed by 16.8 months A safe follow-up is required up to 12 months after the last dose of rituximab or return of ANCA in the. normal range what happens next. The primary objective is to determine the proportion of patients who achieve a BVAS / -WG score of 0 and successful prednisone reduction at 6 months, and without pre-specified adverse events. It is predicted and expected that rituximab (or a humanized 2H7 replaced by rituximab) is effective to induce remission (achieve BVAS / WG ratings of 0) in at least 80% of the patients participating with Wegener's granulomatosis and to allow dose reduction of spheroids on the control arm. BVAS / WG is expected to decrease from the entry rating to approximately 0.2 to 0.4 in week 14. C-reactive protein (mg / L) is expected to decrease from entry level to a range of approximately 3 to 11 per week 14. Average prednisolone dose (mg / day) is expected to decrease from the entry value to a statistically significantly lower value in week 14. Relapse is expected to occur in less than 5 patients after an average of 27 weeks . If the average BVAS / WG input is 3.6, at 6 months of treatment, it is expected to decrease to a statistically significant value of 0.6. Intermittently active disease is expected to be seen in less than 70% of patients. In . In contrast, the control arm is expected to show much lower decrease in BVAS / WG and C-reactive protein and in use of steroids and fewer patients in remission. Example 2 Efficiency Study of Rituximab in Patients with Microscopic Polyangiitis The protocol in Example 1 is followed except that patients are treated for microscopic polyangiitis. Similar results are expected for Wegener's granulomatosis, meaning that remission, as measured by the BVAS / WG score of 0, is expected to occur in at least 80% of the patients treated in the study arm and that the Steroid use is expected to decrease over the course of the study, these results are expected to be much better in a statistically significant sense than the control results.

Example 3 Efficacy Re-treatment Study of Rituximab in Patients with Wegener's Granulomatosis This study estimates the superiority of efficacy and safety of re-treatment with rituximab (MABTHERA® / RITUXAN®) compared to placebo in adult subjects with Wegener's granulomatosis. Study I examines acute disease, either first presentation or relapse (BVAS "ir10; n = 16), study II examines persistent disease (BVAS 14, n = 16). Patients receive rituximab (1 g iv) in three initial doses days 1, 8, and 15 for studies I and II Concomitant therapy in study I includes 1 mg / kg / day of oral prednisone redud according to the regimen in Example I and cyclophosphamide (according to the standard treatment Patients in study II receive rituximab and 1 mg / kg / day of reduced oral prednisone according to the regimen in Example 1. All subjects receive a second infusion course of placebo / rituximab 1000 mg iv separated by days in weeks 24 and 26, respectively, without steroids or cyclophosphamide, whether the patients exhibited symptoms or were in complete remission.Rituximab treatment courses should be separated by a minimum interval of 16 weeks.The experimental regimens are compared with rituximab placebo + the same doses of prednisone and reduced cyclophosphamide (Study I), or reduced oral prednisone (Study II). Changes in immunosuppressive drugs are not allowed during the studies, unless dictated by toxicity, and requests to reduce the drug other than oral prednisone should be discussed in advance with the Medical Monitor. Study staff will train on how to properly administer rituximab. Subjects may be hospitalized for observation, particularly for their first infusion, at the discretion of the investigator. Rituximab should be administered under close supervision and full resuscitation facilities should be available immediately. Patients are monitored monthly for 12 months for disease activity, use of additional immunosuppressants, sudden recurrence or worsening of disease symptoms, use of prednisone, and safety events during the 52 weeks of the study. The extreme point of primary efficacy of the test is 52 weeks, and measures of effectiveness are estimated by a single Examination Advisor who is not involved with the patient's treatment or other study procedures. Patients are estimated by their BVAS / WG ratings and successful prednisone reduction. At the end of 52 weeks, subjects receiving placebo of rituximab or rituximab but demonstrating a BVAS / WG score of 0 and successful prednisone reduction at 6 months will complete the study participation. Subjects receiving rituximab but who have not demonstrated such rating at 52 weeks are observed for 6 months after the last course of rituximab or up to a BVAS / WG score of 0, whichever occurs first. Sites will be informed ie a subject should continue to follow up, but not if the subject receives placebo or rituximab. Safety follow-up is required up to 12 months after the last dose of rituximab or a BVAS / WG rating of 0, whichever occurs later.

