Combination Of A STING Agonist And A Complex Comprising A Cell Penetrating Peptide, A Cargo And A TLR Peptide Agonist

Abstract

The present invention provides a combination of an agonist of stimulator of interferon response cGAMP interactor 1 (STING) and a vaccine including specific antigens or antigenic epitopes, namely, a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist. Such a combination is particularly useful in medicine, in particular in the prevention and/or treatment of cancer. Moreover, the present invention also provides compositions, such as a pharmaceutical compositions and vaccines, which are useful, for example, in the prevention and/or treatment of cancer.

Claims

1. A combination of (i) a Stimulator of Interferon Genes (STING) agonist; and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a Toll Like Receptor (TLR) peptide agonist, wherein the components a)-c) comprised by the complex are covalently linked.

2. The combination according to claim 1, wherein the complex is a recombinant polypeptide or a recombinant protein.

3. The combination according to claim 1, wherein the cell penetrating peptide (1) has a length of the amino acid sequence of said peptide of 15 to 45 amino acids in total; and/or (2) has an amino acid sequence comprising a fragment of the minimal domain of ZEBRA, said minimal domain extending from residue 170 to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO: 3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been substituted, deleted, and/or added without abrogating said peptide's cell penetrating ability.

4.-6. (canceled)

7. The combination according to claim 6, wherein the cell penetrating peptide has an amino acid sequence comprising or consisting of an amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or sequence variants thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity without abrogating said peptide's cell penetrating ability.

8.-11. (canceled)

12. The combination according to claim 1, wherein the at least one antigen or antigenic epitope comprises or consists of at least one tumor or cancer epitope.

13. The combination according to claim 12, wherein the at least one tumor epitope is selected from the group of tumors comprising endocrine tumors, gastrointestinal tumors, genitourinary and gynecologic tumors, breast cancer, head and neck tumors, hematopoietic tumors, skin tumors, and thoracic and respiratory tumors.

14. The combination according to claim 12, wherein the at least one tumor or cancer epitope is selected from the group of tumors or cancers of: gastrointestinal tumors comprising anal cancer, appendix cancer, cholangiocarcinoma, carcinoid tumor, gastrointestinal colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), hepatocellular cancer, pancreatic cancer, rectal cancer, colorectal cancer, and metastatic colorectal cancer.

15. The combination according to claim 12, wherein the at least one antigen or antigenic epitope is selected from a tumor associated antigen, tumor-specific antigen, and tumor neoantigen.

16. The combination according to claim 12, wherein the at least one tumor or cancer epitope is an epitope of an antigen selected from the group consisting of EpCAM, HER-2, MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE, SART, IL13Ralpha2, ASCL2, NY-ESO-1, MAGE-A3, PRAME, and WT1.

17. (canceled)

18. The combination according to claim 1, wherein the complex comprises a multi-antigenic domain, which comprises epitopes of at least two distinct antigens.

19.-43. (canceled)

44. The combination according to claim 18, wherein the multi-antigenic domain comprises one or more epitopes of survivin or sequence variants thereof; one or more epitopes of CEA or sequence variants thereof; and one or more epitopes of ASCL2 or sequence variants thereof.

45.-47. (canceled)

48. The combination according to claim 18, wherein the multi-antigenic domain comprises a peptide consisting of an amino acid sequence according to SEQ ID NO: 32 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity; a peptide consisting of an amino acid sequence according to SEQ ID NO: 18 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity; and a peptide consisting of an amino acid sequence according to SEQ ID NO: 24 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

49. The combination according to claim 18, wherein the multi-antigenic domain of the complex comprises a peptide consisting of an amino acid sequence according to SEQ ID NO: 48 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

50. (canceled)

51. The combination according to claim 1, wherein the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist.

52. (canceled)

53. The combination according to claim 1, wherein the TLR peptide agonist comprises or consists of an amino acid sequence according to SEQ ID NO: 49 or 50; or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

54.-59. (canceled)

60. The combination according to claim 1, wherein the complex is a polypeptide or protein, wherein a) the cell penetrating peptide has an amino acid sequence comprising or consisting of an amino acid sequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14), SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPP8/Z18), or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity (without abrogating said peptide's cell penetrating ability; b) the at least one antigen or antigenic epitope is a peptide, polypeptide or protein; and c) the TLR peptide agonist is a TLR2 peptide agonist and/or a TLR4 peptide agonist.

61. (canceled)

62. The combination according to claim 1, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 54, or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

63. The combination according to claim 1, wherein the STING agonist is a cyclic-dinucleotide (CDN) based STING agonist.

64. The combination according to claim 1, wherein the STING agonist is selected from the group consisting of ADU-S100, MK-1454, E-7766, MK-2118, BMS-986301, IMSA-101, SB-11285, SYNB-1891, GSK-3745417, TAK-676, and TTI-10001.

