KR20070019727A - Preventing autoimmune disease - Google Patents

Abstract

The present application provides a method of preventing autoimmune disease in an asymptomatic subject who is at risk of experiencing one or more symptoms of autoimmune disease by administering the CD20 antibody to the subject in an amount that prevents one or more symptoms of autoimmune disease from being experienced. Describe.

Description

Prevention method of autoimmune disease {PREVENTING AUTOIMMUNE DISEASE}

This application is a non-provisional application that claims priority to US Provisional Application No. 60 / 568,460, filed May 5, 2004, under 35 USC 119, the contents of which are incorporated herein by reference in their entirety.

The present invention is directed to preventing autoimmune diseases in asymptomatic human subjects who are at risk of experiencing one or more symptoms of autoimmune disease.

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

B cells mature in the bone marrow and leave the bone marrow expressing antigen-binding antibodies on the cell surface. When a native B cell first encounters an antigen specific for its membrane-bound antibody, the cell begins to divide rapidly and its progeny are called memory B cells and "trait cells". Begins to differentiate into working cells Memory B cells have a longer survival time and continue to express membrane-bound antibodies with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibodies, but instead produce antibodies in a form that can be secreted. Secreted antibodies are the main working molecule of humoral immunity.

CD20 antigen (also termed human B-lymphocyte-limiting differentiation antigen, Bp35) is a hydrophobic transmembrane protein located on pre-B and mature B lymphocytes and having a molecular weight of approximately 35 kD (Valentine et al. J. Biol . Chem . 264 (19): 11282-11287 (1989); and Einfeld et al. EMBO J. 7 (3): 711-717 (1988)]. Antigens are also expressed on more than 90% of B cell non-Hodgkin's lymphomas (NHL) [Anderson et al. Blood 63 (6): 1424-1433 (1984)], not found on hematopoietic stem cells, pro-B cells, normal plasma cells or other normal tissues [Tedder et al. J. Immunol . 135 (2): 973-979 (1985)]. CD20 regulates early stage (s) of the activation process for cell cycle initiation and differentiation [Tedder et al., Supra] and possibly acts as calcium ion channels [Tedder et al. J. Cell . Biochem . 14D: 195 (1990).

When CD20 is expressed in B cell lymphoma, the antigen may serve as a candidate for "targeting" the lymphoma. In essence, the targeting can be generalized as follows: An antibody specific for the CD20 surface antigen of B cells is administered to the patient. The anti-CD20 antibody specifically binds (surface) to the CD20 antigen of normal and malignant B cells; Antibodies that bind to the CD20 surface antigen can destroy and kill neoplastic B cells. In addition, chemicals or radiolabels having the ability to destroy tumors may be conjugated with anti-CD20 antibodies such that the material can be specifically delivered to neoplastic B cells. Regardless of the above approach, the aim is to destroy the tumor; The specific approach can be determined by the specific anti-CD20 antibody used, and therefore, the approaches that can be used to target the CD20 antigen can vary considerably.

Rituximab (RITUXAN®) antibodies are chimeric murine / human monoclonal antibodies genetically engineered for the CD20 antigen. Rituximab is an antibody named "C2B8" in US Pat. No. 5,736,137 (Anderson), issued April 7, 1998. Rituximab® is indicated for the treatment of patients with alkylating or nonreactive low-grade or vesicles, CD20-positive, B cell non-Hodgkin's lymphomas. In vitro mechanism of action studies have demonstrated that Rituximab® binds to human complement and lyses lymphoid B cell lines through complement-dependent cytotoxicity (CDC) [Reff et al. Blood 83 (2): 435-445 (1994)]. In addition, rituxan shows significant activity in cytotoxicity (ADCC) analysis of antibody-dependent cells. More recently, it has been found that Rituxan (RITUXAN®) has an anti-proliferative effect and directly induces apoptosis in triple hydrogenated thymidine incorporation assays, while other anti-CD19 and CD20 antibodies do not. Blood 88 (10): 637a (1996)]. In addition, synergism between Rituxan (RITUXAN®) and chemotherapy and toxins has been observed experimentally. In particular, Rituxan (RITUXAN®) sensitizes human B cell lymphoma cell lines that are drug-resistant to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and lysine [Demidem et al. Cancer Chemotherapy & Radiopharmaceuticals 12 (3): 177-186 (1997)). In vivo preclinical studies have shown that RITUXAN® kills B cells, possibly through complement and cell-mediated responses, from peripheral blood flow, lymph nodes and bone marrow of cynomolgus monkeys [Reff et al. Blood 83 (2): 435-445 (1994)].

Patents and patent publications related to CD20 antibodies include US Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, and US Patent Application Nos. US 2002/0197255 A1, US 2003/0021781 A1 US 2003/0082172 A1, US 2003/0095963 A1, US 2003/0147885 A1 (Anderson et al.); US Patent Nos. 6,455,043 B1 and WO OO / 09160 (Grillo-Lopez, A.); WO OO / 27428 (Gilo-Lopez and White); WO OO / 27433 (Gilo-Lopez and Leonard); WO OO / 44788 (Braslawsky et al.); WO 01/10462 (Rastetter, W.); WO 01/10461 (lasters and white); WO 01/10460 (white and guillo-lopez); US 2001/0018041 A1, US 2003/0180292 A1, W0 01/34194 (Hanna and Hariharan); US Application Nos. US 2002/0006404 and WO 02/04021 (Hanna and Harhariran); US 2002/0012665 A1 and WO 01/74388 (Hanna, N.); US Application No. US 2002/0058029 A1 (Hanna, N.); US Application No. US 2003/0103971 A1 (Hannah and Harhariran); U.S. Application Nos. US 2002/0009444 A1, and WO 01/80884 (Grillo-Lopez, A.); WO 01/97858 (White, C.); U.S. Application Nos. US 2002/0128488 A1 and WO 02/34790 (Reff, M.); WO 02/060955 (Brasroski et al.); W02 / 096948 (Brassowski et al.); WO 02/079255 (Lev and Davies); US Patent No. 6,171,586 B1, and WO 98/56418 (Lam et al.); WO 98/58964 (Raju, S.); WO 99/22764 (Raju S.); WO 99/51642, US Pat. No. 6,194,551 B1, US Pat. No. 6,242,195 B1, US Pat. No. 6,528,624 B1 and US Pat. No. 6,538,124 (Idusogie et al.); WO OO / 42072 (Presta, L.); WO OO / 67796 (Curd et al.); WO 01/03734 (Gilo-Lopez et al.); US Application Nos. US 2002/0004587 A1 and WO 01/77342 (Miller and Presta); US Application No. US 2002/0197256 (Grewal, I.); US Application No. US 2003/0157108 A1 (Presta L.); US Patent Nos. 6,565,827 B1, 6,090,365 B1, 6,287,537 B1, 6,015,542, 5,843,398, and 5,595,721 (Kaminski et al.); US Patent Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, 6,652,852 B1 (Robinson et al.); US Patent No. 6,410,391 B1 (Raubitschek et al.); US Patent No. 6,224,866 B1 and WO OO / 20864 (Barbera-Guillem, E.); W0 01/13945 (Barbera-Guilm E.); WO OO / 67795 (Goldenberg); US Application Nos. US 2003/0133930 A1 and WO 00/74718 (Goldenberg and Hansen); WO OO / 76542 (Golay et al.); WO 01/72333 (Wolin and Rosenblatt); US Patent No. 6,368,596 B1 (Ghetie et al.); US Patent No. 6,306,393 and US Application No. US 2002/0041847 A1, (Goldenberg D.); US Application No. US 2003/0026801 A1 (Weiner and Hartmann); WO 02/102312 (Engleman, E.); US Patent Application No. 2003/0068664 (Albitar et al.); WO 03/002607 (Leung, S.); WO 03/049694, US 2002/0009427 A1, and US 2003/0185796 A1 (Wolin et al.); WO 03/061694 (Sing and Siegall); US 2003/0219818 A1 (Bohen et al.); US 2003/0219433 A1 and WO 03/068821 (Hansen et al.); US 2003/0219818 A1 (Bohen et al.); US 2002/0136719 A1 (Senoi et al.); WO 2004/032828 (Wahl et al.), Each of which is incorporated herein by reference. See further, US Pat. No. 5,849,898 and EP Application No. 330,191 (Seed et al.); U.S. Patent Nos. 4,861,579 and EP 332,865 A2 (Meyer and Weiss); USP 4,861,579 (Meyer et al.); WO95 / 03770 (Bhat et al.); US 2003/0219433 A1 (Hansen et al.).

Published literature related to therapy with rituximab (Perotta and Abuel "Response of Chronic relapsing ITP of 10 years duration to Rituximab" Abstract # 3360 Blood 10 (1) (part 1-2): p.88B (1998) Stashi et al. "Rituximab Chimeric anti-CD20 monoclonal antibody treatment for adults with Chronic idopathic thrombocytopenic purpura" Blood 98 (4): 952-957 (2001)]; [Matthews, R. "Medical Heretics" New Scientist (7 April, 2001); Leandro et al. "Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion" Ann Rheum Dis 61: 833-888 (2002); Leandro et al. "lymphocyte depletion in rheumatoid arthritis:. early evidence for safety, efficacy and dose response Arthritis and Rheumatism 44 (9): 5370 (2001); Leandro et al. "An open study of B lymphocyte depletion in systemic lupus erythematosus", Arthritis & Rheumatism 46 (1): 2673-2677 (2002); Edwards and Cambridge "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocyte" Rhematology 40: 205-211 (2001); Edwards et al. "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune dieases" Biochem . Soc . Trans. 30 (4): 824-828; Edwards et al. "Efficacy and safety of Rituximab, a B-lymphocyte targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis Arthritis & Rheumatism 46 (9):. S 197 (2002)]; [Levine and Pestronk" IgM antibody -related polyneuropathies: B-cell depletion chemotherapy using Rituximab " Neurology 52: 1701-1704 (1999)]; DeVita et al." Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis " Arthritis & Rheum 46: 2029-2033 (2002)]; [Hidashida et al. "Treatmentof DMARD-Refractory rheumatoid arthritis with rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology ; Oct 24-29; New Orleans, LA 2002; Tuscano, J. "Successful treatment of 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; Specks et al. "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism 44 (12): 2836-2840 (2001).

Arbuckle et al., Arbuckle et al. N. Engl. J. Med . 349 (16): 1526-1533 (2003) described that autoantibodies develop before clinical onset of systemic lupus erythematosus (SLE).

Summary of the Invention

In a first aspect, the present invention provides a subject with systemic lupus erythematosus (SLE), anti-phospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barré syndrome, severe myasthenia gravis, Large vessel vasculitis, intermediate vasculitis, nodular polyarteritis, swelling, scleroderma, Goodpasture syndrome, glomerulonephritis, primary biliary cirrhosis, Graves' disease, membranous nephropathy, autoimmune hepatitis, non-tropic sprue, Addison's disease, multiple Preventing from experiencing one or more autoimmune symptoms selected from myositis / skin myositis, monoclonal gamma disease, factor VIII deficiency, cold globulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and Hashimoto thyroiditis Risk of experiencing one or more symptoms of autoimmune disease, including administering the CD20 antibody to the subject in an amount A method for preventing autoimmune disease in a symptomatic, asymptomatic subject.

In another aspect, the invention has a risk of experiencing one or more autoimmune disease symptoms comprising administering to the subject a CD20 antibody in an amount that prevents the subject from experiencing one or more autoimmune disease symptoms. To a method for preventing autoimmune disease in a subject without.

The invention also provides for the prevention of autoimmune diseases in asymptomatic subjects with abnormal levels of autoantibodies, the method comprising administering to the subject CD20 antibodies in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. It includes a method.

The present invention further

To prevent a subject from experiencing one or more symptoms of autoimmune disease,

(a) a container comprising a composition comprising a CD20 antibody in a container and a pharmaceutically acceptable carrier or diluent; And

(b) a product comprising instructions for administering the composition to an asymptomatic subject who is at risk of experiencing one or more symptoms of autoimmune disease.

1A shows the light chain variable region (V _L ) of each of murine 2H7 (SEQ ID NO: 1), humanized 2H7.v16 variant (SEQ ID NO: 2), and human kappa light chain subgroup I (SEQ ID NO: 3) Sequence sequence for comparing the amino acid sequence of the. CDRs of V _L of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5), and CDR3 (SEQ ID NO: 6).

1B shows the heavy chain variable region (V _H ) of each of murine 2H7 (SEQ ID NO: 7), humanized 2H7.v16 variant (SEQ ID NO: 8), and the human consensus sequence of heavy chain subgroup III (SEQ ID NO: 9) Sequence sequence for comparing amino acid sequences. CDRs of V _H of 2H7 and hu2H7.v16 are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12).

CDR1, CDR2 and CDR3 in each of the chains of FIGS. 1A and 1B are located on the framework site, FR1-FR4 side, as indicated, and are indicated by brackets. 2H7 refers to murine 2H7 antibody. Asterisks between the two sequences arranged indicate different positions in both sequences. Residue numbering is described by Kabat et al. Sequences of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), wherein the insertions are denoted by a, b, c, d, and e.

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

3 shows the amino acid sequence of the mature 2H7.v16 H chain (SEQ ID NO: 14).

4 shows the amino acid sequence of the mature 2H7.v31 H chain (SEQ ID NO: 15). The L chain of 2H7.v31 is the same as for 2H7.v16.

FIG. 5 includes an arrangement of mature 2H7.v16 and 2H7.v511 light chains (SEQ ID NOs: 13 and 16, respectively) including Kabat variable region residue numbering and Eu constant region residue numbering.

FIG. 6 includes an arrangement of mature 2H7.v16 and 2H7.v511 heavy chains (SEQ ID NOs: 14 and 17, respectively) including carbett variable region residue numbering and Eu constant region residue numbering.

