KR20070022045A - Treatment of disorders - Google Patents

Abstract

The present invention relates to the treatment of polychondritis or multiple mononeuritis in a mammal with an effective amount of an antibody binding to CD20, and optionally also an effective amount of another agent for treating this disorder.

CD20, CD20 antibody, polychondritis, multiple mononeuritis, cytotoxic agent, rituximab, humanized 2H7

Description

Treatment of Disorders {TREATMENT OF DISORDERS}

Related Applications

This application claims 35 U.S.C. US Patent Provisional Application Nos. 60 / 563,227, filed April 16, 2004 and 60 / 565,098, filed April 22, 2004, which claim priority under §119. Incorporated herein by reference.

The present invention is directed to treating disorders with antagonists that bind to B-cell surface markers, such as CD19 or CD20, eg, antibodies that bind to CD20.

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

B cells mature in the bone marrow, leaving the bone marrow expressing antigen-binding antibodies on their cell surface. When a naïve B cell first encounters an antigen specific for its membrane-bound antibody, the cell begins to divide rapidly, and its progeny are effectors called memory B cells and "plasma cells". Differentiate into cells. Memory B cells have a longer lifespan and continue to express membrane-bound antibodies with the same specificity as the original parental cells. Plasma cells do not express membrane-bound antibodies, but instead produce antibodies in a form that can be secreted. Secreted antibodies are the major effector molecules of humoral immunity.

The CD20 antigen (human B-lymphocyte-limiting differentiation antigen, also referred to as Bp35) is a hydrophobic transmembrane protein with a molecular weight of about 35 kD located on pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem. 264 (19): 11282-11287 (1989); and Einfeld et al. EMBO J. 7 (3): 711-717 (1988)]. This antigen is expressed on more than 90% B-cell non-Hodgkin lymphoma (NHL) (Anderson et al. Blood 63 (6): 1424-1433 (1984)), but hematopoietic stem cells, It is not found in 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) in the activation process for cell-cycle initiation and differentiation (Tedder et al., Supra), and probably functions as calcium ion channels (Tedder et al. J. Cell Biochem. 14D : 195 (1990)).

Given the expression of CD20 in B-cell lymphomas, these antigens can serve as candidates for the "targeting" of such lymphomas. In essence, such targeting can be generalized as follows: An antibody specific for the CD20 surface antigen of B cells is administered to a patient. Such anti-CD20 antibodies specifically bind to CD20 antigens (on the surface) of both normal and malignant B cells; Antibodies bound to the CD20 surface antigen can lead to the destruction and deficiency of neoplastic B cells. Additionally, chemical agents or radiolabels that have the potential to destroy tumors can be conjugated to anti-CD20 antibodies such that the agents specifically "deliver" to neoplastic B cells. Regardless of the approach, the primary goal is to destroy the tumor; The specific approach can be determined by the specific anti-CD20 antibody used, and thus the available approaches for targeting the CD20 antigen can vary significantly.

CD19 is another antigen expressed on surface cells of the B line. Like CD20, CD19 is found on cells throughout lineage differentiation to some extent just before the final differentiation from the stem cell stage to plasma cells (Nadler, L. Lymphocyte Typing II 2: 3-37 and Appendix, Renling et. al. eds. (1986) by Springer Verlag). However, unlike CD20, antibodies that bind to CD19 cause internalization of the CD19 antigen. The CD19 antigen is specifically identified by the HD237-CD19 antibody (also called "AB4" antibody) (Kiesel et al. Leukemia Research II, 12: 1119 (1987)). CD19 antigen is present on 4-8% of peripheral vascular mononuclear cells and on more than 90% of B cells isolated from peripheral blood vessels, spleen, lymph nodes or tonsils. CD19 is not detected on peripheral vascular T cells, monocytes, or granulocytes. Virtually all non-T-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL) and B-cell lymphoma express CD19 detectable by antibody B4 (Nadler et al. J. Immunol. 131: 244 (1983); and Nadler et al. In Progress in Hematology Vol. XII pp. 187-206, Brown, E. ed. (1981) by Grune & Stratton, Inc.).

Additional antibodies have been identified that recognize differentiation stage-specific antigens expressed by cells of the B-cell lineage. Among them, B2 antibody detected against CD21 antigen; B3 antibody detected against CD22 antigen; And J5 antibodies detected against CD10 antigen (also called CALLA). See, eg, US Pat. No. 5,595,721 to Kaminski et al., Issued January 21, 1997.

Rituximab (RITUXAN®) antibodies are genetically engineered chimeric mouse / human monoclonal antibodies directed against the CD20 antigen. Rituximab is an antibody called "AC2B8" in US Pat. No. 5,736,137 to Anderson et al., Issued April 7, 1998. RITUXAN® has been indicated for the treatment of relapsed or unresponsive hypomalignant or vesicular, CD20 positive, B-cell non-Hodgkin's lymphoma (Maloney et al. Blood 82 (Suppl 1): 445a (1993)); Maloney et al. Proc Am Soc Clin Oncol 13: 993 (1994). In vitro mechanisms of action studies have shown that RITUXAN® binds to human complement and dissolves lymphoid B-cell lines through complement-dependent cytotoxicity (CDC) (Reff et al. Blood 83 (2). 435-445 (1994)). In addition, it has antibody-dependent cellular cytotoxicity (ADCC). More recently, RITUXAN®, unlike other anti-CD19 and CD20 antibodies, has been shown to have an anti-proliferative effect in tritiated thymidine incorporation assays and directly induce apoptosis (Maloney et al. Blood 88 ( 10): 637a (1996)). Synergy between RITUXAN® and chemotherapy and toxins has also been observed experimentally. In particular, RITUXAN® makes drug-resistant human B-cell lymphoma cell lines sensitive to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and lysine (Demidem et al. Cancer Chemotherapy & Radiopharmaceuticals 12 (3): 177-186 (1997); Demidem A et al. FASEB J 9: A206 (1995)]. In vivo preclinical studies have shown that RITUXAN® deficient B cells from peripheral blood vessels, lymph nodes, and bone marrow of cynomolgus monkeys, possibly via complement and cell-mediated processes (Reff et al., Supra) ).

Rituximab has also been studied in various non-malignant autoimmune disorders in which B cells and autoantibodies have been shown to play a role in disease pathophysiology. Edwards et al., Biochem Soc. Trans. 30: 824-828 (2002). Rituximab is described, for example, in rheumatoid arthritis (RA) (Leandro et al., Ann. Rheum. Dis. 61: 883-888 (2002)); Edwards et al., Arthritis Rheum. , 46 (Suppl. 9): S46 (2002); Stahl et al., Ann.Rheum. Dis., 62 (Suppl. 1): OP004 (2003); Emery et al., Arthritis Rheum. 48 (9): S439 (2003)), lupus (Eisenberg, Arthritis. Res. Ther. 5: 157-159 (2003); Leandro et al. Arthritis Rheum. 46: 2673-2677 (2002)); Gorman et al., Lupus, 13: 312-316 (2004)), immune thrombocytopenic purple plaques (D'Arena et al., Leuk. Lymphoma 44: 561-562 (2003)); Blood, 98: 952-957 (2001); Saleh et al., Semin. Oncol, 27 (Supp 12): 99-103 (2000); Zaia et al., Haematolgica, 87: 189-. 195 (2002)]; Ratanatharathorn et al., Ann. Int. Med., 133: 275-279 (2000)], sedative erythroid aplasia (Auner et al., Br. J. Haematol, 116: 725- 728 (2002)); Autoimmune anemia (Zaja et al., Haematologica 87: 189-195 (2002) (errors in Haematologica 87: 336 (2002))), cryoaggregate (Layios et al., Leukemia, 15: 187- 8 (2001); Berentsen et al., Blood, 103: 2925-2928 (2004); Berentsen et al., Br. J. Haematol, 115: 79-83 (2001); Bauduer, Br J. Haematol, 112: 1083-1090 (2001); Damian et al., Br. J. Haematol, 114: 229-234 (2001)), type B syndrome of severe insulin resistance (Coll et al. , N. Engl. J. Med., 350: 310-311 (2004), mixed cold globulinemia (De Vita et al., Arthritis Rheum. 46 Suppl. 9: S206 / S469 (2002)), myasthenia gravis ([ Zaja et al., Neurology, 55: 1062-63 (2000); Wylam et al., J. Pediatr., 143: 674-677 (2003)), Wegener's granulomatosis (Specks et al., Arthritis & Rheumatism 44: 2836-2840 (2001)), unresponsive normal swelling (Dupuy et al., Arch Dermatol, 140: 91-96 (2004)), dermatitis (Levine, Arthritis Rheum., 46 (Suppl. 9): S1299 (2002)), Sjogren's syndrome (Somer et al., Arthritis & Rheumatism, 49: 394-398 (2003)), active type II mixed cold globulinemia (Zaja et al., Blood, 101: 3827-3834 (2003)), normal swelling (Dupay et al., Arch. Dermatol, 140: 91 -95 (2004)), autoimmune neuropathy (Pestronk et al., J. Neurol. Neurosurg. Psychiatry 74: 485-489 (2003)), paraneoplastic choroidal-clonic muscle spasms syndrome (Pranzatelli et al. Neurology 60 (Suppl. 1) PO5.128: A395 (2003)), and recurrent-extensive multiple sclerosis ( RRMS) has been reported to potentially alleviate signs and symptoms. Cross et al. (abstract) "Preliminary Results from a Phase II Trial of Rituximab in MS" Eighth Annual Meeting of the Americas Committees for Research and Treatment in Multiple Sclerosis, 20-21 (2003)].

Phase II study (WA16291) was performed in patients with rheumatoid arthritis (RA) to provide 48-week follow-up data on the stability and efficacy of Rituxima map. Emery et al. Arthritis Rheum 48 (9): S439 (2003); Szczepanski et al. Arthritis Rheum 48 (9): S121 (2003). A total of 161 patients were randomly divided into four treatment stages: methotrexate, rituximab alone, rituximab + methotrexate, and rituximab + cyclophosphamide (CTX). The treatment regimen for rituximab was to administer 1 g intravenously on days 1 and 15. Infusion of rituximab in most patients with rheumatoid arthritis was well tolerated in most patients, with 36% of patients experiencing one or more adverse events during the initial infusion (compared to 30% of patients receiving placebo). Overall, most adverse events were considered mild and moderate in severity and well balanced for all treatment groups. There were 19 serious adverse events in four stages over 48 weeks, which were more frequent in the rituximab / CTX group. The incidence of infection was well balanced for all groups. The average rate of serious infections in this population of rheumatoid arthritis patients was 4.66 per 100 patient-years, which is the proportion of infections requiring hospitalization in patients with rheumatoid arthritis reported in community-based epidemiological studies (per 100 patient-years). Lower than 9.57). Doran et al., Arthritis Rheum. 46: 2287-2293 (2002).

Minority with neurological disorders, including autoimmune neuropathy (Pestronk et al., Supra), ophthalmic-spondylomyopathy syndrome (Pranzatelli et al., Supra), and RRMS (Cross et al., Supra) The reported safety profile of rituximab in patients with was similar to that reported in oncology or rheumatoid arthritis. In an ongoing investigator-sponsored trial (IST) of rituximab in combination with interferon-beta (IFN-β) or glatiramer acetate in RRMS patients (Cross et al., Supra), treated One in ten patients suffered moderate fever and chills after the initial infusion of rituximab and were hospitalized for night observation, the other nine completed four infusion regimens without any reported adverse events.

Patents and patent publications relating to CD20 antibodies and CD20 binding molecules include US Pat. Nos. 5,776,456, 5,736,137, 5,843,439, 6,399,061, and 6,682,734, as well as US 2002/0197255, US 2003/0021781, US 2003. / 0082172, US 2003/0095963, US 2003/0147885 (Anderson et al.); US Patent Nos. 6,455,043 and WO 2000/09160 (Grillo-Lopez, A.); WO 2000/27428 (Grillo-Lopez and White); WO 2000/27433 (Grillo-Lopez and Leonard); Braslawsky et al., WO 2000/44788; WO 2001/10462 (Rastetter, W.); WO01 / 10461 (Rastetter and White); WO 2001/10460 (White and Grillo-Lopez); US 2001/0018041, US 2003/0180292, WO 2001/34194 (Hanna and Hariharan); US 2002/0006404 and WO 2002/04021 (Hanna and Hariharan); US 2002/0012665 and WO 2001/74388 (Hanna, N.); US 2002/0058029 (Hanna, N.); US 2003/0103971 (Hariharan and Hanna); US 2002/0009444 and WO 2001/80884 (Grillo-Lopez, A.); WO 2001/97858 (White, C); US 2002/0128488 and WO 2002/34790 (Reff, M.); Braslawsky et al., WO 2002/060955; Braslawsky et al., WO 2002/096948; WO 2002/079255 (Reff and Davies); US Patent No. 6,171,586 and WO 1998/56418 (Lam et al.); WO 1998/58964 (Raju, S.); WO 1999/22764 (Raju, S.); WO 1999/51642, US Pat. No. 6,194,551, US Pat. No. 6,242,195, US Pat. No. 6,528,624 and US Pat. No. 6,538,124 (Idusogie et al.); WO 2000/42072 (Presta, L.); Curd et al., WO 2000/67796; WO 2001/03734 (Grillo-Lopez et al.); US 2002/0004587 and WO 2001/77342 (Miller and Presta); US 2002/0197256 (Grewal, L); US 2003/0157108 (Presta, L.); US Pat. Nos. 6,565,827, 6,090,365, 6,287,537, 6,015,542, 5,843,398, and 5,595,721, Kaminski et al .; US Pat. Nos. 5,500,362, 5,677,180, 5,721,108, 6,120,767, and 6,652,852 (Robinson et al.); US Patent No. 6,410,391 to Laubitschek et al .; US Pat. Nos. 6,224,866 and WO00 / 20864 (Barbera-Guillem, E.); WO 2001/13945 (Barbera-Guillem, E.); Goldenberg, WO 2000/67795; US 2003/0133930 and WO 2000/74718 (Goldenberg and Hansen); US 2003/0219433 and WO 2003/68821 to Hansen et al .; WO2004 / 058298 (Goldenberg and Hansen); Golay et al., WO 2000/76542; WO 2001/72333 (Wolin and Rosenblatt); US Patent No. 6,368,596 to Ghetie et al .; US Patent No. 6,306,393 and US 2002/0041847 (Goldenberg, D.); US 2003/0026801 (Weiner and Hartmann); WO 2002/102312 to Engleman, E .; US 2003/0068664 to Albitar et al .; WO 2003/002607 (Leung, S.); WO 2003/049694, US 2002/0009427, and US 2003/0185796 (Wolin et al.); WO 2003/061694 (Sing and Siegall); US 2003/0219818 (Bohen et al.); US 2003/0219433 and WO 2003/068821 (Hansen et al.); US 2003/0219818 (Bohen et al.); US2002 / 0136719 (Shenoy et al.); WO 2004/032828 (Wahl et al.); And WO 2002/56910 (Hayden-Ledbetter). See also US Pat. Nos. 5,849,898 and EP 330,191 (Seed et al.); EP332.865A2 (Meyer and Weiss); U.S. Patent 4,861,579 to Meyer et al .; US 2001/0056066 to Bugelski et al .; Bhat et al., WO 1995/03770; US 2003/0219433 Al (Hansen et al.); Teeling et al., WO 2004/035607; Lowman et al., WO 2004/056312; US 2004/0093621 (Shitara et al.); WO 2004/103404 (Watkins et al.); WO 2005/000901 (Tedder et al.); US 2005/0025764 (Watkins et al.); Carr et al., WO 2005/016969; And US 2005/0069545 (Carr et al.). WO 2004/032828 mentions recurrent polychondritis as one of a list of immune disorders to be treated with anti-CD20 antibodies.

Publications related to therapy with rituximab include: [Perotta and Abuel, "Response of chronic relapsing ITP of 10 years duration to rituximab" Abstract # 3360 Blood 10 (1) (part 1-2) : p. 88B (1998); Pierta et al., “Rituxan in the treatment of chronic idiopathic thrombocytopaenic purpura (ITP)”, Blood, 94: 49 (abstract) (1999); 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, supra; Leandro et al., "Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficacy and dose response" Arthritis and Rheumatism 44 (9): S370 (2001); During the 2-cycle period, each patient received two 500-mg injections of rituximab, two 750-mg injections of cyclophosphamide, and a high dose oral corticosteroid, two of each treated 7 Leandro et al., “An open study of B lymphocyte depletion in systemic lupus erythematosus”, Arthritis and Rheumatism, 46: 2673-2677 (2002), recurring at months and 8 months; Patients were treated with rituximab (375 mg / m 2 × 4, repeated at weekly intervals), and additional rituximab application was delivered every 5-6 months, followed by maintenance therapy with rituximab 375 mg / m 2 for 3 months Each patient, and a second patient with unresponsive SLE was successfully treated with rituximab and maintenance therapy was administered every three months, and both patients responded well to ["Successful long-term treatment of systemic lupus erythematosus with rituximab maintenance therapy "Weide et al., Lupus, 12: 779-782 (2003); Edwards and Cambridge, "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes" Rheumatology 40: 205-211 (2001); Cambridge et al., "B lymphocyte depletion in patients with rheumatoid arthritis: serial studies of immunological parameters" Arthritis Rheum., 46 (Suppl. 9): S1350 (2002); Edwards et al., “B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders” Biochem Soc. Trans., Supra; Edwards et al., “Efficacy and safety of retuximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis.Arthritis and Rheumatism 46 (9): S197 (2002)]; Edwards et al., "Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis" N Engl. J. Med. 350: 2572-82 (2004); Pavelka et al., Ami. Rheum. 63: (S1): 289-90 (2004); Emery et al., Arthritis Rheum. 50 (S9): S659 (2004); Levine and Pestronk, "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using rituximab "Neurology 52: 1701-1704 (1999); [DeVita et al.," Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis "Arthritis & Rheum 46: 2029-2033 (2002)]; [Hidashida et al. "Treatment of DMARD-refractory rheumatoid arthritis with rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology; 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; "Pathogenic roles of B cells in human autoimmunity; insights from the clinic" Martin and Chan, Immunity 20: 517-527 (2004); Silerman and Weisman, "Rituximab Therapy and Autoimmune Disorders, Prospects for Anti-B Cell Therapy", Arthritis and Rheumatism, 48: 1484-1492 (2003); Kazkaz and Isenberg, "Anti B cell therapy (rituximab) in the treatment of autoimmune diseases", Current opinion in pharmacology, 4: 398-402 (2004); Virgolini and Vanda, "Rituximab in autoimmune diseases", Biomedicine & pharmacotherapy, 58: 299-309 (2004); Klemmer et al., "Treatment of antibody mediated autoimmune disorders with a AntiCD20 monoclonal antibody Rituximab", Arthritis And Rheumatism, 48: (9) 9, S (SEP), page: S624-S624 (2003); Kneitz et al., “Effective B cell depletion with rituximab in the treatment of autoimmune diseases”, Immunobiology, 206: 519-527 (2002); Arzoo et al., "Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab)" Annals of the Rheumatic Diseases, 61 (10), p922-4 (2002) Comment in Ann Rheum Dis. 61: 863-866 (2002); ["Future Strategies in Immunotherapy" by Lake and Dionne, in Burger's Medicinal Chemistry and Drug Discovery (2003 by John Wiley & Sons, Inc.) Article Online Posting Date: January 15, 2003 (Chapter 2 "Antibody-Directed Immunotherapy")] ; Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section: CD20 as an Immunotherapy Target, article online posting date: 15 January, 2002 entitled "CD20"; Appendix 4A entitled "Monoclonal Antibodies to Human Cell Surface Antigens" by Stockinger et al., Eds: Coligan et al, in Current Protocols in Immunology (2003 John Wiley & Sons, Inc) Online Posting Date: May, 2003; Print Publication Date: February, 2003; Penicet and Morrison, "CD Antibodies / molecules: Definition; Antibody Engineering" in Wiley Encyclopedia of Molecular Medicine Section: Chimeric, Humanized and Human Antibodies; posted online 15 January, 2002; Specks et al. "Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy" Arthritis & Rheumatism 44: 2836-2840 (2001); online abstract submission and invitation Koegh et al., "Rituximab for Remission Induction in Severe ANCA-Associated Vasculitis: Report of a Prospective Open-Label Pilot Trial in 10 Patients", American College of Rheumatology, Session Number: 28-100, Session Title: Vasculitis, Session Type: ACR Concurrent Session, Primary Category: 28 Vasculitis, Session 10/18/2004 (<www.abstractsonline.com/viewer/SearchResults.asp>); Eriksson, "Short-term outcome and safety in 5 patients with ANCA-positive vasculitis treated with rituximab", Kidney and Blood Pressure Research, 26: 294 (2003); Jayne et al., "B-cell depletion with rituximab for refractory vasculitis" Kidney and Blood Pressure Research, 26: 294 (2003); Jayne, poster 88 (11th International Vasculitis and ANCA workshop), 2003 American Society of Nephrology; Stone and Specks, "Rituximab Therapy for the Induction of Remission and Tolerance in ANCA-associated Vasculitis", in the Clinical Trial Research Summary of the 2002-2003 Immune Tolerance Network, <www.immunetolerance.org/research/autoimmune/trials/ stone.html>]. See also, Leandro et al., "B cell repopulation occurs mainly from naive B cells in patient with rheumatoid arthritis and systemic lupus erythematosus" Arthritis Rheum., 48 (Suppl 9): S 1160 (2003).

Sarwal et al. N. Eng. J. Med. 349 (2): 125-138 (July 10, 2003) report molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling.

Recurrent polychondritis is a rare chronic disorder of cartilage, characterized by recurring episodes of inflammation of the cartilage of various tissues of the body. Tissues that contain cartilage that can become inflamed include ears, nose, joints, spine, and breathing organs. Eyes, heart, and blood vessels with cartilage-like biochemical composition can also be affected.

The cause of recurrent polychondritis is unknown. This condition is expected to be caused by an immune system disorder (autoimmune) in which the body's immune system (normally attacks the body's invaders, especially infections) is misinduced. The result is directed inflammation in various body tissues. Relief can be confirmed through anti-inflammatory agents and various steroids.

Multiple mononeuritis is a painful asymmetric asynchronous sensory and motor peripheral neuropathy involving damage isolated in two or more separate nerve regions. Multiple nerves in random areas of the body may be affected. As conditions worsen, they become less polypathic and more symmetric, similar to polyneuropathy. Multiple mononeuropathy syndrome may be distributed bidirectionally, terminally, and towards the body throughout the body. Damage to the nerves is accompanied by the destruction of axons (i.e. parts of nerve cells similar to the copper part of the wire), thus hindering nerve conduction at the site of injury. Common causes include oxygen deficiency due to diabetes and multiple nerve compression, as well as reduced blood flow or inflammation of the blood stream. In about one third of the cases, no cause was identified. Several specific disorders are associated with multiple mononeuritis, including but not limited to vascular diseases such as nodular polyarteritis and other vasculitis diseases, diabetes, and connective tissue diseases such as rheumatoid arthritis or systemic lupus erythematosus. Connective tissue disease is the most common cause in children. Less common causes include: Sjogren's syndrome, Wegener's granulomatosis, hypersensitivity (allergic reactions) that cause inflammation of blood vessels, leprosy, sarcoidosis, amyloidosis, polypathic forms of diabetic neuropathy, and blood disorders (such as Hypereosinophilia and cold globulinemia). For example, the clinicopathologic features of 28 peripheral neuropathy patients associated with Churg-Strauss syndrome were evaluated, and most sensory and motor involvements showed patterns of multiple mononeuritis in the early stages and four limbs. ) And progression to asymmetric polyneuropathy, see Hattori et al. Brain 122 (3): 427-439 (1999). CD20-positive B lymphocytes appeared only occasionally.

