PD-L1 variant immune regulatory protein and its use

Abstract

To provide immunomodulatory proteins comprising variant PD-L1, and nucleic acids encoding such proteins.SOLUTION: Provided herein is a variant PD-L1 polypeptide, containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, where the variant PD-L1 polypeptide contains one or more amino acid modifications in one or more positions in an unmodified PD-L1 or a specific binding fragment thereof corresponding to specific position(s). Also provided is an immunomodulatory protein comprising the polypeptide.SELECTED DRAWING: Figure 2

Claims

1. An immunomodulatory Fc fusion polypeptide comprising: (a) an immunoglobulin superfamily (IgSF) domain derived from CTLA-4; (b) a first peptide linker; (c) an Fc region; (d) a second peptide linker; and (e) a variant PD-L1 polypeptide, wherein the variant PD-L1 polypeptide comprises an amino acid sequence containing an N45D amino acid modification, the amino acids of the amino acid sequence of the variant PD-L1 polypeptide are numbered with reference to SEQ ID NO: 30, and the variant PD-L1 polypeptide specifically binds to the ectodomain of human PD-1 with increased affinity as compared to the binding of unmodified PD-L1 to the ectodomain of human PD-1 An immunomodulatory Fc fusion polypeptide comprising.

2. The variant PD-L1 polypeptide comprises amino acid modifications selected from D43G / N45D / V58A, I20L / E27G / D43G / N45D / V58A / N78I, I20L / D43G / N45D / V58A / N78I, I20L / A33D / D43G / N45D / V58A / N78I, I20L / D43G / N45D / N78I, V11A / I20L / E27G / D43G / N45D / H51Y / S99G, I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins, I20L / I36T / N45D, A33D / D43G / N45D / V58A / S75P, K23R / D43G / N45D, D43G / N45D / L56Q / V58A / G101G-ins, I20L / K23E / D43G / N45D / V58A / N78I, I20L / K23E / D43G / N45D / V50A / N78I, N45D, N45D / K144E, N45D / P198S, N45D / P198T, N45D / R195G, N45D / R195S, N45D / S131F, N45D / V58D, N45D / I148V / R195G, N45D / K111T / R195G, N45D / N113Y / R195S, N45D / N165Y / E170G, N45D / Q89R / I98V, N45D / S131F / P198S, N45D / S75P / P198S, N45D / V50A / R195T, E27D / N45D / T183A / I188V, K23N / N45D / S75P / N120S, N45D / G102D / R194W / R195G, N45D / I148V / R195G / N201D, N45D / K111T / T183A / I188V, N45D / T163I / K167R / R195G, N45D / V50A / I119T / K144E, V11E / N45D / T130A / P198T, K23N / N45D / Q73R / T163I, K28R / N45D / V129D / T163N / R195T, M41K / D43G / N45D / R64S / R195G, M41K / D43G / N45D / R64S / S99G, N45D / R68L / F173L / D197G / P198S, N45D / V50A / I148V / R195G / N201D, M41K / D43G / K44E / N45D / R195G / N201D, and N45D / V50A / L124S / K144E / L179P / R195G.The immunomodulatory Fc fusion polypeptide according to claim 1.

3. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the amino acid modification I20L / D43G / N45D / N78I.

4. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the amino acid modification D43G / N45D / L56Q / V58A / G101G-ins.

5. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the amino acid modification I20L / K28E / D43G / N45D / V58A / Q89R / G101G-ins.

6. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO:

292.

7. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO:

303.

8. The immunomodulatory Fc fusion polypeptide according to claim 2, wherein the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO: 1727.

9. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 8, wherein the IgSF domain derived from CTLA-4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 36 and the IgSF domain derived from CTLA-4 binds to CD80.

10. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 9, wherein each of the first peptide linker and the second peptide linker is GGGGS (4GS; SEQ ID NO: 1942), GGGGSGGGGS (2xGGGGS; SEQ ID NO: 240), GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 239), 4xGGGGS, 5xGGGGS, GSGGGGS (SEQ ID NO: 1941), or a combination thereof.

11. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 10, wherein the Fc region is at least 90% identical to SEQ ID NO:

187.

12. The immunomodulatory Fc fusion polypeptide according to claim 11, wherein the Fc region comprises one or more amino acid modifications selected from the group consisting of deletions of C5S, L19A, L20E, G22A, E141D, M143L, and K232 numbered based on SEQ ID NO:

187.

13. The immunomodulatory Fc fusion polypeptide according to claim 12, wherein the Fc region comprises deletions of amino acid modifications C5S, L19A, L20E, G22A, E141D, M143L, and K232 numbered based on SEQ ID NO:

187.

14. The immunomodulatory Fc fusion polypeptide according to claim 13, wherein the Fc region comprises the sequence of SEQ ID NO: 1715.

15. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 14, wherein the Fc region is located on the N-terminal side with respect to the variant PD-L1 polypeptide.

