CN101151278A - CD20 antibody variants and uses thereof - Google Patents

Abstract

The invention provides improved humanized CD20 binding antibodies for treatment of B cell malignancies and autoimmune diseases.

Description

CD20 antibody variants and application thereof

This application claims the U.S. Provisional Application serial number 60/651 submitted for 7 days 2 months for 2005,111st, the U.S. Provisional Application serial number 60/689 that on June 10th, 2005 submits, the U.S. Provisional Application serial number 60/702 that on July 25th, 404 and 2005 submits, 571 rights and interests, completely take in these and apply for reference herein.

Invention field

The present invention relates to anti-CD 20 antibodies and its they treat B cell relevant disease in terms of purposes.

Background of invention

Lymphocyte is one of several leucocyte groups；Their specific recognitions simultaneously respond exotic antigen.There is three major types lymphocyte：Bone-marrow-derived lymphocyte (B cell), T lymphocytes (T cell) and natural killer (NK) cell.Bone-marrow-derived lymphocyte is responsible for generating antibody and provides the cell of humoral immunity.B cell is ripe in marrow, is then departed from marrow and in its cell surface expression antigen binding antibody.When B progenitor cells meet with for the first time its membrane-bound antibody to special antigen when, the cell starts quick division, and its offspring is divided into memory B cell and is referred to as the effector cell of " thick liquid cell ".Memory B cell has the longer life-span, and continues expression and initial parental cell and have identical specific membrane-bound antibody.Thick liquid cell does not generate membrane-bound antibody, is changed to generate the antibody of secreted form.Circulating antibody is the main effects device molecule of humoral immunity.

(also referred to as human B lymphocyte limits differentiation antigen to CD20 antigens, Bp35 it is) on pre-B lymphocyte (pre-B) and ripe bone-marrow-derived lymphocyte, molecular weight about 35kD hydrophobic transmembrane protein (Valentine et al., J.Biol.Chem.264 (19)：11282-11287(1989)；Einfeld et al., EMBO are J.7 (3)：711-717(1988)).The antigen expresses (Anderson et al., Blood 63 (6) also on the B cell non_hodgkin lymphoma (NHL) more than 90%：1424-1433 (1984)), but do not find (Tedder etal., J.Immunol.135 (2) in candidate stem cell, pro B lymphocyte (pro-B), normal plasma cells or other normal structures：973-979(1985)).Think the early stage step (Tedder et al., supra) in the activation of CD20 regulation cell cycle startings and differentiation, and function (Tedder the et al., J.Cell.Biochem.14D of calcium channel may be played：195(1990)).

Expressed in view of CD20 in B cell lymphoma, the antigen has become the useful therapeutic target for treating such lymthoma.The U.S. has more than 300,000 people with B cell NHL, and newly diagnoses every year more than 56,000.For example, it is used as genetic engineering Chi-meric mice/human monoclonal antibodies for h CD20 antigen, rituximab (RITUXAN ) antibody (can be from Genentech, Inc., South San Francisco, California, U.S. are bought) it is used to treat the patient with recurrent or refractory low grade or follicularis CD20 positive B-cells non_hodgkin lymphoma.Rituximab be exactly on April 7th, 1998 bulletin United States Patent (USP) No.5,736,137 (Anderson et al.) and United States Patent (USP) No.5,776,456 in referred to as " C2B8 " antibody.The in vitro study of mechanism of action shows RITUXAN  combination people complements and dissolves lymphoid B cell lines (Reff et al., Blood83 (2) by complement-dependent cytotoxicity (CDC)：435-445(1994)).In addition, it has remarkable activity in cytotoxicity (ADCC) determination method of antibody dependent cellular.Internal preclinical study shows that RITUXAN  cut down B cell from the peripheral blood, lymph node and marrow of macaque, it may be possible to pass through complement and cell-mediated process (Reff et al., Blood 83 (2)：435-445(1994)).Indicate that other anti-CD 20 antibodies for treating NHL include the mouse antibody Zevalin for being connected with radio isotope Yttrium-90^TM(IDEC Pharmaceuticals, SanDiego, CA), coupling have the antibody Bexxar of I-131 another complete mouse^TM(Corixa, WA).

CD20 still treats the useful target antigen of autoimmunity disease.Also it have studied rituximab in B cell and autoantibody show a variety of non-malignant autoimmunity diseases for being played a role in disease pathology physiology.Edwards et al., Biochem.Soc.Trans.30：824-828(2002).Rituximab is it is reported that potential mitigation such as 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., ArthritisRheum.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)), immunologic thrombocytopenic purpura (D ' Arena et al., Leuk.Lymphoma 44：561-562(2003)；Stasi et al., Blood 98：952-957(2001)；Saleh et al., Semin.Oncol.27 (Supp.12)：99-103(2000)；Zaia et al., Haematolgica 87：189-195(2002)；Ratanatharathorn et al., Ann.Int.Med.133：275-279 (2000)), pure red cell aplasia (Auner et al., Br.J.Haematol.116：725-728 (2002)), autoimmune anemia (Zaja et al., Haematologica 87：Haematologica 87 is shown in 189-195 (2002), corrigenda：336 (2002)), cold agglutinin disease (Layios et al., Leukemia 15：187-8(2001)；Berentsen et al., Blood 103：2925-2928(2004)；Berentsen et al., Br.J.Haematol.115：79-83(2001)；Bauduer, Br.J.Haematol.112：1083-1090(2001)；Damiani et al., Br.J.Haematol.114：229-234 (2001)), severe insulin tolerance Type B syndrome (Coll et al., N.Engl.J.Med.350：310-311 (2004)), Combination cryoglobulinemia (DeVita et al., Arthritis Rheum.46 (Suppl.9)：S206/S469 (2002)), myasthenia gravis (Zaja et al., Neurology 55：1062-63(2000)；Wylam et al., J.Pediatr.143：674-677 (2003)), Wei Genashi granulomas (Speckset al., Arthritis Rheumatism 44：2836-2840 (2001)), intractable pemphigus vulgaris (Dupuy et al., Arch.Dermatol.140：91-96 (2004)), dermatomyositis (Levine, ArthritisRheum.46 (Suppl.9)：S1299 (2002)), siogren's syndrome (Somer et al., Arthritis ＆Rheumatism 49：394-398 (2003)), activity II type Combination cryoglobulinemia (Zaja et al., Blood 101：3827-3834 (2003)), pemphigus vulgaris (Dupay et al., Arch.Dermatol.140：91-95 (2004)), autoimmune neurological disorders (Pestronk et al., J.Neurol.Neurosurg.Psychiatry 74：485-489 (2003)), outer opsoclonus-myoclonic syndrome (Pranzatelli etal., Neurology 60 (Suppl.1) PO5.128 of knurl：A395 (2003)) and recurrence-mitigation type multiple sclerosis (RRMS) (Cross et al., (abstract) " Preliminary Results from a phase II trial ofrituximab in MS ", U.S.'s multiple sclerosis research and the treatment committee the 8th annual meeting, 20-21 (2003)) S＆S.

Having carried out II clinical trial phases in rheumatoid arthritis (RA) patient, there is provided the security about rituximab and 48 weeks tracking datas of effect.Emery et al., Arthritis Rheum.48 (9)：S439(2003)；Szczepanski et al., Arthritis Rheum.48 (9)：S121(2003).Patient is bisected into four treatment groups at random：Methotrexate (MTX), only rituximab, rituximab add methotrexate (MTX) and rituximab plus endoxan (CTX).Rituximab therapeutic scheme is the 1st day and the 15th day intravenous using 1 gram.

Paying close attention to the publication treated with rituximab includes：Perotta and Abuel, " years duration to rituximab ", Abstract # 3360 Blood10 (1) (part 1-2) of Response ofchronic relapsing ITP of 10：p.88B(1998)；Perotta et al., " Rituxan in the treatment of chronicidiopathic thrombocytopenic 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 Blymphocyte depletion ", Ann Rheum Dis, supra；Leandro et al., " Lymphocytedepletion in rheumatoid arthritis：Early evidence for safety, efficacy and doseresponse ", Arthritis and Rheumatism 44 (9)：S370(2001)；Leandro et al., " Anopen study of B lymphocyte depletion in systemic lupus erythematosus ", Arthritis and Rheumatism, 46：2673-2677 (2002), wherein during 2 weeks, every patient receives 500mg rituximab infusions, twice 750mg endoxan infusion and high dose oral corticosteroid twice, wherein two patients receiving treatment are recurred at the 7th and 8 months respectively, and have been controlled again with different schemes；Weide et al., " Successful long-term treatment of systemiclupus erythematosus with rituximab maintenance therapy ", Lupus 12：779-782 (2003), wherein treating a patient (375mg/m with rituximab²× 4, with one week for interval), and more rituximab applications are delivered within individual month per 5-6, then every three months receives 375mg/m²Rituximab maintenance therapy, and the second place patient for suffering from intractable SLE has successfully been treated with rituximab, and every three months receives maintenance therapy, two patients have preferable response to rituximab therapies；Edwards and Cambridge, " Sustained improvement in rheumatoid arthritisfollowing a protocol designed to deplete B lymphocytes ", Rheumatology 40：205-211(2001)；Cambridge et al., " B lymphocyte depletion in patients withrheumatoid arthritis：Serial studies of immunological parameters ", ArthritisRheum.46 (Suppl.9)：S1350(2002)；Edwards et al., " B-lymphocyte depletiontherapy in rheumatoid arthritis and other autoimmune disorders ", supra；Edwardset al., " Efficacy and safety of rituximab, a B-cell targeted chimeric monoclonalantibody：A randomized, placebo controlled trial in patients with rheumatoidarthritis ", Arthritis and Rheumatism 46 (9)：S197(2002)；Levine and Pestronk, " IgM antibody-related polyneuropathies：B-cell depletion chemotherapy usingrituximab ", Neurology 52：1701-1704(1999)；DeVita et al., " Efficacy ofselective B cell blockade in the treatment of rheumatoid arthritis ", Arthritis ＆Rheum 46：2029-2033(2002)；Hidashida et al., " Treatment ofDMARD-refractory rheumatoid arthritis with rituximab ", it is showed in the AnnualScientific Meeting of the American College of Rheumatology, Oct 24-29, NewOrleans, LA 2002；Tuscano, J., " Successful treatment of infliximab-refractoryrheumatoid arthritis with rituximab ", it is showed in the Annual Scientific Meeting ofthe American College of Rheumatology, Oct 24-29, New Orleans, LA 2002；Martin and Chan, " Pathogenic roles of B cells in human autoimmunity；Insightsfrom the clinic ", Immunity 20：517-527(2004)；Silverman and Weisman, " Rituximab Therapy and Autoimmune Disorders, Prospects for Anti-B CellTherapy ", 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 autoimmunedisorders with a AntiCD20 monoclonal antibody Rituximab ", Arthritis AndRheumatism 48：(9) 9, S (SEP), page：S624-S624(2003)；Kneitz et al., " EffectiveB cell depletion with rituximab in the treatment of autoimmune diseases ", Immunobiology 206：519-527(2002)；Arzoo et al., " Treatment of refractoryantibody mediated autoimmune disorders with an anti-CD20 monoclonalantibody (rituximab) ", Annals of the Rheumatic Diseases 61 (10)：922-4(2002)；Comment in Ann Rheum Dis.61：863-866(2002)；Lake and Dionne, " FutureStrategies in Immunotherapy ",《Burger′s Medicinal Chemistry and DrugDiscovery》In (2003by John Wiley ＆ Sons, Inc.), paper pastes the date online：On January 15th, 2003 (Chapter 2 " Antibody-Directed Immunotherapy ")；Liang and Tedder, Wiley Encyclopedia of Molecular Medicine, Section：CD20 as anImmunotherapy Target, paper pastes the date online：15 January, 2002 entitled " CD20 "；Stockinger et al., annex 4A it is entitled " Monoclonal Antibodies to Human Cell SurfaceAntigens ", Coligan et al. compile,《Current Protocols in Immunology》In (2003 JohnWiley ＆ Sons, Inc), the date is pasted online：In May, 2003；The printing and publishing date：2 months 2003；Penichet and Morrison, " CD Antibodies/molecules：Definition；AntibodyEngineering ",《Wiley Encyclopedia of Molecular Medicine Section：Chimeric, Humanized and Human Antibodies》In, the date is pasted online：On January 15th, 2002；Specks et al., " Response of Wegener ' s granulomatosis to anti-CD20 chimericmonoclonal antibody therapy ", Arthritis ＆ Rheumatism 44：2836-2840(2001)；Online summary is submitted and invited, Koegh et al., " Rituximab for Remission Induction inSevere ANCA-Associated Vasculitis：The Patients of Report of a Prospective Open-Label PilotTrial in 10 ", American College of Rheumatology, Session Number：28-100, Session Title：Vasculitis, Session Type：ACR Concurrent Session, Primary Category：(the http of 28 Vasculitis, Session 10/18/2004://www.abstractsonline.com/viewer/SearchResults.asp)；Eriksson, " patients with ANCA-positive vasculitistreated with rituximab ", the Kidney and Blood Pressure Research 26 of Short-term outcome and safety in 5：294(2003)；Jayne et al., " B-cell depletion with rituximab for refractory vasculitis ", Kidneyand Blood Pressure Research 26：294(2003)；Jayne, poster 88 (11^thInternationalVasculitis and ANCA workshop), 2003 American Society of Nephrology；Stoneand Specks, " Rituximab Therapy for the Induction of Remission and Tolerance inANCA-associated Vasculitis ",《the Clinical Trial Research Summary of the2002-2003 Immune Tolerance Network》In, http://www.immunetolerance.org/research/autoimmune/trials/stone.html.Referring also to Leandro et al., " B cell repopulation occurs mainly from

B cells in patientwith rheumatoid arthritis and systemic lupus erythematosus ", Arthritis Rheum.48 (Suppl 9)：S1160(2003).

It is human anti-mouse antibody (HAMA) response (see, for example, Miller using a major limitation of mouse antibody in the treatment of people, R.A.et al., " Monoclonal antibody therapeutic trials in sevenpatients with T-cell lymphoma ", Blood 62：988-995(1983)；Schroff R.W.et al., " Human anti-murine immunoglobulin response in patients receiving monoclonalantibody therapy ", Cancer Res.45：879-885(1985)).Even chimeric molecule, merged with human constant region (C) variable region (V) of wherein rodent antibodies, remain able to trigger significant immune response (HACA, the anti-chimeric antibody of people) (Neuberger et al., Nature (Lond.) 314：268-270(1985)).It is mouse antibody or non-human species antibody's " humanization " (Jones et al., Nature (Lond.) 321 that a kind of these powerful approach limited are overcome in the Clinical practice of monoclonal antibody：522-525(1986)；Riechman et al., Nature (Lond.) 332：323-327(1988)).This antibody-like is estimated to produce the antigenic of bottom line when being applied to patient or without antigenicity, especially for long-term treatment.Humanized anti-cd 20 antibodies have been recorded in WO 03/068821A2 (Hansen et al.)；WO 2004103404(Watkins)；With WO 04/056312 (Adams).Human anti cd 20 antibodies have been recorded in WO2004/035607 (Teeling et al.).

The not only antigenicity with bottom line is provided but also the biological characteristics with improvement and the therapeutic CD20 binding antibodies of clinical efficacy will be beneficial.Present invention accomplishes this demand and other demands.The invention provides anti-CD 20 antibodies, they, which overcome the limitation of Current therapeutic composition and are detailed below there is provided basis, will become apparent to further advantage.

Paying close attention to the patent and patent publications of CD20 antibody includes United States Patent (USP) 5,776,456th, 5,736,137,5,843,439th, 6,399,061 and 6,682,734 and U.S. Patent application US 2002/0197255,00,3/0,021,781 200,3/0,082,172 2003/0095963 A1, US2003/0147885 A1 (Anderson et al.) of A1, US of A1, US of A1, US2；The B1 of United States Patent (USP) 6,455,043 and WO 00/09160 (Grillo-Lopez, A.)；WO 00/27428(Grillo-Lopez and White)；WO 00/27433(Grillo-Lopez and Leonard)；WO 00/44788(Braslawsky et al.)；WO 01/10462 (Rastetter, W.)；WO 01/10461(Rastetter and White)；WO 01/10460(White andGrillo-Lopez)；US 2001/0018041 A1、US 2003/0180292 A1、WO 01/34194(Hanna and Hariharan)；U. S. application US 2002/0006404 and WO 02/04021 (Hannaand Hariharan)；The A1 of U. S. application US 2002/0012665 and WO 01/74388 (Hanna, N.)；The A1 of U. S. application US 2002/0058029 (Hanna, N.)；The A1 of U. S. application US 2003/0103971 (Hariharan and Hanna)；The A1 of U. S. application US 2002/0009444 and WO 01/80884 (Grillo-Lopez, A.)；WO 01/97858 (White, C.)；The A1 of U. S. application US 2002/0128488 and WO 02/34790 (Reff, M.)；WO 02/060955(Braslawsky et al.)；WO 02/096948(Braslawsky et al.)；WO 02/079255(Reff and Davies)；The B1 of United States Patent (USP) 6,171,586 and WO 98/56418 (Lam et al.)；WO 98/58964 (Raju, S.)；WO 99/22764 (Raju, S.)；WO 99/51642, the B1 of United States Patent (USP) 6,194,551, the B1 of United States Patent (USP) 6,242,195, the B1 of United States Patent (USP) 6,528,624 and United States Patent (USP) 6,538,124 (Idusogie et al.)；WO 00/42072 (Presta, L.)；WO 00/67796(Curd et al.)；WO 01/03734(Grillo-Lopez et al.)；U. S. application US2002/0004587 A1 and WO 01/77342 (Miller and Presta)；U. S. application US2002/0197256 (Grewal, I.)；The A1 of U. S. application US 2003/0157108 (Presta, L.)；The B1 of United States Patent (USP) 6,565,827,6,090,365 B1,6,287,537 B1,6,015,542,5,843,398 and 5,595,721 (Kaminski et al.)；The B1 of United States Patent (USP) 5,500,362,5,677,180,5,721,108,6,120,767,6,652,852 (Robinson et al.)；The B1 of United States Patent (USP) 6,410,391 (Raubitschek et al.)；The B1 of United States Patent (USP) 6,224,866 and WO 00/20864 (Barbera-Guillem, E.)；WO 01/13945 (Barbera-Guillem, E.)；WO 00/67795(Goldenberg)；U. S. application US 2003/0133930A1 and WO 00/74718 (Goldenberg and Hansen)；WO 00/76542(Golay et al.)；WO01/72333(Wolin and Rosenblatt)；The B1 of United States Patent (USP) 6,368,596 (Ghetie et al.)；United States Patent (USP) 6,306,393 and the A1 of U. S. application US 2002/0041847 (Goldenberg, D.)；The A1 of U. S. application US 2003/0026801 (Weiner and Hartmann)；WO 02/102312 (Engleman, E.)；U.S. Patent application US 2003/0068664 (Albitar et al.)；WO 03/002607 (Leung, S.)；WO03/049694, US2002/0009427 A1 and US2003/0185796 A1 (Wolin et al.)；WO03/061694(Sing and Siegall)；US 2003/0219818 A1(Bohen et al.)；US2003/0219433 A1 and WO 03/068821 (Hansen et al.)；US 2002/0136719 A1(Shenoy et al.)；WO 2004/032828(Wahl et al.)；WO 2004/035607(Teeling et al.)；US 2004/0093621(Shitara et al.).Referring also to United States Patent (USP) 5,849,898 and European application 330,191 (Seed et al.)；United States Patent (USP) 4,861,579 and the A2 of EP 332,865 (Meyer and Weiss)；WO 95/03770(Bhat et al.)；US 2001/0056066(Bugelski et al.)；WO2004/035607(Teeling et al.)；WO 2004/056312(Lowman et al.)；US2004/0093621(Shitara et al.)；WO 2004/103404(Watkins et al.).Paying close attention to the publication of CD20 antibody includes Teeling, J.et al., " Characterization of new human CD20monoclonal antibodies with potent cytolytic activity against non-Hodgkin ' slymphomas ", Blood, Jun 2004；10.1182.

Summary of the invention

Cut down the invention provides the combination h CD20 listed in table 13 and 14 and in vivo the humanization 2H7 variant antibodies of primate B cells.In specific embodiments, hu2H7 antibody is pattern 472,473,511,523 and 516.In the composition of the present invention, method, product, the preferred embodiment of preparaton, the humanization 2H7 antibody is hu2H7.v511 or hu2H7.v114.

The invention provides the humanization 2H7 antibody or its antigen-binding fragment for combining h CD20, wherein the antibody effectively cuts down primate B cells in vivo, the antibody includes SEQ ID NO.25 light chain variable district (V_L) sequence and SEQ ID NO.8 weight chain variable district (V_H) there is amino acid replacement D56A in sequence, but VH-CDR2, and the N100 in VH-CDR3 is substituted with Y or W.In one embodiment, this antibody also includes the replacement S100aR in VH-CDR3.In the another embodiment of afore mentioned antibodies, antibody, which is also included at least one in Fc areas, improves the amino acid replacement of ADCC and/or CDC activity.In order to improve ADCC and/or CDC effector functions, in one embodiment, afore mentioned antibodies will also include IgG1 Fc and the IgG1 Fc are comprising any in amino acid replacement S298A, E333A, K334A and K326A or K326W.In one embodiment, the antibody display of foregoing embodiments goes out the cytotoxicity (ADCC) of at least 20 times higher than 2H7.v16 of antibody dependent cellular, and shows at least 25 times higher than 2H7.v16 of complement-dependent cytotoxicity.

Or, can have the amino acid change or replacement for improving ADCC activity but reduction CDC activity at least one in Fc comprising the antibody for substituting S100aR.At this point, antibody can comprise at least the amino acid replacement K322A in Fc, and can also include amino acid replacement S298A, E333A, K334A.

In preferred embodiments, the antibody of ADCC and CDC activity with improvement for antibody 2H7.v16 also to improve at least 3 times, preferably at least 6 times of affinity combination h CD20, and the 2H7.v16 has respectively SEQ ID NO.13 and 14 light chain and sequence of heavy chain.

The invention provides the humanization 2H7 antibody for combining h CD20, wherein the antibody is made up of SEQ IDNO.26 sequence of light chain and SEQ ID NO.34 sequence of heavy chain.

Present invention also offers the antibody of any foregoing embodiments, it and cytotoxic agent couplings.In one embodiment, the cytotoxic agent is radio isotope or toxin.

In one embodiment, antibody of the invention is generated in Chinese hamster ovary celI.

Present invention also offers the seperated nuclear acid of the antibody of coding foregoing embodiments, including expression vector.Additionally provide the host cell that the antibody is generated comprising the nucleic acid.In one embodiment, host cell is the cell for generating antibody 2H7.v511.

There is provided the method for generating any afore mentioned antibodies, including cultivate foregoing host cell and antibody is reclaimed from cell culture.

Additionally provide the composition of the 2H7 antibody comprising the present invention and carrier.In one embodiment, the carrier is pharmacy acceptable carriers.

Another embodiment is the composition for including hu2H7.v511, about 80-100% antibody deficiency trehalose wherein in composition.

On the other hand it is product, including container and dress composition in this embodiment, wherein the composition includes the antibody of any foregoing embodiments.The specific processing of composition as needed, the product, which may also include, indicates that said composition is used to treat the package insert of non-hodgkin's (Hodgkin) lymphomas or said composition for treating rheumatoid arthritis.

Another aspect of the present invention is the method for cutting down B cell in vivo using the humanization 2H7 antibody of foregoing embodiments.

Present invention also offers the method for the treatment of CD20 positive cancers, including the humanization 2H7 antibody of the foregoing embodiments of therapeutically effective amount is applied to cancer patient.The preferred B cell lymphoma of CD20 positive cancers or leukaemia.In specific embodiments, the humanization 2H7 antibody and its functional fragment that combine hCD20 are used to treat non_hodgkin lymphoma (NHL), the Hodgkin's disease (LPHD) of lymphocytic predominance, SLL (SLL), chronic lymphocytic leukemia (CLL).In specific embodiments, by humanization CD20 binding antibodies or its functional fragment, particularly v511 and v114, for treating painless (indolent) NHL, includes the painless NHL of painless NHL and the rituximab tolerance (refractory) of recurrence.

In order to treat these cancers, in one embodiment, the antibody is applied through intravenous infusion.Through being transfused applied dose in every dose of about 12.5mg/m²To 800mg/m²In the range of, it is generally one weekly, totally 1,2,3 or 4 doses.

Present invention also offers the method for mitigating autoimmunity disease, including the humanization 2H7 antibody of any foregoing embodiments of therapeutically effective amount is applied to Serum of Patients With Autoimmune Diseases.In preferred embodiments, the antibody is intravenously or subcutaneously applied.The intravenous applied dose of antibody is in the range of every dose of 10mg to 500mg, in specific embodiments, and dosage is 100mg/ agent.

In specific embodiments, the autoimmunity disease is selected from rheumatoid arthritis and juvenile rheumatoid arthritis, systemic loupus erythematosus (SLE) includes lupus nephritis, Wegener (Wegener) family name disease, inflammatory bowel disease, ulcerative colitis, ITP (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephrosis, IgM polyneuropathies, myasthenia gravis, ANCA associated vasculitis, diabetes, Reynolds (Reynaud) Cotard, Si Yegelun (Sjogren) Cotard, neuromyelitis optica (Neuromyelitis Optica, ) and glomerulonephritis NMO.

Hu2H7.v511 and hu2H7.v114 antibody can be used for treating or mitigating autoimmunity disease or the method for B cell tumour, wherein the antibody and second of therapeutic agent with simultaneously, sequential apply or the combination in alternate scheme.In one embodiment, second of therapeutic agent is BAFF antagonists.In certain embodiments, the BAFF antagonists are the antibody or BR3-Fc fusion proteins with reference to people BR3.

In one preferred embodiment of the method for stating disease before the treatment, subject or patient are primates, preferably people.

Liquid adjustments are additionally provided, it is in 20mM sodium acetates, 4% Trehalose Dihydrate, 0.02% polysorbate20, about 20mg/ml humanization 2H7.v511 antibody is contained in pH5.5, applied for intravenous.Liquid adjustments are additionally provided, it is in 20mM sodium acetates, 240mM (8%) Trehalose Dihydrate, 0.02% polysorbate20, about 20mg/ml humanization 2H7.v114 antibody is contained in pH 5.3.Another liquid adjustments available for 2H7.v16 are the 0.02% polysorbate20 (Tween 20 in 20mM sodium acetates pH5.3,4% Trehalose Dihydrate^TM) in contain about 30mg/ml antibody.

Brief description

Figure 1A, which is shown, compares mouse 2H7 (SEQ ID NO.1), humanization 2H7.v16 variants (SEQ IDNO.2) and human kappa light chain subclass I (SEQ ID NO.3) each light chain variable district (V_L) amino acid sequence sequence alignment.2H7 and hu2H7.v16 V_LCDR it is as follows：CDR1 (SEQ ID NO.4), CDR2 (SEQ ID NO.5) and CDR3 (SEQ ID NO.6).

Figure 1B, which is shown, compares mouse 2H7 (SEQ ID NO.7), humanization 2H7.v16 variants (SEQ IDNO.8) and people heavy chain subclass III consensus sequences (SEQ ID NO.9) each weight chain variable district (V_H) amino acid sequence sequence alignment.2H7 and hu2H7.v16 V_HCDR it is as follows：CDR1 (SEQ ID NO.10), CDR2 (SEQ ID NO.11) and CDR3 (SEQ ID NO.12).

In Figure 1A and Figure 1B, as illustrated, CDR1, CDR2 and CDR3 in every chain are trapped among in bracket, flank is framework region FR1-FR4.2H7 refers to mouse 2H7 antibody.Asterisk between two row sequences indicates different positions between two kinds of sequences.Residue numbering is according to Kabat et al., Sequences ofImmunological Interest, the 5th edition, Public Health Service, National Institutes ofHealth, Bethesda, Md. (1991), insertion is shown as a, b, c, d and e.

Fig. 2A shows the sequence alignment of protein for comparing humanization 2H7.v16 and v511 light chain.

Fig. 2 B show the sequence alignment of protein for comparing humanization 2H7.v16 and v511 heavy chain.

Fig. 3 shows the relative combination of the 2H7 variants in the CD20 ELISA of dissolving.Antibody 2H7.v16 (circle), 2H7.v511 (square) and 2H7.v588 (triangle) are compared (see embodiment 12).

Fig. 4 shows the ADCC activity determined using WIL2-S cells and the NK cells of human beings of purifying.The ability that they mediate ADCC in vitro is compared to antibody 2H7.v16 (circle), 2H7.v511 (square) and 2H7.v588 (triangle) (see embodiment 13).

Fig. 5 shows the CDC activity determined using WIL2-S cells and people's complement.The ability that they mediate CDC in vitro is compared to antibody 2H7.v16 (circle), 2H7.v511 (square) and 2H7.v588 (triangle) (see embodiment 14).

Fig. 6 shows the abatement of the 2nd day 2H7 variants v16 and v511 in vivo to B cell in blood (left figure) and cavum peritoneale (right figure) after injection of antibodies (see embodiment 15).

Fig. 7 shows abatements of two days 2H7 variant v16 and v511 to B cell in spleen after injection of antibodies：Spleen B cell (left figure), marginal zone B cells (middle) and follicular B cells (right figure) (see embodiment 15).

The internal B cell that Fig. 8 compares the 2H7.v16 and v511 in expression h CD20 and people CD16 hCD20Tg+/+/mCD16-/-/hCD16 Tg+/- transgenic mice is cut down (see embodiment 15).

Fig. 9 shows the carrier for expressing 2H7.v16 in Chinese hamster ovary celI.

Figure 10 shows the 2H7.v511 and the comparison of other 2H7 variants v16, v31, v114, v138 and v488 CDC activity with WIL2-S raji cell assay Rajis (see embodiment 14).

Figure 11 compares antibody variants 2H7.v511, v16, v31, v114, v138 and v488 for being determined with WIL2-S cells and the NK cells of human beings of purifying ADCC activity (see embodiment 13 and Fig. 4).

The internal B cell that Figure 12 summarises 2H7.v511 and rituximab (Rituxan ) in expression h CD20 and people CD16 hCD20 Tg+/+/mCD16-/-/hCD16Tg+/- transgenic mice cuts down analysis, and the Activity Summary of two kinds of antibody is in table (see embodiment 15).

Figure 13 shows the abatements of the 2nd day 2H7.v511 and rituximab in vivo to B cell in blood (left figure) and cavum peritoneale (right figure) after injection (see embodiment 15).

Figure 14 shows abatements of the 2nd day 2H7.v511 and rituximab to B cell in spleen after injection of antibodies：Spleen B cell (left figure), marginal zone B cells (middle) and follicular B cells (right figure) (see embodiment 15).

Figure 15 A, 15B and 15C show the external CDC expression activitiys for the PBMC for being purified from 3 CLL patients, wherein PBMC 2H7.v114 or Rituxan^TM, and complement processing.

Figure 16 A, 16B and 16C show the external CDC expression activitiys of the PBMC from 3 CLL patients (patient is identical with Figure 15), wherein PBMC 2H7.v511 or Rituxan^TM, and complement processing.

The detailed description of preferred embodiment

" CD20 " antigen is non-glycosylated cross-film phosphoprotein found on the B cell surface more than 90% from peripheral blood or lymphoid organ, molecular weight about 35kD.CD20 is expressed before early stage during B (pre-B) cell development, and is retained to plasma cell differentiation；It can not find on human stem cell, lymphoid progenitors or normal plasma cells.CD20 is present on both normal B cells and malignant B cell.The other titles of CD20 in the literature include " bone-marrow-derived lymphocyte limitation differentiation antigen " and " Bp35 ".CD20 antigens are recorded in such as Clark and Ledbetter, Adv.Can.Res.52：81-149 (1989) and Valentine et al., J.Biol.Chem.264 (19)：11282-11287(1989).

Term " antibody " is used with broadest, monoclonal antibody (including full length monoclonal antibodies), multi-specificity antibody (such as bispecific antibody) and antibody fragment are clearly covered, as long as they show desired biological activity or function.

The biological activity of the humanization CD20 binding antibodies of the present invention will at least include the combination of antibody and h CD20, more preferably and people and other primates (including macaque, rhesus macaque, chimpanzee) CD20 combination.The antibody will be to be not higher than 1 × 10^-8K_dValue, is preferably no greater than about 1 × 10^-9K_dValue combination CD20, and can in vivo kill or cut down B cell, preferably killed or abatement at least 20% compared with the suitable negative control of the unused antibody processing.B cell abatement can be in ADCC, CDC, apoptosis or other mechanism one or more cause.In some embodiments of this paper disease treatments, it may be desirable to which specific effector functions or mechanism are more than others, and preferably humanization 2H7 some variants are to realize those biological functions, such as ADCC.

" antibody fragment " includes the antigen binding domain or variable region of a part for full length antibody, typically antibody.The example of antibody fragment includes Fab, Fab ', F (ab ')₂With Fv fragments；Double antibody；Linear antibodies；Single-chain antibody molecules；And the multi-specificity antibody formed by antibody fragment.

" Fv " is that the minimum antibody fragment with binding site is recognized comprising intact antigen.The fragment is made up of the dimer of close, Non-covalent binding a heavy chain variable domain and a light chain variable domain.Six hypervariable loops (heavy chain and each 3 rings of light chain) are given out from the foldable structure of the two domains, the amino acid residue with reference to antigen is contributed and assigns antibody with antigen-binding specificity.However, even single Variable domain (or only including half of Fv of three CDR to antigen-specific) also has the ability for recognizing and combining antigen, simply affinity is less than entire binding site.

Term " monoclonal antibody " refers to the antibody obtained from the antibody of a group substantially homogeneity as used herein, that is each antibody of composition colony is identical and/or combines same epitope, issuable during except production monoclonal antibody to become external, such variant generally exists with indivisible.Such monoclonal antibody generally includes such antibody, and it includes the peptide sequence for combining target, and wherein target Binding peptide sequence is obtained by the process comprised the following steps, that is, single target Binding peptide sequence is selected in many peptide sequences of comforming.For example, the selection course can be comform it is polyclonal, such as hybridoma clone, phage clone or recombinant DNA clone set in select Unique clones.It should be understood that, selected target binding sequence can be changed further, for example in order to improve the affinity to target, for humanization target binding sequence, in order to improve its yield in cell culture, in order to reduce its immunogenicity in vivo, in order to produce multi-specificity antibody, etc., and the antibody of the target binding sequence comprising change is also monoclonal antibody of the invention.From the typical polyclonal antibody preparations difference included for the different antibodies of different determinants (epitope), every kind of monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Beyond their specificity, the advantage of monoclonal antibody preparations also resides in the pollution that they are generally not affected by other immunoglobulins.Modifier " monoclonal " indicate antibody basically homogeneity antibody population obtain feature, should not be construed as require antibody is generated by any ad hoc approach.For example, the monoclonal antibody used according to the present invention can be prepared by multiple technologies, including such as hybridoma method (such as Kohler et al., Nature 256：495(1975)；Harlow et al., Antibodies：ALaboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1988；Hammerling et al., in：Monoclonal Antibodies and T-Cell Hybridomas 563-681, Elsevier, N.Y., 1981), recombinant DNA method is (see, for example, United States Patent (USP) No.4,816,567), display technique of bacteriophage is (see, for example, Clackson et al., Nature 352：624-628(1991)；Marks etal., J.Mol.Biol.222：581-597(1991)；Sidhu et al., J.Mol.Biol.338 (2)：299-310(2004)；Lee et al., J.Mol.Biol.340 (5)：1073-1093(2004)；Fellouse, Proc.Natl.Acad.Sci.USA 101 (34)：12467-12472(2004)；Lee et al., J.Immunol.Methods284 (1-2)：119-132 (2004)) and technology for generation people's or proper manners the antibody in animal, the animal has the gene of all or part of human immunoglobulin gene's seat or encoding human immunoglobulin's sequence (see, for example, WO 1998/24893；WO 1996/34096；WO 1996/33735；WO1991/10741；Jakobovits et al., Proc.Natl.Acad.Sci.USA 90：2551(1993)；Jakobovits et al., Nature 362：255-258(1993)；Bruggemann et al., Year inImmuno.7：33(1993)；United States Patent (USP) No.5,545,806；5,569,825；5,591,669 (being all GenPharm)；5,545,807；WO 1997/17852；United States Patent (USP) No.5,545,807；5,545,806；5,569,825；5,625,126；5,633,425；With 5,661,016；Marks et al., Bio/Technology10：779-783(1992)；Lonberg et al., Nature 368：856-859(1994)；Morrison, Nature368：812-813(1994)；Fishwild et al., Nature Biotechnology 14：845-851(1996)；Neuberger, Nature Biotechnology 14：826(1996)；And Lonberg and Huszar, Intern.Rev.Immunol.13：65-93(1995).

