Fusion comprising a cell penetrating peptide, a multi epitope and a TLR peptide agonist for treatment of cancer

1. A complex comprising:

a) a cell penetrating peptide;

b) at least three antigenic peptides; and

c) at least one TLR peptide agonist,

wherein the components a) c) are covalently linked, and

wherein the at least three antigenic peptides comprise:

(i) a peptide having an amino acid sequence according to SEQ ID NO: 95;

(ii) a peptide having an amino acid sequence according to SEQ ID NO: 96; and

(iii) a peptide having an amino acid sequence according to SEQ ID NO: 97.

2. The method of claim 1 , wherein the cell penetrating peptide

(i) has a length of the amino acid sequence of said peptide of 5 to 50 amino acids in total, and/or

(ii) has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, and/or added without abrogating said peptide's cell penetrating ability.

3. The complex of claim 1 , wherein the at least one TLR peptide agonist is a TLR2, TLR4 and/or TLR5 peptide agonist.

4. The complex of claim 1 wherein

the cell penetrating peptide has an amino acid sequence according to SEQ ID NO: 6 or a functional sequence variant thereof having at least 90% sequence identity; and

the TLR agonist has an amino acid sequence according to SEQ ID NO: 71 or a functional sequence variant thereof having at least 90% sequence identity.

5. The complex of claim 1 , wherein said complex comprises a polypeptide having at least 80% sequence identity to SEQ ID NO: 89 and comprises SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97.

6. A nucleic acid encoding the complex of claim 1 .

7. A vector comprising the nucleic acid of claim 6 .

8. A host cell comprising the vector as defined in claim 7 .

9. A cell loaded with the complex of claim 1 .

10. A composition comprising at least one of:

(i) the complex of claim 1 ;

(ii) a nucleic acid encoding the complex;

(iii) a vector comprising the nucleic acid;

(iv) a host cell comprising the vector; or

(v) a cell loaded with the complex.

11. A vaccine comprising at least one of:

(i) the complex of claim 1 ;

(ii) a nucleic acid encoding the complex;

(iii) a vector comprising the nucleic acid;

(iv) a host cell comprising the vector; or

(v) a cell loaded with the complex.

12. A combination comprising

(i) the complex of claim 1 ; and

(ii) a chemotherapeutic agent, a targeted drug and an immunotherapeutic agent.

13. A kit comprising the complex of claim 1 , a nucleic acid encoding the complex, a vector comprising the nucleic acid, a cell loaded with the complex, a composition comprising the complex, and/or a vaccine comprising the complex.

14. The complex of claim 1 , wherein the complex comprises a peptide having an amino acid sequence according to SEQ ID NO: 98.

15. The complex of claim 1 comprising

(i) a peptide having an amino acid sequence according to SEQ ID NO: 6;

(ii) a peptide having an amino acid sequence according to SEQ ID NO: 96;

(iii) a peptide having an amino acid sequence according to SEQ ID NO: 95;

(iv) a peptide having an amino acid sequence according to SEQ ID NO: 97; and

(v) a peptide having an amino acid sequence according to SEQ ID NO: 71.

16. The complex of claim 1 comprising in N- to C-terminal direction

(i) a cell penetrating peptide having an amino acid sequence according to SEQ ID NO: 6;

(ii) the peptide having an amino acid sequence according to SEQ ID NO: 96;

(iii) the peptide having an amino acid sequence according to SEQ ID NO: 95;

(iv) the peptide having an amino acid sequence according to SEQ ID NO: 97; and

(v) a TLR peptide agonist having an amino acid sequence according to SEQ ID NO: 71.

17. The complex of claim 1 , wherein the complex comprises a peptide having an amino acid sequence according to SEQ ID NO: 89.

18. The combination of claim 12 , wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

19. The complex of claim 1 , wherein the complex consists of a peptide having an amino acid sequence according to SEQ ID NO: 89.

20. The complex of claim 2 , wherein the cell penetrating peptide has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, or added without abrogating said peptide's cell penetrating ability.

21. The complex of claim 2 , wherein the cell penetrating peptide has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted without abrogating said peptide's cell penetrating ability.