These regimens based on rituximab test the current standard of care, and are expected to demonstrate improved clinical benefit net, with the primary objective of determining the proportion of patients achieving the primary endpoint of a BVAS / G score of 0, and successful prednisone reduction. It is predicted and expected that the administration of rituximab or a humanized 2H7 to the subject in the protocols of Studies I and II previously established, will induce remission (achieving BVAS / WG scores of 0) in at least 80% of the patients participating with Wegener's granulomatosis. and will allow dose reduction of steroids on the control arm. BVAS / WG is expected to decrease from the entry qualification to approximately 0.2 to 0.4 in. week 14. Reactive protine C · (mg / L) is expected to decrease from the entry level to a range of approximately 3 to 11 in week 14. Average spheroid use for both Studies I and II is expected to decrease from value at entry to a significantly lower value from the statistical point of view in week 14. Relapse is expected to occur in less than five patients after an average of 27 weeks. These results are expected to be significantly better than those of the control arms for Studies I and II.

It is also expected that approximately at week 48-54, another dose of 2-g of rituximab delivered immediately or dispersed over approximately 14-16 days in 1-gram amounts will be effective to treat egener's granulomatosis throughout the second year. (causing a BVAS / WG score of 0 in at least 80% of the participating patients), with or without the spheroids and / or other immunosuppressive agents, with a marked improvement over control patients receiving the rituximab placebo instead of rituximab. In this way, rituximab (or humanized 2H7) will be administered initially within approximately a period of 2 weeks, followed by another treatment at approximately 4-8 months, followed by another treatment approximately one year after treatment. ... initial (measured from time in any of the doses given), followed by treatment at approximately two years of initial treatment, with expected success, in approximately one gram x 2-4 doses per treatment, administered together, approximately weekly or approximately every week sautéed for approximately two to four weeks. This re-treatment protocol is expected to be successfully used for several years with few or no adverse effects. Example 4 Second Re-treatment Study of Efficacy of Rituximab in Patients with Wegener's Granulomatosis This study is the same as in Example 3 except that the initial dose of rituximab or placebo rituximab is given as 1000 mg i.v. x 2 (day 0, with the second infusion occurring on Day 15 +/- 1 day), and the subsequent course of rituximab or placebo infusions administered at weeks 24 and 26 and consisting of 1 of 2 biweekly doses administered alone to those subjects in remission, for example, those who do not exhibit increased activity of the disease, such as by increasing ANCA titres, sustained high ANCA titers and other symptoms. All other criteria are the same. It is predicted and expected that the administration of rituximab or a humanized 2H7 to the subject in previously established scheduled re-dosing protocols, will be effective to induce remission (achieving BVAS / G ratings of 0) in at least 80% of the patients participating with granulomatosis. of Wegener and to allow reduction in doses of steroids on the control arm. BVAS / WG is expected to decrease from the entry score to approximately 0.2 to 0.4 in week 14. C-reactive protein (mg / L) is expected to decrease from the entry level to a range of approximately 3 to 11 in the week 14. Average prednisolone dose (mg / day) is expected to decrease from the entry value to a statistically significant lower value in week 14. Relapse is expected to occur in less than five patients after an average of 27 weeks. These results are expected to be significantly better than those of the control. It is also expected that approximately at week 48-54, another dose of 2-g of the 'CD20 antibody (eg, humanized rituximab or 2H7) delivered immediately or dispersed over about 14-16 days in amounts of 1-gram, will be effective to treat Wegener's granulomatosis throughout the second year (with at least 80% of the participating patients having a BVAS / WG score of 0), with or without the reduction of prednisone and methylprednisolone iv and / or other immunosuppressive agents. In this manner, the CD20 antibody will initially be administered within approximately the 2-week time period, followed by another treatment at approximately 4-8 months, followed by another treatment at approximately one year from the initial treatment (measured from time to time). that any of the doses is given), followed by treatment at approximately two years of the initial treatment, with expected success, in approximately one-gram x 2-4 doses per treatment, administered as a whole, approximately on a weekly basis, or approximately every week sautéed in approximately two to four weeks. The results of this treatment are expected to be much better than those of the placebo control. This re-treatment protocol is expected to be used successfully for several years with little or no adverse effects. Example 5 Third Re-treatment Study of Efficacy of Rituximab in Patients with Wegener's Granulomatosis The results of Example 4 are expected to be successful if patients were initially treated with rituximab and then re-treated with rituximab one year after first being treated. , using the same dose and another protocol of Example 4, except that rituximab is given at one year intervals instead of six month intervals.

Example 6 Efficacy Study of Rituximab in Subjects with Vasculiis Associated with Generalized ANCA A double-masked, multi-centered, randomized placebo control test is performed in patients with vasculitis associated with generalized ANCA using rituximab. Two hundred patients were randomized to either (1) conventional treatment (cyclophosphamide and corticosteroids, followed by azathioprine); or (2) rituximab (plus corticosteroids, initially) for induction of remission, using 1 gram of rituximab on day.1 and again on day 15.