Detailed Description of the Preferred Embodiments

I. Definition

An “autoimmune disease” herein is a disease or condition therefor, or a finding or symptom developed therefrom resulting from an individual's own tissue or co-segregate. Examples of autoimmune diseases or diseases include, but are not limited to, arthritis (rheumatic arthritis such as rapid arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, rapid gouty arthritis, rapid immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II Collagen-Induced Arthritis, Infectious Arthritis, Lyme Arthritis, Proliferative Arthritis, Dry Arthritis, Still's Disease, Spinal Arthritis, and Juvenile Rheumatoid Arthritis, Osteoarthritis, Chronic Progressive Arthritis, Deformed Arthritis, Chronic Primary Multiple Arthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, such as psoriasis, such as plaque psoriasis, gutatte psoriasis, pustules psoriasis, and nail psoriasis, atopy, including atopic diseases such as hay fever and occupation Syndrome, contact dermatitis, chronic contact dermatitis, eliminated dermatitis, allergic dermatitis, allergic contact Dermatitis including dermatitis, dermatitis herpes, coin dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper-IgM syndrome, allergic intraocular inflammatory disease, urticaria, eg chronic allergic urticaria And chronic idiopathic urticaria, myositis, polymyositis / skin myositis, combustible dermatitis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), including sclerosis such as systemic sclerosis, multiple sclerosis (MS) Examples include spinal cord visual MS, primary progressive MS (PPMS), and recurrent laryngeal MS (RRMS), systemic progressive sclerosis, atherosclerosis, atherosclerosis, disseminated sclerosis, ataxia, visual neuromyelitis (NMO), inflammatory Intestinal disease (IBD) (eg Crohn's disease, autoimmune-mediated gastrointestinal disease, colitis such as ulcerative colitis, ulcerative colitis, microscopic Enteritis, biliary colitis, colitis polyposis, necrotic colitis, and cirrhosis colitis, and autoimmune inflammatory bowel disease), intestinal inflammation, necrotizing pyoderma, nodular erythema, primary sclerotic cholangitis, adult or rapid respiratory distress syndrome (ARDS) Respiratory distress syndrome, meningitis, inflammation of the entire or part of the uvea, iris, choroiditis, autoimmune blood disease, rheumatoid spondylitis, rheumatoid vasculitis, hereditary angioedema, cerebral nerve damage in meningitis, gestational herpes, gestational ileus, bovine Benign scrotumitis, autoimmune primary ovarian failure, sudden hearing loss due to autoimmune symptoms, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis, such as rasmussen brain m and limbic and / or encephalitis encephalitis, Uveitis, such as anterior uveitis, rapid anterior uveitis, granulomatous uveitis, granulomatous uveitis, pagoantigen uveitis , Posterior uveitis, or autoimmune uveitis, glomerulonephritis with nephrotic syndrome (GN) and glomerulonephritis with nephrotic syndrome, eg chronic or rapid glomerulonephritis, eg primary GN, immune-mediated GN, membranous GN (Membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrane proliferative- or membranous proliferative GN (MPGN), including, for example, type I and II and rapid progressive GN, proliferative nephritis, autoimmune polysecretory endocrine Insufficiency, balanitis, e.g. localized plasma cell blepharitis, scleroderma, central centrifugal lupus erythema, sustained pigmentary erythema, polymorphic erythema, ring granulomas, gloss mammary gland, percutaneous atrophic thyroid, simple chronic gestation, superficial thyroid, squamous thyroid, plaque Young, epidermal fusion hyperkeratosis, precancerous keratosis, necrotizing pyoderma, allergic abnormalities and reactions, allergic reactions, eczema, e.g. allergic or atopic eczema, dry eczema, sweating disorders Gin, and bullous hand, plantar eczema, adrenal glands, such as asthmatic bronchial asthma, bronchial asthmatic nephritis, and abnormalities including autoimmune nephritis, T cells and chronic inflammatory reactive infiltration, ABO in pregnancy Immune response to external antigens such as blood types, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus nephritis, lupus nephritis, lupus encephalitis, pediatric lupus, non-extension lupus, renal lupus, and distant Lupus erythema, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus erythematosus, juvenile onset (type I) diabetes, eg pediatric insulin Dependent diabetes mellitus (IDDM), adult onset diabetes (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic aortic disease Immune responses associated with rapid and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, such as lymphoma granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, such as vasculitis, Vasculitis vasculitis (eg, polymyalgia rheumatoid and giant-cell (Takayasu) arteritis), mesothelial vasculitis (eg, Kawasaki disease and nodular polyarteritis / periarteritis), micro polyarthritis, immune vasculitis, CNS vasculitis, Cutaneous vasculitis, hypervascular vasculitis, necrotic vasculitis, such as systemic necrotic vasculitis, and ANCA-related vasculitis, such as chog-Storau's vasculitis or syndrome (CSS) and ANCA-associated small-vascular vasculitis, temporal arteritis, aplastic Anemia, autoimmune aplastic anemia, Kum's positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia, such as autoimmune hemolytic anemia (AIHA), evil Anemia (hazardous anemia), Addison's disease, sedation erythrocytic anemia or anaplastic (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases including leukocyte leakage, CNS Inflammatory diseases, multiple organ damage syndromes such as sepsis, trauma or bleeding, antigen-antibody complex-mediated disease, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet's disease / syndrome, castle Castleman's Syndrome, Goodpasture Syndrome, Raynaud's Syndrome, Sjogren's Syndrome, Stevens-Johnson Syndrome, Hypoallergenic, for example, Hypoallergenic and dermal Hypoallergenic, Celtic window (eg, vulgaris, deciduous, mucosal) Swelling swelling, and erythema swelling), autoimmune polyendochlorinopathis, lighter disease or syndrome, heat damage, preeclampsia, immune complex diseases such as immune suits Somatic nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, hypothrombocytosis (e.g., caused by myocardial infarction), e.g. thrombosis Platelet purple thrombosis (TTP), post-transfusion purple thrombosis (PTP), heparin-induced hypothrombocytosis, and autoimmune or immune-mediated hypoplatelets, such as idiopathic thrombotic thrombocytopenia (ITP), such as Chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, scleritis, testicular and ovarian autoimmune diseases, such as autoimmune testicular and ovarian inflammation, primary hypothyroidism, parathyroid dysfunction, autoimmune endocrine diseases, For example, thyroid lobes Examples include autoimmune thyroid lobes, Hashimoto's disease, chronic thyroid lobes (Hashimoto's thyroiditis), or subacute thyroid lobes, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polysecretory Syndromes such as autoimmune polysecretory syndrome (or polysecretory endocrine syndrome), neoplastic syndromes, such as neurological neoplastic syndromes, such as Lambert-Eton's Myasthenia Syndrome or Eaton-Lambert syndrome, muscle Stiff-man or muscular stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), severe myasthenia disorder such as thymicoma-related severe myasthenia, cerebellum Degenerative, neuromuscular dystonia, nystagmus or choroidal myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Shh's syndrome, autoimmune hepatitis, chronic hepatitis, lupus hepatitis, macro-cell hepatitis, chronic Active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonia (LIP), obstructive bronchiolitis (non-transplantable) vs NSIP, Guillain-Barré syndrome, Burger disease ( IgA nephropathy), idiopathic IgA nephropathy, gland immunoglobulin A dermatitis, acute febrile neutrophil dermatitis, keratinous hypodermic disease, transient hypercellular dissociative dermatosis, liver cirrhosis, eg primary biliary cirrhosis and menopause, autoimmune enteropathy syndrome , Celiac or celiac disease, non-tropical sprue (gluten enteropathy), unreactive sprue, idiopathic sprue, cold globulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease, such as autoimmune ear deafness (AIED), autoimmune deafness, polychondritis, eg, unresponsive or alkylating or recurrent polychondritis, pulmonary protein, cogan syndrome / Non-toxic interstitial keratitis, facial paralysis, sweet disease / syndrome, rosacea autoimmunity, subject-related disease, amyloidosis, noncancerous lymphocytosis, primary lymphocytosis, e.g. monoclonal B cell lymphocytosis (e.g., positive monocle Ronaldo gamma disease and monoclonal gamma disease of undetermined significance (MGUS), peripheral neuropathy, neoplastic syndrome, channel disease such as epilepsy, migraine, arrhythmia, myopathy, deafness, blindness, periodic paralysis, and channels of the CNS Pathology, autism, inflammatory cardiomyopathy, focal or regional or focal regional glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, choroid retinopathy, autoimmune hepatic disease, fibromyalgia, complex endocrine Gland disorders, Schmidt syndrome, adrenitis, atrophy of the stomach, elderly dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, dressler syndrome, alopecia areata, total alopecia, CREST syndrome (lime Syndrome, Raynaud's phenomenon, esophageal dyskinesia, toe sclerosis, and capillary dilatation), eg, male and female autoimmune infertility due to anti-sperm antibodies, mixed connective tissue vaginalization, Chagas disease, rheumatic fever, relapse Sexual miscarriage, farmer's lung, polymorphic erythema, post-cardiac syndrome, Cushing's syndrome, avian adenocarcinoma, allergic granulomatous vasculitis, benign lymphocyte vasculitis, alpoite syndrome, alveolitis eg allergic alveolitis and fibrotic alveolitis, interstitial lung Diseases, transfusion reactions, leprosy, malaria, parasitic diseases, for example, Rischmann's flagellitis, wave schistosomiasis, schistosomiasis, roundworm, aspergillosis, Sampter syndrome, Kaiplanosis Group, dengue, endocarditis, endocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, ophthalmitis, sustained erythema, fetal erythromatosis, eosinophilic fasciitis, Schulman syndrome, Pelti Syndromes, filamentosis, ciliary salts such as chronic ciliary salts, heterochronic ciliary salts; Iris ciliitis (acute or chronic), or fuchc ciliitis, henohophreat purple pulmonary disease, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, internal Toxinemia, pancreatitis, thyroid poisoning, parvovirus infection, rubella virus infection, vaccination syndrome, congenital rubella infection, Epstein virus infection, mumps, Evans syndrome, autoimmune gonad dysfunction, cydenham chorea, nephritis after streptococcal infection, obstructive raincoat Turance, thyrotoxicosis, spinal syphilis, choroiditis, giant-cell polymyalgia, chronic hyperpneumonia, dry keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephritis syndrome, small change nephropathy, familial and ischemia-reperfusion injury, organ transplantation Reperfusion, retinal autoimmune, joint inflammation, bronchitis, chronic obstructive airway / lung disease, silicosis, aphtha, aphthaic otitis, arteriosclerosis disease, heartless Syndrome, autoimmune hemolysis, Shun's disease, cold globulinemia, dupitren construction, lens hypersensitivity, allergic enteritis, leprosy nodule erythema, idiopathic facial palsy, chronic fatigue syndrome, febrile rheumatism, Hamman-Rich disease, sensorineural hearing loss , Seizure hemochromatosis, hypogonadism, local ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephropathy, sympathetic ophthalmopathy, granulomatous testicles, pancreatitis, acute polyneuropathy, necrotizing pyoderma, querbine thyroiditis Immune Responses Associated with Acute and Delayed Hypersensitivity Induced by Acquired Spanic Atrophy, Nonmalignant Thyroidoma, Vitiligo, Toxic Shock Syndrome, Food Poisoning, Abnormalities Including T Cell Infiltration, Leukocyte Adhesion Deficiency, Cytokines and T-lymphocyte , Diseases including leukocyte leakage, multiple damage syndrome of organ, antigen-antibody complex-mediated disease, anti-glomerular basement membrane disease, allergy Leurgic neuritis, autoimmune polyendochlorinopathis, ovarian inflammation, primary mucin edema, autoimmune atrophic gastritis, sympathetic eye inflammation, rheumatoid disease, mixed connective tissue disease, nephrotic syndrome, pancreatitis, polyendocrine dysfunction, type I autoimmunity Polysecretory syndrome, adult-onset idiopathic hypothyroidism (AOIH), cardiomyopathy, such as dilatation myocardial disease, acquired bullous epidermal detachment (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerotic cholangitis, purulent or non-purulent Sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders such as eosinophilia, pulmonary infiltrating eosinophilia, eosinophilia-muscle pain syndrome, Rheopl syndrome, chronic eosinophilic pneumonia, tropical Granulomas, anaphylaxis, serum, including pulmonary eosinophilia, bronchial pneumonia aspergillosis, aspergillus, or eosinophilia Test negative spondyloarthritis, polyendocrine autoimmune disease, sclerotic cholangitis, sclera, sclera, chronic mucosal candidiasis, Bruton's syndrome, transient gamma globulin hypoemia in early childhood, biscot-oldrich syndrome, capillaries Dilated ataxia syndrome, capillary dilatation, autoimmune diseases associated with collagen disease, rheumatism, nervous system disease, lymphadenitis, decreased blood pressure response, vascular disorders, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral infarction, and vascularization Diseases associated with, allergic hypersensitivity disease, glomerulonephritis, reperfusion injury, ischemic reperfusion disease, reperfusion of myocardium or other tissues, lymphoma bronchitis, inflammatory skin disease, skin disease including acute inflammatory elements, multi-organ failure, bullous disease Nephrotic necrosis, acute purulent meningitis or other central nervous system inflammatory diseases, eye and orbital inflammatory diseases, and Obtain transfusion-associated syndromes, cytokine-induced toxicity include, narcolepsy, acute serious inflammation, chronic intractable inflammation, sinwooyeom, endocarditis arterial hyperplasia, peptic ulcer, valvular salt, and endometriosis.

"B-cells" are lymphocytes that mature in the bone marrow and include natural B cells, memory B cells, or effector B cells (trait cells). B-cells herein can be normal or non-malignant B-cells.

A "B cell surface marker" or "B cell surface antigen" herein is an antigen expressed on a B cell surface that can be labeled with an antagonist that binds thereto. Examples of B cell surface markers are CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82 , CD83, CDw84, CD85 and CD86 leukocyte surface markers (see The leukocyte Antigen Facts Book, 2 ^nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York for explanation). ). Other B cell surface markers are RP 105, FcRH2, B cell CR2, CCR6, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA 232387. In particular, the B cell surface markers of interest may be preferentially expressed on B cells compared to other non-B cell tissues in mammals and expressed in both progenitor B cells and mature B cells. Preferred B cell surface markers herein are CD20.

The "CD20" antigen, or "CD20", is a non-glycosylated phosphoprotein of about 35 kDa present on the surface of more than 90% B cells derived from peripheral blood or lymphoid organs. CD20 is present in both normal and malignant B cells but is not expressed on stem cells. Other names in the literature include "B-lymphocyte-restricting antigen" and "Bp35". CD20 antigens are described, for example, in Clark et al. Proc . Natl . Acad. Sci . (USA) 82: 1766 (1985).

An “antagonist” is one of B cells that, when bound to B cell surface markers on B cells, destroys or kills B cells in a mammal and / or reduces or prevents the humoral response induced by B cells, for example. Molecules interfere with the above functions. Antagonists can be used to remove B cells (ie, reduce levels of circulating B cells) in a treated mammal. The killing can be performed in various ways, 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 (eg, via apoptosis). This can be achieved through a mechanism. Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides and small molecule antagonists that bind to B cell surface markers that are conjugated or fused with cytotoxic agents. Preferred antagonists include antibodies.

"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refers to the binding of nonspecific cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages) that express Fc receptors (FcRs) onto target cells. A cell-mediated reaction that recognizes an antibody and then lyses the target cell. The major cells that mediate ADCC, ie NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR in hematopoietic cells is described by Ravetch and Kinet, Annu . Rev. Immunol . 9: 457-92 (1991), Table 3 on page 464. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, for example, as described in US Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be determined in vivo, eg, in animal models such as those described in Clynes et al., PNAS (USA) 95: 652-656 (1998). Can be evaluated.

A “human effector cell” is a white blood cell that expresses one or more FcRs and performs the function of an effector. Preferably, the cell expresses at least FcγRIII and performs the function of ADCC agonist. Examples of human leukocytes that mediate ADCC include peripheral blood flow mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, of which PBMC and NK cells are preferred.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are receptors (gamma receptors) that bind IgG antibodies and include receptors of the FcγRI, FcγRII and FcγRIII subclasses and allelic variants and other spliced forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”) having similar amino acid sequences that differ primarily in their cytoplasmic regions. The activating receptor FcγRIIA contains an immune receptor tyrosine-based activating motif (ITAM) in its cytoplasmic region. Inhibitor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic region (see Daeron, Annu . Rev. Immunol. 15: 203-234 (1997)). For FcRs see Ravetch and Kinet, Annu . Rev. Immunol . 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al. , J. Lab . Clin . Med . 126: 330-41 (1995), including other FcRs, including those that will be identified later, in the term “FcR” herein. Neonatal receptor, FcRn, which serves to deliver the fetus 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 dissolve a target in the presence of complement. The complement activation pathway is initiated by binding the first component (C1q) of the complement system to molecules (eg, antibodies) complexed with homologous antigens. To assess complement activation, see, eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) may be performed the CDC assay.

An "growth inhibitory" antagonist is one that arrests or slows the proliferation of cells that express the antigen to which the antagonist binds. For example, the antagonist may arrest or slow the proliferation of B cells in vitro and / or in vivo.

Antagonists that "induce apoptosis" are determined by standard apoptosis assays such as binding of Annexin V, fragmentation of DNA, cell contraction, expansion of vesicles, fragmentation of cells and / or formation of membrane vesicles (called apoptotic bodies). As such, for example, those which induce planned apoptosis of B cells.

As used herein, "antibody" is used in its broadest sense and is specifically a monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from at least two complete antibodies (eg, bispecific antibodies), and preferred Antibody fragments are included as long as they exhibit biological activity.

An "antibody fragment" includes a portion of a complete antibody, preferably the antigen binding site or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments; Diabody; Linear antibodies; Single chain antibody molecules; And multispecific antibodies formed from antibody fragments, and the like.

For purposes herein, a "complete antibody" is one that includes heavy and light chain variable regions, and an Fc region.

A "natural antibody" is a heterotetrameric glycoprotein of about 150,000 daltons, generally consisting of two matching light chains (L) and two matching heavy chains (H). Each light chain is linked to the heavy chain by one disulfide covalent bond, but the number of disulfide bonds differs in the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has disulfide bridges within the chain at regular intervals. Each heavy chain has at one end a variable region (V _H ) and a plurality of constant regions subsequent to this region. Each light chain has a variable region (V _L ) at one end and a constant region at the other end, the constant region of the light chain is arranged side by side with the first constant region of the heavy chain, and the light chain variable region is arranged side by side with the variable region of the heavy chain. . Particular amino acid residues are believed to form an interface between the light and heavy chain variable regions.

The term “variable” refers to the fact that the sequence of a particular portion of the variable region between antibodies is greatly different and is used for the binding and specificity of each particular antibody to that particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments called hypervariable sites in both the light and heavy chain variable regions. The highly conserved portion of the variable region is termed the framework region (FR). The variable regions of natural heavy and light chains comprise four FRs, mainly the structure of β-sheets, which are linked by three hypervariable sites that form a loop linkage and in some cases form part of the β-sheet structure. The hypervariable sites in each chain are kept in close proximity to each other by the FRs, and the hypervariable sites in the other chain contribute to the formation of the antigen binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)]. The constant region does not directly participate in antigen binding of the antibody, but shows various functioning factors such as the involvement of the antibody in cytotoxicity (ADCC) of antibody dependent cells.

Digestion of the antibody into papain results in two matching antigen-binding fragments, an "Fab" fragment with each single antigen-binding site immediately, and the remaining "Fc" fragment, named for its ability to readily crystallize. Is generated. Treatment with pepsin results in an F (ab ′) ₂ fragment having two antigen-binding sites and still capable of crosslinking to the antigen.

