Immunogenic Compounds For Treatment Of Adrenal Cancer

Abstract

The present invention relates to antigen-based immunotherapy targeting interleukin 13 receptor alpha 2 (IL13RA2), BIRC5 and/or FOXM1 for treatment of adrenal cancers. In particular, the present invention provides the use of a (poly)peptide comprising an epitope of IL13RA2, BIRC5 and/or FOXM1 or a sequence variant thereof for treatment of an adrenal cancer. Moreover, the present invention also provides combinations of the (poly)peptide comprising an epitope of IL13RA2, BIRC5 and/or FOXM1 or a sequence variant thereof with (poly)peptides comprising other epitopes or sequence variants thereof for treatment of adrenal cancers.

Claims

1. A (poly)peptide comprising an epitope of IL13RA2 or a sequence variant thereof having at least 70% sequence identity for use in prevention and/or treatment of an adrenal cancer.

2. The (poly)peptide for use according to claim 1, wherein the adrenal cancer is malignant pheochromocytoma/paraganglioma (MPP).

3. The (poly)peptide for use according to claim 1 or 2, wherein the (poly)peptide does not bind to and/or inhibit IL-13.

4. The (poly)peptide for use according to any one of claims 1-3, wherein the (poly)peptide has a maximum length of 350 amino acids.

5. The (poly)peptide for use according to any one of claims 1-4, wherein the (poly)peptide consists of the epitope of IL13RA2 or the sequence variant thereof having at least 70% sequence identity.

6. The (poly)peptide for use according to any one of claims 1-5, wherein the epitope of IL13RA2 or the sequence variant thereof has a length of 8-12 amino acids, more preferably of 8-10 amino acids and most preferably of 9 or 10 amino acids.

7. The (poly)peptide for use according to any one of claims 1-6, wherein the epitope of IL13RA2 is an epitope of human IL13RA2.

8. The (poly)peptide for use according to any one of claims 1-7, wherein the (poly)peptide comprises a sequence variant of an epitope of IL13RA2 having at least 70% sequence identity.

9. The (poly)peptide for use according to claim 8, wherein the core sequence of the sequence variant of the IL13RA2 epitope is identical with the core sequence of the IL13RA2 epitope, with the core sequence consisting of all amino acids of the IL13RA2 epitope except the three most N-terminal and the three most C-terminal amino acids.

10. The (poly)peptide for use according to any one of claims 1-9, wherein the sequence variant of the IL13RA2 epitope is a microbiota sequence variant.

11. The (poly)peptide for use according to claim 10, wherein the microbiota sequence variant is a bacterial peptide.

12. The (poly)peptide for use according to any one of claims 1-11, wherein the (poly)peptide does not comprise the amino acid sequence as set forth in SEQ ID NO: 282.

13. The (poly)peptide for use according to any one of claims 1-12, wherein the (poly)peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs 279, 192, 31, 1-30, 32-191, 193-242, 267-274 and 335-344 (or a sequence variant thereof); preferably as set forth in any one of SEQ ID NOs 31, 192, 279, 64, 178, 212 and 267 (or a sequence variant thereof), more preferably as set forth in any one of SEQ ID NOs 31, 279, 64, 178, 212, 335 and 336 (or a sequence variant thereof).

14. The (poly)peptide for use according to any one of claims 1-13, wherein the (poly)peptide comprises an amino acid sequence as set forth in SEQ ID NO: 279, 31, or 192 (or a sequence variant thereof), preferably as set forth in SEQ ID NO: 279 or 31 (or a sequence variant thereof).

15. The (poly)peptide for use according any one of claims 1-14, wherein the (poly)peptide comprises an amino acid sequence as set forth in SEQ ID NO: 279 (or a sequence variant thereof).

16. The (poly)peptide for use according to any one of claims 1-15, wherein the epitope of IL13RA2 has an amino acid sequence as set forth in any one of SEQ ID NOs 243-265, 276-278 and 331-334 (or a sequence variant thereof).

17. A (poly)peptide comprising an epitope of BIRC5 or a sequence variant thereof having at least 70% sequence identity for use in prevention and/or treatment of an adrenal cancer.

18. The (poly)peptide for use according to claim 17, wherein the adrenal cancer is pheochromocytoma.

19. The (poly)peptide for use according to claim 17 or 18, wherein the (poly)peptide has a maximum length of 140 amino acids.

20. The (poly)peptide for use according to any one of claims 17-19, wherein the (poly)peptide consists of the epitope of BIRC5 or the sequence variant thereof having at least 70% sequence identity.

21. The (poly)peptide for use according to any one of claims 17-20, wherein the epitope of BIRC5 or the sequence variant thereof has a length of 8-12 amino acids, more preferably of 8-10 amino acids and most preferably of 9 or 10 amino acids.

22. The (poly)peptide for use according to any one of claims 17-21, wherein the epitope of BIRC5 is an epitope of human BIRC5.

23. The (poly)peptide for use according to any one of claims 17-22, wherein the (poly)peptide comprises a sequence variant of an epitope of BIRC5 having at least 70% sequence identity.

24. The (poly)peptide for use according to claim 23, wherein the core sequence of the sequence variant of the BIRC5 epitope is identical with the core sequence of the BIRC5 epitope, with the core sequence consisting of all amino acids of the BIRC5 epitope except the three most N-terminal and the three most C-terminal amino acids.

25. The (poly)peptide for use according to claim 23 or 24, wherein the sequence variant of the BIRC5 epitope is a microbiota sequence variant.

26. The (poly)peptide for use according to claim 25, wherein the microbiota sequence variant is a bacterial peptide.

27. The (poly)peptide for use according to any one of claims 17-26, wherein the (poly)peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs 287-289 (or a sequence variant thereof).

28. The (poly)peptide for use according any one of claims 17-27, wherein the (poly)peptide comprises an amino acid sequence as set forth in SEQ ID NO: 289 (or a sequence variant thereof).

29. The (poly)peptide for use according to any one of claims 17-28, wherein the epitope of BIRC5 has an amino acid sequence as set forth in SEQ ID NO: 286 (or a sequence variant thereof).

30. A (poly)peptide comprising an epitope of FOXM1 or a sequence variant thereof having at least 70% sequence identity for use in prevention and/or treatment of an adrenal cancer.

31. The (poly)peptide for use according to claim 30, wherein the adrenal cancer is pheochromocytoma.

32. The (poly)peptide for use according to claim 30 or 31, wherein the (poly)peptide has a maximum length of 750 amino acids.

33. The (poly)peptide for use according to any one of claims 30-32, wherein the (poly)peptide consists of the epitope of FOXM1 or the sequence variant thereof having at least 70% sequence identity.

34. The (poly)peptide for use according to any one of claims 30-33, wherein the epitope of FOXM1 or the sequence variant thereof has a length of 8-12 amino acids, more preferably of 8-10 amino acids and most preferably of 9 or 10 amino acids.

35. The (poly)peptide for use according to any one of claims 30-34, wherein the epitope of FOXM1 is an epitope of human FOXM1.

36. The (poly)peptide for use according to any one of claims 30-35, wherein the (poly)peptide comprises a sequence variant of an epitope of FOXM1 having at least 70% sequence identity.

37. The (poly)peptide for use according to claim 36, wherein the core sequence of the sequence variant of the FOXM1 epitope is identical with the core sequence of the FOXM1 epitope, with the core sequence consisting of all amino acids of the FOXM1 epitope except the three most N-terminal and the three most C-terminal amino acids.

38. The (poly)peptide for use according to claim 36 or 37, wherein the sequence variant of the FOXM1 epitope is a microbiota sequence variant.

39. The (poly)peptide for use according to claim 38, wherein the microbiota sequence variant is a bacterial peptide.

40. The (poly)peptide for use according to any one of claims 30-39, wherein the (poly)peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs 302-329 (or a sequence variant thereof).

41. The (poly)peptide for use according any one of claims 30-40, wherein the (poly)peptide comprises an amino acid sequence as set forth in SEQ ID NO: 312 (or a sequence variant thereof).

42. The (poly)peptide for use according to any one of claims 30-42, wherein the epitope of FOXM1 has an amino acid sequence as set forth in any one of SEQ ID NOs 291-301 (or a sequence variant thereof).

43. An immunogenic compound comprising the (poly)peptide as defined in any one of claims 1-42 for use in prevention and/or treatment of an adrenal cancer.

44. The immunogenic compound for use according to claim 43, wherein the (poly)peptide is linked to a carrier protein.

45. The immunogenic compound for use according to claim 43 or 44 comprising or consisting of a (poly)peptide of formula (I):
PepNt-CORE-PepCt  (I), wherein: “PepNt” consists of a polypeptide having an amino acid length varying from 0 to 500 amino acid residues and located at the N-terminal end of the polypeptide of formula (I); CORE consists of the (poly)peptide as defined in any one of claims 1-42; and “PepCt” consists of a polypeptide having an amino acid length varying from 0 to 500 amino acid residues and located at the C-terminal end of the polypeptide of formula (I).