" functional fragment " of the CD20 binding antibodies of the present invention is the fragment that those keep showing the biological activity including abatement B cell with they derivative intact full length molecule with substantially the same affinity combination CD20 and according to the measurement of all determination methods as those described herein of external or in vivoassay method.

Term " variable " refers to the extensive truth of some of variable region section difference in antibody sequence.V structure domain mediate antigen combines and limits specificity of the specific antibodies to its specific antigen.However, variability is not uniformly distributed in 110 amino acid of Variable domain leap.In fact, V areas are by 15-30 amino acid, the referred to as section not made a variation relatively of framework region (FR) and each length for distinguishing framework is 9-12 amino acid, and the shorter region for being referred to as the extreme variation of " hypervariable region " is constituted.Each self-contained four FR of Variable domain of native heavy and light chain, they take beta-pleated sheet conformation mostly, are connected by three hypervariable regions for forming loop connecting and formed in some cases a beta-pleated sheet structure part.Hypervariable region in every chain the keeping together closely by FR, and facilitate together with the hypervariable region of another chain the antigen binding site of antibody formation (referring to Kabat et al.,《Sequences ofProteins of Immunological Interest》, the 5th edition, Public Health Service, NationalInstitutes of Health, Bethesda, MD, 1991).Constant region does not participate in the combination of antibody and antigen directly, but shows the participation of antibody in a variety of effector functions, the cytotoxicity (ADCC) of such as antibody dependent cellular.

Term " hypervariable region " refers to the amino acid residue for being responsible for antigen binding in antibody as used herein.Hypervariable region generally comprises amino acid residue (such as V from " complementary determining region " or " CDR "_LIn residue 24-34 (L1), 50-56 (L2) and 89-97 (L3) nearby and V_HIn residue 31-35 (H1), 50-65 (H2) and 95-102 (H3) near；Kabat et al.,《Sequences of Proteins of ImmunologicalInterest》, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991) and/or those residue (such as V from " hypervariable loop "_LIn residue 26-32 (L1), 50-52 (L2) and 91-96 (L3) and V_HIn residue 26-32 (H1), 53-55 (H2) and 96-101 (H3)；Chothia and Lesk, J.Mol.Biol.196：901-917(1987)).

When referenced herein, " consensus sequence " or shared V structure domain sequence are the artificial sequences compared derived from known human immunoglobulin(HIg) variable region sequences amino acid sequence.Compared based on these, prepare the recombinant nucleic acid sequence of coding V structure domain amino acid sequence, the V structure domain amino acid sequence is the consensus sequence in derived from human κ chains and people's H chain subclass III V structures domain.Shared V sequences do not have any of antibody binding specificity or affinity.

A part for heavy chain and/or light chain is identical or homologous with derived from particular species or the corresponding sequence belonged in the antibody of specific antibodies classification or subclass in " chimeric " antibody (immunoglobulin), and the remainder of chain is identical or homologous with derived from another species or the corresponding sequence belonged in the antibody of another antibody isotype or subclass, and the fragment of this antibody-like, as long as they show desired biological activity (U.S. Patent No. 4,816, No. 567；Morrison et al., Proc.Natl.Acad.Sci.USA 81：6851-6855(1984)).Humanized antibody used herein is a subset of chimeric antibody.

" humanization " form of inhuman (such as mouse) antibody refers to the chimeric antibody that bottom line includes the sequence derived from non-human immunoglobulin.Largely, some hypervariable region residues immunoglobulin that some hypervariable region residues with non-human species' (donor antibody) such as mouse, rat, rabbit or the non-human primate for expecting specificity, affinity and ability are replaced that humanized antibody refers in human immunoglobulin(HIg) (receptor antibody).In some cases, Fv framework regions (FR) residue of human immunoglobulin(HIg) is replaced with corresponding non-human residues.In addition, humanized antibody can be included in the residue not found in receptor antibody or donor antibody.It is to further improve the performance of antibody such as binding affinity to carry out these modifications.In general, humanized antibody will include at least one, usually two substantially whole following variable regions, wherein entirely or substantially upper whole hypervariable loop corresponds to the hypervariable loop of non-human immunoglobulin, and entirely or substantially upper whole FR is the FR of human immunoglobulin sequence, although FR can include the amino acid replacement of one or more improvement binding affinities.The number of the amino acid replacement of these in FR is generally no more than at 6 in heavy chain, is no more than in light chain at 3.Humanized antibody optionally will also include at least part constant region for immunoglobulin (Fc), the typically constant region of human immunoglobulin(HIg).More details are referring to Jones et al., Nature 321：522-525(1986)；Riechmann et al., Nature 332：323-329(1988)；Presta, Curt.Op.Struct.Biol.2：593-596(1992).

Antibody " effector functions " refers to the biological activity that those are attributable to antibody Fc district (native sequences Fc areas or amino acid sequence variation Fc areas).The example of antibody mediated effect device function includes：Clq is combined and complement-dependent cytotoxicity；Fc acceptors are combined；The cytotoxicity (ADCC) of antibody dependent cellular mediation；Phagocytosis；Cell surface receptor (such as B-cell receptor) is lowered；With B cell activation.

" cytotoxicity of antibody dependent cellular mediation " or " ADCC " refer to the secreting type Ig being wherein attached to present on some cytotoxic cells (such as natural killer (NK) cell, neutrophil(e) cell and macrophage) on Fc acceptors (FcR) and enable these cytotoxic effect cells to specifically bind the target cell for carrying antigen, and the cytotoxic form of target cell is then killed with cytotoxin.Antibody " arms " (arm) cytotoxic cell, and such lethal effect definitely requires.Mediation ADCC main cell, NK cells, an expression Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.Ravetch and Kinet, Annu.Rev.Immunol.9：The page tables 3 of 457-92 (1991) the 464th summarize the FcR expression on hematopoietic cell.For the ADCC activity of purpose of appraisals molecule, external ADCC determination methods, such as U.S. Patent No. 5,500,362 or 5 can be carried out, it is described in 821, No. 337 or Presta U.S. Patent No. 6,737,056.Effector cell available for such determination method includes PMNC (PBMC) and natural killer (NK) cell.Or can purpose of appraisals molecule in vivo ADCC activity, such as in animal model, such as Clynes et al., PNAS (USA) 95：Disclosed in 652-656 (1998).If antibody is CD20 binding antibodies, ADCC activity can be tested in the transgenic mice (hCD20+/hCD16+Tg mouse) that expression h CD20 adds CD16 as described below.

" human effector cell " refers to expression one or more FcR and exercises the leucocyte of effector functions.Preferably, the cell at least expresses Fc γ RIII and exercises ADCC effector functions.Mediating the example of ADCC human leukocytes includes PMNC (PBMC), natural killer (NK) cell, monocyte, cytotoxic T cell and neutrophil cell, preferably PBMC and NK cells.Effector cell can separate from its natural origin, such as blood.

The acceptor in " Fc acceptors " or " FcR " description binding antibody Fc areas.It is preferred that FcR be native sequences people FcR.Furthermore it is preferred that FcR be FcR (γ acceptors) with reference to IgG antibody, including Fc γ RI, the acceptor of Fc γ RII and Fc γ RIII subclass include the allelic variant and alternative splice forms of these acceptors.Fc γ RII acceptors include Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" suppression acceptor "), and they have similar amino acid sequence, and difference is main in its cytoplasmic domains.Activated receptor Fc γ RIIA are in its cytoplasmic domains comprising immunity receptor the activation motifs (ITAM) based on tyrosine.Suppress acceptor Fc γ RIIB and suppression motif (ITIM) of the immunity receptor based on tyrosine is included in its cytoplasmic domains (referring to summary Da ё ron, Annu.Rev.Immunol.15：203-234(1997)).FcR summary is referring to Ravetch and Kinet, Annu.Rev.Immunol.9：457-492(1991)；Capel et al., Immunomethods 4：25-34(1994)；De Haas et al., J.Lab.Clin.Med.126：330-41(1995).Term " FcR " covers other FcR herein, including those futures will be identified.The term also include neonatal receptor, FcRn, it is responsible for Maternal immunoglobulin G being transferred to fetus (Guyer et al., J.Immunol.117：587 (1976) and Kim et al., J.Immunol.24：249 (1994)) and adjust the dynamic equilibrium of immunoglobulin.

WO 00/42072 (Presta) describes the antibody variants that the combination to FcR is improved or reduced.The content of the patent application is clearly taken in herein as reference.It also see Shields et al., J.Biol.Chem.9 (2)：6591-6604(2001).

On the binding affinity to FcRn, in one embodiment, the EC50 of antibody or apparent Kd (in pH6.0) are ＜=100nM, more preferably ＜=10nM.As for Fc γ RIII (F158；That is low-affinity isotype) binding affinity that improves, in one embodiment, EC50 or apparent Kd ＜=10nM, and for Fc γ RIII (V158；High-affinity), EC50 or apparent Kd ＜=3nM.It is known (referring to such as Ghetie 1997, Hinton 2004) and described below to measure to the method for FcRn combination.It can determine the Binding in vivo and serum half-life of people's FcRn high-affinities Binding peptide and people FcRn, such as in expression people FcRn transgenic mice or through transfected human cells be, or in the primate that application of Fc variant polypeptides.In certain embodiments, the binding affinity improved to people FcRn is changed and shown to the amino acid that the humanization 2H7 antibody of the present invention is also included in IgG Fc, it is higher than the antibody with wild type IgG Fc at least 60 times, at least 70 times, at least 80 times, more preferably at least 100 times, preferably at least 125 times, even more desirably at least 150 times to about 170 times.

" complement-dependent cytotoxicity " or " CDC " refers to dissolving when there is complement to target cell.The activation of classic complement approach is antibody (suitable subclass) starting combined by the component of complement system first (Clq) with reference to its associated antigen.In order to assess complement activation, CDC determination methods can be carried out, such as such as Gazzano-Santoro et al., J.Immunol.Methods 202：Described in 163 (1996).

The polypeptide variants of the Clq binding abilities of Fc region amino acid sequences and raising or reduction with change are recorded in U.S. Patent No. 6,194,551B1 and WO 99/51642.The content of those patent publications is clearly taken in herein as reference.It also see Idusogie et al., J.Immunol.164：4178-4184(2000).

Make a general survey of present specification and claims, unless otherwise indicated, the residue numbering mode of immunoglobulin heavy chain constant region is according to Kabat et al., Sequences of Proteins of ImmunologicalInterest, the 5th edition, Public Health Service, National Institutes of Health, the numbering of Bethesda, MD (1991) EU indexes, clearly income is used as reference herein." the EU indexes according to Kabat " refers to the residue numbering mode of human IgG1's EU antibody.The residue in V areas is numbered according to Kabat numberings, unless expressly stated order or other numbering systems.

The example of CD20 antibody includes：" C2B8 ", present referred to as " rituximab " (" RITUXAN  ") (United States Patent (USP) 5,736,137)；The 2B8 mouse antibody of yttrium [90] mark, is referred to as " Y2B8 " or " IbritumomabTiuxetan " (ZEVALIN ), can buy (United States Patent (USP) 5,736,137 from IDEC Pharmaceuticals companies；2B8 was preserved in ATCC, numbering HB11388 on June 22nd, 1993)；Mouse IgG2a " B1 ", also referred to as " Tositumomab ", is optionally used¹³¹I marks to produce "¹³¹I-B1 " or " iodine 131 tositumomab " antibody (BEXXAR^TM, can be bought from GlaxoSmithKline, referring also to United States Patent (USP) 5,595,721)；Mouse monoclonal antibody " 1F5 " (Press et al., Blood, 69 (2)：584-591 (1987)) and its variant, including " framework repairing " or humanization 1F5 (WO 2003/002607, Leung, S.；ATCC preserved material HB-96450)；Mouse 2H7 and chimeric 2H7 antibody (United States Patent (USP) 5,677,180)；Humanization 2H7 (WO 2004/056312 (Lowman et al.) and listed hereinafter)；HUMAX-CD20^TMAntibody (Genmab, Denmark of complete people；See, for example, Glennie and van de Winkel, DrugDiscovery Today 8：503-510(2003)；Cragg et al., Blood 101：1045-1052(2003))；Listed human monoclonal antibodies in WO 04/035607 (Teeling et al.)；Fc areas described in US2004/0093621 (Shitara et al.) are combined with the antibody of the sugar chain of complicated N- glucosides connection；Listed AME-133 in the CD20 binding molecules of such as AME series antibodies, such as WO 2004/103404^TMAntibody (Watkins et al., Applied Molecular Evolution)；A20 antibody or its variant, such as chimeric or humanization A20 antibody (being cA20, IMMU-106a.k.a.hA20 respectively) (US 2003/0219433, US 2005/0025764；Immunomedics)；And monoclonal antibody L27, G28-2,93-1B3, B-C1 or NU-B2, can be obtained from international leukocyte differential count seminar (InternationalLeukocyte Typing Workshop) (Valentine et al.,《Leukocyte Typing III》, McMichael compiles, p.440, Oxford University Press, 1987).CD20 antibody preferred herein is humanization, the CD20 antibody of chimeric or people, more preferably humanization 2H7 antibody, rituximab, chimeric or humanization A20 antibody (Immunomedics) and HUMAX-CD20^TMH CD20 antibody (Genmab).

As used herein, " B cell abatement " refers to after medicine or Antybody therapy, compared with the level before treatment, and b cell level is reduced in animal or human body.Well-known determination method can be used to measure for b cell level, such as by obtaining whole blood count, by the facs analysis that is dyed to known B cell mark and by the method described in such as EXPERIMENTAL EXAMPLESThe.B cell abatement can be the complete of part.In one embodiment, the abatement of expression CD20 B cell is at least 25%.In the patient for receiving B cell abatement agent, the duration of B cell abatement is typically the time recovered circulation time and B cell of the medicine in patient's body.

" tumor necrosis factor α (TNF α) " refers to comprising such as Pennica et al., Nature 312：721 (1984) or Aggarwal et al., JBC 260：The human TNF alpha molecule of amino acid sequence described in 2345 (1985)." TNF α inhibitor " is herein defined as suppressing to a certain degree the material of the biological function of TNF α, generally by combining TNF α and neutralizing its activity.The example of the tnf inhibitor specifically considered herein is Etanercept (Etanercept, ENBREL ), Infliximab (infliximab, REMICADE ) and Adalimumab (adalimumab, HUMIRA^TM)。

It is improper to the TNF α inhibitor for treating response being previously or is currently being because of toxicity and/or effect deficiency that statement " it is improper to be responded to TNF α inhibitor " refers to.Improper response can be assessed by discussing clinician known to disease to treatment.Occur the mammal of " effect is not enough " state of an illness after the TNF α inhibitor for treating being previously or is currently being to continue to keep active state.For example, patient may be with still having active disease activity after TNF α inhibitor for treating 3 months.

Term " BAFF ", " BAFF polypeptides ", " TALL-1 " or " TALL-1 polypeptides ", " BLys ", " THANK " cover " native sequences BAFF polypeptides " and " BAFF variants " as used herein." BAFF " is to give the those polypeptides coded by any amino acid sequence shown below

People BAFF sequences (SEQ ID NO：46)

1  mddstereqs rltsclkkre emklkecvsi Iprkespsvr sskdgkllaa tlllallscc

61 Itvvsfyqva alqgdlaslr aelqghhaek lpagagapka gleeapavta glkifeppap

121gegnssqnsr nkravqgpee tvtqdclqli adsetptiqk gsyffvpwll sfkrgsalee

181kenkilvket gyffiygqvl ytdktyamgh liqrkkvhvf gdelslvtlf rciqnmpetl

241pnnscysagi akleegdelq laiprenaqi sldgdvtffg alkll

(sequence that also see Fig. 3 in US2005/0095243A1) and its homologue and fragment and the title of variant, the homologue and fragment and variant have native sequences BAFF biological activity.BAFF biological activity may be selected from：Promote B cell survival, promote B cell ripe and combine BR3.Term " BAFF " includes those polypeptides described in following documents：Shu et al., J.Leukocyte Biol.65：680(1999)；GenBank registration numbers AF136293；WO98/18921, is disclosed on May 7th, 1998；EP 869,180, is disclosed on October 7th, 1998；WO98/27114, is disclosed on June 25th, 1998；WO 99/12964, is disclosed on March 18th, 1999；WO 99/33980, is disclosed on July 8th, 1999；Moore et al., Science 285：260-263(1999)；Schneider et al., J.Exp.Med.189：1747-1756(1999)；Mukhopadhyay etal., J.Biol.Chem.274：15978-15981(1999).

Term " BAFF antagonists " is used with broadest as used herein, combine native sequences BAFF polypeptides including (1) or combine native sequences BR3 polypeptides partially or completely to block BR3 and many peptide interactions of BAFF, and (2) partially or completely block, suppress or neutralization native sequences BAFF signal transductions any molecule.The conduction of native sequences BAFF polypeptide signals promotes B cell survival and B cell maturation etc..Suppression, blocking or the neutralization of BAFF signal transductions cause B cell number reduction etc..BAFF antagonists according to the present invention will block partially or completely in vitro or in vivo, suppress or neutralize one or more biological activities of BAFF polypeptides.In one embodiment, biological activity BAFF strengthens following any one event or its combination in vitro or in vivo：B cell survival is improved, IgG and/or IgM levels are improved, the increase of thick liquid cell number, and NF- κ b2/100 are processed into p52NF- κ b (such as Batten, M.et al., (2000) J.Exp.Med.192 in spleen B cell：1453-1465；Moore et al., (1999) Science 285：260-263；Kayagaki et al., (2002) 10：515-524).

Term " BR3 ", " BR3 polypeptides " or " BR3 acceptors " cover " native sequences BR3 polypeptides " and " BR3 variants " (having further restriction to it herein) as used herein." BR3 " is to give polypeptide and its title of homologue comprising following any amino acid sequence.

(a) people BR3 sequences (SEQ ID NO：47)

1  MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT PRPKPAGASS PAPRTALQPQ

61 ESVGAGAGEA ALPLPGLLFG APALLGLALV LALVLVGLVS WRRRQRRLRG ASSAEAPDGD

121KDAPEPLDKV IILSPGISDA TAPAWPPPGE DPGTTPPGHS VPVPATELGS TELVTTKTAG

181PEQQ

(b) variable people BR3 sequences (SEQ ID NO：48)

1  MRRGPRSLRG RDAPAPTPCV PAECFDLLVR HCVACGLLRT PRPKPAGAAS SPAPRTALQP

61 QESVGAGAGE AALPLPGLLF GAPALLGLAL VLALVLVGLV SWRRRQRRLR GASSAEAPDG

121DKDAPEPLDK VIILSPGISD ATAPAWPPPG EDPGTTPPGH SVPVPATELG STELVTTKTA

181GPEQQ

In some embodiments, the BAFF antagonists according to the present invention include anti-BAFF antibody, BAFF Binding peptides (including immunoadhesin and peptide) and the small molecule for combining BAFF.BAFF antagonists include BAFF binding antibodies (antibody for for example including any amino acid sequence of SEQ IDNO.1-46,321-329,834-872,1563-1595,1881-1905 in table 1) described in WO 02/02641.In another embodiment, immunoadhesin includes the BAFF lands (such as BR3, BCMA or TACI ectodomain) of BAFF-R.In yet another embodiment, the immunoadhesin is BR3-Fc.WO 02/66516, WO 00/40716, WO 01/87979, WO 03/024991, WO 02/16412, WO 02/38766, WO 02/092620, WO 01/12812 are see with reference to other examples of BAFF Fc protein.The method for preparing BAFF antagonists loads on US2005/0095243A1, ibid with US 2005/0163775.

" separation " antibody refer to it is identified and with the/antibody that is separated and/or reclaimed by a kind of composition of its natural surroundings.The contaminant component of its natural surroundings refers to the material of the diagnosis that can disturb the antibody or therapeutical uses, it may include the solute of enzyme, hormone and other oroteins property or non-proteinaceous.In preferred embodiments, by antibody purification to the measure of (1) according to Lowry methods, antibody weight is more than 95%, most preferably weight is more than 99%, (2) the N- ends by using spinning cup sequenator at least 15 residues of acquisition or the degree of internal amino acid sequence are enough, or (3) reach homogeneity according to the SDS-PAGE under the reproducibility or non-reducing conditions using Coomassie blue or preferred Silver stain.Since at least one composition of antibody natural surroundings is not in, then the antibody of separation includes the antibody iM situ in recombinant cell.However, the antibody of separation will generally be prepared by least one purification step.

" separation " nucleic acid molecules refer to the nucleic acid molecules that at least one contaminative nucleic acid molecules being generally associated in identified and natural origin with antibody nucleic acids are separated.The nucleic acid molecules of separation are different from finding in nature at the form of it or background.Therefore the nucleic acid molecules of separation have any different with being present in nucleic acid molecules when in n cell.However, the nucleic acid molecules of separation include being often expressed as the nucleic acid molecules included in the cell of the antibody, such as when the chromosome mapping when the nucleic acid molecules in the cell is different from its chromosome mapping in n cell.

Statement " control sequence " refers to DNA sequence dna necessary to the coded sequence expressed and be operatively connected in specific host organism.For example, the control sequence suitable for prokaryotes includes promoter, optional operator sequence and ribosome bind site.Known eukaryotic utilizes promoter, polyadenylation signal and enhancer.

If one section of nucleic acid is in functional interrelationship with another section of nucleotide sequence, it is " being operatively connected ".If for example, presequence (presequence) or secreting the DNA of leading (secretory leader) and being expressed as participating in the preceding protein (preprotein) of polypeptide secretion, the DNA of it and polypeptide is operatively connected；If promoter or enhancer influence the transcription of coded sequence, it is operatively connected with the sequence；Or, if the position of ribosome bind site promotes translation, it is operatively connected with coded sequence.Generally, " being operatively connected " means that connected DNA sequence dna is adjacent, and means adjacent in the case of secretion is leading and be in read state.However, enhancer need not be adjacent.Connection can be realized by the coupled reaction at convenient restriction site.If without such site, then according to oligonucleotides adapter or joint of the conventional practice using synthesis.

" carrier " includes shuttle vector and expression vector.Typically, plasmid construction thing will also include replication orgin (such as ColE1 replication orgins) and selection marker (such as ampicillin or tetracyclin resistance), be respectively used to duplication and selection of the plasmid in bacterium." expression vector " refers to the carrier that control sequence or controlling element necessary to antibody includes the antibody fragment of the present invention are expressed included in bacterium or eukaryotic.Following discloses suitable carrier.

The cell of humanization CD20 binding antibodies such as humanization 2H7 antibody of the invention is generated by the bacterium of the nucleic acid including wherein having imported encoding said antibody and eukaryotic host cell.Following discloses suitable host cell.

Term " label " refers to the detectable compounds or composition being directly or indirectly coupled with antibody as used herein.Label itself can be by itself just detectable (such as radioisotopic tracer or fluorescent marker), or in the case of enzyme marker, the chemical modification of detectable substrate compounds or composition can be catalyzed.

The compositions and methods of the invention

The invention provides humanization 2H7 antibody, they are 2H7.v16 variants.In a specific embodiment, the variant is hu2H7.v511.

In full length antibody, humanization CD20 binding antibodies of the invention are by the V structure domain comprising connected humanization and the C-structure domain of human immunoglobulin(HIg).In a preferred embodiment, H chains C areas come from human IgG, preferably IgG1 or IgG3.L chain C-structure domain is preferred from people's κ chains.

For this paper purpose, " humanization 2H7 " refers to following complete antibody or antibody fragment, and it includes light chain variable district (V_L) sequence：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKR(SEQ ID NO：2)；

With weight chain variable district (V_H) sequence：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSS(SEQ ID NO：8).

If humanization 2H7 antibody is complete antibody, it is preferred that it includes v16 light-chain amino acid sequences：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAP

SNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGT

KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC(SEQ ID NO：13)；

And heavy chain amino acid sequence：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK(SEQ ID NO：14).

A kind of foregoing humanized 2H7mAb variant is 2H7.v31, and it has and SEQ ID NO above：13 identical L chain-orderings and H chain amino acid sequences：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGDTSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCA

RVVYYSNSYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGIAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP

REEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAK

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN

YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK

SLSLSPGK(SEQ ID NO：15).

Another variant is 2H7.v138, and it has SEQ ID NO.26 heavy chain amino acid sequence.

Another variant of foregoing humanized 2H7 antibody is such a antibody, its SEQ ID NO.25 comprising 2H7.v511 V_LWith SEQ ID NO.33 V_H(being shown in Table 2).

Hu2H7.v511 is total length IgG1.In one embodiment, the antibody includes 2H7.v511 light chains (SEQ ID NO.26)：

DIQMTQSPSSLSASVGDRVTITCRASSSVSYLHWYQQKPGKAPKPLIYAPS

NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWAFNPPTFGQGTK

VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC；

With 2H7.v511 heavy chains (SEQ ID NO.34)：

EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYNMHWVRQAPGKGLEWV

GAIYPGNGATSYNQKFKGRFTISVDKSKNTLYLQMNSLRAEDTAVYYCAR

VVYYSYRYWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNAALPAPIAATISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK。

The variable region of all other variant based on v16 is by the amino acid sequence with v16, except the position of amino acid replacement shown in table 1 below.Unless otherwise indicated, 2H7 variants will have and v16 identical L chains.Humanized antibody 2H7.v16 is also referred to as rhuMAb2H7, PRO70769 or Ocrelizumab.

Table 1

2H7 Pattern	Light chain (VL) Change	Heavy chain (VH) Change	Fc Change
					16 supply Reference			-
31	-	-	S298A, E333A, K334A

73	M32L	N100A
					75	M32L	N100A	S298A, E333A, K334A
96	S92A	D56A, N100A
				114	M32L, S92A	D56A, N100A	S298A, E333A, K334A
115	M32L, S92A	D56A, N100A	S298A, E333A, K334A, E356D, M358L
				116	M32L, S92A	D56A, N100A	S298A, K334A, K322A
138	M32L, S92A	D56A, N100A	S298A, E333A, K334A, K326A
				477	M32L, S92A	D56A, N100A	S298A, E333A, K334A, K326A, N434W
375	-	-	K334L
				511	M32L, S92A	D56A, N100Y,  S100aR	S298A, E333A, K334A, K326A

Table 2

2H7 patterns	VL   SEQ ID NO.	VH   SEQ ID NO.	Whole light chains   SEQ ID NO.	Entire heavy chain   SEQ ID NO.
					16	2	8	13	14
31	2	8	13	15
					73	16	17	18	19
75	16	17	18	20
					96	21	22	23	24
114	25	22	26	27
					115	25	22	26	28
116	25	22	26	29
					138	25	22	26	30
477	25	22	26	31
					375	2	8	13	32
511	25	33	26	34

Residue numbering mode is according to Kabat et al., Sequences of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), insertion represents that breach is represented in sequence chart with dash with a, b, c, d and e.In the CD20 binding antibodies comprising Fc areas, the C- terminal lysines (residue 447 according to EU numbering systems) in Fc areas can remove, for example, transforming the nucleic acid of encoding antibody polypeptide during antibody purification or by recombined engineering.Therefore, humanization 2H7 antibody compositions of the invention can include the antibody with K447, eliminate all K447 antibody or have and antibody without K447 residues mixture.

N- glycosylation sites in IgG are located at the Asn297 in CH2 domains.The humanization 2H7 antibody compositions of the present invention include the composition of any foregoing humanized 2H7 antibody with Fc areas, about 80-100% (preferably approximately 90-99%) antibody includes the ripe core carbohydrate structure for lacking fucose wherein in composition, is attached to the Fc areas of glycoprotein.Such composition confirms to show herein combines the surprising improvement of aspect with Fc γ RIIIA (F158), and Fc γ RIIIA (F158) are effective not as Fc γ RIIIA (V158) in terms of with human IgG interaction.Fc γ RIIIA (F158) are more more conventional than Fc γ RIIIA (V158) in normal, healthy African American and Caucasian.Referring to Lehrnbecher et al., Blood 94：4220(1999).In history, at Chinese hamster ovary cell (CHO), antibody of the antibody generated in one of the most frequently used industrial host comprising about 2-6% is non-fucosylated.However, YB2/0 and Lec13 can generate the antibody with the non-fucosylated types of 78-98%.Shinkawa et al., J.Bio.Chem.278 (5)：The antibody with relatively low FUT8 activity that 3466-347 (2003) reports generated in YB2/0 and Lec13 cells shows notable elevated ADCC activity in vitro.The generation of the antibody of fucose content reduction is also recorded in such as Li et al., (GlycoFi) " the Jan.2006 of Optimization of humanized IgGs in glycoengineered Pichia pastoris ", in NatureBiology online publication 22；Niwa R.et al., Cancer Res.64 (6)：2127-2133(2004)；US 2003/0157108(Presta)；US 6,602,684 and US2003/0175884 (Glycart Biotechnology)；US 2004/0093621, US 2004/0110704, US 2004/0132140 (being all Kyowa Hakko Kogyo).

Bispecific humanization 2H7 antibody covers such antibody, and wherein the one arm of antibody at least has the antigen binding domain of humanization 2H7 heavy chain of antibody of the present invention and/or L chains, and another arm has the V areas binding specificity for the second antigen.In specific embodiments, second antigen is selected from CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.

In certain embodiments, the humanization 2H7 antibody of the present invention further includes the amino acid change in IgG Fc, and show the binding affinity to people FcRn improved than the antibody with wild type IgG Fc, improve at least 60 times, at least 70 times, at least 80 times, more preferably at least 100 times, preferably at least 125 times, even more desirably at least 150 times to about 170 times.

Antibody producing

Monoclonal antibody

Monoclonal antibody can be used initially by Kohler et al., Nature 256：The hybridoma method that 495 (1975) are recorded is generated, or can be generated by recombinant DNA method (United States Patent (USP) No.4,816,567).

In hybridoma method, immune mouse as described above or other suitable host animals, such as hamster, to trigger generation or can generate the lymphocyte of following antibody, the antibody will specifically bind the protein for being used for being immunized.Or, can immunological lymphocyte in vitro.After immune, lymphocyte is separated, is then merged using suitable fusion agent such as polyethylene glycol with myeloma cell line, to form hybridoma (Goding, Monoclonal Antibodies：Principles and Practice, pp.59-103, AcademicPress, 1986).

By thus prepared hybridoma in suitable inoculation of medium and culture, the culture medium preferably comprises the one or more materials for suppressing Parent Myeloma Cell (also referred to as fusion partner) the growth or survival do not merged.For example, if Parent Myeloma Cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then the selective medium for hybridoma will typically contain hypoxanthine, aminopterin-induced syndrome and thymidine (HAT culture mediums), and these materials prevent the growth of HGPRT deficient cells.

It is preferred that fusion partner myeloma cell be that those efficiently merge, support the stable high-caliber generation antibody of selected antibody-producting cell and the myeloma cell sensitive to carrying out the selective medium of selection for the parental cell not merged.It is preferred that myeloma cell line be rat bone marrow tumour system, such as those can be from Sol gram research institute cell distributing center (Salk Institute Cell Distribution Center, SanDiego, California, USA) MOPC-21 the and MPC-11 mouse tumors obtained are derivative and can be from American type culture collection (American Type Culture Collection, Rockville, Maryland, USA) obtain SP-2 and derivative such as X63-Ag8-653.Human myeloma and mouse-people's heteromyeloma cell lines also have record (Kozbor, the J.Immunol.133 for generating human monoclonal antibodies：3001(1984)；Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).

Generation of the culture based assays that hybridoma just can be wherein being grown for the monoclonal antibody of antigen.Preferably, by immunoprecipitation or by external binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), the binding specificity of the monoclonal antibody generated by hybridoma is determined.

The binding affinity of monoclonal antibody can be for example, by Munson et al., Anal.Biochem.107：Scatchard described in 220 (1980) analyzes to determine.

Once identifying hybridoma of the generation with the antibody for expecting specificity, affinity and/or activity, the clone can be subcloned by limiting dilution flow, and be cultivated (Goding, Monoclonal Antibodies by standard method：Principles and Practice, pp.59-103, AcademicPress, 1986).Culture medium suitable for this purpose includes such as D-MEM or RPMI-1640 culture mediums.In addition, hybridoma can in animal as ascites tumor carry out In vivo culture, for example by by cell intraperitoneal injection into mouse.

Flow can be purified by conventional antibody, such as affinity chromatography (such as using albumin A or Protein G-Sepharose), ion-exchange chromatography, hydroxyapatite, gel electrophoresis, dialysis, will suitably it be separated with culture medium, ascites or serum by the monoclonal antibody of subclone secretion.

Encode the DNA old process separation easy to use and sequencing (such as by using the oligonucleotide probe for the gene that can specifically bind coding mouse heavy chain and light chain) of monoclonal antibody.Hybridoma is such DNA preferred source.Once separation, DNA can be placed in expression vector, then the expression vector is transfected into not in the host cell of generation antibody protein in addition, such as Bacillus coli cells, Simian COS cells, Chinese hamster ovary (CHO) cell or myeloma cell, to obtain the synthesis of monoclonal antibody in recombinant host cell.The summary of recombination expressions of the DNA in bacterium on encoding antibody includes Skerra et al., Curr.Opinion in Immunol.5：256-262 (1993) and Pl ü ckthun, Immunol.Revs.130：151-188(1992).

In another embodiment, can be from use McCafferty et al., Nature 348：Monoclonal antibody or antibody fragment are separated in the phage antibody library of technique construction described in 552-554 (1990).Clackson et al., Nature 352：624-628 (1991) and Marks et al., J.Mol.Biol.222：581-597 (1991) describes the separation of the mouse carried out using phage library and human antibody respectively.Subsequent publications describe human antibody (Marks the et al., Bio/Technology 10 for reorganizing generation high-affinity (nM scopes) by chain：779-783 (1992)), and combination infects and In vivo recombination is used as strategy (Waterhouse et al., the Nuc.Acids Res.21 for building very big phage library：2265-2266(1993)).In this way, these technologies are the feasible alternative methods for separating the conventional monoclonal antibody hybridoma technology of monoclonal antibody.

The DNA of encoding antibody can be modified to generate chimeric or fusion antibody polypeptide, for example, pass through employment heavy chain and constant region of light chain (C_HAnd C_L) sequence replacing homologous murine sequences (United States Patent (USP) No.4,816,567；Morrison et al., Proc.Natl.Acad.Sci.USA 81：6851 (1984)), or by the way that immunoglobulin coding sequence is merged with the coded sequence all or in part of NIg polypeptide (heterologous polypeptide).The constant region of NIg polypeptide sequence replacing antibody can be used, or the variable region of an antigen binding site of antibody is substituted with them, to produce chimeric bivalent antibody, it is comprising having a specific antigen binding site to a kind of antigen and have another specific antigen binding site to not synantigen.

Humanized antibody

This area has described the method for humanizing non-human antibodies.Preferably, humanized antibody has one or more amino acid residues introduced from non-people source.These non-human amino acid residues are often referred to as " inputting " residue, and they are typically derived from " input " variable region.Humanization can substantially follow Winter and its method for colleague carries out (Jones et al., Nature 321：522-525(1986)；Reichmann et al., Nature 332：323-327(1988)；Verhoeyen et al., Science239：1534-1536 (1988)), substitute corresponding human antibody sequence by using hypervariable region sequence.Therefore, such " humanization " antibody is chimeric antibody (United States Patent (USP) No.4,816,567), wherein substantially less than whole people variable region is substituted with the corresponding sequence from non-human species.In practice, humanized antibody is typically such human antibody, and some of some hypervariable region residues are substituted with some possible FR residues with the residue in the similar site in rodent antibodies.