65. The combination according to claim 1, wherein the STING agonist is a compound of formula I ##STR00015## wherein R.sup.1 is selected from the group consisting of H, F, —O—C.sub.1-3 alkyl and OH, and R.sup.2 is H, or R.sup.2 is —CH.sub.2— and R.sup.1 is —O—, forming together a —CH.sub.2—O— bridge, and R.sup.3 is a purine nucleobase selected from the group consisting of purine, adenine, guanine, xanthine, and hypoxanthine, connected through its N.sup.9 nitrogen; or a salt thereof.

66. The combination according to claim 65, wherein the STING agonist is a compound of formula Ia ##STR00016## or a salt thereof; or a compound of formula Ib ##STR00017## or a salt thereof.

67. (canceled)

68. The combination according to claim 66, wherein the STING agonist is a compound of formula Ia.1 ##STR00018## or a salt thereof or a compound of formula Ia.2 ##STR00019## or a salt thereof; or a compound of formula Ia.3 ##STR00020## or a salt thereof; or a compound of formula Ib.1 ##STR00021## or a salt thereof.

69. (canceled)

70. (canceled)

71. (canceled)

72. The combination according to claim 1, wherein the STING agonist is a compound of formula II: ##STR00022## wherein Base.sup.1 and Base.sup.2 are independently selected from the group consisting of purine, adenine, guanine, xanthine, and hypoxanthine, connected through their N.sup.9 nitrogen atoms; or a salt thereof.

73. The combination according to claim 72, wherein the STING agonist is a compound of formula II-1 ##STR00023## or a salt thereof; or a compound of formula II-2 ##STR00024## or a salt thereof; or a compound of formula II-3 ##STR00025## or a salt thereof; or a compound of formula II-4 ##STR00026## or a salt thereof.

74. (canceled)

75. (canceled)

76. (canceled)

77. The combination according to claim 1, wherein the STING agonist is a substantially pure (Sp,Sp), (Rp,Rp), (Sp,Rp), or (Rp,Sp) stereoisomer of a compound selected from the group of compounds represented by any one of formula I, Ia, Ia.1, Ia.2, Ia.3, Ib, Ib.1, II, II-1, II-2, II-3 and II-4, which is at least 90% pure relative to the other possible diastereomers, or a salt thereof.

78. The combination according to claim 1, wherein the STING agonist is a substantially pure (Rp,Rp) stereoisomer of a compound selected from the group of compounds represented by any one of formula I, Ia, Ia.1, Ia.2, Ia.3, Ib, Ib.1, II, II-1, II-2, II-3 and II-4, which is at least 90% pure relative to the other possible diastereomers, or a salt thereof.

79. The combination according to claim 1, wherein the STING agonist is a pharmaceutically acceptable salt of a compound selected from the group of compounds represented by any one of formula I, Ia, Ia.1, Ia.2, Ia.3, Ib, Ib.1, II, II-1, II-2, II-3 and II-4, or a substantially pure (Sp,Sp), (Rp,Rp), (Sp,Rp), or (Rp,Sp) stereoisomer thereof, which is at least 90% pure relative to the other possible diastereomers.

80. The combination according to claim 1, wherein the STING agonist is a sodium salt of a compound selected from the group of compounds represented by any one of formula Ia.1, Ia.2, Ia.3, Ib.1, II-1, II-2, II-3 and II 4.

81. (canceled)

82. (canceled)

83. The combination according to claim 1, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is ADU-S100.

84. The combination according to claim 1, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 55 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is one compound selected from the group of compounds represented by formula Ia.1, Ia.2, Ia.3, Ib.1, II-1, II-2, II-3 and II 4, or a salt thereof.

85. The combination according to claim 1, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 54 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is ADU-S100.

86. The combination according to claim 1, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 54 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is one compound selected from the group of compounds represented by formula Ia.1, Ia.2, Ia.3, Ib.1, II-1, II-2, II-3 and II-4, or a salt thereof.

87.-96. (canceled)

97. A kit comprising (i) a STING agonist and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a TLR peptide agonist, wherein the components a)-c) are covalently linked.

98. The kit according to claim 97, wherein the complex comprises or consists of an amino acid sequence according to SEQ ID NO: 54 or a sequence variant thereof having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity, and wherein the STING agonist is one compound selected from the group of compounds represented by formula Ia.1, Ia.2, Ia.3, Ib.1, II-1, II-2, II-3 and II-4, or a salt thereof.

99.-102. (canceled)

103. A composition comprising (i) a STING agonist and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a TLR peptide agonist, wherein the components a)-c) are covalently linked.

104.-109. (canceled)

110. A method for treating cancer or initiating, enhancing or prolonging an anti-tumor-response in a subject in need thereof comprising administering to the subject an effective amount of (i) a STING agonist and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a TLR peptide agonist, wherein the components a)-c) are covalently linked.

