Multivalent and multispecific 41BB-binding fusion proteins
This invention relates generally to molecules that specifically engage 41BB, a member of the TNF receptor superfamily (TNFRSF). More specifically, this invention relates to multivalent and multispecific molecules that bind at least 41BB.
1. An isolated polypeptide comprising at least one VHH domain that binds PDL1 and at least one VHH domain that binds 41BB, wherein each VHH domain that binds PDL1 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 105, a CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and wherein each VHH domain that binds 41BB comprises a CDR1, a CDR2, and a CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 26, 27 and 28; or SEQ ID NOs: 26, 61, and 28; or SEQ ID NOs: 26, 63, and 28; or SEQ ID NOs: 26, 71, and 28; or SEQ ID NOs: 26, 73, and 28; or SEQ ID NOs: 26, 75, and 28; or SEQ ID NOs: 26, 77, and 28; or SEQ ID NOs: 26, 79, and 28; or SEQ ID NOs: 65, 61, and 28; or SEQ ID NOs: 67, 61, and 28; or SEQ ID NOs: 69, 61, and 28.
2. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds PDL1 is humanized.
3. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds PDL1 comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 104 and 119-124.
4. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds PDL1 comprises an amino acid sequence selected from SEQ ID NOs: 104 and 119-124.
5. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds PDL1 comprises the amino acid sequence of SEQ ID NO: 124.
6. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds 41BB is humanized.
7. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds 41BB comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 25, 59, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, and 83.
8. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds 41BB comprises an amino acid sequence selected from SEQ ID NOs: 25, 59, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, and 83.
9. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60.
10. The isolated polypeptide of claim 1 , wherein at least one VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28.
11. The isolated polypeptide of claim 1 , wherein each VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60.
12. The isolated polypeptide of claim 1 , wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28.
13. The isolated polypeptide of claim 12 , wherein the VH H domain that binds 41BB is humanized.
14. The isolated polypeptide of claim 12 , wherein the VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60.
15. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises an Fc region.
16. The isolated polypeptide of claim 15 , wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6.
17. The isolated polypeptide of claim 15 , wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-6.
18. The isolated polypeptide of claim 15 , which forms a homodimer under physiological conditions.
19. The isolated polypeptide of claim 15 , wherein the polypeptide has the structure VHH-Linker-VHH-Linker-Hinge-Fc.
20. The isolated polypeptide of claim 19 , wherein each linker is, independently, 5-20 amino acids in length.
21. The isolated polypeptide of claim 19 , wherein the hinge comprises a sequence selected from SEQ ID NOs: 7-9.
22. The isolated polypeptide of claim 1 , wherein each VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28.
23. The isolated polypeptide of claim 22 , wherein the isolated polypeptide comprises two VHH domains that bind PDL1.
24. The isolated polypeptide of claim 23 , wherein the isolated polypeptide comprises two VHH domains that bind 41BB.
25. The isolated polypeptide of claim 22 , wherein the isolated polypeptide comprises two VHH domains that bind 41BB.
26. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises two VHH domains that bind PDL1.
27. The isolated polypeptide of claim 26 , wherein the isolated polypeptide comprises two VHH domains that bind 41BB.
28. The isolated polypeptide of claim 1 , wherein the isolated polypeptide comprises two VHH domains that bind 41BB.
29. The isolated polypeptide of claim 1 , wherein each VI-11-1 domain of the isolated polypeptide is humanized.
30. The isolated polypeptide of claim 1 , wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB.
31. The isolated polypeptide of claim 1 , wherein the polypeptide comprises one VHH domain that binds PDL1.
32. The isolated polypeptide of claim 31 , wherein the VHH domain that binds PDL1 is humanized.
33. The isolated polypeptide of claim 31 , wherein the VHH domain that binds PDL1 comprises the amino acid sequence of SEQ ID NO: 124.
34. The isolated polypeptide of claim 33 , wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises the amino acid sequence of SEQ ID NO: 60.
35. The isolated polypeptide of claim 34 , wherein the isolated polypeptide comprises an Fc region.
36. The isolated polypeptide of claim 35 , wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6.
37. The isolated polypeptide of claim 35 , wherein the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 1-6.
38. The isolated polypeptide of claim 35 , wherein the polypeptide has the structure VHH-Linker-VHH-Linker-Hinge-Fc.
39. The isolated polypeptide of claim 38 , wherein each linker is, independently, 5-20 amino acids in length.
40. The isolated polypeptide of claim 38 , wherein the hinge comprises a sequence selected from SEQ ID NOs: 7-9.
41. The isolated polypeptide of claim 38 , which forms a homodimer under physiological conditions.
42. The isolated polypeptide of claim 35 , which forms a homodimer under physiological conditions.
43. The isolated polypeptide of claim 31 , wherein the polypeptide comprises one VHH domain that binds 41BB, wherein the VHH domain that binds 41BB comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28.
44. The isolated polypeptide of claim 43 , wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB.
45. A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 43 .
46. The method of claim 45 , wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia.
47. A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 43 .
48. The method of claim 47 , wherein the T cells are CD4+ T cells and/or CD8+ T cells.
49. A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 1 .
50. The method of claim 49 , wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia.
51. A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 1 .
52. The method of claim 51 , wherein the T cells are CD4+ T cells and/or CD8+ T cells.
53. An isolated polypeptide comprising a first VHH domain (VHH 1 ) that binds PDL1 and a second VHH domain (VHH 2 ) that binds 41BB, wherein VHH 1 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 105, a CDR2 comprising the amino acid sequence of SEQ ID NO: 106, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 107; and VHH 2 comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28; and wherein the polypeptide has the structure VHH 1 -Linker-VHH 2 -Linker-Hinge-Fc.
54. The isolated polypeptide of claim 53 , wherein the first VHH domain comprises the amino acid sequence of SEQ ID NO: 124 and the second VHH domain comprises the amino acid sequence of SEQ ID NO: 60, and wherein the Fc region comprises an amino acid sequence that is at least 97% identical to an amino acid sequence selected from SEQ ID NOs: 1-6.
55. The isolated polypeptide of claim 54 , wherein the Fc region comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 2.
56. The isolated polypeptide of claim 54 , wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 2.
57. The isolated polypeptide of claim 53 , wherein the polypeptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 449.
58. The isolated polypeptide of claim 53 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 449.
59. The isolated polypeptide of claim 58 , which forms a dimer at physiological conditions.
60. The isolated polypeptide of claim 53 , wherein the PDL1 is human PDL1, and wherein the 41BB is human 41BB.
61. A method of treating cancer comprising administering to a subject with cancer a polypeptide of claim 53 .
62. The method of claim 61 , wherein the cancer is selected from carcinoma, sarcoma, lymphoma, and leukemia.
63. A method of increasing T cell activation and/or proliferation comprising contacting T cells with a polypeptide of claim 53 .
64. The method of claim 63 , wherein the T cells are CD4+ T cells and/or CD8+ T cells.