The primary clinical comparison is the ability of rituximab in its. dose regimen and corticosteroids to induce remissions of the disease, as measured by cumulative disease activity at six months. Consistent with the standard duration of treatment for ANDA-associated vasculitis, patients in the conventional therapy arm will receive cyclophosphamide for up to 6 months followed by azathioprine, to complete a total treatment duration of 18 months. To estimate the ability of rituximab to restore tolerance of B cells, patients in both arms of the test will be followed for a total of 18 months.

It is expected that rituximab (or a humanized 2H7 replaced by rituximab) will induce stable remissions in patients with vasculitis associated with ANCA and re-establish B cell tolerance to ANCA-targeted antigens in at least two thirds of patients. It is also expected that rituximab or another CD20 antibody is at least as effective as the conventional treatment regimen for induction and maintenance of remission of the disease, offering substantial advantages over standard therapy by virtue of its superior side-effect profile, for example, much less toxic than chemotherapeutics and spheroids, and better to restore tolerance.

Example 7 Re-treatment Studies of Efficacy of Rituximab in Subjects with Severe Wegener's Granulomatosis or Severe Microscopic Polyangiitis Twenty patients with severe Wegener's granulomatosis active or severe microscopic polyangiitis, positive ANCA test, and BVAS / WG score of at least 3, which do not respond to cyclophosphamide or have contraindications to the use of cyclophosphamide, participate. See the definition of severe Wegener's granulomatosis in Example 1. The regimen for induction of remission consists of oral prednisone (1 mg / kg / day) and rituximab (1 gram on day 1 and 1 gram on day 15). In week 4, prednisone is reduced to 40 mg / day. A standardized reduction regimen continues, resulting in complete discontinuation of prednisone over the next 16 weeks. This is compared to the same regimen, but with the placebo of rituximab instead of rituximab (control study). The protocol stipulates re-treatment with the same 6-month remission induction regimen for all patients, whether they experience a sudden recurrence or a worsening of the symptoms of the disease after reconstitution of B cells, whether they are asymptomatic or not. recurrence of ANCA or ANCA titration coincident or following, B cell reconstitution, or if it is in complete remission. This re-treatment regimen includes the 1 g x 2 two weeks spaced by rituximab and placebo of rituximab. A sudden recurrence or worsening of clinical symptoms in the absence of B cells is considered treatment failure. Patients are estimated monthly for one year. It is expected that patients in the treatment arm will tolerate rituximab infusions well and that their B cells will be rapidly depleted and all will achieve full remission (BVAS / WG of 0) at three months. All patients in the treatment arm are expected to complete the glucocorticoid reduction in 6 months. It is expected that after 12 months, no patient in the treatment arm will experience a sudden recurrence or worsening of clinical symptoms, and that B cells will return in most, if not all, of these patients within 12 months. Apart from glucocorticoids, additional immunosuppressive agents are not expected as necessary for induction of remission and maintenance of sustained remission (6 months or more) in patients treated with rituximab. Example 8 Useful Humanized 2H7 Variants Here Useful for purposes herein, are humanized 2H7 antibodies comprising one, two, three, four, five, or six of the following CDR sequences: CDR sequence L RASSSVSYXH where X is M or L (SEQ ID NO: 35), eg, SEQ ID NO: 4 (Figure 1A), CDR Sequence L2 of SEQ ID NO: 5 (Fig. 1A), CDR Sequence L3 QQWXFNPPT where X is S or A (SEQ ID NO: 36), for example, SEQ ID NO: 6 (Figure 1A), CDR Sequence Hl of SEQ ID NO: 10 (Figure IB), CDR H2 Sequence of AIYPGNGXTSYNQKFKG where X is D or A (SEQ ID NO: 37), for example, SEQ ID NO: 11 (Figure IB), and CDR Sequence H3 of VVYYSXXYWYFDV where the X in position 6 is N, A, Y, W, or D, and X in position 7 is S or R (SEQ ID NO: 38), for example, SEQ ID NO: 12 (Figure IB). The 2H7 antibodies humanized herein, include those with heavy chain amino acid sequences containing C-terminal lysine and those without. The above CDR sequences are generally present within. heavy frame sequences of variable and light human variable, such as substantially the FR residues of human consensus of the subgroup. light chain kappa I (VL6l), and substantially the human consensus FR residues of the subgroup of: human heavy chain III (VHIII). See also WO 2004/056312 (Lowman et al.). The variable heavy region can be linked to a human IGG chain constant region, wherein the region for example can be IgG1 or IgG3, including constant regions of native sequence and non-native sequence. In a preferred embodiment, this antibody comprises the variable heavy domain sequence of SEQ ID NO: 8 (vl6, as shown in Figure IB), optionally also comprises the variable light domain sequence of ID NO: 2 (vl6, as is shown in Figure 1A), which optionally comprises one or more amino acid substitutions at positions 56, 100, and / or 100a, for example D56A, NlOOA, or N100Y and / or SlOOaR, in the variable heavy domain and one or more amino acid substitutions at positions 32 and / or 92, for example M32L and / or S92A, in the variable light domain. Preferably, the antibody is an intact antibody comprising the light chain amino acid sequence of SEQ ID NO: 13 or 