Interleukin-2 variant and method of use

Abstract

This provides a polypeptide that shares its primary sequence with human IL-2, except for a few amino acid mutations. [Solution] A pharmaceutical composition is provided for use in the treatment of a disease or disorder selected from the group consisting of autoimmune diseases, organ transplant rejection or related graft-versus-host disease (GvHD), inflammatory diseases, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atopic dermatitis, diabetes mellitus, and immune-related adverse events (irAEs), wherein the pharmaceutical composition comprises a fusion protein containing a specific amino acid sequence mixed with a pharmaceutically acceptable carrier.

Claims

1. An isolated interleukin-2 (IL-2) variant polypeptide comprising the amino acid sequence of SEQ ID NO: 3, wherein one or more amino acid residues at positions L19, D20, L21, Q22, R38, F42, N88, S125, or Q126 are substituted with other amino acids, and having reduced ability to activate the IL-2Rβγ receptor complex compared to the polypeptide represented by SEQ ID NO: 3, but retaining the ability to activate the IL-2Rαβγ receptor complex.

2. The amino acid substitutions are L19D substitution, L19H substitution, L19N substitution, L19R substitution, L19S substitution, L19P substitution, and L19Y substitution at position 19 of Sequence ID No. 3; D20E substitution, D20I substitution, D20N substitution, D20Q substitution, D20S substitution, D20T substitution, and D20Y substitution at position 20; L21S substitution, L21R substitution, and L21N substitution at position 21; Q22N substitution, Q22H substitution, Q22K substitution, Q22Y substitution, and Q22I substitution at position 22; and N88E substitution and N88G substitution at position 88 of Sequence ID No.

3. The IL-2 variant polypeptide according to claim 1, selected from the group consisting of substitution, N88T substitution, N88M substitution, N88Q substitution, N88R substitution, and N88I substitution, S125E substitution, S125K substitution, S125H substitution, S125W substitution, and S125I substitution at position 125, and Q126D substitution, Q126E substitution, Q126H substitution, Q126K substitution, Q126L substitution, Q126M substitution, Q126N substitution, and Q126Y substitution at position 126, or any combination thereof.

3. The IL-2 variant polypeptide according to any one of claims 1 and 2, comprising two amino acid substitutions at the L19 and Q126 amino acid residues of SEQ ID NO:

3.

4. The IL-2 variant polypeptide according to any one of claims 1 and 2, comprising three amino acid substitutions at the L19, S125, and Q126 amino acid residues of SEQ ID NO:

3.

5. The IL-2 variant polypeptide according to any one of claims 3 and 4, wherein the amino acid substitution is selected from the group consisting of L19D substitution, L19H substitution, L19N substitution, L19R substitution, L19S substitution, L19P substitution, and L19Y substitution at position 19 of SEQ ID NO: 3, S125E substitution, S125K substitution, S125H substitution, S125W substitution, and S125I substitution at position 125, and Q126D substitution, Q126E substitution, Q126H substitution, Q126K substitution, Q126L substitution, Q126M substitution, Q126N substitution, and Q126Y substitution at position 126.

6. An IL-2 variant polypeptide according to any one of claims 1 to 5, comprising an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 4-43, 108-146, and 193-197.

7. The IL-2 variant polypeptide according to claim 6, comprising an amino acid sequence selected from the group consisting of amino acid sequences described in SEQ ID NOs. 5-14, 26-43, 108-111, 125-146, and 193-197 (including IL-2 variants).

8. An isolated IL-2 variant polypeptide comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to SEQ ID NO: 3, wherein its ability to activate the IL-2Rβγ receptor complex is reduced compared to the polypeptide represented by SEQ ID NO: 3, but its ability to activate the IL-2Rαβγ receptor complex is retained.

9. 1) an isolated fusion protein comprising an IL-2 variant polypeptide as described in any one of claims 1 to 8, and 2) a heterologous protein.

10. The isolated fusion protein according to claim 9, wherein the IL-2 variant polypeptide is optionally fused to the C-terminal amino acid of the heterologous protein at its N-terminal amino acid via a peptide linker.

11. The isolated fusion protein according to claim 9, wherein the IL-2 variant polypeptide is optionally fused at its C-terminal amino acid to the N-terminal amino acid of the heterologous protein via a peptide linker.

12. The isolated fusion protein according to any one of claims 9 to 11, wherein the heterologous protein extends the cyclic half-life of the IL-2 variant polypeptide.

13. The isolated fusion protein according to claim 12, wherein the heterologous protein is selected from the group consisting of a full-length null antibody or antibody fragment that enables the extension of the half-life.

14. The isolated fusion protein according to any one of claims 9 to 11, wherein the heterologous protein enhances the expression level and total purity of the IL-2 variant polypeptide.

15. The isolated fusion protein according to any one of claims 9 to 11, wherein the heterologous protein functions as a marker, tag, or targeting portion.

16. An isolated fusion protein according to any one of claims 9 to 15, which is either in dimeric or monomeric form.

17. The isolated fusion protein according to any one of claims 9 to 11, wherein the heterologous protein is an Fc domain selected from the group consisting of the Fc domain of human IgG1, human IgG2, human IgG3, human IgG4, IgA, IgD, IgE, IgG, and IgM.

18. The isolated fusion protein according to claim 17, wherein the Fc domain is an Fc domain having an effector function that is silenced and / or has a function that extends the half-life.

19. The isolated fusion protein according to any one of claims 17 to 18, wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs: 44 to 47 and 212 to 213.

20. The isolated fusion protein according to any one of claims 9 to 19, wherein the IL-2 variant polypeptide is fused with the heterologous protein via a peptide linker.

21. The fusion protein according to claim 20, wherein the peptide linker contains 1 to 40 amino acids.

22. The isolated fusion protein according to claim 9, comprising an amino acid sequence selected from the group consisting of the amino acid sequences described in SEQ ID NOs. 69-107, 147-189, and 198-211.

23. A pharmaceutical composition comprising an IL-2 variant polypeptide or isolated fusion protein according to any one of claims 1 to 22, mixed with a pharmaceutically acceptable carrier.

24. A method for treating an autoimmune disease in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 23.

25. The method according to claim 24, further comprising administering a second therapeutic agent or treatment method capable of treating an autoimmune disease in a subject.

26. A method for treating organ transplant rejection or related graft-versus-host disease (GvHD) in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 23 to the subject.

27. A method for treating inflammatory diseases, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atopic dermatitis, diabetes mellitus, and immune-related adverse events (irAEs) in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition described in claim 23 to the subject.

28. The method according to claim 27, further comprising administering a second therapeutic agent or treatment method capable of treating an inflammatory disease in a subject.

29. An isolated nucleic acid molecule encoding an IL-2 variant polypeptide or fusion protein according to any one of claims 1 to 22.

30. An expression vector comprising the nucleic acid molecule described in claim 29.

31. A host cell comprising the nucleic acid molecule described in claim 29 or the expression vector described in claim 30.

32. A method for producing an IL-2 variant polypeptide or fusion protein according to any one of claims 1 to 22, comprising: culturing a host cell according to claim 31 under conditions that promote the expression of the IL-2 variant polypeptide or fusion protein; and recovering the IL-2 variant polypeptide or fusion protein.

33. An isolated IL-2 variant polypeptide or fusion protein prepared by the method described in claim 32.

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