"Fv" is the minimum antibody fragment that contains a full-length antigen recognition and antigen-binding site. This site consists of a dimer consisting of one heavy chain variable region and one light chain variable region that are tightly covalently bonded. In this structure, three hypervariable regions of each variable region interact to form antigen-binding sites on the surface of the V _H -V _L dimer. In conclusion, six hypervariable regions confer antigen-binding specificity to antibodies. However, even a single variable region (or 1/2 of an Fv comprising only three hypervariable regions specific for the antigen) has affinity than the entire binding site but has the ability to recognize and bind the antigen.

The Fab fragment also comprises the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments in that several residues have been added to the carboxy terminus of the heavy chain CH1 region comprising one or more cysteines derived from the antibody hinge region. Fab'-SH herein refers to Fab 'in which the cysteine residue (s) of the constant region bear at least one free thiol group. F (ab ') ₂ antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The “light chain” of an antibody (immunoglobulin) derived from any vertebrate species can be classified into one of two apparently different types, termed kappa (κ) and lambda (λ), based on the amino acid sequence of its constant region. have.

Antibodies can be classified into different classes depending on the amino acid sequence of the constant region of the heavy chain. Complete antibodies include five major classes; IgA, IgD, IgE, IgG and IgM, some of which may be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant regions corresponding to the different classes of antibodies are named α, δ, ε, γ and μ, respectively. Subunit structures and three-dimensional structures for different classes of immunoglobulins are well known.

"Single-chain Fv" or "scFv" antibody fragments comprise the V _H and V _L regions of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide also comprises a polypeptide linker between the V _H and V _L regions which allows the scFv to form the desired structure for antigen binding (see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabody" refers to a small antibody with two antigen-binding sites, comprising a heavy chain variable region (V _H ) linked to a light chain variable region (V _L ) within the same polypeptide chain (V _H -V _L ). Refer to the fragment. By using linkers that are too short to link two regions in the same chain, the regions are forcibly linked with the complementary regions of the other chain to create two antigen-binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11611 and Hollinger et al., Proc . Natl . Acad . Sci . USA , 90: 6444-6448 (1993).

As used herein, the term "monoclonal antibody" excludes possible variants that may be formed in the production of monoclonal antibodies, such as antibodies obtained from a population of substantially homologous antibodies, ie, variants present generally in small amounts. As such, the subject antibody of one population refers to binding to identical and / or identical epitopes. Unlike polyclonal antibody preparations, which typically include different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. Monoclonal antibodies have an advantage in that they are not contaminated by other immunoglobulins in addition to their specificity. The modifier “monoclonal” refers to antibody properties obtained from a substantially homogeneous antibody population and is not considered to be required for the production of antibodies in any particular manner. For example, monoclonal antibodies for use in accordance with the present invention can be prepared by the hybridoma method described first in Kohler et al., Nature , 256: 495 (1975), or by recombinant DNA methods (eg For example, see US Pat. No. 4,816,576. “Monoclonal antibodies” can also be found, for example, in Clackson et al., Nature , 352: 624-628 [1991] and Marks et. al., J. Mol . Biol . , 222: 581-597 (1991)]) can be isolated from phage antibody library.

Monoclonal antibodies herein are specifically matched with the corresponding sequence of an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, provided that it exerts specifically desirable biological activity or Although homologous, the remainder of the chain (s) is a "chimeric" antibody (immunoglobulin) consistent with or homologous to an antibody from another species or an antibody belonging to another antibody class or subclass or fragment of such an antibody. US Pat. No. 4,816,567; Morrison et al., Proc . Natl . Acad . Sci . USA , 81: 6851-6855 (1984)]. Chimeric antibodies of interest herein include variable region antigen-binding sequences and human constant region sequences derived from non-human primates (eg, Old World monkeys such as baboons, rhesus or cynomolgus monkeys). Including “primatized” antibodies (US Pat. No. 5,693,780).

A “humanized” form of a non-human (eg, murine) antibody is a chimeric antibody comprising a minimal sequence derived from an immunoglobulin other than human. In most cases, the humanized antibody can be derived from the hypervariable region of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate with desirable specificity, affinity, and capacity. Human immunoglobulins (recipient antibodies) substituted with residues of In some cases, residues of the framework region (FR) of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies can also include residues that are not found in either the recipient antibody or the donor antibody. Such modifications are further made to improve antibody performance. In general, a humanized antibody will comprise substantially all of at least one, typically two variable regions, wherein all or substantially all hypervariable loops correspond to those of immunoglobulins other than human, and all or substantially Thus all FRs belong to that of human immunoglobulin sequences, except for the above-mentioned FR substitution (s). In addition, the humanized antibody will optionally include at least a portion of an immunoglobulin constant region, typically a portion of a human immunoglobulin. For more details, see Jones et al., Nature , 321: 522525 (1986); Riechmann et al., Nature , 332: 323-329 (1988); and Presta, Curr. Op . Struct . Biol . 2: 593-596 (1992).

As used herein, the term “hypervariable site” refers to the amino acid residue of an antibody that is responsible for antigen-binding. The hypervariable region may be a “complementarity determining region” or “CDR” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable region, and residue 31 of the heavy chain variable region). -35 (H1), 50-65 (H2) and 95-102 (H3); Kabar et al., Sequences of Proteins of Immunological Interests , 5th Ed. Public Health Services, National Institute of Health, Bethesda, MD. (1990) and / or “hypervariable loops” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the light chain variable region, and residue 26 of the heavy chain variable region) -32 (H1), 53-55 (H2) and 96-101 (H3); amino acid residues from Clothia and Lesk, J. Mol. Biol., 196: 901-917 (1987). " Framework ”or“ FR ”residues are those residues of the variable region other than the hypervariable region residues as herein defined.

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

Examples of antibodies that bind the CD20 antigen are "C2B8" (US Pat. No. 5,736,137, herein incorporated by reference), currently designated "rituximab" (RITUXAN® "); or as" Y2B8 "or Yttrium- [90] -labeled 2B8 murine antibody designated “Ibritumomab Tiuxetan” ZEVALIN® (US Pat. No. 5,736,137, herein incorporated by reference); “ ¹³¹ I-B1” Murine IgG2a "B1" (Yodine I131 Tocitumomab, BEXXAR ™), also named "Tositumomab", optionally labeled with ¹³¹ I to generate antibodies (US, incorporated herein by reference) Patent 5,595,721); murine monoclonal antibody “1F5” [Press et al., Blood 69 (2): 584-591 (1987)]; and “Framework patched” or humanized 1F5 (W0 03/002607 , Leung, S .; variants thereof including ATCC Accession No. HB-96450; murine 2H7 and chimeric 2H7 antibodies (references herein) US Pat. No. 5,677,180, cited as; humanized 2H7; huMax-CD20 (Genmab, Denmark; W02004 / 035607); AME-133 (Applied Molecular Evolution); A20 antibody or its Variants such as chimeric or humanized A20 antibodies (cA20, hA20, respectively) (US 2003/0219433, Immunmedics); and monoclonals available from the International Leukocyte Typing Workshop Antibodies L27, G28-2, 93-1B3, B-Cl or NU-B2 (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., P. 440, Oxford University Press (1987))).

The term rituximab or rituxan® as used herein is a genetically engineered chimeric murine / denoted as “C2B8” in US Pat. No. 5,736,137, directed against the CD20 antigen and incorporated herein by reference. Mention is made including human monoclonal antibodies, and fragments thereof which retain the ability to bind CD20 antigens.

For the purposes of this application and unless stated otherwise, "humanized 2H7" refers to a humanized antibody, or antigen-binding fragment thereof, that binds human CD20, wherein the antibody is a Effective for B cell ablation and in its H chain variable region (V _H ) at least the CDR H3 sequence of SEQ ID NO: 12 (FIG. 1B) and human heavy chain subgroup III (V _H III) from an anti-human CD20 antibody As human common framework (FR) residues. In a preferred embodiment, the antibody further comprises an H chain CDR H1 sequence of SEQ ID NO: 10 and a CDR H2 sequence of SEQ ID NO: 11, more preferably, an L chain CDR L1 sequence of SEQ ID NO: 4, CDR L2 sequence of SEQ ID NO: 5, CDR L3 of SEQ ID NO: 6 and substantially human consensus framework (FR) residues of the human light chain κ subgroup I (VκI), wherein the V _H region is a human IgG It may be linked to a chain constant region, which may be, for example, IgG1 or IgG3. In a preferred embodiment, the antibody comprises the V _H sequence of SEQ ID NO: 8 (shown as v16 in FIG. 1B), optionally comprises the V _L sequence of SEQ ID NO: 2 (shown as v16 in FIG. 1B), This includes amino acid substitutions (v96) of D56A in the H chain and S92A in the N100A and L chains. Preferably, the antibody is a complete antibody comprising the light and heavy chain amino acid sequences of SEQ ID NOs: 13 and 14 as shown in FIGS. 2 and 3, respectively. Another preferred embodiment is 2H7.v31 wherein the antibody comprises the light and heavy chain amino acid sequences of SEQ ID NOs: 13 and 15 as shown in FIGS. 2 and 4, respectively. Antibodies herein may further comprise at least one amino acid substitution in the Fc region that improves ADCC and / or CDC activity, for example, the amino acid substitution is S298A / E333A / K334A, more preferably SEQ ID NO: 2H7.v31 having a heavy chain amino acid sequence of 15 (as shown in FIG. 4). Any of the above antibodies may further comprise at least one amino acid substitution at the Fc moiety that further reduces CDC activity, for example, may comprise at least the substitution K322A. U.S. Patent 6,528,624 B1 (Ethersogi et al.).

Preferred humanized 2H7

Variable light sequence:

(SEQ ID NO: 2); And

Variable heavy chain sequence:

A complete antibody or antibody fragment comprising (SEQ ID NO: 8).

If the humanized 2H7 antibody is a complete antibody, preferably, the light chain amino acid sequence is:

(SEQ ID NO: 13); And

Heavy chain amino acid sequence:

(SEQ ID NO: 14) or

Heavy chain amino acid sequence:

(SEQ ID NO: 15).

In a preferred embodiment of the invention, the V region of the variant based on 2H7 version 16 will have the amino acid sequence of v16 excluding the position of the amino acid substitutions shown in the table below. Unless stated otherwise, the 2H7 variant will have the same L chain as that of v16.

An “isolated” antagonist is an antagonist identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with the diagnostic or therapeutic use of antagonists and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antagonist is (1) greater than 95% by weight, and most preferably, greater than 99% by weight of the antagonist, as measured by the Lowry method, and (2) using a spinning cup sequence analyzer. Sufficient to obtain at least 15 of the N-terminal or internal amino acid sequences, or (3) is homologous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue, or preferably silver staining. Will be purified. Isolated antagonist includes the antagonist present in its original location in the recombinant cell since at least one component will not be present in the natural environment of the antagonist. However, isolated antagonists will generally be purified by one or more purification steps.

As used herein, "subject" is a human subject.

A subject who is "no symptoms" herein is a subject who has not experienced any of the symptoms of an autoimmune disease.

A "symptom" of a disease is any pathological phenomenon or departure from the normal structure, function, or sensation that a subject experiences and indicates the disease.

A subject who is at risk for experiencing one or more autoimmune disease symptoms for purposes herein is a subject who is more than normal who is more likely to experience one or more symptom (s) with a subject having similar statistical properties. to be. For risk subjects, for example, the probability of experiencing autoimmune symptom (s) within 0-10 years is about 80-100%.

An “autoantibody” herein is an antibody produced by a subject that binds to a self-antigen produced by the subject.

An "abnormal" autoantibody level is intended for a concentration of autoantibodies that exceeds the concentration of autoantibodies present in normal subjects who are not at risk of experiencing an autoimmune disease of interest.

The expression “effective amount” refers to the amount of antagonist that is effective for preventing the disease in question.

As used herein, the term "immunosuppressant" for adjuvant treatment refers to a substance that acts to inhibit or mask the immune system of the mammal to be treated. These substances include substances that inhibit cytokine production, substances that downregulate or inhibit self-antigen expression, or substances that mask MHC antigens. Examples of such materials include 2-amino-6-aryl-5-substituted pyrimidines (US Pat. No. 4,665,077, incorporated herein by reference); Nonsteroidal anti-inflammatory drugs (NSAIDs); Azathioprine; Cyclophosphamide; Bromocriptine; Danazol; Dapson, glutaraldehyde (masking MHC antigens as described in US Pat. No. 4,120,649); Anti-idiotype antibodies against MHC antigens and MHC fragments; Cyclosporin A; Steroids such as glucocorticosteroids such as prednisone, methylprednisolone, and dexamethasone; Methotrexate (oral or subcutaneous); Hydroxychloroquine; Sulfasalazine; Leflunomide; Anti-interferon-γ, -β, or -α antibody, anti-tumor necrosis factor-α antibody (inpriximab or adalinumab), anti-TNFα immunoahesin (etanercept), anti-tumor necrosis factor-β Cytokine or cytokine receptor antagonists including antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; Anti-LFA-1 antibodies, including anti-CD 11a and anti-CD 18 antibodies; Anti-L3T4 antibodies; Heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; Soluble peptide comprising LFA-3 binding region (WO 90/08187 published July 26, 1990; Streptokinase; TGF-β; Streptodonase; RNA or DNA from host; FK506; RS-61443; Deoxy Spergualin; Rapamycin; T-cell receptor (Cohen et al., US Pat. No. 5,114,721); T-cell receptor fragment (Offner et al., Science , 251: 430-432 (1991)); WO 90 / 11294; Ianeway, Nature , 341: 482 (1989); and WO 91/01133) and T cell receptor antibodies (EP 340,109), for example T10B9.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or inhibits the function of a cell and / or destroys a cell. The term refers to radioisotopes (e.g., radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu), chemotherapy agents and toxins (Eg, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.) Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; Sulfonates such as busulfan, improsulfan and pifosulfan; aziridine such as benzodopa, carbocuone, meturedopa and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylene Ethyleneimine and methylamelamine, including thiophosphoramide and trimethylolomelamine; acetogenin (especially bulatacin and bulatacinone); camptothecin (including the synthetic analog tapotecan); bryostatin; calstatin; CC-1065 (His ah Dozelesin, carzelesin and bizelesin synthetic analogues; Cryptophycin (particularly Cryptophycin 1 and Cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); elute Robin; pankratisstatin; sarcodictin; spongistatin; nitrogen mustard, for example chlorambucil, chlornaphazine, colophosphamide, esturamustine, ifosfamide, mechloretamine, meclo Retamine Oxide Hydrochloride, Melphalan, Norshambicin, Fennesterine, Prednisostin, Trophosphamide, Urasyl Mustard; Nitrosurea, for example, Carmustine, Chlozozotocin, Potetemustine, Romustine Antibiotics, such as enedine antibiotics (eg calcicheamicin, in particular calcicheamicin gamma 1I and calcicheamicin omegal (see eg Agnew, Chem) Intl . Ed . Engl . , 33: 183-186 (1994); Dinemycin, including dinemycin A; Bisphosphonates such as clodronate; Esperamicin; And neocardinostatin chromophores and related chromophore protein edynein antibiotic chromophores), aclacinomycin, actinomycin, auturamycin, azaserine, bleomycin, carcinomycin, carabicin, carminomycin, carzinophil Lean, chromomycin, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN @ doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin , 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olibomycin, Peplomycin, fort pyromycin, puromycin, quelamycin, rhorubicin, streptonigrin, streptozosin, tubercidine, ubenimex, ginostatin, zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denophtherine, methotrexate, pterophtherin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enositabine, phloxuridine; Androgens such as calusosterone, dromostanolone propionate, epithiostanol, mepitiostane, testosterone; Anti-adrenal such as aminoglutetimides, mitotans, trilostane; Folic acid sources such as proline acid; Aceglaton; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Vestravusyl; Bisantrene; Edatraxate; Depopamine; Demecolsin; Diajikuon; Elponnitine; Elftinium acetate; Epotyrones; Etogluside; Gallium nitrate; Hydroxyurea; Lentinane; Rodidamine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Fur mall; Nitracrine; Pentostatin; Penammet; Pyrarubicin; Roxanthrone; Grape filinic acid; 2-ethylhydrazide; Procarbazine; PSK®; Polysaccharide complexes (JHS Natural Products, Eugene, Oregon); Lakamic acid; Lagoxin; Sizopyran; Spirogermanium; Tenuazone acid; Triazcuone; 2,2 ', 2''-trichlorotriethylamine; Tricortesene (particularly T-2 toxin, verraculin A, loridine A and anguidine); urethane; Bindesin; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Fifobroman; Pricktocin; Arabinoxide ("Ara-C");Cyclophosphamide;Thiotepa; Taxoids such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ™ (albumin engineered nanoparticle formulations without chromophores of paclitaxel) American Pharmaceutical Partners, Schaumburg, Ill.), And TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinblastine; platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE® vinorelbine; Novantron; Teniposide; Edda trexate; Daunomycin; Aminopterin; Xceloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoic acid, such as retinoic acid; Capecitabine; And pharmaceutically acceptable salts, acids or derivatives of those mentioned above.