111. A method for increasing the infiltration of a tumor with tumor antigen-specific T-cells in a patient, the method comprising administering to a patient afflicted with a tumor or cancer (i) a STING agonist and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a TLR peptide agonist, wherein the components a)-c) are covalently linked.

112. A combination therapy for preventing and/or treating cancer, wherein the combination therapy comprises administration of (i) a STING agonist and (ii) a complex comprising: a) a cell penetrating peptide; b) at least one antigen or antigenic epitope; and c) a TLR peptide agonist, wherein the components a)-c) are covalently linked.

113. The method of claim 110, wherein the subject suffers from cancer or a tumor.

114. The method of claim 113, wherein the subject suffers from an endocrine tumor, a gastrointestinal tumor, a genitourinary or gynecologic tumor, breast cancer, head and neck tumor, hematopoietic tumor, skin tumor, or thoracic or respiratory tumor.

115.-118. (canceled)

119. The combination according to claim 15, wherein the at least one antigen or antigenic epitope is selected from the group of tumor associated antigens, tumor-specific antigens, or tumor neoantigens of colorectal cancer, or metastatic colorectal cancer.

120. The combination according to claim 60, wherein the at least one antigen or antigenic epitope comprises or consists of at least one cancer epitope.

121. The method of claim 114, wherein the subject suffers from colorectal cancer.

122. The method of claim 121, wherein the subject suffers from metastatic colorectal cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0522] 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.

[0523] Throughout the figure legends, the letter ‘K’ stands for the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist, such as Z13Mad25Anaxa (SEQ ID NO: 55) or ATP128 (SEQ ID NO: 54), as indicated in the respective Examples sections.

[0524] FIG. 1A-1M shows for Example 1 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) modulate both CD4 and CD8 T cell peripheral responses in tumor-free mice. C57BL/6 mice were treated with two administrations of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at two weeks interval. (A) Vaccination schedule. (B) Serum IFN-a level measured 4 and 24 hours post first vaccination. (C) Circulating HPV-E7-specific CD8 T cells measured by multimer staining one week after the second vaccination. Mice were sacrificed one week after the third vaccination and CD8 (D-E) or CD4 (F-K) T cell responses were analyzed by flow cytometry. (D) Frequency of CD8 T cells among splenocytes. (E) Percentage of cytokine-producing PMA-restimulated CD8 T cells. (F) Frequency of CD4 T cells among splenocytes. Frequency of Treg (G), Th17 (H), Th1 (I) and Th2 (J) among splenic CD4 T cells. (K) Ratio of Th1/Th2 splenic CD4 T cells. (L) In vivo cytotoxicity of RAHYNIVTF-specific CD8 T cells as measured by transfer of RAHYNIVTF peptide loaded splenocytes. (M) RAHYNIVTF-specific CD8 T cell TCR avidity as measured by ex vivo ELIspot.

[0525] FIG. 2A-2B shows for Example 2 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) is well tolerated by tumor bearing mice. (A-B) 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with two administrations of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), a STING agonist (STINGa) or a combination thereof at one week interval. Mouse temperature (A) and weight (B) were measured at the indicated time points.

[0526] FIG. 3A-3B shows for Example 3 the phenotype of circulating HPV-specific CD8 T cells in TC-1 tumor bearing mice. 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with two administrations of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at one week interval. One week after the last treatment HPV-specific CD8 T cell responses were analysed in mouse blood. Frequency (A) and number (B) of circulating HPV-specific CD8 T cells as measured by flow cytometry.

[0527] FIG. 4A-4D shows for Example 3 the effects of combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) on CD8 T cells. 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with two administrations of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at one week interval. One week after the last treatment, mice were sacrificed, tumor harvested, and CD8 T cells presence and phenotype was analyzed by FACS staining. Frequency and total number of total (A-B) and HPV-specific (C-D) CD8 T cells among tumor infiltrating leukocytes are shown.

[0528] FIG. 5A-5B shows for Example 3 the functionality of tumor-infiltrating HPV-specific CD8 T cells as monitored by measuring IFNγ, TNFα and degranulating marker CD107α expression after ex vivo stimulation with HPV peptide-loaded bone marrow derived dendritic cells (BMDCs). Tumor infiltrating CD45+ cells were co-cultured ex vivo with HPV peptide-loaded BMDCs for 6 hours. Antigen-specific cytokine production was measured by intracellular staining; representative FACS plots and frequency of cytokine-producing among CD8 T cells are shown.

[0529] FIG. 6A-6D shows for Example 3 the intracellular production of Granzyme B (GzB) following brief ex vivo TILs culture in presence of Golgi inhibitor. CD45+ tumor infiltrating cells were cultured ex vivo with Golgi inhibitor for 4 hours. Granzyme B production was monitored by intracellular staining; frequency and total number of granzyme B-producing total (A-B) and HPV-specific (C-D) CD8 T cells are depicted.