46. A nanoparticle loaded with a. at least one (poly)peptide as defined in any one of claims 1-42, or b. at least one immunogenic compound as defined in any one of claims 43-45; and, optionally, with an adjuvant for use in prevention and/or treatment of an adrenal cancer.

47. A cell loaded with the (poly)peptide as defined in any one of claims 1-42 or the immunogenic compound as defined in any one of claims 43-45 for use in prevention and/or treatment of an adrenal cancer.

48. The cell for use according to claim 47, wherein said cell is an antigen presenting cell, preferably a dendritic cell.

49. A nucleic acid encoding the (poly)peptide as defined in any one of claims 1-42 or the immunogenic compound as defined in any one of claims 43-45 for use in prevention and/or treatment of an adrenal cancer.

50. The nucleic acid for use according to claim 49, wherein the nucleic acid is a DNA molecule or an RNA molecule; preferably selected from genomic DNA; cDNA; siRNA; rRNA; mRNA; antisense DNA; antisense RNA; ribozyme; complimentary RNA and/or DNA sequences; RNA and/or DNA sequences with or without expression elements, regulatory elements, and/or promoters; a vector; and combinations thereof.

51. A host cell comprising the nucleic acid as defined in claim 49 or 50 for use in prevention and/or treatment of an adrenal cancer, wherein the nucleic acid is preferably a vector.

52. The host cell for use according to claim 51, wherein the host cell is a bacterial cell, preferably a gut bacterial cell.

53. A cytotoxic T lymphocyte (CTL) specific for the (poly)peptide according to any one of claims 1-42 for use in prevention and/or treatment of an adrenal cancer.

54. A pharmaceutical composition comprising the (poly)peptide as defined in any one of claims 1-42; the immunogenic compound as defined in any one of claims 43-45; the nanoparticle as defined in claim 46; the cell as defined in claim 47 or 48; the nucleic acid as defined in claim 49 or 50; and/or the host cell as defined in claim 51 or 52; and/or the cytotoxic T lymphocyte as defined in claim 53; and, optionally, one or more pharmaceutically acceptable excipients for use in prevention and/or treatment of an adrenal cancer.

55. The pharmaceutical composition for use according to claim 54 further comprising one or more immunostimulatory agents.

56. The pharmaceutical composition for use according to claim 55, wherein the said immunostimulatory agent is selected in a group consisting of immuno-adjuvants and antigen-presenting cells.

57. The pharmaceutical composition for use according to claim 56, wherein the antigen-presenting cells are dendritic cells.

58. A pharmaceutical composition comprising the (poly)peptide as defined in any one of claims 1-16 and, optionally, one or more pharmaceutically acceptable excipients for use in prevention and/or treatment of an adrenal cancer.

59. The pharmaceutical composition for use according to any one of claims 54-58, wherein the composition comprises (i) a (poly)peptide as defined in any one of claims 1-16; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

60. The pharmaceutical composition for use according to any one of claims 54-59, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279.

61. The pharmaceutical composition for use according to claim 58, wherein the composition comprises the (poly)peptide as defined in any one of claims 17-29.

62. The pharmaceutical composition for use according to claim 58 or 61, wherein the composition comprises the (poly)peptide as defined in any one of claims 30-42.

63. The pharmaceutical composition for use according to any one of claims 59-62, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 17-29; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

64. The pharmaceutical composition for use according to any one of claims 59-63, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 289 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289.

65. The pharmaceutical composition for use according to any one of claims 59-64, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 30-42; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

66. The pharmaceutical composition for use according to any one of claims 59-65, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 312 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312.

67. The pharmaceutical composition for use according to any one of claims 54-57, wherein the composition comprises (i) a (poly)peptide as defined in any one of claims 17-29; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

68. The pharmaceutical composition for use according to any one of claims 54-57 and 67, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 289 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289.

69. The pharmaceutical composition for use according to claim 67 or 68, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 1-16; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

70. The pharmaceutical composition for use according to any one of claims 67-69, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279.

71. The pharmaceutical composition for use according to any one of claims 67-70, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 30-42; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

72. The pharmaceutical composition for use according to any one of claims 67-71, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 312 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312.

73. The pharmaceutical composition for use according to any one of claims 54-57, wherein the composition comprises (i) a (poly)peptide as defined in any one of claims 30-42; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

74. The pharmaceutical composition for use according to any one of claims 54-57 and 73, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 312 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312.

75. The pharmaceutical composition for use according to claim 73 or 74, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 1-16; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

76. The pharmaceutical composition for use according to any one of claims 73-75, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279.

77. The pharmaceutical composition for use according to any one of claims 73-76, wherein the composition further comprises (i) a (poly)peptide as defined in any one of claims 17-29; (ii) an immunogenic compound as defined in any one of claims 43-45 comprising the (poly)peptide as defined in (i); (iii) a nanoparticle as defined in claim 46 loaded with the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); (iv) a nucleic acid as defined in claim 49 or 50 comprising a polynucleotide encoding the (poly)peptide as defined in (i) or the immunogenic compound as defined in (ii); or (v) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in (i).

78. The pharmaceutical composition for use according to any one of claims 73-77, wherein the composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in 289 or with an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; (iv) a nucleic acid comprising a polynucleotide encoding a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289 or an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289.

79. The pharmaceutical composition for use according to any one of claims 54-78, wherein the pharmaceutical composition comprises (i) a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279, a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289, and a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (ii) an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279, an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289, and an immunogenic compound comprising a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iii) a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279, a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289, and a nanoparticle loaded with a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312; (iv) a nucleic acid comprising a polynucleotide encoding an (poly)peptide having an amino acid sequence as set forth in SEQ ID NO: 279, a nucleic acid comprising a polynucleotide encoding an (poly)peptide having an amino acid sequence as set forth in SEQ ID NO: 289, and a nucleic acid comprising a polynucleotide encoding an (poly)peptide having an amino acid sequence as set forth in SEQ ID NO: 312; or (v) a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279, a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289, and a cytotoxic T lymphocyte (CTL) specific for the (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312.

80. The pharmaceutical composition for use according to any one of claims 54-79, wherein the pharmaceutical composition comprises a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 279, a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 289, and a (poly)peptide comprising an amino acid sequence as set forth in SEQ ID NO: 312.

81. A combination of (i) a (poly)peptide as defined in any one of claims 1-16, and (ii) a (poly)peptide as defined in any one of claims 17-29, for use in prevention and/or treatment of an adrenal cancer.

82. The combination of claim 81, further comprising a (poly)peptide as defined in any one of claims 30-42.

83. A combination of (i) a (poly)peptide as defined in any one of claims 1-16, and (ii) a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

84. The combination of claim 83, further comprising a (poly)peptide as defined in any one of claims 17-29.

85. A combination of (i) a (poly)peptide as defined in any one of claims 17-29, and (ii) a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

86. The combination of claim 85, further comprising a (poly)peptide as defined in any one of claims 1-16.

87. A combination of (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29, for use in prevention and/or treatment of an adrenal cancer.

88. The combination of claim 87, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42.

89. A combination of (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

90. The combination of claim 89, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29.

91. A combination of (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

92. The combination of claim 91, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16.

93. A combination of (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29, for use in prevention and/or treatment of an adrenal cancer.

94. The combination of claim 93, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42.

95. A combination of (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

96. The combination of claim 95, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29.

97. A combination of (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

98. The combination of claim 97, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16.

99. A combination of (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29, for use in prevention and/or treatment of an adrenal cancer.

100. The combination of claim 99, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42.

101. A combination of (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

102. The combination of claim 101, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29.

103. A combination of (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

104. The combination of claim 103, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16.

105. A combination of (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29, for use in prevention and/or treatment of an adrenal cancer.

106. The combination of claim 105, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42.

107. A combination of (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

108. The combination of claim 105, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29.

109. A combination of (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42, for use in prevention and/or treatment of an adrenal cancer.

110. The combination of claim 105, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16.

111. The combination for use according to any one of claims 81-110, wherein components (i) and (ii) are comprised in distinct compositions.

112. The combination for use according to any one of claims 81-111, wherein components (i) and (ii) are comprised in the same composition.

113. The combination for use according to any one of claims 81-112, wherein components (i) and (ii) are administered via distinct routes of administration.

114. The combination for use according to any one of claims 81-113, wherein components (i) and (ii) are administered via the same route of administration.

115. The combination for use according to any one of claims 81-111, 113 and 114 wherein components (i) and (ii) are administered consecutively.

116. The combination for use according to any one of claims 81-115 wherein components (i) and (ii) are administered at about the same time.

117. A kit comprising the (poly)peptide as defined in any one of claims 1-42; the immunogenic compound as defined in any one of claims 43-45; the nanoparticle as defined in claim 46; the cell as defined in claim 47 or 48; the nucleic acid as defined in claim 49 or 50; the host cell as defined in claim 51 or 52; the cytotoxic T lymphocyte as defined in claim 53; and/or the pharmaceutical composition as defined in claim 54-80 for use in prevention and/or treatment of an adrenal cancer.