The definition also includes, for example, an article comprising tamoxifen (NOLVADEX® tamoxifen), raloxifene, droxyfen, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onafristone and FARESTON® toremifene Anti-hormonal agents that act to modulate or inhibit hormonal action on the tumor, such as estrogens and selective estrogen receptor modulators (SERMs); Aromatase inhibitors that inhibit the enzyme aromatase, which modulates estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglutetimides, MEGASE® megestrol acetate, AROMASIN® exequestan, formmethanie , Podrozole, RIVISOR® Borazole, FEMARA® Letrozole, and ARIMIDEX® Anastrozole; Anti-androgens such as flutamide, nilutamide, bicalutamide, lutrolide and goserelin; And troxacitabine (1,3-dioxolane nucleoside cytosine analog); Antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Ralf and H-Ras; Vaccines such as gene therapy vaccines, such as the ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; And pharmaceutically acceptable salts, acids or derivatives of those mentioned above.

The term "cytokine" is a generic term for proteins that act on another cell as intercellular mediators released by one cell population. Examples of such cytokines include lymphokine, monocaine; Interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12, IL-15; Tumor necrosis factors such as TNF-α or TNF-β; And other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes small molecule entities synthesized synthetically with biologically active equivalents of proteins and natural sequence cytokines from natural sources or recombinant cell culture, and pharmaceutically acceptable derivatives thereof. It includes.

The term “hormone” generally refers to polypeptide hormones secreted by the glandular organs. Among the hormones, for example, growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxin; Prolylacin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); Prolactin, placental lactogen, mouse gonadotropin-associated peptide, inhibin; Activin; Back tube suppressor; And thrombopoietin. As used herein, the term hormone includes small molecule entities produced synthetically with biologically active equivalents of proteins and natural sequence hormones from natural sources or recombinant cell culture, and includes pharmaceutically acceptable derivatives thereof. .

The term "growth factor" refers to a protein that promotes growth, for example, liver growth factor; Fibroblast growth factor; Vascular endothelial growth factor; Nerve growth factors such as NGF-β; Platelet-derived growth factor; Conversion growth factors (TGFs) such as TGF-α and TGF-β; Insulin-yang growth factor-I and -II; Erythropoietin (EPO); Osteoinduction factors; Interferons such as interferon-α, -β, and -γ; And colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF). As used herein, the term growth factor includes small molecule entities synthesized synthetically with biologically active equivalents of proteins and natural sequence growth factors from natural sources or recombinant cell culture, and includes pharmaceutically acceptable derivatives thereof. Include.

The term “integrin” enables receptors to bind to and respond to extracellular matrix and refers to receptor proteins involved in various cellular actions, such as wound healing, cell differentiation, homing and apoptosis of tumor cells. These are part of the cell adhesion receptor large family involved in cell-extracellular matrix and cell-cell interactions. Functional integrins are called glycoprotein subunits, alpha and beta, and consist of two transmembrane glycoprotein subunits that are noncovalently linked. All alpha subunits share some homology with each other like beta subunits. The receptor always contains one alpha chain and one beta chain. Examples include Alpha6beta1, Alpha3beta1, Alpha7beta1, LFA-1, and the like. As used herein, the term integrin includes small molecule entities produced synthetically with proteins from natural sources or recombinant cell culture and biologically active equivalents of native sequence integrin, and includes pharmaceutically acceptable derivatives thereof. .

For the purposes of this application, "Tumor Necrosis Factor Alpha (TNFα)" is described in Pennica et al., Nature , 312: 721 (1984) or Aggarwal et al., JBC , 260: 2345 (1985). Reference is made to human TNFα molecules comprising the amino acid sequence as described.

“TNFα inhibitors” herein are agents that generally inhibit to some extent the biological function of TNFα by binding to TNFα and neutralizing its activity. Examples of TNF inhibitors specifically contemplated herein include Etanercept (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 metrotraxate), azathioprine, D-pheny Silamine, gold (oral), gold (muscle), minocycline, cyclosporin, streptococcal protein A immunosorbents and the like.

As used herein, the term “prodrug” refers to the form of a precursor or derivative of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be converted into an enzymatically activated or more active parent form. (Wilman, "Pro Drugs in Cancer Chemotherapy", Biochemical Society Transactions , 14: 375-382, 615th Meeting, Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery , Borchardt et al., (Ed.), Pp. 247-276, Humana Press (1985)). Prodrugs of the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D- Amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs. Examples of cytotoxic drugs that can be derivatized in the form of prodrugs for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

"B cell malignancy" is a malignancy comprising B cells. Examples include Hodgkin's disease, including lymphocyte predominant Hodgkin's disease (LPHD); Non-Hodgkin's lymphoma (NHL); Follicle central cell (FCC) lymphoma; Rapid lymphocytic leukemia (ALL); Chronic lymphocytic leukemia (CLL); Hairy cell leukemia; Plasmacytoid lymphoma; Mantle cell lymphoma; AIDS or HIV-related lymphomas; Multiple myeloma; Central nervous system (CNS) lymphoma; Post-transplant lymphoid disease (PTLD); Waldenström macroglobulinemia (lymphoid cellular lymphoma); Mucosal associated lymphoid tissue (MALT) lymphoma; And marginal lymphoma / leukemia.

Non-Hodgkin's lymphoma (NHL) is not limited, but low-malignant / follicular NHL, alkylating or non-reactive NHL, first-line low-malignant NHL, III / IV NHL, chemotherapy resistant NHL, Sorphocytes (SL ) NHL, intermediate grade / vesicle NHL, moderate malignant diffuse NHL, diffuse large cell lymphoma, invasive NHL (invasive NHL and invasive recurrent NHL at first line), recurrent NHL after autologous stem cell transplant or Non-responsive NHL, high-malignant immune blast NHL, high-malignant lymphocytic NHL, high-malignant small non-dividing cell NHL, bulk disease NHL, and the like.

II . Screening for Subjects at Risk

In accordance with a preferred embodiment of the present invention, subjects selected for treatment herein are generally from a cohort of asymptomatic but high-risk individuals who are at high risk of developing moderate-to-severe disease within a defined time frame. Is an object of. For example, in subjects, the likelihood of developing the disease within 0-10 years may be about 80-100%.

As the subject has no symptoms, a substitute label for one or more diseases will be evaluated. For example, autoantibody production can be assessed and / or genetic and / or proteomic signatures can be evaluated to select individuals at high risk. Alternatively, or in addition, autoimmune profiles can be obtained by FACs analysis of B-cell subsets from whole blood.

Samples may be taken from a subject who has experienced one or more diagnostic / prognostic assays to assess the likelihood that the subject will have or develop an autoimmune disease. Body cells, such as those present in blood, tissue biopsies, surgical specimens, or autopsy materials. The sample may be, for example, serum, whole blood, cell lysate, milk, saliva or other secretion but serum is preferred.

Polynucleotide (s) including oligonucleotide sequences, genomic DNA and complementary RNA and DNA molecules. Polynucleotides can be used to detect and quantify gene expression in biopsied tissue after biopsy, where mutation or abnormal expression of the gene (s) is associated with the risk of developing the disease. Genomic DNA used for diagnosis or prognosis can be obtained from being present in body cells, such as blood, tissue biopsies, surgical tests, or autopsy materials. DNA can be isolated and used directly for specific sequence detection or amplified by polymerase chain reaction (PCR) prior to analysis. Similarly, RNA or cDNA with or without PCR amplification can also be used. To detect specific nucleic acid sequences, direct nucleotide sequencing, reverse transcriptase PCR (RT-PCR), hybridization with specific oligonucleotides, restriction enzyme digestion and mapping, PCR mapping, RNAse protection, and various other methods can be used. .

Oligonucleotides specific for a particular sequence can be hybridized to a subject sample in solution or a solution that is chemically synthesized, radioactive or nonradioactively labeled, and immobilized on a membrane or other solid-support. Methods, such as autoradiography, fluorescence, or colorimetry, may then be used to visualize the presence, absence, or excess of expression of the gene (s).

The nucleotide sequences of the gene (s) of the normal sample and the pathological sample from the diseased patient can be compared to provide a criterion for diagnosis or prognosis or a criterion for the risk of developing the disease.

Another method of identifying normal or standard profiles for expression is through survey RT-PCR studies. RNA isolated from body cells of normal individuals, in particular RNA isolated from tumor cells, is subjected to real-time PCR using oligonucleotides that are reverse transcribed and specific for the relevant genes to establish normal levels of gene expression.

Standard values obtained in both examples can be compared with values obtained from samples derived from subjects exhibiting symptoms for the disease. Deviation from the standard value is used to establish susceptibility to the disease.

Once sensitivity to disease is established and treatment protocols are initiated, hybridization assays or quantitative RT-PCR studies can be repeated regularly to determine whether expression levels in a subject are close to those observed in normal subjects. The results obtained from the repeated assays can be used to suggest therapeutic effects over a period of days to months.

In assessing disease susceptibility by nucleic acid studies, the microarray (s) are preferably used to compare the subject's nucleic acid profile and control profile (s). Microarrays can be prepared, used and analyzed using methods known in the art (see, eg, Schena et al.PNAS USA 93: 10614-10619 (1996); Hell et al.,PNAS USA 94: 2150-2155 (1997); And [Heller, M,Annual Review of Biomedical Engineering 4: 129-53 (2002)]. For example, a microarray containing multiple genes may optionally be linked to 3-aminopropyltriethoxysilane (Wisconsin) using a robotic arrayer (Norgren Systems, Mountain View, CA). CDNA clones (Invitrogen, Calif.) On glass slides coated with Aldrich, Milwaukee) and 1,4-phenylenediisothiocyanate (Aldrich, Milwaukee, Wis.) And PCT products derived from Genentech, Inc. RNA isolation can be performed by CsCl step gradients [Kingston,Current Protocols in Molecular Biology 1: 4.2.5-4.2.6 (1998). Probe for measurement assays can be generated by conservative amplification and subsequent labeling as follows: Double-stranded DNA (Invitrogen, Carlsbad, Calif.) Generated from total RNA is a single round. Amplified using a modified in vitro transcription protocol (MEGASCript T7 from Ambion, Austin, Texas [Gelder et al., Proc . Natl . Acad . Sci . USA 87: 1663-1667 (1990)). The resulting cRNA can be used as a template to generate sense DNA probes using random primers and using MMLV-derived reverse transcriptase (Invitrogen, Carlsbad, Calif.). The probe was then hybridized to the array in 50% frucamide / 5XSSC overnight at 37 ° C. and washed the next day by 2 × SSC, 0.2% SDS, then 0.2 × SSC, 0.2% SDS. Images of the array can be collected using a CCD-camera based imaging system (Norgren system, Mountain View, Calif.) Equipped with an appropriate optical filter and Xenon light source for each dye. Full dynamic-range images can be collected (Autograb, Genentech, Inc.) and built on the Matlab (the MathWorks) platform in Nadiq, Massachusetts. Intensities and ratios were extracted using automated automated gridding and data extraction software (glmage, Genentech, Inc.).

Microarray methods are described in US 2003/0219818 A1 to Bohen et al.

In another aspect, subjects susceptible to disease are identified using assays that detect autoantibodies such as those mentioned in the table below. In a preferred embodiment autoantibody production is assessed qualitatively and preferably quantitatively. The autoantibodies or antibodies to be evaluated generally depend on the autoimmune disease to be prevented. Examples of autoantibodies associated with selected autoimmune diseases are reflected in the table below.

Table 1

Autoimmune diseases Autoantibodies ( Ab ) Guillain-Barré Syndrome Cross Reactive Antibodies to GM1 Ganglioside or GQ1b Ganglioside Myasthenia gravis Anti-acetylcholine receptor (AchR) Ab, anti-AchR subtype Ab, anti-MuSK Ab Large vessel vasculitis / giant cell (Takayasu) arteritis Serum Anti-Endothelial Cell Ab Middle Vasculitis / Kawasaki Disease Anti-endothelial Ab, anti-neutrophil cytoplasmic Ab (ANCA)

Nodular polyarteritis Autoantibodies (pANCA) that stain or nucleus neutrophils pemphigus Anti-Desmoglen (Dsg) Ab, including IgG, anti-Dsg 3 (phasic spear), anti-Dsg 1 (deciduous spear), and anti-Dsg 2 Ab Scleroderma Anti-central, anti-topoisomerase-1 (Scl-70) Ab, anti-RNA polymerase or anti-U3-RNP Ab Goodpasture syndrome Anti-glomerular basement membrane (GBM) Ab Rapid Progressive Glomerulonephritis Anti-glomerular basement membrane (GBM) Ab Sjogren's syndrome Anti-La / SSB Ab, Anti-Ro / SSB Ab Primary cholangiocytosis Anti-mitochondrial Ab (AMA), anti-M2 Ab Ulcerative colitis, Crohn's disease Autoantibodies (pANCA), anti-saccharomyces cerevisiae antibodies (ASCA), which stain or nucleus neutrophils Graves' disease Anti-TPO Ab, anti-TG Ab, anti-thyroid stimulating hormone receptor (TSHR) Ab Membranous nephropathy Anti-dsDNA Ab (if associated with lupus nephritis) Autoimmune hepatitis Anti-nuclear (AN) Ab, anti-actin (AA) Ab, anti-ASM Ab Non-tropical Sprue (Gluten Free Disease) IgA anti-myofiber membrane Ab, IgA anti-tissue transglutaminase Ab, IgA anti-gliadin Ab, IgG anti-gliadin Ab

Addison's disease Anti-CYP21 A2 (p450c21 or 21, hydroxylase), anti-CYP11 A1, anti-CYP17 Polymyositis / Skin Myositis Anti-nuclear Ab (ANA), anti-ribonucleic acid protein (RNP) Ab, myositis-specific Ab (anti-Jo-1 Ab, anti-Mi-2 Ab, anti-PM-Scl Ab, anti-Ku Ab) Monoclonal gamma globulin disease Anti-MAG Ab Cold globulinemia Anti-HCV Ab Systemic lupus erythematosus Anti-nuclear Ab (ANA), anti-double strand DNA (dsDNA) Ab, anti-Sm Ab, anti-nucleus ribonucleic acid Ab, anti-phospholipid Ab, anti-ribosome P Ab, anti-Ro / SS-A Ab , Anti-Ro Ab, anti-La Ab Rheumatoid Arthritis (RA) Low Affinity IgM Rheumatoid Factor (RF) Antibodies to Fc of IgG Factor VIII Deficiency Anti-factor VIII Ab Peripheral Neuropathy Anti-GM1 Ab, anti-MAG Ab, anti-SGPG Ab, IgM anti-glycoconjugate Ab IgM polyneuropathy Anti-myelin Related Glycoprotein (MAG) Ab Chronic neuropathy IgM anti-gangliosides Ab Hashimoto thyroiditis Anti-TPO Ab, anti-TG Ab, anti-thyroid stimulating hormone receptor (TSHR) Ab Anti-phospholipid antibody syndrome Anti-phospholipid Ab Multiple sclerosis Anti-myelin basic protein, anti-myelin oligodendrocyte glycoprotein Ab

In general, antibodies or other reagents that bind to autoantibodies of interest are used in the assay. However, detection of autoantibody nucleic acids is another option. Autoantibodies in human body fluids or cell or tissue extracts are evaluated. Antibodies or other reagents that bind autoantibodies can be used with or without modification and can be labeled by covalent or non-covalent association with the reporter molecule.

Various protocols for measuring autoantibodies, including ELISAs, RIAs, and FACS are known in the art and provide criteria for diagnosing altered or abnormal levels of autoantibodies. Normal or reference values for autoantibody levels can be established by assessing autoantibody levels in body fluids or cell extracts taken from normal mammalian subjects, preferably humans. Measurement of autoantibodies in samples from subjects can be compared to standard values. The deviation between the standard value and the subject's value establishes parameters for diagnosing susceptibility to disease.

III . Prophylaxis

The present invention is at risk of experiencing one or more autoimmune disease symptoms comprising administering to the subject an antagonist that binds to B cell surface markers in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. The present invention provides a method for preventing autoimmune diseases in asymptomatic human subjects. Preferably, the B cell surface marker is CD20 and the antagonist is preferably an antibody. Thus, in a preferred embodiment, the present invention is at risk of experiencing one or more autoimmune disease symptoms comprising administering to the subject a CD20 antibody in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease, Provided are methods for preventing autoimmune diseases in asymptomatic human subjects.

The methods herein can prevent "new onset" of a disease (ie, the subject does not experience one or more symptoms of autoimmune disease, or the subject does not experience one or more symptoms of autoimmune disease to be prevented). can not do it). Alternatively, the present invention can prevent the recurrence of autoimmune diseases in subjects at rest during the parenchymal period (eg, upon remission of at least one year, at least two years, for example, for 2-20 years). In addition, the methods herein may prevent subjects who have already experienced one or more symptoms of autoimmune disease from experiencing one or more other different autoimmune disease symptoms.

