CD47 / PD-L1-targeting protein complex and method of use thereof

Abstract

This disclosure relates to protein complexes targeting CD47, PD-L1, and / or TGFβ, and methods of using them. In one embodiment, the protein complex comprises a CD47-binding domain having all or part of the SIRPα extracellular domain, a PD-L1-binding domain having a VHH that binds to PD-L1, and optionally, a TGFβ-binding domain having all or part of the TGFBRII extracellular domain.

Claims

1. (a) Fc and, (b) CD47 binding domain and (c) A PD-L1 (programmed dead ligand 1) binding domain, which is a VHH (heavy chain single variable domain) that binds to PD-L1, and A protein complex containing [the specified ingredient].

2. The protein complex according to claim 1, further comprising a TGFβ (transforming growth factor beta) binding domain.

3. The protein complex according to claim 1 or 2, wherein the CD47-binding domain can bind to cells expressing CD47 (e.g., cancer cells) and / or can block the interaction between CD47 and signal regulatory protein α (SIRPα).

4. The protein complex according to any one of claims 1 to 3, wherein the CD47-binding domain is or comprises a SIRPα extracellular domain.

5. The protein complex according to claim 4, wherein the SIRPα extracellular domain comprises a sequence that is at least 80% identical to SEQ ID NO: 32, and optionally comprises one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56, and / or E70 of SEQ ID NO:

32.

6. The SIRPα extracellular domain, (a) The amino acid corresponding to H24 in SEQ ID NO: 32 is T. (b) The amino acid corresponding to I31 in SEQ ID NO: 32 is Y. (c) The amino acid corresponding to E54 in SEQ ID NO: 32 is R. (d) The amino acid corresponding to G55 in SEQ ID NO: 32 is Q. (e) The amino acid corresponding to H56 in SEQ ID NO: 32 is T, and (f) The amino acid corresponding to E70 in SEQ ID NO: 32 is F. The protein complex according to claim 4 or 5, comprising one or more of the following.

7. The protein complex according to any one of claims 1 to 6, wherein the CD47-binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

7.

8. The protein complex according to any one of claims 1 to 7, wherein the VHH can bind to cells expressing PD-L1 (e.g., cancer cells) and / or can block the interaction between PD-L1 and programmed cell death protein 1 (PD-1).

9. The VHH comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence that is at least 80% identical to the selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80% identical to the selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80% identical to the selected VHH CDR3 amino acid sequence. The selected VHH CDR1, 2, and 3 amino acid sequences are (1) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs. 26, 27, and 28, respectively, and (2) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 29, 30, and 31, respectively. One of them is The protein complex according to any one of claims 1 to 8.

10. The protein complex according to any one of claims 1 to 9, wherein the VHH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

8.

11. The protein complex according to any one of claims 2 to 10, wherein the TGFβ-binding domain can capture TGFβ, thereby increasing the immune response and / or improving the tumor microenvironment.

12. The protein complex according to any one of claims 2 to 11, wherein the TGFβ-binding domain is or comprises a TGFBRII extracellular domain.

13. The protein complex according to any one of claims 2 to 12, wherein the TGFBRII extracellular domain comprises a sequence that is at least 80% identical to SEQ ID NO: 33, and optionally, the TGFBRII extracellular domain comprises one or more amino acid mutations at positions corresponding to S49 and / or D118 of SEQ ID NO:

33.

14. The TGFβ-binding domain, (a) The amino acid corresponding to S49 in Sequence ID No. 33 is E, and (b) The amino acid corresponding to D118 in SEQ ID NO: 33 is E. A protein complex according to any one of claims 2 to 13, comprising one or more of the above.

15. The protein complex according to any one of claims 2 to 14, wherein the TGFβ-binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

9.

16. The protein complex according to any one of claims 1 to 15, wherein the CD47-binding domain is optionally linked to the N-terminus of the CH2 domain in Fc via a hinge region.

17. The protein complex according to claim 16, wherein the PD-L1 binding domain is optionally linked to the N-terminus of the CD47 binding domain via a linker peptide.

18. The protein complex according to any one of claims 1 to 15, wherein the PD-L1 binding domain is optionally linked to the N-terminus of the CH2 domain in Fc via a hinge region.

19. The protein complex according to claim 18, wherein the CD47-binding domain is optionally linked to the N-terminus of the PD-L1-binding domain via a linker peptide.

20. The protein complex according to any one of claims 1 to 19, wherein the Fc is human IgG1 Fc.

21. The protein complex according to any one of claims 1 to 19, wherein the Fc is human IgG4Fc.

22. The protein complex according to any one of claims 16 to 19 and 21, wherein the hinge region is a human IgG4 hinge region having an S228P mutation assigned by EU numbering, which can be optionally selected.

23. The protein complex according to any one of claims 2 to 22, wherein the TGFβ-binding domain is optionally linked to the C-terminus of the CH3 domain in Fc via a linker peptide.

24. (a) A first polypeptide comprising, from the N-terminus to the C-terminus, a first CD47-binding domain, an optional first linker peptide, a first VHH binding to PD-L1, an optional first hinge region, and a first Fc region, (b) A second polypeptide comprising, from the N-terminus to the C-terminus, a second CD47-binding domain, an optional second linker peptide, a second VHH binding to PD-L1, an optional second hinge region, and a second Fc region. A protein complex containing [the specified ingredient].