It is extremely important for reduction antigenicity and HAMA (human anti-mouse antibody) response when antibody is intended for human therapeutic use by the selection of the people variable region for building humanized antibody, including light chain and heavy chains.According to so-called " most suitable " (best-fit) method, the whole library of known people's variable region sequences is screened with the variable region sequences of rodent antibodies.Identify with the immediate people's variable region sequences of rodent and receive people's framework region (FR) therein be used for humanized antibody (Sims et al., J.Immunol.151：2296(1993)；Chothia et al., J.Mol.Biol.196：901(1987)).Another method uses the specific frame area as derived from specific light chain or the consensus sequence of all human antibodies of heavy chain subgroup.Same framework can be used for several different humanized antibody (Carter et al., Proc.Natl.Acad Sci.USA 89：4285(1992)；Presta et al., J.Immunol.151：2623(1993)).

What is more important, antibody keeps the high binding affinity and other favourable biological characteristicses to antigen after humanization.In order to realize this target, according to a kind of preferred method, analyze the method for parental array and various conceptual humanized products to prepare humanized antibody by using the threedimensional model of parent and humanized sequence.Three dimensional immunoglobulin model is typically available, and is familiar with by those skilled in the art.It can be illustrated and be shown the computer program of the possible three-dimensional conformation structure of selected candidate immunoglobulin sequences sequence.Check that these display images allow to analyze possibility effect of the residue in candidate immunoglobulin sequences sequence functions, i.e. analyzing influence candidate immunoglobulin sequences combine the residue of the ability of its antigen.So, FR residues can be selected from acceptor and list entries and are combined, so as to realize desired antibody characteristic, such as the affinity to target antigen is improved.In general, some hypervariable region residues are direct and the most substantial influence being related to antigen binding.

Humanized antibody can be antibody fragment, such as Fab, and it is optionally with one or more cytotoxic agent couplings to generate immune conjugate.Or, humanized antibody can be full length antibody, such as total length IgG1 antibody.

Human antibody and phage display method

As the alternative of humanization, human antibody can be generated.For example, it is now possible to the such transgenic animals (such as mouse) of generation, they in the case of lacking endogenous immunoglobulin generation can when immune generation human antibody full repertoire.For example, having described antibody heavy chain joining region (J in chimeric and germ line mutant mice_H) gene homozygosis delete cause endogenous antibody generate complete inhibition.A large amount of human germline immunoglobulin's gene transfers will be caused into such germ line mutant mice to generate human antibody when antigen is attacked.See, for example, Jakobovits et al., Proc.Natl.Acad.Sci.USA 90：2551(1993)；Jakobovits et al., Nature 362：255-258(1993)；Bruggemann et al., Year inImmuno.7：33(1993)；United States Patent (USP) No.5,545,806,5,569,825,5,591,669 (being all GenPharm)；5,545,807；WO 97/17852.

Or, display technique of bacteriophage (McCafferty et al., Nature 348：552-553 (1990)) it can be used in vitro from immune globulin variable region (V) gene complete or collected works generation human antibody and antibody fragment from epidemic disease donor rather.According to this technology, antibody V domain genes are cloned into filobactivirus such as M13 or fd main or secondary coat protein gene in the way of meeting reading frame, and functional antibody fragment is shown as on phage particle surface.Because filamentous phage particle includes the single-stranded DNA copy of phage genome, the selection carried out based on the functional characteristic of antibody also causes coding to show that the gene of the antibody of those characteristics is selected.In this way, bacteriophage simulates some characteristics of B cell.Phage display can be carried out in a variety of forms, be summarized see, for example, Johnson, Kevin S.and Chiswell, DavidJ., Current Opinion in Structural Biology 3：564-571(1993).The Several sources of V constant gene segment Cs can be used for phage display.Clackson et al., Nature 352：624-628 (1991) isolates a large amount of different anti- oxazolones antibody from the derivative small-sized V genes random combinatorial libraries for hanging oneself immune mice spleen.Marks et al., J.Mol.Biol.222 can substantially be followed：581-597 (1991) or Griffith et al., EMBO are J.12：725-734 (1993) described technology, V genes complete or collected works and Separated pin are built to the largely not antibody of synantigen (including autoantigen) by people donor is not immunized.Referring also to United States Patent (USP) No.5,565,332 and 5,573,905.

As described above, can also pass through vitro activated B cells next life human antibodies (referring to United States Patent (USP) No.5,567,610 and 5,229,275).

Antibody fragment

In some cases, it is advantageous using antibody fragment, rather than complete antibody.The reduced size of fragment allows quick removing, and can cause to be more easily accessible to solid tumor.

The multiple technologies for generating antibody fragment are developed.Traditionally, these fragments are derived by proteolytic digestion complete antibody (see, for example, Morimoto et al., Journal of Biochemicaland Biophysical Methods 24：107-117(1992)；Brennan et al., Science 229：81(1985)).However, these fragments directly can be generated by recombinant host cell now.Fab, Fv and scFv antibody fragment all can so be allowed readily to generate these substantial amounts of fragments in expression in escherichia coli and by E. coli secretion.Can from phage antibody library discussed above isolated antibody fragment.Or, directly it can reclaim Fab '-SH fragments and chemical coupling to form F (ab ') from Escherichia coli₂Fragment (Carter et al., Bio/Technology 10：163-167(1992))., can be directly from recombinant host cell culture separation F (ab ') according to another method₂Fragment.The Fab and F (ab ') of Half-life in vivo comprising salvage receptor binding epitope residue, with extension₂Fragment is recorded in United States Patent (USP) No.5,869,046.Other technologies for generating antibody fragment will be apparent for skilled practitioner.In other embodiments, the antibody of selection is Single-Chain Fv Fragment of Murine (scFv).Referring to WO 93/16185；United States Patent (USP) No.5,571,894；And United States Patent (USP) No.5,587,458.Fv and sFv are the unique types with entire binding site, shortage constant region；In this way, they are suitable to reduce non-specific binding when using in vivo.SFv fusion proteins can be built to generate fusion of the effector protein in sFv amino or carboxyl terminal.Compiled referring to AntibodyEngineering, Borrebaeck, supra.Antibody fragment can also be " linear antibodies ", such as such as United States Patent (USP) No.5, described in 641,870.Such linear antibody fragments can be monospecific or bispecific.

Bispecific antibody

Bispecific antibody refers to the antibody for having binding specificity at least two different epitopes.Exemplary bispecific antibody can combine two kinds of different epitopes of CD20 protein.This other antibody-like can combine the binding site of CD20 binding sites and another protein.Or, the arm of anti-CD20 arms and triggering molecule such as φt cell receptor molecule (such as CD3) or IgG Fc acceptors (Fc γ R) such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16) or NKG2D or other NK cell activation parts on combination leucocyte can be combined so that cellular defence mechanisms focus on and be positioned at CD20 expression cells.Bispecific antibody can be additionally used in the cell that cytotoxic agent is positioned to expression CD20.These antibody possess CD20 combination arms and combine the arm of cytotoxic agent (such as saporin, anti-interferon-α, vinca alkaloids, ricin A chains, methotrexate (MTX) or radioactive isotope hapten).Bispecific antibody can be prepared into full length antibody or antibody fragment (such as F (ab ')₂Bispecific antibody).

WO 96/16673 describes a kind of bispecific anti-ErbB/anti- Fc γ RIII antibody, United States Patent (USP) No.5, and 837,234 disclose a kind of bispecific anti-ErbB/anti- Fc γ RI antibody.WO 98/02463 shows a kind of bispecific anti-ErbB/Fc Alpha antibodies.United States Patent (USP) No.5,821,337 has taught a kind of bispecific anti-ErbB/CD3 antibody.

Method for building bispecific antibody is known in the art.Coexpression of the traditional mode of production of total length bispecific antibody based on two pairs of heavy chain immunoglobulin-light chains, two of which chain has different specificity (Millstein et al., Nature 305：537-539(1983)).Due to being randomly assigned for heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein only a kind of have correct bispecific structure.The purifying of the correct molecule generally carried out by affinity chromatography step is fairly cumbersome and Product yields are low.J.10 similar flow is disclosed in WO93/08829 and Traunecker et al., EMBO：3655-3659(1991).

According to a kind of different method, there will be the antibody variable region for expecting binding specificity (antibody-antigen binding site) to be merged with constant region for immunoglobulin sequence.Preferably, with including at least part hinge, C_H2 and C_HMerged the immunoglobulin heavy chain constant region in 3rd area.It is preferred that there is the first heavy chain constant region (C that necessary site is combined comprising light chain at least one fusions _H1).By encoding immune immunoglobulin heavy chain fusions thing and, when needed, the DNA of light chain immunoglobulin inserts separated expression vector, and cotransfection is into suitable host cell.There is provided when not waited for the three of structure kind polypeptide chain ratio in the embodiment for the optimum point of production for expecting bispecific antibody, this provides greater flexibility to adjust the mutual ratio of three kinds of polypeptide fragments.However, when at least two polypeptide chains cause high yield with same ratio expression or when the ratio is to it is expected that the yield of chain combination has no significant effect, it is possible to which the coded sequence of two kinds or all three polypeptide chains is inserted into single expression vector.

In a preferred embodiment of this method, bispecific antibody has the hybrid immunoglobulin heavy chain of the first binding specificity on an arm, and hybrid immunoglobulin heavy chain-light chain on another arm is constituted to (providing the second binding specificity).Due to the separating pathway that presence of the light chain immunoglobulin only in half of bispecific molecule is provided convenience, consequently found that this dissymmetrical structure is easy to separate desired bispecific compound with undesired immunoglobulin chain combinations.This method is disclosed in WO94/04690.On generating the further details of bispecific antibody see, for example, Suresh et al., Methods in Enzymology 121：210(1986).

According to United States Patent (USP) No.5, another method described in 731,168 can transform the interface between a pair of antibody molecules, by the percent maximum of the heterodimer reclaimed from recombinant cell culture thing.It is preferred that interface include at least part C_H3 domains.In the method, one or more small amino acid side chains of first antibody molecular interface are replaced with larger side chain (such as tyrosine or tryptophan).By the way that big amino acid side chains are replaced with compared with small amino acid side chains (such as alanine or threonine), compensatory " cavity " with the same or similar size of bulky side chain is produced on the interface of secondary antibody molecule.This provides the mechanism that heterodimer yield is improved than other undesired end-products such as homodimer.

Bispecific antibody includes crosslinking or " Heteroconjugate " antibody.For example, a kind of antibody in Heteroconjugate thing can be coupled with avidin, another antibody is coupled with biotin.For example, this antibody-like by immune system cell it has been proposed that for targetting undesired cell (United States Patent (USP) No.4,676,980), and for treating HIV (WO 91/00360, WO 92/200373 and EP 03089).Any easily cross-linking method can be used to prepare Heteroconjugate antibodies.Suitable crosslinking agent is well-known in the art, and United States Patent (USP) No.4,676,980 are disclosed in together with many crosslinking technologicals.

The technology that bispecific antibody is generated by antibody fragment is also described in document.It is connected chemically to prepare bispecific antibody for example, can be used.Brennan et al., Science 229：81 (1985) are described cuts complete antibody to generate F (ab ') by proteolysis₂The flow of fragment.These fragments are reduced in the case where there are two mercaptan complexing agent sodium arsenites, to stablize two neighbouring mercaptan and prevent the formation of intermolecular disulfide bond.Then Fab ' the fragments of generation are changed into thionitrobenzoate ester (TNB) derivative.Then one of Fab '-TNB derivatives are reverted into Fab '-mercaptan by the reduction of mercaptoethylmaine again, and mixed with another Fab '-TNB derivatives of equimolar amounts, to form bispecific antibody.The bispecific antibody of generation can be used as the selective immobilized reagent of enzyme.

Most new progress is easy to directly reclaim Fab '-SH fragments from Escherichia coli, and these fragments are chemically coupled to form bispecific antibody.Shalaby et al., J.Exp.Med.175：217-225 (1992) describes the bispecific antibody F (ab ') of full-length human₂The generation of molecule.Every kind of Fab ' fragments are separately secreted by Escherichia coli, and are oriented chemical coupling in vitro to form bispecific antibody.The bispecific antibody being thusly-formed can combine cell and the normal human T cells for being overexpressed ErbB2 acceptors, and triggering people's cell Cytotoxic Lymphocytes for the dissolving activity of people's lacteal tumor target.

Also describe the multiple technologies for directly generating and separating bispecific antibody fragment from recombinant cell culture thing.For example, generating bispecific antibody using leucine zipper.Kostelny et al., J.Immunol.148 (5)：1547-1553(1992).Leucine zipper peptide from Fos and Jun albumen is connected by Gene Fusion with the Fab ' parts of two kinds of different antibodies.Antibody homodimer is reduced to form monomer in hinge area, then reoxidized to form antibody heterodimer.This method can also be used for generating antibody homodimer.Hollinger et al., Proc.Natl.Acad.Sci.USA90：" double antibody " technology that 6444-6448 (1993) is recorded provides the replacement mechanism for building bispecific antibody fragment.The fragment includes the V being connected by joint_HAnd V_L, the joint too it is short cause same chain on two domains between can not match.Therefore, the V in a fragment is forced_HAnd V_LDomain and the complementary V in another fragment_LAnd V_HDomain is matched, and is consequently formed two antigen binding sites.It is also reported that building another strategy of bispecific antibody fragment by using scFv (sFv) dimer.Referring to Gruber et al., J.Immunol.152：5368(1994).

Contemplate the antibody with more than two potency.For example, three-specific antibody can be prepared.Tutt et al., J.Immunol.147：60(1991).

Multivalent antibody

Multivalent antibody can the internalization (and/or alienation) by the cell for expressing the antibody combination antigen more faster than bivalent antibody.The antibody of the present invention can readily be generated, the multivalent antibody with three or more antigen binding sites (such as tetravalent antibody) by the recombination expression of the nucleic acid of encoding antibody polypeptide chain (beyond IgM classifications).Multivalent antibody can include dimerization domain and three or more antigen binding sites.It is preferred that dimerization domain include (or being made from it) Fc areas or hinge area.In this case, antibody is by three or more antigen binding sites comprising Fc areas and Fc areas amino terminal.Multivalent antibody preferred herein includes (or being made from it) three to about eight, but preferably four antigen binding sites.Multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains.For example, polypeptide chain can include VD1- (X1)_n-VD2-(X2)_n- Fc, wherein VD1 are the first variable domains, and VD2 is the second variable domains, and Fc is a polypeptide chain in Fc areas, X1 and X2 represented amino acids or polypeptide, and n is 0 or 1.For example, polypeptide chain can be included：VH-CH1- flexible joint-VH-CH1-Fc areas chain；Or VH-CH1-VH-CH1-Fc areas chain.Multivalent antibody herein is preferably further comprising at least two (and preferably four) light variable domains polypeptides.Multivalent antibody herein can include e.g., from about two to about eight light variable domains polypeptides.The light variable domains polypeptide contemplated herein includes light variable domains, and optionally further includes CL domains.

Other amino acid sequence modifications

Contemplate the amino acid sequence modifications of CD20 binding antibodies described herein.For example, it may be desirable to improve the binding affinity and/or other biological characteristicses of antibody.By the way that suitable nucleotides change is introduced into anti-CD 20 antibodies nucleic acid or the amino acid sequence variation of anti-CD 20 antibodies is prepared by peptide symthesis.The residue that such modification is included in such as anti-CD 20 antibodies amino acid sequence is deleted and/or insertion and/or replacement.As long as final construction has desired characteristic, any combination that can be deleted, inserted and be substituted is to obtain final construction.Amino acid change can also change the post translational processing of anti-CD 20 antibodies, such as change number or the position of glycosylation site.

Available for as some residues of preferred mutagenesis position or a kind of method in region being referred to as " alanine scanning mutagenesis " in identification anti-CD 20 antibodies, such as Cunningham and Wells, Science244：Described in 1081-1085 (1989).Here, identify a residue or one group of target residue (such as electrically charged residue, such as arginine, aspartic acid, histidine, lysine and glutamic acid), and replaced with neutral or negatively charged amino acid (most preferably alanine or polyalanine), to influence the interaction of amino acid and CD20 antigens.Then by introducing more or other variants or to alternate site, those amino acid positions that function sensitive is shown to replacement are weighed.So, although it is pre-determined to introduce the site of variant amino acid sequence, but the property of mutation itself need not be predetermined.For example, the consequence in order to analyze the mutation to anchor point, carries out Alanine-scanning or random mutagenesis, and expect activity to expressed anti-CD 20 antibodies variant screening in target codon or region.

Amino acid sequence insertion includes the fusion of amino and/or carboxyl terminal, length range from a residue to the polypeptide for including up to a hundred or more residues, and the sequence of single or multiple amino acid residues in insertion.The example of end insertion includes the anti-CD 20 antibodies with N- terminal methionyl residues or the antibody merged with cytotoxic polypeptide.Other insertion variants of anti-CD 20 antibodies molecule are included in N- the or C- terminal fusions enzyme (such as ADEPT) of anti-CD 20 antibodies or improve the polypeptide of antibody serum half-life period.

Another kind of variant is amino acid substitution variant.These variants have at least one amino acid residue to be replaced with different residues in anti-CD 20 antibodies molecule.The most interested site for substitute mutagenesis includes hypervariable region, but also contemplates FR changes.Conservative replacement is shown under title " preferably substituting " in the following table.If such replacement causes the change of biological activity, then can be introduced into table and be referred to as further describing on amino acid classes in the more substantial variation, or following article of " illustrate and substitute ", and screen product.

Amino acid replacement table

Original Residue	Illustrate and substitute	It is preferred that substituting
			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；Nor-leucine	Leu
Leu(L)	Nor-leucine；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；Nor-leucine	Leu

To the substantive sex modification of antibody biological characteristics by selecting dramatically different replacement in the effect for keeping following aspect to complete：(a) structure of the polypeptide backbone of replacement area, such as pleated sheet or helical conformation, (b) target site punishment son electric charge or hydrophobicity, or (c) side chain volume.Based on common side chain properties, naturally occurring residue is grouped as follows：

(1) it is hydrophobic：Nor-leucine, Met, Ala, Val, Leu, Ile；

(2) it is neutral, hydrophilic：Cys、Ser、Thr；

(3) it is acid：Asp、Glu；

(4) it is alkaline：Asn、Gln、His、Lys、Arg；

(5) residue of chain orientation is influenceed：Gly、Pro；With

(6) it is aromatic：Trp、Tyr、Phe.

Non-conservative replacement will need the member with one of these classifications to exchange another classification.

Any cysteine residues for not involving the holding correct conformation of anti-CD 20 antibodies are also alternative, generally with serine, to improve the oxidation stability of molecule and prevent abnormal crosslinking.On the contrary, cysteine key can be added into antibody to improve its stability (particularly when antibody is antibody fragment such as Fv fragments).

Particularly preferred class alternative variations involve the one or more some hypervariable region residues for substituting parental antibody (such as humanization or human antibody).It is typically chosen for the gained variant further developed relative to producing their parental antibody by the biological characteristics with improvement.A kind of facilitated method for producing such alternative variations involves the affinity maturation for using phage display.Briefly, several hypervariable region sites (such as 6-7 site) are mutated, all possible amino acid replacement is produced in each site.The antibody variants so produced are illustrated on filamentous phage particle with monovalent fashion, are used as the fusions with the M13 gene III products of each particle inner packing.Then its biological activity (such as binding affinity) is screened to the variant of phage display as disclosed herein.In order to identify the candidate hypervariable region site for modification, alanine scanning mutagenesis can be carried out to identify some hypervariable region residues that there is antigen binding significant contribution.Or the crystal structure of analysis antigen-antibody complex is probably beneficial with the contact point identified between antibody and h CD20.Such contact residues and neighbouring residue are the candidate locus substituted according to technology detailed in this article.Once producing such variant, this group of variant is screened with regard to as described herein, the antibody with good characteristic in one or more relevant assays, which may be selected, to be used to further develop.

The another kind of amino acid variant of antibody changes the original glycosylation pattern of antibody.Changing means to delete non-existent one or more glycosylation sites in the one or more carbohydrate moieties found in antibody, and/or addition antibody.

The typical N- connections of the glycosylation or O- connections of antibody.N- connections refer to the side chain that carbohydrate moiety is attached to asparagine residue.Tripeptide sequence asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid in addition to proline) are the recognition sequences that carbohydrate moiety enzymatic is attached to asparagine side chain.In this way, any presence of the tripeptide sequence of both in polypeptide generates potential glycosylation site.The glycosylation of O- connections refers to is attached to hydroxy-amino-acid, most commonly serine or threonine by one of sugars N-aceylgalactosamine, galactolipin or xylose, but 5-OxoPro or 5- hydroxylysines can also be used.

It is by changing (glycosylation site for being used for N- connections) that amino acid sequence makes it easily be completed comprising one or more above-mentioned tripeptide sequences that glycosylation site is added into antibody.It can be also changed by the way that one or more serines or threonine residues are added or substituted in the sequence to original antibodies (glycosylation site for being used for O- connections).

It is prepared by a variety of methods that encoding the nucleic acid molecules of the amino acid sequence variation of anti-CD 20 antibodies can be known by this area.These methods include but is not limited to from natural origin separation (in the case of naturally occurring amino acid sequence variation), or carry out oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis and cassette mutagenesis to prepare by the variant or the anti-CD 20 antibodies of non-variant pattern that prepare early stage.

It may want to modify the antibody of the present invention in terms of effector functions, for example, strengthen the cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody dependent cellular mediation of antibody.This can be realized by introducing one or more amino acid replacements in antibody Fc district.Or cysteine residues are introduced in Ke Fc areas, so as to allow to form interchain disulfide bond in this zone.The homodimer antibody so produced can have the cell killing and the cytotoxicity (ADCC) of antibody dependent cellular of the internalization ability of improvement and/or the complement-mediated of raising.Referring to Caron et al., J.Exp.Med.176：1191-1195 (1992) and Shopes, B., J.Immunol.148：2918-2922(1992).Homodimer antibody with enhanced antitumor activity can also be such as Wolff et al., Cancer Research53：In 2560-2565 (1993) prepared by described use heterobifunctional crosslinker.Or, antibody can be transformed into dual Fc areas, thus can have dissolving and the ADCC abilities of enhanced complement-mediated.Referring to Stevenson et al., Anti-Cancer Drug Design 3：219-230(1989).

, can be as described in 739,277 that salvage receptor binding epitope is mixed into antibody (especially antibody fragment) such as such as United States Patent (USP) No.5 in order to extend the serum half-life of antibody.As used herein, term " salvage receptor binding epitope " refers to IgG molecules (such as IgG₁、IgG₂、IgG₃Or IgG₄) Fc areas in be responsible for extension IgG molecule bodies in serum half-life epitope.

Other antibody modifications

Other modifications of antibody have been contemplated herein.For example, one of antibody and a variety of non-proteinaceous polymers can be connected, such as the copolymer of polyethylene glycol, polypropylene glycol, polyoxyalkylene or polyethylene glycol and polypropylene glycol.Antibody can also be contained in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule) or in macro emulsion.Such technology is disclosed in Remington ' sPharmaceutical Sciences, and the 16th edition, Osol, A. is compiled, and 1980.

Antibody of the screening with desired characteristic

The antibody with some biological characteristicses can be selected as described in EXPERIMENTAL EXAMPLESThe.

Method that the growth inhibitory effect of anti-CD 20 antibodies of the present invention can be known by this area is assessed, such as using endogenic or express after being transfected with CD20 genes CD20 cell.For example, can handle tumor cell line and CD20 transfectional cells several days (such as 2-7 days) with the anti-CD-20 monoclonal antibody of the present invention of a variety of concentration, and dyed, or analyzed by some other colorimetric methods with crystal violet or MTT.Another method for measuring propagation will be by comparing the cell handled when existing or lacking anti-CD 20 antibodies of the present invention³H- thymidines are absorbed.After antibody processing, harvesting is simultaneously quantified in scintillation counter to the radioactive amount for mixing DNA.Suitable positive control includes handling the cell line with the known growth inhibiting antibody for suppressing selected cell line growth.

In order to select the antibody of inducing cell death, it can assess and be lost for example, by propidium iodide (PI), trypan blue or the 7AAD film integrality for absorbing display relative to control.PI intakes determination method can be carried out when lacking complement and immune effector cell.Single culture medium or containing concentration for the e.g., from about culture medium of 10 μ g/ml Suitable monoclonal antibodies in incubate expression CD20 tumour cell.By the cell culture time limit of 3 days.After per treatment, cell is cleaned and is distributed to 35mm and is stamped in 12 × 75 pipes of filter screen (strainer-capped) (often pipe 1ml, each treatment group 3 manage) to remove cell mass.Then PI (10 μ g/ml) is added into pipe.FACSCAN can be used^TMFlow cytometer and FACSCONVERT^TMCellQuest softwares (Becton Dickinson) analyze sample.The antibody of the cell death of statistical significant level can be induced to be elected to be the antibody of inducing cell death those measure absorbed according to PI.

In order to screen the antibody of the epitope combined with reference to purpose antibody on CD20, conventional cross can be carried out and block determination method, such as Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane, it is described in 1988.This determination method can be used for determine test antibody whether with the present invention anti-CD 20 antibodies combination same loci or epitope.Or the method that can be known by this area carries out epitope mapping.For example, mutagenesis can be carried out to antibody sequence, such as by Alanine-scanning, to identify contact residues.Test the combination of polyclonal antibody to Mutant Antibodies to ensure correct folding first.In a kind of different method, can by corresponding to the peptide of CD20 different zones and test antibody or with test antibody and with characterized or the antibody of known epitope together be used for competition assay.

Carrier, host cell and recombination method

Recombinant technique present invention also offers the nucleic acid of the separation of encoding humanized 2H7 variant antibodies, the carrier comprising the nucleic acid and host cell and for producing the antibody.

For recombinant production antibody, the nucleic acid of encoding antibody is separated, and inserts replicable vector, for further cloning (DNA cloning) or expressing.Encode the DNA old process separation easy to use of monoclonal antibody and be sequenced (such as by using the oligonucleotide probe that can be combined with the gene specific of encoding antibody heavy and light chain).Many carriers can be obtained.Support element typically includes, but not limited to following one or more：Signal sequence, replication orgin, one or more marker gene, enhancer element, promoter and transcription terminator.

(i) signal sequence component

The present invention humanization 2H7 antibody not only can directly recombinant production, and can be as the fused polypeptide with heterologous polypeptide, the heterologous polypeptide is preferably the signal sequence of the N- ends of mature protein or polypeptide or other polypeptides with specific cleavage site.Selected Heterologous signal sequences are preferably by host cell recognizes and processes and (is cut by signal peptidase).For nonrecognition and the prokaryotic host cell of the natural CD20 binding antibodies signal sequence of processing, the signal sequence is replaced with the prokaryotic signal sequence for being selected from alkaline phosphatase, penicillase, lpp or Thermostable α-amylase II targeting sequencings.For yeast secretary, signal sequences native can be replaced with the signal described in such as yeast invertase leader, α factor leaders (including saccharomyces and genus Kluyveromyces α factor leaders), acid phosphatase leader, Candida albicans glucoamylase leader or WO 90/13646.In mammalian cell expression, it is possible to use mammalian signal sequences and viral secretory leaders, such as herpes simplex gD signal.

DNA by the DNA of such prosoma (precursor region) with encoding humanized 2H7 antibody in the way of meeting reading frame is connected.

(ii) replication orgin component

Expression and cloning vector, which are all included, can make the nucleotide sequence that carrier is replicated in one or more selected host cells.Generally, in cloning vector, this sequence is the sequence that carrier can be made to be replicated independent of host chromosome DNA, including replication orgin or autonomously replicating sequence.It is known that such sequence of various bacteria, yeast and virus.Replication orgin from pBR322 plasmid is suitable for most of gramnegative bacteriums, 2 μ plasmid origins are suitable for yeast, and various viral origins (SV40, polyomavirus, adenovirus, VSV or BPV) are available for the cloning vector in mammalian cell.Generally, mammalian expression vector does not need replication orgin component (use of SV40 starting points generally may be simply because it and include early promoter).

(iii) Select gene component

Expression and cloning vector can include Select gene, also referred to as selection marker.Typical Select gene encodes following protein：(a) resistance to antibiotic or other toxin, such as ampicillin, neomycin, methotrexate (MTX) or tetracycline are assigned；(b) auxotrophic defect is supplied；Or (c) provides the required nutrients that can not be obtained by complex medium, for example for bacillus encoding D-alanine racemase gene.

One example of selection scheme blocks the growth of host cell using medicine.The protein of drug resistance is assigned with those Hemapoiesis of heterologous gene successful conversion, thus survives selection scheme.The example of such dominant selection uses drug neomycin, mycophenolic acid and hygromycin.

Another example suitable for the selection marker of mammalian cell be those can identify the encoding humanized 2H7 antibody of intake of having the ability nucleic acid cell selection marker, DHFR, thymidine kinase, metallothionein-I and the preferred primate metallothionein's genes of-II, adenosine deaminase, ornithine decarboxylase etc..

For example, first by the way that all transformants are identified into the cell converted through DHFR Select genes containing methotrexate (MTX) (Mtx), being cultivated in a kind of culture medium of DHFR competitive antagonist.When using wild type DHFR, suitable host cell is Chinese hamster ovary (CHO) cell line (such as ATCC CRL-9096) of DHFR active defects.

Or, DNA sequence dna conversion or the host cell (wild-type host for particularly including endogenous DHFR) of cotransformation of encoded humanization 2H7 antibody, wild type DHFR protein matter and another selection marker such as aminoglycoside 3 '-phosphotransferase (APH) can be selected by growth of the cell in containing such as aminoglycoside antibiotics of the selective agent for selection marker such as kanamycins, neomycin or G418 culture medium.Refering to United States Patent (USP) No.4,965,199.

It is trp1 genes (Stinchcomb the et al., Nature 282 that is present in yeast plasmid YRp7 suitable for the Select gene of yeast：39(1979)).Trp1 bases are in default of the yeast mutant of the growth ability in tryptophan, and such as ATCC No.44076 or PEP4-1 are there is provided selection marker.Jones, Genetics 85：12(1977).There are trp1 damages in yeast host cell genome to provide therewith for detecting the effective environment of conversion by the growth when lacking tryptophan.Similar, supply Leu2 defective yeasts bacterial strain (ATCC 20,622 or 38,626) with the known plasmid for carrying Leu2 genes.

In addition, the carrier derived from 1.6 μm of cyclic plasmid pKD1 can be used for conversion genus Kluyveromyces yeast.Or, it has been reported that the expression system for the large-scale production restructuring calf chymosin in Kluyveromyces lactis.Van den Berg, Bio/Technology 8：135(1990).Further disclose for the albuminised stable multicopy expression vector of the ripe recombinant human serum of industrial strain secretes by genus Kluyveromyces.Fleer et al., Bio/Technology 9：968-975(1991).

(iv) promoter component

Expression and cloning vector generally comprise the promoter recognized by host organisms, and the nucleic acid of it and encoding humanized 2H7 antibody is operatively connected.Promoter suitable for prokaryotic hosts includes phoA promoters, beta-lactamase and lactose promoter system, alkaline phosphatase promoter, tryptophan (trp) promoter systems and hybrid promoter such as tac promoters.However, other known promoters are also suitable.Promoter for bacterial system is also by comprising with encoding Shine-Dalgarno (S.D.) sequence that the DNA of CD20 binding antibodies is operatively connected.

The promoter sequence of known eukaryotic.In fact, all eukaryotic genes, which all have, is rich in AT areas, it is located at about 25 to 30 bases of site upstream of starting transcription.Another sequence found at the base of transcriptional start point upstream 70 to 80 of many genes is CNCAAT areas, and wherein N can be any nucleotides.It is AATAAA sequences at 3 ' ends of most of eukaryotic genes, it is probably the signal of the 3 ' end addition poly A tails to coded sequence.All these sequences suitably insert carrier for expression of eukaryon.

Include the promoter of glycerol 3-phosphate acid kinase or other glycolytic ferments, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, GPI, 3-phoshoglyceric acid mutase, pyruvate kinase, phosphotriose isomerase, glucose phosphate isomerase and glucokinase suitable for the example of the promoter sequence of yeast host.

It is the promoter region of the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, the digestive enzyme relevant with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase and responsible maltose and galactose utilization as other Yeast promoters of the inducible promoter with the additional advantage that transcription is controlled by growth conditions.Further stated that suitable for the carrier and promoter of Yeast expression in EP 73,657.Yeast enhancers favorably can also be used together with Yeast promoter.

Humanization 2H7 antibody is transcribed by for example from viral such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B and most preferred simian virus 40 (SV40) genome by carrier in mammalian host cell, from heterologous mammal promoter such as actin promoter or immunoglobulin promoter, and the control of the promoter of this heat-shock promoters acquisition, if if such promoter is compatible with host cell systems.

The early and late promoter of SV40 viruses is easily obtained in the form of SV40 restriction fragments, the fragment also includes SV40 virus origin of replication.The immediate early promoter of human cytomegalovirus is easily obtained in the form of HindIII E restriction fragments.The system for using bovine papilloma virus as carrier DNA being expressed in mammalian hosts is disclosed in United States Patent (USP) No.4,419,446.A kind of improvement of the system has been recorded in United States Patent (USP) No.4,601,978.Reyes et al., Nature 297 is also see on control following table intelligent's beta-interferon cDNA in the thymidine kinase promoter from herpes simplex virus in mouse cell：598-601(1982).Or, Rous sarcoma virus LTR can be used as promoter.

(v) enhancer element component

Transcription of the higher eucaryotic cells to the DNA of coding the present inventor source 2H7 antibody is improved often through by enhancer sequence insertion vector.Many enhancer sequences from mammalian genes (globulin, elastoser, albumin, alpha-fetoprotein and insulin) that it is now know that.However, usually using the enhancer from eukaryotic cell virus.Example includes enhancer (bp100-270), the sub- enhancer of cytomegalovirus early promoter, the enhancer and adenovirus cancers of polyomavirus replication orgin late period side of SV40 replication orgins late period side.Reinforcing element on activating eukaryotic promoter also see Yaniv, Nature 297：17-18(1982).Can be by enhancer montage into carrier, positioned at 5 ' or 3 ' positions of CD20 binding antibody coded sequences, it is preferred that positioned at 5 ' sites of promoter.

(vi) tanscription termination component

Expression vector for eukaryotic host cell (yeast, fungi, insect, plant, animal, people or karyocyte from other multicellular organisms) will also be comprising terminating transcription and sequence necessary to stable mRNA.Such sequence can generally be obtained from 5 ' ends of eucaryon or viral DNA or cDNA non-translational regions with 3 ' ends once in a while.These regions are included in the nucleotide segment that polyadenylated fragments are transcribed into the mRNA of coding CD20 binding antibodies untranslated part.A kind of useful tanscription termination component is bovine growth hormone polyadenylation area.Refering to WO 94/11026 and the expression vector wherein disclosed.

(vii) selection and conversion of host cell

Host cell suitable for cloning or expressing the DNA in this paper carriers is above-described prokaryotes, yeast or higher eucaryotic cells.Prokaryotes suitable for this purpose include eubacteria, such as gram negative organism or gram-positive organism, such as enterobacteriaceae, such as Escherichia (Escherichia) such as ETEC (E.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonella (Salmonella) such as salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia) such as serratia marcescens (Serratia marcescans), Shigella (Shigella), and bacillus (Bacilli) such as bacillus subtilis (B.subtilis) and bacillus licheniformis (the B.licheniformis) (DD 266 that on April 12nd, 1 announces, the bacillus licheniformis 41P disclosed in 710), pseudomonas (Pseudomonas) such as pseudomonas aeruginosa (P.aeruginosa), with streptomyces (Streptomyces).A kind of preferred escherichia coli cloning host is the (ATCC 31 of Escherichia coli 294,446), although other bacterial strains such as Escherichia coli B, Escherichia coli X1776 (ATCC31,537) and Escherichia coli W3110 (ATCC 27,325) are also suitable.These examples are exemplary, rather than restricted.

Full length antibody, antibody fragment and antibody fusion protein can be produced in bacterium, particularly when that need not glycosylate with Fc effector functions, such as when being coupled treatment with antibody and cytotoxic agent (such as toxin) and immune conjugate itself shows the effect in terms of destroying tumour cell.Full length antibody has longer half-life period in the circulating cycle.Production in Escherichia coli is more rapidly and less expensive.On expressing antibody fragment and polypeptide in bacterium, see, for example, U.S.5,648,237 (Carter et al.), U.S.5,789,199 (Joly et al.) and U.S.5,840,523 (Simmons et al.), they describe the Translation initiator (TIR) and signal sequence for Optimal Expression and secretion, and these patents are taken in herein as reference.After expression, separation antibody is pasted from Bacillus coli cells in soluble fraction, can be for example, by selecting albumin A or G posts to be purified according to isotype.Final purifying can be similar with the process for purifying the antibody for example expressed in Chinese hamster ovary celI progress.