30, and the heavy chain amino acid sequence of SEQ ID NO: 14, 15, 29, 31, 34, or 39, the sequence of SEQ ID NO: 39 is given below. A preferred humanized 2H7 antibody is ocrelizumab (Genentech, Inc.). The antibody herein may further comprise at least one amino acid substitution in the Fe region that enhances ADCC activity such as one wherein the amino acid substitutions are at positions 298, 333, and 334, preferably S298A, E333A, and K334A, using Eu numbering of heavy chain residues. See also the US patent. No. No. 6,737,056, L. Presta. Any of these antibodies may comprise at least one substitution in the Fe region that improves FcRn linkage or serum half-life, for example, a substitution of heavy chain position 434, such as N434W. See also the US patent. No. 6,737,056, L. Presta. Any of the antibodies may further comprise at least one amino acid substitution in the Fe region that increases CDC activity, for example comprising at least one substitution of position 326, preferably K326A or K326W. See also the US patent. No. 6,528,624, Idusogie et al. Some preferred humanized 2H7 variants are those comprising the variable light domain of SEQ ID NO: 2 and the variable heavy domain of SEQ ID NO: 8, including those with or without substitutions in a Fe region (if present), and those which they comprise a variable heavy domain with alteration in SEQ ID NO: 8 of N100A; or D56A and N100A; or D56A, N100Y, and SlOOaR; and the variable light domain with alteration in SEQ ID NO: 2 of M32L; or S92A; or M32L and S92A. M34 in the variable heavy domain of 2H7.vl6 has been identified as a potential source of antibody stability and another potential candidate for substitution. In a summary of several preferred embodiments of the invention, the variable region of variants based on 2H7.vl6, comprises the amino acid sequences of vl6 except at the amino acid substitution positions indicated in Table 4 below. Unless indicated otherwise, the 2H7 variants will have the same light chain as the vl6 one.

Table 4 Variants of Humanized 2H7 Antibody Exemplary Humanized Preferred comprises Variable Light Domain Sequence 2H7.vl6: DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKR (SEQ ID NO: 2); And the variable heavy domain sequence 2H7.vl6: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSYWYFDVWGQ GTLVTVSS (SEQ ID NO: 8). Wherein the humanized 2H7.vl6 antibody is an intact antibody it can comprise the light chain amino acid sequence; DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ KVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13); And the heavy chain amino acid sequence of SEQ ID NO: 14 or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLE VGAIYPGNGD TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSNSY YFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS NSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15). Another preferred humanized 2H7 antibody comprises the sequence. of variable light domain 2H7.V511:. . · ..;. ·. ·; ·. · DIQMTQSPSSLSASVGDRVTITCRAS.SSVS'YLHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKR (SEQ ID NO: 39) and the variable heavy domain sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGA TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRY YFDVWGQ GTLVTVSS (SEQ ID NO: 40). Where the humanized 2H7.v511 antibody is an intact antibody, it can comprise the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 30) and amino acid sequence of heavy chain SEQ ID NO: 31 or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGA TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEY CKVSNAALPAPIAATIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFMHEALHNHYTQKSLSLSPG (SEQ ID NO: 41). Where Figures 7 and 8 align the light and heavy heavy chains respectively of humanized 2H7.v511 with humanized 2H7.vl6 using the C-terminal lysine sequence with the heavy chain. Wherein the humanized 2H7.v31 antibody is an intact antibody it can comprise the light chain amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASSSVSYLH YQQKPGKAPKPLIYAPSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13) and amino acid sequence of heavy chain SEQ ID NO: 15 or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGD TSYNQKFKGRFTISVDKSKNTLYLQMNS'LRAEDTAVYYCARVVYYSNSYWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN KVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNATYRVVSVLTVLHQD LNGKEYKGKVSNKALPAPIAATIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLV GFYPSDIAVE ESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVF HEALHNHYTQKSLSLSPG (SEQ ID NO: 42) or: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWVGAIYPGNGA TSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARVVYYSYRYWYFDV GQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSR QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 43).

A preferred embodiment herein is when the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 2 and 8 (version 16). Another preferred embodiment herein is when the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 39 and 40 (version 511). It is further preferred when the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 32 and 33 (see Figure 9 re-version .11) such as one comprising the variable light chain domain in SEQ ID NO: 32 and the heavy chain amino acid sequence of SEQ ID NO: 34. It is further preferred when the antibody is humanized .. 2H7 comprising a. heavy chain variable domain with alteration N100A, or. D56A and N100A, or D56A, N100Y, and SlOOaR in SEQ ID NO: 8 - and a variable light chain domain with alteration M32L, or S92A, or M32L and S92A in SEQ ID NO: 2.