118. The kit for use according to claim 117 further comprising a package insert or instruction leaflet with directions to prevent and/or to treat an adrenal cancer by using the (poly)peptide as defined in any one of claims 1-42; the immunogenic compound as defined in any one of claims 43-45; the nanoparticle as defined in claim 46; the cell as defined in claim 47 or 48; the nucleic acid as defined in claim 49 or 50; the host cell as defined in claim 51 or 52; the cytotoxic T lymphocyte as defined in claim 53; and/or the pharmaceutical composition as defined in any one of claims 54-80.

119. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a (poly)peptide as defined in any one of claims 1-16, and (ii) a (poly)peptide as defined in any one of claims 17-29.

120. The kit of claim 119, further comprising a (poly)peptide as defined in any one of claims 30-42.

121. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a (poly)peptide as defined in any one of claims 1-16, and (ii) a (poly)peptide as defined in any one of claims 30-42.

122. The kit of claim 121, further comprising a (poly)peptide as defined in any one of claims 17-29.

123. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a (poly)peptide as defined in any one of claims 17-29, and (ii) a (poly)peptide as defined in any one of claims 30-42.

124. The kit of claim 123, further comprising a (poly)peptide as defined in any one of claims 1-16.

125. The kit for use according to claim 117 or 118, wherein the kit comprises (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29.

126. The kit of claim 125, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42.

127. The kit for use according to claim 117 or 118, wherein the kit comprises (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42.

128. The kit of claim 127, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29.

129. The kit for use according to claim 117 or 118, wherein the kit comprises (i) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 17-29, and (ii) an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 30-42.

130. The kit of claim 129, further comprising an immunogenic compound as defined in any one of claims 43-45 comprising a (poly)peptide as defined in any one of claims 1-16.

131. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29.

132. The kit of claim 131, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42.

133. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42.

134. The kit of claim 133, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29.

135. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 17-29, and (ii) a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 30-42.

136. The kit of claim 135, further comprising a nanoparticle as defined in claim 46 comprising a (poly)peptide as defined in any one of claims 1-16.

137. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29.

138. The kit of claim 137, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42.

139. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42.

140. The kit of claim 139, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29.

141. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 17-29, and (ii) a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 30-42.

142. The kit of claim 141, further comprising a nucleic acid as defined in claim 49 or 50 encoding a (poly)peptide as defined in any one of claims 1-16.

143. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29.

144. The kit of claim 143, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42.

145. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42.

146. The kit of claim 145, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29.

147. The kit for use according to claim 117 or 118, wherein the kit comprises (i) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 17-29, and (ii) a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 30-42.

148. The kit of claim 143, further comprising a cytotoxic T lymphocyte (CTL) as defined in claim 53 specific for the (poly)peptide as defined in any one of claims 1-16.

149. A method for ameliorating, reducing, preventing and/or treating an adrenal cancer or for reducing or preventing its recurrence in a subject comprising administering to the subject the (poly)peptide as defined in any one of claims 1-42; the immunogenic compound as defined in any one of claims 43-45; the nanoparticle as defined in claim 46; the cell as defined in claim 47 or 48; the nucleic acid as defined in claim 49 or 50; the host cell as defined in claim 51 or 52; the cytotoxic T lymphocyte (CTL) as defined in claim 53; the pharmaceutical composition as defined in any one of claims 54-80; and/or the combination as defined in any one of claims 81-112.

150. The method according to claim 149, wherein the adrenal cancer is pheochromocytoma.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0985] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.

[0986] FIG. 1 shows the general protocol for the validation of the proof-of-concept of an antigen-based immunotherapy targeting IL13RA2.

[0987] FIG. 2: shows a schematic view of the immunization scheme. d: day.

[0988] FIG. 3: shows ELISPOT-IFNγ results for group 1 (IL13RA2-B) and group 2 (IL13RA2-A). The peptide used for vaccination (in between brackets under each group) and the stimulus used in the ELISPOT culture (X-axis) are indicated on the graphs. (A) Number of specific ELISPOT-IFNγ spots (medium condition subtracted). Each dot represents the average value for one individual/mouse from the corresponding condition quadruplicate. (B) For each individual, the level of specific ELISPOT-IFNγ response is compared to the ConA stimulation (value: 100%). Statistical analysis: paired t-test for intra-group comparison and unpaired t-test for inter-group comparison; * p<0.05.

[0989] FIG. 4: shows the results of Example 3.

[0990] FIG. 5: shows the results of Example 4.

[0991] FIG. 6: shows for Example 6 ELISPOT results for HLA-A2 transgenic mice vaccinated with the peptide IL13R2A-L as indicated in the figure and cross-reactivity with the human corresponding peptide IL13RA2-H. For each group the normalized number of spot-forming cells (SFC) is shown.

[0992] FIG. 7 shows for Example 7 in vitro affinity for peptides IL13RA2-B and IL13RA2-L in comparison to the corresponding human peptide IL13RA2-H and to the comparative peptide 1A9V.

[0993] FIG. 8: shows for Example 8 in vitro affinity for antigenic peptides IL13RA2-B and IL13RA2-L in comparison to the corresponding human IL13RA2 epitope IL13RA2-H.

[0994] FIG. 9: shows for Example 8 in vitro affinity for antigenic peptides BIRC5-B1, BIRC5-B2 and BIRC5-B3 in comparison to the corresponding human BIRC5 epitope BIRC5-H.

[0995] FIG. 10: shows for Example 8 in vitro affinity for (A) antigenic peptide FOXM1-B in comparison to the corresponding human FOXM1 epitope FOXM1-H and (B) antigenic peptide FOXM1-B2 in comparison to the corresponding human FOXM1 epitope FOXM1-H2.

[0996] FIG. 11: shows for Example 9 ELISPOT results for mice vaccinated with the antigenic peptides as indicated in the figure (BIRC5-H, BIRC5-B1, BIRC5-B2, BIRC5-B3, FOXM1-H2, FOXM1-B2, IL13RA2-H, IL13RA2-B. For each group the normalized number of spot-forming cells (SFC) is shown. Each dot represents the average value for one individual/mouse.

[0997] FIG. 12: shows for Example 10 ELISPOT results for HLA-A2 transgenic mice vaccinated with the antigenic peptide BIRC5-B1 as indicated in the figure and cross-reactivity with the human corresponding peptide BIRC5-H. For each group the normalized number of spot-forming cells (SFC) is shown.

[0998] FIG. 13: shows for Example 11 ELISPOT results for HLA-A2 transgenic mice vaccinated with the antigenic peptide FOXM1-B2 as indicated in the figure and cross-reactivity with the human corresponding peptide FOXM1-H2. For each group the normalized number of spot-forming cells (SFC) is shown.

[0999] FIG. 14: shows for Example 12 in vitro affinity for the antigenic peptides BIRC5-B1 in comparison to the corresponding human BIRC5 epitope BIRC5-H, to the comparative peptide 2M and to the positive control HIV.

[1000] FIG. 15: shows for Example 14 the detection of IL13RA2-L, BIRC5-B1 and FOXM1-B2 peptide-specific CD8+ T cells detected in peripheral blood from HLA-A2 positive healthy donors.

[1001] FIG. 16: shows for Example 14 the cytotoxic capacity of the of IL13RA2-L peptide-specific human T cells clones expanded in vitro by microbiome derived peptide stimulation. IL13RA2-L peptide-specific T cells have the ability to kill T2 cells loaded with bacterial peptide.

EXAMPLES

[1002] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

[1003] Examples 1 and 2 are both linked to the general protocol described in FIG. 1.

Example 1: Identification of a Candidate (Poly)Peptide Having Superior Affinity to the HLA-A*0201 Allele

[1004] This Example provides evidence that the (poly)peptide of sequence SEQ ID NO: 31 («FLPFGFILV» also referred herein as IL13RA2-B) has high affinity to the HLA-A*0201 allele, whereas the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID No 263, also referred herein as IL13RA2-H) has low affinity.

[1005] A. Materials and Methods

[1006] A1. Measuring the Affinity of the Peptide to T2 Cell Line.

[1007] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.

[1008] T2 cells (2.Math.10.sup.5 cells per well) are incubated with decreasing concentrations of peptides from 100 μM to 0.1 μM in a AIMV medium supplemented with 100 ng/μl of human P2m at 37° C. for 16 hours. Cells are then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).

[1009] The analysis is achieved by FACS (Guava Easy Cyte).

[1010] For each peptide concentration, the geometric mean of the labelling associated with the peptide of interest is subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM. The relative affinity is then determined as follows:

relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.

[1011] A2. Solubilisation of Peptides

[1012] Each peptide is solubilized by taking into account the amino acid composition. For peptides which do not include any Cystein, Methionin, or Tryptophane, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are resuspended in water or PBS pH7.4.