Treatment of neuropathy depends on its cause, and many neuropathies can be treated by addressing the underlying cause (such as vitamin deficiency). It can be prevented that nothing else happens. For example, controlling diabetes can prevent diabetic neuropathy. If the tumor or intervertebral disc prolapse is the cause, surgery may be involved in the therapy to remove the tumor or restore the intervertebral disc prolapse. In trapping or compression, neuropathy treatment may consist of splint fixation of the ulnar nerve or median nerve or decomposition by surgery. Calf and radial compression neuropathy may require evasion of pressure. Physical therapy and / or splints can be useful for the prevention of contractions (a condition in which muscle shortening around the joints causes abnormal and sometimes painful posture of the joints). Neuropathy associated with autoimmune diseases can be ameliorated by treatment for abnormal features of the immune system. Such treatments include intravenous immunoglobulins, plasma exchange and immunosuppressive therapies (Cook et al. Neurology 40: 212-214 (1990); Dyck et al. N. Engl. J. Med 325: 1482-). 1486 (1991); Ernerudh et al. J. Neurol. Neurosurg. Psychiatry 55: 930-934 (1992); Blume et al. Neurology 45: 1577-1580 (1995); Pestronk et al. Neurology 44: 2027-2031 (1994)]. They can only produce minimal functional improvement. In addition, such treatment can be expensive and time consuming.

The literature on antibody-directed treatment of B-cell surface membrane markers is very limited. Levine and Pestronk described five neuropathic patients with immunoglobulin M antibodies against GM1 or MAG, treated with rituximab. Within the treatment period of 3-6 months, all five had improved function and lowered titer of serum antibodies (Levine and Pestornk Am. J. Neurol. 52: 1701-1704 (1999)).

If no specific treatment is available, the pain of neuropathy is generally controllable, such as painkillers, pain medications, tricyclic antidepressants, anti-seizure medications, or nerve blockers.

Sutton and Winer, Current Opinion in Pharmacology 2/3: 291-295 (June 1, 2002) states that plasma exchange, intravenous immunoglobulins and corticosteroids continue to be the main treatments for immunological neuropathy. Recent tests indicate that the combination of these therapies is significantly more effective than single-agent treatment. The utility of new immunotherapy and cytotoxic agents is difficult to confirm due to the treatment of a small number of patients in open-label studies.

Lee et al. Bone Marrow Transplantation 30/1: 53-56 (2002) has shown that high-dose chemotherapy and autologous peripheral vascular stem cell (PBSC) transplantation are significant, progressive, and treatment-resistant significant unknown unknown monoclonal gamma globulin disease (MGUS: monoclonal gammopathy It is proposed that it may function in the treatment of peripheral neuropathy secondary to unknown significance. Latov et al. Neurology 52: A551 (1999) discloses that RITUXAN® has shown safe and effective treatment in two neuropathy patients associated with IgM monoclonal gamma globulinopathy and anti-MAG antibody activity. Canavan et al. Neurology 58/7 (Suppl. 3): A233 (April 2002) discloses that RITUXAN® has been associated with continuous clinical improvement in the majority of treated patients with IgM autoantibody-related polyneuropathy.

Regarding monoclonal antibody treatment of interferon-alpha resistant mixed cold globulinemia with anti-CD20 antibodies, see Sansonno et al. Blood 101 (10): 3818-3826 (May 15 2003) discloses treatment of peripheral neuropathy with RITUXAN®.

Hattori et al. Brain 122/3: 427-439 (1999) evaluated the clinicopathologic features of patients with peripheral neuropathy associated with Chug-Strauss syndrome and mentions that CD20-positive B lymphocytes only appeared occasionally.

Zaja et al. Blood 101 (10): 3827-3834 (May 15 2003) discloses that RITUXAN® may represent a safe and effective alternative to standard immunosuppression in type II mixed cold globulinemia (MC). RITUXAN® has been shown to be effective for signs of cutaneous vasculitis (ulcers, purpura, or urticaria), subjective symptoms of peripheral neuropathy, low grade B-cell lymphoma, arthralgia, and fever. Zaidi et al. Leukemia and Lymphoma 45/4: 777-780 (2004), a rare lymphoma proliferative disorder with lymphoma proliferative disorder (LYG) with 60% mortality in the first year of lung, liver, central and peripheral nervous system involvement. It is disclosed that an example of has been effectively treated with RITUXAN®. However, Trojan et al. Annals of Oncology 13/5: 802-805 (2002) discloses that RITUXAN® has not been shown to be effective in patients with peripheral neuropathy due to neurolymphoma. Associative PET-CT imaging performed on an in-line PET-CT system showed multiple small nodule injury expanding along the peripheral nerve corresponding to the initial relapse of transformed B-cell non-Hodgkin's lymphoma. .

Binstadt et al. Journal of Pediatrics 143/5: 598-604 (November 2003)] describes four pediatric patients with multisystem autoimmune diseases that are unresponsive to conventional immunosuppressive agents, each with a central nervous system (CNS) involvement. Has concluded that RITUXAN® is safe and effective. One patient with autoimmune thrombocytopenia and autoimmune CNS and peripheral vascular disease resolved thrombocytopenia and showed improvement in neurological symptoms; He is currently reported not receiving any immunosuppressive drugs. These symptoms improved in two heterozygous motives with lymphoplasmic colitis, pulmonary nodules, and CNS disease. The fourth patient with seizures and chorea secondary to primary antiphospholipid antibody syndrome had improved micro and overall motor function and decreased seizure frequency. Saito et al. Lupus 12/10: 798-800 (2003) discloses that RITUXAN® has been useful in the treatment of patients with systemic lupus erythematosus (SLE) associated with highly active B lymphocytes and patients with unresponsive lupus nephritis.

There is a need in the art for additional drugs to treat various indications such as polychondritis and multiple mononeuritis.

Summary of the Invention

Accordingly, the invention is as claimed. In particular, the present invention provides a method of treating polychondritis or multiple mononeuritis in a mammal, comprising administering to the mammal an effective amount of an antibody that binds CD20 in a first aspect.

In one embodiment of this method, the antibody is not conjugated to another molecule. In another embodiment, the antibody is conjugated to another molecule, eg, a cytotoxic agent such as a radioactive compound, eg, Y2B8 or ¹³¹ I-B1. In another embodiment, the antibody comprises rituximab or humanized 2H7. Humanized 2H7 in one embodiment comprises the variable domain sequences of SEQ ID NOs: 2 and 8. In another embodiment, humanized 2H7 comprises a variable heavy domain having modification (s) N100A or D56A, N100A in SEQ ID NO: 8 and a variable light domain having modification (s) M32L, S92A, or M32L, S92A in SEQ ID NO: 2. . In a further embodiment, humanized 2H7 comprises the light chain variable region (V _L ) sequence of SEQ ID NO: 30 and the heavy chain variable region (V _H ) sequence of SEQ ID NO: 8, wherein the antibody further contains an amino acid substitution of D56A in VH-CDR2 And N100 in VH-CDR3 is substituted with Y or W, more preferably the antibody comprises a v511 light chain sequence of SEQ ID NO: 31 and a v511 heavy chain sequence of SEQ ID NO: 32.

The antibody is preferably administered to the mammal at a dose of about 20 mg / m 2 to about 250 mg / m 2, more preferably about 50 mg / m 2 to about 200 mg / m 2 of antibody. In another preferred embodiment, the methods of the invention comprise administering an initial dose of an antibody followed by a subsequent dose, wherein the mg / m 2 dose of the antibody at the subsequent dose is mg / m 2 of the antibody at the initial dose. Exceed the dose.

In another preferred embodiment, the mammal is a human. The antibody is preferably administered intravenously or subcutaneously.

In a preferred embodiment, the methods of the present invention consist essentially of administering an effective amount of the antibody to a mammal.

In another preferred aspect, the methods of the invention further comprise administering to the mammal an effective amount of an immunosuppressant, anti-pain, or chemotherapeutic agent.

In another embodiment, when treating polychondritis, the methods of the present invention provide a mammal with an effective amount of a non-steroidal anti-inflammatory drug, steroid, or immunosuppressant such as methotrexate, cyclophosphamide, dipson, azathioprine , Penicillamine, or cyclosporin. In the treatment of multiple mononeuritis, the method of the present invention provides a mammal with an effective amount of an anti-pain agent, a steroid, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulin, cyclosporin, or mycophenolate mofetil. Further comprising administration.

In a further aspect, the present invention includes a composition comprising a container and an antibody that binds to CD20 contained in the container, and adds a package insert directing the user of the composition to treat polychondritis or multiple mononeuritis in a mammal. It relates to a manufactured product containing. In a preferred embodiment, the article of manufacture further comprises a container comprising an agent other than the therapeutic antibody and further comprising instructions for treating the mammal with such an agent.

1A is a sequence alignment comparing the amino acid sequences of the light chain variable domains (V _L ) of mouse 2H7 (SEQ ID NO: 1), humanized 2H7.v16 variant (SEQ ID NO: 2), and human kappa light chain subgroup I (SEQ ID NO: 3). 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 compares the amino acid sequences of the heavy chain variable domains (V _H ) of each of mouse 2H7 (SEQ ID NO: 7), humanized 2H7.v16 variant (SEQ ID NO: 8), and human consensus sequence (SEQ ID NO: 9) of heavy chain subgroup III. Sequence alignment. The 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).

In FIGS. 1A and 1B, CDR1, CDR2 and CDR3 in each chain are flanked by framework regions FR1-FR4, as indicated, in parentheses. 2H7 refers to mouse 2H7 antibody. Asterisks between two rows of sequences indicate different positions between the two 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), and insertions are represented by a, b, c, d, and e.

Figure 2 shows the nucleotide sequence of phagemid pVX4 (SEQ ID NO: 13 {5 'sequence} and SEQ ID NO: 14 {3' complement sequence}) used in the construction of the 2H7 Fab plasmid (see Example 1), as well as the CDR-graft anti-IFN The amino acid sequences of the L chain (SEQ ID NO: 15) and H chain (SEQ ID NO: 16) of the Fab for the -a humanized antibody are shown.

Figure 3 shows the nucleotide sequence of the expression plasmid encoding the chimeric 2H7.v6.8 Fab (SEQ ID NO: 17 {5 'sequence} and SEQ ID NO: 18 {3' complement sequence}). The amino acid sequences of the L chain (SEQ ID NO: 19) and H chain (SEQ ID NO: 20) are shown.

4 shows the nucleotide sequence of plasmid pDR1 (SEQ ID NO: 21; 5391 bp) for expression of immunoglobulin light chain as described in Example 1. FIG. pDR1 contains a sequence encoding an unrelated antibody, the light chain of a humanized anti-CD3 antibody (Shalaby et al. J. Exp. Med. 175: 217-225 (1992)), a bold and underlined start and stop codon do.

5 shows the nucleotide sequence of plasmid pDR2 (SEQ ID NO: 22; 6135 bp) for expression of immunoglobulin heavy chains as described in Example 1. FIG. pDR2 contains sequences encoding unrelated antibodies, heavy chains of humanized anti-CD3 antibodies (Shalaby et al., supra), in bold and underlined start and stop codons.

6A and 6B show the amino acid sequence of the 2H7.v16 L chain and FIG. 6A shows the complete L chain (SEQ ID NO: 23) containing the first 19 amino acids before DIQ, a secretory signal sequence not present in the mature polypeptide chain. 6B shows the mature polypeptide L chain (SEQ ID NO: 24).

7A and 7B show the amino acid sequence of the 2H7.v16 H chain, and FIG. 7A shows the complete H chain (SEQ ID NO: 25) containing the first 19 amino acids before EVQ, a secretory signal sequence not present in the mature polypeptide chain. 7B shows the mature polypeptide H chain (SEQ ID NO: 26). Aligning the V _H sequence (SEQ ID NO: 8) in FIG. 1B with the full H chain sequence, the human γ1 constant region is from amino acid positions 114-471 of SEQ ID NO: 25.

8A and 8B show the amino acid sequence of the 2H7.v31 H chain, and FIG. 8A shows the complete H chain (SEQ ID NO: 27) containing the first 19 amino acids before EVQ, a secretory signal sequence not present in the mature polypeptide chain. 8B shows the mature polypeptide H chain (SEQ ID NO: 28). L chain is the same as for 2H7.v16 (see FIG. 6).

9 is a flow chart summarizing amino acid changes from the humanized version of mouse 2H7 to v75 subpopulations.

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

FIG. 11 is a sequence alignment comparing the heavy chain amino acid sequences of humanized 2H7.v16 variant (SEQ ID NO: 8) and humanized 2H7.v138 variant (SEQ ID NO: 30).

I. Definition

A “B-cell surface marker” or “B-cell surface antigen” herein is an antigen expressed on the surface of a B cell to which an antagonist that binds to it can be targeted. Exemplary B-cell surface markers include CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81 , CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers. (For explanation, see The Leukocyte Antigen Facts Book, 2 ^nd Edition. 1997, ed. Barclay et al. Academic Press, Harcourt Brace & Co., New York). Other B-cell surface markers include RP105, FcRH2, CD79A, C79B, B cell CR2, CCR6, CD72, P2X5, HLA-DOB, CXCR5, FCER2, BR3, Btig, NAG14, SLGC16270, FcRH1, IRTA2, ATWD578, FcRH3, IRTA1, FcRH6, BCMA, and 239287_at. Particularly interesting B-cell surface markers are preferentially expressed on B cells as compared to other non-B-cell tissues in mammals and can be expressed in both precursor B cells and mature B cells. Preferred B-cell surface markers herein are CD20 and CD22.

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

The "CD22" antigen or "CD22", also known as BL-CAM or Lyb8, is a type 1 endogenous membrane glycoprotein having a molecular weight of about 130 (reduced) to 14K kD (non-reduced). It is expressed in both the cytoplasm and cell membrane of B-lymphocytes. The CD22 antigen appears early at approximately the same stage as the CD19 antigen in B-cell lymphocyte differentiation. Unlike other B-cell markers, CD22 membrane expression is limited to late expression stages, including between mature B cells (CD22 +) and plasma cells (CD22−). CD22 antigens are described, for example, in Wilson et al. J. Exp. Med. 173: 137 (1991) and Wilson et al. J. Immunol. 150: 5013 (1993).

A "non-malignant disorder" herein is polychondritis or multiple mononeuritis, preferably multiple mononeuritis. In addition, it may include spino-optical multiple sclerosis; Ordinary spear spear; Chug-straus vasculitis or syndrome (CSS); Lupus encephalitis; Lupus nephritis; Cutaneous systemic lupus erythematosus (SLE); IgE-mediated diseases other than asthma, specifically allergic rhinitis, anaphylaxis, or atopic dermatitis; Chronic neuropathy; Ocular band-myoclonus syndrome; Alveolar proteinosis; Scleritis; Microscopic polyangiitis; Neoplastic syndromes such as hypercalcemia, such as hypercalcemia (not including Lambert-Eaton, anemia, or hypoglycemia) caused by tumors; Rasmussen encephalitis; Central nervous system (CNS) vasculitis; Channel disease, a disease with various properties associated with ion channel dysfunction, such as epilepsy, migraine, arrhythmia, muscle disorders, hearing loss, blindness, periodic paralysis, and channel diseases of the CNS (not including CNS inflammatory disorders); Autism; Or neuropathic, myopathy or CNS sarcoidosis.

As used herein, "polychondritis" includes recurrent polychondritis, Von Meyenburg disease, Meyenburg disease or syndrome, Meyenburg Altherr Uehlinger syndrome, multiple chondropathy, askenage (Askenazy), Jaksch Wartenhorst, Meyenburg, or Bon Jaksch Wartenhorst syndrome, cartilage soluble, diffuse or recurrent chondritis, chondromalacia arthritis, or total chondritis Means any polychondritis.

As used herein, “multiple mononeuritis” describes a condition characterized by inflammation caused by several nerves in unrelated parts of the body, ie, nerve damage is isolated in two or more separate nerve regions. Accompanied by damage. When this worsens, it becomes more diffuse and less concentrated in certain areas, similar to polyneuropathy. Symptoms of this class of diseases may include numbness, weakness, burning pain (especially at night), and loss of reflexes. The pain can be severe and can cause disability.

An “antagonist” is one or more substances that, upon binding to a B-cell surface marker, destroys or lacks B cells in a mammal and / or reduces or prevents the humoral response elicited by B cells, for example. It is a molecule that interferes with B-cell function. Preferably the antagonist may lack B cells in the mammal treated with the antagonist (ie, reduce circulating B-cell levels). Such deficiency can be achieved through various mechanisms such as ADCC and / or CDC, inhibiting B-cell proliferation, and / or inducing B-cell death (eg, via apoptosis). Antagonists included within the scope of the present invention include antibodies, synthetic or naturally-sequence peptides and small-molecular antagonists that bind to B-cell markers, optionally conjugated or fused to cytotoxic agents. Preferred antagonists include antibodies, ie antibodies that bind to B-cell surface markers.

"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. It refers to a cell-mediated response that recognizes the antibody and then causes lysis of the target cell. NK cells, primary cells that mediate ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is described by Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991), is summarized in Table 3 on page 464. To assess ADCC activity of the molecule, in vitro ADCC assays such as those described in US Pat. No. 5,500,362 or 5,821,337 can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, ADCC activity of the molecule can be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably such cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include PBMCs, NK cells, monocytes, cytotoxic T cells and neutrophils, with PBMCs and NK cells being 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 naturally-sequence human FcRs. In addition, preferred FcRs are receptors (gamma receptors) that bind IgG antibodies, including receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants of these receptors and alternatively spliced forms. . FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), which mainly have different but similar amino acid sequences in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine based activation motif (ITAM) in the cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); And de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs that are to be identified later are included in the term "FcR" herein. The term also includes neonatal receptor FcRn, which is responsible for delivering maternal IgG 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 the binding of the first component (C1q) of the complement system to a molecule (eg an antibody) complexed with a cognate antigen. To assess complement activation, see, eg, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) can be performed for CDC analysis.

"Growth-inhibiting" antagonists are those which prevent or reduce the proliferation of cells expressing the antigen to which the antagonist binds. For example, antagonists can prevent or reduce proliferation of B cells in vitro and / or in vivo.

Antagonists that “induce apoptosis” include standard apoptosis assays such as binding of Annexin V, fragmentation of DNA, cell contraction, expansion of endoplasmic reticulum, cell fragmentation and / or membrane vesicles (called apoptotic bodies). As determined by the formation of the cells that induce planned cell death, eg, planned cell death of B cells.

The term “antibody” herein is used in its broadest sense and is specifically a multispecific antibody (eg, bispecific antibody) formed from intact monoclonal antibodies, polyclonal antibodies, two or more intact antibodies , And one antibody fragment that exhibits the desired biological activity.

An "antibody fragment" includes a portion of an intact antibody, preferably comprising an antigen-binding or variable region of the intact 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.

For the purposes herein, an "intact antibody" is one that includes the Fc region as well as the heavy and light chain variable domains.

In general, a “natural antibody” is a heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one disulfide covalent bond, and the number of disulfide linkages depends on the heavy chain of several immunoglobulin isotypes. Each heavy and light chain also has regular intervals of intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V _H ) followed by a number of constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

The term “variable” refers to the fact that certain portions of the variable domains differ greatly in sequence between antibodies and are used for binding and specificity of each specific antibody to a specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions in the variable domains are called the framework regions (FRs). The variable domains of each of the natural heavy and light chains are mainly adopting a β-sheet arrangement, which is linked by β-sheet structures, and in some cases by three hypervariable regions that form a loop that forms part of the β-sheet structure. Two FRs. The hypervariable regions in each chain are held together very closely by the FRs and contribute to forming the antigen-binding site of the antibody with the hypervariable regions from the other chain (Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD (1991). The constant domains are not directly involved in binding the antigen to the antigen, but exhibit various effector functions such as the participation of the antibody in ADCC.

Digestion of the antibody with papain results in two identical antigen-binding fragments, each with a single antigen-binding site, referred to as a "Fab" fragment, and the remaining "Fc" fragment, which is easily crystallized. It reflects its power. Treatment with pepsin results in an F (ab ') ₂ fragment having two antigen binding sites and still capable of crosslinking with the antigen.

"Fv" is the minimum antibody fragment with a complete antigen-recognition site and antigen-binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are bound in tight non-covalent bonds. It is within this arrangement that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the V _H -V _L dimer. Collectively, six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv including only three hypervariable regions specific for the antigen), although having a lower affinity than the entire binding site, has the ability to recognize and bind antigen.

In addition, the Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain comprising one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab 'in which the cysteine residue (s) of the constant domains have one or more free thiol groups. F (ab ') ₂ antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinctly different types, called kappa (κ) and lambda (λ), based on the amino acid sequences of the constant domains.

Depending on the amino acid sequence of the constant domain of the heavy chains, antibodies can be assigned to several classes. Intact antibodies have five major classes of IgA, IgD, IgE, IgG and IgM, some of which are further classified as subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2. Can be. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

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

The term "dia body" in the same polypeptide chain, the light chain variable domain (V _L) chain variable domain (V _H), 2 different antigens, including linked to in (V _H -V _L) - refers to a small antibody fragments with a binding site do. By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with the complementary domain of another chain, resulting in two antigen-binding sites. Diabodies are described, for example, in EP 404,097; WO 93/11161; And Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies are directed against a single antigenic site and are highly specific. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include several antibodies directed against several determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture that is not contaminated with other immunoglobulins. The modifier “monoclonal” refers to the properties of the obtained antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring antibody production by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by the hybridoma method first described in Kohler et al., Nature, 256: 495 (1975), or by recombinant DNA methods (eg, For example, US Pat. No. 4,816,567). Also "monoclonal antibodies" are described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and in Marks et al., J. Mol. Biol., 222: 581-597 (1991) can be used to isolate from phage antibody libraries.

Monoclonal antibodies herein include those in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain (s) Particularly included are “chimeric” antibodies (immunoglobulins) identical or homologous to the corresponding sequences of antibodies from other species or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity ( U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). The chimeric antibody herein includes a variable domain antigen-binding sequence and a human constant-region sequence derived from a non-human primate (eg, a continental monkey such as baboon, rhesus monkey or cynomolgus monkey). Primateized "antibodies are included (US Pat. No. 5,693,780).

A “humanized” form of a non-human (eg mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. For most parts, the humanized antibody is a hypervariable region (donor antibody) of a non-human species such as a mouse, rat, rabbit or non-human primate whose residues from the hypervariable region of the recipient have the desired specificity, affinity and ability. Human immunoglobulin (receptor antibody) replaced with a residue from. In some cases, framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies may also include residues that are not found in the recipient antibody or in the donor antibody. Such modifications are made to further refine the performance of the antibody. In general, humanized antibodies comprise substantially all of one or more, typically two, variable domains, where all or substantially all hypervariable loops correspond to hypervariable loops of non-human immunoglobulins and all or substantially All FRs are of human immunoglobulin sequence. Humanized antibodies will also optionally optionally include at least a portion of an immunoglobulin constant region (Fc), typically of human immunoglobulins. Further details are given by Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); And Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

As used herein, the term “hypervariable region” refers to an amino acid residue of an antibody that is responsible for antigen binding. Hypervariable regions include amino acid residues from “complementarity determining regions” or “CDRs” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3), and heavy chains in the light chain variable domain) 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda , MD. (1991)]) and / or residues from “hypervariable loops” (eg, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) within the light chain variable domain). And 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987). “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.

Agents that “bind” to an antigen of interest, eg, a B-cell surface marker, are those capable of binding to the antigen with sufficient affinity and / or binding such that the antagonist is useful as a therapeutic for targeting cells expressing the antigen.

Examples of CD19 antigens are described in Hekman et al. Cancer Immunol. Immunother. 32: 364-372 (1991) and Varsveld et al. Cancer Immunol. Immunother. 40: 37-47 (1995); And Kiese et al. Leukemia Research II, 12: 1119 (1987)].