Claims (124)

1. CLAIMS 1. A method for treating vasculitis associated with antineutrophil cytoplasmic antibodies (vasculitis associated with ANCA) in a patient, which comprises administering a CD20 antibody to the patient in a dose of about 400 mg to 1.3 grams at a frequency of 1 to 3 doses within of a period of approximately one month.
2. 2. The method according to claim 1, characterized in that the dose is approximately 500 mg to 1.2 grams.
3. 3. The method according to claim 1 or 2, characterized in that the dose is approximately 750 mg to 1.1 grams.
4. 4. The method of compliance with any of claims 1 to 3, characterized in that the antibody is administered in 2 to 3 doses.
5. 5. The method according to any of claims 1 to 4, characterized in that the antibody is administered in 3 doses.
6. 6. The method according to any of claims 1 to 5, characterized in that the antibody is administered within a period of about 2 to 3 weeks.
7. 7. The method according to claim 6, characterized in that the period is about 3 weeks.
8. 8. The method according to any of claims 1 to 7, characterized in that the vasculitis associated with ANCA is Wegener's granulomatosis.
9. 9. The method according to any of claims 1 to 7, characterized in that the vasculitis associated with ANCA is microscopic polyangiitis.
10. 10. The method according to any of claims 1 to 9, characterized in that a second medicament is administered in an amount
11. Effective where the CD20 antibody is a. first medication. The method according to claim 10, characterized in that the second medicament is more than one medicament.
12. 12. The method according to claim 10 or 11, characterized in that the second drug is a chemo-therapeutic agent, an immunosuppressive agent, an anti-rheumatic drug that modifies disease (DMARD = disease-modifying anti-rheumatic drug), a cytotoxic agent, an integrin antagonist, a non-spheroidal anti-inflammatory drug (NSAID = non-steroidal anti-inflammatory drug), a cytokine antagonist or a hormone, or a combination thereof.
13. The method according to claim 12, characterized in that the second medicament is a steroid or an immunosuppressant agent or both.
14. 14. The method according to claim 13, characterized in that the second medicament is a steroid.
15. 15. The method according to claim 14, characterized in that the steroid is a corticosteroid.
16. The method according to claim 15, characterized in that the steroid is prednisone, prednisolone, methylprednisolone, hydrocortisone, or dexamethasone.
17. 17. The method according to any of claims 14 to 16, characterized in that the steroid is administered in smaller amounts than those used if the CD20 antibody is not administered to a patient treated with steroid.
18. 18. The method according to claim 13, characterized in that the second drug is an immunosuppressive agent.
19. 19. The method according to claim 18, characterized in that the immunosuppressive agent is cyclophosphamide, chlorambucil, mycophenolate

    • Mofetil, leflunomide, azathioprine or methotrexate.
20. 20. The method according to claim 18, characterized in that the immunosuppressant agent is cyclophosphamide.
21. 21. The method according to claim 13, characterized in that the second drug is an etseroid and an immunosuppressive agent.
22. 22. The method according to any of claims 1 to 21, characterized in that the patient has never been previously treated with a CD20 antibody.
23. 23. The method according to any of claims 1 to 22, characterized in that the patient has not had relapse with vasculitis.
24. 24. The method according to any of claims 1 to 22, characterized in that the antibody is a naked antibody.
25. 25. The method according to any of claims 1 to 22, characterized in that the antibody is conjugated with another molecule.
26. 26. The method according to claim 25, characterized in that the other molecule is a cytotoxic agent.
27. 27. The method of. according to any of claims 1 to 26, characterized in that the antibody is administered intra-venously.
28. 28. The method of. compliance with any of the. : || claims 1 a. 26, characterized in that the antibody is administered subcutaneously.
29. 29. The method according to any one of claims 1 to 28, characterized in that no other medication apart from the CD20 antibody is administered to the subject to treat vasculitis associated with ANCA.
30. 30. The method according to any of claims 1 to 29, characterized in that the antibody is rituximab.
31. 31. The method according to any of claims 1 to 29, characterized in that the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID Nos. 2 and 8.
32. 32. The method according to any one of claims 1 to 29 , characterized in that the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 39 and 40.
33. The method according to any of claims 1 to 29, characterized in that the antibody is humanized 2H7 comprising the sequences of variable domain in SEQ ID NOS: 32 and 33.