[1013] B. Results

[1014] For T2 Cells: Mean fluorescence intensity for variable peptidic concentrations: Regarding the couple IL13RA2 peptides (IL13RA2-H and IL13RA2-B), it appears that the human peptide does not bind to the HLA-A*0201 contrarily to the candidate peptide IL13RA2-B, which binds strongly to HLA-A*0201:112.03 vs 18.64 at 100 μM; 40.77 vs 11.61 at 10 μM; 12.18 vs 9.41 at 1 μM; 9.9 vs 7.46 at 0.1 μM.

[1015] Also, IL13RA2-B at 4.4 μM induces 20% of expression of the HLA-A*0201 (vs 100 μM for IL13RA2-H).

[1016] Similar results were obtained from a second distinct T2 cell clone.

Example 2: Vaccination on Mice with the Candidate (Poly)Peptide Induces Improved T Cell Responses in a ELISPOT-IFNγ Assay

[1017] A. Materials and Methods

[1018] A.1 Mouse Model

[1019] The features of the model used in this project are shown in Table 6.

TABLE-US-00011 TABLE 6 Model features. Mouse Model C57BL/6J B2m.sup.tm1UncIAb.sup.−/−Tg(HLA-DRA HLA- DRB1*0301).sup.#GjhTg(HLA-A/H2-D/B2M).sup.1Bpe Acronym β/A2/DR3 Description Immunocompetent, no mouse class I and class II MHC Housing SOPF conditions (ABSL3) Number of mice 24 adults (>8 weeks of age)

[1020] A.2. Immunization Scheme.

[1021] The immunization scheme is shown in FIG. 2. Briefly, 14 β/A2/DR3 mice were assigned randomly (based on mouse sex and age) to two experimental groups, each immunized with a specific vaccination peptide (vacc-pAg) combined to a common helper peptide (h-pAg) (as outlined in Table 7 below). The vacc-pAg were compared in couples (group 1 vs. group 2). Thereby, both native and optimized versions of a single peptide were compared in each wave.

TABLE-US-00012 TABLE 7 Experimental group composition. h-pAg: ‘helper’ peptide; vacc-pAg: vaccination peptide. The number of boost injections is indicated into brackets. Peptide Helper Animal Group (vacc-pAg) (h-pAg) Prime Boost number 1 IL13RA2-B HHD-DR3 + + (1X) 6 (100 μg) (150 μg) 2 IL13RA2-H HHD-DR3 + + (1X) 6 (100 μg) (150 μg)

[1022] The peptides were provided as follows: [1023] couples of vacc-pAg: IL13RA2-H and IL13RA2-B; all produced and provided at a 4 mg/ml (4 mM) concentration; [1024] h-pAg: HHD-DR3; provided lyophilized (50.6 mg; Eurogentec batch 1611166) and re-suspended in pure distilled water at a 10 mg/mL concentration;

[1025] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 μL of an oil-based emulsion that contained: [1026] 100 μg of vacc-pAg (25 μL of 4 mg/mL stock per mouse); [1027] 150 μg of h-pAg (15 μL of 10 mg/mL stock per mouse); [1028] 10 μL of PBS to reach a total volume of 50 μL (per mouse); [1029] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 μL per mouse).

[1030] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.

[1031] A.3. Mouse Analysis

[1032] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 μm-filtering and Ficoll density gradient purification.

[1033] The splenocytes were immediately used in an ELISPOT-IFNγ assay (Table 8). Experimental conditions were repeated in quadruplets, using 2*105 total splenocytes per well, and were cultured in presence of vacc-pAg (10 μM), Concanavalin A (ConA, 2.5 μg/mL) or medium-only to assess for their capacity to secrete IFNγ. The commercial ELISPOT-IFNγ kit (Diaclone Kit Mujrine IFNγ ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 16 h of incubation.

TABLE-US-00013 TABLE 8 Setup of the ELISPOT-IFNγ assay. Group Stimulus Wells Animal Total 1 IL13RA2-B (10 μM) 4 6 24 IL13RA2-H (10 μM) 4 6 24 ConA (2.5 μg/ml) 4 6 24 Medium 4 6 24 2 IL13RA2-B (10 μM) 4 6 24 IL13RA2-H (10 μM) 4 6 24 ConA (2.5 μg/ml) 4 6 24 Medium 4 6 24

[1034] Spots were counted on a Grand ImmunoSpot® S6 Ultimate UV Image Analyzer interfaced to the ImmunoSpot 5.4 software (CTL-Europe). Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).

[1035] The cell suspensions were also analyzed by flow cytometry, for T cell counts normalization. The monoclonal antibody cocktail (data not shown) was applied on the purified leucocytes in presence of Fc-block reagents targeting murine (1:10 diluted ‘anti-mCD16/CD32 CF11 clone’—internal source) Fc receptors. Incubations were performed in 96-well plates, in the dark and at 4° C. for 15-20 minutes. The cells were washed by centrifugation after staining to remove the excess of monoclonal antibody cocktail, and were re-suspended in PBS for data acquisition.

[1036] All data acquisitions were performed with an LSR-II Fortessa flow cytometer interfaced with the FACS-Diva software (BD Bioscience). The analysis of the data was performed using the FlowJo-9 software (TreeStar Inc.) using a gating strategy (not shown).

TABLE-US-00014 TABLE 9 FACS panel EXP-1. Target Label Clone Provider Dilution mCD3εγ FITC 145-2C11 Biolegend 1/100 mCD4 PE RM4-5 Biolegend 1/100 mCD8α APC 53-6,7 Biolegend 1/100

[1037] B. Results

[1038] A total of 14 β/A2/DR3 mice were used for this experiment (see Table 10). At time of sacrifice, the spleen T cell population was analysed by flow cytometry, showing that the large majority belonged to the CD4+ T cell subset.

TABLE-US-00015 TABLE 10 Individual mouse features (groups 1 & 2). Mouse Age.sup.a Group T cells.sup.b T4.sup.c T8.sup.c ID Sex (wks) (pAg) (%) (%) (%) Note.sup.d 826 M 14 1 (IL13RA2-B) 18.6 72.0 13.7 P1/2 827 M 14 1 (IL13RA2-B) 21.1 82.5 8.7 P1/2 828 M 14 1 (IL13RA2-B) 20.9 78.4 8.6 P1/2 829 F 15 1 (IL13RA2-B) 23.8 67.0 17.5 P1/2 830 F 15 1 (IL13RA2-B) 29.2 73.3 12.5 P1/2 831 F 15 1 (IL13RA2-B) N.A. N.A. N.A. ID tag lost (excluded) 17 M 9 1 (IL13RA2-B) 8.3 83.7 10.4 P5 832 F 15 2 (IL13RA2-H) 28.3 83.4 5.7 P1/2 833 F 15 2 (IL13RA2-H) N.A. N.A. N.A. ID tag lost (excluded) 834 F 15 2 (IL13RA2-H) 27.5 79.7 7.2 P1/2 835 M 13 2 (IL13RA2-H) 33.8 84.2 8.5 P1/2 836 M 13 2 (IL13RA2-H) 31.4 84.7 6.3 P1/2 837 M 15 2 (IL13RA2-H) 30.8 83.4 5.4 P1/2 18 M 9 2 (IL13RA2-H) 11.2 85.9 9.2 P5 Each mouse is identified by a unique ear tag ID number. .sup.aage at onset of the vaccination protocol (in weeks); .sup.bpercentage of T cells in total leukocytes; .sup.cpercentage of CD4+ or CD8+ T cells in total T cells; .sup.dplate (P) number.

[1039] After plating and incubation with the appropriate stimuli, the IFNγ-producing cells were revealed and counted. The data were then normalized as a number of specific spots (the average counts obtained in the ‘medium only’ condition being subtracted) per 10.sup.6 total T cells.

[1040] The individual average values (obtained from the quadruplicates) were next used to plot the group average values (see FIG. 3A). As the functional capacity of T cells might vary from individual to individual, the data were also expressed as the percentage of the ConA response per individual (see FIG. 3B).

[1041] Overall, vaccination with the IL13RA2-B pAg (candidate) peptide induced improved T cell responses in the ELISPOT-IFNγ assay, as compared to IL13RA2-H pA (reference human)-vaccinated animals (group 2). For group 1 (IL13RA2-B), ex vivo restimulation with the IL13RA2-B pAg promoted higher response than with the IL13RA2-H pAg. It was not the case for group 2 (IL13RA2-H). The percentage of ConA-induced response (mean+/−SEM) for each condition was as follows: [1042] Group 1 (IL13RA2-B)/IL13RA2-B pAg: 56.3%+/−18.1 [1043] Group 1 (IL13RA2-B)/IL13RA2-H pAg: 32.3%+/−11.8 [1044] Group 2 (IL13RA2-H)/IL13RA2-B pAg: 2.0%+/−0.8 [1045] Group 2 (IL13RA2-H)/IL13RA2-H pAg: 1.1%+/−0.8

[1046] Accordingly, those results provide experimental evidence that antigen-based immunotherapy targeting IL13RA2 is able to improve T cell response in vivo and that the IL13RA2-B candidate peptide (SEQ ID NO: 31) is particularly efficient for that purpose.

