TNF-alpha VARIANTS PROTEINS FOR THE TREATMENT OF TNF-alpha RELATED DISORDERS

Abstract

The invention relates to novel proteins with TNF-alpha antagonist activity and nucleic acids encoding these proteins. The invention further relates to the use of the novel proteins in the treatment of TNF-alpha related disorders.

1 .- 71 . (canceled)  
     
     
         72 . A method for treating a Tumor necrosis factor (TNF-alpha) related disorder comprising administering to a patient in need of such treatment a non-naturally occurring variant TNF-alpha protein comprising an amino acid sequence that has at least one amino acid substitution in the Large Domain and at least one amino acid substitution in a second domain selected from the group consisting of the DE Loop and the Small Domain as compared to the wild-type TNF-alpha sequence of SEQ ID NO: 3, wherein said variant TNF-alpha protein is capable of interacting with the wild-type TNF-alpha to form mixed trimers having at least a 50% decrease in receptor activation as compared to a homotrimer of wild-type TNF-alpha proteins as determined by a caspase assay.  
     
     
         73 . The method according to  claim 72 , wherein said variant TNF-alpha protein has from 2 to 5 amino acid substitutions as compared to said wild-type TNF-alpha sequence.  
     
     
         74 . The method according to  claim 72 , wherein said Large Domain substitution is at a position selected from the group consisting of 21, 30, 31, 32, 33, 35, 65, 66, 67, 111, 112, 115, 140, 143, 144, 145, 146 and 147  
     
     
         75 . The method according to  claim 74 , wherein said substitution in said Large Domain is at position 145.  
     
     
         76 . The method according to  claim 72 , wherein said substitution in said second domain is a Small Domain substitution at a position selected from the group consisting of 75 and 97.  
     
     
         77 . The method according to  claim 72 , wherein said substitution in said second domain is a DE Loop substitution at a position selected from the group consisting of 84, 86, 87 and 91.  
     
     
         78 . The method according to  claim 72 ,  75  or  77  wherein said variant TNF-alpha protein further comprises at least one additional substitution in the Trimer Interface of said TNF-alpha protein at a position selected from the group consisting of 34, 91 and 57.  
     
     
         79 . The method according to  claim 72 , wherein said variant TNF-alpha protein is capable of interacting with said wild-type TNF-alpha to form mixed trimers having at least a 76% decrease in receptor activation as compared to a homotrimer of wild-type TNF-alpha protein as determined by a caspase assay.  
     
     
         80 . The method according to  claim 79 , wherein said variant TNF-alpha protein is capable of interacting with wild-type TNF-alpha to form mixed trimers having at least a 90% decrease in receptor activation as compared to a homotrimer of wild-type TNF-alpha proteins as determined by a caspase assay.  
     
     
         81 . The method according to  claim 79 , wherein said variant TNF-alpha protein is capable of interacting with said wild-type TNF-alpha to form mixed trimers that are incapable of activating receptor signaling as determined by a caspase assay.  
     
     
         82 . The method according to  claim 74 ,  76  or  77  wherein said Large Domain substitution is selected from the group of substitutions consisting of Q21C, Q21R, N30D, R31C, R31I, R31D, R31E, R32D, R32E, R32S, A33E, A35S, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, A111R, A111E, K112D, K112E, Y115D, Y115E, Y115F, Y115H, Y115I, Y115K, Y115L, Y115M, Y115N, Y115Q, Y115R, Y115S, Y115T, Y115W, D140K, D140R, D143E, D143K, D143L, D143R, D143N, D143Q, D143R, D143S, F144N, A145D, A145E, A145F, A145H, A145K, A145M, A145N, A145Q, A145R, A145S, A145T, A145Y, E146K, E146L, E146M, E146N, E146R, E146S and S147R, and said DE Loop substitution is selected from the group consisting of A84V, S86Q, S86R, Y87H, Y87R and V91E.  
     
     
         83 . The method according to  claim 82 , wherein said Large Domain substitution is selected from the group consisting of Y151, Y115Q, Y115T, D143K, D143R, D143E, A145E, A145R, E146K and E146R.  
     
     
         84 . The method according to  claim 72 , wherein said Large Domain substitution is selected from the group consisting of Y1151, Y115T, D143K, D143R, D143E, A145R, E146K and E146R.  
     
     
         85 . The method according to  claim 82 , wherein said substitutions comprise (a) either Y1151 or Y115T and (b) either A145E or A145R.  
     
     
         86 . The method according to  claim 82 , wherein said substitutions comprise A145R and further comprise at least one of L57F, L75E, L75Q, Y87H, or I97T.  
     