Beyond prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also the suitable clones or expressive host for the carrier for encoding CD20 binding antibodies.Saccharomyces cerevisiae (Saccharomyces cerevisiae) or conventional Saccharomyces cerevisiae are the most frequently used low eucaryon host microorganisms.However, can generally obtain many other genus and species and bacterial strain and available for the present invention, such as grain wine pombe (Schizosaccharomyces pombe)；Kluyveromyces (Kluyveromyces) host, such as Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis) (ATCC 12, 424), Bulgarian kluyveromyces (K.bulgaricus) (ATCC 16, 045), Brunswick kluyveromyces (K.wickeramii) (ATCC 24, 178), K.waltii (ATCC 56, 500), drosophila kluyveromyces (K.drosophilarum) (ATCC 36, 906), Kluyveromyces thermotolerans (K.thermotolerans) and Kluyveromyces marxianus (K.marxianus)；Yarrow saccharomyces (Yarrowia) (EP 402,226)；Pichia pastoris phaff (Pichia pastoris) (EP 183,070)；Candida (Candida)；Filamentous fungi (Trichoderma reesia) (EP 244,234)；Neuraspora crassa (Neurospora crassa)；Perhaps prosperous saccharomyces (Schwanniomyces), all so prosperous yeast (Schwanniomyces occidentalis)；And filamentous fungi, such as Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (Tolypocladium) and aspergillus (Aspergillus) host such as aspergillus nidulans (A.nidulans) and aspergillus niger (A.niger).

Host cell suitable for expression glycosylation humanization 2H7 antibody is derived from multicellular organisms.The example of invertebral zooblast includes plant and insect cell.Many baculoviral strains and variant are identified and have allowed insect host cell accordingly, they are from hosts such as fall army worm Spodoptera frugiperda (caterpillar), Aedes aegypti Aedes aegypti (mosquito), aedes albopictus Aedes albopictus (mosquito), Drosophila melanogaster Drosophila melanogaster (drosophila) and silkworm Bombyx mori.The public, which can obtain a variety of Strain, to be used to transfect, such as autographa california Autographa californica NPV L-1 variants and silkworm Bombyx mori NPV Bm-5 strains, and this viroid can be used as virus herein according to the present invention, particularly for transfecting Spodopterafrugiperda cells.

Also host is used as using the plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco.

However, vertebrate cells are at most paid close attention to, and the breeding of vertebrate cells has become old process in culture (tissue cultures).The example of useful mammalian host cell line is the monkey kidney CV1 systems (COS-7, ATCC CRL 1651) converted with SV40；Human embryonic kidney cell line (293 or in order to suspend culture in growth and be subcloned 293 cells, Graham et al., J.Gen Virol.36：59(1977))；Baby hamster kidney cell (BHK, ATCC CCL 10)；Chinese hamster ovary cell/- DHFR (CHO, Urlaub et al., Proc.Natl.Acad.Sci.USA 77：4216 (1980)) or CHO-OP-12 systems；Mouse Sai Tuoli (sertoli) cells (TM4, Mather, Biol.Reprod.23：243-251(1980))；MK cells (CV1, ATCC CCL70)；African green monkey kidney cell (VERO-76, ATCC CRL-1587)；Human cervical carcinoma cell (HELA, ATCC CCL2)；MDCK (MDCK, ATCC CCL34)；Ox mouse (buffalo rat) liver cell (BRL 3A, ATCC CRL 1442)；Human pneumonocyte (W138, ATCC CCL75)；Human liver cell (Hep G2, HB 8065)；Mouse mammary tumor (MMT 060562, ATCC CCL 51)；TRI cells (Mather et al., Annals N.Y.Acad Sci.383：44-68(1982)；MRC5 cells；FS4 cells；With people's hepatoma system (Hep G2).

Host cell is converted with the expression described above for being used to produce CD20 binding antibodies or cloning vector, and is cultivated in for evoked promoter, the conventional nutrient culture for selecting transformant or the gene of amplification coding expectation sequence and suitably changing.

(viii) host cell is cultivated

The host cell for producing CD20 binding antibodies of the present invention can be cultivated in a variety of culture mediums.Commercially available culture medium such as HamShi F10 (Sigma), minimum essential medium (MEM, Sigma), the EagleShi culture mediums (DMEM, Sigma) of RPMI-1640 (Sigma) and DulbeccoShi changes are suitable to culture host cell.Further, it is possible to use the culture medium of any culture medium described in following documents as host cell：Ham et al., Meth.Enz.58：44(1979)；Barnes et al., Anal.Biochem.102：255(1980)；United States Patent (USP) No.4,767,704；4,657,866；4,927,762；4,560,655；Or 5,122,469；WO 90/03430；WO 87/00195；Or United States Patent (USP) review 30,985.These any culture mediums can hormone supplemented and/or other growth factors (such as insulin, transferrin or EGF), salt (such as sodium chloride, calcium, magnesium and phosphate), buffer (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotic (such as GENTAMYCIN as needed^TMMedicine), trace element (being defined as the inorganic compound generally existed with the final concentration of micro-molar range) and glucose or the equivalent energy.Can also with suitable concentration include those skilled in the art will know that any other required supplement.Condition of culture, temperature, pH etc., previously selected for expression for host cell, this is obvious for those of ordinary skill.

(ix) purifying of antibody

When using recombinant technique, can in the cell, antibody is generated in periplasmic space, or be directly secreted into culture medium.If generating antibody in the cell, then as the first step, the particle debris of host cell or crack fragment is removed for example, by centrifugation or ultrafiltration.Carter et al., Bio/Technology10：163-167 (1992) describes the flow of the antibody for being secreted into colibacillus periplasm space.Briefly, cell paste is made to melt when there is sodium acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) about 30 minutes.Cell fragment can be removed by centrifugation.If by antibody-secreting into culture medium, then generally first by commercialization protein concentration filter, such as supernatant of the Amicon or Millipore Pellicon ultra filtration units concentration from such expression system.In any above-mentioned steps, protease inhibitors such as PMSF can be included to suppress proteolysis, and antibiotic can be included to prevent the growth of external contaminant.

Such as hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography can be used to purify the antibody compositions prepared by cell, purification technique preferably is affinity chromatography.Albumin A depends on the species and isotype of any immunoglobulin fc region present in antibody as the suitability of affinity ligand.Albumin A can be used for antibody (Lindmark et al., J.Immunol.Meth.62 of the purifying based on people γ 1, γ 2 or the heavy chains of γ 4：1-13(1983)).Protein G recommends to be used for all mouse isotypes and people γ 3 that (Guss et al., EMBO are J.5：1567-1575(1986)).Matrix accompanying by affinity ligand is most commonly used that agarose, but can use other matrix.The matrix of physically stable such as controlled pore glass or poly- (styrene divinyl) benzene result in flow velocity more faster than agarose and shorter process time.If antibody includes C_H3 domains, then can be used Bakerbond ABX^TMResin (J.T.Baker, Phillipsburg, NJ) is purified.According to antibody to be recycled, it is possible to use classification, ethanol precipitation, reversed-phase HPLC on other oroteins purification technique, such as ion exchange column, the chromatography on tripoli, heparin SEPHAROSE^TMOn chromatography, anion or cationic ion-exchange resin (such as poly-aspartate post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preparation property purification step, the mixture comprising purpose antibody and pollutant can carry out low pH hydrophobic interaction chromatographies, using the elution buffer between pH about 2.5-4.5, preferably carry out (such as about 0-0.25M salt) in low salt concn.

Antibody coupling matter

Antibody can be coupled with cytotoxic agent such as toxin or radio isotope.In certain embodiments, the toxin is Calicheamicin (calicheamicin), maytansinoids (maytansinoid), dolastatin (dolastatin), auristatin E and the like or derivative.

It is preferred that medicine/toxin include the inhibitor and antimetabolite of DNA damage agent, microtubule polymerization or depolymerization.The preferred classes of cytotoxic agent include such as enzyme inhibitor such as dihydrofolate reductase inhibitor and thymidilate synthase inhibitors, DNA intercalators, DNA cutting agents, topoisomerase enzyme inhibitor, anthracycline antibiotic (anthracycline) medicine family, Changchun class (vinca) medicine, mitomycin (mitomycin), bleomycin (bleomycin), cytotoxic nucleoside, pteridine (pteridine) medicine family, two acetylenic antibiotic (diynenes), podophyllotoxin (podophyllotoxin) and differentiation inductor.The particularly useful member of those classifications includes such as methopterin (methotrexate), methotrexate (MTX) (methopterin), dichloro methopterin (dichloromethotrexate), 5 FU 5 fluorouracil (fluorouracil), Ismipur (mercaptopurine), cytarabine (cytosine arabinoside), melphalan (melphalan), leurosine (leurosine), inrosidine (leurosidine), D actinomycin D (actinomycin), daunorubicin (daunorubicin), Doxorubicin (doxorubicin), N- (5, 5- diacetoxies amyl group) Doxorubicin, morpholino-Doxorubicin, 1- (2- chloroethyls) -1, 2- dimethyl methyls hydrazides (1- (2-choroehthyl) -1, 2-dimethanesulfonyl hydrazide), N⁸- acetyl spermidine (N⁸- acetyl spermidine), aminopterin (aminopterin), methotrexate (MTX) (methopterin), ai sibo mycin (esperamicin), mitomycin C (mitomycin C), Mitomycin A (mitomycin A), D actinomycin D (actinomycin), bleomycin (bleomycin), carminomycin (carminomycin), aminopterin (aminopterin), Talisomycin (tallysomycin), podophyllotoxin (podophyllotoxin) and podophyllotoxin derivative such as Etoposide (etoposide) or etoposide phosphate, vincaleukoblastinum (vinblastine), vincristine (vincristine), eldisine (vindesine), PTX (taxol), taxotere (taxotere), retinoic acid (retinoic acid), butyric acid (butyric acid), N⁸- acetyl spermidine (N⁸- acetyl spermidine), camptothecine (camptothecin), Calicheamicin (calicheamicin), bryostatin (bryostatin), cephalostatins, ansamitocin (ansamitocin), actosin, maytansinoids (maytansinoid) such as DM-1, maytansine (maytansine), maytansinol (maytansinol), N- demethyls -4, 5- removes epoxy maytansinol, C-19- dechlorination maytansinols, C-20- hydroxyl maytansinols, C-20- demethoxylation maytansinols, C-9-SH maytansinols, C-14- alkoxyl-methyl maytansinols, C-14- hydroxyls or acetyl-o-methyl maytansinol, C-15- hydroxyls/acetoxyl group maytansinol, C-15- methoxyl group maytansinols, C-18-N- demethylations maytansinol and 4, 5- deoxidation maytansinols；Auristatin, such as auristatin E, M, PHE and PE；Dolostatin, such as dolostatin A, dolostatin B, dolostatin C, dolostatinD, dolostatin E (20- tables and 11- tables), dolostatin G, dolostatin H, dolostatin I, dolostatin1, dolostatin2, dolostatin3, dolostatin4, dolostatin5, dolostatin6, dolostatin7, dolostatin8, dolostatin9, dolostatin10, deo-dolostatin10, dolostatin11, dolostatin12, dolostatin13, dolostatin14, dolostatin15, dolostatin16, dolostatin17 and dolostatin18；Cephalostatin, such as cephalostatin1, cephalostatin2, cephalostatin3, cephalostatin4, cephalostatin5, cephalostatin6, cephalostatin7, 25 '-table-cephalostatin7, 20- tables-cephalostatin7, cephalostatin8, cephalostatin9, cephalostatin10, cephalostatin11, cephalostatin12, cephalostatin13, cephalostatin14, cephalostatin15, cephalostatin16, cephalostatin17, cephalostatin18 and cephalostatin19.

Maytansinoids are the mitotic inhibitors played a role by suppressing tubulin polymerization.Maytansine is initially from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) isolated (United States Patent (USP) No.3,896,111).It is subsequently found certain micro-organisms and also generates maytansinoids, such as maytansinol and C-3 maytansinols ester (United States Patent (USP) No.4,151,042).The maytansinol and its derivative and analog of synthesis are disclosed in such as United States Patent (USP) No.4,137,230；4,248,870；4,256,746；4,260,608；4,265,814；4,294,757；4,307,016；4,308,268；4,308,269；4,309,428；4,313,946；4,315,929；4,317,821；4,322,348；4,331,598；4,361,650；4,364,866；4,424,219；4,450,254；4,362,663；And 4,371,533, the disclosure of which is clearly taken in herein is used as reference.

By maytansine and maytansinoids and the antibody coupling of specific binding tumor-cell antigen.Immune conjugate and its therapeutical uses comprising maytansinoids are disclosed in such as United States Patent (USP) No.5,208,020；5,416, the 064 and B1 of European patent EP 0 425 235, clearly takes in the disclosure of which and is used as reference herein.Liu et al., Proc.Natl.Acad.Sci.USA 93：8618-8623 (1996) describes the immune conjugate for including the maytansinoids for being referred to as DM1 being connected with the monoclonal antibody C242 for human colorectal cancer.It was found that the conjugate has the high cell toxicity of the colon cancer cell for culture, and show antitumor activity in tumour growth measurement method in vivo.Chari et al., Cancer Research 52：127-131 (1992) describes wherein maytansinoids through disulfde linker and the immune conjugate for combining another mouse monoclonal antibody TA.1 couplings of the mouse antibody A 7 of antigen or combination HER-2/neu oncogenes in human colon cancer cell line.

Know that many linking groups can be used for preparing antibody-maytansinoids conjugate, including such as United States Patent (USP) No.5,208,020 or B1 and Chari et al., the Cancer Research 52 of European patent 0 425 235 in this area：Disclosed in 127-131 (1992).Linking group includes disulphide group, sulfide group, acid-unstable group, photo-labile group, the unstable group of peptase or the unstable group of esterase, as disclosed in above-mentioned patent, preferably disulphide and sulfide group.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and maytansinoids, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (to azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (to diazoniumbenzoyl) ethylenediamines), diisocyanate (such as toluene 2, 6- diisocyanate) and double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.Particularly preferred coupling agent includes N- succinimide bases -3- (2- pyridine radicals two is thio) propionic ester (SPDP) (Carlsson et al., Biochem.J.173：723-737 (1978)) and N- succinimide bases -4- (2- pyridylthios) valerate (SPP), thus disulfide bond is provided.

According to the type of connection, joint can be attached to multiple positions of maytansinoids molecule.For example, conventional coupling techniques can be used to pass through the reaction with hydroxyl to form ester bond.Reaction can occur in the C-3 positions with hydroxyl, C-14 positions, the C-15 positions through hydroxyl modified and the C-20 positions with hydroxyl modified through methylol.In a preferred embodiment, key connection is formed in the C-3 positions of maytansinol or maytansinol analog.

Another immune conjugate interested includes the CD20 binding antibodies being coupled with one or more calicheamicin molecules.Calicheamicin antibiotic family can produce double-strand DNA cleavage in sub- picomolar concentrations.Preparation on Calicheamicin family conjugate is referring to United States Patent (USP) No.5,712,374；5,714,586；5,739,116；5,767,285；5,770,701；5,770,710；5,773,001；5,877,296 (being all Cyanamid companies of the U.S.).Available Calicheamicin analogue includes but is not limited to γ₁ ^I、α₂ ^I、α₃ ^I, N- acetyl group-γ₁ ^I, PSAG and θ^I ₁(Hinman et al., Cancer Research53：3336-3342(1993)；Lode et al., Cancer Research 58：2925-2928(1998)；And the above-mentioned United States Patent (USP) for authorizing Cyanamid companies of the U.S.).Can be QFA with another antineoplastic of antibody coupling, it is a kind of antifolic thing.Calicheamicin and QFA have intracellular action site, and are difficult through plasma membrane.Therefore, these medicaments greatly strengthen their cytotoxic effect via the cellular uptake of antibody-mediated internalization.

Radio isotope

For selective destruction tumour, antibody can include high radioactive atom.A variety of radio isotopes can be used for the anti-CD 20 antibodies of generation radiation coupling.Example includes At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²With Lu radio isotope.When conjugate to be used to detect, it can be studied comprising radioactive atom for scitiphotograph, such as Tc^99mOr I¹²³, or being used for nuclear magnetic resonance (NMR) comprising spin label is imaged (also referred to as magnetic resonance imaging, mri), such as iodo- 123, iodine -131, indium -111, fluoro- 19, carbon -13, nitrogen -15, oxygen -17, gadolinium, manganese or iron.

Radioactively labelled substance or other labels can be mixed into conjugate in a known way.For example, can biosynthesis peptide, or by chemical amino acid synthetic method synthetic peptide, involve for example fluoro- 19 suitable amino group acid precursors for replacing hydrogen wherein using.Label, such as Tc can be adhered to through the cysteine residues in peptide^99mOr I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹.Yttrium-90 can be adhered to through lysine residue.IODOGEN methods (Fraker et al., Biochem.Biophys.Res.Commun.80：49-57 (1978)) it can be used for mixing iodo- 123.Monoclonal Antibodies in Immunoscintigraphy, Chatal, CRC Press, 1989 describe other methods in detail.

A variety of bifunctional protein coupling agents can be used to prepare the conjugate of antibody and cytotoxic agent, such as N- succinimides base -3- (2- pyridine radicals two is thio) propionic ester (SPDP), succinimide base -4- (N- Maleimidomethyls) hexamethylene -1- carboxylates, iminothiolane (IT), imino-ester (such as hydrochloric acid adipyl imido dimethyl phthalate), active esters (such as succinimide base ester of suberic acid two), aldehydes (such as glutaraldehyde), double azido compound (such as double (to azido benzoyl base) hexamethylene diamines), dual azepine derivatives (such as double (to diazoniumbenzoyl)-ethylenediamines), diisothio-cyanate (such as toluene 2, 6- diisocyanate) and double activated fluorine compounds (such as 1, 5- bis- fluoro- 2, 4- dinitro benzenes) dual-function derivative.For example, can be such as Vitetta et al., Science 238：It is described in 1098 (1987) to prepare ricin immunotoxin.The 1- isothiocyanic acid benzyl -3- methyl diethylene-triamine pentaacetic acids (MX-DTPA) of carbon-14 mark are for by the exemplary chelating agent of radioactive nucleotides and antibody coupling.Referring to WO94/11026.Joint can be easy for discharging " the cleavable joint " of cell toxicity medicament in cell.For example, sour unstable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker or containing disulfde linker (Chari et al., Cancer Research 52 can be used：127-131(1992)；United States Patent (USP) No.5,208,020).

Therapeutical uses

The humanization 2H7 CD20 binding antibodies of the present invention include CD20 positive cancers such as B cell lymphoma and leukaemia, and autoimmunity disease available for many pernicious and nonmalignant disease is treated.Stem cell (B cell precursor) in marrow lacks CD20 antigens so that healthy B cell can after treatment regenerate and normal level is returned within the several months.Hu2H7.v511 is by with the preferred antibody for the treatment of method in this article.

CD20 positive cancers refer to such cancer, wherein expressing CD20 abnormal cell proliferation on cell surface.CD20 positive B-cells tumour includes CD20 positive He Jiejinshi (Hodgkin) diseases, includes the Hodgkin's disease (LPHD) of lymphocytic predominance；Non_hodgkin lymphoma (NHL)；Follicular center cells (FCC) lymthoma；Acute lymphatic leukemia (ALL)；Chronic lymphocytic leukemia (CLL)；Hairy cell.

Term " non-Hodgkin's (Hodgkin) lymthoma " or " NHL " refer to the lymphatic system cancer beyond He Jiejin lymphomas as used herein.Generally can by exist in He Jiejin lymphomas it is inner-apply (Reed-Sternberg) cell and make a distinction He Jiejin lymphomas and non_hodgkin lymphoma in the absence of the cell in non_hodgkin lymphoma.The example that non_hodgkin lymphoma is covered when the term is used for this paper includes those skilled in the art (such as oncologist or virologist) will be accredited as such any lymthoma according to classification chart known in the art, such as Color Atlas of ClinicalHematology, 3rd edition, Victor A.Hoffbrand and John E.Pettit are compiled, HarcourtPublishers Ltd., Revised European-American Lymphoma (REAL) scheme (American-European lymthoma correction chart) described in 2000.Referring specifically to the table in Figure 11 .57,11.58 and 11.59.More specific example includes but is not limited to recurrent or intractable NHL,Front (front line) rudimentary NHL,Stage III/IV NHL,Chemotherapy tolerance NHL,Precursor B lymphoblastic leukemias and/or lymthoma,SLL,B cell chronic lymphocytic leukemia and/or pre-lymphocytic leukemia and/or SLL,B cell prolymphocyte lymthoma,Immune cell tumor and/or lympho-plasmacytic (lymphoplasmacytic) lymthoma,Lymphoma lymphoplasmacytic,Marginal zone B-cell lymphoma,Splenic marginal zone lymthoma,Save outer edge area (extranodal marginal zone)-MALT lymthomas,Save marginal zone (nodal marginal zone) lymthoma,Hairy cell,Plasmacytoma and/or plasma cell myeloma,Rudimentary/follicular lymphoma,Middle rank/folliculus NHL,Lymphoma mantle cell,Follicle center lymphoma (folliculus),Intermediate diffusivity NHL,Diffusivity large B cell lymphoid tumor,Aggressiveness (agressive) NHL (including aggressive front NHL and aggressiveness recurrent NHL),Recurrent or intractable NHL after autologous stem cell transplantation,Primary Mediastinal large B cell lymphoid tumor,Lymphoma primary effusion,Senior immunoblast NHL,Senior lymphoblast NHL,Senior small non-cleaved cell NHL,Thesaurismosis (bulky disease) NHL,Bai Jiteshi (Burkitt) lymthoma,The big granular lymphocytic leukemia of precursor (periphery),Mycosis fungoides and/or Sai Zhali (Sezary) syndrome,Skin lymphoma,Primary cutaneous type,Angiocentric lymphoma.

In specific embodiments, humanization CD20 binding antibodies and its functional fragment are used to treat non_hodgkin lymphoma (NHL), the Hodgkin's disease (LPHD) of lymphocytic predominance, SLL (SLL) and chronic lymphocytic leukemia (CLL), include the recurrence of these illness.

Painless lymthoma is a kind of slow-growing, incurable disease, wherein patient Average Survival 6 to 10 years after the multiple state of an illness disappears and recurs.In one embodiment, humanization CD20 binding antibodies or its functional fragment are used to treat painless NHL, including the painless painless NHL of NHL and Rituximab tolerances of recurrent.The painless NHL patient of recurrent can be the Rituximab respondent of the Rituximab of previously-accepting one course for the treatment of of mistake and response more than 6 months.

The humanization 2H7 antibody or its functional fragment of the present invention can be used for such as recurrent or refractory low grade or follicularis CD20 positive B-cells NHL as single pharmaceutical treatment (monotherapy), or drug scheme can be unified into other medicines be applied to patient.

The humanization 2H7 antibody or its functional fragment of the present invention can be used as the first gamma therapy.Present invention further contemplates be used to treat by these antibody the treatment using following any medicine not being responded to or responded CD20 positive B-cells tumor patients that are not enough or recurring after with these drug therapies：rituximab(Genentech)；Ocrelizumab (Genentech, Inc.)；ibritumomab tiuxetan(Zevalin^TM, Biogen Idec)；tositumomab(Bexxar^TM, GlaxoSmithKline)；HuMAX-CD20^TM(GenMab)；IMMU-106 (a kind of Humanized anti-CD 20, also referred to as hA20 or 90Y-hLL2, Immunomedics)；AME-133(Applied Molecular Evolution/Eli Lilly)；gentuzumab ozogamicin(Mylotarg^TM, a kind of antibody of Humanized CD 3-resisting 3, Wyeth/PDL)；alemtuzumab(Campath^TM, a kind of anti-CD 52 antibody, Schering Plough/Genzyme)；epratuzumab(IMMU-103^TM, a kind of humanization anti-CD22 antibody, Immunomedics).

Invention further provides the method for the humanization 2H7 Antybody therapy CLL patients with the present invention, the patient that the patient have failed including the use of the therapy of fludarabine is treated with 2H7.v511 and 2H7.v114 in specific embodiments.

" autoimmunity disease " herein refer to caused by individual autologous tissue and for the disease or illness of individual autologous tissue or its isolate (co-segregate) or performance or resulting illness.The example of autoimmune disease or illness includes but is not limited to arthritis (rheumatoid arthritis such as acute arthritis,Chronic rheumatoid arthritis,Urarthritis,Acute gouty arthritis,Chronic inflammatory arthritis,Degenerative arthritis,Infectional arthritis,Lime (Lyme) arthritis,Hypertrophic arthritis,Psoriasis arthropathica,Arthritis vertebralis and young hair style rheumatoid arthritis,Osteoarthritis,Chronic progressive arthritis (arthritis chronica progrediente),Arthritis deformans,Chronic primary panarthritis,Adjuvant arthritis,And ankylosing spondylitis),Inflammatory hyperproliferative skin disease,Psoriasis such as plaque psoriasis,Psoriasis guttata,Pustular psoriasis and nail psoriasis,Idiocrasy includes atopic diseases such as hay fever and Qiao Bu Shi (Job) syndrome,Dermatitis includes contact dermatitis,Chronic contact dermatitis,Allergic dermatitis,Allergic contact dermatitis,Dermatitis herpetiformis and atopic dermatitis,High IgM syndromes chain x,Such as chronic allergic urticaria of nettle rash and chronic idiopathic urticaria include chronic auto-immune nettle rash,Polymyositis/dermatomyositis,Adolescent dermatomyositis,Toxic epidermal necrolysis,Chorionitis (including systemic scleroderma),Harden such as systemic sclerosis,Multiple sclerosis (MS) such as spinal cord-eye (spino-optical) MS,Primary progressive MS (PPMS) and recurrent regression (relapsing remitting) MS (RRMS),Progressive systemic sclerosis,Atherosclerosis,Artery sclerosis,Disseminated sclerosis (sclerosis disseminata) and incoordination (ataxic) hardening,Inflammatory bowel disease (IBD) (such as Chron (Crohn) disease,The gastrointestinal disease of autoimmunity mediation,Colitis such as ulcerative colitis (colitis ulcerosa),Microcosmic (microscopic) colitis,Collagenous colitis,Colitis polyposa,Necrotizing enterocolitis and transmural colitis,With autoimmune inflammatory enteropathy),Gangrenous pyaphysia,Erythema nodosum,Primary sclerotic cholangitis,Episcleritis,Respiratory Distress Syndrome(RDS) includes adult type or ARDS (ARDS),Meningitis,The inflammation of uvea all or in part,Iritis,Choroiditis,Autoimmune hematological illness,Rheumatoid,Sudden hearing loss,The disease such as allergic reaction and allergia of IgE mediations and atopic rhinitis,Silent Sen Shi (Rasmussen) encephalitis in encephalitis such as Lars and edge system and/or BBE,Uveitis such as anterior uveitis,Acute anterior uveitis,Granulomatous uveitis,Nongranulomatous uveitis,Phacoantigenic uveitis,Posterior uveitis or Autoimmune uveitis,With and without the glomerulonephritis (GN) of nephrotic syndrome is such as chronic or acute glomerulonephritis such as primary GN,Immune-mediated GN,Film GN (membranous nephropathy),Idiopathic film GN or idiopathic membranous nephropathy,Film proliferative or film proliferative GN (MPGN) include I types and II types,With radical property GN,Allergia illness and response,Allergic reaction,Eczema includes allergia or atopic eczema,Asthma such as bronchial astehma (asthma bronchiale) and autoimmune asthma,It is related to the illness of T cell infiltration and chronic inflammatory response,For the immune response of exotic antigen such as gestation fetus A-B-O blood groups,Chronic pulmonary inflammatory disease,Autoimmune myocarditis,Leukocyte adhesion deficiency,Systemic loupus erythematosus (SLE) (systemic lupus erythematodes) such as skin SLE,Subacute cutaneous lupus erythema tosus,Neonatal lupus syndrome (NLE),Lupus erythematosus disseminatus,Lupus (including lupus nephritis,Lupus encephalitis,Paediatrics lupus,Non- kidney lupus,The outer lupus of kidney,Discoid lupus,Lupus alopecia),Young hair style (I types) diabetes include paediatrics insulin-dependent diabetes mellitus (IDDM),The diabetes (type ii diabetes) of adult onset,Autoimmune diabetes,Idiopathic diabetes insipidus,With cell factor and the acute immune response relevant with delayed hypersensitivity of T- cell mediateds,Tuberculosis,Sarcoidosis,Granulomatosis includes lymphomatoid granulomatosis,Wei Genashi (Wegener) granulomatosis,Agranulocytosis,Vasculitides includes vasculitis (including big vessel vasculitis (including polymyalgia rheumatica and giant cell (high iS-One (Takayasu)) arteritis),Medium vessels vasculitis (including Chuan Qishi (Kawasaki) diseases and PAN/periarteritis nodosa),Microcosmic (microscopic) panarteritis,CNS vasculitises,Gangrenosum acne,Cutaneous or allergic angiitis,Systemic necrotizing vasculitis,With ANCA relevant blood vessels inflammation such as Qiu-apply Er Shi (Churg-Strauss) vasculitises or syndrome (CSS)),Temporal arteritis,Alpastic anemia,LADA alpastic anemia,Claire (Coombs) positive anemia,Dai-cloth Er Shi (DiamondBlackfan) anaemia,Hemolytic anemia or immune hemolytic anemia include autoimmune hemolytic anemia (AIHA),Pernicious anaemia (anemia perniciosa),A Disenshi (Addison) diseases,Simple erythrocyte anemia or aregeneratory (PRCA),Factor IX lacks,Hemophilia A,LADA Neutropenia,Pancytopenia,Leukopenia,It is related to the disease of leukocyte infiltration,CNS inflammatory conditions,Multiple organ injury's syndrome such as those septicemia,Wound or bleeding are secondary,The disease of antigen-antibody complex mediation,Anti-GBM disease,Antiphospholipid antibody syndrome,Allergic neuritis,Bei Qieteshi (Bechet or Behcet) diseases,Ka Siermanshi (Castleman) syndrome,Gu Depasiqiushi (Goodpasture) syndrome,Lei Nuoshi (Reynaud) syndrome,Siogren (Sjogren) syndrome,Shi-about Er Shi (Stevens-Johnson) syndrome,Pemphigoid such as bullous pemphigoid and skin pemphigoid,Pemphigus (including pemphigus vulgaris,Pemphigus foliaceus,Mucosal pemphigus type pemphigus and pemphigus erythematosus),Autoimmune polyendocrinopathy,Lay Te Shi (Reiter) diseases or syndrome,Immune complex nephritis,Antibody-mediated ephritis,Neuromyelitis optica,Polyneuropathy,Chronic neuropathic such as IgM polyneuropathies or the neuropathy of IgM mediations,Thrombopenia (for example myocardial infarction patient occurs) includes thrombotic thrombocytopenic purpura (TTP),Post-transfusion purpura (PTP),The thrombopenia that heparin induces,Include chronic or acute ITP with autoimmunity or immune-mediated thrombopenia such as ITP (ITP),The autoimmunity disease of testis and ovary includes LADA orchitis and oaritis,Primary hypothyroidism,Hypoparathyroidism,Autoimmune endocrinopathy includes thyroiditis such as autoimmune thyroiditis,Hashimoto (Hashimoto) disease,Chronic thyroiditis (Hashimoto (Hashimoto) thyroiditis) or subacute thyroiditis,AITD,Idiopathic hypothyroidism,Graves (Graves) disease,Polyglandular syndrome such as autoimmune polyglandular syndrome (or pluriglandular endocrine disease syndrome),Paraneoplastic syndrome includes neurology paraneoplastic syndrome such as Lambert-Eton (Lambert-Eaton) myasthenic syndrome or Eaton-Lambert (Lambert-Eaton) syndrome,Stiff body or stiff man syndrome,Encephalomyelitis such as allergic encephalitis (encephalomyelitis allergica) and experimental allergic encephalomyelitis (EAE),Myasthenia gravis such as thymoma associated myasthenia gravis,Cerebellar degeneration,Neuromyotonia,Opsoclonus or opsoclonus myoclonic syndrome (OMS),And esthesioneurosis,Many focus motor neuropathies,Seat Han Shi (Sheehan) syndrome,Oneself immunity hepatitis,Chronic hepatitis,Lupoid hepatitis,Giant cell hepatitis,CAH or autoimmune chronic active hepatitis,Lymphoid interstitial pneumonia (LIP),Bronchiolitis obliterans (Nonimplantation) is to NSIP,Ge-bar Er Shi (Guillain-Barr é) syndrome,Bei Geershi (Berger) diseases (IgA nephrosis),Idiopathic IgA nephrosis,Linear IgA skin diseases,PBC,Pneumonocirrhosis,Auto immune enteropathy syndrome,Chylous diarrhea,Abdominal disease,Sprue (gluten enteropathy),Intractable sprue,Idiopathic sprue,Cryoglobulinemia,ALS (ALS) (Lu Geli kirschner (Lou Gehrig) disease),Coronary artery disease,LADA otopathy such as Autoimmune Inner Ear Disease (AIED),LADA anaudia,Opsoclonus myoclonic syndrome (OMS),Polychondritis such as intractable or relapsing polychondritis,Pulmonary alveolar proteinosis,Amyloidosis,Sclerotitis,Non-cancerous lymphocytosis,Primary lymphocytosis includes monoclonal B cell lymphocytosis (the undetermined monoclonal gamma globulin disease of such as benign monoclonal gammopathy and property (MGUS)),Peripheral nerve disease,Paraneoplastic syndrome,Passage disease such as epilepsy,Antimigraine,Arrhythmia cordis,Disorder of muscle,Become deaf,Blindness,Periodic paralysis and CNS passage disease,Autism,Inflammatory myopathy,Focal segmental glomerulosclerosis (FSGS),Endocrine ophthalmopathy,Uveoretinitis,Choroidoretinitis,LADA hepatology illness,Fibromyalgia,Multiple Endocrine exhaustion,Shi Miteshi (Schmidt) syndrome,Paranephritis,Lipogastry,Alzheimer's disease,Demyelinating disease such as LADA demyelinating disease and chronic inflammatory demyelinating polyneuropathy,Diabetic nephropathy,De Leisileshi (Dressler) syndrome,Alopecia areata,CREST syndrome (calcinosises,Lei Nuoshi (Raynaud) phenomenon,Esophageal dysmotility,Sclerodactyly and capillarectasia),Masculinity and femininity LADA is infertile,MCTD,Just add Si Shi (Chagas) diseases,Rheumatic fever,Habitual abortion,Farmer lung,Erythema multiforme,Postcardiotomy syndrome,Ke Xing Shi (Cushing) syndrome,Bird breeders' lung,Allergic granulomatous angiitis,Benign lymphocytic vasculitis,A Erboteshi (Alport) syndrome,Pulmonary alveolitis such as allergic pulmonary alveolitis and FA,Interstitial lung disease,Transfusion reaction,Leprosy,Malaria,Leishmaniasis,Trypanosomiasis (kypanosomiasis),Snail fever,Roundworm disease,Aspergillosis,Sampter Cotards,Kapp Lan Shi (Caplan) syndrome,Dengue,Endocarditis,Endomyocardial fibrosis,Diffusivity pulmonary interstitial fibrosis,Interstitial pulmonary fibrosis,Pulmonary fibrosis,Idiopathic pulmonary fibrosis,Cystic fibrosis,Entophthamia,Erythema elevatum diutinum (erythema elevatum et diutinum),Fetal erythrocytosis,Eosinophilic fascitis (faciitis),Shu Er Man (Shulman) syndrome,Fil Ti Shi (Felty) syndrome,flariasis,Cyclitis such as chronic cyclitis,Heterochronia cyclitis,Iridocyclitis (acute or chronic) or FuchShi cyclitises,Heng Nuo-Xu Lan Er Shi (Henoch-Schonlein) purpura,Human immunodeficiency virus (HIV) infects,Echovirus infects,Cardiomyopathy,Alzheimers (Alzheimer) disease,Parvovirus infections,Rubella virus infection,Syndrome after vaccination,Congenital rubella infects,Epstein-Ba Er (Epstein-Barr) virus infection,Parotitis,Evans (Evans) syndrome,LADA gonadal failure,Xi Denghamushi (Sydenham) chorea,Ephritis after streptococcus,Buerger's disease (thromboangitis ubiterans),Thyrotoxicosis,Tabetic crisis,Choroiditis,Megaloblastic polymyalgia,Endocrine ophthalmopathy,Chronic hypersensitivity pneumonitis,Keratoconjunctivitis sicca,Epidemic keratoconjunctivities,Idiopathic nephritic syndrome,Minute nephropathy,Benign familial and ischemia reperfusion injury,Retina autoimmunity,Arthritis,Bronchitis,Chronic obstructive airway disease,Silicosis,Aphtha,Aphthous stomatitis,Arteriosclerotic illness,Without spermatogenesis (aspermiogenese),Autoimmune hemolytic anemia,Primary kirschner (Boeck) disease,Cryoglobulinemia,Dupp Yi Telunshi (Dupuytren) contracture,Phacoanaphylaxis entophthamia (endophthalmiaphacoanaphylactica),Allergia enteritis (enteritis allergica),Leprosy section erythema nodosum,Idiopathic facial palsy,Chronic Fatigue Syndrome,Rheumatic fever (febris rheumatica),Ha-inner Er Shi (Hamman-Rich) diseases,Sensory neural hearing loss,Paroxysmal hemoglobinuria (haemoglobinuriaparoxysmatica),Hypogonadism,Regional enteritis (ileitis regionalis),Leukopenia,Infectious mononucleosis,Transverse (traverse) myelitis,Primary essential myxoedema,Nephrosis,Sympathetic ophthalmia (ophthalmia symphatica),Granulomatous orchitis (orchitisgranulomatosa),Pancreatitis,Acute polyradiculitis,Gangrenous pyaphysia,Kui Erwanshi (Quervain) thyroiditis,Acquired splenatrophy,Due to the sterility of anti-spermatozoon antibody,Non-malignant thymoma,Leucoderma,SCID and Epstein-Ba Er (Epstein-Barr) virus associated-diseases,Acquired immunodeficiency syndrome (AIDS),Parasitic disease such as Leishmania disease,TSS,Food poisoning,It is related to the illness of T cell infiltration,Leukocyte adhesion deficiency,With cell factor and the acute immune response relevant with delayed hypersensitivity (DH) of T- cell mediateds,It is related to the disease of leukocyte infiltration,Multiple organ injury's syndrome,The disease of antigen-antibody complex mediation,Anti-GBM disease,Allergic neuritis,Autoimmune polyendocrinopathy,Oaritis,Primary myxedema,Autoimmune atrophic gastritis,Sympathetic ophthalmia,Rheumatism,MCTD,Nephrotic syndrome,Inflammation of pancreatic islet,Many endocrinasthenias,Peripheral nerve disease,Autoimmune polyglandular syndrome I types,The Idiopathic hypoparathyroidism (AOIH) of adult onset,Whole alopecia,Dilated cardiomyopathy,Epidermolysis bullosa acquisita (epidermolisis bullosa acquisita,EBA),Hematochromatosis,Myocarditis,Nephrotic syndrome,Primary sclerotic cholangitis,Suppurative or apyetous nasosinusitis,Acute or chronic nasosinusitis,Eso-ethmoiditis,Frontal sinusitis,Maxillary sinusitis or sphenoiditis,Eosinocyte associated conditions such as eosinophilia,Ensinophilosis infiltrates,Eosinophilia-myalgia syndrome,Lv Fuleshi (Loffler) syndrome,Chronic eosinophilic pneumonia,Tropical ensinophilosis,Bronchial aspergillosis,Aspergilloma,Or the granuloma containing eosinocyte,Allergic reaction,Seronegativity arthritis vertebralis disease,Polyendocrine autoimmune disease,Sclerosing cholangitis,Sclera,Episclera,Chronic mucocutaneous candidiasis,Bruton's (Bruton) syndrome,Infancy transient hypogammaglobulinemia,Neat (Wiskott-Aldrich) syndrome in prestige Scott-Ao Er Delhis,Incoordination capillarectasia,The autoimmune conditions relevant with the following：Collagen disease, rheumatism, neurological disease, lymphnoditis, ischemia-reperfusion is disorderly, blood pressure response reduces (reduction in blood pressureresponse), dysfunction of blood vessel, capillarectasia (antgiectasis), tissue damage, Cardiovascular ischemia, hyperalgia, cerebral ischemia and the disease with vascularization, allergia supersensitivity illness, glomerulonephritis disease, reperfusion injury, the reperfusion injury of myocardium or other tissues, skin disease with acute inflammatory components, acute purulent meningitis or other central nervous system inflammatory conditions, eye and socket of the eye inflammatory conditions, granulocyte Transfusion related syndromes, the poisoning that cell factor induces, acute severe inflammation, chronic and refractory inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, diabetic keratopathy main artery illness, intra-arterial hyperplasia, peptic ulcer, cardiovalvulitis, and endometriosis.