“Antibody that binds to CD20” refers to an antibody that binds to the CD20 antigen with sufficient affinity and / or binding so that the antagonist is useful as a therapeutic agent for targeting cells that express or overexpress the CD20 antigen. Examples of such antibodies include "C2B8" (US Pat. No. 5,736,137), now referred to as "rituximab"("RITUXAN®"); Yttrium- [90] -labeled 2B8 mouse antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN® (US Pat. No. 5,736,137); "Toshio tube momaep (Tositumomab)," also referred mouse IgG2a "B1" (an optionally labeled with ¹³¹ I ¹³¹ I-B1 "antibody (iodine I131 Toshio tube momaep, BEXXAR ™) generated) by (U.S. Patent No. 5595721 No.) Mouse monoclonal antibody "1F5" (Press et al. Blood 69 (2): 584-591 (1987)) and "Framed patched" or humanized 1F5 (WO03 / 002607, Leung, S.) ATCC deposited HB-96450; mouse 2H7 and chimeric 2H7 antibodies (US Pat. No. 5,677,180); humanized 2H7; huMax-CD20 (Genmab, Denmark); AME-133 (Applied Molecular Evolution); and International Leukocyte Typing Workshop Monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available from Valentine et al., In: Leukocyte Typing III, (McMichael, Ed., P. 440, Oxford University Press) (1987)))).

The terms “rituximab” and “RITUXAN®” herein refer to a genetically engineered chimeric mouse / human monoclonal antibody, directed against the CD20 antigen and named “C2B8” in US Pat. No. 5,736,137, and CD20 Fragments thereof whose ability to bind to is retained.

Purely for the purposes herein, “humanized 2H7” refers to a humanized antibody, or antigen-binding fragment thereof, that binds human CD20, wherein the antibody is effective to deplete primate B cells in vivo and the antibody is Within the H chain variable region (V _H ) at least the CDR3 sequence of SEQ ID NO: 12 (FIG. 1B) from an anti-human CD20 antibody and substantially the human consensus framework (FR) residues of human heavy chain subgroup III (V _H III) . In a preferred embodiment, such an antibody further comprises an H chain CDR1 sequence of SEQ ID NO: 10 and a CDR2 sequence of SEQ ID NO: 11, more preferably an L chain CDR1 sequence of SEQ ID NO: 4, a CDR2 sequence of SEQ ID NO: 5, a CDR3 sequence of SEQ ID NO: 6 And substantially a human consensus framework (FR) residue of human light chain κ subgroup I (VκI), wherein the V _H region may be linked to a human IgG chain constant region, which region may be, for example, IgG1 or IgG3. Can be. In a preferred embodiment, such an antibody comprises the V _H sequence of SEQ ID NO: 8 (v16, shown in FIG. 1B) and optionally also comprises the V _L sequence of SEQ ID NO: 2 (v16, shown in FIG. 1A), which is in the H chain. Amino acids substitutions of D56A and N92A and S92A in the L chain (v. 96). More preferred such antibodies are 2H7.v16 having the light and heavy chain amino acid sequences of SEQ ID NOs: 24 and 26 shown in Figures 6B and 7B, respectively. Another preferred embodiment is that the antibody is 2H7.v31 having the light and heavy chain amino acid sequences of SEQ ID NOs: 24 and 28 shown in FIGS. 6B and 8B, respectively. The antibodies herein may further comprise one or more amino acid substitutions in the Fc region that improve ADCC and / or CDC activity, such as where the amino acid substitutions are S298A / E333A / K334A, more preferably SEQ ID NO: 28 (FIG. 8B). 2H7.v31 having a heavy chain amino acid sequence of Any such antibodies may further comprise one or more amino acid substitutions in the Fc region that reduce CDC activity, for example, may comprise one or more substitutions K322A. Preferably such an antibody is 2H7.v114 or 2H7.v115 with at least 10-fold improvement in ADCC activity over RITUXAN®.

Preferred humanized 2H7 is a variable light chain sequence:

And variable heavy chain sequences:

Intact antibodies or antibody fragments comprising a.

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

And heavy chain amino acid sequences:

Or heavy chain amino acid sequence:

It includes.

An “isolated” antagonist is one that has been identified and separated and / or recovered from components of the natural environment. Contaminant components of the natural environment are substances that interfere with the use of antagonists in diagnosis or treatment and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antagonist is (1) to more than 95 weight percent of the antagonist, most preferably to more than 99 weight percent, as measured by the Lowry method, by (2) using a spinning cup sequencer Sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, or (3) homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver staining. . Isolated antagonists include in situ antagonists in recombinant cells since at least one component of the antagonist's natural environment will not be present. Ordinarily, however, isolated antagonist will be prepared by one or more purification steps.

"Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and animal husbandry, zoos, sports or pets such as dogs, horses, cats, cattle and the like. Preferably, the mammal is a human.

"Treatment" refers to both therapeutic treatment and prophylactic or prophylactic measures. Subjects in need of treatment include those already suffering from the disease or disorder, as well as those who wish to prevent the disease or disorder. Thus, the mammal may have been diagnosed with a disease or disorder, or may be susceptible to the disease or disorder.

The expression “effective amount” refers to the amount of the antagonist effective to prevent, ameliorate or treat the autoimmune disease in question.

The term "immunosuppressant" as used herein for adjuvant therapy refers to a substance that acts to inhibit or mask the immune system of the mammal to be treated herein. Substances that inhibit cytokine production, downregulate or inhibit self-antigen expression, or mask MHC antigens will be included. Examples of such immunosuppressants include 2-amino-6-aryl-5-substituted pyrimidines (US Pat. No. 4,665,077); Mycophenolate mofetil such as CELLCEPT®; Azathioprine (IMURAN®, AZASAN® / 6-mercaptopurine; bromocriptine; danazole; dapson; glutaraldehyde (masking MHC antigens as described in US Pat. No. 4,120,649); MHC antigens and MHC Anti-idiotypic antibodies to fragments; cyclosporin A; steroids such as corticosteroids and glucocorticosteroids such as prednisone, prednisolone such as PEDIAPRED® (prednisolone sodium phosphate) or ORAPRED® (prednisolone sodium phosphate oral solution), methyl Prednisolone, and dexamethasone; methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALL ™); hydroxychloroquine / chloroquine; sulfasalazine; leflunomide; anti-interferon-γ, -β, or -α antibody, anti- Tumor necrosis factor-α antibodies (infliximab or adalimumab), anti-TNFα immunoadhesin (ENBREL® itanercept), Cytokine or cytokine receptor antagonists, including tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1, including anti-CD11a and anti-CD18 antibodies Antibody; anti-L3T4 antibody; heterologous anti-lymphocyte globulin; polyclonal or pan-T antibody, or monoclonal anti-CD3 or anti-CD4 / CD4a antibody; soluble peptide containing LFA-3 binding domain (1990 WO 1990/08187 published July 26); streptokinase; TGF-β; streptodonase; RNA or DNA from a host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor ( Cohen et al., US Pat. No. 5,114,721; T-cell receptor fragments (Offner et al. Science, 251: 430-432 (1991); WO 1990/11294; Ianeway, Nature, 341: 482 (1989) And WO 1991/01133) T cell receptor antibodies (EP 340,109) such as T10B9; Cyclophosphamide (CYTOXAN®); Dapson; Penicillamine (CUPRIMINE®); Plasma exchange; Or intravenous immunoglobulin (IVIG). They may be used alone or in combination with each other, in particular the steroid and another immunosuppressant may be combined, or after such a combination, a non-steroidal agent is provided in a maintenance dose to reduce the need for steroids.

"Anti-pain agents" are non-steroidal drugs that include drugs that inhibit or inhibit pain, such as over-the-counter painkillers or prescription pain medications to control neuralgia, such as ibuprofen (MOTRIN®), naproxen (NAPROSYN®) Refers to sex anti-inflammatory drugs (NSAIDs), as well as a variety of other agents used to reduce the stinging pain that may occur, including anticonvulsants (gabapentin, phenytoin, carbamazepine) or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin (NEURONTIN®), (E) -N-vanylyl-8-methyl -6-nonaneamide (CAPSAICIN®), or nerve blockers.

As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents the function of a cell and / or causes cell destruction. This 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), chemotherapeutic agents, and bacteria Toxins, such as enzymatically active toxins or small-molecular toxins of fungal, plant or animal origin, or fragments thereof.

“Chemotherapeutic agents” are chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; Alkyl sulfonates such as busulfan, improsulfan and pifosulfan; Aziridine such as benzodopa, carboquinone, meturedopa, and uredopa; Ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylololomeramine; Acetogenin (particularly bulatacin and bulatacinone); Camptothecins (including the synthetic analog topotecan); Bryostatin; Calistatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); Cryptopycin (particularly cryptotopin 1 and cryptotopin 8); Dolastatin; Duocarmycin (including the synthetic analogues KW-2189 and CBl-TM1); Eleuterobins; Pankratisstatin; Sarcodictin; Spongestatin; Nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, esturamustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, normovicin, fensterrin, prednimo Stin, trophosphamide, uracil mustard; Nitrosureas such as carmustine, chlorozotocin, potemustine, lomustine, nimustine, and rannimustine; Antibiotics such as endyne phosphorus (eg, calicheamicin, in particular calicheamicin gamma II and calicheamycin omega II (see, eg, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994) Dynemycin, including dynemycin A; bisphosphonates such as clodronate; esperamycin; as well as neochrominostatin chromophores and related chromoprotein endyne antibiotic chromophores), aclacinomycin, malignant Tinomycin, otramycin, azaserine, bleomycin, coctinomycin, carabicin, carminomycin, carcinophylline, chromomomycinis, dactinomycin, daunorubicin, dextrubicin, 6-diazo -5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,Darvisin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olibomycin, peplomycin, potyrromycin, puromycin, kelamycin, rhorubicin, streptonicin, streptozosin, Tubercidine, ubenimex, ginostatin, zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denophtherine, methotrexate, putrophtherin, 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 agents such as aminoglutetimide, mitotan, trilostane; Folic acid supplements such as proline acid; Aceglaton; Aldophosphamide glycosides; Aminolevulinic acid; Eniluracil; Amsacrine; Vestravusyl; Bisantrene; Edda trexate; Depopamine; Demecolsin; Diaziquiones; Elponnitine; Elftinium acetate; Epothilones; Etogluside; Gallium nitrate; Hydroxyurea; Lentinane; Ronidinin; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Fur coat; Nitraerin; Pentostatin; Penammet; Pyrarubicin; Roxanthrone; Grape filinic acid; 2-ethylhydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); Lakamic acid; Lysine; Sizopyran; Spirogermanium; Tenuazone acid; Triaziquion; 2,2 ', 2 "-trichlorotriethylamine; tricothecene (especially T-2 toxin, veracrine A, loridine A and anaguidine); urethane; bindesin; dacarbazine; mannosestin; mitobronitol; Mitolactol; Picobroman; Gazaitocin; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoids, for example TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ .), ABRAXANE ™ Cremophor-free, albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); phosphamide; mitoxantrone; vincristine ; NAVELBINE® vinorelbine; novantron; teniposide; edretrexate; dow Mycin; aminopterin; geloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and any of the foregoing materials Pharmaceutically acceptable salts, acids or derivatives are included.

This definition includes anti-hormonal agents that act to modulate or inhibit hormonal action in tumors such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxyphene, keoxyphene Anti-estrogens and selective estrogen receptor modulators (SERMs) including, for example, LY1 17018, onapristone and FARESTON toremifene; Aromatase inhibitors that inhibit the enzyme aromatase that modulates estrogen production in the adrenal glands, such as 4 (5) -imidazole, aminoglutetimides, MEGASE® megestrol acetate, AROMASIN® exemestane, formes Tanière, Padrosol, RIVISOR® Borazole, FEMARA® Letrozole, and ARIMIDEX® Anastrozole; And anti-androgens such as flutamide, nilutamide, bikallutamide, leuprolide, and goserelin; As well as troxacitabine (1,3-dioxolane nucleoside cytosine analogue); Antisense oligonucleotides, especially those that inhibit expression of genes in signaling pathways implicated in abnormal cell proliferation, such as PKC-alpha, Ralf and H-Ras; Ribozymes such as VEGF expression inhibitors (eg, ANGIOZYME® ribozymes) and HER2 expression inhibitors; Vaccines such as gene therapy vaccines such as ALLOVECTIN® vaccines, LEUVECTIN® vaccines, and VAXID® vaccines; PROLEUKTN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitors; ABARELIX® rmRH; And pharmaceutically acceptable salts, acids or derivatives of any of the above materials.

The term “cytokine” is a generic name for a protein released by one cell population that acts as an intercellular mediator on another cell. Examples of such cytokines include lymphokine, monocaine, interleukin (IL) 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 ligands (KL). As used herein, the term cytokine includes biologically active equivalents of proteins and natural-sequence cytokines from natural sources or from recombinant cell culture.

The term “hormone” refers to a polypeptide hormone, which is generally secreted by the ductal gland 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; Mullerian-inhibiting substance; And thrombopoietin.

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; Converting growth factors (TGF) such as TGF-α and TGF-β; Insulin type growth factor-I and -II; Erythropoietin (EPO); Osteoinductive factor; 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).

The term “integrin” refers to a receptor protein that allows cells to bind and respond to extracellular matrix and is involved in various cellular functions such as wound healing, cell differentiation, homing of tumor cells and apoptosis. These are part of the large family of cell-attached receptors involved in the extracellular matrix and cell-cell interactions. Functional integrins consist of two transmembrane glycoprotein subunits called alpha and beta, which are non-covalently bound. All of the alpha subunits share some homology with each other, as are the beta subunits. The receptor always contains one alpha chain and one beta chain. Examples include alpha 6beta1, alpha3beta1, and alpha7beta1.

As used herein, the term “prodrug” refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells and can be converted to an enzymatically activated or more active parental form than a parental drug. Refer. See, eg, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 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. 247-267, Humana Press (1985). Prodrugs of the invention include phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acids- which can be converted to more active cytotoxic free drugs 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 include, but are not limited to. Examples of cytotoxic drugs that can be derivatized in prodrug form for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.

“Liposomes” are vesicles composed of various types of lipids, phospholipids and / or surfactants useful for delivering drugs (such as the antagonists disclosed herein, and optionally chemotherapeutic agents) to a mammal. The components of liposomes are generally arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes.

The term “packaging insert” is used to refer to instructions that are customarily included in the commercial packaging of a therapeutic product and refer to indications, usage, dosage, administration, contraindications and / or warnings related to the use of such therapeutic product. Contains information about

II . Production of antagonists

The methods and articles of manufacture of the invention use antagonists that bind to B-cell surface markers, or incorporate antagonists. Thus, methods of producing such antagonists will be described below.

The B-cell surface marker to be used in the production or screening of the antagonist may be, for example, an soluble form of the antigen, or part thereof, containing the desired epitope. Alternatively or additionally, cells expressing B-cell surface markers on the cell surface can be used to generate or screen antagonists. Other forms of B-cell surface markers useful for the production of antagonists will be apparent to those skilled in the art. Preferably, the B-cell surface marker is a CD19 or CD20 antigen.

Preferred antagonists are antibodies, but antagonists other than antibodies are embodied herein. For example, the antagonist may include small molecule antagonists optionally fused or conjugated to a cytotoxic agent (such as those described herein). To identify small molecules that bind to the antigen, libraries of small molecules can be screened for such B-cell surface markers herein. Small molecules may further be screened for antagonistic properties and / or conjugated to cytotoxic agents.

The antagonist may also be a peptide produced by rational design or by phage display (see, eg, WO 1998/35036 published August 13, 1998). In one embodiment, the selected molecule can be a “CDR mimic” or antibody analog designed based on the CDRs of the antibody. Although such a peptide itself can be antagonistic, the peptide can be added or enhances the antagonistic properties of the peptide. May optionally be fused to a cytotoxic agent.

Exemplary techniques for the production of antibody antagonists for use in accordance with the present invention are described below.

(i) Polyclonal  Antibodies.

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Difunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide esters (conjugation via cysteine residues), N-hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ Or a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine tyroglobulin, or using R ¹ N═C═NR where R and R ¹ are different alkyl groups It may be useful to conjugate the relevant antigen to soybean trypsin inhibitor.

For example, antigen or immunogenic conjugates may be combined by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with a threefold dose of Freund's complete adjuvant and injecting this solution into the dermal at multiple sites. Or animals are immunized against the derivative. After one month, the animal is boosted by subcutaneous injection of the peptide or conjugate in Freund's complete adjuvant into multiple sites at 1/5 to 1/10 the original amount. After 7-14 days, animals are bled and serum is analyzed for antibody titers. The animal is boosted until the titer reaches the plateau. Preferably, animals are boosted with conjugates of the same antigen conjugated to different proteins and / or through different crosslinking reagents. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, coagulants such as alum are used appropriately to enhance the immune response.

( ii ) Monoclonal  Antibodies.

Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, ie the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in trace amounts. Thus, the modifier “monoclonal” refers to the characteristics of an antibody as not being a mixture of individual antibodies.

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

In the hybridoma method, mice or other suitable host animals such as hamsters are immunized as described above to produce lymphocytes capable of producing or producing antibodies that will specifically bind to the protein 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 grown by inoculation into an appropriate culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells are free of hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) enzymes, the culture medium for hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium These substances prevent the growth of HGPRT-deficient cells.

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

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

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

After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by a limiting dilution procedure and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.

Monoclonal antibodies secreted by the subclones are prepared by conventional immunoglobulin purification procedures such as, for example, protein A-SEPHAROSE ™ agarose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Appropriate isolation from culture medium, ascites fluid or serum.

Monoclonal antibodies can also be produced recombinantly. Easily isolate and sequence DNA encoding monoclonal antibodies using conventional methods (e.g., using oligonucleotide probes that can specifically bind genes encoding the heavy and light chains of a mouse antibody). . Hybridoma cells function as a preferred source of such DNA. Once isolated, DNA can be inserted into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins unless transfected. Infection, yields the production of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Plueckthun, Immunol. Revs. 130: 151-188 (1992).

In additional embodiments, the antibody or antibody fragment can be isolated from the antibody phage library using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describes the isolation of mouse and human antibodies using phage libraries, respectively. Subsequent documents include the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as very large phage libraries. Combination infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993)) as a strategy for construction are described. Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

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

Typically, such non-immunoglobulin polypeptides replace the constant domains of an antibody, or the variable domains of one antigen-binding site of an antibody, thereby allowing specificity for one antigen-binding site and a different antigen with specificity for the antigen. Chimeric bivalent antibodies are generated comprising another antigen-binding site having.

( iii A) humanized antibody

Methods for humanizing non-human antibodies are known in the art. Preferably, the humanized antibody has one or more amino acid residues derived from a non-human source and introduced into the humanized antibody. Such non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is performed by Winter and co-investigators (Jones et al., Nature, 321: 522-525 (1986)) by substituting the corresponding sequences of human antibodies with hypervariable regions (Riechmann et al., 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 sequences than intact human variable domains are substituted with corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from similar sites in rodent antibodies.

Selecting the human variable domains (both light and heavy chains) used to prepare humanized antibodies is very important for reducing antigenicity. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable-domain sequences. The human sequence closest to that of the rodent is then accepted as the human framework region (FR) for humanized antibodies (Sims et al., J. Immunol, 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)]. Another method uses a specific framework region 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 many different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immnol., 151: 2623 (1993)].

It is also important to humanize antibodies while maintaining high affinity for the antigen and other beneficial biological properties. To achieve this goal, humanized antibodies are prepared by an analytical process of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences, in accordance with a preferred method. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these displays can analyze the possible role of residues in the functionalization of candidate immunoglobulin sequences, ie, analyze residues that affect the ability of candidate immunoglobulins to bind antigen. In this way, FR residues can be selected and combined from recipient and import sequences such that the desired antibody properties, such as increased affinity for the target antigen (s), are achieved. In general, hypervariable regions 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 produce transnasal animals (eg mice) that, upon immunization, can produce the entire repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain linkage region (J _H ) gene in chimeric and germline mutant mice completely inhibits the production of endogenous antibodies. Transferring the human germline immunoglobulin gene array to such germline mutant mice will produce human antibodies upon antigen inoculation. See, eg, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); And US Pat. Nos. 5,591,669, 5,589,369, and 5,545,807.

Alternatively, human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors using phage display technology (McCafferty et al., Nature 348: 552-553 (1990)). Can produce. According to this technique, antibody V domain genes are cloned in-frame into major or minor coat protein genes of fibrous bacteriophages such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since the fibrous particles contain a single-stranded DNA copy of the phage genome, genes encoding antibodies exhibiting these properties are selected by selection based on the functional properties of the antibody. Thus, phage mimics some of the properties of B-cells. Phage display can be performed in a variety of formats; For an overview of this see, eg, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Various sources of V-gene segments can be used for phage display. In Clackson et al., Nature 352: 624-628 (1991), various arrays of anti-oxazolone antibodies were isolated from a small random combinatorial library of V genes derived from the spleen of immunized mice. In essence, Marks et al., J. Mol. Biol. 222: 581-597 (1991), or according to the techniques described in Griffith et al., EMBO J. 12: 725-734 (1993), a repertoire of V genes from unimmunized human donors can be constructed. And antibodies against various arrays of antigens (including self-antigens). See also US Pat. Nos. 5,565,332 and 5,573,905.

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

(v) antibody fragments

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived through proteolytic degradation of intact antibodies (eg, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al. , Science 229: 81 (1985). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically coupled to form F (ab ') ₂ fragments (Carter et al, Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In another embodiment, the antibody of choice is a single chain Fv fragment (scFv). WO 1993/16185; US Patent No. 5,571,894; And US Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody", eg, as described in US Pat. No. 5,641,870. Such linear antibody fragments 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. Another such antibody may bind to a first B-cell surface marker and further bind to a second B-cell surface marker. Alternatively, the anti-B-cell surface marker binding arm allows the Fc receptor (FcγR) to IgG such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so that the cellular defense mechanism is concentrated on B cells. ), Or an arm that binds to a triggering molecule on white blood cells, such as a T-cell receptor molecule (eg, CD2 or CD3). Bispecific antibodies can also be used to localize cytotoxic agents to B-cells. These antibodies bind to B-cell surface marker-binding arms, and cytotoxic agents (eg, saporin, anti-interferon-α, vinca alkaloids, lysine A chains, methotrexate, or radioisotope hapten). Have arms. 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. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). . Due to the random organization of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography steps, is rather cumbersome and the product yield is low. Similar procedures are disclosed in WO 1993/08829 and in Traunecker et al., EMBO J., 10: 3655-3659 (1991).

According to another approach, antibody variable domains (antibody-antigen binding sites) with the desired binding specificities are fused to immunoglobulin constant domain sequences. Fusion with an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions is preferred. It is preferred that the first heavy chain constant region (CH1) containing the site necessary for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusions, and, if desired, the DNAs encoding the immunoglobulin light chains, are inserted into separate expression vectors and co-transfected into appropriate host cells. This provides great flexibility in controlling the mutual ratios of the three polypeptide fragments in embodiments in which unequal proportions of the three polypeptide chains used for construction provide optimal yields. However, if high yields are obtained by expression of two or more polypeptide chains in the same proportion, or if the proportions do not have special significance, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector. .

In a preferred embodiment of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair in the other arm, providing a second binding specificity. It has been found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since it provides an easy separation mode where only one half of the bispecific molecule is present with an immunoglobulin light chain. It became. This approach is disclosed in WO 1994/04690. For further details for the generation of bispecific antibodies, see, eg, Suresh et al., Methods in Enzymmology, 121: 210 (1986).

According to another approach described in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least a portion of the C _H 3 domain of the antibody constant domains. 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). By replacing large amino acid side chains with smaller amino acid side chains (eg, alanine or threonine) a compensating "cavity" of the same or similar size for the large side chain (s) is created on the interface of the second antibody molecule. . This provides a mechanism for increasing the yield of heterodimers over other unwanted 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 to biotin. Such antibodies have been proposed, for example, for targeting immune system cells to unwanted cells (US Pat. No. 4,676,980), and for the treatment of HIV infection (WO 1991/00360, WO 1992/200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Along with many crosslinking techniques, suitable crosslinkers are well known in the art and are described, for example, in US Pat. No. 4,676,980.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a method for proteolytically cleaving intact antibodies to produce F (ab ') ₂ fragments. This fragment is reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction to mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as agents for the selective fixation of enzymes.

Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F (ab ') ₂ molecule. Each Fab 'fragment was secreted separately from E. coli and directionally chemically coupled in vitro to form a bispecific antibody. The bispecific antibody thus formed was able to bind cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques have also been described for preparing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies were produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were linked by gene fusion to the Fab 'portion of two different antibodies. The antibody homodimer was reduced in the hinge region to form a monomer and then oxidized to form the antibody heterodimer. Such methods can also be used for the production of antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provided an alternative manufacturing mechanism for bispecific antibody fragments. The fragment comprises a heavy chain variable domain (V _H ) linked to the light chain variable domain (V _L ) by a linker that is too short to pair two domains on the same chain. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of another fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments using single chain Fv (sFv) dimers has also been reported. Gruber et al., J. Immunol. 152: 5368 (1994).

Antibodies in excess of bivalent are also implemented. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).

III . Other Modifications of Conjugates and Antagonists

Antagonists used in or included in the methods herein are optionally conjugated to cytotoxic agents.

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

Conjugates of antagonists with one or more small-molecular toxins such as calicheamicin, maytansine (US Pat. No. 5,208,020), tricotene and CC1065 are also embodied herein. In one embodiment of the invention, the antagonist may be conjugated 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 is reduced to May-SH3 and reacted with a modified antagonist (Chari et al., Cancer Research 52: 127-131 (1992), May Tansinoid-antagonist conjugates may be generated.

Alternatively, the antagonist is conjugated with one or more calicheamicin molecules. Antibiotics of the calicheamicin family can cause double-stranded DNA breakage at sub-picomole concentrations. Structural analogs 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 ^I ₁ (Hinman et. al., Cancer Research 53: 3336-3342 (1993) and Lode et al., Cancer Research 58: 2925-2928 (1998).

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chains, unbound active fragments of diphtheria toxins, exotoxin A chains ( Pseudomonas aeruginosa ), lysine A chain, abrin A chain, modesin A chain, alpha-sarsine, alleurites fore fordii ) protein, diantine protein, phytolaca americana americana ) protein (PAPI, PAPII and PAP-S), Momordica Charantia inhibitor, Cursin, Crotin, Safaonaria Offisinalis Inhibitor, Geronine, Mitogeline, Restrictocin, Phenomycin, Eno Mycin and trichotheneses are included. See, eg, WO 1993/21232, published October 28, 1993.

In the present invention also antagonists conjugated with compounds exhibiting nucleolytic activity (eg ribonuclease or DNA endonucleases such as deoxyribonuclease; DNase) are also embodied.

Various radioisotopes are available for 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.

Various difunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxyl Latex, iminothiolane (IT), difunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suverate), aldehydes (such as glutaraldehyde), bis-azido compounds (Such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and Bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) can be used to make conjugates of antagonists with cytotoxic agents. Lysine immunotoxins can be prepared, for example, as described in Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 1994/11026. 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 (Chari et al., Cancer Research 52: 127-131 (1992)) can be used.

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

In another embodiment, an antagonist can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, wherein the antagonist-receptor conjugate is administered to the patient, and then the conjugate that is not bound using a scavenger. Is removed from the circulatory system and then administered "ligand" (eg, avidin) conjugated to a cytotoxic agent (eg radionucleotide).

Antagonists of the invention may also be conjugated to prodrug-activating enzymes that convert prodrugs (eg, peptidyl chemotherapeutic agents, see WO 1981/01145) into active anticancer drugs. See, for example, WO 1988/07378 and US Pat. No. 4,975,278.

Enzymatic components of such conjugates include any enzyme that can act on the prodrug in such a way as to convert the prodrug into its more active cytotoxic form.

Enzymes useful in the methods of the invention include alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; Arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; Cytosine deaminase useful for converting non-toxic 5-fluorocytosine into anticancer drug 5-fluorouracil; Proteases useful for converting peptide-containing prodrugs into free drugs, such as seratia proteases, thermolysine, subtilisin, carboxypeptidase and cathepsins (such as 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, useful for converting drugs derivatized from amine nitrogen with phenoxyacetyl or phenylacetyl groups, respectively, to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as “abzyme,” can be used to convert prodrugs of the invention to free active drugs (eg, Massey, Nature 328). : 457-458 (1987). Antagonist-Abzyme conjugates can be prepared as described herein for delivery of azyme to tumor cell populations.

The enzymes of the present invention can be covalently linked to the antagonist by techniques well known in the art, such as by using the heterobifunctional crosslinking reagents discussed above. Alternatively, a fusion protein comprising at least the antigen-binding region of the antagonist of the invention linked to at least the functionally active portion of the enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (eg See Neuberger et al., Nature 312: 604-608 (1984).

Another variation of the antagonist is implemented herein. For example, the antagonist can be linked to one of various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol.

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

Particularly useful liposomes can be produced by reverse phase evaporation methods using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Via disulfide interchange reaction [Martin et al., J. Biol. Chem., 257: 286-288 (1982), Fab 'fragments of antibodies of the invention can be conjugated to liposomes. Chemotherapeutic agents may optionally be contained within the liposomes. See 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 implemented. 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 and / or insertions and / or substitutions of residues in the amino acid sequence of the antagonist. Any combination of deletions, insertions, and substitutions is made under the condition that the final construct has the desired properties to reach the final construct. Amino acid changes can also alter the post-translational process of the antagonist, such as changing the number or location of glycosylation sites.

A useful method to identify specific residues or regions of antagonists that are preferred positions for mutagenesis is called "alanine-scanning mutagenesis" as described in Runningham and Wells, Science, 244: 1081-1085 (1989). Become. Here, a group of residues or target residues are identified (eg, charged residues such as arg, asp, his, lys and glu) and substituted with neutral or negatively charged amino acids (most preferably alanine or polyalanine) This affects the interaction of amino acids with antigens. Subsequently, amino acid positions that exhibit functional sensitivity to substitution are refined by introducing additional or other variants at or about the substitution site. Thus, the site for introducing amino acid sequence variation is predetermined, but there is no need to predetermine the nature of the mutation itself. For example, to analyze the performance of mutations at a given site, ala scanning or random mutagenesis is performed at the target codon or region and the expressed antagonist variants are screened for the desired activity.

Amino acid sequence insertions include amino- and / or carboxy terminal fusions ranging in length from one residue to one or more residues in a polypeptide, as well as intrasequence insertion of single or multiple amino acid residues. Examples of terminal insertions include antagonists with N-terminal methionyl residues or antagonists fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include those in which the polypeptide or enzyme that increases the serum half-life of the antagonist is fused to the N- or C-terminus of the antagonist.

Another type of variant is an amino acid substitution variant. Such variants are those in which one or more amino acid residues in an antagonist molecule are replaced with different residues. Sites most interesting for substitutional mutagenesis of antibody antagonists include hypervariable regions, but FR alterations are also implemented. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, as referred to in Table 1 as “exemplary substitutions” or further described below with respect to amino acid classes, may be introduced and the product screened.

Original residues Exemplary Substitution Preferred Substitution Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp; lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gln asp Gly (G) ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; Norleucine leu Leu (L) Norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; Norleucine leu

Substantial changes in the biological properties of the antagonist include (a) the structure of the polypeptide backbone within the substitutional region, such as in sheet or helical form; (b) the charge or hydrophobicity of the molecule at the target site; Or (c) the effect of maintaining the bulk of the side chain is carried out by selecting substitutions that differ significantly. Naturally occurring residues are classified into the following groups based on 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 exchanging a member of one of these classes for another class.

Any cysteine residue that is not involved in maintaining the proper form of the antagonist, in general, may also be substituted with serine to improve the oxidative stability of the molecule and to 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 involve the substitution of one or more hypervariable region residues of the parental antibody. In general, production variant (s) selected for further development will have improved biological properties compared to the parental antibody from which they are derived. A convenient way to generate such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (eg 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus produced are each displayed in a monovalent fashion from fibrous phage particles as fusions to the gene III product of M13 packaged in the particles. Phage-displayed variants are then screened for their biological activity (eg, binding affinity) as described herein. To identify candidate hypervariable region sites for modification, alanine-scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to confirm the location of contact between the antibody and the antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques described above herein. Once such variants are generated, variant panels can be screened as described herein, and antibodies with superior properties in one or more related assays can be selected for further development.

Another type of amino acid variant of the antagonist alters the original glycosylation pattern of the antagonist. By altering is meant deleting one or more carbohydrate moieties found in the antagonist and / or adding one or more glycosylation sites that are not present in the antagonist.

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

Adding a glycosylation site to the antagonist is conveniently accomplished by altering the amino acid sequence such that the antagonist contains one or more of the above described tripeptide sequences (for N-linked glycosylation sites). Alterations can also be made by addition or substitution of one or more serine or threonine residues to the sequence of the original antagonist (for O-linked glycosylation sites).

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

With regard to effector function, it may be desirable to modify the antagonists of the invention, for example, such that the ADCC and / or CDC of the antagonist are enhanced. This can be accomplished by introducing one or more amino acid substitutions in the Fc region of the antibody antagonist. Alternatively or additionally, the cysteine residue (s) may be introduced into the Fc region to allow interchain disulfide bonds to form within this region. Homodimeric antibodies so produced may have improved internalization capacity and / or increased complement-mediated cell death and ADCC. See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, B. J. Immunol., 148: 2918-2922 (1992). Wolf et al. Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional cross-linkers as described in Cancer Research, 53: 2560-2565 (1993). Alternatively, engineering antibodies with double Fc regions can enhance complement lysis and ADCC ability. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).

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 “salvage receptor binding epitope” refers to the epitope of the Fc region of an IgG molecule (eg, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ) which serves to increase serum half-life in vivo of the IgG molecule. Refers to.

IV . Pharmaceutical formulation

Therapeutic formulations of antagonists used according to the present invention may be prepared by any pharmaceutically acceptable carrier, excipient or stabilizer of any pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)) for storage. 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 (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclo Hexanol, 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) polypeptide; 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 non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

Exemplary anti-CD20 antibody formulations are described in WO 1998/56418. This publication discloses a liquid multidose comprising 40 mg / ml rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, and 0.02% POLYSORBATE ™ 20 (polyoxyethylene sorbitan monooleate) at pH 5.0. Formulations are described and their minimum shelf life is 2 years at 2-8 ° C. Another interesting anti-CD20 formulation is 10 mg / ml rituximab in 9.0 mg / ml sodium chloride, 7.35 mg / ml sodium citrate dihydrate, 0.7 mg / ml POLYSORBATE ™ 80 (polyoxyethylene sorbitan monooleate), and Sterile water for injection, pH 6.5.

Lyophilized formulations adapted for subcutaneous administration are described in WO 1997/04801. Such lyophilized formulations can be reconstituted with an appropriate diluent at high protein concentrations and the reconstituted formulations can be administered subcutaneously to the mammal to be treated herein.

The formulations herein may also contain one or more active compounds, preferably those with complementary activities that do not adversely affect each other when necessary for the particular indication to be treated. For example, cytotoxic agents, chemotherapeutic agents, cytokines, or immunosuppressive agents (e.g., those that act on T cells, such as cyclosporin or antibodies that bind T cells, e.g., bind to LFA-1 It may be desirable to further provide). The effective amount of such other agents depends on the amount of antagonist present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. They are generally used at the same dosage as previously used and at or from the route of administration or at about 1-99% of the dosage used so far.

The active ingredients are for example microcapsules prepared by coacervation technology or interfacial polymerization, for example colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nanoparticles and Nanocapsules) or in hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. Ed. (1980).

Sustained release formulations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, 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-glutamic acid Copolymers of γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT® (injectable microspheres consisting of lactic acid-glycolic acid copolymers and leuprolide acetate) , And poly-D-(-)-3-hydroxybutyric acid.

Formulations to be used for in vivo administration must be sterile. Sterilization is readily performed by filtration through sterile filtration membranes.

V. Treatment with antagonists

Compositions comprising antagonists that bind to B-cell surface antigens can be formulated, formulated, and administered in a fashion consistent with good medical practice. Factors contemplated in this situation include the particular disease or disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and the physician known to the physician. Other factors are included. The therapeutically effective amount of the antagonist to be administered will depend on such considerations.

As a general suggestion, a therapeutically effective amount per dose of an antagonist administered parenterally will range from about 0.1 to 20 mg / kg body weight of the patient per day, with a typical initial range of antagonists used being in the range of about 2 to 10 mg / kg. .

Preferred antagonists are antibodies which are not conjugated to cytotoxic agents, for example antibodies such as RITUXAN®. Appropriate dosages for the unconjugated antibody are, for example, in the range of about 20 mg / m 2 to about 1000 mg / m 2. In one embodiment, the dosage of the antibody is different from what is currently recommended for RITUXAN®. For example, the patient may be administered one or more doses of an antibody substantially less than 375 mg / m 2, eg, the dose may be from about 20 mg / m 2 to about 250 mg / m 2, eg, about 50 mg / M 2 to about 200 mg / m 2.

In addition, one or more initial dose (s) of the antibody may be administered followed by one or more subsequent dose (s), wherein the mg / m 2 dose of the antibody at the subsequent dose (s) is determined by the initial dose (s). Exceeds the mg / m 2 dose of the antibody. For example, the initial dose may range from about 20 mg / m 2 to about 250 mg / m 2 (eg, about 50 mg / m 2 to about 200 mg / m 2), with subsequent doses ranging from about 250 mg / m 2 to about 1000 mg / m 2.

However, as mentioned above, this suggested amount of antagonist is highly dependent on therapeutic discretion. The main factor in selecting the appropriate dose and schedule is the result obtained, as indicated above. For example, relatively high doses may be needed initially for ongoing and acute diseases. To obtain the most effective results, depending on the disease or disorder, the antagonist is administered as close as possible to the initial sign, diagnosis, appearance, or occurrence of the disease or disorder or while the disease or disorder is alleviated.

Antagonists are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, inhalation, intradural, intraarticular and intranasal, and if desired for local immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the antagonist may be appropriately administered by pulse infusion, for example while attenuating the dose of the antagonist. Depending in part on whether the administration is short or long term, preferably the dose is given by injection, most preferably intravenously or subcutaneously.

One or more other compounds, such as cytotoxic agents, chemotherapeutic agents, immunosuppressants, anti-pain agents, hormones, integrins, growth factors, and / or cytokines may be administered with the antagonists herein or are known to those skilled in the art Various other therapies can be applied. Preferably, for example, depending on the type of indication, the severity and severity of the indication, and the type of antagonist, the other compound administered is an immunosuppressive, anti-pain, or chemotherapeutic agent.

 When polychondritis such as recurrent polychondritis is treated, preferably another compound is used to control inflammation, if the symptoms are not severe, ibuprofen (MOTRIN®), naproxen (NAPROSYN®) or sulindac (CLINORIL®) Non-steroidal anti-inflammatory drugs (NSAIDs). However, in general, there is a need for corticosteroid-related drugs, such as steroids such as prednisone and prednisolone. High dose steroids are often needed early, especially if eye or respiratory tract is involved. In addition, most patients require long-term use of steroids.

Another preferred compound that can be used in combination with antagonists for the treatment of polychondritis is methotrexate (RHEUMATREX®, TREXALL ™), which, in combination with steroids, has shown potential as a treatment for recurrent polychondritis as well as maintenance therapy. Indicated the possibility. The findings indicate that methotrexate may help reduce steroid demand. Other preferred compounds include cyclophosphamide (CYTOXAN®), dapson, azathioprine (IMURAN®, AZASAN®), penicillamine (CUPRIMINE®), cyclosporine (NEORAL®, SANDIMMUNE®), and these drugs Combinations of steroids are included.

With regard to treating multiple mononeuritis with another agent, the pain of neuropathy can generally be controlled when no specific treatment is available. The simplest treatment is the use of over-the-counter painkillers such as acetaminophen (TYLENOL®), NSAIDs such as ibuprofen as mentioned above, or aspirin followed by prescription pain medications. Tricyclic antidepressants such as amitriptyline (ELAVIL®) and anti-seizure agents such as carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), or gabapentin (NEURONTIN®) are used to alleviate the pain of neuropathy. come. CAPSAICIN® ((E) -N-Vanyl-8-methyl-6-Noneamide), the chemical responsible for the pungent taste of chili peppers, is used as a cream to help alleviate the pain of peripheral neuropathy. In addition, nerve blockers may be effective in alleviating pain. Other preferred compounds for the treatment of peripheral neuropathy include autologous PBSC transplantation, steroids such as corticosteroids (including pulse therapy thereof) and prednisone, prednisolone and methyl-prednisolone (including pulse therapy thereof), methotrexate, cyclophosphamide (eg For example, CYTOXAN®) (including intravenous cyclophosphamide pulse therapy), plasma exchange or plasmapheresis, intravenous immunoglobulins, cyclosporins such as cyclosporin A, mycophenolate mofetil (e.g., CELLCEPT® ), Or chemotherapeutic agents (including high dose chemotherapeutic agents), including those that lower IgM concentrations, such as FLUDARA® (fludarabine phosphate) or LEUKERAN® (chlorambucil). Other compounds particularly preferred for such indications are anti-pain agents, steroids, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulins, cyclosporin, or mycophenolate mofetil.

Combination administrations include co-administration using separate formulations or a single pharmaceutical formulation, and continuous administration in any order, with the time intervals in which both (or all) active agents exert their biological activity simultaneously. It is preferable to exist.

In addition to administering a protein antagonist to a patient, the present application implements the administration of the antagonist by gene therapy. Such administration of nucleic acids encoding antagonists is included in the expression "administering an effective amount of an antagonist." See, eg, WO 1996/07321, published March 14, 1996, regarding the use of gene therapy for the production of intracellular antibodies.

There are two main approaches regarding the incorporation of nucleic acid (optionally contained in a vector) into a patient's cell: in vivo and ex vivo. For in vivo delivery, the nucleic acid is usually injected directly into the patient at the site where the antagonist is needed. For ex vivo treatment, the cells of the patient are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered directly to the patient or, for example, encapsulated in a porous membrane that is implanted into the patient. (See, eg, US Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into living cells. This technique depends on whether the nucleic acid is delivered in vitro into cultured cells or in vivo to cells of the intended host. Techniques suitable for in vitro delivery of nucleic acid into mammalian cells include the use of liposomes, electrophoresis, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, and the like. A commonly used vector for ex vivo delivery of genes is a retrovirus.

Currently preferred in vivo nucleic acid delivery techniques include viral vectors (such as adenoviruses, herpes simplex type I viruses, or adeno-associated viruses) and lipid-based systems (lipids useful for lipid-mediated delivery of genes include, for example, DOTMA, DOPE and DC-Chol). In some situations, it is desirable to provide a nucleic acid source with agents that target the target cell, such as cell-surface membrane proteins or antibodies specific for the target cell, ligands for receptors on the target cell, and the like. If liposomes are used, proteins that bind to cell-surface membrane proteins associated with endocytosis, such as capsid proteins or fragments that are flexible for a particular cell type, antibodies to proteins that internalize in circulation, and Proteins that target intracellular localization and enhance intracellular half-life and the like can be used for targeting and / or to facilitate uptake. Techniques for receptor-mediated endocytosis are described, for example, by Wu et al., J. Biol. Chem, 262: 4429-4432 (1987) and Waggner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990). For an overview of the currently known gene marking and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). See also WO 1993/25673 and references cited therein.

VI . Manufactured goods

In another embodiment of the present invention, an article of manufacture containing materials useful for the treatment of the diseases or disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or in combination with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from various materials such as glass or plastic. The container may contain or contain a composition effective for the treatment of the selected disease or disorder, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper penetrated by a hypodermic needle). Can be). At least one agent in the composition is an antagonist that binds to B-cell surface markers. The label or package insert indicates that the composition is used to treat a patient suffering from or prone to autoimmune diseases such as those listed herein. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable diluent buffer such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. Other materials may be further included that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Further details of the invention are illustrated by the following non-limiting examples. The description of all citations in the specification is expressly incorporated herein by reference.

Example  One

2 H7  term- CD20  mouse Monoclonal  Humanization of Antibodies

Humanization of the mouse anti-human CD20 antibody 2H7 (also referred to herein as m2H7; m refers to mice) was performed in a series of site-directed mutagenesis steps. Chimeric 2H7 with mouse 2H7 antibody variable region sequences and mouse V and human C have already been described; For example, US Pat. Nos. 5,846,818 and 6,204,023. The CDR residue of 2H7 represents the amino acid sequence of the mouse 2H7 variable domain (disclosed in US Pat. No. 5,846,818) of the known antibody (Kabat et al. Sequences of proteins of immunological interest, Ed. 5. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). CDR regions for the light and heavy chains are defined based on sequence hypervariability (Kabat et al., Supra), and are shown in FIGS. 1A and 1B, respectively. Using synthetic oligonucleotides (Table 2), all six mouse 2H7 CDR regions were identified using plasmid pVX4 (FIG. 2) using site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci 82: 488-492 (1985)). 2) was introduced into the complete human Fab framework corresponding to the consensus sequences _Vκ I, V _H III (V _L kappa subgroup I, V _H subgroup III) contained on.

Phagemid pVX4 (FIG. 2) was used for expression of F (ab) in E. coli as well as mutagenesis. Based on phagemid pb0720, a derivative of pB0475 (Cunningham et al. Science 243: 1330-1336 (1989)), pVX4 is a humanized consensus κ-subgroup I light chain (V _L κI-C _L ) and a humanized consensus subgroup III heavy chain ( V _H III-C _H 1) contains a DNA fragment encoding an anti-IFN-α (interferon-α) antibody. pVX4 also has an alkaline phosphatase promoter and Shine-Dalgarno sequences, all of which are another previously described pUC119-based plasmid pAK2 (Carter et al. Proc. Natl. Acad. Sci. USA 89: 4285 (1992). A unique Spel restriction site was introduced between the DNA encoding the F (ab) light and heavy chains. The first 23 amino acids of both the anti-IFN-α heavy and light chains are STII secretory signal sequences (Chang et al. Gene 55: 189-196 (1987)).

To construct a CDR-exchange version of 2H7 (2H7.v2), site-directed mutagenesis was performed on a deoxyuridine-containing template of pVX4; All six CDRs of anti-IFN-α were changed to mouse 2H7 CDRs. The resulting molecule is referred to as humanized 2H7 version 2 (2H7.v2), or “CDR-exchange version” of 2H7; It has an m2H7 CDR residue with the consensus human FR residue shown in FIGS. 1A and 1B. Humanized 2H7.v2 was used for further humanization.

Table 2 shows the oligonucleotide sequences used to generate each of the mouse 2H7 (m2H7) CDRs in the H and L chains. For example, CDR-H1 oligonucleotides were used to regenerate m2H7 H chain CDR1. CDR-H1, CDR-H2 and CDR-H3 refer to H-chain CDR1, CDR2 and CDR3, respectively; Similarly, CDR-L1, CDR-L2 and CDR-L3 refer to the respective L-chain CDRs. Substitution in CDR-H2 was made in two steps by two oligonucleotides CDR-H2A and CDR-H2B.

Oligonucleotide sequences used to construct CDR-exchange of mouse 2H7 CDRs into the human framework in pVX4. The residues changed by each oligonucleotide are underlined.

For comparison with humanized constructs, plasmids expressing chimeric 2H7 Fabs (containing mouse V _L and V _H domains, and human C _L and CH ₁ domains) were synthesized by site-directed mutagenesis (Kunkel, supra). Constructed using nucleotides, mouse framework residues were introduced into 2H7.v2. The sequence of the resulting plasmid construct for expression of chimeric Fab, known as 2H7.v6.8, is shown in FIG. 3. Each coated chain of Fab has a STII secretion signal sequence of 23 amino acids as described for pVX4 (FIG. 2) above.

