Single-chain and multi-chain chimeric polypeptides and methods of use thereof

Abstract

To provide single-chain and multi-chain chimeric polypeptides useful for treating cancer, aging-related disease or other conditions.SOLUTION: The present invention provides single-chain and multi-chain chimeric polypeptides having a linker domain positioned between two target-binding domains that are useful for stimulating an immune cell, inducing or increasing proliferation of an immune cell, inducing differentiation of an immune cell, or treating a subject. In one aspect, provided herein is a method of promoting the activation and proliferation of a natural killer cell or a T cell that include the steps for contacting a natural killer cell or a T cell in a liquid culture medium comprising effective amounts of (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain, and (ii) an IgG1 antibody construct that comprises at least one antigen-binding domain that binds specifically to the linker domain, under conditions that allow for the activation and proliferation of the natural killer cell or the T cell.SELECTED DRAWING: Figure 5

Claims

1. 1. A method for promoting activation and proliferation of memory T cells, comprising: an effective amount (i) a single-chain chimeric polypeptide comprising a first target-binding domain, a linker domain, and a second target-binding domain; and (ii) an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to said linker domain; contacting the memory T cells in a liquid culture medium comprising, under conditions that allow activation and proliferation of said memory T cells; The method of claim 1, wherein the first target binding domain and the second target binding domain are each independently selected from the group consisting of a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor, and a ligand of a costimulatory molecule.

2. 2. The method of claim 1, wherein the first target binding domain and the linker domain are immediately adjacent to each other.

3. The method of claim 1 , wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain.

4. The method of any one of claims 1 to 3, wherein the linker domain and the second target binding domain are immediately adjacent to each other.

5. The method of any one of claims 1 to 3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target binding domain.

6. 1. A method for promoting activation and proliferation of memory T cells, comprising the steps of: （１） (a) (i) a first target binding domain; (ii) a linker domain, and (iii) the first domain of a pair of affinity domains a first chimeric polypeptide comprising: (b) (i) the second domain of the pair of affinity domains, and (ii) a second target-binding domain and a second chimeric polypeptide comprising: Including, the first chimeric polypeptide and the second chimeric polypeptide are associated via binding between the first domain and the second domain of the pair of affinity domains; an effective amount of a multi-chain chimeric polypeptide, and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the linker domain; contacting the memory T cells in a liquid culture medium comprising under conditions that allow activation and proliferation of said memory T cells.

7. The method of claim 6, wherein the first target binding domain and the linker domain are immediately adjacent to each other within the first chimeric polypeptide.

8. 7. The method of claim 6, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain within the first chimeric polypeptide.

9. The method of any one of claims 6 to 8, wherein the linker domain and the first domain of the pair of affinity domains are immediately adjacent to each other in the first chimeric polypeptide.

10. The method of any one of claims 6 to 8, wherein the first chimeric polypeptide further comprises a linker sequence between the linker domain in the first chimeric polypeptide and the first domain of the pair of affinity domains.

11. 11. The method of any one of claims 6 to 10, wherein the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other in the second chimeric polypeptide.

12. The method of any one of claims 6 to 10, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

13. The method of any one of claims 1 to 12, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

14. One or both of the first target binding domain and the second target binding domain may bind to any of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, and CD86. , PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5 AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TY RO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHC I ligand, scMHC II ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor 14. The method of any one of claims 1 to 13, wherein the antibody specifically binds to a target selected from the group consisting of IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

15. 13. The method of any one of claims 1 to 12, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein.

16. 16. The method of claim 15, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

17. 13. The method of any one of claims 1 to 12, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor.

18. 13. The method of any one of claims 1 to 12, wherein one or both of the first target binding domain and the second target binding domain is soluble TGF-β receptor II (TGF-β RII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28.

19. The method of any one of claims 1 to 18, wherein the linker domain is a soluble human tissue factor domain.

20. 20. The method of claim 19, wherein the soluble human tissue factor domain does not stimulate blood clotting.

21. The method of any one of claims 1 to 12, wherein one or both of the first target binding domain and the second target binding domain is a ligand for a costimulatory molecule.

22. The method of any one of claims 1 to 21, wherein the memory T cells have been previously obtained from the subject.

23. The memory T cells include Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, effector T cells, and CD8. ＋ T cells, CD4 ＋ The method according to any one of claims 1 to 22, wherein the T cells are peripheral blood memory T cells selected from the group consisting of T cells.

24. The method of any one of claims 1 to 23, wherein the memory T cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

25. 24. The method of any one of claims 1 to 23, further comprising, after the contacting step, introducing into the memory T cells a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor.

