TNF-α variants
The invention relates to novel proteins with TNF-α antagonist activity and nucleic acids encoding these proteins. The invention further relates to the use of the novel proteins in the treatment of TNF-α related disorders, such as rheumatoid arthritis.
1. A recombinant nucleic acid encoding a variant TNF-α protein comprising an amino acid substitution of amino acids 1–157 of SEQ ID NO: 2, said substitution at a position selected from the group consisting of positions 21, 30, 31, 32, 35, 66, 84, 111, 112, 115, and 140, wherein said variant protein interacts with a human TNF-α protein to form a mixed miner that has a reduced capacity to effect TNF-α receptor signaling in a caspase assay.
2. A recombinant nucleic acid according to claim 1 , wherein said variant TNF-α protein has between 2 and 5 amino acid substitutions at positions selected from the group consisting of positions 21, 30, 31, 32, 35, 66, 84, 111, 112, 115, and 140.
3. A recombinant nucleic acid according to claim 1 , wherein said substitution is selected from the group consisting of K112D, Y115l, Y115T, end A84V.
4. A vector composition comprising the recombinant nucleic acid of claim 1 .
5. A host cell comprising the recombinant nucleic acid of claim 1 .
6. A method of producing a protein composition comprising culturing a host cell of claim 5 under conditions suitable for the expression of said nucleic acids.
7. A method of forming a TNF-α heterotrimer comprising:
a) expressing a human TNF-α amino acid sequence comprising the amino acid sequence of amino acids 1–157 of SEQ ID NO: 2;
b) expressing a variant TNF-α amino acid sequence comprising an amino acid substitution of amino acids 1–157 of SEQ ID NO: 2, said substitution at a position selected from the group consisting of positions 21, 30, 31, 32, 33, 35, 65, 66, 67, 84, 111, 112, 115, 140, 143, 144, 145, 146 and 147;
wherein the TNF-α amino acid interacts with the variant TNF-α sequence to form the TNF-α heterotrimer having a reduced capacity to effect TNF-α receptor signaling in a caspase assay.
8. The method according to claim 7 , wherein said variant TNF-α sequence has between 2 and 5 amino acid substitutions at positions selected from the group consisting of positions 21, 30, 31,32, 33, 35, 65, 66, 67, 84, 111, 112, 115, 140, 143, 144, 145, 146 and 147.
9. The method according to claim 7 , wherein said substitution is selected from the group consisting of K112D, Y151l, Y115T, D143E, D143K, D143R, D143N, D143S, A145R A145K, A145E, E146K, E146R, and A84V.
10. The method according to claim 7 , wherein said substitution comprises A145R.
11. The method according to claim 7 , wherein said sequence comprises at least two amino acid substitutions selected from the group consisting of A84V, D143S; A145E and E146K.
12. The method according to claim 7 , wherein said TNF-α heterotrimer comprises one said variant TNF-α sequence.
13. The method according to claim 7 , wherein said TNF-α heterotrimer comprises two said variant TNF-α sequences.