OX40-binding polypeptides and uses thereof
Provided herein are VHH-containing polypeptides that bind OX40. In some embodiments, VHH-containing polypeptides that bind and agonize OX40 are provided. Uses of the VHH-containing polypeptides are also provided.
1. A polypeptide comprising at least one VHH domain that binds OX40, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and wherein the VHH domain comprises a FR2 comprising the amino acid sequence of SEQ ID NO: 22.
2. The polypeptide of claim 1 , wherein the VHH domain comprises a FR3 comprising the amino acid sequence of SEQ ID NO: 23.
3. The polypeptide of claim 1 , wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 9.
4. The polypeptide of claim 1 , comprising two VHH domains.
5. The polypeptide of claim 4 , wherein each VHH domain binds OX40.
6. The polypeptide of claim 5 , wherein each VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and wherein each VHH domain comprises a FR2 comprising the amino acid sequence of SEQ ID NO: 22.
7. The polypeptide of claim 6 , wherein the polypeptide comprises an Fc domain.
8. The polypeptide of claim 7 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
9. A pharmaceutical composition comprising the polypeptide of claim 7 and a pharmaceutically acceptable carrier.
10. The polypeptide of claim 5 , wherein each VHH domain comprises the amino acid sequence of SEQ ID NO: 9.
11. The polypeptide of claim 10 , wherein the polypeptide comprises an Fc domain.
12. The polypeptide of claim 11 , which forms a dimer under physiological conditions.
13. The polypeptide of claim 11 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
14. A pharmaceutical composition comprising the polypeptide of claim 11 and a pharmaceutically acceptable carrier.
15. The polypeptide of claim 5 , wherein the polypeptide comprises an Fc domain.
16. The polypeptide of claim 1 , comprising three VHH domains.
17. The polypeptide of claim 16 , wherein each VHH domain binds OX40.
18. The polypeptide of claim 17 , wherein each VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and wherein each VHH domain comprises a FR2 comprising the amino acid sequence of SEQ ID NO: 22.
19. The polypeptide of claim 18 , wherein the polypeptide comprises an Fc domain.
20. The polypeptide of claim 19 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
21. A pharmaceutical composition comprising the polypeptide of claim 19 and a pharmaceutically acceptable carrier.
22. The polypeptide of claim 17 , wherein each VHH domain comprises the amino acid sequence of SEQ ID NO: 9.
23. The polypeptide of claim 17 , wherein the polypeptide comprises an Fc domain.
24. The polypeptide of claim 1 , comprising four VHH domains.
25. The polypeptide of claim 24 , wherein each VHH domain binds OX40.
26. The polypeptide of claim 25 , wherein each VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and wherein each VHH domain comprises a FR2 comprising the amino acid sequence of SEQ ID NO: 22.
27. The polypeptide of claim 26 , wherein the polypeptide comprises an Fc domain.
28. The polypeptide of claim 27 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
29. A pharmaceutical composition comprising the polypeptide of claim 27 and a pharmaceutically acceptable carrier.
30. The polypeptide of claim 25 , wherein each VHH domain comprises the amino acid sequence of SEQ ID NO: 9.
31. The polypeptide of claim 30 , wherein the polypeptide comprises an Fc domain.
32. The polypeptide of claim 31 , which forms a dimer under physiological conditions.
33. The polypeptide of claim 31 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
34. A pharmaceutical composition comprising the polypeptide of claim 31 and a pharmaceutically acceptable carrier.
35. The polypeptide of claim 25 , wherein the polypeptide comprises an Fc domain.
36. The polypeptide of claim 1 , wherein each VHH domain binds OX40.
37. The polypeptide of claim 36 , wherein each VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, and wherein each VHH domain comprises a FR2 comprising the amino acid sequence of SEQ ID NO: 22.
38. The polypeptide of claim 37 , wherein each VHH domain comprises a FR3 comprising the amino acid sequence of SEQ ID NO: 23.
39. The polypeptide of claim 36 , wherein each VHH domain comprises the amino acid sequence of SEQ ID NO: 9.
40. The polypeptide of claim 1 , wherein the polypeptide comprises an Fc domain.
41. The polypeptide of claim 40 , wherein the Fc domain comprises an amino acid sequence selected from SEQ ID NOs: 25 and 26.
42. The polypeptide of claim 40 , which forms a dimer under physiological conditions.
43. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 14.
44. The polypeptide of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 15.
45. The polypeptide of claim 1 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
46. The polypeptide of claim 45 , wherein the increase occurs in the presence of Treg cells.
47. The polypeptide of claim 45 , wherein the increase is in in vitro and is by at least 1.5-fold or by at least 2-fold.
48. The polypeptide of claim 45 , wherein the increase is determined as an average of results from T cells of at least five or at least ten different healthy human donors.
49. The polypeptide of claim 1 , which is an agonist of OX40 biological activity.
50. The polypeptide of claim 1 , wherein the OX40 is human OX40.
51. The polypeptide of claim 50 , wherein the polypeptide binds human OX40 with an affinity (K D ) of less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM.
52. The polypeptide of claim 51 , wherein the polypeptide binds cynomolgus monkey OX40 with an affinity (K D ) of less than 10 nM, less than 5 nM, less than 2 nM, or less than 1 nM.
53. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
54. The polypeptide of claim 22 , wherein the polypeptide comprises an Fc domain.
55. The polypeptide of claim 54 , which forms a dimer under physiological conditions.
56. The polypeptide of claim 54 , wherein the polypeptide:
a) increases CD4 + and/or CD8 + T cell proliferation in vitro and/or in vivo;
b) increases CD25 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
c) increases CD71 expression on CD4 + and/or CD8 + T cells in vitro and/or in vivo;
d) increases NFκB signaling in CD4 + and/or CD8 + T cells in vitro and/or in vivo; and/or
e) increases IFNγ expression in CD4 + and/or CD8 + T cells in vitro and/or in vivo.
57. A pharmaceutical composition comprising the polypeptide of claim 54 and a pharmaceutically acceptable carrier.
58. A polypeptide that binds OX40 consisting of the amino acid sequence of SEQ ID NO: 15.
59. The polypeptide of claim 58 , which forms a dimer under physiological conditions.
60. A pharmaceutical composition comprising the polypeptide of claim 58 and a pharmaceutically acceptable carrier.