[0530] FIG. 7A-7B shows for Example 3 the phenotype of circulating HPV-specific CD8 T cells in TC-1 tumor bearing mice, namely, that a very low frequency of cytokine- or GzB-producing splenic HPV-specific CD8 T cells was observed in all the different treatments. To this end, splenocytes were restimulated ex vivo with HPV-derived peptides. Frequency of cytokine-producing (A) and Granzyme B secreting (B) HPV-specific CD8 T cells are shown.

[0531] FIG. 8A-8I shows for Example 3 the expression of activation and exhaustion markers by total (A) or HPV-specific (B) CD8 T cells was measured by flow cytometry. Frequency of CD38 (C-D), NKg2 Da (E-F) or TCF-1 (G-H) expression by total (C-E-G) or HPV-specific (D-F-H) CD8 T cells. Co-expression of PD-1, Granzyme B and TCF-1 on HPV-specific CD8 T cells (I).

[0532] FIG. 9 shows for Example 3 the phenotype of circulating HPV-specific CD8 T cells in TC-1 tumor bearing mice, namely, the expression of activation/exhaustion markers by circulating HPV-specific CD8 T cells as measured by flow cytometry.

[0533] FIG. 10A-10L shows for Example 4 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) modulates intra-tumoral CD4 T cells in TC-1 model. 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with 2 administrations of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at one week interval. One week post the last treatment, mice were sacrificed, blood and tumor harvested, and CD4 T cells presence and phenotype was analyzed by FACS staining. Frequency and total number of total (A-B) and Treg (C-D) CD4 T cells among tumor infiltrating leukocytes. Ratio between tumor infiltrating CD8 T cells and total (E) or Treg (F) CD4 T cells. (G) Frequency of Treg and non-Treg among tumor infiltrating CD4 T cells. Frequency of Th1 (H), Th2 (I) and Th17 (K) among tumor infiltrating CD4 T cells. (J) ratio between Th1 and Th2 tumor infiltrating CD4 T cells. (L) Tumor infiltrating CD45+ cells were co-cultured ex vivo with HPV peptide-loaded BMDCs for 6 hours. Antigen-specific cytokine production was measured by intracellular staining; frequency of cytokine-producing among CD4 T cells is shown.

[0534] FIG. 11A-11H shows for Example 5 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) modulates the tumor microenvironment (TME). 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with 2 administrations of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at one week interval. One week post the last treatment, mice were sacrificed, tumor harvested, and tumor microenvironment was analyzed by FACS staining. (A) Proportion of different cell populations among CD45+ tumor-infiltrating cells, every circle represent 1% of the CD45+ population. (B) Proportion of different dendritic cell populations. Proportion of type 1 (C) or type 2 (D) tumor associate macrophages (TAM) among CD45+ tumor-infiltrating cells. (E) Ratio between TAM1 and TAM2. Proportion of monocytic myeloid-derived suppressor cells mMDSC (F), granulocytic MDSC (G) and neutrophils (H) among CD45+ tumor-infiltrating cells.

[0535] FIG. 12A-12H shows for Example 6 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) modulates intra-tumoral expression of PD-L1 and MHC-I. 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with 2 administrations of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two at one week interval. One week post the last treatment, mice were sacrificed, tumor harvested, and tumor microenvironment was analyzed by FACS staining. Expression level (mean MFI) of PD-L1 on CD45− (A) and CD45+(B). % of PD-L1 among infiltrating TAM1 (C) and TAM2 (D). Expression level of H2-Kb (E) and H2-db (F) on CD45− tumor infiltrating cells. Expression level (G) and frequency (H) of MHC-II.sup.hi among CD11b+ cells. A pool of two independent experiment is shown (n=7), Mann-Whitney test *p<0.05, **p<0.01, ***p<0.001.

[0536] FIG. 13A-13B shows for Example 7 the antitumoral effect of the combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa). 10.sup.5 TC-1 cells (A-B) were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated twice at one week interval and tumor growth (A) and mouse survival (B) were monitored.

[0537] FIG. 14A-14B shows for Example 8 the anti-tumoral effect of the combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa 2). 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated twice at one week interval with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) and/or at days 6, 10, 13 and 17 with the STING agonist (STINGa 2) administered systemically. Tumor growth (A) and mouse survival (B) were monitored.

[0538] FIG. 15A-15B shows for Example 8 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa 2) modulates CD8 T cell peripheral responses. Circulating HPV-specific CD8 T cells measured by multimer staining one week after the second vaccination are shown as % among CD8+ T cells (A) and number of HPV-specific CD8 T cells/ml blood (B).