25. The protein complex according to claim 24, wherein the first VHH and / or the second VHH include sequences that are at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

8.

26. The protein complex according to claim 24 or 25, wherein the first CD47-binding domain and / or the second CD47-binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

7.

27. The protein complex according to any one of claims 24 to 26, wherein the first hinge region and / or the second hinge region include a sequence that is at least 80% identical to sequence number 14.

28. The protein complex according to any one of claims 24 to 27, wherein the first Fc region and / or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO:

15.

29. The protein complex according to any one of claims 24 to 26, wherein the first hinge region and / or the second hinge region include a sequence that is at least 80% identical to sequence number 16.

30. The protein complex according to any one of claims 24 to 26 and 29, wherein the first Fc region and / or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO:

17.

31. The protein complex according to any one of claims 24 to 30, wherein the first linker peptide and / or the second linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

12.

32. The protein complex according to any one of claims 24 to 31, wherein the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO: 4 or 5.

33. The protein complex according to any one of claims 24 to 32, wherein the first polypeptide further comprises a first TGFβ-binding domain, and the second polypeptide further comprises a second TGFβ-binding domain.

34. The protein complex according to claim 33, wherein the first TGFβ-binding domain and / or the second TGFβ-binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

9.

35. The protein complex according to claim 33 or 34, wherein the first TGFβ-binding domain is optionally linked to the C-terminus of the first Fc region via a third linker peptide, and the second TGFβ-binding domain is optionally linked to the C-terminus of the second Fc region via a fourth linker peptide.

36. The protein complex according to claim 35, wherein the third linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

13.

37. The protein complex according to any one of claims 33 to 36, wherein the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO: 1 or 2.

38. (a) A first polypeptide comprising, from the N-terminus to the C-terminus, a first VHH that binds to PD-L1, an optional first linker peptide, a first CD47 binding domain, an optional first hinge region, and a first Fc region, (b) A second polypeptide comprising, from the N-terminus to the C-terminus, a second VHH that binds to PD-L1, an optional second linker peptide, a second CD47 binding domain, an optional second hinge region, and a second Fc region. A protein complex containing [the specified ingredient].

39. The protein complex according to claim 38, wherein the first VHH and / or the second VHH include sequences that are at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

8.

40. The protein complex according to claim 38 or 39, wherein the first CD47-binding domain and / or the second CD47-binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

7.

41. The protein complex according to any one of claims 38 to 40, wherein the first hinge region and / or the second hinge region include a sequence that is at least 80% identical to SEQ ID NO:

16.

42. The protein complex according to any one of claims 38 to 41, wherein the first Fc region and / or the second Fc region includes a sequence that is at least 80% identical to SEQ ID NO:

17.

43. The protein complex according to any one of claims 38 to 42, wherein the first linker peptide and / or the second linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

12.

44. The protein complex according to any one of claims 38 to 43, wherein the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

6.

45. The protein complex according to any one of claims 38 to 44, wherein the first polypeptide further comprises a first TGFβ-binding domain, and the second polypeptide further comprises a second TGFβ-binding domain.

46. The protein complex according to claim 45, wherein the first TGFβ-binding domain and / or the second TGFβ-binding domain comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

9.

47. The protein complex according to claim 45 or 46, wherein the first TGFβ-binding domain is optionally linked to the C-terminus of the first Fc region via a third linker peptide, and the second TGFβ-binding domain is optionally linked to the C-terminus of the second Fc region via a fourth linker peptide.

48. The protein complex according to claim 47, wherein the third linker peptide and / or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO:

13.

49. The protein complex according to any one of claims 45 to 48, wherein the first polypeptide and / or the second polypeptide comprises a sequence that is at least 80%, 90%, 95%, or 100% identical to SEQ ID NO:

3.

50. A nucleic acid comprising a polynucleotide encoding a protein complex according to any one of claims 1 to 49.

51. The nucleic acid according to claim 50, wherein the nucleic acid is DNA (e.g., cDNA) or RNA (e.g., mRNA).

52. A vector comprising one or more nucleic acids according to claim 50 or 51.

53. A cell comprising the vector according to claim 52.

54. The cell according to claim 53, which is a CHO cell.

55. A cell comprising one or more nucleic acids according to claim 50 or 51.

56. A method for producing a protein complex, (a) Culturing the cells described in any one of claims 53 to 55 under conditions sufficient to produce the protein complex, (b) Collecting the protein complex produced by the cells and The method, including the method described above.

57. A protein conjugate comprising a protein complex according to any one of claims 1 to 49, covalently bound to a therapeutic agent.

58. The protein conjugate according to claim 57, wherein the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor.

59. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of a composition comprising a protein complex according to any one of claims 1 to 49 or a protein conjugate according to claim 57 or 58.

60. The method according to claim 59, wherein the subject has cancer cells expressing CD47 and / or PD-L1.

61. The method according to claim 59 or 60, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, bile duct cancer, head and neck cancer, hematological cancer, or a combination thereof.

62. A method for reducing the rate of tumor growth, Contacting tumor cells with an effective amount of a composition comprising the protein complex described in any one of claims 1 to 49 or the protein conjugate described in claim 57 or 58. The method, including the method described above.

63. A method for killing tumor cells, Contacting tumor cells with an effective amount of a composition comprising the protein complex described in any one of claims 1 to 49 or the protein conjugate described in claim 57 or 58. The method, including the method described above.

64. A pharmaceutical composition comprising a protein complex according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier.

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