Methods of treating cancer cells expressing tumor-associated integrins

1. A method for treating a cancer comprising tumor cells expressing a tumor-associated integrin in a subject, comprising administering to the subject a therapeutically effective amount of:

(a) interleukin (IL)-2;

(b) an integrin-binding-Fc fusion protein which binds to the tumor cells expressing the tumor-associated integrin; and

(c) a cancer vaccine,

wherein the integrin-binding-Fc fusion protein comprises (i) an integrin-binding polypeptide comprising an integrin-binding loop and a knottin polypeptide scaffold, and (ii) an immunoglobulin Fc domain, wherein the integrin-binding polypeptide is operably linked to the Fc domain.

2. The method of claim 1 , wherein the IL-2 is an extended pharmacokinetic (PK) IL-2.

3. The method of claim 2 , wherein the extended-PK IL-2 comprises a fusion protein.

4. The method of claim 3 , wherein the fusion protein comprises an IL-2 moiety and a moiety selected from the group consisting of an immunoglobulin fragment, serum albumin, transferrin, and Fn3, or variants thereof.

5. The method of claim 1 , wherein the IL-2 comprises an IL-2 moiety conjugated to a non-protein polymer.

6. The method of claim 5 , wherein the non-protein polymer is polyethylene glycol.

7. The method of claim 4 , wherein the fusion protein comprises an IL-2 moiety operably linked to an immunoglobulin Fc domain or human serum albumin.

8. The method of claim 1 , wherein the tumor-associated integrin is selected from the group consisting of αvβ3, αvβ5, and α5β1, or combination thereof.

9. The method of claim 1 , wherein the knottin polypeptide scaffold comprises at least three cysteine disulfide linkages or crosslinked cysteine residues, and wherein the integrin-binding loop is adjacent to cysteine residues of the knottin polypeptide scaffold.

10. The method of claim 9 , wherein the integrin-binding loop comprises an RGD peptide sequence.

11. The method of claim 9 , wherein the knottin polypeptide scaffold is derived from a knottin protein selected from the group consisting of EETI-II, AgRP, and agatoxin.

12. The method of claim 1 , wherein the knottin polypeptide scaffold is derived from EETI-II and the integrin-binding loop comprises the sequence X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 X 11 , wherein each X represents any amino acid, wherein the loop is inserted between 2 cysteine residues in the EETI-II sequence and replaces the native EETI-II sequence.

13. The method of claim 1 , wherein the integrin-binding polypeptide comprises the amino acid sequence set forth SEQ ID NO: 42 or 43, wherein X 1 is selected from the group consisting of A, V, L, P, F, Y, S, H, D, and N; X 2 is selected from the group consisting of G, V, L, P, R, E, and Q; X 3 is selected from the group consisting of G, A, and P; X 7 is selected from the group consisting of W and N; X 8 is selected from the group consisting of A, P, and S; X 9 is selected from the group consisting of P and R; X 10 is selected from the group consisting of A, V, L, P, S, T, and E; and X 11 is selected from the group consisting of G, A, W, S, T, K, and E.

14. The method of claim 1 , wherein the integrin-binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-133.

15. The method of claim 1 , wherein the Fc domain is a human IgG1 Fc domain.

16. The method of claim 1 , wherein the integrin-binding polypeptide is operably linked with or without a linker to the Fc domain.

17. The method of claim 1 ,

wherein the integrin-binding polypeptide is operably linked to the N-terminus of the Fc domain; or

wherein the integrin-binding polypeptide is operably linked to the C-terminus of the Fc domain.

18. The method of claim 1 , wherein the integrin-binding-Fc fusion protein comprises the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51.

19. The method of claim 1 , wherein the cancer vaccine is a population of cells comprising antigen presenting cells immunized in vitro with a tumor antigen and administered to the subject.

20. The method of claim 1 , wherein the cancer vaccine is an amphiphilic peptide conjugate comprising a tumor-associated antigen, and a lipid component, and optionally a linker, wherein the amphiphilic peptide conjugate binds albumin under physiological conditions.

21. The method of claim 20 , wherein the tumor-associated antigen is conjugated to a lipid via a linker.

22. The method of claim 20 , wherein the lipid is a diacyl lipid.

23. The method of claim 20 , wherein the cancer vaccine further comprises an adjuvant.

24. The method of claim 23 , wherein the adjuvant is an amphiphilic oligonucleotide conjugate comprising an immunostimulatory oligonucleotide conjugated to a lipid with or without a linker, and optionally a polar compound, wherein the conjugate binds albumin under physiological conditions.

25. The method of claim 24 , wherein the molecular adjuvant is an immunostimulatory oligonucleotide that can bind a pattern recognition receptor.

26. The method of claim 24 ,

wherein the immunostimulatory oligonucleotide comprises CpG; or

wherein the immunostimulatory oligonucleotide is a ligand for a toll-like receptor.

27. The method of claim 24 , wherein the linker is an oligonucleotide linker.

28. The method of claim 24 , wherein the oligonucleotide linker comprises “N” consecutive guanines, wherein N is between 0-2.

29. The method of claim 24 , wherein the lipid is a diacyl lipid.

30. The method of claim 1 , wherein IL-2 or extended-PK IL-2, integrin-binding-Fc fusion protein, and cancer vaccine are administered simultaneously or sequentially.

31. The method of claim 1 , wherein the subject has a tumor.

32. The method of claim 31 , wherein the treatment increases the number of interferon gamma expressing CD8+ T cells in the tumor.

33. The method of claim 31 , wherein the treatment increases the ratio of CD8+ T cells to T regulatory cells in the tumor.

34. The method of claim 1 , wherein the cancer is selected from the group consisting of melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

35. A method for inhibiting growth and/or proliferation of tumor cells expressing a tumor-associated integrin in a subject comprising administering to the subject an effective amount of (a) an integrin-binding-Fc fusion protein which binds to the tumor cells expressing the tumor-associated integrin, wherein the integrin-binding-Fc fusion protein comprises (i) an integrin-binding polypeptide comprising an integrin-binding loop and a knottin polypeptide scaffold, and (ii) an immunoglobulin Fc domain, wherein the integrin-binding polypeptide is operably linked to the Fc domain; and (b) a cancer vaccine.