In one embodiment, the subject has not been previously treated with drug (s), eg, immunosuppressant (s), and / or has not been previously treated with an antagonist to B-cell surface markers to treat autoimmune diseases ( For example, no treatment with CD20 antibody).

Examples of autoimmune diseases to be prevented herein include systemic lupus erythematosus (SLE), anti-phospholipid antibody syndrome, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, Sjogren's syndrome, Guillain-Barré syndrome, severe muscle Asthenia, macrovascular vasculitis, intermediate vasculitis, nodular polyarteritis, pemphigus, scleroderma, goodpasture syndrome, glomerulonephritis, primary biliary cirrhosis, Graves' disease, membrane nephropathy, autoimmune hepatitis, nontropical sprue, Addison's disease , Polymyositis / dermatitis, monoclonal gamma disease, factor VIII deficiency, cold globulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and Hashimoto thyroiditis.

In one embodiment, it is determined that a subject to be treated herein produces autoantibodies in abnormal amounts. Accordingly, the present invention provides a method for preventing autoimmune disease in a subject with no symptoms having abnormal autoantibody levels, comprising administering to the subject a CD20 antibody in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. To provide.

Once identified as a subject at risk, the subject is treated with an antagonist that binds B cell surface markers, preferably an antibody that binds CD20, in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. do.

Compositions comprising an antagonist are formulated in a manner consistent with good medical practice and administered by determining the dosage. Factors contemplated herein include the specific disease or condition to be treated, the particular mammal to be treated, the clinical symptoms of the individual patient, the cause of the disease or condition, the site to which the drug is delivered, the method of administration, the schedule of administration, and the skilled person in the medical arts. Other arguments are included. The therapeutically effective amount of the antagonist to be administered will be determined in consideration of these factors.

According to a general proposal, the effective amount of parenteral administered antagonist per dose will range from about 20 mg / m 2 to about 10,000 mg / m 2 (patient body weight) by one or more dosages. IV dosing regimens for complete antibodies include 375 mg / m ² × 4, 1000 mg × 2 (eg, days 1 and 15), or 1 gram × 3 per week.

However, as mentioned above, the amount of antagonist proposed above requires a great deal of care in terms of treatment. The key factor in selecting the appropriate dosage and in planning the administration is the result obtained as described above. For example, relatively high dosages may be required for early treatment of advanced and rapid disease. To obtain the most effective outcome depending on the disease or condition, the antagonist is administered as close as possible to the first sign, diagnosis, aspect or occurrence of the disease or condition, or while the disease or condition is alleviating.

Antagonists are administered by any suitable method including parenteral, topical, subcutaneous, intraperitoneal, pulmonary and intranasal, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Intradural administration is also being observed. In addition, the antagonist may be appropriately administered by pulse infusion, for example, with a reduced dose of the antagonist. Preferably the injection is administered by intravenous injection.

Other compounds, such as cytotoxic agents, chemotherapeutic agents, immunosuppressants, cytokines, cytokine antagonists or antibodies, growth factors, integrins, integrin antagonists or antibodies, etc., can be administered with the antagonists herein. For example, antagonists include TNF-inhibitors, disease-modified antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids (through joint injections), low dose prednisone, glucocorticoids / prednisone / methylprednisone ( Glucocorticoids), intravenous immunoglobulins (gamma globulins), plasmaferases, levothyroxine, cyclosporin A, somatostatin analogs, cytokine antagonists, anti-metabolites, immunosuppressants, cytotoxic agents such as chlorine Rambusil, cyclophosphamide, azathioprine), rehabilitation surgery, radioiodine, thyroidectomy, and the like. Combination administration may be by co-administration using a separate formulation or a single pharmaceutical formulation, and preferably in any order, with time intervals while both (or both) active agents simultaneously exert their biological activity. Sequential administration.

Apart from administering a protein antagonist to a subject, the present application contemplates administering the antagonist by gene therapy. Administration of nucleic acids encoding antagonists includes those expressed as "effective amount administration of antagonists." See, eg, WO 96/07321 published March 14, 1996, relating to the use of gene therapy to generate intracellular antibodies.

There are two main ways of introducing nucleic acids (optionally included in a vector) into the subject's cells in vivo and ex vivo. For in vivo delivery, nucleic acids are usually injected directly into the site of the subject in need of an antagonist. For ex vivo treatment, the subject's cells are removed and nucleic acids are introduced into these isolated cells and the mutated cells are administered directly to the subject or encapsulated, for example, in a porous membrane implanted in the subject (eg See, for example, US Pat. Nos. 4,892,538 and 5,283,187. There are a variety of techniques available for introducing nucleic acids into viable cells. This technique depends on whether the nucleic acid is delivered to cells cultured in vitro or to cells in vivo in the host of interest. Techniques suitable for delivering nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-textran, calcium phosphate precipitation, and the like. A vector commonly used for ex vivo delivery of genes is a retrovirus.

Currently preferred in vivo nucleic acid delivery techniques include transfections using viral vectors (eg, adenoviruses, herpes simplex I virus or adeno-associated viruses) and lipid-based systems (lipids useful for lipid-mediated delivery of genes, for example). , DOTMA, DOPE and DC-Chol). In some cases, it is desirable to provide a nucleic acid source with a substance that targets the target cell, such as cell membrane proteins or antibodies specific for the target cell, ligands for receptors on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis are used for targeting and / or, for example, capsid proteins or fragments thereof that are friendly to a particular cell type, endocytosis cycle. It can be used to facilitate the influx of antibodies to proteins entering into the cell, and proteins that enhance intracellular half-life by targeting intracellular localization. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al., J. Biol . Chem . 262: 4429-4432 (1987); and Wagner et al., Proc . Natl . Acad . Sci . USA 87: 3410-3414 (1990). See Anderson et al., Science 256: 808-813 (1992) for details on currently known gene labels and gene therapy protocols. See also WO 93/25673 and references cited herein.

IV . Antagonist production

The methods and products of the present invention use or include antagonists that bind to B cell surface markers. Therefore, a method of producing such antagonists will now be described.

The antigen used for the production or screening of the antagonist (s) can be, for example, a soluble form of B cell surface marker or portion thereof comprising the desired epitope. Alternatively, or in addition, cells expressing B cell surface markers at their cell surface may be used to generate or screen antagonists. Other forms of B cell surface markers useful for producing antagonists are apparent to those skilled in the art.

Preferred antagonists are antibodies, but antagonists other than antibodies are also considered herein. For example, the antagonist may optionally include small molecule antagonists that fuse to or bind to a cytotoxic agent (eg, those described herein).

Small molecule libraries of B cell surface markers of interest herein can be screened to identify small molecules that bind the antigen. These small molecules may be further screened for their antagonist properties and / or combined with cytotoxic agents.

The antagonist may be a peptide produced by rational design or phage display (see, eg, WO 98/35036 published August 13, 1998). In one embodiment, the selection molecule can be a “CDR mimic” or antibody analog designed based on the CDRs of the antibody. These peptides may themselves be antagonistic, but the peptides may optionally be fused to cytotoxic agents to add or enhance the antagonist properties of the peptide.

In the following, exemplary techniques for the production of antibody antagonists for use in accordance with the present invention are described.

(i) Polyclonal  Antibodies

Polyclonal antibodies are preferably produced by several subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in an animal. Difunctional or derivatizing materials such as maleimidobenzoyl sulfosuccinimideesters (conjugation via cysteine residues), N-hydroxysuccinimides (linked via lysine residues), glutaraldehyde, Proteins that are immunogenic in species to be immunized with the relevant antigen using succinic anhydride, SOCl ₂ , or R ¹ N═C═NR where R and R ¹ are other alkyl groups, eg, keyhole limpet hemocyanin, It may also be useful to conjugate with serum albumin, bovine thyroglobulin or soybean trypsin inhibitor.

Antigens were challenged by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with Freund's complete adjuvant corresponding to three times their amount and injecting the solution into the skin at various sites. , Immunogenic conjugates or derivatives. One month thereafter, 1/5 to 1/10 of the original amount of peptide or conjugate was placed in the Freund's complete adjuvant and subcutaneously injected at various sites to antigen stimulate the animal. After 7-14 days, animals were bled and serum was analyzed for antibody titers. Animals were antigen stimulated until titer retention. Antigen stimulation is preferably with conjugates of the same antigen linked to other proteins and / or bound by different cross-linking reagents. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, flocculants such as alum are suitably used to boost the immune response.

( ii ) Monoclonal  Antibodies

Monoclonal antibodies are obtained substantially from a population of homologous antibodies, i.e., each antibody constituting the population is identical to the same epitope, except for possible variants produced in the production of monoclonal antibodies, such as those typically present in small amounts. Matches and / or combines with it. Thus, the modifier “monoclonal” characterizes the antibody, not as a separate or mixture of polyclonal antibodies.

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

In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as mentioned above to induce lymphocytes capable of producing or producing antibodies that specifically bind to proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding,Monoclonal Antibodies . Principles and Practice , pp. 59-103 (Academic Press, 1986)].

The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium, preferably comprising one or more substances that inhibit the proliferation or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas is typically hypo Forc, a substance that inhibits the proliferation of HGPRT-deficient cells. Xanthine, aminopterin and thymidine (HAT medium).

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

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

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

After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, clones were subcloned by limiting dilution methods and cultured by standard methods. [Goding, Monoclonal Antibodies : Principles and Practice , pp. 59-103 (Academic Press, 1986)]. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be cultured in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclones are suitably, for example, conventional immunonoids such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It is isolated from the culture medium, ascites fluid or serum by the globulin purification method.

DNA encoding monoclonal antibodies is readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells serve as a preferred source of such DNA. After isolation, the DNA can be placed in an expression vector, which then does not produce immunoglobulin proteins. Host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells are transfected to synthesize monoclonal antibodies in recombinant host cells. For studies on recombinant expression of DNA encoding such antibodies in bacteria, see Skerra et al, Curr. Opinion in Immunol ., 5: 256-262 (1993) and Pluckthun , Immunol . Revs ., 130: 151-188 (1992).

In a further embodiment, the antibody or antibody fragment is described using McGafferty et al, Nature, 348: 552-554 (1990), using phage libraries to describe the isolation of each of murine and human antibodies. Clackson et al, Nature, 352: 624. -628 (1991) and Marks et al., J. Mol Biol., 222: 581-597 (1991)) can be isolated from antibody phage libraries obtained. Subsequent literature describes chain shuffling [Marks et al, Biol. Technology, 10: 779-783 (1992), as well as combinatorial infection and in vivo recombination, a method for producing very large phage libraries [Waterhouse et al., Nuc. Acids . Res ., 21: 2265-2266 (1993), for the production of high affinity (nM range) human antibodies. Thus, these techniques are available as an alternative to conventional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

Also, for example, DNA can be substituted for sequences encoding human heavy and light chain constant regions instead of homologous murine sequences (US Pat. No. 4,816,567; Morrison et al., Pro . Natl Acad . Sci . USA , 81: 6851 (1984)], or all or part of the coding sequence for a non-immunoglobulin polypeptide can be mutated by covalently binding to an immunoglobulin coding sequence.

Typically, the non-immunoglobulin polypeptide is substituted for the constant region of the antibody or substituted for the variable region of one antigen-binding site of the antibody to have one antigen-binding site and another antigen having specificity for one antigen. Chimeric bivalent antibodies comprising other antigen-binding sites with specificity for can be generated.

( iii A) humanized antibody

Methods for humanizing antibodies other than humans are well known in the art. Preferably the humanized antibody has one or more amino acid residues introduced into the antibody derived from a non-human source. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from "import" variable sites. Humanization consists essentially of the method of Winter et al. (Jones et al., Nature , 321: 522-525 (1986)); Riechmann et al. By substituting the hypervariable region sequences in place of the corresponding sequences of human antibodies. , Nature , 332: 323-327 (1988); Verhoeyen et al., Science , 239: 1534-1536 (1988)). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially fewer intact human variable regions are replaced by corresponding sequences from species other than human. In practice, humanized antibodies are typically human antibodies in which some hypervariable site residues and possibly some FR residues are substituted by residues from the cognate sites of rodent antibodies.

The choice between human light chain variable region and human heavy chain variable region to be used to prepare a humanized antibody is of great importance for reducing antigenicity. According to the so-called "most suitable" method, the sequence of variable regions of rodent antibodies is screened against the entire library of known human variable-region sequences. The human sequence closest to the rodent sequence is then adopted as the human framework site (FR) for humanized antibodies (Sims et al, J. Immunol , 151: 2296 (1993); Chothia et al., J. Mol . Biol , 196: 901 (1987)]. Another method uses specific framework sites derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for different humanized antibodies (Carter et al., Proc. Natl . Acad . Sci . USA , 89: 4285 (1992); Presta et al, J. Immunol . , 151) . : 2623 (1993)].

It is also important to humanize the antibodies to have high affinity for antigens and other beneficial biological properties. To achieve this object, according to a preferred method, humanized antibodies are prepared by methods of analysis of the parental sequences and various theoretical humanized products, using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commercially available and known to those skilled in the art. Computer programs are available that illustrate and show possible three-dimensional structures of selected candidate immunoglobulin sequences. Examining the display of such computer programs allows for the analysis of possible roles of residues in the functionalization of candidate immunoglobulin sequences, ie, the analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the acceptor sequence and the import sequence to obtain the properties of the desired antibody, such as increased affinity for the target antigen (s). In general, residues at hypervariable sites are directly and most substantially involved in influencing antigen binding.

( iv A) human antibody

As an alternative to humanization, human antibodies can be generated. For example, it is currently possible to make transgenic animals (eg mice) capable of producing the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production upon immunization. For example, it has been described that homozygous deletion of the antibody heavy chain binding site (J _H ) gene in chimeric and germ cell mutant mice completely inhibits endogenous antibody production. Delivery of the human germline immunoglobulin gene sequence into such germline mutant mice will produce human antibodies upon antigen exposure (see, eg, Jakobovitset al, Proc . Natl . Acad . Sci . USA , 90. : 2551 (1993); Jakobovits et al, Nature , 362: 255-258 (1993); Bruggermann et al, Year in Immuno ., 7:33 (1993); And US Pat. Nos. 5,591,669, 5,589,369 and 5,545,807).

Alternatively, human antibodies and antibody fragments from immunoglobulin variable (V) region gene repertoires obtained from unimmunized donors using phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) were tested. Can be produced in the building. According to this technique, antibody V region genes are cloned in-frame into the major coat protein or minority coat protein genes of fibrous bacteriophages such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particles. Since the fibrous particles contain a single stranded DNA copy of the phage genome, selection based on the functional properties of the antibody allows the selection of genes encoding antibodies exhibiting these properties. Thus, phages mimic some of the properties of B cells. Phage display can be performed in a variety of formats (see, eg, Johnson, Kevin S. and Chiswell, David J., Current). Opinion in Structural Biology 3: 564-571 (1993). Several sources of V-gene fragments can be used for phage display. Clackson et al., Nature , 352: 624-628 (1991) isolated various arrays of anti-oxazolone antibodies from a small random combination library of V genes derived from the spleen of immunized mice. A repertoire of V genes from non-immunized human donors was constructed and constructed essentially from Marks et al., J. Mol Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993), US Pat. Nos. 5,565,332 and 5,573,905, can also be used to isolate antibodies against a wide array of antigens (including self-antigens).

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

(v) antibody fragments

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

( vi ) Bispecific  Antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of B cell surface markers. Alternatively, the anti-B cell surface marker binding arm may be a triggering molecule on leukocytes, such as a T-cell receptor molecule (eg CD2 or CD3), or an Fc receptor (FcγR) on IgG, eg For example, it can be combined with arms that bind FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) to focus on cellular defense mechanisms against B cells. Bispecific antibodies can also be used to target cytotoxic agents to B cells. These antibodies have B cell surface marker-binding arms and arms that bind to cytotoxic agents (eg, saporin, anti-interferon-α, vinca alkaloids, lysine A chain, methotrexate, or radioisotope hapten). . Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab ') ₂ bispecific antibodies).

Methods of making bispecific antibodies are known in the art. Conventional methods for producing full-length bispecific antibodies are based on the expression of two immunoglobulin heavy chain-light chain pairs with different specificities together (Milstein et al., Nature , 305: 537-539 (1983)). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce potential mixtures of 10 different antibody molecules, only one of which has an accurate bispecific structure have. Purification of these exact molecules, usually done by affinity chromatography steps, is a rather cumbersome process and the product yield is low. Similar procedures are described in WO 93/08829; and Traunecker et al., EMBO J. , 10: 3655-3659 (1991).

