PD-l1 variant immunomodulatory proteins and uses thereof

Abstract

To provide particular variants of PD-L1 that exhibit improved binding, such as improved binding affinity or selectivity, for one or more of the cognate binding partner proteins PD-1 and CD80.SOLUTION: Provided are variant PD-L1 polypeptides comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.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) 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 that includes a D43G amino acid modification, wherein the amino acids of the amino acid sequence of the variant PD-L1 polypeptide are numbered based on SEQ ID NO: 30, and wherein the variant PD-L1 polypeptide specifically binds to the ectodomain of human PD-1 with increased affinity compared to the binding of unmodified PD-L1 to the ectodomain of human PD-1.

1. An immunomodulatory Fc fusion polypeptide comprising:

2. The immunomodulatory Fc fusion polypeptide of claim 1, wherein the unmodified PD-L1 polypeptide comprises (i) an amino acid sequence set forth in SEQ ID NO: 30, or (ii) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

30.

3. The immunomodulatory Fc fusion polypeptide of claim 1 or claim 2, wherein the variant PD-L1 polypeptide comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acid modifications.

4. The immunomodulatory Fc fusion polypeptide of any one of claims 1 to 3, wherein the variant PD-L1 polypeptide comprises amino acid modifications selected from among M18I / M41K / D43G / H51R / N78I, I20L / E27G / D43G / N45T / V50A, I20L / D43G, I36T / D43G, D43G / N45T, D43G / V50A, D43G / V58A, D43G / S75P, D43G / N78I, E27G / D43G, A33D / D43G, D43G / M97L, D43G / S99G, D43G / R195G, D43G / P198S, and D43G / R198T.

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

6. The immunomodulatory Fc fusion polypeptide of claim 1, wherein each of the first peptide linker and the second peptide linker is GGGGS (4GS; SEQ ID NO: 1942), GGGGSGGGGGS (2xGGGGGS; SEQ ID NO: 240), GGGGSGGGGSGGGGS (3xGGGGGS; SEQ ID NO: 239), 4xGGGGGS, 5xGGGGGS, GSGGGGS (SEQ ID NO: 1941), or a combination thereof.

7. The immunomodulatory Fc fusion polypeptide described in claim 1, wherein the Fc region is at least 90% identical to sequence number 187.

8. The immunomodulatory Fc fusion polypeptide of claim 7, 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.

9. An immunomodulatory Fc fusion polypeptide as described in claim 8, wherein the Fc region comprises the sequence of SEQ ID NO: 1715.

10. The immunomodulatory Fc fusion polypeptide of claim 1, wherein the Fc region is located N-terminal to the variant PD-L1 polypeptide.

11. The immunomodulatory Fc fusion polypeptide of claim 10, comprising, in an N-terminal to C-terminal direction, an IgSF domain derived from the CTLA-4, the first peptide linker, the Fc region, the second peptide linker, and the variant PD-L1 polypeptide.

12. An immunomodulatory Fc fusion polypeptide described in claim 10 or claim 11, wherein the first peptide linker, the second peptide linker, or the first peptide linker and the second peptide linker is GSGGGGS (sequence number 1941).

13. An immunomodulatory Fc fusion polypeptide according to any one of claims 10 to 12, wherein the CTLA-4-derived IgSF domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 36, the CTLA-4-derived IgSF domain binds to CD80, the first peptide linker is GSGGGGS (SEQ ID NO: 1941), the Fc region comprises the sequence of SEQ ID NO: 1715, and the second peptide linker is GGGGSGGGGGSGGGGS (3xGGGGGS; SEQ ID NO: 239).

14. The immunomodulatory Fc fusion polypeptide of claim 1, wherein the Fc region is located N-terminal to both the CTLA-4-derived IgSF domain and the variant PD-L1 polypeptide.

15. The immunomodulatory Fc fusion polypeptide of claim 14, comprising, from the N-terminus to the C-terminus, the Fc region, the first peptide linker, the CTLA-4-derived IgSF domain, the second peptide linker, and the variant PD-L1 polypeptide.

