Anti-cd40 antibodies and uses thereof

Abstract

The present invention relates to antibodies specific for a particular epitope on CD40 and antibodies that bind CD40 and have particular functional characteristics. The present invention also relates to fragments of these antibodies, uses of the antibodies for reduction or treatment of transplant rejection and graft-versus-host disease, and methods for making the antibodies.

What is claimed is: 
     
         1 . An isolated antibody, or antigen-binding fragment thereof, that specifically binds to an epitope present on CD40, wherein said epitope is recognized by the 2C10 antibody. 
     
     
         2 . The antibody of  claim 1 , wherein said antibody blocks B lymphocyte activation by CD154-expressing Jurkat cells in vitro. 
     
     
         3 . The antibody of  claim 1 , wherein said antibody inhibits B lymphocyte CD23, CD80, or CD86 expression. 
     
     
         4 . The antibody of  claim 2  or  3 , wherein said B lymphocyte is a rhesus B lymphocyte. 
     
     
         5 . The antibody of  claim 1 , wherein said CD40 is rhesus CD40. 
     
     
         6 . The antibody of  claim 1 , wherein said CD40 is human CD40. 
     
     
         7 . The antibody of  claim 1 , wherein the constant regions of said antibody are human constant regions. 
     
     
         8 . The antibody of  claim 1 , wherein said antibody is a humanized antibody. 
     
     
         9 . The antibody of  claim 1 , wherein said antibody is a human antibody. 
     
     
         10 . The antibody of any of  claims 1 - 9 , wherein said antibody is a monoclonal antibody. 
     
     
         11 . The antibody of  claim 10 , wherein said antibody is the 2C10 antibody. 
     
     
         12 . The antibody of any of  claims 1 - 10 , wherein the heavy chain variable region of said antibody comprises amino acids 20-132 of SEQ ID NO:2, or an antibody-binding portion or fragment thereof. 
     
     
         13 . The antibody of any of  claims 1 - 12 , wherein the light chain variable region of said antibody comprises the sequence of 23-128 of SEQ ID NO:4, or an antibody binding portion or fragment thereof. 
     
     
         14 . The antibody of  claim 1 , wherein the heavy chain variable region of said antibody comprises amino acids 20-132 of SEQ ID NO:2 and the light chain variable sequence of said antibody comprises amino acids 23-128 of SEQ ID NO:4. 
     
     
         15 . The antibody binding fragment of  claim 1 , wherein said antigen-binding fragment is an antibody that lacks the Fc portion or is a F(ab′) 2 , a Fab, an Fv, or an scFv structure. 
     
     
         16 . A polynucleotide encoding the antibody or antibody fragment of any of  claims 1 - 15 . 
     
     
         17 . A vector encoding the polynucleotide of  claim 16 . 
     
     
         18 . A cell comprising the vector of  claim 17 . 
     
     
         19 . The cell of  claim 18 , wherein said cell is eukaryotic. 
     
     
         20 . The cell of  claim 19 , wherein said cell is mammalian. 
     
     
         21 . The cell of  claim 19 , wherein said cell is prokaryotic. 
     
     
         22 . A method of suppressing the immune system in a subject, said method comprising administering to said subject an effective amount of an antibody, or antigen-binding fragment thereof, of any of  claims 1 - 15  to said subject. 
     
     
         23 . A method of treating or treating prophylactically transplant rejection or increasing the duration of time before transplant rejection occurs in a subject in need thereof, said method comprising administering an effective amount of an antibody, or antigen-binding fragment thereof, of any of  claims 1 - 15  to said subject. 
     
     
         24 . The method of  claim 22  or  23 , wherein said subject has received, or is in need of, an organ transplant. 
     
     
         25 . The method of  claim 24 , wherein said organ is selected from the group consisting of heart, kidney, lung, liver, pancreas, intestine, and thymus, or a portion thereof. 
     
     
         26 . The method of  claim 23 , wherein said subject has received, or is in need of a tissue transplant. 
     
     
         27 . The method of  claim 26 , wherein said tissue is bone, tendon, cornea, skin, heart valve, vein, or bone marrow. 
     