In specific embodiments, humanization 2H7 antibody and its functional fragment are used to treat rheumatoid arthritis and juvenile rheumatoid arthritis, systemic loupus erythematosus (SLE) includes lupus nephritis, Wei Genashi (Wegener) diseases, inflammatory bowel disease, ulcerative colitis, ITP (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis includes recurrence retentivity MS, psoriasis, IgA nephrosis, IgM polyneuropathies, myasthenia gravis, ANCA relevant blood vessels are scorching, diabetes, Lei Nuoshi (Reynaud) syndrome, Siogren (Sjogren) syndrome, neuromyelitis optica (NMO) and glomerulonephritis.

" processing " or " treatment " or " mitigation " refer to therapeutic treatment, and wherein target is the recurrence for slowing down (mitigation) targeted pathological condition or illness if it can not cure or preventing illness.If after the humanization CD20 binding antibodies of the present invention of therapeutic dose are received according to the method for the present invention, patient shows observable and/or measurable reduction or disappearance in one or more of S＆S of specified disease, then subject success " treatment " autoimmunity disease or CD20 positive B-cells malignant tumours.For example, for cancer, significant cancer cell number is reduced or cancer cell disappears；Tumor mass reduction；Metastases are suppressed (i.e. a certain degree of to slow down, preferably to stop)；Tumour growth is suppressed by a certain degree of；Paracmasis extends；And/or the one or more symptoms relevant with particular cancers obtain a certain degree of mitigation；Morbidity and mortality are reduced；And quality of life is improved.The mitigation of the S or S of disease can also by patient perceptions to.Complete response can be achieved in treatment, and all signs for being defined as cancer disappear, or partial response, wherein tumor mass reduction, preferably greater than 50%, more preferably 75%.If the state of an illness of patient obtains stabilization, it is also considered as patient and obtains medical treatment.In a standard, h2H7 antibody of the invention realizes ＞ 95% peripheral blood B cell abatement and B cell returns to the 25% of baseline.In preferred embodiments, the treatment carried out with antibody of the present invention effectively causes the cancer of cancer patient not develop within 4 months after the treatment, 6 months after preferred therapeutic, more preferably 1 year, even more preferably 2 years or more years.These parameters for assessing the successful treatment of disease and improving are easy to measure by old process known to the competent internist in this area.

" therapeutically effective amount " refers to the amount of the disease or illness in antibody and medicine effectively " treatment " subject.For cancer, the therapeutically effective amount of medicine can reduce the number of cancer cell；Reduce the size of tumour；Suppress and (slow down to a certain extent, preferably prevent) cancer cell infiltration into peripheral organs；Suppress and (slow down to a certain extent and preferably prevent) metastases；Suppress tumour growth to a certain extent；And/or mitigate one or more symptoms relevant with cancer to a certain extent.Referring to the definition of foregoing " treatment ".For autoimmunity disease, the antibody or other medicines of therapeutically effective amount effectively mitigate the S＆S of disease.

To be known to the internist that is competent in terms of the corresponding disease for assessing the therapeutic efficiency of knurl or the parameter of achievement.Generally, competent internist will expect the mitigation of the S＆S of specified disease.Parameter may include stable disease, regression time, the Median Time of disease development.

Lymthoma and CLL, their diagnosis, treatment and standard medical flow for measuring therapeutic efficiency are described below with reference to document：Canellos GP, Lister, TA, Sklar JL, The Lymphomas, W.B.Saunders Company, Philadelphia, 1998；Van Besien K and Cabanillas, F, Clinical Manifestations, Staging and Treatment of Non-Hodgkin ' s Lymphoma, Chap.70, pp 1293-1338, in：Hematology, Basic Principles and Practice, the 3rd edition, Hoffman et al. (eds.), Churchill Livingstone, Philadelphia, 2000；And Rai, K andPatel, D, Chronic Lymphocytic Leukemia, Chap.72, pp 1350-1362, in：Hematology, Basic Principles and Practice, the 3rd edition, Hoffman et al. (eds.), Churchill Livingstone, Philadelphia, 2000.

To be known to the internist that is competent in terms of the corresponding disease for assessing the therapeutic efficiency of autoimmunity disease or autoimmune-associated diseases or the parameter of achievement.Generally, competent internist can expect the mitigation of the S＆S of specified disease.It is exemplified below.

In one embodiment, humanization 2H7 antibody and specific hu2H7.v511 and its functional fragment are used to treat rheumatoid arthritis.

RA is influence more than 2,000,000 Americans and the debilitating autoimmunity disease of obstruction victim's daily routines.Occurs RA when the improper attack joints tissue of the self immune system of body and when causing chronic inflammation and the intraarticular damage of destruction health tissues.Symptom includes Joint Inflammation, swelling, stiff and pain.Further, since RA is systemic disease, it may have an impact to other tissue such as lung, eye and marrow.Have no knowledge about healing.Treatment includes a variety of steroids and nonsteroid anti-inflammatory drugs, immunodepressant, the antirheumatic drug (DMARD) and biological agent for mitigating disease.However, many patients continue not enough to treatment response.

Antibody can be used as the gamma therapy in early stage RA (i.e. not used methopterin (MTX)) patient, as monotherapy, or joint or after such as MTX or endoxan.Or, antibody can be used to treat DMARD and/or MTX resistant patients as second-line therapy, be used as monotherapy or joint such as MTX.Humanization CD20 binding antibodies can be used for preventing and controlling joint injury, delay structure damage, mitigate the pain with the inflammation-related in RA, and generally mitigate S＆S of the moderate into severe RA.RA patient can be before with other medicines (conjoint therapy that the see below) treatment used in treatment RA, afterwards or together with which with humanization CD20 Antybody therapies.In one embodiment, the antirheumatic drug for previously having mitigated disease with the humanization CD20 binding antibodies treatment of the present invention have failed and/or single methopterin is responded not enough patient.In an embodiment of this treatment, patient received single humanization CD20 binding antibodies (the 1st day and the 15th day intravenous infusion 1g) at 17 days in therapeutic scheme；CD20 binding antibodies+endoxan (the 3rd day and the 17th day intravenous infusion 750mg)；Or CD20 binding antibodies+methopterin.

Because body produces tumor necrosis factor α (TNF α) during RA, TNF α inhibitor has been used for treating the disease.However, TNF α inhibitor such as Etanercept (Etanercept, ENBREL ), infliximab (Infliximab, REMICADE ) and adalimumab (Adalimumab, HUMIRA^TM) side effect of passiveness can be produced, such as infection, heart failure and demyelinate.Therefore, in one embodiment, humanization CD20 binding antibodies or its biological function fragment can be used to treating RA patient as a such as gamma therapy produces the risks of these passive side effects using TNF α inhibitor medicaments to reduce, or for treating the patient for such as cardiotoxicity of thinking to be prone to be poisoned.Humanization CD20 binding antibodies or its biological function fragment can be additionally used in the method for treating following RA patient：With TNF α inhibitor for treating but do not responded to, not enough (TNF-IR patient) responded to TNF α inhibitor, either after response a period of time disease relapse or be defined as the RA patient of unlikely response TNF α inhibitor therapy.In one embodiment, first TNF-IR is treated with low dosage such as less than 100mg before with TNF α inhibitor for treating.

Based on a kind of method of therapeutic efficiency in assessment RA is with rheumatology association of the U.S. (AmericanCollege of Rheumatology, ACR) standard, it measures improvement percentage of tenderness and swollen joint etc..Compared with (such as the baseline of before processing) or placebo treatment are handled without antibody, RA patient can score as such as ACR 20 (20% improves).Assessing the other manner of Antybody therapy effect includes the Sharp X-rays scoring that X-ray scores such as the scoring that narrows to structural damage such as bone erosion and articular cavity.Can also based on treatment during or after health evaluating questionnaire (Health AssessmentQuestionnaire, the HAQ) score in each period, AIMS scores, the SF-36 prevention or improvement that are disabled to patient evaluation.ACR20 standards may include to touch a tender spot in (pain) joint number and 20% improving both swollen joint number+following 5 extra measurements and the 20% of at least 3 improve：

1. according to the Patient Pain Assessment (VAS) of intuitive analog scale,

2. patient's overall evaluation (VAS) of disease activity,

3. doctor's overall evaluation (VAS) of disease activity,

4. the patient's self-assessment measured by health evaluating questionnaire disables, and

5. acute phase reactant, CRP or ESR.

ACR50 and 70 defines similar.Preferably, a certain amount of CD20 binding antibodies of the present invention are applied to patient, it is enough to realize at least ACR20 score, most preferably at least preferably at least ACR30, more preferably at least ACR50, even more desirably at least ACR70, ACR75 and Geng Gao.

Psoriatic arthritis has unique and unique radiograph feature.For psoriatic arthritis, also joint erosion can be assessed by Sharp scores and joint space narrows.The humanization CD20 binding antibodies of the present invention can be used for prevention joint injury and mitigate the disease signs and symptom of illness.

Another aspect of the present invention is come sanatory method by the humanization CD20 binding antibodies of the present invention to patient therapeuticallv's effective dose with SLE or lupus nephritis.SLEDAI scores provide the numerical quantization of disease activity.SLEDAI is the Weighted Index of known 24 clinics relevant with disease activity and laboratory parameters, number range 0-103.Referring to Bryan Gescuk ＆ John Davis, " Novel therapeutic agent for systemic lupus erythematosus ", in：Current Opinionin Rheumatology 2002,14：515-521.Other methods of marking score including BILAG.Think that the antibody for being directed to double-stranded DNA causes other performances of kidney rubescent (renal flares) and lupus.Kidney rubescent time (time to renal flare) can be reached to the patient-monitoring for receiving Antybody therapy, this is defined as significant, the reproducible rise of blood in serum creatinine, urine protein or urine.Or the level of antibody that can be to patient-monitoring antinuclear antibodies and for double-stranded DNA.SLE treatment includes the corticosteroid and/or endoxan (HDCC) of high dose.Here, the successful treatment of lupus will mitigate rubescent (flare), that is, reduce seriousness and/or be reduced to the time of next time rubescent (flare).

Spondyloarthropathy is one group of disorder of joint, including ankylosing spondylitis, psoriatic arthritis and Chron (Crohn) disease.Treatment achievement can be determined by the patient by checking and doctor's total evaluation survey tool.

As for vasculitis, about 75% systemic vasculitis patient there is anti-neutrophil cell cytoplasmic antibody and belong to influence it is small/one of three kinds of illness of median size blood vessel：Wei Genashi granulomas (Wegener ' s granulomatosus, WG), microcosmic Polyangiitis (microscopic polyangiitis,) and two Cotards (Churg Strauss syndrome are applied on mound MPA, CSS), it is referred to as ANCA relevant blood vessels scorching (AAV).

The therapeutic efficiency of psoriasis is assessed by the clinical sign and symptom of monitoring of diseases than the change of baseline condition, including doctor's overall evaluation (PGA) change and psoriasis area and severity index (PASI) score, psoriasis symptom assessment (PSA).Can the humanization CD20 binding antibodies such as hu2H7.v511 of the periodic measurement present invention is treated in the intuitive analog scale for indicating the degree of itching that particular point in time is subjected to during whole treatment psoriatic.

Patient may they first infusion of therapeutic with antibody when be subjected to infusion reaction or infusion related symptoms.The order of severity of these symptoms is different and can generally be reversed by medical intervention.These symptoms include but is not limited to the heating of influenza sample, shiver with cold/stiff, nauseous, nettle rash, headache, bronchial spasm, angioedema.Expect that infusion reaction is minimized for the methods for the treatment of diseases of the present invention.In order to mitigate or minimize the antibody of such adverse events, the acceptable initial adaptation of patient or tolerance dose, treatment effective dose is then only.Adapt to dosage (conditioning dose) and will be less than treatment effective dose so that patient adapts to and is resistant to higher doses.

Dosage is administered

The factor relevant with dosage administration according to known to indication to be treated and the skilled internist in this area, will apply antibody of the invention effectively to treat the indication dosage that Side effect is minimized simultaneously.Preferable dosage is likely to be dependent on the order of severity, the stage of disease, the desired B cell regulation and control level of disease and disease, and other factorses known to the skilled internist in this area.

For the treatment of autoimmunity disease, it may be desirable to regulate and control the degree of B cell abatement by adjusting the dosage of humanization 2H7 antibody according to the order of severity of illness in disease and/or individual patient.B cell abatement can with and it is nonessential be complete.Or, whole B cell abatements are may expect in initial treatment, but can adjust dosage to reach that only part is cut down in successive treatment.In one embodiment, B cell abatement is at least 20%, i.e., retain 80% or less CD20 positive B-cells compared with the baseline values before treatment.In other embodiments, B cell abatement is 25%, 30%, 40%, 50%, 60%, 70% or more.Preferably, B cell abatement is enough to prevent advancing of disease, more preferably mitigates the S＆S of the disease specific in treatment, even more preferably cures disease.

Genentech and Biogen Idec clinical investigation have evaluated using multi-agent scope from the low treatment effect for reaching one anti-CD20 (hu2H7.v16 and Rituximab) the treatment autoimmunity diseases of 10mg up to 1g (see the background parts studied on Rituximab；And WO 04/056312, embodiment 16).In general, applying two doses of antibody in these clinical investigations, it is spaced about two weeks.The example for the therapeutic scheme studied in clinical investigation includes：It is 2 × 10mg (2 doses, every dose of 10mg that humanization CD20 antibody 2H7.v16, which is used for during rheumatoid arthritis,；For body weight 70kg, the patient of 67 inches of height, accumulated dose~10.1mg/m²), 2 × 50mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose 55mg/m²), 2 × 200mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose 220mg/m²), 2 × 500mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose~550mg/m²) and 2 × 1000mg (for body weight 70kg, the patient of 67 inches of height, accumulated dose~1100mg/m²)；And Rituxan be 2 × 500mg (for body weight 70kg, the patient of 67 inches of height, accumulated dose~550mg/m²), 2 × 1000mg is (for body weight 70kg, the patient of 67 inches of height, accumulated dose~1100mg/m²).In these each dosage, bone-marrow-derived lymphocyte is cut down in substantial circulation is observed after applying first dose of antibody.At present, the 10mg to 2000mg applied by 1 time or 2 intravenous infusions dosage range is also explored.

In the inventive method that autoimmunity disease and abatement B cell are treated in Serum of Patients With Autoimmune Diseases, in one embodiment, 1 dose of flat dosage by 0.1mg of scope to 1000mg applies humanization 2H7.v511 antibody.We have found that realizing substantial B cell abatement less than 300mg, or even 10mg flat dosage.In this way, in the B cell abatement and treatment method of the present invention, in different embodiments, hu2H7.v511 antibody is applied with 0.1,0.5,1,5,10,15,20,25,30,40,50,75,100,125,150,200 or 250mg dosage.If target is part or short-term B cell abatement, then can use relatively low-dose, such as 20mg, 10mg or lower.

For the treatment of CD20 positive cancers, it may be desirable to the abatement maximization of the B cell of anti-CD 20 antibodies target of the present invention will be used as.So, for the treatment of CD20 positive B-cells tumours, wish that B cell abatement is at least enough to prevent advancing of disease, this can be assessed by the skilled internist in this area, for example other S＆Ss by monitoring tumour growth (size), the propagation of cancerous cell type, transfer, specific cancer.Preferably, B cell abatement is enough in the time of at least two month to prevent advancing of disease, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 months or more months.In even more preferably embodiment, B cell abatement is enough regression time extending at least six month, more preferably 9 months, more preferably 1 year, more preferably 2 years, more preferably 3 years, even more preferably 5 years or more years.In a most preferred embodiment, B cell abatement is enough to cure disease.In preferred embodiments, the B cell abatement in cancer patient is at least about the 75% and more preferably 80%, 85%, 90%, 95%, 99% and even 100% of the preceding baseline values for the treatment of.

Hu2H7 antibody includes v16 and v511 and is recorded in hereafter EXPERIMENTAL EXAMPLESThe 18-20 for treating NHL the dosage therapeutic regimen of clinical test and the example of dosage.

Dosage 50 in terms of mg/ agent, 75,100,125,150,200,250,300,350mg/ agent can also be used for B cell malignant tumour such as NHL maintenance therapy.

Administration frequency can change with a number of factors.In general, generally at least 2 doses humanization 2H7CD20 binding antibodies will be applied to patient, 2-4 agent, 2-8 agent, 2-10 agent are subjected in different embodiments.Generally, 2 doses were applied in one month, 1 is typically spaced, 2 or 3 weeks.In a kind of therapeutic scheme for oncology, it is contemplated to 8 intravenous infusion 200mg/m²To 750mg/m²Between dosage.In a kind of dosage regimen for RA, 2 each 500mg humanized antibodies such as v16, v114 or v511 were applied to patient in every 6 months.Another dosage regimen for RA is every 9 months 2 times each 1000mg.The third dosage regimen for RA is every 6 months 2 times each 10mg v16, v114 or v511.According to the level of amelioration of disease or recurrence, more multi-agent or the maintenance therapy as disease can be entirely being applied during one's sickness.

One or more Current Therapies are invalid, the autoimmunity disease or B cell malignant tumor patient of Intolerance or taboo can be treated with any dosage regimen of the present invention.For example, present invention contemplates be used to the treatment method of the present invention respond not enough RA patient to TNF (TNF) inhibitor therapy or to antirheumatic drug (DMARD) therapy for mitigating disease.

" long-term " apply refers to short term patterns on the contrary, medicament is applied in a continuous mode, so that initial treatment effect (activity) is maintained into longer period of time.It is not the treatment free of discontinuities being carried out continuously that " interval " administration, which refers to, but substantially periodic.

Administration route

Humanization 2H7 antibody is applied to human patientses according to known method, such as applied by intravenous, for example inject or by continuous pouring for a period of time, by in subcutaneous, intramuscular, intraperitoneal, myelencephalon, intra-articular, intrasynovial, it is intrathecal or suction path, generally by intravenously or subcutaneously applying.

Typically for oncology indication, humanization 2H7 antibody formulations are such as transfused to apply by intravenous lines.In one embodiment, humanization 2H7 antibody is applied using 0.9% sodium chloride solution as infusion medium by intravenous infusion.In the I/II clinical trial phases of rheumatoid arthritis, antibody formulations are by intravenous infusion come using (see embodiment 20).

In another embodiment, humanization 2H7 antibody specifics are applied by being subcutaneously injected.

Conjoint therapy

When stating B cell tumour in the treatment, patient can be treated with the humanization 2H7 antibody of the present invention with the drug scheme of one or more therapeutic agent such as chemotherapeutic agents.Humanization 2H7 antibody can with chemotherapeutics simultaneously, it is sequential or replace administration, or applied after other therapies are without response.Standard chemotherapeutic for lymphoma treating may include endoxan, cytarabine, melphalan (melphalan) and mitoxantrone (mitoxantrone)+melphalan.CHOP is one of the most frequently used chemotherapy regimen for treating non_hodgkin lymphoma.The following is the medicine for CHOP schemes：Endoxan (trade mark cytoxan, neosar)；Adriamycin (Doxorubicin/hydroxyl Doxorubicin)；Vincristine (Oncovin)；With prednisolone (sometimes referred to as Deltasone or Orasone).In specific embodiments, CD20 binding antibodies are applied to required patient with one or more following chemotherapeutic agents：Doxorubicin, endoxan, vincristine and prednisolone.In a specific embodiment, combined to treat lymthoma (such as non_hodgkin lymphoma) patient with CHOP (endoxan, Doxorubicin, vincristine and prednisolone) therapy with the humanization 2H7 antibody of the present invention.In another embodiment, cancer patient can combine CVP (endoxan, vincristine and prednisolone) chemotherapy to treat with the humanization 2H7CD20 binding antibodies of the present invention.In a specific embodiment, CD20 positive NHL patients apply humanization 2H7.v511 or v114 and combine CVP, such as per 8 circulations of progress once in three weeks.In a treatment CLL specific embodiment, hu2H7.v511 antibody using the chemotherapy combined of one or both of fludarabine (fludarabine) and endoxan (cytoxan) with being applied.

" chemotherapeutics " refers to the chemical compound available for treating cancer.The example of chemotherapeutics includes alkylating agents (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) and CYTOXAN  endoxan (cyclophosphamide)；Alkylsulfonates (alkyl sulfonates), such as busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridines (aziridines), such as Benzodepa (benzodepa), carboquone (carboquone), meturedepa (meturedepa) and uredepa (uredepa)；Ethylenimines (ethylenimines) and methylamelamines (methylamelamines), including hemel (altretamine), triethylenemelamine (triethylenemelamine), triethylphosphoramide (triethylenephosphoramide), triethylene thiophosphamide (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine)；TLK286(TELCYTA^TM)；Annonaceousacetogenicompounds (acetogenins) (especially bullatacin (bullatacin) and bullatacinone (bullatacinone))；Delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (Dronabinol (dronabinol), MARINOL )；β-lapachol (lapachone)；Lapachol (lapachol)；Colchicines (colchicines)；Betulic acid (betulinic acid)；Camptothecine (camptothecin) (including synthetic analogues Hycamtin (topotecan) (HYCAMTIN ), CPT-11 (Irinotecan (irinotecan), CAMPTOSAR ), acetyl camptothecine, scopoletin (scopoletin) and 9-aminocamptothecin)；Bryostatin (bryostatin)；callystatin；CC-1065 (including its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues)；Podophyllotoxin (podophyllotoxin)；Podophyllic acid (podophyllinic acid)；Teniposide (teniposide)；Cryptophycins (cryptophycins) (particularly cryptophycin 1 and cryptophycin 8)；Dolastatin (dolastatin)；Duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1)；Eleutherobin (eleutherobin)；pancratistatin；sarcodictyin；Spongistatin (spongistatin)；Nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), cholophosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), mustron (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard)；Nitrosourea (nitrosoureas), such as BCNU (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine)；Diphosphonates (bisphosphonates), such as clodronate (clodronate)；Antibioticses, such as Enediyne Antibiotic (enediyne) (such as Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 are (see, for example, Agnew, Chem.Intl.Ed.Engl.33：183-186 (1994)) and anthracycline antibiotic (anthracyclines) such as annamycin,AD32,alcarubicin,Daunorubicin (daunorubicin),Dexrazoxane (dexrazoxane),DX-52-1,Epirubicin (epirubicin),GPX-100,Idarubicin (idarubicin),KRN5500,Menogaril (menogaril),Dynemicin includes dynemicin A,Ai sibo mycin (esperamicin),Neocarzinostatin (neocarzinostatin) chromophore and related chromoprotein Enediyne Antibiotic chromophore,Aclacinomycin (aclacinomycin),D actinomycin D (actinomycin),Anthramycin (anthramycin),Azaserine (azaserine),Bleomycin (bleomycin),Act-C (cactinomycin),carabicin,Carminomycin (carminomycin),Cardinophyllin (carzinophilin),Chromomycin (chromomycin),Actinomycin D (dactinomycin),Detorubicin (detorubicin),6- phenodiazine -5- oxygen-L- nor-leucines,ADRIAMYCIN  Doxorubicins (doxorubicin) (including morpholino Doxorubicin,Cyanomorpholino Doxorubicin,2- pyrroles is for Doxorubicin,Mycocet and deoxydoxorubicin),Esorubicin (esorubicin),Marcellomycin (marcellomycin),Mitomycin (mitomycins) such as mitomycin C,Mycophenolic acid (mycophenolic acid),Nogalamycin (nogalamycin),Olivomycin (olivomycin),Peplomycin (peplomycin),potfiromycin,Puromycin (puromycin),Triferricdoxorubicin (quelamycin),Rodorubicin (rodorubicin),Streptonigrin (streptonigrin),Streptozotocin (streptozocin),Tubercidin (tubercidin),Ubenimex (ubenimex),Zinostatin (zinostatin) and zorubicin (zorubicin)；Folacin, such as denopterin (denopterin), pteropterin (pteropterin) and Trimetrexate (trimetrexate)；Purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), thiapurine (thiamiprine) and thioguanine (thioguanine)；Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6- azauridines (azauridine), Carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine) and floxuridine (floxuridine)；Androgens, such as calusterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane) and Testolactone (testolactone)；Anti- adrenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane) and Trilostane (trilostane)；Folic acid supplement, such as folinic acid (folinicacid) (leucovorin)；Aceglatone (aceglatone)；Anti- folic acid antitumor agent; such as ALIMTA , LY231514 pemetrexeds (pemetrexed), dihydrofolate reductase inhibitor such as methopterin (methotrexate), antimetabolic species; such as 5 FU 5 fluorouracil (fluorouracil) (5-FU) and its prodrug such as UFT, S-1 and capecitabine (capecitabine), and thymidilate synthase inhibitors and glycinamide ribonucleotide transformylase inhibitor such as Raltitrexed (raltitrexed) (TOMUDEX^TM, TDX)；Dihydropyrimidine dehydrogenase inhibitor such as eniluracil (eniluracil)；Aldophosphamideglycoside (aldophosphamide glycoside)；Amino-laevulic acid (aminolevulinic acid)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (bisantrene)；Edatrexate (edatraxate)；Defosfamide (defosfamide)；Demecolcine (demecolcine)；Diaziquone (diaziquone)；elfornithine；Elliptinium Acetate (elliptinium acetate)；Epothilones (epothilone)；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxyurea (hydroxyurea)；Lentinan (lentinan)；Lonidamine (lonidamine)；Maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；Mopidamol (mopidamol)；C-283 (nitracrine)；Pentostatin (pentostatin)；Phenamet (phenamet)；THP (pirarubicin)；Losoxantrone (losoxantrone)；2- ethylhydrazides (ethylhydrazide)；Procarbazine (procarbazine)；PSK  polysaccharide compounds (JHS NaturalProducts, Eugene, OR)；Razoxane (razoxane)；Rhizomycin (rhizoxin)；Sizofiran (sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2 ', 2 "-trichlorotriethylamines；Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and snake rhzomorph (anguidin))；Urethane (urethan)；Eldisine (vindesine) (ELDISINE , FILDESIN )；Dacarbazine (dacarbazine)；Mannomustine (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Cytarabine (arabinoside) (" Ara-C ")；Endoxan (cyclophosphamide)；Phosphinothioylidynetrisaziridine (thiotepa)；Taxoids (taxoids) and taxanes (taxanes), such as TAXOL  taxols (paclitaxel) (Bristol-MyersSquibb Oncology, Princeton, N.J.), ABRAXANE^TMWithout cremophor (Cremophor), nano particle formulation taxol (the American Pharmaceutical Partners of albumin transformation, Schaumberg, Illinois) and TAXOTERE  Taxoteres (docetaxel) (- PoulencRorer, Antony, France)；Chlorambucil (chlorambucil)；Gemcitabine (gemcitabine) (GEMZAR )；6- thioguanines (thioguanine)；Purinethol (mercaptopurine)；Platinum (platinum)；Platinum analogs or the analog based on platinum, such as cis-platinum (cisplatin), oxaliplatin (oxaliplatin) and carboplatin (carboplatin)；Vincaleukoblastinum (vinblastine) (VELBAN )；Etoposide (etoposide) (VP-16)；Ifosfamide (ifosfamide)；Mitoxantrone (mitoxantrone)；Vincristine (vincristine) (ONCOVIN )；Vinca alkaloids (vinca alkaloid)；Vinorelbine (vinorelbine) (NAVELBINE )；NSC-279836 (novantrone)；Edatrexate (edatrexate)；Daunomycin (daunomycin)；Aminopterin (aminopterin)；Xeloda (xeloda)；Ibandronate (ibandronate)；Topoisomerase enzyme inhibitor RFS2000；DFMO (DMFO)；Retinoic acid-like (retinoid), such as retinoic acid (retinoic acid)；Pharmacy acceptable salt, acid or the derivative of any of above material；And the combination of two or more above-mentioned substances, such as CHOP (endoxan, Doxorubicin, the abbreviation of vincristine and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN^TM) joint 5-FU and folinic acid therapeutic scheme abbreviation).

This definition also includes acting as the antihormone agent of regulation or inhibitory hormone to function of tumor, such as anti-estrogens and SERM class (SERM), including such as TAM (tamoxifen) (including NOLVADEX  TAMs), Raloxifene (raloxifene), Droloxifene (droloxifene), 4-hydroxytamoxifen, Trioxifene (trioxifene), that Lip river former times sweet smell (keoxifene), LY117018, Onapristone (onapristone) and FARESTON  Toremifenes (toremifene)；Suppress the aromatase inhibitor of the aromatase enzyme of regulation estrogen production in adrenal gland, such as 4 (5)-imidazoles, aminoglutethimide (aminoglutethimide), MEGASE  megestrol acetates (megestrolacetate), AROMASIN  Exemestanes (exemestane), formestane (formestane), Fadrozole (fadrozole), RIVISOR  Vorozoles (vorozole), FEMARA  Letrozoles (letrozole) and ARIMIDEX  Anastrozoles (anastrozole)；Anti-androgens, such as Drogenil (flutamide), Nilutamide (nilutamide), bicalutamide (bicalutamide), Leuprorelin (leuprolide) and Goserelin (goserelin)；And troxacitabine (troxacitabine) (DOX nucleosides analogue of cytosine)；ASON, particularly those suppression are related to gene expression of the signal of adherent cell propagation in, such as PKC- α, Raf, H-Ras and EGF-R ELISA (EGF-R)；Vaccine, such as gene therapy vaccine, such as ALLOVECTIN  vaccines, LEUVECTIN  vaccines and VAXID  vaccines；PROLEUKIN  rIL-2；The inhibitor of LURTOTECAN  topoisomerases 1；ABARELIX  rmRH；And pharmacy acceptable salt, acid or the derivative of any of above material.

In addition, hu2H7 antibody and its functional fragment can be used for the B cell tumour (such as NHL) for treating expression CD20 with antineoplastic vascular propellant such as VEGF (VEGF) antagonist combination." antiangiogenic agent " or " angiogenesis inhibitor " refers to or the small molecular weight material of direct or indirect suppression angiogenesis (angiogenesis, vasculogenesis) or undesired vasopermeability, polynucleotides, polypeptide, the protein of separation, recombinant protein, antibody or its conjugate or fusion protein.For example, antiangiogenic agent is the antibody or other antagonists of anti-angiogenesis agent defined above, the small molecule (such as PTK787/ZK2284, SU6668) that such as VEGF antibody, the antibody of vegf receptor, blocking VEGF acceptor signal." VEGF antagonist " refers to neutralize, block, suppress, eliminate, reduce or disturb VEGF activity, including its molecule for being combined with one or more vegf receptors.In one embodiment, the patient with such B cell tumour combines Avastin  (bevacizumab with 2H7.v511 or 2H7.v114；Genentech) treated.Anti-VEGF antibody " bevacizumab " (bevacizumab, BV), also known as " rhuMAb VEGF " or " Avastin  " are according to Presta et al., Cancer Res.57：The recombinant humanized Anti-X activity that 4593-4599 (1997) is produced.

Hu2H7 antibody and its functional fragment, it is 2H7.v511 and 2H7.v114 in specific embodiments, the method that TRAIL combines the B cell tumour for treating expression CD20 can be also also known as with member's such as Apo2L/TRAIL (Apo2L) of TNF cytokine families.Total length native sequences people's Apo2L/TRAIL is that length is 281 amino acid, the II type transmembrane proteins of TNF cytokine family.The Apo2L/TRAIL of soluble form, such as includes those of ectodomain (ECD) or part thereof, it has been found that have various active, including apoptosis activity in mammalian cancer cells.Apo2L/TRAIL (being recorded in WO 97/01633 and WO 97/25428) is the soluble human protein as ECD fragment, includes the amino acid/11 14-281 of total length Apo-2L protein.