Conventional humanized antibodies (Carter et al Proc Natl Acad Sci USA 89:..... 4285-4289 (1992)) , and human V _κ I, V _H III consensus framework (Figures 1A and 1B) and the mouse 2H7 framework Based on the sequence comparison of the residues, several framework mutations were introduced by site-directed mutagenesis into the 2H7.v2 Fab construct. As a result of this mutation, at sites that may affect CDR conformation or antigen contact, certain human consensus framework residues have been changed to those found in the mouse 2H7 framework. Version 3 contained V _H (R71V, N73K), version 4 contained V _H (R71V), version 5 contained V _H (R71V, N73K) and V _L (L46P), and version 6 contained V _H (R71V, N73K) and V _L (L46P, L47W).

Humanized and chimeric Fab versions of the m2H7 antibody were expressed and purified in E. coli as follows. Plasmids were transformed into E. coli strain XL-1 Blue (Stratagene, San Diego, Calif.) For the preparation of double- and single-stranded DNA. For each variant, both light and heavy chains were fully sequenced using the dideoxynucleotide method (SEQUENASE®-labeled primer cycle sequencing, US Biochemical Corp.). Plasmids were transformed into E. coli strain 16C9, a derivative of MM294, plated on LB plates containing 5 μg / ml carbenicillin, and single colonies were selected for protein expression. Single colonies were grown at 37 ° C. for 5-8 hours in 5 ml LB-100 μg / ml carbenicillin. 5 ml culture was added to 500 ml AP5-100 μg / ml carbenicillin and grown at 37 ° C. in a 4-L shake flask for 16 hours. AP5 medium contains 1.5 g glucose in 1 L water, 11.0 g HYCASE SF ™ (casein hydrolysate), 0.6 g yeast extract (certified), 0.19 g anhydrous MgSO ₄ , 1.07 g NH ₄ Cl, 3.73 g KCl, 1.2 g NaCl, It was composed of 120 mL 1 M triethanolamine (pH 7.4) and then sterile filtered through a 0.1 μm SEAKLEEN® biocide filter.

Cells were harvested by 3000 × g centrifugation in 1-L centrifuge (Nalgene) and supernatant was removed. After freezing for 1 hour, the pellet was resuspended in 25 ml cold 10 mM MES-10 mM EDTA, pH 5.0 (Buffer A). 250 μl of 0.1 M phenylmethylsulfonyl fluoride (PMSF) (Sigma) was added to inhibit proteolysis, and 3.5 mg of mother liquor 10 mg / ml hen egg white lysozyme (Sigma) was added to aid in lysis of bacterial cell walls. After gentle shaking on ice for 1 hour, the samples were centrifuged at 40,000 × g for 15 minutes. The supernatant was brought to 50 mL with Buffer A and loaded onto a 2-mL DEAE column equilibrated with Buffer A. Penetration was then added to protein G-SEPHAROSE CL-4B ™ Pharmacia chromatography column (0.5-ml bed volume) equilibrated with buffer A. The column was washed with 10 ml buffer A and eluted into 1.25 ml 1 M TRIS (pH 8.0) using 3 ml 0.3 M glycine (pH 3.0). The buffer of F (ab) was then exchanged for phosphate-buffered saline (PBS) using a CENTRICON-30® centrifugal filter instrument (Amicon) and concentrated to a final volume of 0.5 ml. Purity was confirmed by running all F (ab) SDS-PAGE gels, and the molecular weight of each variant was confirmed by electrospray mass spectroscopy.

In cell-based ELISA binding assays (described below), it was difficult to detect the binding of the Fab containing the chimeric 2H7 Fab to CD20. Thus, the 2H7 Fab version was reset to full length IgG1 antibody for analysis and further mutagenesis.

Plasmids for expression of full length IgG were constructed by subcloning the chimeric 2H7 (v6.8) Fab as well as the V _L and V _H domains of humanized Fab versions 2 to 6 into the previously described pRK vectors for mammalian cell expression (Gorman et al. DNA Prot. Eng. Tech. 2: 3-10 (1990)). Briefly, each Fab construct was digested with EcoRV and BlpI to cleave the V _L fragment and cloned into the EcoRV / BlpI site of plasmid pDR1 (FIG. 4) for expression of the complete light chain (V _L -C _L domain). . Additionally, each Fab construct was digested with PvuII and ApaI to cleave the V _H fragment, which was then digested with PvuII of plasmid pDR2 (FIG. 5) for expression of the complete heavy chain (VH-CH ₁ -hinge-CH ₂ -CH ₃ domain). Cloned into / ApaI site. For each IgG variant, the light chain expression plasmid and heavy chain expression plasmid were co-transformed into adenovirus-transformed human embryonic kidney cell line, 293 (Graham et al. J. Gen. Virol. 36: 59-74 (1977)). Transfection was performed by infection. In summary, 293 cells were split the day before transfection and plated in serum-containing medium. The next day, double-stranded DNA prepared as calcium phosphate precipitate, followed by PADVANTAGE ™ DNA (Promega, Madison, Wis.) Was added and the cells were incubated overnight at 37 ° C. Cells were cultured in serum free medium and harvested after 4 days. After purifying the antibody from the culture supernatant using protein A-SEPHAROSE CL-4B ™ agarose chromatography, the buffer is exchanged with 10 mM sodium succinate, 140 mM NaCl (pH 6.0), and the CENTRICON-10® centrifugal filter Centrifugation was carried out using an instrument (Amicon). Protein concentration was determined by quantitative amino acid analysis.

To determine the relative binding affinity for the CD20 antigen, a cell-based ELISA assay was developed. Human B-lymphoblastoid WIL2-S cells (ATCC CRL 8885, American Type Culture Collection, Manassas, VA) were treated with 2 mM L-glutamine, 20 mM HEPES (pH 7.2) in a humidified 5% CO ₂ incubator and Growth was at RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Cells were washed with PBS (assay buffer) containing 1% fetal bovine serum (FBS) and seeded on 96-well round bottom plates (Nunc, Roskilde, Denmark) at 250-300,000 cells / well. Two-fold step diluted standards (15.6-1000 ng / ml 2H7 v6.8 chimeric IgG) and three-step step diluted samples (2.7-2000 ng / ml) in assay buffer were added to the plate. The plate was buried in ice and incubated for 45 minutes. To remove unbound antibody, 0.1 ml assay buffer was added to the wells. The plate was centrifuged and the supernatant removed. Cells were washed twice with 0.2 ml assay buffer. Peroxidase-conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, West Grove, PA) was added to the plate to detect the antibody bound to the plate. After 45 min incubation, cells were washed as described above. TMB substrate (3,3 ', 5,5'-tetramethyl benzidine; Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the plate. 1 M phosphoric acid was added to stop the reaction. Titration curves were fitted with a 4-parameter nonlinear regression curve-fitting program (KALEIDAGRAPH ™, Synergy software, Reading, PA). The absorbance at the midpoint of the titration curve (mid-OD) and its corresponding concentration of standard were determined. The concentration of each variant in this mid-OD was then determined and the concentration of the standard was divided by the concentration of each variant. Thus, this value is the ratio of binding of each variant to the standard. The standard deviation (equivalent concentration) in relative affinity was generally +/− 10% between experiments.

As shown in Table 3, the binding of CDR-exchange variants (v.2) was greatly reduced compared to chimeric 2H7 (v.6.8). However, versions 3 to 6 showed improved binding. To determine the minimum number of mutations required to restore binding affinity to the level of chimeric 2H7, additional mutations and combinations of mutations were constructed by site-directed mutagenesis, resulting in variants 7-17 as indicated in Table 4. Produced. In particular, these include V _H mutations A49G, F67A, I69L, N73K, and L78A; And V _L mutations M4L, M33I, and F71Y. Versions 16 and 17 showed the best relative binding affinity within 2 times the affinity of the chimeric version without significant difference between the two (sd = + / − 10%). To minimize the number of mutations, version 16 (Table 4) with only four mutations of the human framework residues relative to the mouse framework residues was selected as the humanized form for further characterization.

Relative binding affinity of humanized 2H7 IgG variants for CD20 compared to chimeric 2H7 using cell based ELISA. Relative binding is expressed as the concentration of variant required for binding dividing equivalent of the concentration of chimeric 2H7; Thus, a ratio less than 1 indicates a weaker affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%. Framework substitutions in the variable domains follow Kabat's numbering system and are associated with CDR-exchange versions (Kabat et al., Supra).

Oligonucleotide sequences used to construct mutant VH (A49G, R71V, N73K) and VL (L46P) in humanized 2H7 version 16 (2H7.v16). Underlined codons encode the indicated amino acid substitutions. For V _H (R71V, N73K) and V _L (L46P), oligonucleotides are presented as sense strands because they were used for mutagenesis on Fab templates, whereas for V _H (A49G) this is pRK (IgG heavy chain). ) Is presented as an anti-sense strand because it is used with a template. The protein sequence of version 16 is shown in FIGS. 6 and 7.

Example  2

2 H7 Antigen-binding determinants of Paratope )

Alanine substitutions (Cunningham & Wells, Science 244: 1081-1085 (1989)) were made in 2H7.v16 or 2H7.v17 to test the contribution of the individual side chains of the antibody in binding to CD20. IgG variants were expressed from pDR1 and pDR2 vectors in 293 cells, purified and analyzed as described above for relative binding affinity. Several alanine substitutions significantly reduced the relative binding to CD20 on WIL-2S cells (Table 5).

Effect of alanine substitution in the CDR region of humanized 2H7.v16 measured using cell based ELISA (WIL2-S cells). Relative binding is expressed as the concentration of variant required for the concentration division equivalent binding of 2H7.v16 parents; Thus, a ratio below 1 indicates a weaker affinity for the variant and a ratio above 1 indicates a higher affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%. Framework substitutions in the variable domains follow Kabat's numbering system and are related to 2H7.v16 (Kabat et al., Supra). NBD means no detectable binding. Two numbers for version 45 were obtained from separate experiments.

Example  3

2 H7 CDR  Additional mutations in the region

Combinations of substitutions and substitutions of additional residues at CDR positions identified as important by Ala-scanning were also tested. Several combination variants, especially v.96, have been shown to bind more tightly than v.16.

Combination of mutations in the CDR regions of humanized 2H7.v16 and the effect of non-alanine substitutions measured using cell based ELISA (WBL2-S cells). Relative binding to CD20 is expressed as the concentration of variant required for concentration division equivalent binding of 2H7.v16 parents; Thus, a ratio below 1 indicates a weaker affinity for the variant and a ratio above 1 indicates a higher affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%. Framework substitutions in the variable domains follow Kabat's numbering system and are related to 2H7.v16 (Kabat et al., Supra).

Example  4

Framework  Mutations at Humanized Substitution Sites

Substitution of additional residues at changed framework positions during humanization was also tested in the 2H7.v16 background. In particular, alternative framework substitutions not found in mouse 2H7 parental or human consensus frameworks were made in V _L (P46) and V _H (G49, V71, and K73).

In general, such substitutions show little change in relative binding (Table 7), indicating that there is some flexibility in framework residues at these positions.

Relative binding in (WEL2-S) assays based on cells of framework substitution. IgG variants are shown with mutations in the 2H7.v16 background. Relative binding is expressed as the concentration of variant required for binding dividing equivalent of the concentration of 2H7.v6.8 chimera; Thus, a ratio below 1 indicates a weaker affinity for the variant and a ratio above 1 indicates a higher affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%. Framework substitutions in the variable domains follow Kabat's numbering system and are related to 2H7.v16 (Kabat et al., Supra).

(*) Variants analyzed with 2H7.v16 by standard comparator; Relative values are normalized to the chimera's value.

Example  5

Effector  Enhanced Humanization 2 H7 Variant

Since 2H7 can mediate lysis of B cells via both CDC and ADCC, variants of humanized 2H7.v16 with improved CDC and ADCC activity were investigated. Mutations of certain residues within the Fc region of other antibodies for improving CDC through enhanced binding to complement component C1q are described (Idusogie et al. J. Immunol. 166: 2571-2575 (2001)). Mutations for improving ADCC through enhanced IgG binding to activating Fcγ receptors and reduced IgG binding to inhibitory Fcγ receptors are also described (Shields et al. J. Biol. Chem. 276: 6591-6604). (2001); Presta et al. Biochem. Soc. Trans. 30: 487-490 (2002)). Idusogie et al., Supra (2001); As described in Shields et al., Supra, three mutations have been identified, particularly to improve CDC and ADCC activity: S298A / E333A / K334A (also referred to herein as a triple-Ala mutant or variant). Numbering in the Fc region is according to the EU numbering system (Kabat et al., Supra).

To enhance CDC and ADCC activity of 2H7, triple-Ala mutants of 2H7 Fc were constructed. Humanized variants of the anti-HER2 antibody 4D5 with mutant S298A / E333A / K334A were produced and known as 4D5Fc110 (ie, anti-p ¹⁸⁵ HER2 IgG1 (S298A / E333A / K334A); Shields et al., Supra). It is. Plasmid p4D5Fc110 encoding the antibody 4D5Fc110 (Shields et al., Supra) was digested with ApaI and HindIII, and the Fc-fragment (containing mutations S298A / E333A / K334A) was ligated into the ApaI / HindIII site of the 2H7 heavy chain vector pDR2-v16. To produce pDR2-v31. The amino acid sequence of the version 31 full H chain is shown in FIG. 8. L chain is the same as v16.

The constant domain of the Fc region of an IgG1 antibody is relatively conserved in certain species, but allelic variants exist (Lefranc and Lefranc, The Human IgG Subclasses: molecular analysis of structure, function, and regulation, pp. 43-78, F. Shakib (ed.), Pergamon Press, Oxford (1990)).

Effect of Substitution in the Fc Region on CD20 Binding. Relative binding to CD20 was measured in (WIL2-S) assays based on cells of framework substitution. Fc mutations (*) appear as EU numbering (Kabat, supra) and are related to 2H7.v16 parents. The combination of three Ala changes in the Fc region of v.31 is described as "Fc110". IgG variants are shown with mutations in the 2H7.v16 background. Relative binding is expressed as the concentration of variant required for binding dividing equivalent of the concentration of 2H7.v6.8 chimera; Thus, a ratio less than 1 indicates a weaker affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%.

Example  6

Stability-Enhanced Humanization 2 H7 Variant

For development as a therapeutic protein, it is desirable to select variants that continue to be stable with respect to oxidation, deaminoation, and other processes that can affect product quality in appropriate formulation buffers. In 2H7.v16 several residues have been identified as possible sources of instability: VL (M32) and VH (M34, N100). Thus, mutations were introduced at these sites for comparison with v16.

Relative binding of 2H7 variants, designed for enhanced stability and / or enhanced effector function for CD20 in cell-based (WIL2-S) assays. IgG variants are shown with mutations in the 2H7.v16 background. Relative binding is expressed as the concentration of variant required for binding dividing equivalent of the concentration of 2H7.v6.8 chimera; Thus, a ratio less than 1 indicates a weaker affinity for the variant. The standard deviation in the determination of relative affinity was mean +/- 10%. Framework substitutions in the variable domains follow Kabat's numbering system and are associated with 2H7.v16, with FC mutations (*) appearing in EU numbering (Kabat et al., Supra).

(**) variants analyzed with 2H7.v16 by standard comparator; Relative binding values are normalized to the chimera's value.

Based on previously reported mutations, additional Fc mutations were combined with stability- or affinity-enhancing mutations to alter or enhance effector function (Idusogie et al. J. Immunol. 164: 4178-4184 (2000)); Idusogie et al. J. Immunol. 166: 2571-2575 (2001); Shields et al. J. Biol. Chem. 276: 6591-6604 (2001). Such changes include S298, E333A, K334A as described in Example 5; K322A is for reducing CDC activity, D265A is for reducing ADCC activity, K326A or K326W is for enhancing CDC activity, and E356D / M358L is for testing the effect of allogeneic changes in the Fc region. . None of these mutations caused a significant change in CD20 binding affinity.

To test the stability effect of mutations on proteolysis rate, 2H7.v16 and 2H7.v73 were formulated at 12-14 mg / ml in 10 mM histidine, 6% sucrose, 0.02% POLYSORBATE 20 ™ emulsifier (pH 5.8). And incubated at 40 ° C. for 16 days. The incubated samples are then analyzed by ion exchange chromatography, aggregation, and fragmentation by size exclusion chromatography for changes in charge variants, and relative binding is analyzed by cell based (WIL2-S) assay. It was.

The results indicate that 2H7 v.73 is more stable compared to 2H7 v.16 with respect to the reduction in the fraction of the main peak by ion exchange chromatography under accelerated stability conditions. No significant changes were observed with regard to aggregation, fragmentation or binding affinity.

Example  7

WIL2 On -S cells CD20 Of antibody binding to Skachard  analysis

Radiolabeled 2H7 IgG was used to determine the equilibrium dissociation constant (K _d ) for 2H7 IgG variant binding to WIL2-S cells. IgG variants were produced in CHO cells. RITUXAN® (source for all experiments is Genentech (S. San Francisco, CA)) and mouse 2H7 (BD PharMingen, San Diego, CA) were used for comparison with humanized variants. Mouse 2H7 antibodies can be obtained from other sources, such as eBioscience and Calbiochem (both San Diego, CA), Accurate Chemical & Scientific Corp. (Westbury, NY), Ancell (Bayport, MN), and Vinci-Biochem (Vinci, Italy). All dilutions were performed in binding assay buffer (DMEM medium containing 1% bovine serum albumin, 25 mM HEPES, pH 7.2), and 0.01% sodium azide. Aliquots of ¹²⁵ I-2H7.v16 (iodinated with lactoperoxidase) at a concentration of 0.8 nM (0.025 ml) were dispensed into a V-shaped 96-well microassay plate, and a step dilution of cold antibody ( 0.05 mL) was added and mixed. WIL2-S cells (60,000 cells in 0.025 mL) were then added. The plate was sealed and incubated for 24 hours at room temperature and then centrifuged at 3,500 RPM for 15 minutes. The supernatant was then aspirated, the cell pellet washed and centrifuged. The supernatant was aspirated again and the pellet dissolved in 1 N NaOH and transferred to a tube for gamma counting. Data was used for Scatchard analysis (Munson and Rodbard Anal. Biochem. 107: 220-239 (1980)) using the program Ligand (McPherson Comput. Programs Biomed. 17: 107-114 (1983)). The results presented in Table 10 indicate that the humanized 2H7 variants had similar CD20 binding affinity and similar binding affinity for RITUXAN® as compared to mouse 2H7. Based on the combinations shown in Table 8 above, it is expected that 2H7.v31 will have a K _d very similar to v.16.

Equilibrium Binding Affinity of 2H7 Variants from Scatchard Analysis

Example  8

Complement -Dependent cytotoxicity ( CDC ) analysis

In essence, Idusogie et al. J. Immunol. 164: 4178-4184 (2000); Idusogie et al. J. Immunol. 166: 2571-2575 (2001), 2H7 IgG variants were analyzed for their ability to mediate complement-dependent lysis of WIL2-S cells, a CD20-expressing lymphoblastoid B-cell line. Antibodies were diluted 1: 3 steps from 0.1 mg / ml mother liquor. A 0.05 ml aliquot of each dilution was added to a 96-well tissue culture plate containing 0.05 ml of a solution of normal human complement (Quidel, San Diego, Calif.). To this mixture, 50,000 WIL2-S cells were added in a 0.05 ml volume. After incubation at 37 ° C. for 2 hours, 0.05 ml of a solution of ALAMAR BLUE ™ resazurin (Accumed International, Westlake, OH) was added and the incubation was continued for another 18 hours at 37 ° C. The cover was then removed from the plate and shaken for 15 minutes at room temperature on a rotary shaker. The relative fluorescence unit (RFU) was read using a 530-nm excitation filter and a 590-nm emission filter. EC ₅₀ was calculated by fitting RFU as a function of concentration for each antibody using KALEID AGRAPH ™ software.

The results (Table 11) show a remarkable improvement in CDC by humanized 2H7 antibody, with relative efficacy similar to RITUXAN® for v.73, 3 times stronger than RITUXAN® for v.75, v.16 Was about three times weaker than RITUXAN®.

CDC activity of 2H7 antibody compared to RITUXAN®. Numbers greater than 1 indicate CDC activity is less potent than RITUXAN®, and numbers less than 1 indicate more potent activity than RITUXAN®. Antibodies were produced from stable CHO cell lines except those marked with (*).

Example  9

Antibody-dependent cell type cytotoxicity ( ADCC ) analysis

Lactate dehydrogenase (LDH) readings were used essentially to [Shields et al. J. Biol. Chem. 276: 6591-6604 (2001), 2H7 IgG variants were analyzed for their ability to mediate NK-cell lysis of WIL2-S cells, a CD20-expressing lymphoblastoid B-cell line. NK cells were prepared from 100 ml of heparinized blood diluted with 100 ml PBS obtained from normal human donors that are isotype for FcγRIII (also known as CD16) (Koene et al. Blood 90: 1109-1114 (1997) ). In this experiment, NK cells were obtained from human donors that are heterozygous for CD16 (F158 / V158). Diluted blood was layered on 15 ml of lymphocyte-separation medium (ICN Biochemical, Aurora, Ohio) and centrifuged at 2000 RPM for 20 minutes. The leukocytes at the interlayer interface were dispensed into four clean 50-ml tubes, filled with RPMI medium containing 15% fetal bovine serum. The tube was centrifuged at 1400 RPM for 5 minutes and the supernatant was removed. The pellet was resuspended in MACS buffer (0.5% BSA, 2 mM EDTA) and NK cells were purified using beads (NK Cell Isolation Kit, 130-046-502) according to the manufacturer's protocol (Miltenyi Biotech.). NK cells were diluted to 2 × 10 ⁶ cells / ml in MACS buffer.

Antibodies in assay medium (F12 / DMEM 50:50 without glycine, 1 mM HEPES buffer, pH 7.2), penicillin / streptomycin (100 units / ml; Gibco), glutamine, and 1% heat-inactivated fetal bovine serum) 0.05 mL) step dilutions were added to a 96-well round bottom tissue culture plate. WIL2-S cells were diluted to a concentration of 4 × 10 ⁵ cells / ml in assay buffer. WIL2-S cells (0.05 ml / well) were mixed with diluted antibody in 96-well plates and incubated for 30 minutes at room temperature to allow the antibody to bind to CD20 (opsonized).

ADCC reactions were initiated by adding 0.1 ml of NK cells to each well. To the control wells, 2% TRITON® X-100 alkylaryl polyether alcohol was added. Plates were then incubated at 37 ° C. for 4 hours. Levels of released LDH were measured using the cytotoxicity (LDH) detection kit (Kit # 1644793, Roche Diagnostics, Indianapolis, Indiana) according to the manufacturer's instructions. 0.1 ml of LDH developer was added to each well and then mixed for 10 seconds. The plate was then covered with aluminum foil and incubated for 15 minutes at room temperature in the dark. The optical density at 490 nm was then read and used to calculate% dissolution by dividing by the total LDH measured in control wells. Lysis was plotted as a function of antibody concentration and EC ₅₀ concentrations were determined using a 4-parameter curve fit (KALEIDAGRAPH ™ software).

The results showed that the humanized 2H7 antibody was active in ADCC and the relative potency was 20 times higher than RITUXAN® for v.31 and v.75, 5 times stronger for v.16, and for v.73 It was almost four times higher than RITUXAN®.

n ADCC activity of 2H7 antibody against WIL2-S cells compared to 2H7.v16 based on experiment. (Values greater than 1 indicate lower efficacy than 2H7.v16, values less than 1 indicate greater efficacy)

Additional ADCC analysis was performed to compare combinatorial variants of 2H7 with RITUXAN®. The results of this analysis indicated that 2H7.v114 and 2H7.v115 had more than 10-fold improved ADCC efficacy compared to RITUXAN® (Table 13).

n ADCC activity of 2H7 antibody against WIL2-S cells compared to RITUXAN® based on experiments. (Values greater than 1 indicate lower efficacy than RITUXAN®, values less than 1 indicate greater efficacy)

Example  10

Cynomolgus From monkey Pilot  2 in the study H7 Variant In vivo  effect

To assess in vivo activity, 2H7 variants produced by transient transfection of CHO cells were isolated from normal male cynomolgus ( Macaca). fascicularis ) in monkeys. Other anti-CD20 antibodies, such as C2B8 (RITUXAN®), have demonstrated the ability to lack B-cells in normal primates (Reff et al. Blood 83: 435-445 (1994)).

