AFFINITY MATURED CRIg VARIANTS

Abstract

The present invention concerns affinity matured CRIg variants. In particular, the invention concerns CRIg variants having increased binding affinity to C3b and retaining selective binding to C3b over C3.

What is claimed is: 
     
         1 . A method for the prevention or treatment of a complement-associated disease or condition, comprising administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of a CRIg variant comprising an amino acid substitution in a region selected from the group consisting of E8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111 of the amino acid sequence of SEQ ID NO: 68, or an immunoadhesin comprising such variant. 
     
     
         2 . The method of  claim 1 , wherein said variant selectively binds to C3b over C3, or a fragment thereof 
     
     
         3 . The method of  claim 1 , wherein said variant has increased binding affinity to C3b over native sequence human CRIg of SEQ ID NO: 2. 
     
     
         4 . The method of  claim 3 , wherein the binding affinity is increased by at least 2 fold, or by at least 5 fold, or by at least 10 fold, or by at least 90 fold. 
     
     
         5 . The method of  claim 1 , wherein said variant is more potent inhibitor of the alternative complement pathway than native sequence human CRIg of SEQ ID NO: 2. 
     
     
         6 . The method of  claim 5 , wherein said variant is at least 2-time more potent, or at least 5-time more potent, or at least 10-times more potent than native sequence human CRIg of SEQ ID NO: 2. 
     
     
         7 . The method of  claim 1 , wherein said variant comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of positions 8, 14, 18, 42, 44, 45, 60, 64, 86, 99, 105,and 110 n the amino acid sequence of SEQ ID NO: 2. 
     
     
         8 . The method of  claim 1 , wherein sa id variant comprises an amino acid substitution at one or more of amino acid positions 64, 86, 99, 105 and 110 in the amino acid sequence of SEQ ID NO: 2. 
     
     
         9 . The method of  claim 1 , wherein said variant comprises one or more substitutions selected from the group consisting of E8W, W14F, E84Y/W14F; P45F; G42D/D44H/P45F; Q60I; Q64R; Q60I/Q64R; M86Y; M86W, M86F, M86W/Q9R; M86F/Q99R; K110D, K11N; Q105R/K110N; Q105R/K110Q; and Q105K/K110D. 
     
     
         10 . The method of  claim 1 , wherein said variant comprises one or more substitutions selected from the group consisting of Q64R/M86Y; Q601/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y; Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N; Q60I/Q105R/K110N; M86Y/E8Y; M86Y/G42D/D44H/P45F; M86Y/P45F; M86Y/G42D/D44H/P45F; and M86Y/Q99K/M86Y/Q99R/M86Y/Q105R/M86Y/Q105K/M86Y/Q105R/K110N. 
     
     
         11 . The method of  claim 1 , wherein said variant comprises one or more substitutions selected from the group consisting of Q60I; Q64R; Q60I/Q64R; M86Y; Q99L; Q105K/K110D; E8W/Q105R/K110N; Q64R/M86Y; Q60I/Q64R/E8Y; Q60I/Q64R/G42D; Q60I/Q64R/P45F; Q60I/Q64R/G42D/D44H/P45F; Q60I/Q64R/M86Y; Q60I/Q64R/Q105R; Q60I/Q64R/Q105K; Q60I/Q64R/K110N; M86Y/P45F; and M86Y/Q105K. 
     
     
         12 . The method of  claim 1 , wherein said variant comprises a Q60I/Q64R/M86Y or Q60I/Q64R/G42D/D44H/P45F substitution. 
     
     
         13 . The method of  claim 1 , wherein said variant is shorter than the mature full-length CRIg of SEQ ID NO: 68. 
     
     
         14 . The method of  claim 1 , wherein said immunoadhesin comprises the extracellular domain of said CRIg variant. 
     
     
         15 . The method of  claim 1 , wherein said complement-associated disease is an inflammatory disease or an autoimmune disease. 
     
     
         16 . The method of  claim 15 , wherein said complement-associated disease is selected from the group consisting of rheumatoid arthritis (RA), adult respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dematomyositis, pemphigus, lupus nephritis and sesultant glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulemia, antiphospholipid syndrom, age-related macular degeneration, uveitis, diabetic retinopathy, allo-transplantation, hyperacute rejection, hemodialysis, chronic occlusive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, utricaria, chronic idiopathic utricaria, hemolytic uremic syndrome, endometriosis, cardiogenic shock, ischemia reperfusion injury, and multiple schlerosis (MS). 
     
     
         17 . The method of  claim 15 , wherein said complement-associated disease is selected from the group consisting of inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropaties, systemic sclerosis (scleoderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tubulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other nonhepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis), gluten-sensitive enteropathy, Whipple's disease, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection, graft-versus host disease, Alzheimer's disease, paroxysmal nocturnal hemoglobinurea, hereditary angioedema, atherosclerosis and type II membranoproliferative glomerulonephritis. 
     
     
         18 . The method of  claim 15 , wherein said complement-associated disease is rheumatoid arthritis (RA). 
     
     
         19 . The method of  claim 15 , wherein said complement-associated disease is a complement-associated eye condition. 
     
     
         20 . The method of  claim 19 , wherein said complement-associated eye condition is selected from the group consisting of all stages of age-related macular degeneration (AMD), uveitis, diabitic and other ischemia-related retinopathies, endophthalmitis, and other intraocular neovascular diseases. 
     
     
         21 . The method of  claim 20  wherein the intraocular neovascular disease is selected from the group consisting of diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. 
     
     
         22 . The method of  claim 19  wherein said complement-associated eye condition is selected from the group consisting of age-related macular degeneration (AMD), choroidal neovascularization (CNV), diabetic retinopathy (DR), and endophthalmitis. 
     
     
         23 . The method of  claim 22  wherein said AMD is wet AMD. 
     
     
         24 . The method of  claim 23  wherein said AMD dry or atrophic AMD. 
     
     
         25 . The method of  claim 1  wherein said subject is a mammal. 
     
     
         26 . The method of  claim 25  wherein said mammal is a human. 
     
     
         27 . A method for inhibition of the production of C3b complement fragment in a mammal comprising administering to said mammal an effective amount of a CRIg variant comprising an amino acid substitution in a region selected from the group consisting of E8-K15, R41-T47, S54-Q64, E85-Q99, and Q105-K111 of the amino acid sequence of SEQ ID NO: 68, or an immunoadhesin comprising said variant.