22. The complex of claim 4 , wherein the cell penetrating peptide has an amino acid sequence according to SEQ ID NO: 6.

23. The complex of claim 4 , wherein the TLR agonist has an amino acid sequence according to SEQ ID NO: 71.

24. The complex of claim 4 , wherein the cell penetrating peptide has an amino acid sequence according to SEQ ID NO: 6 and wherein the TLR agonist has an amino acid sequence according to SEQ ID NO: 71.

25. The complex of claim 4 , wherein components (a)-(c) are linked by a linker or spacer.

26. The complex of claim 5 , wherein said complex consists of a polypeptide having at least 80% sequence identity to SEQ ID NO: 89 and comprises SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97.

27. A combination comprising (i) the complex of claim 1 ; and (ii) a chemotherapeutic agent, a targeted drug, or an immunotherapeutic agent.

28. The complex of claim 15 , wherein components (i) - (v) are linked by a linker or spacer.

29. A method for treating colorectal cancer in a subject in need thereof, the method comprising administering to the subject: (i) the copmlex of claim 1 ; (ii) a nucleic acid encoding the complex; (iii) a vector comprising the nucleic acid; (iv) a host cell comprising the vector; or (v) a cell loaded with the complex.

30. The complex of claim 29 , wherein the cell penetrating peptide

(i) has a length of the amino acid sequence of said peptide of 5 to 50 amino acids in total; and/or

(ii) has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, and/or added without abrogating said peptide's cell penetrating ability.

31. The method of claim 29 , wherein the cell penetrating peptide has an amino acid sequence comprising or consisting of an amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a sequence variant thereof sharing at least 90 sequence identity without abrogating said peptide's cell penetrating ability.

32. The method of claim 29 , wherein the complex comprises a peptide having an amino acid sequence according to SEQ ID NO: 98.

33. The method of claim 29 , wherein the at least one TLR peptide agonist is a TLR2, TLR4 and/or TLR5 peptide agonist.

34. The method of claim 29 , wherein the at least one TLR peptide agonist comprises an amino acid sequence according to SEQ ID NO: 15 or 71; or of a sequence variant thereof sharing at least 90% sequence identity without abrogating said peptide's TLR agonist ability.

35. The method of claim 29 , wherein the complex comprises

(i) a peptide having an amino acid sequence according to SEQ ID NO: 6;

(ii) a peptide having an amino acid sequence according to SEQ ID NO: 96;

(iii) a peptide having an amino acid sequence according to SEQ ID NO: 95;

(iv) a peptide having an amino acid sequence according to SEQ ID NO: 97; and

(v) a peptide having an amino acid sequence according to SEQ ID NO: 71.

36. The method of claim 29 , wherein said complex comprises an amino acid sequence according to SEQ ID NO: 89.

37. The method of claim 30 , wherein the cell penetrating peptide has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, or added without abrogating said peptide's cell penetrating ability.

38. The method of claim 30 , wherein the cell penetrating peptide has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein 0, 1, 2, 3, 4, or 5 amino acids have been substituted without abrogating said peptide's cell penetrating ability.

39. The method of claim 31 , wherein the cell penetrating peptide has an amino acid sequence comprising an amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13).

40. The method of claim 31 , wherein the cell penetrating peptide has an amino acid sequence consisting of an amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13).

41. The method of claim 34 , wherein the at least one TLR peptide agonist consists of an amino acid sequence according to SEQ ID NO: 15 or 71.

42. The method of claim 34 , wherein the at least one TLR peptide agonist comprises an amino acid sequence according to SEQ ID NO: 71.

43. The method of claim 36 , wherein said complex consists of an amino acid sequence according to SEQ ID NO: 89.

44. A method for preventing or treating colorectal cancer or initiating, enhancing or prolonging an anti-tumor-response in a subject in need thereof comprising administering to the subject:

(i) the complex of claim 1 ;

(ii) a nucleic acid encoding the complex;

(iii) a vector comprising the nucleic acid;

(iv) a host cell comprising the vector; or

(v) a cell loaded with the complex.