In one study, humanized 2H7 variants were compared. In a parallel study, RITUXAN® was also tested in cynomolgus monkeys. Four monkeys were used in each of the five dose groups: (1) vehicle, (2) 0.05 mg / kg hu2H7.v16, (3) 10 mg / kg hu2H7.v16, (4) 0.05 mg / kg hu2H7 v31, and (5) 10 mg / kg hu2H7.v31. Antibodies were administered intravenously at concentrations of 0, 0.2, or 20 mg / ml, with two doses, one on study day 1 and another on day 8. The first day of dosing was named 1 day and the day before was named -1; The first day of recovery (two animals for each group) was named 11 days. Blood samples were collected at −19, −12, 1 (prior to administration), and 6 hours after the first dose, 24 hours after, and 72 hours after. Additional samples were collected on day 8 (prior to administration), day 10 (prior to sacrifice two animals per group), and on days 36 and 67 (for recovered animals).

Peripheral B-cell concentrations were determined by the FACS method of counting CD3- / CD40 + cells. The percentage of CD3-CD40 + B cells of total lymphocytes in monkey samples was obtained by the following gating strategy. Lymphocyte populations were marked on forward scatter / side scatter plots to define region 1 (R1). Using events at R1, fluorescence intensity dot plots were displayed for CD40 and CD3 markers. Fluorescently labeled isotype controls were used to determine the respective cutoff points for CD40 and CD3 positives.

The results indicated that both 2H7.v16 and 2H7.v31 could cause global peripheral B-cell deficiency at the 10 mg / kg dose, and partial peripheral B-cell deficiency at the 0.05 mg / kg dose. The time course and extent of B-cell deficiency measured during the first 72 hours of administration were similar for both antibodies. Subsequent studies of restorative animals indicated that animals treated with 2H7.v31 showed longer-term B-cell deficiency than animals treated with 2H7.V16. In particular, for recovered animals treated with 10 mg / kg 2H7.v16, B-cells showed substantial B-cell recovery at some time between 10-day and 36-day sampling. However, in recovered animals treated with 10 mg / kg 2H7.v31, B-cells did not show recovery until some time between 36 and 67 days. This suggests a longer overall deficiency period of about 1 month for 2H7.v31 when compared to 2H7.v16.

No toxicity was observed in monkeys at low or high doses and overall pathology was normal. In another study, after two doses of intravenous administration given at two week intervals in these monkeys v16 was well tolerated to the highest dose evaluated (100 mg / kg × 2 = 1200 mg / m 2 × 2).

Data in cynomolgus monkeys with 2H7.v16 vs. RITUXAN® show that a 5-fold reduction in CDC activity does not adversely affect efficacy. Antibodies with potent ADCC activity but reduced CDC activity may have a more desirable stability profile with respect to the initial infusion response than with greater CDC activity.

Example  11

Effector  Augmented Fucos Deficit 2 H7 Variant  Antibodies

Normal CHO and HEK293 cells add fucose to IgG oligosaccharides to a high degree (97-98%). IgG from serum is also highly fucosylated.

Foucault misfire water soluble (competent) dihydro folate-reductase-minus (DHFR ^-) CHO cell line, using the DP12, and the Lec13 protein Foucault misfire-deficient cell lines such research was to produce antibodies. The CHO cell line Pro-Lec13.6a (Lec13) was obtained from Professor Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University. Parental cell lines are Pro- (proline nutrient composition) and Gat- (glycine, adenosine, thymidine nutrient composition). The CHO-DP12 cell line is a derivative of the CHO-K1 cell line (ATCC # CCL-61), which is dehydrofolate reductase deficient, and the demand for insulin is reduced. Cell lines were transfected with cDNA using the SUPERFECT ™ transfection reagent method (Qiagen, Valencia, CA). Selection of Lec13 cells expressing the transfected antibody was performed with L-glutamine, ribonucleoside and deoxyribonuine supplemented with 10% inactive FBS (Gibco), 10 mM HEPES, and 1 × penicillin / streptomycin (Gibco). 10 μg / ml of furomycin dihydrochloride (Calbiochem, San Diego, Calif.) Was performed in growth medium containing MEM alpha medium with cleoside (GIBCO-BRL, Gaithersburg, MD). Low glucose without glycine, with NaHCO3 supplemented with 5% FBS (Gibco), 10 mM HEPES, 2 mM L-glutamine, 1 × GHT (glycine, hypoxanthine, thymidine), and 1 × penicillin / streptomycin DMEM: CHO cells were similarly selected in growth medium containing Ham's F12 without GHT.

Colonies formed within two to three weeks and were pooled for expansion and protein expression. The cell pools were initially seeded at 3 × 10 ⁶ cells per 10 cm of plate for small batch protein expression. Once grown to 90-95% densities, cells were converted to serum-free medium, 3-5 days later cell supernatants were collected and tested in Fc IgG- and intact IgG-ELISA to assess protein expression levels. One day before conversion to PS24 production medium supplemented with 10 mg / L recombinant human insulin and 1 mg / L trace elements, Lec13 and CHO cells were seeded at about 8 × 10 ⁶ cells per 15 cm of plate.

Lec13 cells and DP12 cells were maintained for 3-5 days in serum-free production medium. Supernatants were collected and clarified by centrifugation in 150-ml centrifuge tubes to remove cells and debris. Add a protease inhibitor PMSE and aprotinin (Sigma, St. Louis, MO) and use a MWCO30 ™ filter (Amicon, Beverly, MA) before immediate purification using protein G chromatography (Amersham Pharmacia Biotech, Piscataway, NJ) The supernatant was concentrated 5 times on the stirred cells. All proteins were buffer exchanged into PBS using a CENTRIPREP-SO ™ concentrator (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentration was determined using A280 absorbance values and verified using amino acid composition analysis.

CHO cells were transfected and selected with vectors expressing humanized 2H7v16, 2H7v.31 as described above. The 2H7v.16 antibody retained the wild type Fc region, whereas v.31 (see Example 5, Table 8) had an Fc region consisting of three amino acid changes (S298A, E333A, K334A), resulting in an FcγRIIIa receptor. Higher affinity (Shields et al. J. Biol. Chem. 276 (9): 6591-6604 (2001)). After transfection and selection, individual colonies of cells were isolated, evaluated for protein expression levels, and the highest producers were selected by methotrexate to select cells that produced higher levels of antibody with increased plasmid copy number. Cells were grown and transferred to serum-free medium for a period of 7 days, then the medium was collected and loaded onto a Protein A column and antibodies were eluted using standard techniques. Final concentrations of antibodies were determined using ELISA to measure intact antibodies. All proteins were buffer exchanged into PBS using a CENTRIPREP-30 ™ concentrator (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis.

Matrix-assisted laser desorption / ionization time of asparagine-linked oligosaccharides (MALDI-TOF: Matrix - Assisted Laser Desorption Of Ionization Time - of - flight A) mass spectral analysis

N-linked oligosaccharides are described in Papac et al. Glycobiology 8: 445-454 (1998)] was used to release from recombinant glycoproteins. In summary, the wells of a 96-well polyvinylidene difluoride (PVDF) -line microtiter plate (Millipore, Bedford, Mass.) Were drawn on a PVDF membrane by applying a vacuum to a Millipore MULTISCREEN ™ vacuum manifold. Conditioned with μl methanol. Conditioned PVDF membranes were washed with 3 × 250 μl water. Between all cleaning steps, the wells were drained completely by applying an appropriate vacuum to the manifold. The membrane was washed with reduction and carboxymethylation buffer (RCM) consisting of 6 M guanidine hydrochloride, 360 mM TRIS, 2 mM EDTA, pH 8.6. Glycoprotein samples (50 μg) were added to individual wells and again drawn to the PVDF membrane by appropriate vacuum and the wells washed with 2 × 50 μl RCM buffer. The fixed sample was reduced by adding 50 μl of 0.1 M dithiothreitol (DTT) solution to each well and incubating the microtiter plate at 37 ° C. for 1 hour. DTT was removed by vacuum and the wells were washed with 4 × 250 μl water.

Cysteine residues were carboxymethylated by adding 50 μl of a 0.1 M iodoacetic acid (IAA) solution freshly prepared in 1 M NaOH and diluted to 0.1 M with RCM buffer. Carboxymethylation was achieved by incubation at room temperature in the dark for 30 minutes. The plate was vacuumed to remove the IAA solution and the wells washed with 4 × 250 μl purified water. PVDF membranes were blocked by adding 100 μl of 1% PVP-360 (polyvinylpyrrolidone 360,000 MW) (Sigma) solution and incubating at room temperature for 1 hour. The PVP-360 solution was removed by appropriate vacuum and the wells were washed with 4 x 250 μl water. 25 μl of PNGASE F ™ amidase (New England Biolabs, Beverly, MA) digestion solution per ml solution in 10 mM TRIS acetate, pH 8.4 was added to each well and digestion proceeded at 37 ° C. for 3 hours. . After digestion, the samples were transferred to 500 μl Eppendorf tubes and 2.5 μl of 1.5 M acetic acid solution was added to each sample. The acidified sample was incubated at room temperature for 3 hours to convert oligosaccharides from glycosylamine to hydroxyl form. Prior to MALDI-TOF mass spectral analysis, the released oligosaccharides are slurry-packed cation-exchange resins (hydrogen form AG50W-X8 ™ resin) in a compact-reaction tube (US Biochemical, Cleveland, OH) (Bio-Rad, Hercules, CA Desalinated using a 0.7-ml layer).

For MALDI-TOF mass spectral analysis of the sample in positive mode, desalted oligosaccharides (0.5 μl aliquots) were added with 2 mg of 2,5 dihydroxybenzoic acid and 0.1 ml of 5-methoxysalicylic acid with 1 ml of ethanol. 0.5 μl of 2,5 dihydroxybenzoic acid matrix (sDHB) prepared by dissolving in 10 mM sodium chloride 1: 1 (v / v) was added to the stainless target. The sample / matrix mixture was dried by vacuum. For analysis in the negative mode, desalted N-linked oligosaccharides (0.5 μL aliquots) were prepared in 2 ', 4', 6'- prepared in 1: 3 (v / v) acetonitrile / 13.3 mM ammonium citrate buffer. 0.5 μl of trihydroxyacetophenone matrix (THAP) was added to the stainless target. The sample / matrix mixture was vacuum dried and then allowed to absorb atmospheric moisture prior to analysis. The released oligosaccharides were analyzed on a PERSEPTIVE BIOSYSTEMS ™ VOYAGER-DE ™ mass spectrometer by MALDI-TOF. The mass spectrometer was operated at 20 kV in positive or negative mode using delayed extraction in a linear configuration. Data was acquired in a data accumulation mode (240 scans) using a laser power of 1300 to improve the signal-to-noise ratio. The device was calibrated with a mixture of standard oligosaccharides and data was leveled using a 19-point Savitsky-Golay algorithm before assigning mass. Integration of mass spectral data was achieved using the CAESAR 7.0 ™ data analysis software package (SciBridge Software).

NK  cell ADCC

ADCC analysis was performed as described in Example 9. The NK-to-target cell (WIL2-S) ratio was 4 to 1, the assay was run for 4 hours, and toxicity was measured using the lactose dehydrogenase assay as before. Target cells were opsonized at the concentration of antibody indicated for 30 minutes prior to addition of NK cells. The RITUXAN® antibodies used were obtained from Genentech (S. San Francisco, CA).

The results indicate that underfucosylated antibodies mediate NK-cell target cell death more efficiently than antibodies fully supplemented with fucose. The less fucosylated antibody 2H7v.31 is most efficient for target cell killing. Such antibodies are effective at lower concentrations and can mediate a greater percentage of target cells at higher concentrations than other antibodies. The activity of the antibodies is as follows: Lec13-derived 2H7v31> Lec13-derived 2H7v16> Dp12-derived 2H7v31> Dp12-derived 2H7v16> RITUXAN®. Protein and carbohydrate alterations are additive. Comparison of carbohydrates found on native IgG from Lec13-producing IgG and CHO-producing IgG showed no detectable difference in the degree of galactosylation, so the result is only due to the presence / absence of fucose Can be.

Example  12

Cynomolgus  monkey CD20 of Cloning  And antibody binding

Between nomol between the isolation of a cDNA encoding the CD20 of splenic that nomol Gus Gus cDNA library from monkeys (Macaca fascicularis ) CD20 DNA sequence was determined. Libraries were constructed using slightly modified [SUPERSCRIPT ™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat # 18248-013, Invitrogen, Carlsbad, Calif.). The cDNA library was ligated into the pRK5E vector using restriction sites XhoI and NotI. mRNA was isolated from spleen tissue (California Regional Research Primate Center, Davis, CA). Primers for amplifying cDNA encoding CD20 were designed based on the non-coding sequence of human CD20. Cynomolgus monkey CD20 by polymerase chain reaction (PCR) using N-terminal region primer 5'-AGTTTTGAGAGCAAAATG-3 '(SEQ ID NO: 41) and C-terminal region primer 5'-AAGCTATGAACACTAATG-3' (SEQ ID NO: 42) The cDNA coding for was cloned. PCR reactions were performed using the PLATINUM TAQ DNA POLYMERASE HIGH FIDELITY ™ system according to the manufacturer's instructions (Gibco, Rockville, MD). PCR products were subcloned into the PCR®2.1-TOPO® vector (Invitrogen) and transformed into XL-1 Blue Escherichia coli (Stratagene. La Jolla, Calif.). Plasmid DNA containing the ligated PCR product was isolated from individual clones and sequenced.

The amino acid sequence for cynomolgus monkey CD20 is shown in FIG. 19. 20 shows a comparison of cynomolgus with human CD20. Cynomolgus monkey CD20 was only 8 different, 97.3% similar to human CD20. The extracellular domain contains one change in V157A and the remaining seven residues can be found in the cytoplasmic or transmembrane region.

Antibodies directed against human CD20 were analyzed for the ability to bind and substitute FITC-conjugated mouse 2H7 to bind cynomolgus monkey cells expressing CD20. 20 ml blood was drawn into sodium heparin from two Cynomolgus monkeys (California Regional Research Primate Center, Davis, Calif.) And delivered directly to Genentech, Inc. On the same day, blood samples were pooled and diluted 1: 1 by the addition of 40 ml PBS. 20 ml of diluted blood is layered on 4 × 20 ml of FICOLL-PAQUE ™ PLUS (Amersham Biosciences, Uppsala, Sweden) in a 50 ml centrifuge tube (Cat # 352098, Falcon, Franklin Lakes, NJ) and 1300 rpm Were centrifuged in a SOR VAL ™ 7 centrifuge (Dupont, Newtown, CT) for 30 minutes at room temperature. The PBMC layer was isolated and washed with PBS. Erythrocyte cells were lysed in 0.2% NaCl solution, restored to isotonic with an equivalent volume of 1.6% NaCl solution and centrifuged at 1000 RPM for 10 minutes. PBMC pellets were resuspended in RPMI 1640 (Gibco, Rockville, MD) containing 5% FBS and dispensed at 37 ° C. for 1 hour into 10-cm tissue culture dishes. Non-adherent B- and T-cell populations were removed by aspiration, centrifuged and counted. A total of 2.4 × 10 ⁷ cells were recovered. Resuspended PBMCs were distributed into 20 12 × 75-mm culture tubes (Cat # 352053, Falcon), each containing 1 × 10 ⁶ cells in 0.25 ml volume. The culture tubes were divided into four sets of five culture tubes. To each set was added medium (RPMI1640, 5% FBS), titrated amount of control human IgG ₁ antibody, RITUXAN®, 2H7.v16, or 2H7.v31. Final concentrations of each antibody were 30, 10, 3.3 and 1.1 nM. Each culture tube was also provided with 20 μl of fluorosane isothiocyanate (FITC) -conjugated anti-human CD20 (Cat # 555622, BD Biosciences, San Diego, CA). The cells were gently mixed and incubated on ice for 1 hour before being washed twice with cold PBS. Cell surface staining was analyzed on an EPIC XL-MCL ™ flow cytometer (Coulter, Miami, FL) and geometric mean was derived and plotted against antibody concentration (KALEIDAGRAPH ™, Synergy Software, Reading, PA).

The data indicated that 2H7 v.16 and 2H7 v.31 competitively substituted FITC-mouse 2H7 binding to cynomolgus monkey cells. In addition, RITUXAN® also substituted FITC-mouse 2H7 binding, indicating that both 2H7 and RITUXAN® bind to overlapping epitopes on CD20. The data also show that IC ₅₀ values for 2H7v.16, 2H7v.31 and RITUXAN® are similar and fall in the 4-6 nM range.

Example  13

Medium  To serious Rheumatism  In arthritis rhuMAb  2 H7  (2 H7 . v16 Phase I / II Studies

Protocol Overview

Randomized, placebo-controlled, multicenter, blind phase I / II phase safety of stepwise elevated doses of PRO70769 (rhuMAb 2H7) in moderate to severe rheumatoid arthritis patients receiving stable doses of incidental methotrexate (MTX) Research

goal

The primary goal of this study is to evaluate the safety and tolerability of a progressively elevated intravenous (IV) dose of PRO70769 (rhuMAb 2H7) in patients with moderate to severe rheumatoid arthritis (RA).

Research design

This study is a blind, randomized, placebo-controlled, multicenter, blind phase I / II study for both the investigator and patient for the safety of the stepped escalation dose of PRO70769 in combination with MTX in moderate to severe RA patients. . The study consists of a dose-phase escalation phase and a second phase in which a greater number of patients are enrolled. The sponsor will not continue to be involved in the treatment task.

Moderate to severe RA patients with one to five disease-modified anti-rheumatic drugs or biologics that are currently unsatisfactory in clinical response to treatment with MTX will be enrolled.

Patients will be required to receive MTX for at least 12 weeks in the range of 10-25 mg weekly prior to initiation of the study, and to remain stable for at least 4 weeks prior to receiving an initial dose of study drug (PRO70769 or placebo). Patients may also be given stable doses of oral corticosteroids (up to 10 mg daily, or equivalent prednisone) and stable doses of non-steroidal anti-inflammatory drugs (NSAIDs). Patients will receive two intravenous infusions of the indicated doses of PRO70769 or placebo equivalents on Days 1 and 15, in accordance with the following dose-step escalation scheme.

Dose escalation will occur after evaluation of acute toxicity according to certain criteria and after review of safety data by the Internal Stability Data Review Committee and 72 hours after the second infusion in the last patient treated in each cohort. After the dose-stage escalation phase, 40 additional patients (32 active and 8 placebo) will be randomly selected for each of the following dose levels: 2 × 50 mg, 2 × 200 mg, 2 × 500 mg, and 2 1000 mg (if the dose level proved to be acceptable in the dose-gradation stage). Approximately 205 patients will be enrolled in the study.

B-cell counts will be obtained and recorded. B-cell counts will be assessed using flow cytometry within the 48-week follow-up period after the 6-month efficacy assessment. B-cell deficiency will not be considered dose-limiting toxicity (DLC), but rather the expected pharmacokinetic outcome of PRO70769 treatment.

In any substudy, blood for serum and RNA analysis as well as urine samples will be obtained from the patient at various time points. These samples can be used to identify biomarkers that may be a precursor to the response to PRO70769 treatment in moderate to severe RA patients.

Measuring results

 The primary outcome measure for this study is the safety and tolerability of PRO70769 in patients with moderate to severe RA.

Study treatment

The patient's cohort will be injected intravenously with either PRO70769 or placebo equivalents at the indicated doses on Days 1 and 15, according to the following dose-step escalation scheme:

10 mg PRO70769 or placebo equivalent: 4 active drugs, 1 control

50 mg PRO70769 or placebo equivalent: 8 active drugs, 2 controls

200 mg PRO70769 or placebo equivalent: 8 active drugs, 2 controls

500 mg PRO70769 or placebo equivalent: 8 active drugs, 2 controls

1000 mg PRO70769 or placebo equivalent: 8 active drugs, 2 controls

efficacy

The efficacy of PRO70769 will be measured by ACR response. The percentage of patients who achieved ACR20, ACR50, and ACR70 responses will be summarized by treatment group, and a 95% confidence interval will be generated for each group. The components of these responses and their changes from baseline will be summarized by treatment and visit.

Example  1-13 results

The data indicated success in producing humanized CD20 binding antibodies, particularly humanized 2H7 antibody variants, with retained biological properties and even enhanced. The humanized 2H7 antibodies of the present invention bound to CD20 with similar affinities as mouse donors and chimeric 2H7 antibodies and were effective for B-cell killing in primates, leading to B-cell deficiency. Certain variants exhibit enhanced ADCC compared to chimeric anti-CD20 antibodies currently used to treat non-Hodgkin's lymphoma (NHL), allowing lower doses of therapeutic antibodies to be used in patients. In addition, chimeric antibodies with mouse FR residues may need to be administered at an effective dose to achieve complete B-cell deficiency to prevent antibody responses thereto, but the humanized antibodies of the present invention may be directed to certain diseases and patients. For several time periods as required for the administration, the dosage can be administered to achieve partial or complete B-cell deficiency. In addition, these antibodies showed stability in solution. This property of humanized 2H7 antibodies makes them ideal for use as immunotherapeutic agents in the treatment of CD20-positive autoimmune diseases, which antibodies are not expected to be immunogenic in human patients, or are completely mouse-like or chimeric anti-CD20 antibodies. At least less immunogenic.

Example  14

Preparation of Additional Humanized Antibodies

The antibody 2H7.v31 comprising the light chain and heavy chain amino acid sequences of SEQ ID NO: 24 and 28, respectively, such as 2H7.v31 having an amino acid substitution of S298A / E333A / K334A, more preferably having a heavy chain amino acid sequence of SEQ ID NO: 28 is a Fc It may further comprise one or more amino acid substitutions in the region that improve ADCC and / or CDC activity. The antibody may be 2H7.v138 comprising the light- and heavy chain amino acid sequences of SEQ ID NOs: 29 and 30 shown in FIGS. 10 and 11, respectively, and FIGS. 10 and 11 show the corresponding light and heavy chain amino acid sequences of 2H7.v16 and 2H8. alignment of the light and heavy chain amino acid sequences of v138. Alternatively, such a preferred intact humanized 2H7 antibody is 2H7.v477, which has the light and heavy chain sequences of 2H7.v138 except for the amino acid substitution of N434W. Any such antibodies may further comprise one or more amino acid substitutions in the Fc region that reduce CDC activity, eg, include one or more substitutions K322A. See US Pat. No. 6,528,624B1 to Idusogie et al.

Some preferred humanized 2H7 variants include those having the variable light chain domain of SEQ ID NO: 2 and the variable heavy chain domain of SEQ ID NO: 8, ie those having or without substitutions in the Fc region, and variable heavy chain domains and sequences having altered N100A or D56A and N100A in SEQ ID NO: 8 Alterations in 2 M32L, or S92A, or those with the variable light domain with M32L and S92A, ie those with or without substitution in the Fc region. If substitutions are made in the Fc region, they are preferably one of those listed in the table below.

In a summary of some various preferred embodiments of the invention, the V region of the variant based on 2H7 version 16 will have the amino acid sequence of v16 except for the position of the amino acid substitutions set forth in the table below. Unless otherwise indicated, 2H7 variants will have the same L chain as v16.

In addition to these variants, intact humanized 2H7 antibodies have the following light chain amino acid sequences:

And the heavy chain amino acid sequence of:

It may be a version 138 including.