34. 34. The method according to any of claims 1 to 29 ,. characterized in that the antibody is humanized 2H7 comprising a variable heavy chain domain with alteration N100A, or D56A and N100A, or D56A, N100Y, and SlOOaR in SEQ ID NO: 8 and a variable light chain domain with M32L alteration, or S92A , or M32L and S92A in SEQ ID NO: 2.
35. 35. The method according to any of claims 1 to 34, characterized in that the patient has a BVAS / WG score of 0 to 6 months after administration of the antibody.
36. 36. The method according to any of claims 1 to 35, characterized in that the patient has a high level of antinuclear antibodies (ANA), anti-rheumatoid factor (RF) antibodies, blood urea nitrogen, anti-endothelial antibodies, antibodies anti-neutrophil cytoplasmic (ANCA) or a combination thereof.
37. 37. An article of manufacture, characterized in that it comprises: a) a container comprising an antibody CD20; Y. b) a package insert with instructions for treating vasculitis associated with cytoplasmic antibodies .anti-neutrophils (vasculitis associated with ANCA). in a patient, where the instructions indicate that a dose of the CD20 antibody of approximately .400 mg to 1.3 grams at a frequency of 1 to 3 doses is administered to the patient within a period of approximately one month.
38. 38. The article according to claim 37, characterized in that it further comprises a container comprising a second medicament, wherein the CD20 antibody is a first medicament, further comprising instructions in the package insert for treating the patient with the second medicament.
39. 39. The article according to claim 38, characterized in that the second drug is a chemo-therapeutic agent, an immunosuppressive agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist or a hormone.
40. 40. The article according to claim 38 or 39, characterized in that the second drug is a steroid or an immunosuppressive agent or both.
41. 41. A method for treating vasculitis associated with cytoplasmic anti-neutrophil antibodies (vasculitis associated with || ANCA) in a subject, characterized in that it comprises. administering an effective amount of a 'CD20 antibody to the subject, to provide an exposure of' initial antibody followed by a second exposure of antibody, wherein the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure.
42. 42. The method according to claim 41, characterized in that each of the antibody exposures is provided to the subject as a single dose or as 2 or 3 separate doses of antibody.
43. 43. The method according to claim 41 or 42, characterized in that the second exposure is not provided until approximately 20 to 30 weeks from the initial exposure.
44. 44. The method according to any of claims 41 to 43, characterized in that the second exposure is not provided until approximately 40 to 54 weeks from the initial exposure.
45. 45. The method according to any of claims 41 to 44, characterized in that each of the initial and second antibody exposures are provided in amounts ... of ... approximately 0.5 to 4 grams. | . ..|| '·
46. 46. The method · of. according to any of claims 41 to 45, characterized in that each of the initial and second antibody exposures are provided in amounts of about 1.5 to 3.5 grams.
47. 47. The method according to any of claims 41 to 46, characterized in that each of the initial and second antibody exposures is provided in amounts of about 1.5 to 2.5 grams.
48. 48. The method according to any of claims 41 to 47, characterized in that it additionally comprises administering to the subject an effective amount of the CD20 antibody to provide a third antibody exposure, wherein the third exposure is not provided until about 46 to 60 weeks of the initial exposure.
49. 49. The method according to claim 48, characterized in that the third antibody exposure is provided in an amount of about 46 to 60 weeks.
50. 50. The method. from . according to claim 48 or 49, characterized in that the. Third exposure of antibody is provided in an amount of about 1.5 to 3.5 grams.
51. 51. The method according to any of claims 48 to 50, characterized in that the third antibody exposure is provided in an amount of about 1.5 to 2.5 grams.
52. 52. The method according to any of claims 48 to 51, characterized in that the third exposure is not provided until approximately 46 to 55 weeks from the initial exposure.
53. 53. The method according to any of claims 48 to 52, characterized in that no further exposure of antibody is provided until at least about 70 and 75 weeks of initial exposure. 5 .
54. The method according to claim 53, characterized in that no additional antibody exposure is provided up to about 74 to 80 weeks of the initial expression.
55. 55. The method according to any of claims 41 or 43 to 54, characterized in that one or more of the exposures of the antibody is provided to the subject as a single dose of antibody.
56. 56. The method. of conformity. with. any of claims 41 to 55, wherein each antibody exposure is provided to the subject as a single dose of antibody.
57. 57. The method according to any of claims 41 or 43 to 54, characterized in that one or more of the antibody exposures is provided to the subject as separate doses of the antibody.
58. 58. The method according to any of claims 41 to 54 or 57, characterized in that each antibody exposure is provided as separate doses of the antibody.
59. 59. The method according to claim 57, characterized in that the separate doses are approximately 2 to 3 doses.
60. 60. The method according to any of claims 57 to -59, characterized in that the separate doses constitute a first and a second dose.