Example 3: Candidate (Poly)Peptides Having Superior Affinity to the HLA-A*0201 Allele

[1047] This Example provides further evidence that the (poly)peptide of sequence SEQ ID NO: 31 («FLPFGFILV», also referred to herein as IL13RA2-B) has high affinity to the HLA-A*0201 allele, whereas the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID No 263, also referred to herein as IL13RA2-H) has low affinity. Moreover, this Example provides evidence that the (poly)peptide of sequence SEQ ID NO: 192 («YLYTFLIST», also referred to herein as IL13RA2-B2) has high affinity to the HLA-A*0201 allele, whereas the corresponding reference human peptide derived from IL13RA2 («CLYTFLIST», SEQ ID NO: 245, also referred to herein as IL13RA2-H2) has low affinity.

[1048] A. Materials and Methods

[1049] A1. Measuring the Affinity of the Peptide to T2 Cell Line.

[1050] The experimental protocol was similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides was achieved with the human tumor cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.

[1051] T2 cells (2.Math.10.sup.5 cells per well) were incubated with decreasing concentrations of peptides from 100 μM to 0.1 μM in a AIMV medium supplemented with 100 ng/μl of human P2m at 37° C. for 16 hours. Cells were then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).

[1052] The analysis was achieved by FACS (Guava Easy Cyte).

[1053] For each peptide concentration, the geometric mean of the labelling associated with the peptide of interest was subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM. The relative affinity is then determined as follows:

relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.

[1054] A2. Solubilisation of Peptides

[1055] Each peptide was solubilized by taking into account the amino acid composition. For peptides which do not include any Cystein, Methionin, or Tryptophane, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are resuspended in water or PBS pH7.4.

[1056] B. Results

[1057] Results are shown in FIG. 4. Regarding the two couples of IL13RA2 peptides ((i) IL13RA2-H and IL13RA2-B, and (ii) IL13RA2-H2 and IL13RA2-B2), the human peptides do not bind to or show much lower affinity to HLA-A*0201, whereas the candidate peptides IL13RA2-B and IL13RA2-B2, bind strongly to HLA-A*0201. Moreover, both candidate peptides bind to HLA-A*0201 with higher affinity than the peptide “1A9V” (as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891). Reference peptide HIV pol 589-597 (“HIV”) served as positive control.

[1058] Similar results were obtained from a second distinct T2 cell clone.

Example 4: Candidate (Poly)Peptide Provides In Vitro Cytotoxicity Against Cells Expressing IL13RA2

[1059] This Example provides evidence that the (poly)peptide of sequence SEQ ID NO: 31 («FLPFGFILV», also referred to herein as IL13RA2-B) provides in vitro cytotoxicity against U87 cells, which express IL13RA2. In contrast, the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID No 263, also referred to herein as IL13RA2-H) does not provide in vitro cytotoxicity against U87 cells.

[1060] Methods:

[1061] Briefly, CD8 T cells from mice immunized with IL13RA2-H or IL13RA2-B were used. These cells were obtained after sorting of splenocyte from immunized mice and were placed on top of U87 cells (expressing IL13RA2).

[1062] In more detail, CD3+ T cells were purified from splenocytes of HHD mice immunized with IL13RA2-H (WLPFGFILI, SEQ ID No 263) or IL13RA2-B (FLPFGFILV). To this end, B6 β2m.sup.ko HHD/DR3 mice were injected s.c. at tail base with 100 μL of an oil-based emulsion containing vaccination peptide plus helper peptide plus CFA (complete Freund's adjuvant), at day 0 and day 14. On d21, i.e. seven days after the boost injection, the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ. CD3+ purification was performed using the mouse total T cells isolation kit from Miltenyi biotec using the recommended procedure. Efficient purification of cells and viability was validated by cytometry using appropriate marker for viability, CD8, CD4, CD3, and CD45.

[1063] U87-MG cells were seeded at 6×10.sup.5 cells/well in flat-bottomed 24-well culture plates and incubated for 24 h at 37° C. in DMEM (Dulbecco's Modified Eagle Medium) containing 10% of FCS (fetal calf serum) and antibiotics. After 24 hours, culture media were removed and replaced with media containing purified T CD3+ cells. The following ratios of T cells vs. U87-MG cells were used: 1/0.5, 1/1 and 1/5.

[1064] 72 hours after co-culture of U87-MG cells and CD3+ T cells, all cells from the wells were harvested and specific U87-MG cell death was evaluated after immunostaining of CD45 negative cells with DAPI and fluorescent annexin V followed by cytometry analysis.

[1065] Results:

[1066] Results are shown in FIG. 5. In general, U87 cell lysis was observed after treatment with IL13RA2-B, but not with IL13RA2-H.

Example 5: Candidate (Poly)Peptide has Superior Affinity to the HLA-A*0201 Allele

[1067] This Example provides evidence that the (poly)peptide of sequence SEQ ID NO: 31 («FLPFGFILV», also referred to herein as IL13RA2-B) has higher affinity to the HLA-A*0201 allele than other sequence variants of the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID No 263, also referred to herein as IL13RA2-H). In this experiment, the (poly)peptide of sequence SEQ ID NO: 31 was compared to [1068] the peptide “1A9V” (SEQ ID NO: 282), as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891, in which the tryptophan at position 1 of SEQ ID No 263 was substituted by alanine (1A) and the isoleucine at position 9 of SEQ ID No 263 was substituted by valine (9V); [1069] peptide “119A”, wherein the tryptophan at position 1 of SEQ ID No 263 was substituted by isoleucine (11) and the isoleucine at position 9 of SEQ ID No 263 was substituted by alanine (9A); and [1070] peptide “1F9M”, wherein the tryptophan at position 1 of SEQ ID No 263 was substituted by phenylalanine (1F) and the isoleucine at position 9 of SEQ ID No 263 was substituted by methionine (9M).

[1071] A. Materials and Methods

[1072] The experimental protocol, materials and methods correspond to those outlined in Example 3, with the only difference that the above mentioned (poly)peptides were used.

[1073] B. Results

[1074] The following in vitro binding affinities were obtained:

TABLE-US-00016 TABLE 11 Peptide In vitro binding affinity IL13RA2-B (SEQ ID NO: 31) 0.49 1A9V 3.06 1I9A 2.22 1F9M 2.62

[1075] Accordingly, the (poly)peptide according to the present invention (IL13RA2-B (SEQ ID NO: 31)) showed considerably higher binding affinity to HLA-A*0201 than all other peptides tested, whereas the peptide “1A9V”, as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891, showed the lowest affinity of the peptides tested.

Example 6: Immunogenicity of IL13R2A-L in HLA-A2 Transgenic Mice and Cross-Reactivity with the Corresponding Human Peptide

[1076] A. Materials and Methods

[1077] The (poly)peptide of the present invention IL13RA2-L (SEQ ID NO: 279) and the corresponding human reference peptide IL13RA2-H (SEQ ID NO: 263) were tested in distinct groups of male and female HHD DR3 mice expressing human HLA-A2 and HLA-DR3 MHC and lacking the murine H-2 class I and class II MHCs. Groups of 5 mice (male and female) were subcutaneously injected on days 0 and 14 with 100 μg of IL13RA2-L (SEQ ID NO: 279) or IL13RA2-H (SEQ ID NO: 263), 150 μg of helper peptide (DR3) and IFA. On day 21, the mice were euthanized and splenocytes were prepared and stimulated in vitro with IL13RA2-L or the human corresponding peptide IL13RA2-H to assess their capacity to secrete IFN- as assessed by ELISpot. Concanavalin A (ConA) was used as a positive control.

[1078] B. Results

[1079] The number of spot forming cells (SFC) (normalized to the number of CD8 cells) are depicted in FIG. 6. Results are shown for mice immunized with IL13RA2-L. The results show that immunisation of mice with IL13RA2-L allows to induce T-cells that are able to react strongly after challenge with either IL13RA2-L or the human corresponding peptide. Thus, IL13RA2-L is strongly immunogenic and is able to drive an effective immune response against the corresponding human peptide. As expected, the immunisation of mice with the human corresponding peptide IL13RA2-H does not induce an immune response after challenge with either IL13RA2-L or the human corresponding peptide IL13RA2-H (data not shown).

[1080] These results were confirmed in HHD DR1 mice expressing human HLA-A2 and HLA-DR1 MHC and lacking the murine H-2 class I and class II MHCs (groups of 5 mice).

Example 7: IL13RA2-L has Superior Affinity to the HLA-A*0201 Allele

[1081] This Example provides evidence that the (poly)peptide of the invention as set forth in SEQ ID NO: 279 (also referred to herein as IL13RA2-L) has a similarly high affinity to the HLA-A*0201 allele as the (poly)peptide of the invention as set forth in SEQ ID NO: 31 (FLPFGFILV, also referred to herein as IL13RA2-B)—and a higher affinity than the corresponding reference human peptide derived from IL13RA2 (IL13RA2-H, WLPFGFILI, SEQ ID NO: 263) and other sequence variants thereof. In this experiment, the (poly)peptide of sequence SEQ ID NO: 279 (IL13RA2-L) was compared to [1082] the comparative peptide “1A9V”, as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891, in which the tryptophan at position 1 of SEQ ID NO: 263 was substituted by alanine (1A) and the isoleucine at position 9 of SEQ ID NO: 263 was substituted by valine (9V); [1083] the (poly)peptide of the invention as set forth in SEQ ID NO: 31 (IL13RA2-B); [1084] the corresponding reference human peptide IL13RA2-H (SEQ ID NO: 263); and [1085] a positive control (HIV).