According to different methods, antibody variable regions having the desired binding specificities (antibody-antigen binding sites) are fused to immunoglobulin constant region sequences. This fusion preferably uses an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2 and CH3 sites. It is preferred to have a first heavy chain constant region (CH1) containing a site necessary for light chain binding, present in one or more of these fusions. The immunoglobulin heavy chain fusions are encoded, and if desired, the DNA encoding the immunoglobulin light chain is inserted into separate expression vectors and they are transfected together into a suitable host organism. This provides great flexibility in controlling the mutual ratios of these three polypeptide fragments in embodiments in which unequal proportions of the three polypeptide chains used in the construct provide optimal yields. However, if the expression of at least two polypeptide chains in equal proportions yields high yields or if these proportions have no particular significance, the coding sequences for two or all of the three polypeptide chains are inserted into one expression vector. It is possible to do

In a preferred embodiment of this method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair on the other arm (provides a second binding specificity). It consists of. This asymmetric structure has been found to facilitate the separation of the desired bispecific compounds from the undesirable immunoglobulin chain combinations, since the immunoglobulin light chain is only present in half of these bispecific molecules. One separation method is provided. This method is described in WO 94/04690. Further details on bispecific antibody production can be found in, for example, Suresh et al., Methods in Enzymology , 121: 210 (1986).

According to another method described in US Pat. No. 5,731,168, the interface between antibody molecule pairs can be genetically engineered to maximize the percentage of heterodimer recovered from recombinant cell culture. Preferred interfaces include at least a portion of the C _H 3 region of the antibody constant region. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensation of large side chain (s) with "cavities" of the same or similar size occurs at the interface of the second antibody molecule by replacing the large amino acid side chain with smaller amino acids (eg alanine or threonine). This provides a mechanism for increasing the yield of heterodimer over other undesirable end products such as homodimers.

Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Such antibodies are proposed for example to target immune system cells with unwanted cells (US Pat. No. 4,676,980) and to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using conventional crosslinking methods. Suitable crosslinkers are well known in the art with many crosslinking techniques and are described in US Pat. No. 4,676,980.

Techniques for generating bispecific antibodies from antibody fragments have also been reported in the literature. For example, bispecific antibodies can be prepared using chemical crosslinking. Brennan et al., Science , 229: 81 (1985) describe the cleavage of a complete antibody with proteolytic enzymes to produce F (ab ') ₂ fragments. These fragments are reduced in the presence of a dithiol binder former sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragment thus generated is then converted to thionitrobenzoate (TNB) derivatives. Subsequently, one of the Fab'-TNB derivatives is converted back to Fab'-thiol by reduction with mercapto ethylamine, which is mixed with an equimolar amount of other Fab'-TNB derivatives to form a bispecific antibody. The bispecific antibodies thus produced can be used as substances for the selective immobilization of enzymes.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers were used to generate bispecific antibodies [Kostelny et al., J. Immunol . , 148 (5): 1547-1553 (1992). The leucine zipper peptide from Fos and Jun proteins was linked to the Fab 'portion of two different antibodies by gene fusion. This antibody homodimer was reduced at the hinge site to form a monomer and then reoxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. Hollinger et al., Proc . Natl . Acad . Sci . USA , 90: 6444-6448 (1993), provides another mechanism for making bispecific antibody fragments. The fragment comprises a heavy chain variable region (V _H ) linked to the light chain variable region (V _L ) by a linker that is too short to form a pair between two regions of the same chain. Thus, by the V _H and V _L regions of a single fragment to form complementary V _H and V _L region and a pair of another fragment, to form two antigen-binding site it may leave the focus. Another method for preparing bispecific antibody fragments by using single chain Fv (sFv) dimers is also reported [Grubber et al., J. Immunol . , 152: 5368 (1994).

Bivalent or higher antibodies are also contemplated. For example, trispecific antibodies can be prepared [Tutt et al., J. Immunol . 147: 60 (1991).

V. Conjugate  And other variants of antagonists

Antagonists used in the methods of the present invention or included in the products herein are optionally conjugated to cytotoxic agents. For example, the antagonist may be conjugated to the drug as described in WO 2004/032828.

Chemotherapeutic agents useful for the production of such antagonist-cytotoxic agent conjugates are described above.

Also included herein are conjugates with antagonists and one or more small molecule toxins such as calicheamicin, maytansine (US Pat. No. 5,208,020), tricotene, and CC1065. In one preferred embodiment of the invention, the antagonist is bound to one or more maytansine molecules (eg, about 1 to about 10 maytansine molecules per antagonist molecule). Maytansine can be converted, for example, to May-SS-Me, which can be reduced to May-SH3, and can react with a modified antagonist to form a maytansinoid-antagonist conjugate [Chari et al. Cancer Research 52: 127-131 (1992).

Alternatively, the antagonist is bound to one or more calicheamicin molecules. Antibiotics of the calicheamicin family that can produce double stranded DNA are destroyed at concentrations below picomolar. Structural homologues of calicheamicin that may be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG and θ ₁ ^I [Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1988).

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain ( Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modesin A chain, alpha-sarsine, Aleurites Fordia fordii ) protein, diantine protein, Phytolaca americana americana ) protein (PAPI, PAPII and PAP-S), Momordica Charantia inhibitor, Cursin, Crotin, Sapaonaria Officinalis Inhibitor, Geronine, Mitogeline, Restrictocin, Phenomycin, Eno Mycin and tricortesene (see, for example, WO 93/21232 published October 28, 1993).

The invention also encompasses antagonists conjugated with compounds having nuclease activity (eg ribonuclease or DNA endonucleases such as deoxyribonuclease; DNase).

Various radioactive isotopes may be used in the production of radioconjugated antagonists. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu.

Conjugates of antagonists with cytotoxic agents include 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), difunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimi Dill suverate), aldehyde (eg glutaraldehyde), bis-azido compound (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivative (eg bis- (p- Diazonium benzoyl) -ethylenediamine), diisocyanate (eg tolyene 2,6-diisocyanate) and bis-active fluorine compounds (eg 1,5-difluoro-2,4-dinitrobenzene) It can be prepared using. For example, lysine immunotoxins are described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DPTA) is an exemplary chelating agent for binding of radionucleotides to antagonists (see WO 94/11026). ). The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, dimethyl linkers or disulfide-containing linkers can be used (Chari et al., Cancer Research 52: 127-131 (1992).

Alternatively, fusion proteins comprising antagonists and cytotoxic agents can be prepared, for example, by recombinant techniques or peptide synthesis.

In another embodiment, the antagonist may be conjugated to a "receptor" (eg, streptavidin) for use in tumor pretargeting, wherein the antagonist-receptor conjugate is administered to the subject, followed by a wash Is used to remove unbound conjugate from the circulatory system, followed by administration of a "ligand" (eg, avidin) that is bound to a cytotoxic agent (eg, a radionucleotide).

Antagonists of the invention may also be conjugated with prodrug-activating enzymes that convert prodrugs (eg, peptidyl chemotherapeutic agents; see WO 81/01145) into active anticancer agents (eg, WO 88/07378). And US Pat. No. 4,975,278).

Enzyme components of such conjugates include enzymes that can act on prodrugs in a way that converts them into more active and cytotoxic forms.

Alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs, including but not limited to enzymes useful in the methods of the invention; Arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; Cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anticancer agent, 5-fluorouracil; Proteases useful for converting peptide-containing prodrugs to free drugs, such as seratia proteases, thermolysine, subtilisin, carboxypeptidase and cathepsins (eg, cathepsin B and L); D-alanylcarboxypeptidase useful for converting prodrugs containing D-amino acid substituents; Carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; β-lactamase useful for converting drugs derivatized with β-lactams into free drugs; And penicillin amidases such as penicillin V amidase or penicillin G amidase, which are useful for converting drugs derivatized with phenoxy acetyl or phenyl acetyl groups in amine nitrogen to free drugs, respectively. Alternatively, antibodies with enzymatic activity, also known as "abzyme", can be used to convert prodrugs of the invention to free active drugs. Massey, Nature 328: 457-458 ( 1987). Antagonist-Abzyme conjugates for delivering azyme to tumor cell populations can be prepared as described herein.

Enzymes of the invention can be covalently linked to the antagonist by techniques well known in the art, such as using the heterodifunctional crosslinked reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding site of the antagonist of the invention linked to at least the functionally active portion of the enzymes of the invention can be prepared using recombinant DNA techniques well known in the art [Neuberger et al. , Nature 312: 604-608 (1984).

Other variations of the antagonist are included herein. For example, the antagonist can be linked to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. Antibody fragments, such as Fab ', linked to one or more PEG molecules are a particularly preferred embodiment of the present invention.

Antagonists disclosed herein may also be formulated as liposomes. Liposomes containing antagonists can be prepared using methods known in the art, for example, Epstein et al., Proc. Natl . Acad . Sci . USA , 82: 3688 (1985); Hwang et al., Proc . .... Natl Acad Sci USA , 77: 4030 (1980); prepared by the method described in October 1997 and published May 23, WO 97/38731); U.S. Patent No. 4,485,045 and No. 4,544,545 arc. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of constant pore size to produce liposomes with the desired diameter. Fab 'fragments of the antibodies of the invention were subjected to disulfide exchange reactions by Martin et al., J. Biol . Chem . 257: 286-288 (1982). Optionally, chemotherapeutic agents are included in these liposomes [Gabizon et al., J. National Cancer Inst . 81 (19) 1484 (1989).

Amino acid sequence modification (s) of the protein or peptide antagonists described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antagonist. Amino acid sequence variants of the antagonist are prepared by introducing appropriate nucleotide changes into the antagonist nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from residues within the amino acid sequence of the antagonist, and / or insertion into and / or substitution of residues. Any combination of deletions, insertions, and substitutions to reach the final construct may be possible, but the final construct must possess the desired characteristics. Such amino acid changes may also alter the post-translational process of the antagonist, eg, the number or location of glycosylation sites.

Methods useful for identifying specific residues or sites of antagonists, which are preferred locations for mutagenesis, are called "alanine scanning mutagenesis" described in Cunningham and Wells, Science , 244: 1081-1085 (1989). Here, by identifying a target residue or group of residues (eg, charged residues such as arg, asp, his, lys and glu) and replacing them with neutral or negatively charged amino acids (most preferably alanine or polyalanine) Affects the interaction of the amino acid with the antigen. Subsequently, amino acid moieties that exhibit functional sensitivity to such substitutions are defined by introducing other variants or other variants at or instead of substitution sites. Thus, while sites for introducing amino acid sequence variations are intended, the nature of the mutations need not be intended. For example, to analyze the performance of mutations at a given site, ala scanning or random mutagenesis is performed at the target codon or site and the expressed antagonist variants are screened for the desired activity.

Amino acid sequence inserts include amino- and / or carboxyl-terminal fusions of polypeptides of length containing from one residue to several hundred or more residues, as well as intrasequence inserts of single or multiple amino acid residues. Examples of terminal inserts include antagonists with N-terminal methionyl residues or antagonists fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include fusions with the N- or C-terminus of the antagonist to a polypeptide or enzyme that increases the serum half-life of the antagonist.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antagonist molecule, replaced with a different residue. Sites of greatest interest for substitutional mutagenesis include hypervariable sites, but FR mutations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions." If such substitutions result in a change in biological activity, more substantial changes, as referred to in Table 2 as “exemplary substitutions” or further described below with reference to the amino acid class, can be introduced and the product can be screened.

TABLE 2

Substantial changes in the biological properties of the antagonist include (a) their effect on maintaining the structure of the polypeptide backbone, eg, sheet or helix conformation, in the substitution region, (b) the molecule at the target site Their effect in maintaining the charge or hydrophobicity of, or (c) their effect in maintaining the bulk of the side chain is carried out by selecting significantly different substitutions. Naturally occurring residues are classified into the following groups on the basis of common side chain properties:

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

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

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

(5) residues affecting chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by replacing one member of these classes with another.

Any cysteine residue that is not involved in maintaining the proper conformation of the antagonist can generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bond (s) can be added to the antagonist to improve its stability (especially when the antagonist is an antibody fragment such as an Fv fragment).

Particularly preferred types of substitutional variants include substituting one or more hypervariable site residues of the parent antibody. In general, the resultant variant (s) selected for further development will have improved biological properties compared to the parent antibody that produced them. A convenient way to generate such substitutional variants is the affinity maturation process using phage display. Briefly, several hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed in a monovalent fashion from filamentary phage particles as a fusion with the gene III product of M13 packed in each particle. Phage displayed variants are then screened for their biological activity (eg, binding affinity) as described herein. To identify candidate hypervariable region sites suitable for modification, alanine scanning mutagenesis can be performed to identify hypervariable site residues that substantially contribute to antigen binding. Alternatively or in addition, it may be advantageous to identify the contact point between the antibody and antigen by analyzing the crystal structure of the antigen-antibody complex. Such contact residues and neighboring residues are candidates for substitution according to the techniques reviewed herein. Once such variants have been generated, a panel of variants can be screened as described herein, and antibodies exhibiting excellent properties in one or more related assays can be selected for further development.

Other types of amino acid variants of the antagonist alter the glycosylation pattern inherent in the antagonist. By change is meant the deletion of one or more carbohydrate moieties found in the antagonist and / or the addition of one or more glycosylation sites that are not present in the antagonist.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to the asparagine side chains. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxy Lysine can also be used.

Adding a glycosylation site to the antagonist is conveniently accomplished by changing the amino acid sequence to contain one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may also be made by adding or replacing one or more serine or threonine residues with the sequence of the original antibody (for O-linked glycosylation sites).

If the antibody comprises an Fc moiety, the carbohydrate attached to it can be changed. For example, antibodies with mature carbohydrate structures lacking fucose attached to the Fc region of the antibody are described in US Patent Application No. US 2003/0157108 A1 to Presta, L. Antibodies comprising bisecting N-acetylglucosamine (GlcNAc) in carbohydrates attached to the Fc region of an antibody are described in WO 03/011878 by Jean-Mairet et al. And Umana et al. See patent 6,602,684. Antibodies comprising at least one galactose residue of an oligosaccharide attached to the Fc region of the antibody are reported in Pat. Et al. WO 97/30087. See also WO 98/58964 (Raju, S.) and WO 99/22764 (Razues) with respect to antibodies comprising altered carbohydrates attached to their Fc sites.

Nucleic acid molecules encoding amino acid sequence variants of the antagonist are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation methods from natural sources (for naturally occurring amino acid sequence variants), or oligonucleotide mediated (or site-specific) mutagenesis of variants or non-variant variants of previously prepared antagonists, Production methods by PCR mutagenesis and cassette mutagenesis.

For example, it may be desirable to alter the function of the antagonist to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antagonist of the invention. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody antagonist. Alternatively or additionally, the cysteine residue (s) can be introduced into the Fc site to form interchain disulfide bonds at this site. The resulting homodimeric antibodies may have improved internal transduction capacity and / or increased complement mediated cell death and antibody-dependent cellular cytotoxicity (ADCC) [Caron et al., J. Exp Med . 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with improved antitumor activity are also described in Wolf et al., Cancer Research 53: 2560-2565 (1993), which may also be prepared using heterobifunctional crosslinking agents. Alternatively, genetic engineering of an antibody with a double Fc site may result in improved complement lysis and ADCC ability [Stevenson et al., Anti - Cancer Drug Design 3: 219-230 (1989).

WO OO / 42072 (Presta L) describes antibodies with improved ADCC function in the presence of human effector cells when the antibody comprises amino acid substitutions in its Fc region. Preferably, the antibody with improved ADCC comprises substitutions at positions 298, 333, and / or 334 of the Fc region. Preferably, the altered Fc moiety is a human IgG1 Fc moiety comprising or consisting of a substitution at one, two, or three said positions.

Antibodies with altered Clq binding and / or complement dependent cytotoxicity (CDC) are described in WO 99/51642, US Pat. No. 6,194,551 B1, US Pat. No. 6,242,195 B1, US Pat. No. 6,528,624 B1 and US Pat. No. 6,538,124 (Ether Idusogie et al.). The antibody comprises an amino acid substitution at one or more of the amino acid positions 270, 322, 326, 327, 329, 313, 333 and / or 334 of its Fc region.

To increase the serum half-life of antagonists, salvage receptor binding epitopes can be incorporated into antagonists (especially antibody fragments), as described, for example, in US Pat. No. 5,739,277. As used herein, the term “rescue receptor binding epitope” refers to the epitope of the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is involved in increasing the serum half-life of an IgG molecule in vivo. To mention. Antibodies comprising substitutions at their Fc sites and increased serum half-life are also described in WO OO / 42072 (Presta El).

Engineered antibodies comprising three or more (preferably four) functional antigen binding sites are also contemplated (US Application No. US 2002/0004587 to Miller et al.).