61. 61. The method of compliance with any of the. claims .57 to 59, acterized because the separate doses constitute a first, second, and third dose.
62. 62. The method according to any of claims 57 to 61, characterized in that a subsequent dose is administered approximately 1 to 20 days from the time in which the previous dose was administered.
63. 63. The method according to any of claims 57 to 62, characterized in that a subsequent dose is administered approximately 6 to 16 days from the time in which the previous dose was administered.
64. 6 The method according to any of claims 57 to 63, characterized in that a subsequent dose is administered at approximately 14 to 16 days from the time before the dose is administered.
65. 65. The method according to any of claims 57 to 64, characterized in that the separate doses are administered within a total period of between about one day and four weeks.
66. 66. The method according to any of claims 57 to 65, characterized in that the separate doses are administered within a total period of between about 1 to 25 days.
67. 67. The method according to any of claims 57 to 66, characterized in that the separate doses are administered approximately weekly with the second dose that is administered approximately one week after the first dose and any subsequent dose is administered approximately one week after the first dose. week of the previous dose.
68. 68. The method according to any of claims 57 to 67, characterized in that each separate dose of antibody is about 0.5 to 1.5 grams.
69. 69. The method according to any of claims 57 to 68, characterized in that each separate dose of antibody is approximately 0.75 to 1.3 grams.
70. 70. The method according to any of claims 41 to 69, characterized in that 4 to 20 antibody exposures are administered to the subject.
71. 71. The method according to any of claims 41 to 70, characterized in that a second drug is administered in an effective amount with an antibody exposure, wherein the CD20 antibody is a first drug. : t .--
72. 72. The method of compliance ·. with the

    • Claim 71, characterized in that the second medicament is administered with the initial exposure .-. ··
73. 73. The method according to claim 71 or 72, characterized in that the second medicament is administered with the initial and second exposures.
74. 74. The method according to any of claims 71 to 73, characterized in that the second drug is administered with all the exposures.
75. 75. The method according to any of claims 71 to 74, characterized in that the second drug is a chemotherapeutic agent, an immunosuppressive agent, a disease-modifying anti-rheumatic drug (DMARD), a cytotoxic agent, an integrin antagonist, a non-spheroidal anti-inflammatory drug (NSAID), a cytokine antagonist or a hormone or a combination thereof.
76. 76. The method according to any of claims 71 to 75, characterized in that the

    • second drug comprises a spheroid or an immunosuppressive agent or both.
77. 77. The method according to any of claims | 71 to 76, characterized in that the second medicament is a spheroid. · . .
78. 78. The method of compliance with the. claim 77, characterized in that the spheroid is a corticosteroid.
79. 79. The method according to claim 78, characterized in that the spheroid is prednisone, prednisolone, methylprednisolone, hydrocortisone, or dexamethasone.
80. 80. The method according to any of claims 77 to 79, characterized in that the steroid is administered in smaller amounts than those used if the CD20 antibody is not administered to a steroid treated subject.
81. 81. The method according to any of claims 71 to 76, characterized in that the second medicament is an immunosuppressive agent.
82. 82. The method according to claim 81, characterized in that the immunosuppressive agent is cyclophosphamide, chlorambucil, leflunomide, mycophenolate mofetil, azathioprine, or methotrexate.
83. 83. The method according to claim 82, characterized in that the immunosuppressive agent is cyclophosphamide.
84. 84. The method according to any of claims 71 .. to 76,. characterized in that the second medicament comprises a steroid and an immunosuppressive agent.
85. 85. The method according to claim 72, characterized in that the second drug is not administered with the second exposure or is administered in smaller amounts than those used with the initial exposure.
86. 86. The method according to any of claims 41 to 85, characterized in that approximately 2 to 3 grams of the CD20 antibody is administered as the initial exposure.
87. 87. The method according to claim 86, characterized in that approximately 1 gram of the CD20 antibody is administered weekly for approximately three weeks as the initial exposure.
88. 88. The method according to claim 86 or 87, characterized in that the second exposure is approximately six months from the initial exposure and is administered in an amount of approximately 2 grams.
89. 89. The method according to any of claims 86 to 88, characterized in that the second exposure is approximately six months from the initial exposure and is administered as approximately 1 gram of the antibody followed in about two weeks by another approximately gram of the antibody.
90. 90. The method according to claim 86, characterized in that approximately 1 gram of the CD20 antibody is administered followed in about two weeks by another approximate gram of the antibody as the initial exposure.
91. 91. The method according to claim 90, characterized in that the second exposure is approximately six months from the initial exposure and is administered in an amount of approximately 2 grams.
92. 92. The method according to claim 90 or 91, characterized in that the second exposure is approximately six months from the initial exposure and is administered as approximately 1 gram of the antibody followed in about two weeks by another approximate gram of the antibody.