[1086] A. Materials and Methods

[1087] The experimental protocol, materials and methods correspond to those outlined in Example 2, with the only difference that the above mentioned (poly)peptides were used.

[1088] B. Results

[1089] The following in vitro binding affinities were obtained:

TABLE-US-00017 TABLE 12 Concentration of peptide that In vitro SEQ induces 20% of HLA-A2 expression binding Peptide ID NO (μM) affinity IL13RA2-H 263 ND ND IL13RA2-B 31 2.9 0.3 IL13RA2-L 279 3.2 0.3 1A9V 282 36.5 3.6

[1090] Accordingly, the (poly)peptides according to the present invention (IL13RA2-B; SEQ ID NO: 31 and IL13RA2-L; SEQ ID NO: 279) showed considerably higher binding affinity to HLA-A*0201 than the corresponding human peptide (IL13RA2-H) and the comparative peptide “1A9V”, as described by Eguchi Junichi et al., 2006, Identification of interleukin-13 receptor alpha 2 peptide analogues capable of inducing improved antiglioma CTL responses. Cancer Research 66(11): 5883-5891. In particular, the (poly)peptide IL13RA2-L (SEQ ID NO: 279) shows a strong binding affinity to HLA-A*0201, namely, 69% of maximum HIV pol 589-597 binding activity at 100 μM; 96% at 25 μM and 43% at 6.25 μM. Results are also shown in FIG. 7.

Example 8: IL13RA2, BIRC5 and FOXM1 Peptides have Superior Affinity to the HLA-A*0201 Allele

[1091] Binding affinity of various selected sequence variants of epitopes and of the corresponding fragments of human tumor antigens (human reference peptides) to the HLA-A*0201 allele was confirmed in vitro. Namely, the antigenic peptides of sequence SEQ ID NO: 289 («FMLGEFLKL» also referred herein as BIRC5-B1); SEQ ID NO: 287 («YTLGEFLYI» also referred herein as BIRC5-B2); and SEQ ID NO: 288 («GLLGEFLQI» also referred herein as BIRC5-B3) were compared to the corresponding reference human peptide derived from BIRC5 («LTLGEFLKL», SEQ ID NO: 286, also referred herein as BIRC5-H). Moreover, the antigenic peptides of sequence SEQ ID NO: 314 («RLSSYLVEI» also referred herein as FOXM1-B) and sequence SEQ ID NO: 312 («LMDLSTTEV» also referred herein as FOXM1-B2) were compared to the corresponding reference human peptides derived from FOXM1 («RVSSYLVPI», SEQ ID NO: 294, also referred herein as FOXM1-H and «LMDLSTTPL», SEQ ID NO: 293, also referred herein as FOXM1-H2, respectively). Moreover, the antigenic peptides of sequence SEQ ID NO: 31 («FLPFGFILV» also referred herein as IL13RA2-B) and sequence SEQ ID NO: 279 («FLPFGFILPV» also referred herein as IL13RA2-L) were compared to the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID NO: 263, also referred herein as IL13RA2-H).

[1092] A. Materials and Methods

[1093] A1. Measuring the Affinity of the Peptide to T2 Cell Line.

[1094] The experimental protocol is similar to the one that was validated for peptides presented by the HLA-A*0201 (Tourdot et al., A general strategy to enhance immunogenicity of low-affinity HLA-A2.1-associated peptides: implication in the identification of cryptic tumor epitopes. Eur J Immunol. 2000 December; 30(12):3411-21). Affinity measurement of the peptides is achieved with the human tumoral cell T2 which expresses the HLA-A*0201 molecule, but which is TAP1/2 negative and incapable of presenting endogenous peptides.

[1095] T2 cells (2.Math.10.sup.5 cells per well) are incubated with decreasing concentrations of peptides from 100 μM to 1.5625 μM in a AIMV medium supplemented with 100 ng/μl of human P2m at 37° C. for 16 hours. Cells are then washed two times and marked with the anti-HLA-A2 antibody coupled to PE (clone BB7.2, BD Pharmagen).

[1096] The analysis is achieved by FACS (Guava Easy Cyte).

[1097] For each peptide concentration, the geometric mean of the labelling associated with the peptide of interest is subtracted from background noise and reported as a percentage of the geometric mean of the HLA-A*0202 labelling obtained for the reference peptide HIV pol 589-597 at a concentration of 100 μM. The relative affinity is then determined as follows:

relative affinity=concentration of each peptide inducing 20% of expression of HLA-A*0201/concentration of the reference peptide inducing 20% of expression of HLA-A*0201.

[1098] A2. Solubilisation of Peptides

[1099] Each peptide is solubilized by taking into account the amino acid composition. For peptides which do not include any Cystein, Methionin, or Tryptophane, the addition of DMSO is possible to up to 10% of the total volume. Other peptides are resuspended in water or PBS pH7.4.

[1100] B. Results

[1101] The mean relative fluorescence intensity values (data are normalized to the mean fluorescence of HIV peptide, i.e. a value of 100 is equal to the best binding observed with HIV peptide) of T2 cells obtained for the various concentrations of each peptide are shown in Table 13 below:

TABLE-US-00018 TABLE 13 Peptide Peptide concentration (μM) Name SEQ ID NO. 100 50 25 6.25 3.125 1.5625 BIRC5-H 286 35.6 18.9 9.8 10.8 1.4 1.7 BIRC5-B1 289 117.0 77.1 61.7 36.1 22.3 1.9 BIRC5-B2 287 58.0 54.4 29.6 9.0 6.6 nd BIRC5-B3 288 35.0 29.8 20.9 nd 8.9 9.4 FOXM1-H 294 83.8 30.7 10.5 0.0 0.0 0.0 FOXM1-B 314 47.5 21.3 7.6 0.0 0.0 0.0 FOXM1-H2 293 77.6 62.5 65.4 19.7 0.9 5.3 FOXM1-B2 312 105.0 91.5 98.2 33.5 12.7 7.2 IL13RA2-H 263 26.5 14.2 11.2 9.6 3.7 3.0 IL13RA2-B 31 128.4 112.7 86.5 40.8 15.7 14.8 IL13RA2-L 279 107.7 85.5 77.3 30.4 19.8 13.3

[1102] Table 14 below summarizes for each tested peptide the concentration required to induce 20% of HLA-A2 expression and the in vitro binding affinity.

TABLE-US-00019 TABLE 14 Concentration of peptide that In vitro induces 20% of HLA-A2 expression binding Peptide (μM) affinity BIRC5-H 53.0 16.1 BIRC5-B1 2.7 0.8 BIRC5-B2 14.7 4.5 BIRC5-B3 22.9 7.0 FOXM1-H 37.6 3.7 FOXM1-B 46.7 4.6 FOXM1-H2 12.6 2.5 FOXM1-B2 6.7 1.3 IL13RA2-H ND ND IL13RA2-B 2.9 0.3 IL13RA2-L 3.2 0.3 ND—not determined

[1103] In addition, FIGS. 8-10 illustrate the results for selected examples, namely for antigenic peptides IL13RA2-B and IL13RA2-L in comparison to the corresponding human IL13RA2 fragment IL13RA2-H (FIG. 8), for antigenic peptides BIRC5-B1, BIRC5-B2 and BIRC5-B3 in comparison to the corresponding human BIRC5 fragment BIRC5-H (FIG. 9), and for antigenic peptide FOXM1-B in comparison to the corresponding human FOXM1 fragment FOXM1-H (FIG. 10A) and antigenic peptide FOXM1-B2 in comparison to the corresponding human FOXM1 fragment FOXM1-H2 (FIG. 101B).

[1104] In summary, the results show that the exemplified IL13RA2, BIRC5 and FOXM1 peptides show at least similar binding affinity to HLA-A*0201 as the corresponding human tumor antigen fragments. In most cases, the binding affinity observed for the antigenic peptides according to the present invention was stronger than that of the corresponding human epitopes. Without being bound to any theory it is assumed that such a strong binding affinity of the antigenic peptides according to the present invention reflects their ability to raise an immune response (i.e., their immunogenicity).