In another embodiment, a humanized 2H7 antibody may contain the light chain variable region (V _L ) sequence of SEQ ID NO: 43 and heavy chain variable region (V _H ) sequence of SEQ ID NO: 8, wherein the antibody comprises an amino acid substitution of D56A in VH-CDR2 Further contains, wherein N100 in VH-CDR3 is substituted with Y or W, wherein SEQ ID NO: 43 has the following sequence:

In one embodiment of this last humanized 2H7 antibody, N100 is substituted with Y. In another embodiment, N100 is substituted with W. In addition, in a further embodiment, the antibody comprises a substitution S10OOR in VH-CDR3, preferably ADCC and / or CDC activity, such as comprising an IgG1 Fc comprising amino acid substitutions S298A, E333A, K334A, K326A. One or more amino acid substitutions to improve are further included within the Fc region. Alternatively, the antibody comprises a substitution S10OOR in VH-CDR3, preferably ameliorates ADCC, but at least includes amino acid substitution K322A, as well as further comprising amino acid substitutions S298A, E333A, K334A. The activity further comprises one or more amino acid substitutions in the Fc region that reduce.

In one particularly preferred embodiment, the antibody is version 511 and has the following 2H7.v511 light chain sequence:

And the following 2H7.v511 heavy chain sequences:

It includes.

Example  15

In polychondritis Rituximab  Clinical research

Patients diagnosed with polychondritis were treated with RITUXAN® antibody. The treated patient will not have a B-cell malignancy.

RITUXAN® is administered intravenously (IV) to a patient according to any of the following dosing schedules:

(A) 50 mg / m 2 IV per day

150 mg / m 2 IV on days 8, 15 & 22

(B) 150 mg / m 2 IV per day

375 mg / m 2 IV on days 8, 15 & 22

(C) 375 mg / m 2 IV at 1, 8, 15 & 22 days

Additional adjuvant therapy (such as immunosuppressive agents as mentioned above) can be combined with RITUXAN® therapy, but preferably the patient is treated with RITUXAN® as a single agent throughout the course of therapy.

The overall response rate is determined based on the reduction in inflammation of the cartilage tissue determined by standard chemical parameters. Administration of RITUXAN® will ameliorate any one or more symptoms of polychondritis in a patient as described above.

Example  16

Multiple In mononeuritis Rituximab  Clinical research

Patients clinically diagnosed with multiple mononeuritis as defined herein were treated with Rituximab (RITUXAN®) antibody, optionally in combination with steroid therapy. The treated patient will not have a B-cell malignancy. Detailed and complete history is very important in determining the possible root cause of the disorder. Pain often begins at the lower back or hips and spreads to the thighs and knees on one side. Pain in general is characterized by deep, tingling pain in addition to the most severe jab at night. In people with diabetes, unilateral severe thigh pain typically begins acutely, followed by a rapid collapse of the anterior thigh muscles and lack of atrophy and knee reflexes. Other possible symptoms that may be reported by the patient include: numbness, numbness, abnormal sensations, burning sensations-abnormalities, difficulty moving body parts-paralysis, lack of control of body parts. Sensory and motor loss can be associated with dysfunction of certain nerves. The inspection shows the preservation of reflections and good intensity except in the more deeply affected areas. Some common findings of multiple mononeuritis may include the following (not listed in order of frequency): sciatic nerve dysfunction, thigh nerve dysfunction, calf neural dysfunction, axillary nerve dysfunction, radial Nerve dysfunction, median nerve dysfunction, ulnar nerve dysfunction, and autonomic nervous system, ie dysfunction of parts of the nervous system that control unconscious body functions such as the secretory organ and the heart.

Positive response to therapy plans improvement in two of the four parameters listed below that account for its changes, and is also based on existing treatment studies in diabetic neuropathy (Jaradeh et al. Journal of Neurology , Neurosurgery and Psychiatry 67: 607-612 (1999)). Patients should have measurable neuropathy as defined by electrophysiological tests. Known diabetic or hereditary neuropathy patients are excluded.

Patients must have suitable organ function as measured by the following criteria (values must be measured within 2 months prior to enrollment): Liver: AST <3 x upper limit of laboratory standard and bilirubin <2.0 mg / dL. Kidney: creatinine <3.0 mg / dL.

Rituximab will be administered intravenously in an outpatient setting. No in-line filter is needed. The initial rate is 50 mg / hr for the first hour. If no toxicity is observed, the rate is raised in steps of 50 mg / hour increments at 30 minute intervals up to 400 mg / hr. If the initial dose is well tolerated, the initial rate for subsequent dosing is 100 mg / hour and is incrementally increased by 100 mg / hour increments at 30 minute intervals so as not to exceed 400 mg / hour. If the patient experiences fever and chills, the antibody infusion is discontinued. The severity of the side effects should be assessed. If symptoms improve, the infusion is continued initially at half the previous rate. After antibody injection, intravenous lines should be maintained for dosing if necessary. If there is no complication after 1 hour of observation, intravenous can be discontinued.

All patients enrolled in this study will receive rituximab weekly for four consecutive weeks. The dose is based on the actual surface area. The dosing schedule is an intravenous infusion of 375 mg / m 2 rituximab four times per week on days 1, 8, 15 and 22. To reduce adverse events 30-60 minutes prior to treatment, all patients should be pre-administered intravenously or orally with 650 mg of TYLENOL® Pain Relief and 50 mg of BENADRYL® Allergy. Agents for the treatment of hypersensitivity reactions such as epinephrine, antihistamines and corticosteroids should be available for immediate use in case of hypersensitivity reactions during administration. In addition, anti-pain agents such as acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin (NEURONTIN®), (E) -N-vanylyl -8-methyl-6-nonaneamide (CAPSAICIN®), or nerve blockers, can be used with rituximab.

Neuropathy will be assessed by several different parameters: 1) EMG / NCS, 2) Quantitative Sensory Test, 3) Neuropathy Injury Score, 4) Neuropathy Symptoms and Change Questionnaire.

EMG / NCS: EMG and nerve conduction velocity measurements will be performed by the same EMG and technician 3 months, 6 months and 12 months after rituximab injection. Summary data from each study, including mean sensory nerve action potentials (calf, median and ulna), mean compound motor neuron action potentials (calf, shin, ulnar and decoy in anterior shin), and mean conduction velocity of motor nerves Will be used for comparison with the initial value. The mean F-wave latency and the proportion of motion amplitude from proximal to distal will also be calculated. Targeted reactions will require at least 10% improvement from baseline. Stable disease will not indicate a significant change in neuropathy (+/− ≧ 10). Ongoing disease will indicate exacerbation of neuropathy (> 10% from baseline).

Quantitative sensory test: Quantitative sensations with variation in detection threshold (VDT), cooling detection threshold (CDT), and thermal pain detection threshold (HPT) on the instep and back of the hand, in addition to the toe and fingertips sweat secretion axon reflex test (QSART) The test will be performed by the same technician after 3 months, 6 months and 12 months after rituximab injection. Abnormality in such a test can be transformed into points based on percentile scores associated with standard deviations. Changes in percentile 2 from preliminary study measurements will be considered significant.

Neuropathy Injury Score (NIS): This test measures reflexes, sensory and muscle strength. Functional assessments of the lower extremities are made with toes, walking on the heel, and kneeling. Scoring will be performed 3 months, 6 months and 12 months after rituximab injection by the same neurologist throughout the study. Improvement will be defined as a reduction of at least 5 points in NIS (Dyck "Quantitating severity of neuroopathy" In: Dyck et al. Eds. Peripheral Neurophaty. Philadelphia: WB Saunders, 686-697 (1993)).

Neuropathy Symptoms and Change Questionnaire (NSC): This questionnaire consists of 38 items that are questioned in an authentic manner. It assesses the presence or absence of neuropathy symptoms, their severity and changes over time. This will be done by the same neurologist for each patient throughout the study. A 10% change from baseline will be considered significant.

The primary outcome measure of the study is patient improvement. Patients are classified as improved if they show a significant improvement in two of the four parameters listed above without decay in other measurements. Based on this response classification, an accurate 95% confidence interval is computerized for the response rate based on the binomial calculation. For 14 patients, the width of this interval will be less than about 50% when the intrinsic response rate is between 30-70% and less than about 40% when the rate is between 70-90% or 10-30% and the rate is> 90. % Or <10% will be less than about 30%.

Point estimates and 95% confidence intervals will be computerized for the proportion of patients whose results for each parameter are successful using the correct binomial interval. For each successive or conventional measurement, the exact 95% confidence interval will be computerized by the Hodges-Lehmann statistics and the Tukey interval for changes from baseline (Hollender and Wolfe). Nonparametric Statistical Methods 2nd Edition, Wiley, New York, 1999 p51-56). The calculation will be made using the STATEXACT ™ (Cytel) statistical software package.

The neuropathy damage score test will provide a single score of neuropathy deficiency and a subset score related to cranial nerve function, muscle weakness, reflexes and sensations. Deficiency will be scored by the investigator when compared to age and sex-related patients taking into account height, weight and physical health. Muscle breakdown is normal at 0, 25% breakdown 1, 50% breakdown 2, 75% breakdown 3, muscle movement against gravity 3.25, gravity is removed 3.5, movement is removed It will be scored 3.75 if there is no flicker and 4 if there is a general paralysis. It can be applied to cranial nerves III, VI, VII, X and XII. Individual muscle groups tested for strength include breathing, neck bending, shoulder abduction, elbow bending, upper arm radius, elbow straightening, wrist bending and straightening, finger bending and spreading, thumb abduction, hip flexion and straightening, knee for a total of 24 items Bending and straightening, ankle bend, ankle foot flexion, toe extensor and flexors. Each group will be tested on the right and left.

Reflexes will be scored as 0 = normal, 1 = reduced, 2 = absent. Fiber-tendon reflexes will be tested on each side including biceps, triceps, brachial radius, quadriceps, and lower triceps. For patients 60 years of age or older, ankle reflex reduction will be rated 0 and its absence will be graded 1.

Sensory tests will be performed on the dorsal side of the fingers and the big toe. The contact pressure will be measured by using a long cotton wool. A straight pin will be used to assess the feeling of stabbing. Vibration sensations will be tested with a 165 Hz tuning fork and joint position will be tested by moving the last phalanges of the index finger and big toe. Tests will be made on each limb and scoring will be 0 = normal, 1 = decrease and 2 = absent.

Rituximab or humanized 2H7 will show patient improvement as defined above compared to the control (no such antibody) and is therefore expected to treat multiple mononeuritis.