93. 93. The method according to any of claims 86 to 92, characterized in that a steroid is administered to the subject before or with the initial exposure. 9.
94. The method according to claim 93, characterized in that the steroid is not administered with the second exposure or is administered with the second exposure but in smaller quantities than those used with the initial exposure.
95. 95. The method according to claim 93 or 94, characterized in that the steroid is not administered with the third or subsequent exposures.
96. 96. The method according to any of claims 41 to 95, characterized in that the subject has never been previously treated with a CD20 antibody.
97. 97. The method according to any of claims 41 to 96, characterized in that the subject is in remission after initial or subsequent antibody challenge.
98. 98. The method according to any of claims 41 to 96, characterized in that the subject is in remission when the second antibody exposure is provided.
99. 99. The method according to claim 98, characterized in that the subject is in remission when all antibody exposures are provided.
100. 100. The method according to any of claims 41 to 99, characterized in that the initial and second antibody exposures are within the same CD20 antibody.
101. 101. The method according to any of claims 41 to 100, characterized in that all the antibody exposures are within the same CD20 antibody.
102. 102. The method according to any of claims 41 to 101, characterized in that the antibody is a naked antibody.
103. 103. The method according to any of claims 41 to 101, characterized in that the antibody is conjugated with another molecule.
104. 104. The method according to claim 103, characterized in that the other molecule is a cytotoxic agent.
105. 105. The method according to any of claims 41 to 104, characterized in that the antibody is administered intravenously.
106. 106. The method according to claim 105, characterized in that the antibody is administered intravenously for each antibody exposure.
107. 107. The method according to any of claims 41 to 104, characterized in that the antibody is administered subcutaneously.
108. 108. The method according to claim 107, characterized in that the antibody is administered subcutaneously for each antibody exposure.
109. 109. The method according to any of claims 41 to 108, characterized in that no other drug of the CD20 antibody is administered to the subject to treat vasculitis associated with ANCA.
110. 110. The method according to any of claims 41 to 109, characterized in that the vaitis associated with ANCA is Wegener's granulomatosis.
111. 111. The method according to any of claims 41-109, characterized in that the vaitis associated with ANCA is microscopic polyangiitis.
112. 112. The method according to any of claims 41 to 111, characterized in that the antibody is rituximab.
113. 113. The method according to any of claims 41 'to 111, characterized in that the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID Nos. 2 and 8.
114. 114. The method according to any of the claims 41 to 111, characterized in that the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 39 and 40.
115. 115. The method according to any of claims 41 to 111, characterized in that the antibody is humanized 2H7 comprising the variable domain sequences in SEQ ID NOS: 32 and 33.
116. 116. The method according to any of claims 41 to 111 , characterized in that the antibody is humanized 2H7 comprising a variable heavy chain domain with alteration N100A, or D56A and N100A, or D56A, N100Y, and SlOOaR in SEQ ID NO: 8 and a variable light chain domain with M32L alteration, or S92A, or M32L and S92A in SEQ ID NO: 2.
117. 117. The method according to any of claims 41 to 116, characterized in that the subject has a BVAS / WG score from 0 to six months after administration of the antibody.
118. 118. The method according to any of claims 41 to 1.17, characterized in that the subject has a high level of antinuclear antibodies (ANA), anti-rheumatoid factor (RF) antibodies, creatinine, blood urea nitrogen, anti-human antibodies, -endothelial, anti-neutrophil cytoplasmic antibodies (ANCA), or a combination thereof.
119. 119. A manufacturing article, characterized in that it comprises: a. a container comprising an antibody CD20; and b. a packaging insert with instructions for treating vaitis associated with cytoplasmic anti-neutrophil antibodies (vaitis associated with ANCA) in a subject, wherein the instructions indicate that an amount of the antibody is administered to the subject that is effective to provide an initial antibody challenge followed by a second antibody exposure, where the second exposure is not provided until approximately 16 to 54 weeks from the initial exposure.
120. 120. The article according to claim 119, characterized in that each of the antibody exposures is provided to the subject as a single dose or as two or three separate doses of antibody.
121. 121. The article according to claim 119 or 120, characterized in that each of the initial and second antibody exposures are provided in amounts of 0.5 to 4 grams.
122. 122. The article according to any of claims 119 to 121, characterized in that it further comprises a container comprising a second medicament, wherein the CD20 antibody is a first medicament and further comprises instructions in the packaging insert to treat the subject with the second medication
123. 123. The article according to claim 122, characterized in that the second drug is a chemotherapeutic agent, an immunosuppressive agent, a cytotoxic agent, an integrin antagonist, a cytokine antagonist or a hormone.
124. 124. The article according to claim 123, characterized in that the second medicament is a steroid, or an immunosuppressive agent or both.