Example 9: Vaccination of Mice with Antigenic Peptides According to the Present Invention Induces Improved T Cell Responses in ELISPOT-IFNγ Assay

[1105] A. Materials and Methods

[1106] A.1 Mouse Model

[1107] The immunization scheme is the same as described above and shown in FIG. 2. Briefly, HLA-A2 humanized mice (HLA-A2 (CB6F1-Tg(HLA-A*0201/H2-K.sup.b)A*0201) were assigned randomly (based on mouse sex and age) to experimental groups, wherein each group was immunized with a specific vaccination peptide (vacc-pAg) combined to a common helper peptide (h-pAg T13L; sequence: TPPAYRPPNAPIL; SEQ ID NO: 280; Bhasin M, Singh H, Raghava G P (2003) MHCBN: a comprehensive database of MHC binding and non-binding peptides. Bioinformatics 19: 665-666) (as outlined in Table 15 below). The vacc-pAg were compared in couples (group 1 vs. group 2, group 1 vs. group 3; group 1 vs. group 4; group 5 vs. group 6; group 7 vs. group 8). Thereby, both native and optimized versions of a single peptide were compared in each wave.

TABLE-US-00020 TABLE 15 Experimental group composition Peptide Helper Animal Group (vacc-pAg) (h-Ag) Prime Boost Number 1 BIRC5-H T13L + +(1X) 5 (100 μg) (150 μg) 2 BIRC5-B1 T13L + +(1X) 5 (100 μg) (150 μg) 3 BIRC5-B2 T13L + +(1X) 5 (100 μg) (150 μg) 4 BIRC5-B3 T13L + +(1X) 5 (100 μg) (150 μg) 5 FOXM1-H2 T13L + +(1X) 5 (100 μg) (150 μg) 6 FOXM1-B2 T13L + +(1X) 5 (100 μg) (150 μg) 7 IL13RA2-H T13L + +(1X) 5 (100 μg) (150 μg) 8 IL13RA2-B T13L + +(1X) 5 (100 μg) (150 μg) h-pAg: ′helper′ peptide; vacc-pAg: vaccination peptide. The number of boost injections is indicated into brackets.

[1108] The peptides were provided as follows: [1109] vacc-pAg: BIRC5-H, BIRC5-B1, BIRC5-B2, BIRC5-B3, FOXM1-H2, FOXM1-B2, IL13RA2-H and IL13RA2-B; all produced and provided at a 4 mg/ml (4 mM) concentration; [1110] h-pAg: T13L; Eurogentec batch 1713334 re-suspended in pure distilled water at a 10 mg/mL concentration

[1111] The animals were immunized on day 0 (d0) with a prime injection, and on d14 with a boost injection. Each mouse was injected s.c. at tail base with 100 μL of an oil-based emulsion that contained: [1112] 100 μg of vacc-pAg (25 μL of 4 mg/mL stock per mouse); [1113] 150 μg of h-pAg (15 μL of 10 mg/mL stock per mouse); [1114] 10 μL of PBS to reach a total volume of 50 μL (per mouse); [1115] Incomplete Freund's Adjuvant (IFA) added at 1:1 (v:v) ratio (50 μL per mouse).

[1116] A separate emulsion was prepared for each vacc-pAg, as follows: IFA reagent was added to the vacc-pAg/h-pAg/PBS mixture in a 15 mL tube and mixed on vortex for repeated cycles of 1 min until forming a thick emulsion.

[1117] A.2 Analysis

[1118] Seven days after the boost injection (i.e. on d21), the animals were euthanized and the spleen was harvested. Splenocytes were prepared by mechanical disruption of the organ followed by 70 μm-filtering and Ficoll density gradient purification.

[1119] The splenocytes were immediately used in an ELISPOT-IFNγ assay (Table 16). Experimental conditions were repeated in triplicates, using 2*105 total splenocytes per well, and were cultured in presence of vacc-pAg (10 μM), lonomycin (0.1 μM) plus PMA (1 μM) or medium-only to assess for their capacity to secrete IFNγ. The commercial ELISPOT-IFNγ kit (Diaclone Kit Mujrine IFNγ ELISpot) was used following the manufacturer's instructions, and the assay was performed after about 19 h of incubation.

TABLE-US-00021 TABLE 16 Setup of the ELISPOT-IFNγ assay. Group Stimulus Wells Animal Total 1 BIRC5-H (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 2 BIRC5-B1 (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 3 BIRC5-B2 (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 4 BIRC5-B3 (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 5 FOXM1-H2 (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 6 FOXM1-B2 (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 7 IL13RA2-H (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15 8 IL13RA2-B (10 μM) 3 5 15 Ionomycin (0.1 μM) PMA 1 μM) 3 5 15 Medium 3 5 15

[1120] Spots were counted on a CTL ELISpot reader. Data plotting and statistical analysis were performed with the Prism-5 software (GraphPad Software Inc.).

[1121] B. Results

[1122] A total of 40 HLA-A2 (CB6F1-Tg(HLA-A*0201/H2-K.sup.b)A*0201) mice were used for these experiment. All mice were aged of 6 to 9 weeks at the experiment starting date. Both males and females were used in the study. Animals have been housed in groups of 5 per cage at maximum. At time of sacrifice, the spleen T cell population was analysed by flow cytometry, showing that the large majority belonged to the CD4+ T cell subset.

[1123] After plating and incubation with the appropriate stimuli, the IFNγ-producing cells were revealed and counted. The data were then normalized as a number of specific spots (the average counts obtained in the ‘medium only’ condition being subtracted) per 50*103 total T cells.

[1124] The individual average values (obtained from the triplicates) were next used to plot the group average values. As the functional capacity of T cells might vary from individual to individual, the data were also expressed as the percentage of the ionomycin plus PMA response per individual (see FIG. 11).

[1125] Overall, vaccination with the antigenic peptides according to the present invention (BIRC5-B1, BIRC5-B2, BIRC5-B3, FOXM1-B2 and IL13RA2-B) induced improved T cell responses in the ELISPOT-IFNγ assay, as compared to the respective human reference epitopes (BIRC5-H, FOXM1-H2 and IL13RA2-H).

Example 10: Immunogenicity of BIRC5-B1 in HLA-A2 Transgenic Mice and Cross-Reactivity with the Corresponding Human Peptide

[1126] A. Materials and Methods

[1127] The antigenic peptide of the invention BIRC5-B1 (SEQ ID NO: 289) and the corresponding human peptide BIRC5-H (SEQ ID NO: 286) were tested in distinct groups of male and female HHD DR3 mice expressing human HLA-A2 and HLA-DR3 MHC and lacking the murine H-2 class I and class II MHCs. Groups of 5 mice (male and female) were subcutaneously injected on days 0 and 14 with 100 μg of BIRC5-B1 or BIRC5-H, 150 μg of helper peptide (DR3) and IFA. On day 21, the mice were euthanized and splenocytes were prepared and stimulated in vitro with BIRC5-B1 or the human peptide BIRC5-H to assess their capacity to secrete IFN- as assessed by ELISpot. ConA was used as a positive control.

[1128] B. Results

[1129] The number of SFC (normalized to the number of CD8 cells) are depicted in FIG. 12. Results are shown for mice immunized with BIRC5-B1. The results show that immunisation of mice with BIRC5-B1 allows to induce T-cells that are able to react strongly after challenge with either BIRC5-B1 or the human corresponding peptide BIRC5-H. Thus, BIRC5-B1 is strongly immunogenic and is able to drive an effective immune response against human corresponding peptide. Immunisation of mice with the human corresponding peptide BIRC5-H does not induce any immune response against BIRC5-B1 or the human corresponding peptide (data not shown).

[1130] These results were confirmed in HHD DR1 mice expressing human HLA-A2 and HLA-DR1 MHC and lacking the murine H-2 class I and class II MHCs (groups of 5 mice).

Example 11: Immunogenicity of FOXM1-B2 in HLA-A2 Transgenic Mice and Cross-Reactivity with the Corresponding Human Peptide

[1131] A. Materials and Methods

[1132] The antigenic peptide of the invention FOXM1-B2 (SEQ ID NO: 312) and the corresponding human peptide FOXM1-H2 (SEQ ID NO: 293) were tested in distinct groups of male and female HHD DR3 mice expressing human HLA-A2 and HLA-DR3 MHC and lacking the murine H-2 class I and class II MHCs. Groups of 5 mice (male and female) were subcutaneously injected on days 0 and 14 with 100 μg of FOXM1-B2 or FOXM1-H2, 150 μg of helper peptide (DR3) and IFA. On day 21, the mice were euthanized and splenocytes were prepared and stimulated in vitro with FOXM1-B2 or the human corresponding peptide FOXM1-H2 to assess their capacity to secrete IFN- as assessed by ELISpot. ConA was used as a positive control.

[1133] B. Results

[1134] The number of SFC (normalized to the number of CD8 cells) are depicted in FIG. 13. Results are shown for mice immunized with FOXM1-B2. The results show that immunisation of mice with FOXM1-B2 allows to induce T-cells that are able to react strongly after challenge with either FOXM1-B2 or human corresponding peptide. Thus, FOXM1-B2 is strongly immunogenic and is able to drive an effective immune response against human corresponding peptide FOXM1-H2. Immunisation of mice with the human corresponding peptide FOXM1-H2 does not induce immune response against FOXM1-B2 or the human corresponding peptide (data not shown).

[1135] These results were confirmed in HHD DR1 mice expressing human HLA-A2 and HLA-DR1 MHC and lacking the murine H-2 class I and class II MHCs (groups of 5 mice).