     
         28 . The method of any of  claims 23 - 27 , wherein said administration is commenced prior to said transplantation or said graft. 
     
     
         29 . The method of any of  claims 23 - 28 , wherein said administration continues for at least one month following said transplantation or said graft. 
     
     
         30 . The method of  claim 29 , wherein said administration continues for at least six months following said transplantation of said graft. 
     
     
         31 . A method of treating or treating prophylactically graft-versus-host disease in a subject in need thereof, said method comprising administering an effective amount of an antibody, or an antigen-binding fragment thereof, of any of  claims 1 - 15  to said subject. 
     
     
         32 . A method of treating or treating prophylactically an autoimmune disorder in a subject in need thereof, said method comprising administering an effective amount of an antibody, or an antigen-binding fragment thereof, of any of  claims 1 - 15  to said subject. 
     
     
         33 . The method of  claim 32 , wherein said autoimmune disorder is associated with or caused by the presence of an autoantibody. 
     
     
         34 . The method of  claim 32 , wherein said disorder is selected from the group consisting of systemic lupus erythematosus (SLE), CREST syndrome (calcinosis, Raynaud's syndrome, esophageal dysmotility, sclerodactyl, and telangiectasia), opsoclonus, inflammatory myopathy (e.g., polymyositis, dermatomyositis, and inclusion-body myositis), systemic scleroderma, primary biliary cirrhosis, celiac disease (e.g., gluten sensitive enteropathy), dermatitis herpetiformis, Miller-Fisher Syndrome, acute motor axonal neuropathy (AMAN), multifocal motor neuropathy with conduction block, autoimmune hepatitis, antiphospholipid syndrome, Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, rheumatoid arthritis, chronic autoimmune hepatitis, scleromyositis, myasthenia gravis, Lambert-Eaton myasthenic syndrome, Hashimoto's thyroiditis, Graves' disease, Paraneoplastic cerebellar degeneration, Stiff person syndrome, limbic encephalitis, Isaacs Syndrome, Sydenham's chorea, pediatric autoimmune neuropsychiatric disease associated with  Streptococcus  (PANDAS), encephalitis, diabetes mellitus type 1, and Neuromyelitis optica. 
     
     
         35 . The method of  claim 32 , wherein said disorder is selected from the group consisting of pernicious anemia, Addison's disease, psoriasis, inflammatory bowel disease, psoriatic arthritis, Sjögren's syndrome, lupus erythematosus (e.g., discoid lupus erythematosus, drug-induced lupus erythematosus, and neonatal lupus erythematosus), multiple sclerosis, and reactive arthritis. 
     
     
         36 . The method of  claim 32 , wherein said disorder is selected from the group consisting of polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, adrenalitis, thyroiditis, autoimmune thyroid disease, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, presenile dementia, demyelinating diseases, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, alopecia arcata, pemphigoid, scleroderma, progressive systemic sclerosis, adult onset diabetes mellitus (e.g., type II diabetes), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, Crohn's disease, mixed connective tissue disease, polyarteritis nedosa, systemic necrotizing vasculitis, juvenile onset rheumatoid arthritis, glomerulonephritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, allergic disease, allergic encephalomyelitis, toxic epidermal necrolysis, alopecia, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, human immunodeficiency virus infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post vaccination syndromes, congenital rubella infection, Hodgkin's and non-Hodgkin's lymphoma, renal cell carcinoma, multiple myeloma, Eaton-Lambert syndrome, relapsing polychondritis, malignant melanoma, cryoglobulinemia, Waldenstrom's macroglobulemia, Epstein-Barr virus infection, mumps, Evan's syndrome, and autoimmune gonadal failure. 
     
     
         37 . The method of any of  claims 22 - 36 , wherein said subject is a mammal. 
     
     
         38 . The method of  claim 37 , wherein said subject is a human. 
     
     
         39 . The method of any of  claims 22 - 38 , wherein said administration is parenteral, intravenous, subcutaneous, oral, topical, intrathecal, or local. 
     