16. An immunomodulatory Fc fusion polypeptide described in claim 14 or claim 15, wherein the first peptide linker, the second peptide linker, or the first peptide linker and the second peptide linker are GGGGSGGGGGSGGGGS (3xGGGGGS; sequence number 239).

17. An immunomodulatory Fc fusion polypeptide according to any one of claims 14 to 16, wherein the Fc region comprises the sequence of SEQ ID NO: 1715, the first peptide linker is GGGGSGGGGGSGGGGS (3xGGGGGS; SEQ ID NO: 239), the CTLA-4-derived IgSF domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 36, the CTLA-4-derived IgSF domain binds to CD80, and the second peptide linker is GGGGSGGGGGSGGGGS (3xGGGGGS; SEQ ID NO: 239).

18. An immunomodulatory Fc fusion polypeptide as described in claim 1, which is a multimer.

19. A nucleic acid molecule encoding the immunomodulatory Fc fusion polypeptide described in claim 1.

20. A vector comprising the nucleic acid molecule described in claim 19.

21. A cell comprising the vector described in claim 20.

22. A method for producing an immunomodulatory Fc fusion polypeptide, comprising introducing the nucleic acid molecule described in claim 19 or the vector described in claim 20 into a host cell in vitro under conditions in which the immunomodulatory Fc fusion polypeptide is expressed in the host cell.

23. The method of claim 22, further comprising isolating or purifying the immunomodulatory Fc fusion polypeptide from the host cell.

24. A pharmaceutical composition comprising the immunomodulatory Fc fusion polypeptide of claim 1 and a pharmaceutically acceptable excipient.

25. The pharmaceutical composition of claim 24 for use in modulating an immune response in a subject.

26. The pharmaceutical composition for use according to claim 25, wherein modulating the immune response comprises reducing the immune response.

27. ​​A pharmaceutical composition for use as described in claim 25 or claim 26, wherein modulating the immune response treats a disease or condition in the subject.

28. The pharmaceutical composition for use according to claim 27, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

29. A pharmaceutical composition for use according to claim 27 or claim 28, wherein the disease or condition is antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, vasculitis, an autoimmune skin disease, transplant rejection, a rheumatic disease, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory lung disease, an inflammatory endocrine disease, or an autoimmune blood disease.

30. A pharmaceutical composition for use according to claim 27 or claim 28, wherein the disease or condition is inflammatory bowel disease, transplant rejection, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

31. A variant PD-L1 polypeptide or its specific PD-1-binding fragment, comprising an IgV domain or its specific PD-1-binding fragment, or both an IgV domain or its specific PD-1-binding fragment and an IgC domain or its specific PD-1-binding fragment, the variant PD-L1 polypeptide or the specific PD-1-binding fragment thereof comprises a D43G modification in unmodified PD-L1, based on the numbering of SEQ ID NO: 30; A variant PD-L1 polypeptide or specific PD-1-binding fragment thereof, wherein the variant PD-L1 polypeptide or specific PD-1-binding fragment thereof specifically binds to the ectodomain of PD-1 with increased affinity compared to the binding of the unmodified PD-L1 to the ectodomain of human PD-1.

32. The unmodified PD-L1 polypeptide, 32. The variant PD-L1 polypeptide or specific PD-1-binding fragment thereof of claim 31, comprising: (i) a sequence of amino acids set forth in SEQ ID NO: 30; or (ii) a sequence of amino acids having at least 95% sequence identity to SEQ ID NO:

30.

33. A variant PD-L1 polypeptide or specific PD-1-binding fragment thereof according to claim 31 or claim 32, comprising up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acid modifications.

34. A variant PD-L1 polypeptide or specific PD-1-binding fragment thereof according to any one of claims 31 to 33, comprising amino acid modifications selected from among M18I / M41K / D43G / H51R / N78I, I20L / E27G / D43G / N45T / V50A, I20L / D43G, I36T / D43G, D43G / N45T, D43G / V50A, D43G / V58A, D43G / S75P, D43G / N78I, E27G / D43G, A33D / D43G, D43G / M97L, D43G / S99G, D43G / R195G, D43G / P198S, and D43G / R198T.