26. The method of any one of claims 6 to 12, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting.

27. 27. The method of any one of claims 1 to 26, wherein the contacting step is carried out for a period of from 2 hours to 20 days.

28. The method of any one of claims 1 to 5, wherein the liquid culture medium comprises the single-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of 0.5:1 to 2:

1.

29. The method of any one of claims 6 to 12, wherein the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of 0.5:1 to 2:

1.

30. the linker domain comprises a sequence at least 90% identical to SEQ ID NO: 1; the first domain of the pair of affinity domains comprises a sequence at least 90% identical to SEQ ID NO: 39; the second domain of the pair of affinity domains comprises a sequence at least 90% identical to SEQ ID NO: 10; and (A) the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 135, and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 124; (B) the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 124, and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 135; (C) the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 124, and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 60; or (D) the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 60, and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

60. The method according to any one of claims 6 to 12.

31. 31. The method of claim 30, wherein the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 135 and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

124.

32. the first target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 135; the linker domain comprises a sequence at least 95% identical to SEQ ID NO: 1; the first domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 39; the second domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 10; the second target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 124; 32. The method of claim 31 .

33. the first target binding domain comprises SEQ ID NO: 135; the linker domain comprises SEQ ID NO: 1; the first domain of the pair of affinity domains comprises SEQ ID NO: 39; the second domain of the pair of affinity domains comprises SEQ ID NO: 10; the second target binding domain comprises SEQ ID NO: 124; 32. The method of claim 31 .

34. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 141 and the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

145.

35. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 141 and the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

145.

36. 7. The method of claim 6, wherein the first chimeric polypeptide comprises SEQ ID NO: 141 and the second chimeric polypeptide comprises SEQ ID NO:

145.

37. 31. The method of claim 30, wherein the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 124 and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

135.

38. the first target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 124; the linker domain comprises a sequence at least 95% identical to SEQ ID NO: 1; the first domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 39; the second domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 10; the second target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 135; 38. The method of claim 37.

39. the first target binding domain comprises SEQ ID NO: 124; the linker domain comprises SEQ ID NO: 1; the first domain of the pair of affinity domains comprises SEQ ID NO: 39; the second domain of the pair of affinity domains comprises SEQ ID NO: 10; the second target binding domain comprises SEQ ID NO: 135; 38. The method of claim 37.

40. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 126 and the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

137.

41. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 126 and the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

137.

42. 7. The method of claim 6, wherein the first chimeric polypeptide comprises SEQ ID NO: 126 and the second chimeric polypeptide comprises SEQ ID NO:

137.

43. 31. The method of claim 30, wherein the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO: 124 and the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

60.

44. the first target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 124; the linker domain comprises a sequence at least 95% identical to SEQ ID NO: 1; the first domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 39; the second domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 10; the second target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 60; 44. The method of claim 43.

45. the first target binding domain comprises SEQ ID NO: 124; the linker domain comprises SEQ ID NO: 1; the first domain of the pair of affinity domains comprises SEQ ID NO: 39; the second domain of the pair of affinity domains comprises SEQ ID NO: 10; the second target binding domain comprises SEQ ID NO: 60; 44. The method of claim 43.

46. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 126 and the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

132.

47. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 126 and the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

132.

48. 7. The method of claim 6, wherein the first chimeric polypeptide comprises SEQ ID NO: 126 and the second chimeric polypeptide comprises SEQ ID NO:

132.

49. 31. The method of claim 30, wherein the first target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 60, and the second target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO:

60.

50. the first target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 60; the linker domain comprises a sequence at least 95% identical to SEQ ID NO: 1; the first domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 39; the second domain of the pair of affinity domains comprises a sequence at least 95% identical to SEQ ID NO: 10; the second target binding domain comprises a sequence at least 95% identical to SEQ ID NO: 60; 50. The method of claim 49.

51. the first target binding domain comprises SEQ ID NO: 60; the linker domain comprises SEQ ID NO: 1; the first domain of the pair of affinity domains comprises SEQ ID NO: 39; the second domain of the pair of affinity domains comprises SEQ ID NO: 10; the second target binding domain comprises SEQ ID NO: 60; 50. The method of claim 49.

52. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 161 and the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

130.

53. 7. The method of claim 6, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 161 and the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO:

130.

54. 7. The method of claim 6, wherein the first chimeric polypeptide comprises SEQ ID NO: 161 and the second chimeric polypeptide comprises SEQ ID NO: 130.

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