[1136] Altogether, these immunogenicity studies described in Examples 6, 10 and 11 performed in HHD DR3 and HHD DR1 mice showed that the three antigenic peptides of the invention, IL13RA2-L, BIRC5-B1 and FOXM1-B2, induced strong immune responses. Cross-reactivity of the T cells generated against IL13RA2-L, BIRC5-B1 and FOXM1-B2 for the corresponding human peptides was shown in HHD DR3 and HHD DR1 mice.

[1137] Accordingly, those results provide experimental evidence that antigen-based immunotherapy is able to improve T cell response in vivo and that the antigenic peptides according to the present invention are particularly efficient for that purpose.

Example 12: BIRC5-B1 has Superior Affinity to the HLA-A*0201 Allele

[1138] This Example provides evidence that the peptide of the invention as set forth in SEQ ID NO: 289 (also referred to herein as BIRC5-B1) has a higher affinity than the corresponding reference human peptide derived from BIRC5 (BIRC5-H, SEQ ID NO: 286) and a comparative sequence variant thereof (“2M”; SEQ ID NO: 330). In this experiment, the peptide of sequence SEQ ID NO: 289 (BIRC5-B1) was compared to [1139] the peptide “2M” (LMLGEFLKL; SEQ ID NO: 330), in which the threonine at position 2 of SEQ ID NO: 593 was substituted by methionine (2M); [1140] the corresponding reference human peptide BIRC5-H (SEQ ID NO: 286); and [1141] a positive control (HIV).

[1142] A. Materials and Methods

[1143] The experimental protocol, materials and methods correspond to those outlined in Example 8, with the only difference that the above mentioned (poly)peptides were used.

[1144] B. Results

[1145] The following in vitro binding affinities were obtained:

TABLE-US-00022 TABLE 17 Concentration of peptide that In vitro SEQ induces 20% of HLA-A2 expression binding Peptide ID NO (μM) affinity BIRC5-H 286 95.9 112.82 BIRC5-B1 289 1.24 1.46 2M 330 2.87 3.38 HIV 0.85 1.00

TABLE-US-00023 TABLE 18 Peptide Peptide concentration (μM) Name SEQ ID NO. 100 10 1 0.1 HIV 100 84.725 22.14 2.405 BIRC5-H 286 20.545 3.515 0 0 BIRC5-B 289 101.845 65.06 17.42 1.07 2M 330 75.22 48.465 8.37 0.76

[1146] In summary, the antigenic peptide according to the present invention (BIRC5-B1; SEQ ID NO: 289) showed considerably higher in vitro binding affinity to HLA-A*0201 than the corresponding human epitope (BIRC5-H) and the comparative peptide “2M”. Results are also shown in FIG. 14.

Example 13: Candidate (Poly)Peptides has Superior Affinity to the HLA-A*0201 Allele

[1147] This Example provides evidence that the (poly)peptides of sequence SEQ ID NO: 31 («FLPFGFILV», also referred to herein as IL13RA2-B), SEQ ID NO: 279 («FLPFGFILPV», also referred to herein as IL13RA2-L), SEQ ID NO: 64 («FMPFGFILV», also referred to herein as IL13RA2-B2), SEQ ID NO: 336 («FMPFGFILGV», also referred to herein as IL13RA2-B3), SEQ ID NO: 212 («YMPFGFILV», also referred to herein as IL13RA2-B4), SEQ ID NO: 178 («YLPFGFILV», also referred to herein as IL13RA2-B5) and SEQ ID NO: 335 («FMPFGFILPI», also referred to herein as IL13RA2-B6) have higher affinity to the HLA-A*0201 allele than other sequence variants of the corresponding reference human peptide derived from IL13RA2 («WLPFGFILI», SEQ ID No 263, also referred to herein as IL13RA2-H). Synthetic peptide “1A9V” (SEQ ID NO: 282), as described above and by Eguchi Junichi et al., 2006 was used as comparative peptide.

[1148] A. Materials and Methods

[1149] The experimental protocol, materials and methods correspond to those outlined in Example 3, with the only difference that the above mentioned (poly)peptides were used.

[1150] B. Results

[1151] The following in vitro binding affinities were obtained:

TABLE-US-00024 TABLE 19 Concentration of peptide that In vitro SEQ induces 20% of HLA-A2 expression binding Peptide ID NO (μM) affinity IL13RA2-B 31 1.9 2.48 IL13RA2-L 279 0.4 0.53 IL13RA2-H 263 62.3 78.77 1A9V 282 20.9 27.68 IL13RA2-B2 64 4.5 5.97 IL13RA2-B3 336 3.3 4.39 IL13RA2-B4 212 4.5 5.24 IL13RA2-B5 178 1.5 1.76 IL13RA2-B6 335 5.9 6.86 HIV 1

[1152] Accordingly, the (poly)peptides according to the present invention (IL13RA2-B (SEQ ID NO: 31), IL13RA2-L (SEQ ID NO: 279), IL13RA2-B2 (SEQ ID NO: 64), IL13RA2-B3 (SEQ ID NO: 336), IL13RA2-B4 (SEQ ID NO: 212), IL13RA2-B5 (SEQ ID NO: 178) and IL13RA2-B6 (SEQ ID NO: 335)) showed considerably higher binding affinity to HLA-A*0201 than the corresponding reference human peptide or the peptide variant “1A9V” described by Eguchi Junichi et al., 2006.

Example 14: Identification of IL13RA2-L, BIRC5-B1 and FOXM1-B2 Peptide-Specific CD8 T Cells in Humans and Ex Vivo Cytotoxic Effects of IL13RA2-L Peptide Specific CD8 Human T Cells

[1153] Multiple investigations support the notion of presence of a repertoire of specific T cells against microbial peptides. The number of microbial specific T-cells against peptides is expected to be low, but sufficient to be re-activated by a vaccine challenge.

[1154] To identify, amplify and functionally characterize circulating IL13RA2-L, BIRC5-B1 and FOXM1-B2 peptide-specific T cells in humans, an in vitro amplification protocol has been developed in order to detect T cells specific for each antigenic peptide and investigate their cytotoxic capacity.

[1155] 3.1 Identification of Antigenic Peptide-Specific CD8 T Cells in Human

[1156] Peripheral blood mononuclear cells (PBMC) from several HLA-A*02 healthy donors were subjected to multiple rounds of in vitro stimulation with cells presenting IL13RA2-L, BIRC5-B1 and FOXM1-B2 peptides in HLA-A2 dependent context (as peptide loaded T2 cells). Identification and quantification of T cells specific clones were performed using peptide MHC multimers (pMHC). pMHC multimers are recombinantly produced and coupled with fluorescent labels. Binding of pMHC multimers to T cells allows identification and sorting by flow cytometry of specific T cells harboring TCRs able to specifically recognize selected pMHC complexes.

[1157] In vitro amplification method and specific pMHC multimers have been used for identification of IL13RA2-L-, BIRC5-B1- and FOXM1-B2-specific T cells. pMHC multimers were generated for all the bacteria peptides and their respective human counterpart. PBMCs from several HLA-A*02 healthy donors (up to 19 donors) were collected, enriched after CD137 and CD8 selection and subjected to multiple rounds of in vitro amplification with bacteria peptides loaded T2 cells to increase the number of specific T cell clones. Detection of bacteria and human peptide-specific CD8 T cells using cytometry analysis with the fluorescent multimer was performed on enriched CD8 T cell populations.

[1158] FIG. 15 exemplifies results obtained with one HLA-A2 healthy donor. For this donor, cell amplification allows detection of IL13RA2-L specific cells (0.94%), BIRC5-B1 specific cells (0.098%) and FOXM1-B2 specific cells (0.15%). We also detect IL13RA2-H specific cells (0.22%), BIRC5-H1 specific cells (0.064%) and FOXM1-H2 specific cells (0.19%) CD8 T cells.

[1159] These results demonstrate the presence of CD8 T cells in the blood of healthy HLA-A2 donors that can recognize the microbiome-derived peptides, and importantly also the human counterpart peptides.

[1160] 3.2 Antigenic Peptide-Specific CD8 T Cytotoxicity Functions

[1161] CD8+ T cells expanded per above were used to perform cytotoxic assays in presence of different ratios of target and effector cells to assess their cytotoxic capacity, using flow cytometry readout. Target cells were T2 cell lines loaded with bacterial peptide. Negative control was T2 cells unloaded and T2 cells loaded with irrelevant peptide. As shown in FIG. 16, IL13RA2-L peptide-specific human T cell clones expanded in vitro have the capacity to kill T2 cells loaded with the bacteria peptide, IL13RA2-L.

[1162] Overall, these results demonstrate the presence of T cell clones in healthy volunteers able to recognize microbial peptide and to kill target with microbial peptides and human counterparts. These data are particularly encouraging as T cell clones have been obtained in healthy donors, therefore we could expect that specific T cell clones could be efficiently amplified in patients exposed to the immunization by antigenic peptides of the invention.