[0539] FIG. 16A-16C shows for Example 9 the ATP128 immunogenicity tested in a mouse model, and that the combination of STINGa and ATP128 (K) induces a CEA-specific CD8 T cells response. Female C57BL/6J mice were implanted with 5*10.sup.5 MC38-CEA tumor cells subcutaneously on the back of the mouse. At day 6 and day 13 post tumor implantation, mice were vaccinated with 10 nmoles of ATP128, 25 μg of a STING agonist (ADU-S100) or a combination of the two. Both the ATP128 and the STING agonist were injected subcutaneously at the base of the tail. One week after the second vaccination, mouse blood was collected from the tail vein and the frequency and the total number of CEA-specific CD8 T cells was analyzed by flow cytometry using a custom designed multimer, wherein the specific epitope from CEA in C57BL/6 mice was predicted and designed. ATP128 vaccination elicits CEA-specific CD8 T cells, which can be monitored using a custom dextramer staining (A). Custom CEA-dextramer staining was performed 1 week after the 2nd vaccination. The combination of STINGa and ATP128 induces a CEA-specific CD8 T cells response (B,C).

[0540] FIG. 17A-17C shows for Example 10 that combined administration of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) enhances functionality of CD8 T and CD4 T cell peripheral responses in tumor-free mice. (A) C57BL/6 mice were treated with two administrations of Z13Mad39Anaxa vaccine, STING agonist or a combination of the two at two weeks interval. One week after the second vaccination, circulating (left) and splenic (right) SIINFEKL-specific CD8 T cells were measured by multimer staining. (B) SIINFEKL-specific CD8 T cell TCR avidity was measured by ex vivo ELIspot (upper graph). Antigen-specific cytokine production by CD8 T cells was measured by intracellular staining after ex vivo stimulation with SIINFEKL peptide (lower graph). (C) Frequency of Treg (FoxP3.sup.+), Th1 (T-bet.sup.+), Th2 (GATA-3.sup.+) among splenic CD4 T cells and Th1/Th2 ratio was measured by flow cytometry one week after the second vaccination. Antigen-specific cytokine production by CD4 T cells was measured by intracellular staining after ex vivo stimulation with ISQAVHAAHAEINEAGR (OVA-CD4) peptide. One representative experiment is shown (n=5), Mann-Whitney test or Two-way ANOVA *p<0.05, **p<0.01, ***p<0.001.

[0541] FIG. 18A-18D shows for Example 11 that the combination of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K) with a STING agonist (STINGa) inhibits B16-OVA tumor growth. 10.sup.5 B16-OVA cells were injected intravenously into C57BL/6 mice. At day 3 and 10 post tumor injection, mice were treated with two administrations of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope and a TLR peptide agonist (K), STING agonist (STINGa) or a combination of the two. At day 20, lungs were perfused to eliminate blood, the number of tumor metastasis was counted and lung infiltrating lymphocytes were analysed. (A) Vaccination schedule. (B) Number of metastatic nodules per lung and representative pictures. (C) Frequency of SIINFEKL (OVA)-specific CD8 T cells among tumor infiltrating leukocytes and expression of Granzyme B was measured by flow cytometry. Antigen-specific cytokine production by CD8 T cells was measured by intracellular staining after ex vivo stimulation with SIINFEKL peptide (SEQ ID NO: 57) in presence of Golgi inhibitor. Antigen-specific cytokine production was measured by intracellular staining; frequency of cytokine-producing among CD8 T cells is shown. (D) Frequency of Treg (FoxP3.sup.+) and Th1/Th2 ratio were measured by flow cytometry. Antigen-specific cytokine production by CD4 T cells was measured by intracellular staining after ex vivo stimulation with ISQAVHAAHAEINEAGR (OVA-CD4) peptide (SEQ ID NO: 59) in presence of Golgi inhibitor. Antigen-specific cytokine production was measured by intracellular staining; frequency of cytokine-producing among CD4 T cells is shown. A pool of two independent experiments (B) or one representative experiment (C-D) are shown (n≥7), Mann-Whitney test *p<0.05, **p<0.01, ***p<0.001.

[0542] FIG. 19A-19C shows for Example 11 the phenotype and functionality of peripheral antigen-specific T cells in B16-OVA tumor bearing mice. One week after the last treatment, antigen-specific CD8 T cell responses were analyzed in mouse blood and spleen. (A) Frequency and number of circulating SIINFEKL (OVA)-specific CD8 T cells was measured by flow cytometry. (B) Splenocytes were stimulated ex vivo with (or without for granzyme B) SIINFEKL (OVA) peptide (SEQ ID NO: 57) and cytokine and granzyme B production by CD8 T cells was measured by intracellular staining. (C) Splenocytes were stimulated ex vivo with ISQAVHAAHAEINEAGR (OVA-CD4) peptide (SEQ ID NO: 59) and antigen-specific cytokine production was measured by intracellular staining. One representative experiment is shown (n=7) *p<0.05, **p<0.01, ***p<0.001.

EXAMPLES

[0543] 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.