In the treatment when the literary autoimmunity disease or autoimmunity related disorders, patient can combine second of therapeutic agent such as immunodepressant with one or more hu2H7 antibody such as hu2H7.v511, such as be treated with drug scheme.Hu2H7 antibody can with immunodepressant simultaneously, it is sequential or replace administration, or applied after other therapies are without response.Immunodepressant can be applied with the dosage identical or relatively low with listed by this area.It is preferred that skeptophylaxis inhibitor depend on many factors, including sanatory type and patient medical history.

" immunodepressant " is used to refer to the material for acting as suppressing or covering the immune system of patient during complementary therapy herein.Such medicament is by including suppressing cell factor generation, lowering or suppressing autoantigen expression or cover the material of MHC antigens.The example of such medicament includes steroids, such as glucocorticosteroid, such as metacortandracin (prednisone), methylprednisolone (methylprednisolone) and dexamethasone (dexamethasone)；The pyrimidine of 2- amino -6- aryl -5- substitutions (referring to United States Patent (USP) No.4,665,077)；Imuran (azathioprine) (or endoxan (cyclophosphamide), if having adverse reaction to imuran)；Bromocriptine (bromocryptine)；Glutaraldehyde (it covers MHC antigens, such as United States Patent (USP) No.4, described in 120,649)；For MHC antigens and the anti-idiotype of MHC fragments；Cyclosporin A；Cell factor or cytokine receptor antagonist, including anti-interferon-γ ,-β or-Alpha antibodies；Anti-tumor necrosis factor-Alpha antibodies；Anti-tumor necrosis factor-β antibody；Anti- proleulzin antibody and anti-IL-2 receptor antibodies；Anti- L3T4 antibody；Heterologous antilymphocyte globulin (ALG)；General (pan) T antibody, preferably AntiCD3 McAb or anti-CD4/CD4a antibody；Soluble peptide containing LFA-3 binding domain (WO90/08187 is published on July 26th, 90)；Streptokinase；TGF-β；Dornase；RNA or DNA from host；FK506；RS-61443；Deoxyspergualin (deoxyspergualin)；Rapamycin (rapamycin)；φt cell receptor (United States Patent (USP) No.5,114,721)；φt cell receptor fragment (Offneret al., Science 251：430-432(1991)；WO 90/11294；WO 91/01133)；And φt cell receptor antibody (EP 340,109), such as T10B9.

For the treatment of rheumatoid arthritis, patient can combine one or more following medicines with CD20 binding antibodies (such as rituximab or ocrelizumab or its variant) and be treated：DMARD (antirheumatic drug for mitigating disease) (such as methopterin), NSAI or NSAID (nonsteroid anti-inflammatory drugs), immunodepressant (such as imuran (azathioprine)；MMF (mycophenolatemofetil, CellCept ；Roche)), antalgesic, glucocorticosteroid, endoxan, HUMIRA^TM(adalimumab, adalimumab；Abbott Laboratories), ARAVA  (leflunomide, leflunomide), REMICADE  (infliximab, infliximab；Centocor Inc., Malvern, Pa), ENBREL (Etanercept, etanercept；Immunex, WA), ACTEMRA (tocilizumab；Roche, Switzerland), cox 2 inhibitor.The DMARD for being generally used for RA is HCQ (hydroxychloroquine), SASP (sulfasalazine), methopterin (methotrexate), leflunomide (leflunomide), Etanercept (etanercept), infliximab (infliximab), imuran (azathioprine), Beracilline, golden (Gold) (oral), golden (Gold) (intramuscular), minocycline (minocycline), cyclosporin (cyclosporine), staphylococcal protein A immuno absorbence.

Adalimumab (Adalimumab) is the human monoclonal antibodies with reference to TNF α.Infliximab (Infliximab) is gomphosis mouse-human monoclonal antibodies with reference to TNF α.It is to treat RA and the immunosuppressive drug of Crohn's disease, prescription medicine.Infliximab is connected with lethal reaction, such as heart failure and infection, including tuberculosis, and cause MS demyelinate.Actemra (tocilizumab) is Humanized anti-human interleukin-6 (IL-6) acceptor.

Etanercept (Etanercept) is " immunoadhesin " fusion protein, is connected with the Fc parts of human IgG1 by the extracellular ligand binding moiety of people 75kD (p75) Tumor Necrosis Factor Receptors (TNFR) and is constituted.Etanercept (ENBREL ) is to ratify the injectable drug for activity RA therapies in the U.S..Etanercept combination TNF α simultaneously undertakes the most of TNF α of the removing from joint and blood, thus prevents TNF α from promoting the effect of other symptoms of inflammation and rheumatoid arthritis.The medicine and negative side-effects are connected, including serious infection and pyemia, neurological conditions such as multiple sclerosis (MS).See, for example, www.remicade-infliximab.com/pages/enbrel_embrel.html.

For RA conventional therapy, see, for example, " Guidelines for the management ofrheumatoid arthritis ", Arthritis ＆ Rheumatism 46 (2)：328-346 (February, 2002).In a specific embodiment, RA patient is treated with the antibody combined methopterins of hu2H7CD20 (MTX) of the present invention.MTX Exemplary doses are about 7.5-25mg/kg/ weeks.MTX can pass through oral and subcutaneous administration.

In one embodiment, patient also receives adjoint MTX (10-25mg/ weeks, oral or parenteral), and the following corticosteroid formulation of composition：Methylprednisolone, 100mg is intravenous, 30 minutes and metacortandracin before infusion CD20 antibody, 60mg, orally, the 2-7 days；30mg, orally, the 8-14 days；Baseline dose is returned to, by the 16th day.Patient can also receive folic acid (5mg/ weeks), as single dose or be used as separated daily dose.Patient optionally continues to receive any background corticosteroid (10mg/ days metacortandracins or equivalent) during whole treatment.

For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, such as Remicade  (infliximab infliximab can be combined with the CD20 binding antibodies of the present invention；CentocorInc., Malvern, Pa), ENBREL (Etanercept etanercept；Immunex, WA) treat patient.

SLE treatment includes combining for CD20 antibody and high dose corticosteroid and/or endoxan (HDCC).Patient with SLE, AAV and NMO can be treated with the antibody combined any following medicines of 2H7 of the present invention：Corticosteroid, NSAID, antalgesic, cox 2 inhibitor, glucocorticosteroid, routine DMARD (such as methopterin, SASP, HCQ, leflunomide), the biological such as anti-Blys (such as belimumab) of DMARD, anti-IL6R such as tocilizumab, CTLA4-Ig (abatacept), anti-CD22 such as epratuzumabs (epratuzumab), immunodepressant (such as imuran；MMF (CellCept ；)) and cytotoxic agent (such as endoxan) Roche.

For the treatment of psoriasis, patient can apply the antibody combined local treatments of humanization 2H7, such as topical steroids, anthralin (anthralin), calcipotriene (calcipotriene), clobetasol (clobetasol) and tazarotene (tazarotene), or joint methopterin, retinoic acid-like, cyclosporin, PUVA and UVB therapies.In one embodiment, it is sequential or simultaneously use humanization 2H7 antibody and cyclosporin therapy psoriatic.

The hu2H7 antibody of the present invention, particularly hu2H7.v511 and hu2H7.v114 can combine BAFF antagonists simultaneously, it is sequential or be used to treat autoimmunity disease or B cell tumour in alternate scheme.BAFF and BAFF antagonists are as defined above.In one embodiment, BAFF antagonists are the antibody with reference to people BR3, the antibody be preferably humanization, people's or chimeric.In another embodiment, BAFF antagonists are BR3-Fc fusion proteins.Synergy of the anti-CD 20 antibodies with BAFF antagonists in terms of B cell is cut down is had been reported (see, for example, WO05/000351, supra；Gonget al., J.Immunol.174：817-826(2005)).

In order to which toxicity is minimized, traditional systemic therapy can be implemented with samsara, sequential, united or interval therapeutic scheme, or hu2H7CD20 binding antibodies composition uses the relatively low-dose scheme for combining of present dose.

Medicinal proportional preparation

The treatment preparaton of the hu2H7CD20 binding antibodies used according to the present invention passes through with the antibody and the optional acceptable carrier of pharmacy, excipient or stabilizer (Remington ' sPharmaceutical Sciences for expecting purity, 16th edition, Osol, A. (1980) are compiled) mixing, prepared in the form of freeze-dried formulation or the aqueous solution.Acceptable carrier, excipient or stabilizer are nontoxic, including buffer, such as phosphate, citrate and other organic acids to recipient in the dosage and concentration used；Antioxidant, including ascorbic acid and methionine；Preservative (such as octadecyl dimethyl benzyl ammonium chloride；Hexamethonium chloride；Benzalkonium chloride, benzethonium chloride；Phenol, butanol or phenmethylol；Alkyl paraben, such as methyl p-hydroxybenzoate or propyl ester；Catechol；Resorcinol；Cyclohexanol；3- amylalcohols；And metacresol)；Low molecule amount (less than about 10 residues) polypeptide；Protein, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer, such as polyvinylpyrrolidone；Amino acid, such as glycine, glutamine, asparagine, histidine, arginine or lysine；Monose, disaccharides and other carbohydrate, including glucose, mannose or dextrin；Chelating agent, such as EDTA；Carbohydrate, such as sucrose, mannitol, trehalose or sorbierite；Into salt gegenion, such as sodium；Metal composite (such as Zn- protein complexes)；And/or nonionic surfactant, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Exemplary hu2H7 antibody formulations are recorded in WO 98/56418, and clearly income is used as reference herein.Another preparaton is liquid multi-agent preparaton, comprising 40mg/mL hu2H7 antibody, 25mM acetates, 150mM trehaloses, 0.9% phenmethylol, 0.02% polysorbate20 pH5.0, and minimum storage life is preserved 2 years at 2-8 DEG C.Another anti-CD 20 antibodies preparaton interested is in 9.0mg/mL sodium chloride, the citric acid monohydrate sodium of 7.35mg/mL bis-, and 10mg/mL antibody is included in 0.7mg/mL polysorbate80s, and Injectable sterile water pH6.5.Also a kind of aqueous medicinal proportional preparation includes 10-30mM sodium acetates, about pH4.8 to the preferred pH5.5 of about pH5.5, it is used as the polysorbate of the about 0.01-0.1%v/v amounts of surfactant, the trehalose of about 2-10%w/v amounts, with the phenmethylol (U.S.6 as preservative, 171,586).Freeze-dried formulation suitable for subcutaneous administration is recorded in WO 97/04801.Such freeze-dried formulation can use suitable solvent to rebuild to increased protein concentration, and the preparaton of reconstruction can subcutaneous administration mammal to be treated in this article.

In a specific embodiment, a kind of humanization 2H7.v16 intravenous preparaton is：20mM sodium acetates, 4% Trehalose Dihydrate, the 0.02% poly- (Tween 20 of sorbitol ester 20^TM) 30mg/ml antibody in pH5.3.The intravenous preparaton can be applied to oncology indication such as NHL and for rheumatoid arthritis.A kind of preparaton of humanization 2H7.v511 variants is 10mM sulfuric acid histidines, 60mg/ml sucrose (6%), the poly- sorbitol esters 20 (0.02%) of 0.2mg/ml, and the 15-30mg/ml antibody in Injectable sterile water pH5.8, preferably 20mg/mL antibody.In another embodiment, the preparaton of 2H7 variants, specifically 2H7.v511 is 20mg/ml2H7, and 20mM sodium acetates, 4% Trehalose Dihydrate, 0.02% poly- sorbitol ester 20pH5.5 is applied for intravenous.The preparaton can be used in azygos vein being transfused.2H7.v114 a kind of preparaton be 20mM sodium acetates, 240mM (8%) Trehalose Dihydrate, the 15-25mg/ml antibody in 0.02% poly- sorbitol ester 20pH5.3, preferably 20mg/ml.

Preparaton herein can also contain have more than one kind treat reactive compound necessary to specific indication, preferably those complementary activities and the compound not adversely affected each other.For example, it may be desirable to further provide for cytotoxic agent, chemotherapeutics, cell factor or immunodepressant (such as acting on the immunodepressant of T cell, such as cyclosporin or the antibody of combination T cell, such as antibody with reference to LFA-1).The effective dose of such other medicaments depends on amount, disease or the illness of antibody or the type for the treatment of and other factorses discussed above present in preparaton.These be typically with identical dosage described herein, used with administration route described herein, or the dosage used so far about 1-99%.

Active component can be also contained in the microcapsules prepared for example by condensation technique or by interfacial polymerization (being for example hydroxymethyl cellulose or gelatin-microcapsule and poly- (methyl methacrylate) microcapsules respectively), in colloidal drug delivery system (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule), or in macro emulsion.Such technology is disclosed in such as Remington ' sPharmaceutical Sciences, and the 16th edition, Osol, A. compiles (1980).

Extended release preparation can be prepared.The suitable example of extended release preparation includes the solid hydrophobic polymers semipermeable matrices containing antagonist, and the matrix is the form of approved product, such as film or microcapsules.The example of sustained-release matrix includes polyester, hydrogel (such as poly- (2- ethoxys-methacrylate) or poly- (vinyl alcohol)), polyactide (United States Patent (USP) No.3,773,919), the copolymer of Pidolidone and Pidolidone γ ethyl esters, nondegradable ethane-acetic acid ethyenyl, degradable lactic acid-ethanol copolymer such as LUPRON DEPOT^TM(the Injectable microspheres body being made up of lactic acid-ethanol copolymer and leuprorelin acetate) and poly- D- (-) -3-hydroxybutyrate.

Preparaton for applying in vivo must be sterile.This readily can be realized by using sterilised membrane filter filtering.

Product and kit

Another embodiment of the invention is the product for including the material that can be used for treatment autoimmunity disease and related disorders and CD20 positive cancers such as non_hodgkin lymphoma.The product includes on container and container or the label or package insert related to container.Suitable container is included such as medicine bottle, pencil, syringe.The container can be made of multiple material, such as glass or plastics.The container is equipped with the composition for effectively treating the illness, can have sterile access port (such as described container can be the medicine bottle of intravenous solution bag or the plug that can pierce with hypodermic needle).At least one of composition active agents are hu2H7 antibody, hu2H7.v511 of the invention.The label or package insert indicate that said composition is used to treat the specific illness.The label or package insert further include the specification on applying the antibody compositions to patient.

Package insert refers to the specification being typically included in during treatment is packed with product ommercialization, it include used about such treatment with product indication, usage, dosage, using, avoid and/or warning message.In one embodiment, package insert indicates that said composition is used to treat non_hodgkin lymphoma.

In addition, the product can further comprise second container, wherein being subjected to buffer solution, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods grignard (Ringer) solution and dextrose solution equipped with pharmacy.It can further comprise the other materials from business and user's position needs, including other buffer solutions, diluent, filter, pin and syringe.

The kit available for various purposes is additionally provided, for example, kills determination method, the positive control as apoptosis determination method, for purifying or immunoprecipitation CD20 from cell for B cell.In order to separate and purify CD20, the kit can include the hu2H7.v511 antibody being coupled with pearl (such as sepharose pearls).It can provide comprising CD20 vitro detections and quantitative antibody is used for, for example, be carried out in ELISA or western blot.As product, the kit includes on container and container or the label or package insert related to container.The container is equipped with the composition for including at least one anti-CD 20 antibodies of the present invention.It may include such as diluent and buffer solution, other container of control antibodies are housed.The label or package insert can provide the description of said composition and be intended to the specification that external or diagnosis is used.

EXPERIMENTAL EXAMPLESThe

Material

Mouse：H CD20 (hCD20)⁺The generation of mouse is realized by using the bacterial artificial chromosome (BAC) for being mixed with hCD20 locus.BAC clone is injected the blastocyst derived from FVB mouse to produce expression hCD20 transgenosis (Tg) creator's strain.Refering to Gong et al. (2005) J.Immunol.174：Relevant hCD20Tg in 817-826⁺The generation of mouse and the detailed description of sign.hCD20Tg⁺Mouse is then and hCD16Tg⁺mCD16^-/-Mate to produce hCD20Tg⁺mCD16^-/-hCD16Tg⁺Mouse, for research described below.

Antibody：All antibody for facs analysis are purchased from BD PharMingen.

Equipment/software：Facs analysis is carried out using FACScan or FACSCalibur machines and using purchased from BectonDickinson CellQuest softwares.

Conventional method

The preparation of single cell suspension：50 μ l mouse bloods are collected from eye socket with the pipe equipped with EDTA, then with ACK lysis buffer solution (being purchased from Biosource) lysed erythrocyte.After they are removed, mouse spleen particle is melted into single cell suspension with frosted glass slide (frosted glass slide).Cell pellet is washed out, is resuspended and filters.With the abdominal cavity of every mouse of PBS cold 5ml.Irrigating solution is reclaimed with 5ml syringes then to centrifuge.The leucocyte from mouse blood, spleen and peritonaeum and counting are washed out, then dyeing supplies facs analysis.

FACS is dyed：To about 1 × 10⁶Individual cell shows that label (referring to the definition in facs analysis) is dyed in the albumin (Sigma) of 100 μ l phosphate buffered saline (PBS)s+1% for various cells.After standing for 30 min on ice, dyed cell is washed and is resuspended, then carry out facs analysis.Facs analysis：Facs analysis is carried out using FACScan or FACSCalibur and CellQuest softwares.Blood B cells are defined to CD21⁺CD23⁺；Peritoneal B cells are defined to CD19⁺；Splenic B cells are defined to B220⁺；MZ (marginal zone) B cell is defined to CD21^{It is high}CD23^{It is low}；And FO (folliculus) B cell is defined to CD21⁺CD23^{It is high}。

Statistics：Data are expressed as mean+/-standard error (n=5).Statistical analysis examines (two-tail unpaired T test) to carry out using the not paired T of double track.

Embodiment 1：The humanization of the anti-CD20 mouse monoclonal antibodies of 2H7

Mouse anti-humen CD 20 antibody 2H7 (referring to mouse referred to herein as m2H7, m) humanization is carried out with a series of direct mutagenesis steps.Mouse 2H7 antibody variable sequences and chimeric 2H7 with mouse V areas and people C areas have been described, see, for example, United States Patent (USP) 5,846,818 and 6,204,023.U.S.5 (is disclosed in by the amino acid sequence for comparing mouse 2H7 variable domains, 846,818) with sequence (the Kabat et al. of known antibodies, Sequences of proteins of immunological interest, Ed.5.PublicHealth Service, National Institutes of Health, Bethesda, MD (1991)) identify 2H7 CDR residues.The CDR region of light chain and heavy chain is defined based on sequence denatured (Kabat et al., together above) and distinguished as shown in FIG. 1A and 1B.Using the oligonucleotides (table 1) of synthesis, pass through direct mutagenesis (Kunkel, Proc.Natl.Acad.Sci.82：488-492 (1985)) all 6 Ge Shu 2H7CDR areas are imported into included in plasmid pVX4 (referring to WO 04/056342 Fig. 2, the PCT publication is completely taken in herein as reference) and consensus sequence V_κI、V_HIII(V_Lκ hypotypes I, V_HHypotype III) corresponding whole person's Fab frameworks.

Phasmid pVX4 is used for mutagenesis and in expression in escherichia coli F (ab) s.With phasmid pb0720, pB0475 (Cunningham et al., Science 243：1330-1336 (1989)) a kind of derivative based on, pVX4 include encoding humanized shared κ hypotypes I light chains (V_LκI-C_L) and the shared hypotype III heavy chains (V of humanization_HIII-C_H1) DNA fragmentation of anti-IFN-α (interferon-' alpha ') antibody.PVX4 also has alkaline phosphatase promoter and Shine-Dalgamo sequences, and the two is derived from another plasmid pAK2 (Carter the et al., Proc.Natl.Acad.Sci.USA89 based on pUC119 of art heretofore taught：4285(1992)).Unique Spel restriction sites are introduced between coding F (ab) light chains and the DNA of heavy chain.Preceding 23 amino acid of both anti-IFN-α heavy chain and light chain is StII secretory signal sequences (Chang et al., Gene 55：189-196(1987)).

In order to build the 2H7 (2H7.v2) that CDR exchanges pattern, direct mutagenesis is carried out with the pVX4 templates containing BrdU；All 6 CDR of anti-IFN-α become mouse 2H7CDR.Gained molecule is referred to as humanization 2H7 patterns 2 (2H7.v2) or 2H7 " CDR exchanges pattern "；It has the m2H7CDR residues and joint owner's FR residues shown in Figure 1A and 1B.Humanization 2H7.v2 is used for further humanization.

Table 3 shows the oligonucleotide sequence for creating each mouse 2H7 (m2H7) CDR in H and L chains.For example, CDR-H1 oligonucleotides is used to rebuild m2H7H chains CDR1.CDR-H1, CDR-H2 and CDR-H3 points separately refer to H chains CDR1, CDR2 and CDR3；Likewise, CDR-L1, CDR-L2 and CDR-L3 refer to each L chain CDR.Replacement in CDR-H2 is carried out with two kinds of oligonucleotides CDR-H2A and CDR-H2B with two steps.

Table 3：Oligonucleotide sequence for mouse 2H7CDR to be exchanged to people's framework in incorporation pVX4 by CDR.The residue changed by each oligonucleotides is indicated with underscore.

Substitute	Oligonucleotide sequence
		CDR-H1	C TAC ACC TTC ACG AGC TAT AAC ATG CAC TGG   GTC CG(SEQ ID NO.35)
CDR-H2A	G ATT AAT CCT GAC AAC GGC GAC ACG AGC TAT   AAC CAG AAG TTC AAG GGC CG(SEQ ID NO.36)
		CDR-H2B	GAA TGG GTT GCA GCG ATC TAT CCT GGC AAC GGC   GAC AC(SEQ ID NO.37)
CDR-H3	AT TAT TGT GCT CGA GTG GTC TAC TAT AGC AAC   AGC TAC TGG TAC TTC GAC GTC TGG GGT CAA

	GGA  (SEQ ID NO.38)
		CDR-L1	C TGC ACA GCC AGC TCT TCT GTC AGC TAT ATG   CAT TG(SEQ ID NO.39)
CDR-L2	AA CTA CTG ATT TAC GCT CCA TCG AAC CTC GCG   TCT GGA GTC C(SEQ ID NO.40)
		CDR-L3	TAT TAC TGT CAA CAG TGG AGC TTC AAT CCG CCC   ACA TTT GGA CAG(SEQ ID NO.41)

In order to be compared with humanized constructs, the chimeric 2H7Fab of expression is constructed by direct mutagenesis (Kunkel, with above) and (includes mouse V_LAnd V_HDomain and people C_LAnd C _H1 domain) plasmid, wherein using synthesis oligonucleotides by mouse framework residue introduce 2H7.v2.Gained Plasmid Constructs are used to express the chimeric Fab for being referred to as 2H7.v6.8, as shown in Fig. 3 in WO 04/056342.Chain coded by each of Fab has the StII secretory signal sequences above for 23 amino acid described by pVX4.

According to mouse 2H7 Framework residues and people V_κI、V_HIII has framework (Figure 1A and 1B) and previous humanized antibody (Carter et al., Proc.Natl.Acad.Sci.USA 89：4285-4289 (1992)) comparison, by direct mutagenesis by some framework mutations introduce 2H7.v2Fab constructs.These mutation cause the shared Framework residues of some people in site of CDR conformations or antigen contact may influenceed to change over those residues found in mouse 2H7 frameworks.Pattern 3 includes V_H(R71V, N73K), pattern 4 includes V_H(R71V), pattern 5 includes V_H(R71V, N73K) and V_L(L46P), and pattern 6 include V_H(R71V, N73K) and V_L(L46P, L47W).

The humanization and chimeric Fab pattern of m2H7 antibody in expression in escherichia coli and are purified as follows.Plasmid is transformed into coli strain XL-1 Blue (Stratagene, San Diego, CA), for preparing double-strand and single stranded DNA.For every kind of variant, light chain and heavy chains are completely sequenced with dideoxyribonucleoside acid system (Sequenase, U.S.Biochemical Corp.).Plasmid is transformed into coli strain 16C9, MM294 a kind of derivative strain, is laid on the LB flat boards containing 5 μ g/ml carbenicillins, picking individual colonies are used for protein expression.Single bacterium colony is cultivated 5-8 hours in 5ml LB-100 μ g/ml carbenicillins in 37 DEG C.5ml cultures are added into 500ml AP5-100 μ g/ml carbenicillins and it is grown 16 hours in 37 DEG C in 4L band flask with indentation.The composition of AP5 culture mediums is：1.5g glucose, 11.0 Hycase SF, 0.6g yeast extracts (through calibrating), the anhydrous MgSO of 0.19g₄, 1.07gNH₄Cl, 3.73g KCl, 1.2g NaCl, 120ml 1M triethanolamines, pH7.4 add water to 1L, are then filtered by 0.1 μm of Sealkeen filter sterility.

By in lL centrifugal bottles (Nalgene) so that 3000xg centrifugations are come harvesting and remove supernatant.After freezing 1 hour, sediment is resuspended in cold 10mM the MES-10mM EDTA, pH5.0 (buffer A) of 25ml.250 μ l 0.1M PMSF (Sigma) are added to suppress proteolysis, and add 3.5ml liquid storage 10mg/ml hen egg-white lysozymes (Sigma) to help bacteria cell wall to dissolve.After gentle shake on ice 1 hour, sample is centrifuged 15 minutes with 40,000xg.Supernatant is adjusted to 50ml with buffer A and is added on the 2ml DEAE posts balanced with buffer A.Then efflux is added on Protein G-Sepharose CL-4B (Pharmacia) post (0.5ml bed volumes) balanced with buffer A.Pillar is cleaned with 10ml buffer As and with the 3ml 0.3M glycine in 1.25ml 1M Tris, pH8.0, pH3.0 elutions.Then F (ab) buffer-exchanged into PBS and is concentrated into final volume 0.5ml with Centricon-30 (Amicon).All F (ab) s carry out SDS-PAGE to determine purity, and confirm by electrospray mass spectrometry the molecular weight of each variant.

In the ELISA binding assays (being described below) based on cell, Fab, including chimeric combinations of the 2H7Fab to CD20 are difficult to detect.Therefore, 2H7Fab patterns are reconstructed into total length IgG1 antibody, for determining and further mutagenesis.

By by chimeric 2H7 (v6.8) Fab and humanized Fab pattern 2 to 6 V_LAnd V_MDomain is subcloned into pRK carriers (Gorman et al., the DNA Prot.Eng.Tech.2 for mammalian cell expression of art heretofore taught：3-10 (1990)), construct the plasmid for expressing total length IgG.In brief, digest every kind of Fab constructs to cut out V with EcoRV and BlpI_LFragment, is cloned into for expressing Whole light chains (V_L-C_LDomain) plasmid pDR1 (WO 04/056312 Fig. 4) EcoRV/BlpI sites.In addition, digesting every kind of Fab constructs to cut out V with PvuII and ApaI_HFragment, is cloned into for expressing entire heavy chain (V_H-C_H1- hinges-C_H2-C_H3 domains) plasmid pDR2 (WO 04/056312 Fig. 5) PvuII/ApaI sites.For every kind of IgG variants, by the way that light chain expression plasmid and heavy chain expression plasmid cotransfection to be entered to adenovirus transfected human embryonic kidney cell 293 (Graham et al., J.Gen.Virol.36：59-74, (1977)) transiently transfected.In brief, 293 cells are distributed in the day before transfection, and be laid in the culture medium containing serum.Next day, the double-stranded DNA for being prepared into calcium phosphate precipitation is added, pAdVAntage is then added^TMDNA (Promega, Madison, WI), cell is in 37 DEG C of incubated overnights.Cell is cultivated in serum free medium and is harvested after 4 days.With albumin A-Sepharose CL-4B antibody purifications from culture supernatants, then buffer-exchanged in 140mM NaCl, pH6.0, and is concentrated with Centricon-10 (Amicon) to 10mM sodium succinates.Protein concentration is determined by quantitative amino acid analysis.

In order to measure the RA with CD20 antigens, the ELISA determination methods based on cell are developed.By people's B lymphoblast sample WIL2-S cells (ATCC CRL8885, American type culture collection American Type Culture Collection, Rockville, MD 2mM Glus) are being supplemented with, in moist 5%CO in the RPMI 1640 of 20mM HEPES, pH7.2 and 10% heat-inactivated fetal bovine serum₂Cultivated in incubator.Cell is cleaned with the PBS (measure buffer solution) containing 1%FBS and with 250-300, and 000 cells/well is inoculated in 96 hole round bottom plates (Nunc, Roskilde, Denmark).By in buffer solution is determined the standard items (15.6-1000ng/ml 2H7v6.8 are fitted together to IgG) of twice of serial dilution and the sample (2.7-2000ng/ml) of three times serial dilution be added on plate.Plate is embedded in ice and placed 45 minutes.In order to remove uncombined antibody, 0.1mL measure buffer solutions are added in hole.Plate is centrifuged and supernatant is removed.Buffer solution, which is determined, with 0.2mL cleans cell again twice.The antibody with hardened conjunction is detected by the way that the goat anti-human Fc antibodies (Jackson ImmunoResearch, West Grove, PA) of peroxidase conjugate are added on plate.After insulation 45 minutes, cell is cleaned as previously described.By tmb substrate (3,3 ', 5,5 '-tetramethyl benzidine；Kirkegaard ＆ Perry Laboratories, Gaithersburg, MD) it is added on plate.By adding 1M phosphoric acid come terminating reaction.Titration curve is fitted with four parameter nonlinear regression curve fitting procedures (KaleidaGraph, Synergy software, Reading, PA).Bioassay standard product are in the absorbance (mid-OD) of titration curve midpoint and its corresponding concentration.Then concentration of every kind of variant at this mid-OD is determined, and by the concentration of standard items divided by the concentration of each variant.Therefore the numerical value is combining ratio of each variant relative to standard items.The standard deviation of relative affinity (equivalent concentration) is typically +/- 10% between experiment.

As shown in table 4, the CD20 of CDR exchange variants (v.2) is combined has very big reduction compared with chimeric 2H7 (v.6.8).Made moderate progress however, pattern 3 to 6 shows to combine.In order to determine the minimum mutation number that the binding affinity that binding affinity is reverted into chimeric 2H7 may need, the combination of other mutation and mutation is constructed by direct mutagenesis, variant 7 to 17 as shown in table 5 is produced.Specifically, these include V_HIt is mutated A49G, F67A, I69L, N73K and L78A；And V_LIt is mutated M4L, M33I and F71Y.Pattern 16 and 17 shows best RA, within 2 times of the affinity of chimeric pattern, therebetween without significant difference (s.d.=+/- 10%).In order that mutation number is minimized, therefore select the pattern 16 (table 5) for only having 4 people's Framework residues to be mutated into mouse framework residue as humanization form for other sign.

Table 4：Compare humanization 2H7IgG variants and RAs of the chimeric 2H7 to CD20 with the ELISA based on cell.Relative combine is expressed as being fitted together to ratio of the 2H7 concentration relative to the equivalent variant concentration with reference to needed for；Therefore ratio ＜ 1 represents that the affinity of the variant is weaker.Standard deviation average out to +/- 10% in relative affinity measure.It is to exchange pattern relative to CDR that framework in variable domain, which is substituted, according to Kabat numbering systems (Kabat et al., with above).

2H7 patterns	Heavy chain (VH) substitute	Light chain (VL) substitute	It is relative to combine
				6.8	(block polymer)	(block polymer)	-1-
2	(CDR exchanges)	(CDR exchanges)	0.01
				3	R71V, N73K	(CDR exchanges)	0.21
4	R71V	(CDR exchanges)	0.21
				5	R71V, N73K	L46P	0.50
6	R71V, N73K	L46P, L47W	0.58
				7	R71V	L46P	0.33
8	R71V, L78A	L46P	0.19
				9	R71V, F67A	L46P	0.07
10	R71V, F67A, I69L	L46P	0.12
				11	R71V, F67A, L78A	L46P	0.19
12	R71V	L46P, M4L	0.32
				13	R71V	L46P, M33I	0.31
14	R71V	L46P, F71Y	0.25
				15	R71V	L46P, M4L, M33I	0.26
16	R71V, N73K, A49G	L46P	0.65
				17	R71V, N73K, A49G	L46P, L47W	0.67

Table 5：For building the mutation V in humanization 2H7 patterns 16 (2H7.v16)_H(A49G, R71V, N73K) and V_L(L46P) oligonucleotide sequence.The amino acid replacement that the codon coding underlined is indicated.For V_H(R71V, N73K) and V_L(L46P), oligomer is shown as sense strand, because these are the mutagenesis in Fab templates, and for V_H(A49G), oligomer is shown as antisense strand, because this is used together with pRK (IgG heavy chains) template.The protein sequence of pattern 16 sees above " composition " part.

Substitute	Oligonucleotide sequence
		VH(R71V, N73K)	GT TTC ACT ATA AGT GTC GAC AAG TCC AAA   AAC ACATT(SEQ ID NO.42)
VH(A49G)	GCCAGGATAGATGGCGCCAACCCATTCCAGG   CC(SEQ ID NO.43)
		VL(L46P)	AAGCTCCGAAACCACTGATTTACGCT(SEQ

ID NO.44)

Embodiment 2：Other mutation in 2H7CDR areas

Also substituted and alternative combinations to being accredited as the other residue of important CDR position measurements by Alanine-scanning.V.96, some combinatory variants, particularly show closer than v.16 combining.

Table 6：With mutation and the influence of non-alanine alternative combinations in the CDR region of ELISA (WIL2-S cells) the measurement humanizations 2H7.v16 based on cell.It is relative with reference to being expressed as ratio of the 2H7.v16 parents concentration relative to the equivalent variant concentration with reference to needed for CD20's；Therefore ratio ＜ 1 represents that the affinity of the variant is weaker；Ratio ＞ 1 represents that the affinity of the variant is higher.The standard deviation average out to +/- 10% that relative affinity is determined.It is relative to 2H7.v16, according to Kabat numbering systems (Kabat et al., with above) that framework in variable domain, which is substituted,.

2H7 patterns	Heavy chain is substituted	Light chain is substituted	It is relative to combine
16	-	-	-1-
				96	D56A, N100A	S92A	3.5
97	S99T, N100G, Y100bI	-	0.99
				98	S99G, N100S, Y100bI	-	1.6
99	N100G, Y100bI	-	0.80
				101	N54S, D56A	-	1.7
102	N54K, D56A	-	0.48
				103	D56A, N100A	-	2.1
104	S99T, N100G	-	0.81
				105	S99G, N100S	-	1.1
106	N100G	-	~1
				167	S100aG, Y100bS	-
136	D56A, N100A	S56A, S92A	2.6
				137	D56A, N100A	A55G S92A	2.1
156	D56A, N100A	S26A, S56A, S92A	2.1
				107	D56A, N100A, Y100bI	S92A	Do not express

182	Y27W	-
				183	Y27F	-
184	F29Y	-
				185	F29W	-
186	Y32F	-
				187	Y32W	-
188	N33Q	-
				189	N33D	-
190	N33Y	-
				191	N33S	-
208	H35S	-
209	A50S	-
				210	A50R	-
211	A50V	-
				212	A50L	-
168	Y52W	-
				169	Y52F	-	0.75
170	N54D	-	0.25
				171	N54S	-	1.2
172	D56K	-	1
				173	D56R	-
174	D56H	-	1.5
				175	D56E	-	1.2
213	D56S	-
				214	D56G	-
215	D56N	-
				216	D56Y	-
176	Y59W	-
				177	Y59F	-

180	K62R	-
				181	K62D	-
178	F63W	-
				179	F63Y	-
				157	Y97W	-	0.64
158	Y97F	-	1.2
				159	Y98W	-	0.64
160	Y98F	-	0.88
				106	N100G	-
161	W100cY	-	0.05
				162	W100cF	-	0.27
163	F100eY	-	0.59
				164	F100eW	-	0.71
165	D101N	-	0.64
				166	S99G, N100G, S100aD, Y100b is deleted	-	0.99
217	V102Y	-	1.0
207	-	H34Y
192	-	Q89E
				193	-	Q89N
194	-	Q90E
				195	-	Q90N
196	-	W91Y
				197	-	W91F
205	-	S92N
				206	-	S92G
198	-	F93Y

199	-	F93W
				204	-	F93S, N94Y
200	-	P96L
				201	-	P96Y
202	-	P96W
				203	-	P96R

Embodiment 3：Humanization 2H7 variants with enhanced effector functions

Because 2H7 can be by the dissolving of the two mediate B cell of complement-dependent cytotoxicity (CDC) and the cytotoxicity (ADCC) of antibody dependent cellular, it is intended to CDC and ADCC activity of the generation with improvement humanization 2H7.v16 variant.Mutation (Idusogie et al., the J.Immunol.166 of some residues in the other antibody Fc districts for having described to improve CDC by the combination of enhancing and complement component Clq and carry out：2571-2575(2001)).Also describe and mutation (Shieldset al., J.Biol.Chem.276 to improve ADCC are combined with inhibition FcY acceptors in order to be combined and weakened IgG by strengthening IgG with reactivity Fc γ acceptors：6591-6604(2001)；Presta et al., Biochem.Soc.Trans.30：487-490(2002)).Specifically, (the Idusogie et al., with above (2001) as described in document；Shields et al., with above) identified for improve CDC and ADCC activity three at be mutated：S298A/E333A/K334A (is also known as three alanine mutants or variant herein；Numbering in Fc areas is according to EU numbering systems；Kabat et al., with above).

