Full-length interferon gamma polypeptide variants
View Patent ↗The present invention relates to novel full-length interferon gamma (IFNG) polypeptide variants having interferon gamma activity. The full-length interferon gamma polypeptide variants of the invention are obtained by performing selected modifications in the C-terminal part of the molecule. The full-length interferon gamma polypeptide variants of the invention are useful in therapy, in particular for the treatment of interstitial pulmonary diseases, such as idiopathic pulmonary fibrosis.
1. A full-length variant of the interferon gamma (IFNG) polypeptide of SEQ ID NO: 1, said variant exhibiting IFNG receptor binding activity and consisting of up to 10 residue modifications in SEQ ID NO: 1, and wherein up to 10 residue modifications includes:
(a) at least one amino acid substitution in a position selected from the group consisting of S132 and S142; and
(b) at least one amino acid substitution in a position selected from the group consisting of R137, R139 and R140.
2. The full-length variant according to claim 1 , wherein said amino acid substitution is selected from the group consisting of S132P, S142P and S132P+S142P.
3. The full-length variant according to claim 2 , wherein said amino acid substitution is S132P.
4. The full-length variant according to claim 2 , wherein said amino acid substitution is S142P.
5. The full-length variant of claim 2 , wherein at least one non-positively charged amino acid residue is introduced by substitution in a position selected from the group consisting of R137, R139 and R140.
6. The full-length variant according to claim 5 , wherein said non-positively charged amino acid residue is a proline residue.
7. The full-length variant of claim 5 , wherein said variant comprises the following substitutions: R137P+R139P+S142P.
8. The full-length variant of claim 5 , wherein said variant comprises the following substitutions: R137P+S142P.
9. The full-length variant of claim 5 , wherein said variant comprises the following substitutions: S132P+R137P+R140P.
10. The full-length variant of claim 5 , wherein said variant comprises the following substitutions: S132P+R140P.
11. A full-length variant of the interferon gamma (IFNG) polypeptide of SEQ ID NO: 1, said variant exhibiting IFNG receptor binding activity and consisting of up to 10 residue modifications in SEQ ID NO: 1, wherein up to 10 residue modifications an amino acid substitution in position R137 and an amino acid substitution in position R140.
12. The full-length variant according to claim 11 , wherein said variant comprises the substitutions R137X+R140P, wherein X is any amino acid residue, except arginine and lysine.
13. The full-length variant according to claim 11 , wherein said variant comprises the substitutions R137P+R140X, wherein X is any amino acid residue, except arginine.
14. The full-length variant of claim 11 , wherein said variant comprises the substitutions R137P+R140P.
15. The full-length variant of claim 11 , wherein said variant comprises at least one further modification in the C-terminal part from amino acid residue S132 to amino acid residue Q143.
16. The full-length variant according to claim 15 , wherein said further modification comprises introduction of at least one cysteine residue.
17. The full-length variant according to claim 16 , wherein said cysteine residue is covalently attached to a polymer molecule.
18. The full-length variant according to claim 17 , where said polymer molecule is a linear or branched polyethylene glycol.
19. The full-length variant according to claim 11 , wherein said modifications are substitutions.
20. The full-length variant according to claim 19 , wherein said variant comprises the substitution S99T.
21. The full-length variant of claim 1 , wherein said up to 10 residue modifications comprises at least one introduced and/or at least one removed amino acid residue comprising an attachment group for a non-polypeptide moiety.
22. The full-length variant according to claim 21 , wherein said up to 10 residue modifications comprises at least one introduced glycosylation site.
23. The full-length variant according to claim 22 , wherein said glycosylation site is an N-glycosylation site.
24. The full-length variant according to claim 23 , wherein said N-glycosylation site is introduced in a position comprising an amino acid residue having at least 25% of its side chain exposed to the surface.
25. The full-length variant according to claim 24 , wherein said N-glycosylation site is introduced in a position comprising an amino acid residue having at least 50% of its said chain exposed to the surface.
26. The full-length variant of claim 23 , wherein said N-glycosylation site is introduced by substitution.
27. The full-length variant according to claim 1 , wherein said up to 10 residue modifications is a substitution selected from the group consisting of G18S, G18T, E38N, E38N+S40T, K61S, K61T, S65N+Q67S, S65N+Q67T, N85S, N85T, K94N, Q106S and Q106T.
28. The full-length variant according to claim 27 , wherein said substitution is selected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T, N85T, K94N and Q106T.
29. The full-length variant according to claim 28 , wherein said substitution is selected from the group consisting of G18T, E38N+S40T, K61T, S65N+Q67T and N85T.
30. The full-length variant according to claim 29 , wherein said substitution is E38N+S40T.
31. The full-length variant according to claim 21 , wherein said up to 10 residue modifications comprises an introduced cysteine residue.
32. The full-length variant according to claim 31 , wherein said cysteine residue is introduced in a position comprising an amino acid residue having at least 25% of its side chain exposed to the surface.
33. The full-length variant according to claim 32 , wherein said cysteine residue is introduced in a position comprising an amino acid residue having at least 50% of its side chain exposed to the surface.
34. The full-length variant according to claim 31 , wherein said cysteine residue is introduced by substitution.
35. The full-length variant according to claim 31 , wherein said up to 10 residue modifications is a substitution selected from the group consisting of N10C, N16C, E38C, N59C, N83C, K94C, N104C and A124C.
36. The full-length variant according to claim 35 wherein said substitution is selected from the group consisting of N16C, N59C and N16C+N59C.
37. The full-length variant of claim 31 , wherein said cysteine residue is covalently attached to a polymer molecule.
38. The full-length variant according to claim 37 , wherein said polymer molecule is a linear or branched polyethylene glycol.
39. The full-length variant according to claim 21 , wherein said up to 10 residue modifications comprises at least one introduced N-glycosylation site and at least one introduced cysteine residue.
40. The full-length variant of claim 1 , wherein said variant comprises an amino acid sequence, which is identical to the amino acid sequence from residue 1 to residue 131 of huIFNG of SEQ ID NO: 1.
41. The full-length variant according to claim 40 , wherein said variant is un-glycosylated.
42. The full-length variant of claim 31 , wherein said variant is glycosylated.
43. A nucleotide sequence encoding the full-length variant of claim 1 .
44. An expression vector comprising a nucleotide sequence as defined in claim 43 .
45. An isolated host cell comprising a nucleotide sequence as defined in claim 43 or an expression vector according to claim 44 .
46. A composition comprising a full-length IFNG variant of claim 1 and a carrier.
47. A pharmaceutical composition comprising a full-length variant of claim 1 and a pharmaceutically acceptable diluent, carrier or adjuvant.
48. A method for producing a full-length IFNG polypeptide, said method comprising
i) cultivating a host cell as defined in claim 45 under conditions suitable for production of the IFNG polypeptide, and
ii) recovering the IFNG polypeptide.