Methods

Mice

[0544] Female C57BL/6J mice were purchased from Charles River Laboratories (L'arbresles, France). All animals used were between 6 and 10 weeks old at the time of experiments. These studies have been reviewed and approved by the institutional and cantonal veterinary authorities in accordance with Swiss Federal law on animal protection.

Vaccines

[0545] Vaccine constructs were designed in-house and produced in E. coli by Genscript. Vaccines were prepared by dilution in vaccine buffer and administered by subcutaneously (s.c.) injection of 10 nmoles in 100 μl volume. Z13Mad25Anaxa (SEQ ID NO: 55) contains CD4 and CD8 epitopes issued from HPV-16 and was utilized in TC-1 tumor model. ATP128 (SEQ ID NO: 54) contains CEA, survivin and ASCL2 epitopes and was utilized in a MC-38 CEA tumor model.

STING Agonist

[0546] The following STING agonists were used: ADU-S100 (Aduro; also referred to as “STINGa”) in Examples 1-7 and 9; and a distinct STING agonist (referred to as “STINGa 2”) in Example 8.

[0547] STINGa (ADU-S100) has the following structural formula (III):

##STR00013##

[0548] STINGa 2 has the following structural formula Ia.2:

##STR00014##

[0549] ADU-S100 (Aduro) was resuspended in DMSO and diluted in 1× phosphate buffer saline (PBS, Gibco) prior to injection. STINGa 2 was resuspended in 1× phosphate buffer saline (PBS, Gibco) prior to injection.

Cell Lines

[0550] TC-1 cells, a cell line derived from lung epithelial cells transfected with HPV16 E6/E7 and c-H-ras oncogenes, were maintained in RPMI 1640 Glutamax™ supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml Penicillin/Streptomycin (P/S), 1 mM Sodium Pyruvate, MEM NEAA and 0.4 mg/ml geneticin G418.

[0551] The MC-38 C57BL/6 mouse colon adenocarcinoma cell line has been transduced with a retroviral construct containing cDNA encoding the human carcinoembryonic antigen (CEA) gene. The CEA expressed by the MC-38-cea-1 clone had a molecular mass of 180 kDa, similar to that of native CEA. This MC-38-cea-1 clone, used here, expresses high levels of CEA on their cell surface (Hand et al. 1993, Cancer Immunol Immunother. 36:65-75). MC-38-CEA-1 cells were cultured in DMEM Medium 1640 Glutamax (Life Technology) with 10% high inactivated fetal calf serum (Life technologies) using standard laboratory techniques (MC-38-CEA-1 culture for tumor implantation_190115).

[0552] The B16-OVA cell line was provided by Bertrand Huard, University of Grenoble-Alpes, France). This cell line derived from mouse melanoma cells transfected with OVA, was maintained in RPMI 1640 Glutamax™ supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/ml Penicillin/Streptomycin (P/S), 1 mM Sodium Pyruvate, MEM NEAA and 1 mg/ml geneticin G418.

In Vivo Tumor Experiments

[0553] C57BL/6 mice were implanted s.c. with 1×10.sup.5 TC-1 tumor cells in the back and mice were stratified according to tumor size on day 6 tumor implantation. Alternatively, C57BL/6J mice were injected i.v. with 1×10.sup.5 B16-OVA cells. Mice were vaccinated two times (at day 6 and 13 post tumor implantation) by s.c. injection of 10 nmoles of vaccine at the tail base. At the same time of vaccination, mice received 25 μg of STING agonist administered via 2×50 μl s.c. injections in each side of the low back. Alternatively, at the same time of vaccination, mice received 10 μg of STING agonist 2 administered via 2×100 μl s.c. injections in each side of the low back.

[0554] Alternatively, female C57BL/6J mice were implanted with 5×10.sup.5 MC38-CEA tumor cells subcutaneously on the back of the mouse and vaccinated twice at the base of the tail (at day 6 and 13 post tumor implantation) by s.c. injection of 10 nmoles of vaccine, 25 μg of STINGa (ADU-S100) or a combination of the two.

[0555] Tumor size was measured with a caliper and mice were euthanized when tumor reached a volume of 1000 mm.sup.3. Tumor volume was calculated with the following formula:

V=length×length×width×Pi/6

[0556] B16-OVA tumor bearing mice were sacrificed at day 20, lungs were perfused with a saline solution and the number of lung metastasis was counted.

Cell Preparation

[0557] Bone marrow derived DCs (BMDCs) were prepared from C57BL/6 mice by extracting bone marrow from tibias and femurs and culturing DCs in BMDC medium (DMEM Glutamax supplemented with 10% FCS, 100 U/ml P/S, 50 μM β-Mercaptoethanol, 10 mM HEPES, 0.116 mg/ml of L-Arginine, MEM NEAA and 10 ng/ml of GM-CSF). After 3 days at 37° C., 5% CO.sub.2, half a volume of fresh medium was added. At day 6, floating cells were recovered, resuspended in BMDCs medium and cultured separately. BMDCs were harvested at day 9 and used for ex vivo T cells stimulation.