In order to strengthen 2H7 CDC and ADCC activity, 2H7Fc three alanine mutants are constructed.Anti-HER 2 4d5 humanization variants are generated, it has mutation S298A/E333A/K334A and referred to as 4D5Fc110 (i.e. anti-p¹⁸⁵HER2 IgG1(S298A/E333A/K334A)；Shields et al., with above).With ApaI and HindIII digestion encoding antibodies 4D5Fc110 plasmid p4D5Fc110 (Shields et al., with above), Fc fragments (including mutation S298A/E333A/K334A) are connected into 2H7 heavy chain vectors pDR2-v16 ApaI/HindIII sites, pDR2-v31 is generated.The amino acid sequence of the complete H chains of pattern 31 sees above composition part.L chains are identical with v16's.

Although the constant domain of IgG1 antibody Fc districts is guarded relatively in given species, but there are still allelic variation (see summary Lefranc and Lefranc, in：The human IgG subclasses：Molecular analysis of structure, function, and regulation, pp.43-78, F.Shakib (ed.), Pergammon Press, Oxford (1990)).

Table 7：The influence that replacement in Fc areas is combined to CD20.The determination method based on cell (WIL2-S) substituted with framework measures the relative combination to CD20.Fc mutation (^*) (Kabat, with above) is represented with EU numberings and be relative to 2H7.v16 parents.V.31 the combination of three alanine change in Fc areas is recited as " Fc110 ".IgG variants show the mutation relative to 2H7.v16 backgrounds.Relative combine is expressed as ratio of the chimeric bulk concentrations of 2H7.v6.8 relative to the equivalent variant concentration with reference to needed for；Therefore ratio ＜ 1 shows that the affinity of the variant is weaker.The standard deviation average out to +/- 10% that relative affinity is determined.

2H7 patterns	Fc is substituted*	It is relative to combine
			6.8	-	-1-
16	-	0.65
			31	S298A, E333A, K334A	0.62

Embodiment 4：Humanization 2H7 variants with enhanced stability

In order to develop into treatment protein, it is desirable to which selection for oxidation, deamidation or may influence other processes of product quality to keep the variant of stabilization in buffer solution is suitably prepared.In 2H7.v16, it is probably instable source to identify some residues：VL (M32) and VH (M34, N100).Therefore, introduce and be mutated in these sites, be compared with v16.

Table 8：In the measure based on cell (WIL2-S), to improve the relative combination of 2H7 variants that stability and/or effector functions design to CD20.IgG variants show the mutation relative to 2H7.v16 backgrounds.Relative combine is expressed as ratio of the chimeric bulk concentrations of 2H7.v6.8 relative to the equivalent variant concentration with reference to needed for；Therefore ratio ＜ 1 represents that the affinity of the variant is weaker.The standard deviation average out to +/- 10% that relative affinity is determined.The framework of variable domain substitute be relative to 2H7.v16, according to Kabat numbering systems, and Fc mutation (^*) indicated with EU numbering systems (Kabat et al., ibid).(^**) variant is measured using 2H7.v16 as standard reference thing；Relative value is with the standard on data of the chimera.

Based on mutation (the Idusogie et al. (2000) previously reported；Idusogie et al.(2001)；Shields et al. (2001)), by other Fc mutation with improving the mutation combination of stability or affinity to change or enhancement effect device function.These changes include S298, E333A, K334A as described above；Reduce the K322A of CDC activity；Reduce the D265A of ADCC activity；Improve the K326A or K326W of CDC activity；And for testing the E356D/M358L of the influence that allograft changes in Fc areas.These mutation do not cause the significant difference of CD20 binding affinities.

2H7 Pattern

Heavy chain (VH) Change

Light chain (VL) Change

Fc Change*

Relatively With reference to

6.8	(chimera)	(chimera)	-	-1-
					16	-	-	-	0.65
62	-	M32I	-	0.46
					63	M34I	-	-	0.49
64	N100A	-	-
					65	N100A	L47W	-	0.74
66	S99A	L47W	-	0.62
					67	N54A	-	-
68	-	M32I	-	0.48
					69	-	M32L	-	0.52
70	N100A	-	S298A, E333A, K334A	0.80
					71	N100D	-	S298A, E333A, K334A	0.44
72	N100A	M32I	-	0.58
					73	N100A	M32L	-	0.53
74	N100A	M32I	S298A, E333A, K334A	0.61
					75	N100A	M32L	S298A, E333A, K334A	0.60
113	-	-	E356D, M358L	0.60**
					114	D56A, N100A	M32L, S92A	S298A, E333A, K334A	1.2**
115	D56A, N100A	M32L, S92A	S298A, E333A, K334A, E356D, M358L	1.4**
					116	D56A, N100A	M32L, S92A	S298A, K334A, K322A	1.2**
134	D56A, N100A	M32L, S92A	E356D, M358L, D265A	1.5**
					135	D56A, N100A	M32L, S92A	E356D, M358L, D265A,   K326W	0.95**
138	D56A, N100A	M32L, S92A	S298A, E333A, K334A,   K326A	1.2**
					139	D56A, N100A	M32L, S92A	S298A, E333A, K334A, K326A, E356N, M358L	1.1**
154	-	-	D265A	0.70**
					155	-	-	S298A, K322A, K334A	0.70**

(^**) variant that is measured using 2H7.v16 as reference substance；

Standard on data of the Relative binding values to chimera.

For the influence of measuring stability mutations on protein degradation rate, 2H7.v16 and 2H7.v73 is formulated in 10mM histidines, 6% sucrose, 0.02% polysorbate20, pH5.8 with 12-14mg/mL and 16 days are incubated in 40 DEG C.Then the sample after insulation is determined, the charge variation of variant is determined by ion-exchange chromatography, aggregation is determined by size exclusion chromatography and fracture changes, and relative combination is determined by the determination method based on cell (WIL2-S) and is changed.

As a result there is higher stability in terms of showing the main peak fraction loss that 2H7v.73 is determined under the conditions of the accelerated stability compared with 2H7v.16 by ion-exchange chromatography.Significant difference is not observed in terms of aggregation, fracture or binding affinity.

Embodiment 5：Complement-dependent cytotoxicity (CDC) determination method

(Idusogie et al., J.Immunol.164 substantially as described in document：4178-4184(2000)；Idusogie et al., J.Immunol.166：2571-2575 (2001)), becoming body measurement to 2H7IgG, they mediate WIL2-S cells, a kind of ability of the complement-dependent lysis of the lymphoblast sample B cell system of expression CD20.Antibody is subjected to 1: 3 serial dilution from 0.1mg/mL liquid storage.The 0.05mL aliquots of each dilution factor are added into the 96 hole tissue culturing plates equipped with 0.05mL normal person's complement solution (Quidel, San Diego, CA).50,000 WIL2-S cells of 0.05mL volumes are added into this mixture.After 37 DEG C are incubated 2 hours, 0.05mL Alamar blue solution (AccumedInternational, Westlake, OH) is added, 37 DEG C of insulation 18 hours in addition are continued at.Then take the capping of flat board away, they are shaken 15 minutes on orbital shaker in room temperature.Relative fluorescence units (RFU) are read with 530nm exciter filters and 590nm launching filters.RFU is fitted to the function of concentration for every kind of antibody with KaleidaGraph softwares, EC is thus calculated₅₀。

As a result (table 9) shows that the CDC of humanization 2H7 antibody has surprising improvement, wherein v.73 having the relative effectivenes similar to Rituxan , effect v.75 is higher than Rituxan  3 times, and 3 times weaker than Rituxan  of effect v.16.

Table 9：The CDC expression activitiys of 2H7 antibody and Rituxan.Digital ＞ 1 shows that CDC activity is weaker than Rituxan , and numeral ＜ 1 shows that activity is better than Rituxan .Antibody is generated by stable Chinese hamster ovary celI system, except those with (^*) indicate instantaneously generate outside.

Antibody variants	n	EC50(variant)/EC50(Rituxan)
			Rituxan 	4	-1-

2H7.v16	4	3.72；4.08
			2H7.v31*	4	2.21
2H7.v73	4	1.05
			2H7.v75	4	0.33
2H7.v96*	4	0.956
			2H7.v114*	4	0.378
2H7.v115*	4	0.475
			2H7.v116 *	1	＞ 100
2H7.v135*	2	0.42

Embodiment 6：Cytotoxicity (ADCC) determination method of antibody dependent cellular

(Shields et al., J.Biol.Chem.276 substantially as described in document：6591-6604 (2001)), utilize lactic dehydrogenase (LDH) reading, becoming body measurement to 2H7IgG, they mediate WIL2-S cells, a kind of ability of natural killer cell (NK cells) dissolving of the lymphoblast sample B cell system of expression CD20.NK cells are prepared from the 100mL heparinized bloods diluted with 100mL PBS (phosphate buffered saline (PBS)), the blood is obtained from for Fc γ RIII, also known as CD16 (Koene et al., Blood90：1109-1114 (1997)) identify the normal human donor of isotype.In this experiment, NK cells come from people's donor of CD16 heterozygosis (F158/V158).The blood of dilution is added to stratification in 15mL lymphocyte separation mediums (ICN Biochemical, Aurora, Ohio), and centrifuged 20 minutes with 2000RPM.The leucocyte at interface between two layers is distributed into 4 clean 50mL pipes, pipe the RPMI culture mediums containing 15% hyclone are housed.Supernatant is abandoned after pipe is centrifuged 5 minutes with 1400RPM.Sediment is resuspended in MACS buffer solutions (0.5%BSA, 2mM EDTA), scheme (Miltenyi Biotech) pearl (NK Cell Isolation Kit, 130-046-502) purified NK cells according to manufacturer.NK cells are diluted to 2 × 10 in MACS buffer solutions⁶Individual cell/mL.

Will determine culture medium (F12/DMEM50: 50 without glycine, 1mM HEPES buffer solution pH7.2, penicillin/streptomycin (100 units/mL；Gibco), glutamine, and 1% heat-inactivated fetal bovine serum) in serial dilution antibody (0.05mL) add 96 hole round bottom tissue culturing plates.WIL2-S cells are diluted to concentration 4 × 10 in buffer solution is determined⁵/mL.WIL2-S cells (0.05mL is per hole) are mixed with dilution antibody in 96 orifice plates and are incorporated in incubation at room temperature 30 minutes so that antibody binding CD20 (opsonic action) (opsonization).

ADCC reactions are originated by adding 0.1mL NK cells into each hole.In control wells, 2%Triton X-100 are added.Then flat board is incubated 4 hours in 37 DEG C.LDH levels with cytotoxicity (LDH) detection kit (Kit#1644793, Roche Diagnostics, Indianapolis, Indiana.) according to the specification measurement release of manufacturer.0.1mL LDH developers are added into each hole, then mixing 10 seconds.Then cover flat board with aluminium foil and placed 15 minutes in room temperature is dark.Then read 490nm optical density and by divided by control wells in total LDH for measuring and be used to calculate dissolving percentage.Using dissolving situation as the function construction of antibody concentration, EC is determined with 4 parameter curves (KaleidaGraph)₅₀Concentration.

As a result show that humanization 2H7 antibody is active in ADCC, wherein relative effectivenes v.31 and v.75 are higher than Rituxan  20 times, effect v.16 is higher than Rituxan  5 times, and effect v.73 is higher than Rituxan  almost 4 times.

Table 10：Based on n experiment, compare 2H7 antibody and 2H7.v16 to the ADCC activities of WIL2-S cells (numerical value ＞ 1 represents that effect is weaker than that 2H7.v16 is low, and the expressions of numerical value ＜ 1 effect is better than 2H7.v16).

Antibody variants	n	EC50(variant)/EC50(2H7.v16)
			Rituxan 	4	5.3
2H7.v16	5	1
			2H7.v31	1	0.24
2H7.v73	5	1.4
			2H7.v75	4	0.25

Other ADCC is carried out to determine to compare 2H7 variant thereofs and Rituxan .These results determined show that 2H7.v114 and 2H7.v115 ADCC effect is improved than Rituxan  and are more than 10 times (table 11).

Table 11：Based on n experiment, compare 2H7 antibody and ADCC activities of the Rituxan  to WIL2-S cells (numerical value ＞ 1 represents that effect is weaker than Rituxan , and numerical value ＜ 1 represents that effect is better than Rituxan ).

Antibody variants	n	EC50(variant)/EC50(Rituxan)
			Rituxan 	2	-1-
2H7.v16	2	0.52
			2H7.v96	2	0.58
2H7.v114	2	0.093
			2H7.v115	2	0.083

2H7.v116

2

0.30

Embodiment 7：FcRn binding assays

To there is soluble Fc variants (such as N434W, N434Y, N434F of amino acid change in Fc, N434A, N434H, N434A+E380A+T307A) expression, purifying, and determine in BIAcore binding assays their affinity to people, macaque, rat and mouse FcRn.Also analyze Fc variants to determine their aggregation tendency by size exclusion chromatography.

In order to express variant Fc fragments, 34B8 Bacillus coli cells are converted with saltant type pW0437 phasmids, the expression to induce Fc genes in 24 hours are cultivated in 30 DEG C in phosphate free culture medium, then harvesting.By cytoplasm freeze overnight, then dissolved in 10mM Tris, 1mM EDTA by osmotic shock (osmotic shock).Lysate is clarified by centrifugation and is added on albumin A post.Post is washed with PBS, solubility Fc is eluted with albumin A citrate elution buffer (0.1M citrates, pH3.0), and neutralized with Tris pH7.5.With Amicon Centriprep concentration solubilities Fc.

FcRn from people, macaque, rat or mouse is fixed on Biacore CM5 chips by NHS chemical methods with different densities (100-3000RU).By Fc variants in pH6.0 PBS from 10 μM of serial dilutions to 1nM, combined with time supervision.For parent's hinge-less Fc (i.e. wild type), huFcRn almost reaches that balance is combined immediately, shows that it has very fast association and dissociation speed, the Kd that equilibrium analysis is determined is about 700nM.For N434W variants, association rate significantly slows down, and dissociation rate is extremely slow.By to inject N434W compared with the slug flow fast long period, what equilibrium analysis was possible to, and Kd is about 4nM.Variant N434W, N434F, N434A and three mutant the N434A+E380A+T307A affinity in pH6.0 substantially increase about 170 times, about 9 times, about 2.7 times and about 14 times respectively relative to wild type Fc.On the contrary, in pH7.4, N434 variants are too low to measure in this determination method really to huFcRn affinity.Combination improvement relative to wild type of the N434W to macaque FcRn be not so significantly compare rat FcRn combination only show it is high about 10 times, and to mouse FcRn with reference to then actually such as wild type.Therefore the improvement obtained by this mutation is special to people FcRn.

The human IgG1's variant for analyzing 2H7.v138 in ELISA with biotinylation FcRn is combined to people FcRn pH dependences.The 2 μ g/ml NeutrAvidin (Pierce, Rockford, IL) that the hole microwell plates (Nunc, Roskilde, Denmark) of MaxiSorp 96 are formulated in 50mM carbonate buffer solutions pH9.6 with 100 μ l/ holes are stayed overnight in 4 DEG C of coatings.Washed, and closed with the PBS pH7.4 containing 0.5%BSA with 150 μ l/ holes with PBS (lavation buffer solution) pH 7.4 containing 0.05% polysorbate.In after incubation at room temperature 1 hour, with lavation buffer solution pH7.4 washing flat boards.With biotin-X-NHS (ResearchOrganics, Cleveland, OH) by people's FcRn biotinylations.By biotinylated FcRn with 2 μ g/ml, 100 μ l/ holes are added on flat board, and it is formulated in containing 0.5%BSA, in PBS (sample buffer) pH7.4 of 0.05% poly- sorbitol ester 20.Flat board is incubated 1 hour and washed with lavation buffer solution pH6.0.The IgG antibody (3.1-200ng/ml) of 7 twice of serial dilutions in sample buffer pH6.0 is added on flat board.After insulation 2 hours, with lavation buffer solution pH6.0 washing flat boards.By the goat F (ab ') for adding the peroxidase labelling that 100 μ l/ holes are formulated in sample buffer pH6.0₂Anti-human igg F (ab ')₂(Jackson ImmunoResearch, West Grove, PA) detects combined IgG.After insulation 1 hour, with lavation buffer solution pH6.0 washing flat boards, 100 μ l/ bottom holes things 3,3 ', 5,5 '-tetramethyl benzidine (TMB) (Kirkegaard ＆ Perry Laboratories) are added.By adding 100 μ l/ holes 1M phosphoric acid come terminating reaction.The absorbance at 450nm is read on multiskan Ascent reader (Thermo Labsystems, Helsinki, Finland).Calculate the absorbance (mid-OD) of standard curve midpoint.Respective concentration using four parameter nonlinear regression curve fitting procedures (KaleidaGraph, Synergy software, Reading, PA) from titration curve bioassay standard product and sample at this mid-OD.Relative activity is calculated by using the mid-OD concentration divided by sample of standard items.

In order to assess dissociation of the combined IgG and FcRn in pH6.0 or pH7.4, similar carry out is described to be determined, and 100 μ l/ holes sample buffer pH6.0 or 7.4 are simply added after sample incubation step and in washing flat board.Flat board is incubated 45 minutes and washed.Then proceed to be measured as described above.

Embodiment 8：Sign with the FcRn Humanized anti-CD 20 IgG1 variants being mutated

Their effects in complete antibody 2H7.v138 background also are tested to the people Fc mutation identified by phage display.2H7.v138 is the anti-CD 20 antibodies of humanization, and wherein Fc has carried out modification to improve ADCC and CDC activity by following mutation：S298A、K326A、E333A、K334A.Position N434 mutation is introduced into this background, IgG (table 12) is prepared as described previously by 293 cells are transiently transfected.In each case, according to size exclusion chromatography as described above, the IgG variants of purifying are shown with low-level protein aggregation.

Table 12：Humanized anti-cd 20 antibodies 2H7.v138 variant

2H7 variants	FcRn is mutated
		138	-
364	N434A
		477	N434W

478	N434F
		479	N434Y

The human IgG1's variant for analyzing 2H7.v138 in ELISA with biotinylated FcRn is combined to people FcRn pH dependences.The 2 μ g/ml NeutrAvidin (Pierce, Rockford, IL) that MaxiSorp96 holes microwell plate (Nunc, Roskilde, Denmark) is formulated in 50mM carbonate buffer solutions pH9.6 with 100 μ l/ holes are stayed overnight in 4 DEG C of coatings.Washed, and closed with the PBS pH7.4 containing 0.5%BSA with 150 μ l/ holes with PBS (lavation buffer solution) pH 7.4 containing 0.05% polysorbate.In after incubation at room temperature 1 hour, with the washing flat boards of lavation buffer solution pH 7.4.With biotin-X-NHS (ResearchOrganics, Cleveland, OH) by people's FcRn biotinylations.By biotinylated FcRn with 2 μ g/ml, 100 μ l/ holes are added on flat board, and it is formulated in containing 0.5%BSA, in PBS (sample buffer) pH7.4 of 0.05% poly- sorbitol ester 20.Flat board is incubated 1 hour and washed with lavation buffer solution pH6.0.The IgG antibody (3.1-200ng/ml) of 7 twice of serial dilutions in sample buffer pH6.0 is added on flat board.After insulation 2 hours, with lavation buffer solution pH6.0 washing flat boards.By the goat F (ab ') for adding the peroxidase labelling that 100 μ l/ holes are formulated in sample buffer pH6.0₂Anti-human igg F (ab ')₂(Jackson ImmunoResearch, West Grove, PA) detects combined IgG.After insulation 1 hour, with lavation buffer solution pH6.0 washing flat boards, 100 μ l/ bottom holes things 3,3 ', 5,5 '-tetramethyl benzidine (TMB) (Kirkegaard ＆ Perry Laboratories) are added.By adding 100 μ l/ holes 1M phosphoric acid come terminating reaction.The absorbance at 450nm is read on multiskan Ascent reader (Thermo Labsystems, Helsinki, Finland).Calculate the absorbance (mid-OD) of standard curve midpoint.Respective concentration using four parameter nonlinear regression curve fitting procedures (KaleidaGraph, Synergy software, Reading, PA) from titration curve bioassay standard product and sample at this mid-OD.Relative activity is calculated by using the mid-OD concentration divided by sample of standard items.

In order to assess dissociation of the combined IgG and FcRn in pH6.0 or pH7.4, similar carry out is described to be determined, and 100 μ l/ holes sample buffer pH6.0 or 7.4 are simply added after sample incubation step and in washing flat board.Flat board is incubated 45 minutes and washed.Then proceed to be measured as described above.

As a result show that RA is similar to observed by Fc variants.In pH6.0, RA is v477 ＞ v478=v479 ＞ v364 ＞ v138.In pH7.4, the RA being weaker than in pH6.0 of RA unanimously, with the relative combination of identical：V477 ＞ v478=v479 ＞ v364 ＞ v138.

These Fc mutation can be widely applied to human IgG antibody.

Embodiment 9：Vivo efficacy of the 2H7 variants in Primary Study in macaque

Test by transiently transfecting the 2H7 variants of Chinese hamster ovary celI generation to assess their activity in vivo in normal male macaque (Macaca fascicularis).Other anti-CD 20 antibodies, such as C2B8 (Rituxan ) confirm ability (Reff the et al., Blood83 for cutting down B cell in normal primate：435-445(1994)).

In one is studied, the 2H7 variants of humanization are compared.In parallel study, Rituxan  are also tested in macaque.5 dosage administration groups, every group uses 4 monkeys：(1) medium, (2) 0.05mg/kg hu2H7.v16, (3) 10mg/kg hu2H7.v16, (4) 0.05mg/kg hu2H7.v31, and (5) 10mg/kg hu2H7.v31.Antibody is applied with intravenous, and concentration is 0,0.2 or 20mg/mL, two doses altogether, one the 1st day in research, and another dose at the 8th day.The first day of dosage administration is appointed as the 1st day, and the previous day is appointed as the -1st day；The first day for reclaiming (every group of two animals) is appointed as the 11st day.6 the -19th, after -12,1 day (before dosage administration) and first dose, 24 and 72 hours collection blood samples.In the 8th day (before dosage medication), the 10th day (before putting to death every group of two animals) and the 36th and 67 day other sample of (for reclaiming animal) collection.

By determining periphery B cell concentration to the FACS methods of CD3-/CD40+ cell counts.The percentage of the CD3-CD40+B cells of total lymphocyte is obtained by following gating strategy in monkey sample.Lymphocyte populations are marked on forward scattering/sidewise scattered scatter diagram, region 1 (R1) is defined to.Using the event in R1, the fluorescence intensity dot plots of CD40 and CD3 marks are shown.The positive respective retention points of CD40 and CD3 are determined with the isotype controls of fluorescence labeling.

As a result show that both 2H7.v16 and 2H7.v31 can produce complete periphery B cell abatement in 10mg/kg dosage, and the periphery B cell abatement of part is produced in 0.05mg/kg dosage.The time course for the B cell abatement that two kinds of antibody are measured during first 72 hours after dosage is administered is similar with degree.Subsequent recovery animal analysis shows compared with the animal for taking 2H7.v16, and B cell abatement extension is shown with the 2H7.v31 animals handled.Specifically, for the recovery animal handled with 10mg/kg2H7.v16, some time showings between B cell was sampled at the 10th day and the 36th day go out substantial B cell recovery.However, for the recovery animal handled with 10mg/kg 2H7.v31, B cell just shows recovery until some times between the 36th day and the 67th day.This explanation is compared with 2H7.v16, and the 2H7.v31 complete abatement duration is longer, about 1 month.

Toxicity is not observed in low or high dose monkey is studied, and gross pathological (grosspathology) is normal.In other researchs, interval intravenous two doses of administration in two weeks is followed in these monkeys, until the maximum dose level (100mg/kg × 2=1200mg/m assessed²× 2), v16 is resistant to well.

2H7.v16 is compared to Rituxan  as shown by data in macaque, and CDC activity decreases 5 are not adversely influenced to effect again.Antibody with strong ADCC activity but CDC activity reductions may have more favourable security features in terms of infusion first than the antibody with higher CDC activity.

Embodiment 10：The internal suppression of tumour growth

RhuMAb2H7.v16 is assessed in naked (athymia) mouse of Balb/c and suppresses Raji human B cells, a kind of ability of lymphoma cell line (ATCC CCL86) growth.Raji cells express CD20, and refuse to be reported in growth in nude mice, produce metastatic disease；Tumour growth is suppressed (Clynes et al., Nature Medicine 6 by Rituxan ：443-446(2000)).56 8-10 week old Balb/c nude mices are divided into 7 groups (A-G), every group is made up of 8 mouse.At the 0th day, every mouse got an injection under the skin 5 × 10 in side⁶Individual Raji B lymphoma cells.Since the 0th day, every mouse received 100uL negative control solutions (PBS；Phosphate buffered saline (PBS)), Rituxan  or 2H7.v16.Dosage determines by body weight, and medicine through tail vein with intravenous delivery.A group mouse receive PBS.B-D groups receive 5.0mg/kg, 0.5mg/kg and 0.05mg/kg Rituxan  respectively.E-G group mouse receive 5.0mg/kg, 0.5mg/kg and 0.05mg/kg2H7.v16 respectively.Duplicate injection weekly, totally 6 weeks.Injection site, which can touch the presence situation of tumour, to be checked to every mouse for interval with one week during treating, if there is the volume for just measuring and recording tumour.Last inspection was carried out (after two weekly intervals of no processing) at the 8th week.

The result of this research shows that both rhuMAb2H7.v16 and Rituxan  effectively suppress subcutaneous Raji cell tumours growth in nude mice.Tumour growth was observed in PBS control group since the 4th week.However, tumour growth is not observed in the group handled with 5mg/kg or 0.5mg/kg Rituxan  or 2H7.v16 in 8 week duration of research.In low dosage 0.05mg/kg treatment groups, tumour is observed in an animal of 2H7 groups and an animal of Rituxan  groups.

Embodiment 11：I/II phases of the rhuMAb 2H7 (2H7.v16) in moderate into severe rheumatoid arthritis is studied

Purpose

The main purpose of this research is to assess security and tolerance that intravenous (IV) dosage of rhuMAb2H7 is incrementally increased in moderate to severe rheumatoid arthritis (RA) patient.

Research and design

This is to combine the safety research that MTX incrementally increases rhuMAb 2H7 (PRO70769) dosage into severe RA subject in moderate, is random, by control of placebo, polycentric, the blind test I/II phases, investigator and subject's blind test.The second stage that this research incrementally increases stage and recruitment greater number subject by dosage is constituted.Sponsor still knows (unblinded) for referral.

The antirheumatic drug or biological agent for mitigating disease with moderate to severe RA, 1-5 kind is raised to have failed, treat the subject with unsatisfied clinical response to MTX at present.

It is required that subject receives weekly the MTX at least 12 weeks in the range of 10-25mg before research is entered, and receiving their predose and studying medicine (PRO70769 or placebo) MTX before dose stability at least 4 weeks.Subject is also subjected to the oral corticosteroids (up to daily 10mg, or prednisone equivalent) of consistent dose and the nonsteroid anti-inflammatory drugs (NSAID) of consistent dose.Subject incrementally increased plan according to following dosage at the 1st day and the 15th day and receives the PRO70769 of intravenous infusion prescribed dose or placebo equivalent twice.

According to specific standard, check data of safety through IGP data commission for inspecting discipline and assess and carry out dosage after the acute toxicity of 72 hours after the whipper-in subject treated in every group second is transfused and incrementally increase.If dosage level incrementally increases the stage confirms it is tolerable in dosage, after dosage incrementally increases the stage, 40 subjects are added at random in following each dosage level (32 receive activating agent, and 8 receive placebo)：2 × 50mg, 2 × 200mg, 2 × 500mg and 2 × 1000mg.About 205 subjects are raised in research.

Obtain and record B cell counting.After the efficacy assessment of 6 months, counted in 48 weeks interim use hybridoma supematant assesse B cells of tracking.B cell, which is cut down, will not be considered dose-limiting toxicity (DLC), but the expection pharmacodynamic results of PRO70769 processing.

In sub- research, blood and the urine sample for serum and RNA analysis are obtained from subject in Each point in time.These samples can be used for identification that moderate may be indicated to biomarker of the severe RA subject to the PRO70769 responses treated.

Research is handled

Each group subject incrementally increased plan the receiving PRO70769 of intravenous infusion prescribed dose or placebo coordinate twice at the 1st day and the 15th day according to following：

- 10mg PRO70769 or placebo coordinate：4 subjects receive activating agent, 1 control

- 50mg PRO70769 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 200mg PRO70769 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 500mg PRO70769 or placebo coordinate：8 subjects receive activating agent, 2 control -1000mg PRO70769 or placebo coordinate：8 subjects receive activating agent, 2 controls

Effect

PRO70769 effect is measured by ACR responses.The percentage for the subject for obtaining ACR20, ACR50 and ACR70 response is summarized by treatment group, and produces every group of 95% confidential interval.Composition and its change away from baseline that these are responded are summarized by treating and accessing.

Embodiment 12

Alanine-scanning data are combined according to the inspection to 2H7.v16 crystal structures, it appears that including N100 (V_H) and the CDR-H3 areas of neighbouring residue may relate to antigen binding.Therefore we focus on N100 (V_H), many amino acid replacements are generated in 2H7.v16 background.As a result (table 13) shows that Tyr or Trp is substituted improves the situation of combination relative to v16.

Table 13：The relative combination of the 2H7 variants designed for the binding affinity strengthened to CD20, body measurement is become with the determination method based on cell (WIL2-S) with the IgG produced in 293 cells of transfection.Indicate mutation (Kabat numbering) of the IgG variants relative to 2H7.v16 backgrounds.It is relative with reference to being expressed as ratio of the 2H7.v16 concentration relative to the equivalent variant concentration with reference to needed for；Therefore, ratio ＞ 1 shows that the variant is improved compared with pattern 16 to CD20 binding affinity.

2H7 patterns	Heavy chain (VH) change	It is relative to combine
			16	-	-1-
460	N100R	1.44
			461	N100Q	1.15
462	N100E	0.8
			463	N100G	0.76
464	N100H	1.17
			465	N100I	0.86
466	N100L	0.81
			467	N100K	0.74
468	N100F	1.21
			469	N100P	0.9
470	N100S	1.08
			471	N100T	1
472	N100Y	1.53

473	N100W	1.91
			474	N100V	0.79
475	N100M	0.75

Improved in order to which whether the replacement determined by S100a (VH) place can obtain other affinity, many mutation have been carried out in this position, be for background in this case with 2H7.v472 (table 13).Combination result of the comparison (table 14) based on cell shows the raisings that is combined with more than 3 times of the 2H7.v511 to CD20 on cell.

The gene of 2H7.v511 heavy chains is constructed by direct mutagenesis, wherein using the plasmid ssDNA for encoding 2H7.v138 heavy chains as template, and CA1568 5 '-phosphorylation deoxy-oligonucleotide is named as：5′-CCA GAC GTC GAA GTA CCA GTA GCG GTA GCT ATA GTA TAC GACGCG-3′(SEQ ID NO.45).The nucleotides for marking underscore corresponds to the codon that coding CDR-H3 in antisense DNA chain changes N100Y, S100aR.2H7.v511 light chain is identical with 2H7.v138's, and the light chain is expressed using identical plasmid.

Hu2H7 variants such as v138 and v511 can be expressed in Chinese hamster ovary celI, use the carrier for being used to express 2H7.v16 shown in Fig. 9.

Table 14：The relative combination of the 2H7 variants designed for the binding affinity strengthened to CD20, body measurement is become with the determination method based on cell (WIL2-S) with the IgG produced in 293 cells of transfection.Indicate mutation (Kabat numbering) of the IgG variants relative to 2H7.v16 backgrounds at CDR sites.In addition, compared with 2H7.v16, all variants are comprising Fc change S298A, K326A, E333A and K334A (EU numberings).It is relative with reference to being expressed as ratio of the 2H7.v16 concentration relative to the equivalent variant concentration with reference to needed for；Therefore, ratio ＞ 1 shows that the variant is improved compared with pattern 16 to CD20 binding affinity.

2H7 Pattern	VL   32	VL   92	VH   56	VH   100	VH   100a	Relatively With reference to
							16	M	S	D	N	S	-1-
590	L	A	A	W	R
							511	L	A	A	Y	R	3.55
512	L	A	A	Y	N	2.12
							513	L	A	A	Y	D	1.65
515	L	A	A	Y	E	1.92
							516	L	A	A	Y	G	2.34
517	L	A	A	Y	H	2.2

518	L	A	A	Y	I	2.0
							519	L	A	A	Y	L	1.7
520	L	A	A	Y	K	1.55
							521	L	A	A	Y	F	2.21
522	L	A	A	Y	P	1.57
							523	L	A	A	Y	T	2.41
524	L	A	A	Y	V	1.54
							525	L	A	A	Y	M	2.31

Because these variants also change comprising Fc, we measure the combination situation in competitive Scatchard determination methods to determine apparent equilibrium dissociation constant (K in addition_d)。

Humanized anti-CD 20 monoclonal antibody for combining WIL2s-S cells, equilibrium dissociation constant (K is determined with radiolabeled antibody 2H7.v511 and 2H7.v138IgG_d).Anti-CD-20 monoclonal antibody is generated in Chinese hamster ovary celI.All dilutions are carried out in combination mensuration buffer solution (the DMEM culture mediums for containing 0.5% bovine serum albumin, 25mM HEPES pH7.2 and 0.01% sodium azide).It is 0.1nM's by concentration¹²⁵I-2H7.v511 and¹²⁵I-2H7.v138 (with lactoperoxidase iodate) aliquot (0.05mL) is dispensed into the hole of the hole microtiter plate of V bottoms 96.Add the serial dilution (0.05mL) of unmarked antibody and mix.30,000 WIL2-S cells of 0.05mL volumes are added into each hole.Seal plate is simultaneously placed 24 hours in room temperature, is then centrifuged 15 minutes with 2,500RPM.Then supernatant is suctioned out, and cell pellet is cleaned and centrifuged.Supernatant is suctioned out again, and sediment is dissolved in 1N NaOH and is transferred in pipe for Gama Count.Data are used for Scatchard and analyze (Munsonand Rodbard, Anal.Biochem.107：220-239 (1980)), wherein using Ligand programs (McPherson, Comput.Programs Biomed.17：107-114(1983)).The CD20 that result shown in table 15 below shows anti-CD-20 monoclonal antibody 2H7.v511 and 2H7.v138 to be expressed on high-affinity combination WIL2-S cells, and 2H7.v511 binding affinity is higher than 2H7.v16 2.2 times to 7.3 times.With marked¹²⁵I-2H7.v511 and¹²⁵Difference between the numerical value that I-2H7.v138 is obtained may reflect the disturbance of antigen binding caused by Tyr iodate new in 2H7.v511 CDR-H3.So it seems that being possible to¹²⁵I-2H7.v138 result more accurately reflects RA.

Table 15：Balance binding affinity (standard error of +/- fitting) from the Scatchard Humanized anti-CD 20 monoclonal antibodies analyzed.