35. The variant PD-L1 polypeptide or its specific PD-1-binding fragment according to claim 31, wherein the variant PD-L1 polypeptide comprises the IgV domain or its specific PD-1-binding fragment.

36. The variant PD-L1 polypeptide or specific PD-1-binding fragment thereof of claim 31, wherein the IgV domain or specific PD-1-binding fragment thereof is the only PD-L1 IgSF domain of the variant PD-L1.

37. The variant PD-L1 polypeptide or its specific PD-1-binding fragment of claim 31, wherein the variant PD-L1 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity and specifically binds to the ectodomain of CD80 with decreased affinity compared to the unmodified PD-L1 for the same ectodomain.

38. The variant PD-L1 polypeptide or specific PD-1-binding fragment thereof is a soluble protein; the variant PD-L1 polypeptide or the specific PD-1-binding fragment thereof lacks the PD-L1 transmembrane domain and the intracellular signaling domain; and / or 32. The variant PD-L1 polypeptide or specific PD-1-binding fragment thereof of claim 31, wherein the variant PD-L1 polypeptide or specific PD-1-binding fragment thereof is incapable of being expressed on the cell surface.

39. The variant PD-L1 polypeptide or its specific PD-1-binding fragment of claim 31, which is a transmembrane immunomodulatory protein further comprising a transmembrane domain directly or indirectly linked to the extracellular domain (ECD) of the variant PD-L1 polypeptide or its specific PD-1-binding fragment.

40. The variant PD-L1 polypeptide or specific binding fragment thereof of claim 39, further comprising a cytoplasmic signaling domain directly or indirectly linked to the transmembrane domain.

41. An immunomodulatory protein comprising the variant PD-L1 polypeptide of claim 31 or a specific PD-1-binding fragment thereof linked directly or indirectly via a linker to a second polypeptide comprising an IgSF domain of an immunoglobulin superfamily (IgSF) member.

42. The immunomodulatory protein of claim 41, wherein the IgSF domain is an affinity-modified IgSF domain, and the affinity-modified IgSF domain comprises one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member.

43. The immunomodulatory protein of claim 41 or claim 42, wherein the IgSF domain is affinity engineered to exhibit increased binding to one or more of its cognate binding partner(s) compared to binding of the unmodified or wild-type IgSF domain of the IgSF family member to the same cognate binding partner(s), wherein the variant PD-L1 polypeptide is capable of specifically binding to PD-1, and wherein the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than that specifically bound by the variant PD-L1 polypeptide.

44. A nucleic acid molecule encoding the variant PD-L1 polypeptide or its specific PD-1-binding fragment of claim 31.

45. A vector comprising the nucleic acid molecule described in claim 44.

46. A cell comprising the vector described in claim 45.

47. A method for producing a variant PD-L1 polypeptide or its specific PD-1-binding fragment, or an immunomodulatory protein, comprising introducing the nucleic acid molecule of claim 44 or the vector of claim 45 into a host cell in vitro under conditions for expressing the protein in the host cell, and further comprising isolating or purifying the variant PD-L1 polypeptide or its specific PD-1-binding fragment, or the immunomodulatory protein from the host cell.

48. A pharmaceutical composition comprising the variant PD-L1 polypeptide or its specific PD-1-binding fragment of claim 31, and a pharmaceutically acceptable excipient.

49. An article of manufacture comprising the pharmaceutical composition of claim 48, or a kit comprising the pharmaceutical composition of claim 48 and instructions for use.

50. The pharmaceutical composition of claim 48 for use in modulating an immune response in a subject.

51. The pharmaceutical composition for use according to claim 50, wherein modulating the immune response treats a disease or condition in the subject.

52. The pharmaceutical composition of claim 48 for use in the manufacture of a medicament for modulating an immune response in a subject.

53. The pharmaceutical composition for use according to claim 52, wherein the subject has an inflammatory or autoimmune disease or condition.