16. The immunomodulatory Fc fusion polypeptide according to claim 15, wherein the direction from the N-terminus to the C-terminus is the IgSF domain derived from CTLA-4, the first peptide linker, the Fc region, the second peptide linker, and the variant PD-L1 polypeptide.

17. The immunomodulatory Fc fusion polypeptide according to claim 15 or 16, wherein the first peptide linker is GSGGGGS (SEQ ID NO: 1941).

18. The immunomodulatory Fc fusion polypeptide according to any one of claims 15 to 17, wherein the second peptide linker is GGGGSGGGGSGGGGS (3xGGGGS; SEQ ID NO: 239).

19. The IgSF domain derived from CTLA-4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 36, the IgSF domain derived from CTLA-4 binds to CD80, the first peptide linker is GSGGGGS (SEQ ID NO: 1941), the Fc region comprises the sequence of SEQ ID NO: 1715, the second peptide linker is GGGGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239), and the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO:

292. The immunomodulatory Fc fusion polypeptide according to any one of claims 15 to 18.

20. The IgSF domain derived from CTLA-4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 36, the IgSF domain derived from CTLA-4 binds to CD80, the first peptide linker is GSGGGGS (SEQ ID NO: 1941), the Fc region comprises the sequence of SEQ ID NO: 1715, the second peptide linker is GGGGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239), and the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO:

303. The immunomodulatory Fc fusion polypeptide according to any one of claims 15 to 18.

21. The IgSF domain derived from CTLA-4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 36, the IgSF domain derived from CTLA-4 binds to CD80, the first peptide linker is GSGGGGS (SEQ ID NO: 1941), the Fc region comprises the sequence of SEQ ID NO: 1715, the second peptide linker is GGGGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239), and the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO: 1727. The immunomodulatory Fc fusion polypeptide according to any one of claims 15 to 18.

22. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 14, wherein the Fc region is located on the N-terminal side with respect to both the IgSF domain derived from CTLA-4 and the variant PD-L1 polypeptide.

23. The immunomodulatory Fc fusion polypeptide according to claim 22, wherein the direction from the N-terminus to the C-terminus is the Fc region, the first peptide linker, the IgSF domain derived from CTLA-4, the second peptide linker, and the variant PD-L1 polypeptide.

24. The immunomodulatory Fc fusion polypeptide according to claim 22 or 23, wherein the first peptide linker is GGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239).

25. The immunomodulatory Fc fusion polypeptide according to any one of claims 22 to 24, wherein the second peptide linker is GGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239).

26. The immunomodulatory Fc fusion polypeptide according to any one of claims 22 to 25, wherein the Fc region comprises the sequence of SEQ ID NO: 1715, the first peptide linker is GGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239), the IgSF domain derived from CTLA-4 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 36, the IgSF domain derived from CTLA-4 binds to CD80, the second peptide linker is GGGGSGGGGGSGGGGGS (3xGGGGGS; SEQ ID NO: 239), and the variant PD-L1 polypeptide comprises the sequence of SEQ ID NO: 292, 303, or 1727.

27. The immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 26, which is a multimer.

28. The immunomodulatory Fc fusion polypeptide according to claim 27, wherein the multimer is a dimer.

29. The immunomodulatory Fc fusion polypeptide according to claim 27 or 28, which is a homodimer.

30. A nucleic acid molecule encoding the immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 26.

31. A vector comprising the nucleic acid molecule according to claim 30.

32. The vector according to claim 31, which is an expression vector.

33. A cell comprising the vector according to claim 31 or 32.

34. A method for producing an immunomodulatory Fc fusion polypeptide, comprising introducing the nucleic acid molecule according to claim 30, or the vector according to claim 31 or claim 32, in vitro into a host cell under conditions such that the immunomodulatory Fc fusion polypeptide is expressed in the host cell.

35. The method according to claim 34, further comprising isolating or purifying the immunomodulatory Fc fusion polypeptide from the host cell.

36. A pharmaceutical composition comprising the immunomodulatory Fc fusion polypeptide according to any one of claims 1 to 29 and a pharmaceutically acceptable excipient.

37. The pharmaceutical composition according to claim 36, for use in modulating an immune response in a subject.

38. The pharmaceutical composition for use according to claim 37, wherein modulating the immune response comprises reducing the immune response.

39. The pharmaceutical composition for use according to claim 37 or 38, wherein modulating the immune response treats a disease or condition in the subject.

40. The pharmaceutical composition for use according to claim 39, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

41. The pharmaceutical composition for use according to claim 39 or 40, wherein the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, autoimmune skin disease, transplant rejection, rheumatic disease, inflammatory gastrointestinal disease, inflammatory eye disease, inflammatory nerve disease, inflammatory lung disease, inflammatory endocrine disease, or autoimmune blood disease.

42. The pharmaceutical composition for use according to claim 39 or 40, wherein the disease or condition is inflammatory bowel disease, transplant rejection, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

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