                            <110> BRUNETTA, PAUL G       SEWELL, KATHRYN L. <120> Treatment of Disorders <130> P2102R1 <140> US 11 / 106,820 <141> 2005-04-15 <150> US 60 / 563,227 <151> 2004-04-16 <150> US 60 / 565,098 <151> 2004-04-22 <160> 45 <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> Homo sapiens <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 Thr 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> Homo sapiens <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 Tyr 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> 5679 <212> DNA <213> Artificial Sequence <220> <223> sequence is synthesized <400> 13  gaattcaact tctccatact ttggataagg aaatacagac atgaaaaatc 50  tcattgctga gttgttattt aagcttgccc aaaaagaaga agagtcgaat 100  gaactgtgtg cgcaggtaga agctttggag attatcgtca ctgcaatgct 150  tcgcaatatg gcgcaaaatg accaacagcg gttgattgat caggtagagg 200  gggcgctgta cgaggtaaag cccgatgcca gcattcctga cgacgatacg 250  gagctgctgc gcgattacgt aaagaagtta ttgaagcatc ctcgtcagta 300  aaaagttaat cttttcaaca gctgtcataa agttgtcacg gccgagactt 350  atagtcgctt tgtttttatt ttttaatgta tttgtaacta gaattcgagc 400  tcggtacccg gggatcctct agaggttgag gtgattttat gaaaaagaat 450  atcgcatttc ttcttgcatc tatgttcgtt ttttctattg ctacaaacgc 500  gtacgctgat atccagatga cccagtcccc gagctccctg tccgcctctg 550  tgggcgatag ggtcaccatc acctgcagag ccagtcagag cgtgtcgact 600  agctcttata gctatatgca ctggtatcaa cagaaaccag gaaaagctcc 650  gaaactactg atttactatg ctagcaacct cgagtctgga gtcccttctc 700  gcttctctgg atccggttct gggacggatt tcactctgac catcagcagt 750  ctgcagccag aagacttcgc aacttattac tgtcaacact cttggggtat 800  tccgcgcaca tttggacagg gtaccaaggt ggagatcaaa cgaactgtgg 850  ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 900  ggaactgctt ctgttgtgtg cctgctgaat aacttctatc ccagagaggc 950  caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg 1000  agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 1050  accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg 1100  cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag agcttcaaca 1150  ggggagagtg ttaagctgat cctctacgcc ggacgcatcg tggccctagt 1200  acgcaagttc acgtaaaaag ggtatctaga ggttgaggtg attttatgaa 1250  aaagaatatc gcatttcttc ttgcatctat gttcgttttt tctattgcta 1300  caaacgcgta cgctgaggtt cagctggtgg agtctggcgg tggcctggtg 1350  cagccagggg gctcactccg tttgtcctgt gcagcttctg gctacacctt 1400  caccgaatat atcatccact gggtccgtca ggccccgggt aagggcctgg 1450  aatgggttgc atcgattaat cctgactacg acatcacgaa ctataaccag 1500  cgcttcaagg gccgtttcac tataagtcgc gacgattcca aaaacacatt 1550  atacctgcag atgaacagcc tgcgtgctga ggacactgcc gtctattatt 1600  gtgctcgatg gatcagcgat ttcttcgact actggggtca aggaaccctg 1650  gtcaccgtct cctcggcctc caccaagggc ccatcggtct tccccctggc 1700  accctcctcc aagagcacct ctgggggcac agcggccctg ggctgcctgg 1750  tcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc 1800  ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggact 1850  ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc ttgggcaccc 1900  agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 1950  aagaaagttg agcccaaatc ttgtgacaaa actcacacat gaccaccgca 2000  tgcaccagta tcgtccattc cgacagcatc gccagtcact atggcgtgct 2050  gctagcgccg ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc 2100  atggagccgg gccacctcga cctgaatgga agccggcggc acctcgctaa 2150  cggattcacc actccaagaa ttggagccaa tcaattcttg cggagaactg 2200  tgaatgcgca aaccaaccct tggcagaaca tatccatcgc gtccgccatc 2250  tccagcagcc gcacgcggcg catctcgggc agcgttgggt cctggccacg 2300  ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg 2350  gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga 2400  agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt 2450  cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct 2500  gcaccattat gttccggatc tgcatcgcag gatgctgctg gctaccctgt 2550  ggaacaccta catctgtatt aacgaagcgc tggcattgac cctgagtgat 2600  ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac 2650  gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc 2700  ctctctcgtt tcatcggtat cattaccccc atgaacagaa attccccctt 2750  acacggaggc atcaagtgac caaacaggaa aaaaccgccc ttaacatggc 2800  ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc 2850  tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct 2900  gatgagcttt accgcagcat ccggaaattg taaacgttaa tattttgtta 2950  aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc 3000  cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 3050  tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac 3100  tccaacgtca aagggcgaaa aaccgtctat cagggctatg gcccactacg 3150  tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac 3200  taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 3250  ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc 3300  tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 3350  ccgcgcttaa tgcgccgcta cagggcgcgt ccgcatcctg cctcgcgcgt 3400  ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 3450  cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 3500  cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt 3550  agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 3600  gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag 3650  gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc 3700  tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 3750  gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 3800  agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3850  cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 3900  tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 3950  tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4000  ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4050  agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4100  gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4150  gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4200  gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4250  tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag 4300  tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4350  ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4400  tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4450  ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 4500  taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4550  tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 4600  cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 4650  atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 4700  aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 4750  cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 4800  gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 4850  ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 4900  atagtttgcg caacgttgtt gccattgctg caggcatcgt ggtgtcacgc 4950  tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 5000  agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 5050  ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5100  atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 5150  ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 5200  ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa taccgcgcca 5250  catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 5300  aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 5350  ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 5400  gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 5450  gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 5500  gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 5550  tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 5600  acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 5650  ggcgtatcac gaggcccttt cgtcttcaa 5679 <210> 14 <211> 5679 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 14  ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 50  tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 100  cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 150  gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 200  agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 250  gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 300  agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 350  tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 400  atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 450  tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 500  acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 550  acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 600  ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 650  ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 700  gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc 750  agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 800  tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 850  ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 900  atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 950  cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1000  gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 1050  gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1100  ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 1150  tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 1200  agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 1250  ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1300  gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 1350  tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1400  agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 1450  ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 1500  taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 1550  gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1600  accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 1650  cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1700  gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 1750  cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 1800  gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 1850  ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 1900  tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 1950  cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2000  cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 2050  tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata 2100  gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 2150  gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 2200  tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 2250  tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc aggatgcgga 2300  cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2350  gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 2400  ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2450  tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 2500  ccaaaaaact tgattagggt gatggttcac gtagtgggcc atagccctga 2550  tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2600  actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2650  ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 2700  ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 2750  aatttccgga tgctgcggta aagctcatca gcgtggtcgt gaagcgattc 2800  acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa 2850  gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 2900  tcctgtttgg tcacttgatg cctccgtgta agggggaatt tctgttcatg 2950  ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac 3000  tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg 3050  cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag 3100  cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 3150  tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt 3200  ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag 3250  gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg 3300  tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc 3350  tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg 3400  cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat 3450  gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat 3500  tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt 3550  ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg 3600  aggcagacaa ggtatagggc ggcgctagca gcacgccata gtgactggcg 3650  atgctgtcgg aatggacgat actggtgcat gcggtggtca tgtgtgagtt 3700  ttgtcacaag atttgggctc aactttcttg tccaccttgg tgttgctggg 3750  cttgtgattc acgttgcaga tgtaggtctg ggtgcccaag ctgctggagg 3800  gcacggtcac cacgctgctg agggagtaga gtcctgagga ctgtaggaca 3850  gccgggaagg tgtgcacgcc gctggtcagg gcgcctgagt tccacgacac 3900  cgtcaccggt tcggggaagt agtccttgac caggcagccc agggccgctg 3950  tgcccccaga ggtgctcttg gaggagggtg ccagggggaa gaccgatggg 4000  cccttggtgg aggccgagga gacggtgacc agggttcctt gaccccagta 4050  gtcgaagaaa tcgctgatcc atcgagcaca ataatagacg gcagtgtcct 4100  cagcacgcag gctgttcatc tgcaggtata atgtgttttt ggaatcgtcg 4150  cgacttatag tgaaacggcc cttgaagcgc tggttatagt tcgtgatgtc 4200  gtagtcagga ttaatcgatg caacccattc caggccctta cccggggcct 4250  gacggaccca gtggatgata tattcggtga aggtgtagcc agaagctgca 4300  caggacaaac ggagtgagcc ccctggctgc accaggccac cgccagactc 4350  caccagctga acctcagcgt acgcgtttgt agcaatagaa aaaacgaaca 4400  tagatgcaag aagaaatgcg atattctttt tcataaaatc acctcaacct 4450  ctagataccc tttttacgtg aacttgcgta ctagggccac gatgcgtccg 4500  gcgtagagga tcagcttaac actctcccct gttgaagctc tttgtgacgg 4550  gcgagctcag gccctgatgg gtgacttcgc aggcgtagac tttgtgtttc 4600  tcgtagtctg ctttgctcag cgtcagggtg ctgctgaggc tgtaggtgct 4650  gtccttgctg tcctgctctg tgacactctc ctgggagtta cccgattgga 4700  gggcgttatc caccttccac tgtactttgg cctctctggg atagaagtta 4750  ttcagcaggc acacaacaga agcagttcca gatttcaact gctcatcaga 4800  tggcgggaag atgaagacag atggtgcagc cacagttcgt ttgatctcca 4850  ccttggtacc ctgtccaaat gtgcgcggaa taccccaaga gtgttgacag 4900  taataagttg cgaagtcttc tggctgcaga ctgctgatgg tcagagtgaa 4950  atccgtccca gaaccggatc cagagaagcg agaagggact ccagactcga 5000  ggttgctagc atagtaaatc agtagtttcg gagcttttcc tggtttctgt 5050  tgataccagt gcatatagct ataagagcta gtcgacacgc tctgactggc 5100  tctgcaggtg atggtgaccc tatcgcccac agaggcggac agggagctcg 5150  gggactgggt catctggata tcagcgtacg cgtttgtagc aatagaaaaa 5200  acgaacatag atgcaagaag aaatgcgata ttctttttca taaaatcacc 5250  tcaacctcta gaggatcccc gggtaccgag ctcgaattct agttacaaat 5300  acattaaaaa ataaaaacaa agcgactata agtctcggcc gtgacaactt 5350  tatgacagct gttgaaaaga ttaacttttt actgacgagg atgcttcaat 5400  aacttcttta cgtaatcgcg cagcagctcc gtatcgtcgt caggaatgct 5450  ggcatcgggc tttacctcgt acagcgcccc ctctacctga tcaatcaacc 5500  gctgttggtc attttgcgcc atattgcgaa gcattgcagt gacgataatc 5550  tccaaagctt ctacctgcgc acacagttca ttcgactctt cttctttttg 5600  ggcaagctta aataacaact cagcaatgag atttttcatg tctgtatttc 5650  cttatccaaa gtatggagaa gttgaattc 5679 <210> 15 <211> 241 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 15  Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe    1 5 10 15  Ser Ile Ala Thr Asn Ala Tyr Ala Asp Ile Gln Met Thr Gln Ser                   20 25 30  Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr                   35 40 45  Cys Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met                   50 55 60  His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile                   65 70 75  Tyr Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser                   80 85 90  Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu                   95 100 105  Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Trp Gly                  110 115 120  Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg                  125 130 135  Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu                  140 145 150  Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn                  155 160 165  Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala                  170 175 180  Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser                  185 190 195  Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys                  200 205 210  Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His                  215 220 225  Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu                  230 235 240  Cys      <210> 16 <211> 248 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 16  Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe    1 5 10 15  Ser Ile Ala Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser                   20 25 30  Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys                   35 40 45  Ala Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile Ile His Trp Val                   50 55 60  Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile Asn                   65 70 75  Pro Asp Tyr Asp Ile Thr Asn Tyr Asn Gln Arg Phe Lys Gly Arg                   80 85 90  Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln                   95 100 105  Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala                  110 115 120  Arg Trp Ile Ser Asp Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu                  125 130 135  Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro                  140 145 150  Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu                  155 160 165  Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser                  170 175 180  Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala                  185 190 195  Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr                  200 205 210  Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val                  215 220 225  Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro                  230 235 240  Lys Ser Cys Asp Lys Thr His Thr                  245 <210> 17 <211> 5678 <212> DNA <213> Artificial Sequence <220> <223> sequence is synthesized <400> 17  gaattcaact tctccatact ttggataagg aaatacagac atgaaaaatc 50  tcattgctga gttgttattt aagcttgccc aaaaagaaga agagtcgaat 100  gaactgtgtg cgcaggtaga agctttggag attatcgtca ctgcaatgct 150  tcgcaatatg gcgcaaaatg accaacagcg gttgattgat caggtagagg 200  gggcgctgta cgaggtaaag cccgatgcca gcattcctga cgacgatacg 250  gagctgctgc gcgattacgt aaagaagtta ttgaagcatc ctcgtcagta 300  aaaagttaat cttttcaaca gctgtcataa agttgtcacg gccgagactt 350  atagtcgctt tgtttttatt ttttaatgta tttgtaacta gaattcgagc 400  tcggtacccg gggatcctct agaggttgag gtgatttatg aaaaagaata 450  tcgcatttct tcttgcatct atgttcgttt tttctattgc tacaaacgcg 500  tacgctcaga tagtactgtc ccagtccccg gctatcctgt ccgcctctcc 550  tggcgagaag gtcactatga cctgcagagc cagctcttct gtgagctata 600  tgcattggta tcaacagaaa ccaggaagct ctccgaaacc atggatttac 650  gctccatcga acctcgcgtc tggagtccct gcgcgcttct ctggatccgg 700  ttctgggact agttactctc tgaccatcag cagagtggag gcagaagacg 750  ccgcaactta ttactgtcaa cagtggagct tcaatccgcc cacatttgga 800  gccggcacca agctggagct caaacgaact gtggctgcac catctgtctt 850  catcttcccg ccatctgatg agcagttgaa atctggaact gcttctgttg 900  tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 950  gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca 1000  ggacagcaag gacagcacct acagcctcag cagcaccctg acgctgagca 1050  aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 1100  ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttaagc 1150  tgatcctcta cgccggacgc atcgtggccc tagtacgcaa gttcacgtaa 1200  aaagggtatc tagaggttga ggtgatttta tgaaaaagaa tatcgcattt 1250  cttcttgcat ctatgttcgt tttttctatt gctacaaacg cgtacgctca 1300  ggcttatctg cagcagtctg gcgccgagct ggtgcggcca ggagctagcg 1350  tcaagatgtc ctgtaaagct tctggctaca ccttcaccag ctataacatg 1400  cattgggtca agcagacacc gaggcaaggc ctggaatgga ttggagcgat 1450  ctatcctggc aacggcgaca cgagctataa ccagaagttc aagggcaagg 1500  ccactctgac tgtggacaag tccagcagta ctgcctacat gcaactgagc 1550  agcctgactt ctgaggacag cgctgtctac ttttgtgctc gcgtggtcta 1600  ctatagcaac agctactggt acttcgacgt ctggggtacc ggaaccacag 1650  tcaccgtctc ctcggcctcc accaagggcc catcggtctt ccccctggca 1700  ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt 1750  caaggactac ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc 1800  tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 1850  tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca 1900  gacctacatc tgcaacgtga atcacaagcc cagcaacacc aaggtggaca 1950  agaaagttga gcccaaatct tgtgacaaaa ctcacacatg accaccgcat 2000  gcaccagtat cgtccattcc gacagcatcg ccagtcacta tggcgtgctg 2050  ctagcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca 2100  tggagccggg ccacctcgac ctgaatggaa gccggcggca cctcgctaac 2150  ggattcacca ctccaagaat tggagccaat caattcttgc ggagaactgt 2200  gaatgcgcaa accaaccctt ggcagaacat atccatcgcg tccgccatct 2250  ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg 2300  gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg 2350  ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa 2400  gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc 2450  ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg 2500  caccattatg ttccggatct gcatcgcagg atgctgctgg ctaccctgtg 2550  gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt 2600  tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg 2650  ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc 2700  tctctcgttt catcggtatc attaccccca tgaacagaaa ttccccctta 2750  cacggaggca tcaagtgacc aaacaggaaa aaaccgccct taacatggcc 2800  cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct 2850  ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg 2900  atgagcttta ccgcagcatc cggaaattgt aaacgttaat attttgttaa 2950  aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc 3000  gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 3050  gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 3100  ccaacgtcaa agggcgaaaa accgtctatc agggctatgg cccactacgt 3150  gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 3200  aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 3250  cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct 3300  agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 3350  cgcgcttaat gcgccgctac agggcgcgtc cgcatcctgc ctcgcgcgtt 3400  tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 3450  acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 3500  gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta 3550  gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 3600  tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 3650  agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct 3700  gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 3750  taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3800  gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 3850  gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 3900  caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 3950  ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 4000  cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4050  gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4100  ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4150  taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 4200  cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4250  acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 4300  atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4350  gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4400  gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4450  tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 4500  aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 4550  ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 4600  ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 4650  tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 4700  actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 4750  gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 4800  ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 4850  cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 4900  tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct 4950  cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5000  gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 5050  tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 5100  tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 5150  tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 5200  gcgaccgagt tgctcttgcc cggcgtcaac acgggataat accgcgccac 5250  atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5300  aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 5350  tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 5400  ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 5450  acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 5500  catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 5550  agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 5600  cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 5650  gcgtatcacg aggccctttc gtcttcaa 5678 <210> 18 <211> 5678 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 18  ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 50  tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 100  cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc 150  gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 200  agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 250  gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 300  agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 350  tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 400  atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 450  tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 500  acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 550  acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 600  ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 650  ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 700  gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc 750  agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 800  tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 850  ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 900  atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 950  cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 1000  gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 1050  gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1100  ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 1150  tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 1200  agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 1250  ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1300  gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 1350  tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 1400  agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 1450  ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 1500  taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 1550  gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1600  accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 1650  cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1700  gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 1750  cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 1800  gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 1850  ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 1900  tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 1950  cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2000  cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 2050  tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata 2100  gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc 2150  gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 2200  tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 2250  tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc aggatgcgga 2300  cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 2350  gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 2400  ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2450  tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 2500  ccaaaaaact tgattagggt gatggttcac gtagtgggcc atagccctga 2550  tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2600  actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 2650  ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag 2700  ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac 2750  aatttccgga tgctgcggta aagctcatca gcgtggtcgt gaagcgattc 2800  acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa 2850  gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 2900  tcctgtttgg tcacttgatg cctccgtgta agggggaatt tctgttcatg 2950  ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac 3000  tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg 3050  cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag 3100  cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 3150  tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt 3200  ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag 3250  gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg 3300  tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc 3350  tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg 3400  cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat 3450  gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat 3500  tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt 3550  ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg 3600  aggcagacaa ggtatagggc ggcgctagca gcacgccata gtgactggcg 3650  atgctgtcgg aatggacgat actggtgcat gcggtggtca tgtgtgagtt 3700  ttgtcacaag atttgggctc aactttcttg tccaccttgg tgttgctggg 3750  cttgtgattc acgttgcaga tgtaggtctg ggtgcccaag ctgctggagg 3800  gcacggtcac cacgctgctg agggagtaga gtcctgagga ctgtaggaca 3850  gccgggaagg tgtgcacgcc gctggtcagg gcgcctgagt tccacgacac 3900  cgtcaccggt tcggggaagt agtccttgac caggcagccc agggccgctg 3950  tgcccccaga ggtgctcttg gaggagggtg ccagggggaa gaccgatggg 4000  cccttggtgg aggccgagga gacggtgact gtggttccgg taccccagac 4050  gtcgaagtac cagtagctgt tgctatagta gaccacgcga gcacaaaagt 4100  agacagcgct gtcctcagaa gtcaggctgc tcagttgcat gtaggcagta 4150  ctgctggact tgtccacagt cagagtggcc ttgcccttga acttctggtt 4200  atagctcgtg tcgccgttgc caggatagat cgctccaatc cattccaggc 4250  cttgcctcgg tgtctgcttg acccaatgca tgttatagct ggtgaaggtg 4300  tagccagaag ctttacagga catcttgacg ctagctcctg gccgcaccag 4350  ctcggcgcca gactgctgca gataagcctg agcgtacgcg tttgtagcaa 4400  tagaaaaaac gaacatagat gcaagaagaa atgcgatatt ctttttcata 4450  aaatcacctc aacctctaga tacccttttt acgtgaactt gcgtactagg 4500  gccacgatgc gtccggcgta gaggatcagc ttaacactct cccctgttga 4550  agctctttgt gacgggcgag ctcaggccct gatgggtgac ttcgcaggcg 4600  tagactttgt gtttctcgta gtctgctttg ctcagcgtca gggtgctgct 4650  gaggctgtag gtgctgtcct tgctgtcctg ctctgtgaca ctctcctggg 4700  agttacccga ttggagggcg ttatccacct tccactgtac tttggcctct 4750  ctgggataga agttattcag caggcacaca acagaagcag ttccagattt 4800  caactgctca tcagatggcg ggaagatgaa gacagatggt gcagccacag 4850  ttcgtttgag ctccagcttg gtgccggctc caaatgtggg cggattgaag 4900  ctccactgtt gacagtaata agttgcggcg tcttctgcct ccactctgct 4950  gatggtcaga gagtaactag tcccagaacc ggatccagag aagcgcgcag 5000  ggactccaga cgcgaggttc gatggagcgt aaatccatgg tttcggagag 5050  cttcctggtt tctgttgata ccaatgcata tagctcacag aagagctggc 5100  tctgcaggtc atagtgacct tctcgccagg agaggcggac aggatagccg 5150  gggactggga cagtactatc tgagcgtacg cgtttgtagc aatagaaaaa 5200  acgaacatag atgcaagaag aaatgcgata ttctttttca taaatcacct 5250  caacctctag aggatccccg ggtaccgagc tcgaattcta gttacaaata 5300  cattaaaaaa taaaaacaaa gcgactataa gtctcggccg tgacaacttt 5350  atgacagctg ttgaaaagat taacttttta ctgacgagga tgcttcaata 5400  acttctttac gtaatcgcgc agcagctccg tatcgtcgtc aggaatgctg 5450  gcatcgggct ttacctcgta cagcgccccc tctacctgat caatcaaccg 5500  ctgttggtca ttttgcgcca tattgcgaag cattgcagtg acgataatct 5550  ccaaagcttc tacctgcgca cacagttcat tcgactcttc ttctttttgg 5600  gcaagcttaa ataacaactc agcaatgaga tttttcatgt ctgtatttcc 5650  ttatccaaag tatggagaag ttgaattc 5678 <210> 19 <211> 236 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 19  Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe    1 5 10 15  Ser Ile Ala Thr Asn Ala Tyr Ala Gln Ile Val Leu Ser Gln Ser                   20 25 30  Pro Ala Ile Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr                   35 40 45  Cys Arg Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln                   50 55 60  Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr Ala Pro Ser Asn                   65 70 75  Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly                   80 85 90  Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu Asp Ala                   95 100 105  Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe                  110 115 120  Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro                  125 130 135  Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly                  140 145 150  Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu                  155 160 165  Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn                  170 175 180  Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr                  185 190 195  Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys                  200 205 210  His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                  215 220 225  Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys                  230 235 <210> 20 <211> 253 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 20  Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe    1 5 10 15  Ser Ile Ala Thr Asn Ala Tyr Ala Gln Ala Tyr Leu Gln Gln Ser                   20 25 30  Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Met Ser Cys                   35 40 45  Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val                   50 55 60  Lys Gln Thr Pro Arg Gln Gly Leu Glu Trp Ile Gly Ala Ile Tyr                   65 70 75  Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Lys                   80 85 90  Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln                   95 100 105  Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala                  110 115 120  Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp                  125 130 135  Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly                  140 145 150  Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly                  155 160 165  Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu                  170 175 180  Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val                  185 190 195  His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu                  200 205 210  Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr                  215 220 225  Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp                  230 235 240  Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr                  245 250 <210> 21 <211> 5391 <212> DNA <213> Homo sapiens <400> 21  ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat 50  tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 100  ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 150  acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 200  ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 250  atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 300  aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 350  tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 400  ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 450  tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 500  aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 550  aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 600  ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct 650  ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca 700  ttggaacgcg gattccccgt gccaagagtg acgtaagtac cgcctataga 750  gtctataggc ccaccccctt ggcttcgtta gaacgcggct acaattaata 800  cataacctta tgtatcatac acatacgatt taggtgacac tatagaataa 850  catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc 900  acctcggttc tatcgattga attccaccat gggatggtca tgtatcatcc 950  tttttctagt agcaactgca actggagtac attcagatat ccagatgacc 1000  cagtccccga gctccctgtc cgcctctgtg ggcgataggg tcaccatcac 1050  ctgccgtgcc agtcaggaca tccgtaatta tttgaactgg tatcaacaga 1100  aaccaggaaa agctccgaaa ctactgattt actatacctc ccgcctggag 1150  tctggagtcc cttctcgctt ctctggttct ggttctggga cggattacac 1200  tctgaccatc agtagtctgc aaccggagga cttcgcaact tattactgtc 1250  agcaaggtaa tactctgccg tggacgttcg gacagggcac caaggtggag 1300  atcaaacgaa ctgtggctgc accatctgtc ttcatcttcc cgccatctga 1350  tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact 1400  tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 1450  tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac 1500  ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac 1550  acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc 1600  acaaagagct tcaacagggg agagtgttaa gcttggccgc catggcccaa 1650  cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 1700  atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 1750  aaactcatca atgtatctta tcatgtctgg atcgatcggg aattaattcg 1800  gcgcagcacc atggcctgaa ataacctctg aaagaggaac ttggttaggt 1850  accttctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg 1900  tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 1950  tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 2000  agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc 2050  cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 2100  cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc 2150  tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 2200  aggcttttgc aaaaagctgt taacagcttg gcactggccg tcgttttaca 2250  acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 2300  cacatccccc cttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 2350  cgcccttccc aacagttgcg tagcctgaat ggcgaatggc gcctgatgcg 2400  gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa 2450  gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 2500  tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2550  cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 2600  tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac 2650  ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 2700  ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 2750  ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 2800  cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2850  ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 2900  attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg 2950  ccgcatagtt aagccaactc cgctatcgct acgtgactgg gtcatggctg 3000  cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 3050  ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 3100  gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagtattctt 3150  gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 3200  ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 3250  cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3300  tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 3350  agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 3400  attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 3450  atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3500  aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 3550  gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgatg 3600  acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 3650  ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 3700  agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 3750  ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3800  ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 3850  gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgccagcag 3900  caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 3950  gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 4000  accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4050  ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4100  gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 4150  aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4200  ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 4250  gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 4300  tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4350  cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4400  ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 4450  ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4500  gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 4550  ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 4600  gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4650  ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4700  tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 4750  cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg 4800  aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 4850  gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 4900  tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 4950  caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 5000  ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 5050  ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 5100  ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 5150  gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 5200  ttaatccagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 5250  gcaacgcaat taatgtgagt tacctcactc attaggcacc ccaggcttta 5300  cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 5350  atttcacaca ggaaacagct atgaccatga ttacgaatta a 5391 <210> 22 <211> 6135 <212> DNA <213> Homo sapiens <400> 22  attcgagctc gcccgacatt gattattgac tagttattaa tagtaatcaa 50  ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 100  cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 150  gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 200  attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta 250  catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg 300  taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 350  ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 400  cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 450  atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 500  aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 550  caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg 600  tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 650  tccatagaag acaccgggac cgatccagcc tccgcggccg ggaacggtgc 700  attggaacgc ggattccccg tgccaagagt gacgtaagta ccgcctatag 750  agtctatagg cccaccccct tggcttcgtt agaacgcggc tacaattaat 800  acataacctt atgtatcata cacatacgat ttaggtgaca ctatagaata 850  acatccactt tgcctttctc tccacaggtg tccactccca ggtccaactg 900  cacctcggtt ctatcgattg aattccacca tgggatggtc atgtatcatc 950  ctttttctag tagcaactgc aactggagta cattcagaag ttcagctggt 1000  ggagtctggc ggtggcctgg tgcagccagg gggctcactc cgtttgtcct 1050  gtgcagcttc tggctactcc tttaccggct acactatgaa ctgggtgcgt 1100  caggccccag gtaagggcct ggaatgggtt gcactgatta atccttataa 1150  aggtgttact acctatgccg atagcgtcaa gggccgtttc actataagcg 1200  tagataaatc caaaaacaca gcctacctgc aaatgaacag cctgcgtgct 1250  gaggacactg ccgtctatta ttgtgctaga agcggatact acggcgatag 1300  cgactggtat tttgacgtct ggggtcaagg aaccctggtc accgtctcct 1350  cggcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag 1400  agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt 1450  ccccgaaccg gtgacggtgt cgtggaactc aggcgccctg accagcggcg 1500  tgcacacctt cccggctgtc ctacagtcct caggactcta ctccctcagc 1550  agcgtggtga ctgtgccctc tagcagcttg ggcacccaga cctacatctg 1600  caacgtgaat cacaagccca gcaacaccaa ggtggacaag aaagttgagc 1650  ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1700  ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac 1750  cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga 1800  gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 1850  gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta 1900  ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg ctgaatggca 1950  aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 2000  aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac 2050  cctgccccca tcccgggaag agatgaccaa gaaccaggtc agcctgacct 2100  gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 2150  aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc 2200  cgacggctcc ttcttcctct acagcaagct caccgtggac aagagcaggt 2250  ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 2300  aaccactaca cgcagaagag cctctccctg tctccgggta aatgagtgcg 2350  acggccctag agtcgacctg cagaagcttg gccgccatgg cccaacttgt 2400  ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc 2450  acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact 2500  catcaatgta tcttatcatg tctggatcga tcgggaatta attcggcgca 2550  gcaccatggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt 2600  ctgaggcgga aagaaccatc tgtggaatgt gtgtcagtta gggtgtggaa 2650  agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 2700  tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 2750  tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 2800  ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc 2850  catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 2900  ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 2950  tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc 3000  gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 3050  ccccccttcg ccagttggcg taatagcgaa gaggcccgca ccgatcgccc 3100  ttcccaacag ttgcgtagcc tgaatggcga atggcgcctg atgcggtatt 3150  ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac 3200  catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 3250  acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 3300  cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 3350  ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 3400  ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 3450  gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3500  atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc 3550  tattcttttg atttataagg gattttgccg atttcggcct attggttaaa 3600  aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 3650  cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 3700  tagttaagcc aactccgcta tcgctacgtg actgggtcat ggctgcgccc 3750  cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 3800  ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 3850  agaggttttc accgtcatca ccgaaacgcg cgaggcagta ttcttgaaga 3900  cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 3950  aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa 4000  cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 4050  agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 4100  gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 4150  gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 4200  gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 4250  cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4300  gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgatgacgcc 4350  gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 4400  tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 4450  gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 4500  ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 4550  caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4600  atgaagccat accaaacgac gagcgtgaca ccacgatgcc agcagcaatg 4650  gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 4700  ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 4750  ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 4800  gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 4850  taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 4900  tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 4950  cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 5000  aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 5050  atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 5100  gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 5150  cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5200  gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 5250  agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 5300  ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 5350  tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 5400  ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 5450  ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 5500  tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 5550  agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 5600  cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 5650  ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 5700  gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 5750  ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5800  attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 5850  cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 5900  gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 5950  ccaactggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 6000  gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt 6050  tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 6100  acacaggaaa cagctatgac catgattacg aatta 6135 <210> 23 <211> 232 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 23  Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr    1 5 10 15  Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu                   20 25 30  Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser                   35 40 45  Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys                   50 55 60  Ala Pro Lys Pro Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly                   65 70 75  Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr                   80 85 90  Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr                   95 100 105  Cys Gln Gln Trp Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr                  110 115 120  Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile                  125 130 135  Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val                  140 145 150  Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln                  155 160 165  Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser                  170 175 180  Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser                  185 190 195  Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr                  200 205 210  Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys                  215 220 225  Ser Phe Asn Arg Gly Glu Cys                  230 <210> 24 <211> 213 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 24  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> 25 <211> 471 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 25  Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr    1 5 10 15  Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu                   20 25 30  Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                   35 40 45  Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro                   50 55 60  Gly Lys Gly Leu Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly                   65 70 75  Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser                   80 85 90  Val Asp Lys Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu                   95 100 105  Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr                  110 115 120  Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr                  125 130 135  Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe                  140 145 150  Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala                  155 160 165  Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                  170 175 180  Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                  185 190 195  Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val                  200 205 210  Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn                  215 220 225  Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu                  230 235 240  Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala                  245 250 255  Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                  260 265 270  Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys                  275 280 285  Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn                  290 295 300  Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro                  305 310 315  Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu                  320 325 330  Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                  335 340 345  Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                  350 355 360  Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu                  365 370 375  Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr                  380 385 390  Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp                  395 400 405  Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                  410 415 420  Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                  425 430 435  Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser                  440 445 450  Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu                  455 460 465  Ser Leu Ser Pro Gly Lys                  470 <210> 26 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 26  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> 27 <211> 471 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 27  Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr    1 5 10 15  Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu                   20 25 30  Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly                   35 40 45  Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro                   50 55 60  Gly Lys Gly Leu Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly                   65 70 75  Asp Thr Ser Tyr Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser                   80 85 90  Val Asp Lys Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu                   95 100 105  Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr                  110 115 120  Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr                  125 130 135  Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe                  140 145 150  Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala                  155 160 165  Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                  170 175 180  Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro                  185 190 195  Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val                  200 205 210  Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn                  215 220 225  Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu                  230 235 240  Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala                  245 250 255  Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys                  260 265 270  Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys                  275 280 285  Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn                  290 295 300  Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro                  305 310 315  Arg Glu Glu Gln Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu                  320 325 330  Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                  335 340 345  Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile                  350 355 360  Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu                  365 370 375  Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr                  380 385 390  Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp                  395 400 405  Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                  410 415 420  Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                  425 430 435  Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser                  440 445 450  Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu                  455 460 465  Ser Leu Ser Pro Gly Lys                  470 <210> 28 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 28  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> 29 <211> 213 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 29  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> 30 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 30  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 Ala 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  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> 31 <211> 36 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 31  ctacaccttc acgagctata acatgcactg ggtccg 36 <210> 32 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Sequence is synthesized <400> 32  gattaatcct gacaacggcg acacgagcta taaccagaag ttcaagggcc 50  g 51 <210> 33 <211> 38 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 33  gaatgggttg cagcgatcta tcctggcaac ggcgacac 38 <210> 34 <211> 65 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 34  attattgtgc tcgagtggtc tactatagca acagctactg gtacttcgac 50  gtctggggtc aagga 65 <210> 35 <211> 36 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 35  ctgcacagcc agctcttctg tcagctatat gcattg 36 <210> 36 <211> 42 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 36  aactactgat ttacgctcca tcgaacctcg cgtctggagt cc 42 <210> 37 <211> 45 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 37  tattactgtc aacagtggag cttcaatccg cccacatttg gacag 45 <210> 38 <211> 37 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 38  gtttcactat aagtgtcgac aagtccaaaa acacatt 37 <210> 39 <211> 33 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 39  gccaggatag atggcgccaa cccattccag gcc 33 <210> 40 <211> 26 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 40  aagctccgaa accactgatt tacgct 26 <210> 41 <211> 18 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 41  agttttgaga gcaaaatg 18 <210> 42 <211> 18 <212> DNA <213> Artificial sequence <220> <223> sequence is synthesized <400> 42  aagctatgaa cactaatg 18 <210> 43 <211> 107 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 43  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> 44 <211> 213 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 44  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> 45 <211> 452 <212> PRT <213> Artificial sequence <220> <223> sequence is synthesized <400> 45  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         

Claims (21)

A method of treating polychondritis or multiple mononeuritis in a mammal comprising administering to the mammal an effective amount of an antibody binding to CD20. The method of claim 1, wherein the antibody is not conjugated to another molecule. The method of claim 1, wherein the antibody is conjugated to another molecule. The method of claim 3, wherein the other molecule is a cytotoxic agent. The method of claim 4, wherein the cytotoxic agent is a radioactive compound. 6. The method of claim 4 or 5, wherein the cytotoxic agent comprises Y2B8 or ¹³¹ I-B1. The method of claim 1, wherein the antibody comprises rituximab. The method of claim 1, wherein the antibody comprises humanized 2H7. The method of claim 1, comprising administering to the mammal at a dose of about 20 mg / m 2 to about 250 mg / m 2 of antibody. The method of claim 9, wherein the dose is about 50 mg / m 2 to about 200 mg / m 2. The method of any one of claims 1 to 10, comprising administering a subsequent dose following administration of the initial dose of the antibody, wherein the mg / m 2 dose of the antibody at the subsequent dose is mg / d of the antibody at the initial dose. Method of exceeding m 2 capacity. The method of any one of claims 1-11, wherein the mammal is a human. The method of claim 1, wherein the antibody is administered intravenously. The method of claim 1, wherein the antibody is administered subcutaneously. The method of claim 1, further comprising administering to the mammal an effective amount of an immunosuppressant, anti-pain, or chemotherapeutic agent. The method of claim 1, wherein the polychondritis is treated. The method of claim 16, further comprising administering to the mammal an effective amount of a non-steroidal anti-inflammatory drug, a steroid, methotrexate, cyclophosphamide, dipson, azathioprine, penicillamine, or cyclosporin. Way. 16. The method of any one of claims 1-15, wherein multiple mononeuritis is treated. The method of claim 18, further comprising administering to the mammal an effective amount of an anti-pain agent, a steroid, methotrexate, cyclophosphamide, plasma exchange, intravenous immunoglobulin, cyclosporine, or mycophenolate mofetil. Way. An article of manufacture comprising a container, and a composition comprising an antibody that binds to CD20 contained in the container, the package further comprising a package insert directing a user of the composition to treat polychondritis or polymyelitis in a mammal. The article of manufacture of claim 20, further comprising a container comprising an agent other than a therapeutic antibody and further comprising instructions for treating the mammal with such an agent.

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