[0558] TC-1 tumors were harvested at day 20 post implantation and tumor-infiltrating leucocytes (TIL) were purified using Miltenyi tumor dissociation kit following manufacturer instruction. Briefly, tumor tissues were cut into small pieces, and resuspended in DMEM medium containing tumor dissociating enzymes (Miltenyi). Tumors were digested on a Gentle MACS with heating system (Miltenyi) using solid tumor program. Enzymatic digestion was stopped by adding cold PBS 0.5% BSA solution and keeping cells on ice. Digested tumors were passed through a 70 μm to eliminate remaining undigested tissue. CD45+ cells were purified using CD45 TIL microbeads (Miltenyi) following manufacturer protocol. Purified CD45+ cells were used for flow cytometry staining or ex vivo T cells stimulation.

[0559] B16-OVA tumor bearing mice were perfused with a saline solution to eliminate blood from the lungs before their collection. Lung-infiltrating leucocytes (LILs) were purified using mouse tumor dissociation kit from Miltenyi, following manufacturer instruction.

[0560] Peripheral blood and spleen mononuclear cell suspensions from mice were isolated using Ficoll-Paque gradient (GE Healthcare) before flow cytometry analysis, ex vivo stimulation or TCR avidity assay.

Ex Vivo T Cell Restimulation

[0561] TILs, LILs or splenocytes were numerated and 1×10.sup.5 or 2×10.sup.6 cells were plated per condition, respectively. Cells were incubated with HPV-CD4, HPV-CD8, OVA-CD8 or OVA-CD4 epitope peptide, with PMA/ionomycin as a positive control or without any stimulant as a negative control, in presence of Golgi stop (BD biosciences) and anti-CD107a for 6 hours. After washing, cells were stained for cells surface antigens and fixable viability dye, then, after fixation and permeabilization according to manufacturer's instructions (BD biosciences), cells were stained for intracellular cytokines.

In Vivo Cytotoxicity Assay

[0562] Naive splenocytes were harvested and incubated for 1.5 h in DMEM complete medium at 37° C. with or without HPV-E7 CD8 epitope peptide (SEQ ID NO: 56). Then, loaded and non-loaded splenocytes were stained with cell tracer violet (CTV) or CFSE (both from ThermoFisher Scientific), respectively, following manufacturer instruction. Splenocytes were then mixed at a 1:1 ratio and a total of 5×10.sup.6 cells were transferred by intravenous injection into previously vaccinated mice. 20 hours post cell transfer, splenocytes were harvested and the survival of CTV or CFSE stained cells was assessed by flow cytometry. The percentage of antigen-specific killing was calculated with the following formula: % antigen-specific killing=(1-(ratio peptide.sup.+:peptide.sup.− vaccinated/ratio peptide.sup.+:peptide.sup.− naive))*100.

Ex Vivo TCR Avidity Assay

[0563] One week after the second vaccination, spleens were harvested and splenocytes isolated (see above). 1×10.sup.6 cells/well were seeded in a IFN-γ ELIspot plate and stimulated 0/N with decreasing concentrations of RAHYNIVTF (SEQ ID NO: 56) or SIINFEKL (SEQ ID NO: 57) peptide. ELIspot plates were then revealed following manufacturer instruction and the percentage of maximal response calculated relatively to the highest concentration of stimulating peptide.

Antibodies and Flow Cytometry

[0564] The following antibodies were used: CD45 (30-F11), CD11b (M1/70), KLRG1 (2F1), CD103 (M290), NKg2a (20d5), Ly6C (AL-21), Ly6G (1A8), PD-L1 (MIH5), I-A/I-E (M5/114), CD11c (HL3), PDCA1 (927), CD64 (X54-5/7.1), B220 (RA3-6B2), CD24 (M1/69), CD4 (GK1.5), CD25 (3C7), CD3 (500A2), NKp46 (29A1.4), TNF-α (MP6-XT22), IFN-γ (XMG1.2), H2-Kb (AF6-88.5) and H2-db (28-14-8) were from BD Biosciences; Tim3 (RMT3-23), PD-1 (29F.1A12), CD38 (90), Gr-1 (RB6-8C5), CD206 (C068C2), CD68 (FA-11) were from BioLegend; Ki67 (solA15), FoxP3 (FJK-16s), T-bet (4B10), GATA-3 (TWAJ) and RORγt (AFKJS-9) were from ThermoFisher Scientific; Granzyme B (REA226) was from Miltenyi; CD8 (KT15) was from MBL. Dead cells were stained with LIVE/DEAD Yellow or Aqua fluorescent reactive dye (Life Technologies) and excluded from analyses. Murine MHC-peptide multimers were from Immudex (Copenhagen, Denmark). Cells were analyzed using an Attune NxT flow cytometer (ThermoFisher Scientific) Kaluza (Beckman Coulter) software.