IV . Pharmaceutical preparations

The therapeutic preparation of the antagonists used in accordance with the present invention, pharmaceutically acceptable antagonists having the desired purity optionally carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), for storage in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol Cyclohexanol; 3-pentanol; and m-cresol); Small molecule (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates, including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter-ions such as sodium; Metal complexes (eg, Zn-protein complexes); And / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations are described in WO 98/56418, which is incorporated by reference. The publication contains 40 mg / mL rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 (pH 5.0) with a minimum half-life of 2 years at 28 ° C. A liquid formulation for several administrations is described. Other anti-CD20 antibody formulations of interest include 10 mg / mL rituximab in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate hydrate, 0.7 mg / mL polysorbate 80 and for injection. Sterile water (pH 6.5).

Lyophilized formulations adapted for subcutaneous administration are described in US Pat. No. 6,267,958 to Andya et al. The lyophilized formulation can be reconstituted with high concentrations of protein using an appropriate diluent and the reconstituted formulation can be administered subcutaneously to the mammal to be treated herein.

Crystalline antibodies or antagonists have also been observed. See, eg, US 2002/0136719 A1 (Shenoy et al.).

The formulations herein may also include one or more active compounds, preferably those having complementary activity that are free from side effects, as needed for the particular indication to be treated. For example, cytotoxic agents, chemotherapy agents, immunosuppressants, cytokines, cytokine antagonists or antibodies, growth factors, integrins, integrin antagonists or antibodies, TNF-inhibitors, disease-modifying antirheumatic drugs (DMARDs), Nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, low-dose prednisone, glucocorticoids / prednisone / methylprednisone (glucocorticoids), intravenous immunoglobulins (gamma globulins), levothyroxine, cyclosporine A, somatostatin It may be desirable to further provide analogs, anti-metabolites, immunosuppressants, cytotoxic agents such as chlorambucil, cyclophosphamide, azathioprine) and the like. The effective amount of such other agents depends on the amount of antagonist present in the formulation, the type of disease or disorder or method of treatment, and other factors discussed above. They are generally used at the same dosage and route of administration as used previously or at about 1-99% of the dosage previously used.

The active ingredient may also be used in a colloidal drug delivery system (eg liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsion by coacervation techniques or by interfacial polymerization, eg For example, it may be entrapped in microcapsules prepared from hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing an antagonist, which matrices are in the form of shaped articles, eg, films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactide (US Pat. No. 3,773,919), L- Copolymers of glutamic acid with γ ethyl-L-glutamate, non-degradable ethylene-vinylacetate, degradable lactic acid-glycolic acid copolymers, for example LUPRON DEPOT ™ (injection consisting of lactic acid-glycolic acid copolymers and leuprolide acetate Possible microspheres), and poly-D-(-)-3-hydroxybutyric acid. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

VII . product

In another embodiment of the invention, there is provided a product containing a substance useful for the treatment of the disease or disorder described above. Preferably, the product comprises (a) a composition comprising a B cell surface marker (eg, a CD20 antibody) and a pharmaceutically acceptable carrier or diluent in a container to prevent the subject from experiencing one or more symptoms of autoimmune disease. Container comprising a; And (b) instructions for administering the composition to an asymptomatic subject who is at risk of experiencing one or more symptoms of autoimmune disease.

Such products include containers and labels or package inserts on or associated with such containers. Suitable containers include, for example, bottles, vials, syringes, and the like. Such containers can be formed from various materials such as glass or plastic. The container may contain or contain a composition effective for treating the disease or condition, and may have a sterile doorway (eg, such a container may be an intravenous solution bag with a stopper that can be penetrated with a hypodermic needle, or May be vials). At least one active agent in such a composition is an antagonist that binds to B cell surface markers. The label or package insert indicates that the composition is used to prevent autoimmune diseases in subjects at risk of developing autoimmune diseases. The product may further comprise a second container comprising a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. The product may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The detailed description of the invention is illustrated by the following non-limiting examples. The disclosures of all documents cited in the specification are herein incorporated by reference.

Example  One

Rheumatism  Prevention of Arthritis

Rheumatoid arthritis (RA) develops when the body's immune system invades and destroys the tissues that make up its joints. Joints swell and stiffen and cause pain. In later steps, the joint may be deformed. Other parts of the body, including the lungs, heart, blood vessels, and eyes, may also be damaged. About 1% of the US population suffers from RA. It typically occurs in the 30s to 60s, but can occur at any age.

Symptoms of RA include stiffness, swelling, and pain in and around certain joints, especially after a period of inactivity (eg, when awake). Affected joints typically include hands, fingers, wrists, ankles, legs, elbows, and knees. In general, when the right joint of the body is damaged, the same joint on the left is also damaged. In addition, a person suffering from RA may feel tired and guided by swelling lymph nodes, mild fever, loss of appetite, and weight loss. Small bumps may occur under the skin near the affected arthritis.

In order to prevent irreversible regression resulting from RA, this example provides a method for preventing RA in a subject found to be at risk of developing RA. In addition, treatment with non-toxic Rituxan® or humanized 2H7 drugs will prevent subjects from progressing to moderate-to-severe diseases that require therapy with highly toxic drugs such as methotrexate or cyclosporamide. will be.

In the first step, assess the susceptibility of the subject to whom the RA will occur. Accordingly, serum samples are taken from human subjects with consent. The presence of an IgM rheumatoid factor (RF) antibody directed against the Fc region of IgG in the serum sample is determined and compared to normal or baseline levels of the antibody. The RF antibody is measured using standard assays using labeled reagents, generally, antibodies that bind to human RF antibodies, such as immunofluorescence, or enzyme immunoassay.

While the subject does not experience the clinical symptoms of rheumatoid arthritis (RA), elevated RF antibody levels relative to baseline (normal) levels indicate that the subject is at risk of developing rheumatoid arthritis in the next 0-10 years. As identified, subjects at "at risk" use a dosing regimen selected from 375 mg / m ² x 4, 1000 mg x 2 (day 1 and 15), or 1 gram x 3 per week. Prophylactically with rituximab (commercially available from Genentech) or humanized 2H7 (same as above). Other agents used to treat RA optionally, such as one or more immunosuppressive agents, chemotherapeutic agents, methotrexate, prednisone, cytoic acid, Mycophenolate Mofetil (CellCept), cyclo Phosphamide, azathioprine, hydroxychloroquine, CNI, anti-CD4 antibody, anti-CD5 antibody, anti-CD40L antibody, human recombinant DNase, TNF inhibitor, DMARD (s), NSAID (s), LJP-394, Treatment with anti-C5a antibodies, anti-IL-10 antibodies, BlyS inhibitors, CTLA-4Ig, LL2IgG, Limpostat-B, Plaqueenyl, and the like.

When the subject is administered a CD20 antibody, this will prevent clinical symptoms of any one or more rheumatoid arthritis from the subject.

Example 2

Systemic erythema Lupus  prevention

Lupus is an autoimmune disease that includes antibodies that invade connective tissue. The disease is predicted to affect nearly 1 million Americans, primarily women in their 20s and 40s. The main form of lupus is systemic (systemic lupus erythematosus; SLE). SLE is associated with the production of antinuclear antibodies, the circulation of immunocomplexes, and the activation of the complement system.

Untreated Lupus can be fatal because Lupus invades skin and joints and progresses to internal organs that include the lungs, heart, and kidneys (including kidney disease of primary concern). Lupus includes intervening periods with little or no disease manifestation and often appear as a series of flare-ups.

Tan et. al. "The Revised Criteria for the Classification of SLE". Arth Rheum 25 (1982)] is used for the diagnosis of the fit loops.

Buccal rash

-Isthmus rash

Discoid rash

Red raised patches

Photosensitivity

-Reaction to sunlight which causes or promotes skin rash

Mouth ulcers

-Generally painless, nose or mouth ulcers

arthritis

Nonerosive arthritis including two or more peripheral braces (arthritis in which the bones around the joint are not destroyed)

Meningitis

Pleurisy or pericarditis

Kidney disease

Excess protein in urine (greater than 0.5 gm / day or 3+ on test sticks) and / or cell cast (abnormal element derived from urine and / or leukocytes and / or renal tubule cells)

Neurological

Seizure (convulsions) and / or psychosis in the absence of drugs or metabolic disorders known to cause such actions

Hematological

-Hemolytic anemia or leukopenia (leukocyte count less than 4,000 cells per mm ³ ) or lymphocytosis (less than 1,500 lymphocytes per mm ³ ) or hypoplatelet (less than 100,000 platelets per mm ³ ). Leukopenia and lymphocytosis should be measured in two or more cases. Hypothelium should be measured in the absence of a drug known to induce it.

Lupus is generally treated with an immunosuppressive therapy, mainly using prednisone, for example, a corticosteroid, which may be administered during a seizure dilatation period or for those who frequently experience seizure dilatation. Even treatments that are effective in lowering symptoms and extending lifespan can lead to severe disability and death if the disease symptoms continue to be low along with the side effects of the drug. Recent therapeutic therapies include cyclophosphamide, methotrexate, antimalarial agents, hormonal therapies (eg DHEA), and anti-hormonal therapies (eg antiprolactin bromo kryptine).

Due to the severity of the SLE, the ability to prevent it may be desirable, which can be achieved by pre-emptive therapies as described herein. In a first step, subjects are identified at risk of developing one or more SLE symptoms. Serum samples are taken from human subjects, anti-nuclear antibodies (ANA), anti-double stranded DNA (dsDNA) antibodies, anti-Smith antigen (Sm) antibodies, anti-nuclear ribonucleic acid antibodies, antiphospholipid proteins, anti- Ribosome P antibodies, anti-Ro / SS-A antibodies, anti-Ro antibodies, and / or anti-La antibodies are standard assays, eg, using antibodies that bind labeled reagents, generally autoantibodies to be evaluated. Measurement is performed using immunofluorescence or enzyme immunoassay. As a literature example, Arbuckle et al. New Eng . J. Med . 349 (16): 1526 (2003). Subjects who assessed the levels of autoantibodies relative to baseline levels and showed a marked increase in these levels are at risk of developing SLE in the next 0-10 years.

Subsequently, subjects identified as having a risk of developing SLE but not otherwise experiencing disease symptoms from 375 mg / m ² x 4, 1000 mg x 2 (day 1 and 15), or 1 gram x 3, every week The dosing regimen of choice is used to treat rituximab or humanized 2H7. Antibodies may optionally include one or more nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., acetylsalicylic acid, ibuprofen, naproxen, indomethacin, sulindac, tolmethine), acetaminophen, corticosteroid, antimalarial agent (e.g., chloroquine or hydride) Oxychloroquine), immunosuppressive agents, methotrexate, prednisone, cyclopo spamide (cytoxane), mycophenolate mofetil (selcept), azathioprine, hydroxychloroquine, CNI, anti-CD4 antibody, anti-CD5 antibody, Additional such as anti-CD40L antibody, human recombinant DNase, TNF inhibitor, LJP-394, anti-C5a antibody, anti-IL-10 antibody, BlyS inhibitor, CTLA-4Ig, LL2IgG, lymphpostat-B, flakuenyl, etc. Combined with the drug (s).

When subjects are administered rituximab or humanized 2H7, this will prevent them from experiencing any one or more symptoms of SLE from the subject.

Example 3

Prevention of Ulcerative Colitis

It is estimated that 500,000 ulcerative colitis (UC) patients have recurrent episodes of intramucosal inflammation in the United States. Clinical symptoms include rectal bleeding, frequent bowel movements, and advancement symptoms such as fever, weight loss, and anemia. Podolsky, D. NEJM 347: 417-429 (2002). Symptoms of patients with mild UC include proctitis, rectal S colitis, distal colitis, intermittent rectal bleeding, mucus passage, mild diarrhea, abdominal pain. Patients with moderately severe disease may experience symptoms including left colitis, frequent bloody diarrhea (10 / day), mild anemia, mild fever and abdominal pain accompanied by sustained nutrition. Symptoms observed in UC patients with severe disease include more than 10 hematomas / day of colitis, severe spasms, high fever, bleeding requiring transfusion, weight loss, toxic megacolonosis, and perforation (associated with 50% mortality) It includes.

Most doctors use a stepwise treatment logic in UC treatment. The first line of therapy generally includes oral and / or topical 5-ASAs. The second line of treatment includes oral and / or topical steroids, but 50% of first time users use steroids become dependent or unresponsive after one year. The third line of therapy is achieved by the administration of immunosuppressive agents (eg, azathioprine, 6 mercaptopurine, cyclosporin). Finally, the treatment for the fourth gland is surgery (whole colonic resection).

This embodiment provides a means of preventing UC. First, identify human subjects at risk of developing UC. Serum samples from subjects were examined for the presence of autoantibodies (pANCA), or anti-Saccharomyces cerevisiae antibodies (ASCA), which stain the nucleus of the neutrophils or the region around the nucleus of the neutrophils. Try it. Increased or abnormal pANCA or ASCA levels suggest that there is a risk of developing UC in the subject, thus initiating therapy with CD20 antibodies.

While the subject does not develop symptoms of UC, the subject is administered a dosing regimen selected from 375 mg / m ² x 4, 1000 mg x 2 (day 1 and 15), or 1 gram x 3, every week to prevent the onset of the disease. Treatment with rituximab or humanized 2H7.

Apart from CD20 antibodies, oral and / or topical 5-ASAs, oral and / or topical steroids, one or more immunosuppressive agents such as azathioprine, 6-mercaptopurine, cyclosporin), MLN-02, antibiotics, mesalamin , Prednisone, TNF-inhibitor, cortisone cream, hydrocortisone enema, sulfasalazine, alsalazine, valsalazide, methylprednisolone, drocortisone, ACTH, intravenous corticosteroids, GelTex, Vicilizumab, OPC-6535, CBP 1011, Subjects can be treated with thalidomide, ISIS 2302, BXT-51072, Refifermin (KGF-2), RPD-58, Antgren, FK-506, Lviv, Natalizumab, and the like.

When a CD20 antibody is administered as described above, this will prevent any one or more of the symptoms of UC from developing.

Example 4

Humanized 2 H7  Variant

This example describes humanized 2H7 antibody variants for using the methods described herein. Humanized 2H7 antibodies preferably comprise one, two, three, four, five or six of the following CDR sequences:

CDR L1 sequence RASSSVSYXH, where X is M or L (SEQ ID NO: 18), for example SEQ ID NO: 4 (FIG. 1A),

CDR L2 sequence of SEQ ID NO: 5 (FIG. 1A),

CDR L3 sequence QQWXFNPPT (where X is S or A (SEQ ID NO: 19)), for example SEQ ID NO: 6 (FIG. 1A),

CDR H1 sequence of SEQ ID 10 (FIG. 1B),

CDR H2 sequence AIYPGNGXTSYNQKFKG (where X is D or A (SEQ ID NO: 20)), eg, SEQ ID NO: 11 (FIG. 1B), and

CDR H3 sequence VVYYSXXYWYFDV (where X in position 6 is N, A, Y, W or D and X in position 7 is S or R (SEQ ID NO: 21)), eg, SEQ ID NO: 12 (FIG. 1B).

The CDR sequences are generally human variable light and variable heavy chain framework sequences, such as the human consensus FR residues of substantially human light chain kappa subgroup I (V _L 6I), and substantially human heavy chain subgroup III (V _H III). ) In the human consensus FR residues. See WO 2004/056312 (Lowman et al.).

The variable heavy chain region can bind to a human IgG chain constant region, including, for example, IgG1 or IgG3, including the native sequence and variant constant regions.

In a preferred embodiment, the antibody comprises the variable heavy chain region sequence of SEQ ID NO: 8 (v16, shown in Figure 1B) and optionally also comprises the variable light chain region sequence of SEQ ID NO: 2 (v16, shown in Figure 1A). And optionally one or more amino acid substitution (s) at positions 56, 100, and / or 100a of the variable heavy chain region, eg, D56A, N100A or N100Y, and / or S32aR and 32 of the variable light chain region. And / or one or more amino acid substitution (s) at position 92, such as M32L and / or S92A. The antibody is an intact antibody comprising a light chain amino acid sequence of SEQ ID NO: 13 or 16 and a heavy chain amino acid sequence of SEQ ID NO: 14, 15, 17, 22 or 25. Preferred humanized 2H7 antibody is ocrelizumab (Genentech).

Antibodies herein have amino acid substitutions at positions 298, 333, and 334 using at least one amino acid substitution in the Fc region that further improves ADCC activity, eg, Eu numbering of heavy chain residues. , Preferably, S298A, E333A, and K334A. See, US Pat. No. 6,737,056 B1 (Presta).

Any of the above antibodies may comprise at least one substitution in the Fc region, for example at position 434 of the heavy chain, eg, N434W, to improve FcRn binding or serum half-life. See also US Pat. No. 6,737,056 B1 (Presta).

Any of the above antibodies may further comprise at least one amino acid substitution in the Fc region that improves CDC activity, eg, at least one substitution at position 326, preferably K326A or K326W. . See also US Pat. No. 6,528,624 B1 (Ethersogi et al.).