     
         40 . The method of any of  claims 22 - 39 , wherein said method further comprises administration of second agent within six months of said antibody, wherein said second agent is an immunosuppressant. 
     
     
         41 . The method of  claim 40 , wherein said second agent is selected from the group consisting of a calcineurin inhibitor, tacrolimus, an mTor inhibitor, fingolimod, myriocin, alemtuzumab, rituximab, an anti-CD4 monoclonal antibody, an anti-LFA1 monoclonal antibody, an anti-LFA3 monoclonal antibody, an anti-CD45 antibody, an anti-CD19 antibody, monabatacept, belatacept, indolyl-ASC; azathioprine, lymphocyte immune globulin and anti-thymocyte globulin [equine], mycophenolate mofetil, mycophenolate sodium, daclizumab, basiliximab, cyclophosphamide, prednisone, prednisolone, leflunomide, FK778, FK779, 15-deoxyspergualin, busulfan, fludarabine, methotrexate, 6-mercaptopurine, 15-deoxyspergualin, LF15-0195, bredinin, brequinar, and muromonab-CD3. 
     
     
         42 . The method of  claim 41 , wherein said calcineurin inhibitor is cyclosporin A or cyclosporine G. 
     
     
         43 . The method of  claim 41 , wherein said mTor inhibitor is sirolimus, temsirolimus, zotarolimus, or everolimus. 
     
     
         44 . The method of  claim 41 , wherein said anti-CD45 antibody is an anti-CD45RB antibody. 
     
     
         45 . The method of  claim 41 , wherein said second agent is belatacept. 
     
     
         46 . The method of any of  claims 40 - 43 , wherein said antibody and said second agent are administered within one month of each other. 
     
     
         47 . The method of  claim 44 , wherein said antibody and said second agent are administered within one week of each other. 
     
     
         48 . A method of making an antibody, said method comprising:
 (a) administering to a mammal a polypeptide comprising a CD40 fragment that includes the epitope recognized by the 2C10 antibody, but not the full length CD40 protein in a manner sufficient to generate an immune response to said fragment;   (b) isolating spleen cells from said mammal;   (c) forming a hybridoma between said spleen cells and myeloma cells; and   (d) purifying said antibody produced by said hybridoma.   
     
     
         49 . The method of  claim 46 , wherein said polypeptide comprises a CD40 fragment that is 8-40 amino acids in length. 
     
     
         50 . The method of  claim 46  or  47 , wherein said polypeptide is a fusion protein. 
     
     
         51 . The method of  claim 46 , wherein said mammal is a mouse or a rabbit. 
     
     
         52 . An antibody produced by the method of  claim 46  or  49 . 
     
     
         53 . A fragment of CD40 fewer than 100 amino acids in length that is bound by the 2C11 antibody. 
     
     
         54 . The fragment of  claim 51 , wherein said fragment is fewer than 50 amino acids in length. 
     
     
         55 . The fragment of  claim 52 , wherein said fragment is fewer than 30 amino acids in length. 
     
     
         56 . The fragment of  claim 53 , wherein said fragment is fewer than 15 amino acids in length. 
     
     
         57 . The fragment of  claim 54 , wherein said fragment is fewer than 10 amino acids in length. 
     
     
         58 . The fragment of  claim 51 , wherein said fragment is 7-50 amino acids in length. 
     
     
         59 . The fragment of  claim 56 , wherein said fragment is 8-10 amino acids in length. 
     
     
         60 . The fragment of any of  claims 51 - 57 , wherein said CD40 is fragment is from the extracellular domain of CD40. 
     
     
         61 . The fragment of  claim 58 , wherein said fragment is a fragment of the human CD40 extracellular domain (SEQ ID NO:6) 
     
     
         62 . The fragment of  claim 58 , wherein said fragment is a fragment of the  Macacca mulatta  CD40 extracellular domain (SEQ ID NO:5) 
     
     
         63 . A fusion protein comprising:
 (a) a fragment of any of  claims 51 - 60 ; and   (b) a heterologous sequence.