Compete antibody	Labelled antibody	Apparent Kd(nM)	Relative affinity
				2H7.v16	125I-2H7.v138	0.80±0.07	-1-

2H7.v511	125I-2H7.v138	0.11±0.05	7.3
				2H7.v16	125I-2H7.v511	0.98±0.12	-1-
2H7.v511	125I-2H7.v511	0.45±0.05	2.2

Supplement as 2H7 variant binding affinities compares, and ELISA is carried out with the CD20 of dissolving.In order to be compared, CDR region identical with 2H7.v16 is constructed and Fc areas and 2H7.v511 identical another variants 2H7.v588.

With the 2.5 μ g/ml solubility CD20 (Genentech for being dissolved in PBS；Prepare as previously described) by NUNC Maxisorp^TMFlat board is stayed overnight in 4 DEG C of coatings, is then closed 1 hour in room temperature with 0.5%BSA.The sample of the serial dilution in 0.5%BSA is incubated 2 hours on flat board.After washing, anti-human Fab antibody (the Goat anti human Ig being coupled with HRP, Fab ' 2-HRP conjugates, from NB) the combined antibody of detection, wherein with 3,3 ', 5,5 '-tetramethyl benzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD) is used as substrate.Read 450nm absorbance.Use four parameter nonlinear regression curve fitting procedure (KaleidaGraph；Synergy Software, Reading, PA) fitting titration curve.Calculate the concentration of antibody variants corresponding with standard items titration curve midpoint absorbance.As a result (Fig. 3) shows that 2H7.v588 and 2H7.v16 has similar binding affinity；However, 2H7.v511 shows the combination to soluble CD20 and improves about 90 times in this determination method.

Embodiment 13：ADCC activity

The NK cells purified as previously described with WIL2-S cells and from normal human donor assess ADCC activity, Fig. 4 effector：The ratio of target is about 4, and the ratio of Figure 11 determination methods is 5: 1.Result shown in Fig. 4 and Figure 11 shows that 2H7.v511 ADCC effect and maximum activity are improved compared with 2H7.v16 and other 2H7 variants.2H7.v588 shows the effect similar to 2H7.v511 and maximum activity (Fig. 4), and ADCC activity may not be significantly improved by showing that further CD20 affinity improves.

Embodiment 14：CDC activity

CDC activity is have evaluated with WIL2-S cells and people's complement as also described above.Result shown in Fig. 5 shows that compared with 2H7.v588 2H7.v511 CDC effect is improved, compared with 2H7.v16, and 2H7.v511 effect is substantially increased.As being observed with previously described variant, improve CD20 affinity and seem to enhance external CDC activity.In relatively 2H7.v511 and other 2H7 variants CDC separated are determined, v511 also shows the CDC activity higher than v31, v488 and v114, as shown in Figure 10.CDC determination methods in Figure 10 are carried out with 96 micro well formats basically described above, are amended as follows.By 50 μ L since the 2H7.v511 (and Rituxan in the experiment summarized in the Figure 12 of embodiment 15) of the serial dilution 300-1000nM and 50 μ L normal B cells (1 × 10⁶/ ml, per 50,000, hole cell) or 50 μ L WIL2-S cells (30,000) be incubated together with 50 μ L 1: the 4 normal human serum complements (Quidel) diluted.With negative selection (Rosettesep, StemCell Technologies) and Ficoll-Paque Plus (Amersham Biosciences) gradient separations normal B cells are separated from the whole blood extracted added with heparin.After 37 DEG C are incubated 2 hours, add 50 μ L Alamar Blue (BiosourceInternational) and be simultaneously incubated 18 hours again in 37 DEG C.Flat board is gently shaken 15 minutes, then (reading is excited in 535, transmitting 595) to determine Relative fluorescence units (RFU) in fluorimeter plate reader.It will be observed that RFU values mapped relative to mAb concentration in KaleidaGraph, and draw curve with 4 parameter fittings.

Embodiment 15：Internal B cell abatement

Compare 2H7.v511 and 2H7.v16 activity in vivo in the transgene mouse model that B cell is cut down.Give transgenic mice (hCD20Tg^+/+mCD16^-/-hCD16Tg^+/-), every group 5, every mouse vein is interior to inject 125 μ g or 12.5 μ g (equivalent to 5 and 0.5mg/kg) antibody.Each hIgG1 (hollow strips) is Isotype control antibodies in Fig. 6,7,13 and 14.2nd day, the B cell number in blood and selected tissue compartment is counted by facs analysis.As a result (Fig. 6) shows B cells of the 2H7.v511 in low dosage and high dose all than 2H7.v16 in more effective abatement blood and abdominal cavity.In spleen (Fig. 7), 2H7.v511 shows faint to significantly more abatement in low dosage, but this difference is not observed in high dose.Fig. 8 shows the schematic diagram of experiment and summarizes the CD20 between v16 and v511 with reference to the level with Fc functions.

2H7.v511 and rituximab (Rituxan ) B cell abatement situation is also compared in mouse model；General description of experiments is in Figure 12.As shown in figure 13,2H7.v511 shows the blood higher than rituximab and abdominal cavity B cell abatement in low dosage and high dose.Figure 14 shows the B cell abatement situation in spleen.In 0.5mg/kg (12.5 μ g in figure) low dosage, with v511 than with the more effective abatements of rituximab it is total and folliculus spleen B cell；However, v511 is shown in terms of marginal zone B cells abatement is mediated, efficiency is similar or slightly lower compared with rituximab.

2H7.v511 vivo efficacy is also have studied in macaque substantially as described in example 9 above.

Embodiment 16：The internal suppression of tumour growth

2H7.v511 is have evaluated in naked (athymia) mouse of Balb/c according to flow described in example 10 above and suppresses Raji human B cells, a kind of ability of lymphoma cell line (ATCC CCL 86) growth.

Embodiment 17：FcRn combines the in vivo studies influenceed on pharmacokinetics

FcRn is improved with reference to the influence to these Fc variant antibodies Internal pharmacokinetics in order to determine, every kind of antibody variants are injected intravenously to macaque (Macaca fascicularis) or other primate species, blood sample is collected over time to monitor the removing of antibody.Several animals are injected in one or more dosage levels.In one is tested, 1-20mg/kg is injected in single intravenous in time zero and the 1st day.6 hours, 24 hours and 72 hours collection blood (serum) samples before dosage administration and after dosage administration.Other sample was collected at the 8th day, the 10th day, the 30th day and the 60th day.The antibody concentration in blood serum sample is determined with ELISA.With Standard pharmacological technology (Shargel and Yu, Applied Pharmaceutics andPharmacokinetics, Fourth edition, pp.67-98, Appleton and Lange, Stamford, CT (1999)) time dependence of antibody concentration in serum is reduced modeled.Illustrate initial distribution (α stage) of the antibody between tissue using two compartment model (two-compartment model), be followed by terminating or the elimination stage (β stages).Elimination half-life period (the t so calculated_1/2 ^β) effect that FcRn combines raising is disclosed, because FcRn serves the effect for maintaining the IgG in circulation.

Embodiment 18：To ADCC, CDC activity of the cell from CLL patient

In this research, complement activities of the antibody 2H7.v511 and v114 to the B cell from CLL patient is tested.PBMC is isolated from the positive CLL patients of three CD20 (in CLL patient, the most cells found in PBMC fractions are B cells).In 96 hole micro well formats, by 50 from every patient, 000 PBMC and 50 μ l since the 1000nM and the antibody of 1: 3 times of serial dilution titration mAb (v114, v511 or Rituxan^TM) mixing.Each hole receives the normal human serum complement of 50 μ l 1: 4 dilutions.Mixture is incubated 2 hours in 37 DEG C, 50 μ l metabolism dye indicator Alamar Blue (cytolytic indicator) is then added and continues at 37 DEG C of incubated overnights (about 16 hours).96 orifice plates are shaken 1 minute in room temperature and on fluorimeter plate reader in 535nm/590nm readings.Relative fluorescence units are mapped relative to antibody concentration, obtain EC50.From Figure 15 of the CDC activity for comparing v114 and Rituxan and compared the result that v511 and Rituxan Figure 16 is shown, both v511 and v114 are roughly the same to the effect of all 3 patients, but are significantly better than Rituxan and v16.

In separated research, 2H7.v511 and Rituxan are compared in vitro in determination method to MEC1B-CLL cell lines (Stacchini, A et al. (1999) Leukemia Res.23：127-136) to the ADCC effect of the B cell from B-CLL (B cell related CLL) patient.Experiment is carried out as follows.The scheme negative selection recommended according to manufacturer (RosetteSep, StemCell Technologies) separates NK cells from 100mL normal person's whole blood.Whole blood donor is varied from NK cells in terms of CD16 phenotypic expression.Allelic differences betide the 158th, including valine-valine, valine-phenylalanine and phenylalanine-phenylalanine phenotype.Typically, it is measured with valine-phenylalanine phenotype.It is measured as follows in the hole microwell plate of round bottom 96.By 50 μ L, the mAb of serial dilution is incubated together with 50 μ L target cells since the 10-100nM.Target cell includes B-CLL MEC1 cell lines (10,000 every 50 μ L) and the PBMC (30,000 every 50 μ L) from B-CLL patient.PBMC is separated with standard Ficoll-Paque Plus gradient separations from the whole blood of CLL donors.4 hours are incubated in after incubation at room temperature 30 minutes, adding 50 μ L NK cells (50,000) and continue at 37 DEG C with the antibody of serial dilution.Flat board is centrifuged 10 minutes with 1500rpm and 100uL cell culture mediums are removed.Cell level of dissolution is determined by measuring the amount (LDH kits, Roche) of the lactic dehydrogenase discharged by the cell dissolved.Determine the dissolving percentage relative to mAb concentration and mapped with 4 parameter curves in KaleidaGraph.Report the IC obtained using MEC1 cell lines₅₀Value.CLL PBMC data are drawn with the line chart of point to point and are reported in 30,5 and 0.8nM killing percentage.

As a result the ADCC activity for being shown in 2H7.v511 in the sample of all three patients is above Rituxan.In 30nM, 5nM and 0.8nM antibody, raisings of the 2H7.v511 than Rituxan is 2.8 times, 6.2 times and 5.4 times respectively.NK cells are VF phenotypes.

In terms of the cell dissolving of mediation MEC1 cells, 2H7.v511 ADCC activity is also above Rituxan.2H7.v511 activity is taller and bigger than Rituxan in 13 times, 2H7.v511 IC₅₀It is 0.95pM and Rituxan is 0.012nM.NK cells are VV phenotypes.

Conclusion

In a word, compared with 2H7.v16,2H7.v511 and v114 show active elevated CD20 binding affinities, elevated CDC, elevated ADCC activity and the elevated internal effect for cutting down in B cell body.

Embodiment 19

Studied to assess security, the PK in primate and internal Normal B-cell depletion in macaque with 2H7.v511 and v114 according to the research and design shown in following table.The animal receives 4 weekly dosage medications in each dosage level.V511 and v114, which cuts down periphery B cell, to be shown to the analysis that the animal of the dosage of 2.5 or 50mg/kg × 4 administration is carried out.In the Primary Study carried out with v511, in 50mg/kg, periphery B cell is quickly and completely cut down.Found according to postmortem, tissue B cells also have notable abatement in this dosage.

Group Number	Male/female Number	Dosage level   (mg/kg)	Be euthanized number：
					29th day	254th day
			1	4/4			0 (control)	2/2	2/2
2	4/4	2.5			2/2	2/2
			3	4/4			5	2/2	2/2
4	4/4	10			2/2	2/2
			5	4/4			25	2/2	2/2
6	4/4	50			2/2	2/2

Embodiment 20：NHL I/II phase clinical researches

Following table shows an I/II phase clinical study design on treatment non_hodgkin lymphoma (NHL), wherein using hu2H7.v511 in parallel study using antibody hu2H7.v16 in being studied at one.Antibody is applied through intravenous infusion.This dosage regimen can also be used for other hu2H7 variants of the invention described above.

2H7I/II phases research-dosage/infusion rates of non_hodgkin lymphoma

Packet	1st week	2nd week	3rd week	4th week	Accumulated dose
						A	100mg/m2 Rituxan samples Infusion	100mg/m2 Rituxan samples Infusion	100mg/m2 Rituxan samples Infusion	100mg/m2 Rituxan samples Infusion	400mg/m2
B	250mg/m2 Rituxan samples Infusion	250mg/m2 Rituxan samples Infusion	250mg/m2 Rituxan samples Infusion	250mg/m2 Rituxan samples Infusion	1000mg/m2
						C	375mg/m2 Rituxan samples Infusion	375mg/m2 Rituxan samples Infusion	375mg/m2 Rituxan samples Infusion	375mg/m2 Rituxan samples Infusion	1500mg/m2
D	375mg/m2 Rituxan samples Infusion	375mg/m2 Accelerate infusion	375mg/m2 Accelerate infusion	375mg/m2 Accelerate infusion	1500mg/m2

E

100 or 250   mg/m2 Rituxan samples Infusion

375mg/m2 Accelerate infusion

375mg/m2 Accelerate infusion

375mg/m2 Accelerate infusion

1225 or 1375   mg/m2

Embodiment 21：NHL I phases clinical research 2

Following table shows another I phase clinical study design on treatment non_hodgkin lymphoma (NHL), wherein using hu2H7.v511 in parallel study using antibody hu2H7.v16 in being studied at one.Antibody is applied through intravenous infusion.This dosage regimen can also be used for other hu2H7 variants of the invention described above.Every dose is separated by administration in 3 weeks, altogether 8 doses.

3 groups of (10 patient/groups) q3wksx8

Packet (accumulated dose)   mg/m2	1st dose   (mg/m2)	The   2 Agent	The   3 Agent	The   3 Agent	The   4 Agent	The   5 Agent	The   6 Agent	The   7 Agent	The   8 Agent
										A1600	200	200	200	200	200	200	200	200	200
B3000	375	375	375	375	375	375	375	375	375
										C5625	375	750	750	750	750	750	750	750	750

Embodiment 22：NHL I/II phase dosage incrementally increases packet

Following table shows another I/II phase clinical study design on treating non_hodgkin lymphoma (NHL) using antibody hu2H7.v511.The weekly medication of patient, totally 4 weeks.See actual shrinkage to measure effect for example, by CT scan.

(every group of 3-6 patients)

	1st week	2nd week	3rd week	4th week	Accumulated dose
						A	12.5mg/m2	12.5mg/m2	12.5mg/m2	12.5mg/m2	50mg/m2
B	25mg/m2	25mg/m2	25mg/m2	25mg/m2	100mg/m2
						C	50mg/m2	50mg/m2	50mg/m2	50mg/m2	200mg/m2
D	100mg/m2	100mg/m2	100mg/m2	100mg/m2	400mg/m2
						E	200mg/m2	200mg/m2	200mg/m2	200mg/m2	800mg/m2
F	400mg/m2	400mg/m2	400mg/m2	400mg/m2	1600mg/m2
						G	800mg/m2	800mg/m2	800mg/m2	800mg/m2	3200mg/m2

Embodiment 23：NHL I/II phase dosage incrementally increases packet

Following table shows another I/II phase clinical study design on treating non_hodgkin lymphoma (NHL) using antibody hu2H7.v511.The weekly medication of patient, totally 4 weeks.See actual shrinkage to measure effect for example, by CT scan.

	1st week	2nd week	3rd week	4th week	Accumulated dose
						A	25mg/m2	25mg/m2	25mg/m2	25mg/m2	100mg/m2
B	25mg/m2	50mg/m2	50mg/m2	50mg/m2	175mg/m2
						C	50mg/m2	100mg/m2	100mg/m2	100mg/m2	350mg/m2
D	100mg/m2	200mg/m2	200mg/m2	200mg/m2	700mg/m2
						E	200mg/m2	400mg/m2	400mg/m2	400mg/m2	1,400mg/m2
F	400mg/m2	800mg/m2	800mg/m2	800mg/m2	2,800mg/m2

Embodiment 24：I/II phases of the 2H7.v511 in moderate into severe rheumatoid arthritis is studied

Study the design of processing

Each group subject plans to receive intravenous infusion hu2H7.v511 or placebo coordinate twice at the 1st day and the 15th day with prescribed dose according to following incrementally increase：

- 10mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 50mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 100mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 200mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 300mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

- 500mg hu2H7.v511 or placebo coordinate：8 subjects receive activating agent, 2 controls

Effect

2H7.v511 effect is measured by ACR responses.The percentage for the subject for obtaining ACR20, ACR50 and ACR70 response is summarized by treatment group, and produces every group of 95% confidential interval.

Bibliography

The bibliography quoted in the application, including patent, the application announced and other publications, income is used as reference herein.

Unless otherwise indicated, by using the routine techniques of molecular biology etc. in implementation of the invention, these are within the skill of the art.Such technology has complete elaboration in the literature.Refering to for exampleMolecular Cloning：A Laboratory Manual(J.Sambrook et al., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y, 1989)；Current Protoeols in Molecular Biology(F.Ausubel et al., eds., 1987 updated)；Essential MolecularBiology (T.Brown ed., IRL Press 1991)；Gene Expression Technology(Goeddeled., Academic Press 1991)；Methods for Cloning and Analysis of Eukaryotic Genes(A.Bothwell et al.eds., Bartlett Publ.1990)；Gene Transfer and Expression(M.Kriegler, Stockton Press 1990)；Recombinant DNA Methodology II(R.Wu et al.eds., Academic Press 1995)；PCR：A Practical Approach(M.McPherson et al., IRL Press at Oxford University Press 1991)；Oligonucleotide Synthesis(M.Gait ed., 1984)；Cell Culture for Biochemists(R.Adams ed., Elsevier Science Publishers 1990)；Gene Transfer Vectors for Mammalian Cells(J.Miller ＆ M.Calos eds., 1987)；Mammalian Cell Biotechnology(M.Butler ed., 1991)；Animal Cell Culture(J.Pollard et al.eds., Humana Press 1990)；Culture of Animal Cells, 2nd Ed. (R.Freshney et al.eds., Alan R.Liss 1987)；Flow Cytometry and Sorting(M.Melamed et al.eds., Wiley-Liss 1990)；Book seriesMethods in Enzymology(Academic Press, Inc., Wirth M.and Hauser H. (1993)；Immunochemistry in Practice, 3rd edition, A.Johnstone ＆ R.Thorpe, BlackwellScience, Cambridge, MA, 1996；Techniques in Immunocytochemistry(G.Bullock ＆ P.Petrusz eds., Academic Press 1982,1983,1985,1989)；Handbook of Experimental Immunology(D.Weir ＆ C.Blackwell, eds.)；Current Protocols in Immunology(J.Coligan et al.eds.1991)；Immunoassay(E.P.Diamandis ＆T.K.Christopoulos, eds., Academic Press, Inc., 1996)；Goding(1986)Monoclonal Antibodies：Principles and Practice(2d ed) Academic Press, NewYork；Ed Harlow and David Lane,Antibodies A laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, New York, 1988；Antibody Engineering, 2nd edition (C.Borrebaeck, ed., Oxford University Press, 1995)；Book seriesAnnual Review of Immunology；And book seriesAdvances in Immunology。

Sequence table

<110>Genentech Inc (Genentech, Inc.)

Adams, Camellia W.

Lowman, Henry B.

Nakamura, Gerald R.

<120>CD20 antibody variants and application thereof

<130>P2200R2 PCT

<150>US 60/651,111

<151>2005-02-07

<150>US 60/689,404

<151>2005-06-10

<150>US 60/702,571

<151>2005-07-25

<160>48

<210>1

<211>107

<212>PRT

<213>House mouse (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>This sequence is synthesis

<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>The mankind (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 Ile Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser

                 50                  55                  60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

                 65                  70                  75

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

                 80                  85                  90

Tyr Asn Ser Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu

                 95                 100                 105

Ile Lys Arg

<210>4

<211>10

<212>PRT

<213>House mouse

<400>4

Arg Ala Ser Ser Ser Val Ser Tyr Met His

          5                          10

<210>5

<211>7

<212>PRT

<213>House mouse

<400>5

Ala Pro Ser Asn Leu Ala Ser

                  5

<210>6

<211>9

<212>PRT

<213>House mouse

<400>6

Gln Gln Trp Ser Phe Asn Pro Pro Thr

                  5

<210>7

<211>122

<212>PRT

<213>House mouse

<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>This sequence is synthesis

<400>8

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

                 95                 100                 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

                110                 115                 120

Ser Ser

<210>9

<211>119

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<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 Tle 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>House mouse

<400>10

Gly Tyr Thr Phe Thr Ser Tyr Asn Met His

                  5                  10

<210>11

<211>17

<212>PRT

<213>House mouse

<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>House mouse

<400>12

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val

                  5                  10

<210>13

<211>213

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

  1               5                  10                  15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

                 20                  25                  30

Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

                 35                  40                  45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

                 50                  55                  60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

                 65                  70                  75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

                 80                  85                  90

Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

                 95                 100                 105

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

                110                 115                 120

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

                125                 130                 135

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

                140                 145                 150

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

                155                 160                 165

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

                170                 175                 180

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val

                185                 190                 195

Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

                200                 205                 210

Gly Glu Cys

<210>14

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  l               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

                 95                 100                 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

                110                 115                 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

                125                 130                 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

                140                 145                 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

                155                 160                 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

                170                 175                 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

                185                 190                 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

                200                 205                 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

                215                 220                 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

                230                 235                 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

                245                 250                 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

                260                 265                 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

                275                 280                 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

                290                 295                 300

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

                305                 310                 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

                320                 325                 330

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

                335                 340                 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

                350                 355                 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                365                 370                 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

                380                 385                 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                395                 400                 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                410                 415                 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

                425                 430                 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

                440                 445                 450

Gly Lys

<210>15

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Asn Ser

                 95                 100                 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

                110                 115                 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

                125                 130                 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

                140                 145                 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

                155                 160                 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

                170                 175                 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

                185                 190                 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

                200                 205                 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

                215                 220                 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

                230                 235                 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

                245                 250                 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

                260                 265                 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

                275                 280                 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

                290                 295                 300

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

                305                 310                 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

                320                 325                 330

Lys Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys

                335                 340                 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

                350                 355                 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                365                 370                 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

                380                 385                 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                395                 400                 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                410                 415                 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

                425                 430                 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

                440                 445                 450

Gly Lys

<210>16

<211>107

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

  1               5                  10                  15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

                 20                  25                  30

Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro

                 35                  40                  45

Leu Ile Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ser Arg

                 50                  55                  60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

                 65                  70                  75

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp

                 80                  85                  90

Ser Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

                 95                 100                 105

Lys Arg

<210>17

<211>122

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly 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 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

<210>18

<211>213

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>18

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

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>19

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>19

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 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 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>20

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>20

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 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

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>21

<211>107

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>21

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

Ala Phe Asn Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

                 95                 100                 105

Lys Arg

<210>22

<211>122

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly 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

<210>23

<211>213

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

  1               5                  10                  15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser

                 20                  25                  30

Tyr 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

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>24

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser 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 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>25

<211>107

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>25

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>26

<211>213

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>26

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>27

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>27

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

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>28

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<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 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

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

Asp Glu Leu 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>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>29

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 Ala Val Ser Asn

                320                 325                 330

Lys Ala Leu Pro Ala Pro Ile Glu 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>30

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<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>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>31

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 Trp His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

                440                 445                 450

Gly Lys

<210>32

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>32

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 Leu 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>33

<211>122

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>33

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg

                 95                 100                 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

                110                 115                 120

Ser Ser

<210>34

<211>452

<212>PRT

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>34

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly

  1               5                  10                  15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr

                 20                  25                  30

Ser Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

                 35                  40                  45

Glu Trp Val Gly Ala Ile Tyr Pro Gly Asn Gly Ala Thr Ser Tyr

                 50                  55                  60

Asn Gln Lys Phe Lys Gly Arg Phe Thr Ile Ser Val Asp Lys Ser

                 65                  70                  75

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

                 80                  85                  90

Thr Ala Val Tyr Tyr Cys Ala Arg Val Val Tyr Tyr Ser Tyr Arg

                 95                 100                 105

Tyr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

                110                 115                 120

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

                125                 130                 135

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

                140                 145                 150

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

                155                 160                 165

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

                170                 175                 180

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

                185                 190                 195

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

                200                 205                 210

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

                215                 220                 225

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

                230                 235                 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

                245                 250                 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

                260                 265                 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

                275                 280                 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

                290                 295                 300

Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

                305                 310                 315

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

                320                 325                 330

Ala Ala Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys

                335                 340                 345

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

                350                 355                 360

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

                365                 370                 375

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

                380                 385                 390

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

                395                 400                 405

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

                410                 415                 420

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

                425                 430                 435

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

                440                 445                 450

Gly Lys

<210>35

<211>36

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>35

ctacaccttc acgagctata acatgcactg ggtccg 36

<210>36

<211>51

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>36

gattaatcct gacaacggcg acacgagcta taaccagaag ttcaagggcc50

g51

<210>37

<211>38

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>37

gaatgggttg cagcgatcta tcctggcaac ggcgacac 38

<210>38

<211>65

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>38

attattgtgc tcgagtggtc tactatagca acagctactg gtacttcgac 50

gtctggggtc aagga65

<210>39

<211>36

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>39

ctgcacagcc agctcttctg tcagctatat gcattg 36

<210>40

<211>42

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>40

aactactgat ttacgctcca tcgaacctcg cgtctggagt cc42

<210>41

<211>45

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>41

tattactgtc aacagtggag cttcaatccg cccacatttg gacag45

<210>42

<211>37

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>42

gtttcactat aagtgtcgac aagtccaaaa acacatt37

<210>43

<211>33

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>43

gccaggatag atggcgccaa cccattccag gcc 33

<210>44

<211>26

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>44

aagctccgaa accactgatt tacgct 26

<210>45

<211>45

<212>DNA

<213>Artificial sequence

<220>

<223>This sequence is synthesis

<400>45

ccagacgtcg aagtaccagt agcggtagct atagtatacg acgcg 45

<210>46

<211>285

<212>PRT

<213>The mankind

<400>46

Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys

  1               5                  10                  15

Leu Lys Lys Arg Glu Glu Met Lys Leu Lys Glu Cys Val Ser Ile

                 20                  25                  30

Leu Pro Arg Lys Glu Ser Pro Ser Val Arg Ser Ser Lys Asp Gly

                 35                  40                  45

Lys Leu Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys Cys

                 50                  55                  60

Leu Thr Val yal Ser Phe Tyr Gln Val Ala Ala Leu Gln Gly Asp

                 65                  70                  75

Leu Ala Ser Leu Arg Ala Glu Leu Gln Gly His His Ala Glu Lys

                 80                  85                  90

Leu Pro Ala Gly Ala Gly Ala Pro Lys Ala Gly Leu Glu Glu Ala

                 95                 100                 105

Pro Ala Val Thr Ala Gly Leu Lys Ile Phe Glu Pro Pro Ala Pro

                110                 115                 120

Gly Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val

                125                 130                 135

Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile

                140                 145                 150

Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe

                155                 160                 165

Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu

                170                 175                 180

Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile

                185                 190                 195

Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

                200                 205                 210

Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser

                215                 220                 225

Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu

                230                 235                 240

Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu

                245                 250                 255

Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile

                260                 265                 270

Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu

                275                 280                 285

<210>47

<211>184

<212>PRT

<213>The mankind

<400>47

Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala

  1               5                  10                  15

Pro Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg

                 20                  25                  30

His Cys Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro

                 35                  40                  45

Ala Gly Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro Gln

                 50                  55                  60

Glu Ser Val Gly Ala Gly Ala Gly Glu Ala Ala Leu Pro Leu Pro

                 65                  70                  75

Gly Leu Leu Phe Gly Ala Pro Ala Leu Leu Gly Leu Ala Leu Val

                 80                  85                  90

Leu Ala Leu Val Leu Val Gly Leu Val Ser Trp Arg Arg Arg Gln

                 95                 100                 105

Arg Arg Leu Arg Gly Ala Ser Ser Ala Glu Ala Pro Asp Gly Asp

                110                 115                 120

Lys Asp Ala Pro Glu Pro Leu Asp Lys Val Ile Ile Leu Ser Pro

                125                 130                 135

Gly Ile Ser Asp Ala Thr Ala Pro Ala Trp Pro Pro Pro Gly Glu

                140                 145                 150

Asp Pro Gly Thr Thr Pro Pro Gly His Ser Val Pro Val Pro Ala

                155                 160                 165

Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr Lys Thr Ala Gly

                170                 175                 180

Pro Glu Gln Gln

<210>48

<211>185

<212>PRT

<213>The mankind

<400>48

Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala

  1               5                  10                  15

Pro Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg

                 20                  25                  30

His Cys Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro

                 35                  40                  45

Ala Gly Ala Ala Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro

                 50                  55                  60

Gln Glu Ser Val Gly Ala Gly Ala Gly Glu Ala Ala Leu Pro Leu

                 65                  70                  75

Pro Gly Leu Leu Phe Gly Ala Pro Ala Leu Leu Gly Leu Ala Leu

                 80                  85                  90

Val Leu Ala Leu Val Leu Val Gly Leu Val Ser Trp Arg Arg Arg

                 95                 100                 105

Gln Arg Arg Leu Arg Gly Ala Ser Ser Ala Glu Ala Pro Asp Gly

                110                 115                 120

Asp Lys Asp Ala Pro Glu Pro Leu Asp Lys Val Ile Ile Leu Ser

                125                 130                 135

Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala Trp Pro Pro Pro Gly

                140                 145                 150

Glu Asp Pro Gly Thr Thr Pro Pro Gly His Ser Val Pro Val Pro

                155                 160                 165

Ala Thr Glu Leu Gly Ser Thr Glu Leu Val Thr Thr Lys Thr Ala

                170                 175                 180

Gly Pro Glu Gln Gln

                185

Claims (56)

1. combining the humanization 2H7 antibody or its antigen-binding fragment of h CD20, the antibody includes SEQID NO.25 light chain variable district (V_L) sequence and SEQ ID NO.8 weight chain variable district (V_H) there is amino acid replacement D56A in sequence, but VH-CDR2, and the N100 in VH-CDR3 is substituted with Y or W.

2. the antibody of claim 1, wherein N100 are substituted with Y.

3. the antibody of claim 1, wherein N100 are substituted with W.

4. the antibody of claim 1, also comprising the replacement S100aR in VH-CDR3.

5. the antibody of claim 4, also comprising improving the amino acid replacement of ADCC and/or CDC activity in IgG Fc areas at least one.

6. the antibody of claim 5, comprising IgG1 Fc and the IgG1 Fc include amino acid replacement S298A, E333A, K334A, K326A.

7. the antibody of claim 4, also comprising improving the amino acid replacement of ADCC activity but reduction CDC activity in Fc areas at least one.

8. the antibody of claim 7, including at least amino acid replacement K322A.

9. the antibody of claim 8, also comprising amino acid replacement S298A, E333A, K334A.

10. the antibody of claim 6, the sequence of light chain comprising SEQ ID NO.26 and SEQ ID NO.34 sequence of heavy chain.

11. the antibody of claim 4, it for antibody 2H7.v16 to improve at least 3 times of affinity combination h CD20.

12. the antibody of claim 4, it for antibody 2H7.v16 to improve at least 6 times of affinity combination h CD20.

13. the antibody of claim 6, it shows the cytotoxicity (ADCC) of at least 20 times higher than 2H7.v16 of antibody dependent cellular.

14. the antibody of claim 6, it shows at least 25 times higher than 2H7.v16 of complement-dependent cytotoxicity.

15. any one of claim 1-14 antibody, it and cytotoxic agent couplings.

16. the antibody of claim 15, wherein the cytotoxic agent is radio isotope or toxin.

17. any one of claim 1-14 antibody, the antibody is generated in Chinese hamster ovary celI.

18. encode the nucleic acid of the separation of the antibody of claim 1.

19. encode the expression vector of the antibody of claim 1 or claim 10.

20. the host cell of the nucleic acid comprising claim 18.

21. the host cell of claim 20, it generates any one of claim 1-14 antibody.

22. the host cell of claim 20, it generates antibody and sequence of light chain of the antibody comprising SEQ ID NO.26 and SEQ ID NO.34 sequence of heavy chain.

23. the host cell of claim 22, it is Chinese hamster ovary celI.

24. generating the method for any one of claim 1-14 antibody, including cultivate the host cell of claim 21 and antibody is reclaimed from cell culture.

25. the composition of antibody and carrier comprising claim 1.

26. the composition of claim 25, antibody and pharmaceutical acceptable carrier comprising claim 10.

27. product, including container and dress composition in this embodiment, wherein the composition includes any one of claim 1-17 antibody.

28. the product of claim 27, in addition to indicate that said composition is used for the package insert for treating non_hodgkin lymphoma, wherein sequence of light chain of the antibody comprising SEQ ID NO.26 and SEQ IDNO.34 sequence of heavy chain.

29. the product of claim 27, in addition to indicate that said composition is used for the package insert for treating rheumatoid arthritis.

30. treating the method for CD20 positive cancers, including give humanization 2H7 antibody of the cancer patient using any one of the claim 1-17 of therapeutically effective amount.

31. the method for claim 30, wherein the CD20 positive cancers are B cell lymphoma or leukaemia.

32. the method for claim 31, wherein the CD20 positive cancers are non_hodgkin lymphoma (NHL).

33. the method for claim 32, wherein the cancer is chronic lymphocytic leukemia (CLL) or SLL.

34. the method for claim 32 or claim 33, wherein light-chain amino acid sequence of the antibody comprising SEQ ID NO.26 and SEQ ID NO.34 heavy chain amino acid sequence.

35. the method for claim 31, wherein the antibody is applied through intravenous infusion.

36. the method for claim 35, wherein the antibody is with every dose of about 100mg/m²To 375mg/m²In the range of dosage apply.

37. the method for claim 36, wherein the antibody is with every dose of 375mg/m²Dosage apply at least 4 doses.

38. the method for claim 30, in addition at least one chemotherapeutics is applied to patient.

39. the method for claim 38, wherein the cancer is non_hodgkin lymphoma (NHL) and the chemotherapeutics is selected from Doxorubicin, endoxan, vincristine and prednisolone.

40. mitigating the method for autoimmunity disease, including any one of the claim 1-14 of therapeutically effective amount humanization 2H7 antibody is applied to Serum of Patients With Autoimmune Diseases.

41. the method for claim 40, wherein light-chain amino acid sequence of the antibody comprising SEQ ID NO.26 and SEQ ID NO.34 heavy chain amino acid sequence.

42. the method for claim 41, wherein the autoimmunity disease be selected from rheumatoid arthritis and juvenile rheumatoid arthritis, systemic loupus erythematosus (SLE) include lupus nephritis, Wei Genashi diseases, inflammatory bowel disease, ulcerative colitis, ITP (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephrosis, IgM polyneuropathies, myasthenia gravis, ANCA associated vasculitis,

Diabetes, Raynaud's syndrome, siogren's syndrome, neuromyelitis optica (NMO) and glomerulonephritis.

43. the method for claim 42, wherein the autoimmunity disease is rheumatoid arthritis.

44. the method for claim 43, in addition to second of therapeutic agent is applied to patient.

45. the method for claim 44, wherein second of therapeutic agent is immunodepressant.

46. the method for claim 45, wherein the immunodepressant is methotrexate (MTX).

47. the method for claim 43, wherein the antibody is intravenously or subcutaneously applied.

48. the method for claim 43, wherein the antibody in the dose intravenous in the range of every dose of 10mg to 500mg to apply.

49. the method for claim 48, wherein the antibody is applied with the dosage of 100mg/ agent.

50. the method for claim 40, in addition to second of therapeutic agent is applied to patient.

51. the method for claim 50, wherein second of therapeutic agent is BAFF antagonists.

52. the method for claim 51, wherein the BAFF antagonists are anti-BR3 antibody or BR3-Fc fusion proteins.

53. the method for claim 30, in addition to apply VEGF antagonist to patient.

54. liquid adjustments, it is in 20mM sodium acetates pH5.5,4% Trehalose Dihydrate, contains about 20mg/ml humanization 2H7.v511 antibody in 0.02% polysorbate20, is applied for intravenous.

55. liquid adjustments, it contains about 20mg/ml humanization 2H7.v114 antibody in 20mM sodium acetates pH5.3,240mM (8%) Trehalose Dihydrate, 0.02% polysorbate20.

56. liquid adjustments, it is in 20mM sodium acetates pH5.3,4% Trehalose Dihydrate, contains about 30mg/ml humanization 2H7.v16 antibody in 0.02% polysorbate20, is applied for intravenous.

Images