Some preferred humanized 2H7 variants include a variable light chain region of SEQ ID NO: 2 and a variable heavy chain region of SEQ ID NO: 8, including, with or without substitutions in the Fc region, and Change to SEQ ID NO: 8 N100A; Or D56A and N100A; Or the variable heavy chain region having D56A, N100Y, and S100aR and SEQ ID NO: double modified M32L; Or S92A; Or a variable light chain region having M32L and S92A.

M34 in the variable heavy chain region of 2H7.v16 has been identified as a potential source of antibody stability, which is another potential candidate for substitution.

In a summary of several various preferred embodiments of the invention the variable region of the variant based on 2H7.v16 comprises the amino acid sequence of v16 excluding the position of the amino acid substitutions set forth in the table below. Unless stated otherwise, the 2H7 variant will have the same light chain as that of v16.

Exemplary Humanized 2 H7  Antibodies Variant

One preferred humanized 2H7 is

2H7.v16 variable light chain region sequence:

(SEQ ID NO: 2); And

2H7.v16 variable heavy chain region sequence:

(SEQ ID NO: 8).

If the humanized 2H7.v16 antibody is a complete antibody,

Light chain amino acid sequence:

(SEQ ID NO: 13); And

Heavy chain amino acid sequence of SEQ ID NO: 14:

(SEQ ID NO: 22).

Another preferred humanized 2H7 antibody is the 2H7.v511 variable light chain region sequence:

(SEQ ID NO: 23) and

2H7.v511 variable heavy chain region sequence:

(SEQ ID NO: 24).

If the humanized 2H7.v511 antibody is a complete antibody, the light chain amino acid sequence is:

(SEQ ID NO: 16)

A heavy chain amino acid sequence of SEQ ID NO: 17 or:

(SEQ ID NO: 25).

<110> BRUNETTA, PAUL G.       GREWAL, IQBAL S.       WALICKE, PATRICIA A. <120> PREVENTING AUTOIMMUNE DISEASE <130> P2079R2 <140> US 11 / 120,338 <141> 2005-05-03 <150> US 60 / 568,460 <151> 2004-05-05 <160> 25 <210> 1 <211> 107 <212> PRT <213> Mus musculus <400> 1  Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro    1 5 10 15  Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser                   20 25 30  Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro                   35 40 45  Trp Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg                   50 55 60  Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser                   65 70 75  Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp                   80 85 90  Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu                   95 100 105  Lys arg          <210> 2 <211> 107 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 2  Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser                   20 25 30  Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro                   35 40 45  Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg                   50 55 60  Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                   65 70 75  Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp                   80 85 90  Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile                   95 100 105  Lys arg          <210> 3 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> sequence is synthesized <400> 3  Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser                   20 25 30  Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys                   35 40 45  Leu Leu Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser                   50 55 60  Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile                   65 70 75  Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                   80 85 90  Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu                   95 100 105  Ile Lys Arg              <210> 4 <211> 10 <212> PRT <213> Mus musculus <400> 4  Arg Ala Ser Ser Ser Val Ser Tyr Met His                    5 10 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5  Ala Pro Ser Asn Leu Ala Ser                    5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6  Gln Gln Trp Ser Phe Asn Pro Pro Thr                    5 <210> 7 <211> 122 <212> PRT <213> Mus musculus <400> 7  Gln Ala Tyr Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly    1 5 10 15  Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Lys Gln Thr Pro Arg Gln Gly Leu                   35 40 45  Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser                   65 70 75  Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp                   80 85 90  Ser Ala Val Tyr Phe Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Thr Gly Thr Thr Val Thr Val                  110 115 120  Ser Ser          <210> 8 <211> 122 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 8  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser          <210> 9 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> sequence is synthesized <400> 9  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser                   20 25 30  Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Ala Val Ile Ser Gly Asp Gly Gly Ser Thr Tyr Tyr                   50 55 60  Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser                   65 70 75  Lys Asn Thr Leu Thr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Val Gly Tyr Ser Leu                   95 100 105  Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser                  110 115 <210> 10 <211> 10 <212> PRT <213> Mus musculus <400> 10  Gly Tyr Thr Phe Thr Ser Tyr Asn Met His                    5 10 <210> 11 <211> 17 <212> PRT <213> Mus musculus <400> 11  Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe    1 5 10 15  Lys gly          <210> 12 <211> 13 <212> PRT <213> Mus musculus <400> 12  Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val                    5 10 <210> 13 <211> 213 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 13  Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser                   20 25 30  Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro                   35 40 45  Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg                   50 55 60  Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                   65 70 75  Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp                   80 85 90  Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile                   95 100 105  Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser                  110 115 120  Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu                  125 130 135  Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp                  140 145 150  Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln                  155 160 165  Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu                  170 175 180  Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val                  185 190 195  Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg                  200 205 210  Gly glu cys              <210> 14 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 14  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro                  125 130 135  Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu                  140 145 150  Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser                  155 160 165  Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                  170 175 180  Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser                  185 190 195  Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys                  200 205 210  Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys                  215 220 225  Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                  230 235 240  Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                  245 250 255  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp                  260 265 270  Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp                  275 280 285  Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln                  290 295 300  Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His                  305 310 315  Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                  320 325 330  Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys                  335 340 345  Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg                  350 355 360  Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                  365 370 375  Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                  380 385 390  Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                  395 400 405  Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                  410 415 420  Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                  425 430 435  Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro                  440 445 450  Gly lys          <210> 15 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 15  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro                  125 130 135  Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu                  140 145 150  Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser                  155 160 165  Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                  170 175 180  Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser                  185 190 195  Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys                  200 205 210  Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys                  215 220 225  Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                  230 235 240  Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                  245 250 255  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp                  260 265 270  Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp                  275 280 285  Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln                  290 295 300  Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His                  305 310 315  Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                  320 325 330  Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys                  335 340 345  Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg                  350 355 360  Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                  365 370 375  Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                  380 385 390  Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                  395 400 405  Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                  410 415 420  Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                  425 430 435  Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro                  440 445 450  Gly lys          <210> 16 <211> 213 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 16  Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser                   20 25 30  Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro                   35 40 45  Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg                   50 55 60  Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                   65 70 75  Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp                   80 85 90  Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile                   95 100 105  Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser                  110 115 120  Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu                  125 130 135  Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp                  140 145 150  Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln                  155 160 165  Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu                  170 175 180  Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val                  185 190 195  Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg                  200 205 210  Gly glu cys              <210> 17 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 17  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro                  125 130 135  Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu                  140 145 150  Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser                  155 160 165  Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                  170 175 180  Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser                  185 190 195  Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys                  200 205 210  Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys                  215 220 225  Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                  230 235 240  Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                  245 250 255  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp                  260 265 270  Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp                  275 280 285  Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln                  290 295 300  Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His                  305 310 315  Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                  320 325 330  Ala Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys                  335 340 345  Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg                  350 355 360  Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                  365 370 375  Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                  380 385 390  Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                  395 400 405  Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                  410 415 420  Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                  425 430 435  Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro                  440 445 450  Gly lys          <210> 18 <211> 10 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> Unsure <222> 9 <223> Unknown amino acid <400> 18  Arg Ala Ser Ser Ser Val Ser Tyr Xaa His                    5 10 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> Unsure <222> 4 <223> Unknown amino acid <400> 19  Gln Gln Trp Xaa Phe Asn Pro Pro Thr                    5 <210> 20 <211> 17 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> Unsure <222> 8 <223> Unknown amino acid <400> 20  Ala Ile Tyr Pro Gly Asn Gly Xaa Thr Ser Tyr Asn Gln Lys Phe    1 5 10 15  Lys gly          <210> 21 <211> 13 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <220> <221> Unsure <222> 6-7 <223> Unknown amino acid <400> 21  Val Val Tyr Tyr Ser Xaa Xaa Tyr Trp Tyr Phe Asp Val                    5 10 <210> 22 <211> 451 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 22  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro                  125 130 135  Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu                  140 145 150  Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser                  155 160 165  Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                  170 175 180  Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser                  185 190 195  Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys                  200 205 210  Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys                  215 220 225  Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                  230 235 240  Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                  245 250 255  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp                  260 265 270  Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp                  275 280 285  Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln                  290 295 300  Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His                  305 310 315  Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                  320 325 330  Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys                  335 340 345  Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg                  350 355 360  Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                  365 370 375  Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                  380 385 390  Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                  395 400 405  Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                  410 415 420  Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                  425 430 435  Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro                  440 445 450  Gly      <210> 23 <211> 107 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 23  Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val    1 5 10 15  Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser                   20 25 30  Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro                   35 40 45  Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg                   50 55 60  Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                   65 70 75  Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp                   80 85 90  Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile                   95 100 105  Lys arg          <210> 24 <211> 122 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 24  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser          <210> 25 <211> 451 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 25  Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly    1 5 10 15  Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr                   20 25 30  Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu                   35 40 45  Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr                   50 55 60  Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser                   65 70 75  Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp                   80 85 90  Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg                   95 100 105  Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val                  110 115 120  Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro                  125 130 135  Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu                  140 145 150  Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser                  155 160 165  Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln                  170 175 180  Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser                  185 190 195  Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys                  200 205 210  Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys                  215 220 225  Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                  230 235 240  Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                  245 250 255  Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp                  260 265 270  Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp                  275 280 285  Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln                  290 295 300  Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His                  305 310 315  Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn                  320 325 330  Ala Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys                  335 340 345  Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg                  350 355 360  Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                  365 370 375  Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly                  380 385 390  Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser                  395 400 405  Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser                  410 415 420  Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu                  425 430 435  Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro                  440 445 450  Gly     

Claims (71)

Systemic Lupus Erythematosus (SLE), Anti-phospholipid Antibody Syndrome, Multiple Sclerosis, Ulcerative Colitis, Crohn's Disease, Rheumatoid Arthritis, Sjogren's Syndrome, Guillain-Barré Syndrome, Severe Myasthenia, Macrovascular Vasculitis, Middle Vasculitis, Nodular Polyarthritis , Celtic swelling, scleroderma, Goodpasture syndrome, Glomerulonephritis, Primary biliary cirrhosis, Graves' disease, Membrane nephropathy, Autoimmune hepatitis, Non-tropical sprue, Addison's disease, Multiple myositis / skin myositis, Monoclonal gamma disease, The subject is administered CD20 antibodies in an amount that prevents them from experiencing one or more symptoms of autoimmune disease selected from the group consisting of Factor VIII deficiency, cold globulinemia, peripheral neuropathy, IgM polyneuropathy, chronic neuropathy, and Hashimoto's thyroiditis Have no symptoms, with the risk of experiencing one or more symptoms of autoimmune disease, including How to prevent a person from self-immune diseases. The method of claim 1, wherein the subject produces autoantibodies in abnormal amounts. The method of claim 1, wherein the subject has not experienced one or more symptoms of autoimmune disease. The method of claim 1, wherein the subject has not previously been treated with a CD20 antibody. The method of claim 1, wherein the subject is about 80-100% likely to experience one or more symptoms of autoimmune disease in the next 0-10 years. The method of claim 1, wherein the subject has a risk of experiencing one or more systemic lupus erythematosus (SLE) symptoms. The method of claim 6, wherein the subject has abnormal levels of anti-nucleus, anti-double strand DNA (dsDNA), anti-Smith antigen (Sm), anti-nucleus ribonucleic acid protein, anti-phospholipid, anti-ribosome P, anti- Methods with Ro / SS-A, anti-Ro, or anti-La antibodies. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of anti-phospholipid antibody syndrome. The method of claim 8, wherein the subject has anti-phospholipid antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of ulcerative colitis or Crohn's disease. The method of claim 10, wherein the subject has an autoantibody (pANCA), or an anti-Saccharomyces cerevisiae antibody (ASCA), which stains the nucleus of the neutrophils or the region around the nucleus of the neutrophils at abnormal levels. . The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of Guillain-Barré syndrome. The method of claim 12, wherein the subject has cross reactive antibodies against GM1 gangliosides or GQ1b gangliosides at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of severe myasthenia. The method of claim 14, wherein the subject has anti-acetylcholine receptor (AchR), anti-AchR subtype, or anti-muscle specific tyrosine kinase (MuSK) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of macrovascular vasculitis. The method of claim 16, wherein the subject has serum anti-endothelial cell antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of intermediate vasculitis. The method of claim 18, wherein the subject has anti-endothelial or anti-neutrophil cytoplasmic (ANCA) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of nodular polyarteritis. The method of claim 20, wherein the subject has an autoantibody (pANCA) that stains the nucleus of neutrophils or the region around the nucleus of neutrophils at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of sky sores. 23. The method of claim 22, wherein the subject has an IgG or anti-desmogloin (Dsg) antibody at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of scleroderma. The subject of claim 24, wherein the subject has anti-central, anti-topoisomerase-1 (Scl-70), anti-RNA polymerase, or anti-U3-ribonucleic acid protein (U3-RNP) antibody at abnormal levels. Way. The method of claim 1, wherein the subject has a risk of experiencing one or more Goodpasture syndrome symptoms. The method of claim 26, wherein the subject has anti-glomerular basement membrane (GBM) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of glomerulonephritis. The method of claim 28, wherein the subject has anti-glomerular basement membrane (GBM) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of primary biliary cirrhosis. 31. The method of claim 30, wherein the subject has anti-mitochondrial (AMA) or anti-mitochondrial M2 antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more Graves' disease symptoms. 33. The method of claim 32, wherein the subject has anti-thyroid peroxidase (TPO), anti-tyroglobin (TG) or anti-thyroid stimulating hormone receptor (TSHR) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of membranous nephropathy. The method of claim 34, wherein the subject has anti-double stranded DNA (dsDNA) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of autoimmune hepatitis. The method of claim 36, wherein the subject has anti-nucleus (AN), anti-actin (AA) or anti-smooth muscle antigen (ASM) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more non-tropical sprue symptoms. The method of claim 38, wherein the subject has IgA anti-muscular fibrous membrane, IgA anti-tissue transglutaminase, IgA anti-gliadine, or IgG anti-gliadine antibody at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of Addison's disease. The method of claim 40, wherein the subject has anti-CYP21A2, anti-CYP11A1 or anti-CYP17 antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of polymyositis / dermatitis. The method of claim 42, wherein the subject has anti-nucleus (ANA), anti-ribonucleic acid protein (RNP), or myositis-specific antibody at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of monoclonal gamma disease. 45. The method of claim 44, wherein the subject has anti-myelin related glycoprotein (MAG) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of cold globulinemia. The method of claim 46, wherein the subject has anti-hepatitis C virus (HCV) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of peripheral neuropathy. 49. The method of claim 48, wherein the subject has abnormal levels of anti-GM1 gangliosides, anti-myelin related glycoproteins (MAG), anti-sulfate-3-glycuronyl paraglobosides (SGPG), or IgM anti- A method having a glycoconjugate antibody. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of IgM polyneuropathy. 51. The method of claim 50, wherein the subject has anti-myelin related glycoprotein (MAG) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of chronic neuropathy. The method of claim 52, wherein the subject has IgM anti-ganglioside antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of Hashimoto's thyroiditis. 55. The method of claim 54, wherein the subject has anti-thyroid peroxidase (TPO), anti-tyroglobin (TG) or anti-thyroid stimulating hormone receptor (TSHR) antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of multiple sclerosis. The method of claim 56, wherein the subject has anti-myelin-based protein or anti-myelin oligodendrocyte glycoprotein antibody at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of rheumatoid arthritis. 59. The method of claim 58, wherein the subject has IgM rheumatoid factor antibodies directed against the Fc portion of IgG at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more Sjogren's syndrome symptoms. 61. The method of claim 60, wherein the subject has anti-La / SSB or anti-Ro / SSB antibodies at abnormal levels. The method of claim 1, wherein the subject has a risk of experiencing one or more symptoms of factor VIII deficiency. 61. The method of claim 60, wherein the subject has anti-Factor VIII antibodies at abnormal levels. The method of claim 1, wherein the antibody is a naked antibody. The method of claim 1, consisting essentially of administering the antibody to the subject. The method of claim 1, wherein the antibody is Rituximab. The method of claim 1, wherein the antibody is humanized 2H7 comprising variable region sequences in SEQ ID NOs: 2 and 8. 3. The method of claim 1, wherein the antibody is humanized 2H7 comprising variable region sequences in SEQ ID NOs: 23 and 24. 3. Autoimmune disease in asymptomatic subjects, at risk of experiencing one or more autoimmune disease symptoms, comprising administering to the subject a CD20 antibody in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. How to prevent. A method of preventing autoimmune disease in an asymptomatic subject having an abnormal level of autoantibody, comprising administering to the subject an CD20 antibody in an amount that prevents the subject from experiencing one or more symptoms of autoimmune disease. To prevent a subject from experiencing one or more symptoms of autoimmune disease, (a) a container comprising a composition comprising a CD20 antibody in a container and a pharmaceutically acceptable carrier or diluent; And (b) a product comprising instructions for administering the composition to an asymptomatic subject at risk of experiencing one or more symptoms of autoimmune disease.

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