     
         87 . The method according to  claim 86 , wherein said substitutions comprise A145R and Y87H.  
     
     
         88 . The method according to  claim 86 , wherein said substitutions comprise A145R and I97T.  
     
     
         89 . The method according to  claim 82 , wherein said substitutions comprise L57F and A145E.  
     
     
         90 . A method for treating a Tumor necrosis factor I. (TNF-alpha) related disorder comprising administering to a patient in need of such treatment a non-naturally occurring variant TNF-alpha protein comprising an amino acid sequence that has at least one amino acid substitution in the Large Domain and at least one amino acid substitution in a second domain selected from the group consisting of the DE Loop and the Small Domain as compared to the wild-type TNF-alpha sequence of SEQ ID NO: 3, wherein said variant TNF-alpha protein forms mixed trimers capable of interacting with a receptor interface at the receptor binding site to reduce receptor activation by at least 50% as compared to a homotrimer of wild-type TNF-alpha proteins as determined by a caspase assay.  
     
     
         91 . The method according to  claim 90 , wherein said variant TNF-alpha protein blocks the receptor binding site.  
     
     
         92 . The method according to  claim 72  or  90 , wherein said variant TNF-alpha protein is PEGylated.  
     
     
         93 . The method according to  claim 92 , wherein said TNF-alpha protein is PEGylated at positions selected from the group consisting of 10, 21 23, 24, 25, 27, 31, 42, 44, 45, 46, 86, 87, 88, 90, 107, 108, 110, 128, 140 and 145.  
     
     
         94 . The method according to  claim 93 , wherein said TNF-alpha protein is PEGylated at positions selected from the group consisting of 21, 23, 31 and 45.  
     
     
         95 . The method according to  claim 92 , wherein said PEGylated TNF-alpha protein disrupts receptor binding.  
     
     
         96 . The method according to  claim 72 , wherein from about 1 to about 55 amino acids from the N-terminus, the C-terminus or both the N- and C-termini of said variant TNF-alpha protein are deleted.  
     
     
         97 . The method according to  claim 96 , wherein the C-terminal leucine is deleted.  
     
     
         98 . The method according to  claim 72  or  90 , wherein two or more substituted domains of said variant TNF-alpha protein are covalently linked via at least one disulfide bond.  
     
     
         99 . The method according to  claim 72  or  90 , wherein two or more substituted domains of said variant TNF-alpha protein are covalently linked via chemical cross linking.  
     
     
         100 . The method according to  claim 72  or  90 , wherein two or more of said variant TNF-alpha proteins are covalently linked by a linker peptide.  
     
     
         101 . The method according to  claim 100 , wherein said linker peptide comprises at least one and not more than about 30 amino acid residues.  
     
     
         102 . The method according to  claim 100 , wherein said linker peptide comprises one or more of the following amino acid residues: Gly, Ser, Ala and Thr.  
     
     
         103 . The method according to  claim 72  or  90 , wherein said variant TNF-alpha protein further comprises a methionine (M) residue at position 1.  
     
     
         104 . The method according to  claim 72 , wherein said TNF-alpha related disorder is an autoimmune disease.  
     
     
         105 . The method according to  claim 72 , wherein said TNF-alpha related disorder is spondyloarthritis.  
     
     
         106 . A method according to  claim 72 , wherein said TNF-alpha related disorder is inflammatory bowel disease.  
     
     
         107 . A method according to  claim 72 , wherein said TNF-alpha related disorder is psoriasis.  
     
     
         108 . A method according to  claim 72 , wherein said TNF-alpha related disorder is graft versus host disease (GVHD).  
     
     
         109 . A method according to  claim 72 , wherein said TNF-alpha related disorder is a hematologic malignancy.  
     
     
         110 . A method according to  claim 109 , wherein said TNF-alpha related disorder is multiple myeloma (MM), myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML).  
     
     
         111 . The method according to  claim 72 , wherein said condition is rheumatoid arthritis.  
     
     
         112 . The method according to  claim 72 , wherein said disorder is Crohn's disease.  
     
     
         113 . The method according to  claim 72 , wherein said disorder is sepsis or septic shock.  
     
     
         114 . The method according to  claim 72 , wherein said disorder is peripheral nerve injury.  
     
     
         115 . The method according to  claim 114 , wherein said disorder is Chronic Constriction Injury.  
     
     
         116 . The method according to  claim 72 , wherein said disorder is a demyelinating disease.  
     
     
         117 . The method according to  claim 116 , wherein said disorder is Guillain-Barre Syndrome.  
     
     
         118 . The method according to  claim 116 , wherein said disorder is Multiple Sclerosis.  
     
     
         119 . The method according to  claim 72 , wherein said disorder is cancer.