Quantification of Serum Interferon-α

[0565] Blood was collected from mouse tail vein and serum was isolated by centrifugation using Starstedt tubes. The concentration of IFN-α cytokine was measured using commercial ELISA kits according to the manufacturer's recommendations (PBL Assay Science).

Statistical Analysis

[0566] Statistical analyses were performed using Prism software (GraphPad) and considered statistically significant if p<0.05.

Example 1: Combinations of STING Agonists and Vaccine Complexes Modulate T Cell Responses

[0567] In preclinical tumor model and on-going clinical trials, STING agonists are usually administered by intra-tumoral (i.t.) injection in order to inflame the tumor microenvironment (TME). In contrast thereto, in the present experiments systemic administration of the STING agonist in combination with a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and at least one TLR peptide agonist was investigated.

[0568] In order to evaluate the immunogenicity of the combination, tumor-free C57BL/6 mice were vaccinated twice at 2 weeks interval (at day 0 and 14) by s.c. injection of 10 nmoles of Z13Mad25Anaxa (an exemplified a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, which contains human papilloma virus (HPV)-derived CD4 and CD8 epitopes. At about the same time of vaccination, mice received 25 μg of STING agonist ADU-S100 administered via 2×50 μl s.c. injections in each side of the low back. Serum was collected 4 and 24 hours after the first vaccination and IFN-α concentration was measured by ELISA. Whole blood was collected at day 21 and used for antigen-specific CD8 T cells measurement by multimer flow cytometry staining. At the same time, spleens were harvested and splenocytes used for ex vivo stimulation and intracellular cytokine production was analyzed by flow cytometry. Alternatively, splenocytes were used for TCR avidity assay.

[0569] The timeline and results are shown in FIG. 1. FIG. 1A illustrates the timeline of the experiment. Differently from vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, which induces only local inflammation, systemic treatment with the STINGa induced a potent but short lived systemic type I interferon response, characterized by high IFN-α serum levels peaking 4 hours post injection and decreasing already 24 hours later (FIG. 1B). This systemic response was not affected by concomitant injection of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist. While vaccination with Z13Mad25Anaxa is able to elicit circulating HPV-E7-specific CD8 T cells, the data show that combination with STINGa treatment further increases the frequency of antigen-specific CD8 T cells (FIG. 1C). In addition, STINGa treatment resulted in higher proportion of splenic CD8 T cells and increased cytokine secretion after ex vivo PMA/Ionomycin restimulation, indicating a better cell functionality (FIG. 1 D-E). Moreover, combination of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with a STING agonist modulated also CD4 T cells response, slightly increasing splenic proportion and deeply changing their polarization. In fact, a significantly higher proportion of T helper 1 (Th1) and Th17 and lower proportion of Treg and Th2 CD4 T cells was found in combination treated mice, resulting in positive Th1/Th2 and Th17/Th2 ratios (FIG. 1 F-K). Altogether, combination of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with STING agonist improves both CD4 and CD8 T cells response boosting antigen-specific CD8 T cells. In addition to their frequency, combination treatment of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with STINGa also highly enhanced the effector function of antigen-specific CD8 T cells. In vivo killing assay performed one week after vaccination revealed a significant 2.5-fold increase of antigen-specific cytotoxicity in mice treated with a combination of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with STINGa (FIG. 1 L). Furthermore, ex vivo stimulation with decreasing concentration of HPV-CD8 peptide showed significantly higher TCR avidity on complex—STINGa primed T cells (FIG. 1 M).

Example 2: Safety and Tolerability of Combined Administration of STING Agonists and Vaccine Complexes

[0570] Systemic injections of a STING agonist lead to a potent systemic type I interferon response. As this may result in undesired side effects, safety and tolerability of combined administration of STING agonists and vaccine complexes were investigated.

[0571] To this end, 10.sup.5 TC-1 tumor cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with two administrations of (i) a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa); (ii) a STING agonist; or (iii) a combination of both at one week interval, essentially as described in Example 1. Mouse temperature and weight were measured at the time points indicated in FIG. 2.

[0572] Results are shown in FIG. 2. Neither single nor combination treatment caused significant variation of body temperature (FIG. 2A) or weight (FIG. 2B) shortly after administration to TC-1 tumor-bearing mice, confirming the safety and tolerability of combined administration of a STING agonist and a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist.

Example 3: Combinations of STING Agonists and Vaccine Complexes Improve Antigen-Specific CD8 T Cell Responses in TC-1 Tumor Bearing Mice

[0573] TC-1 is a well-known cold tumor model, which is characterized by very low CD4 and CD8 T cells infiltration. To investigate the effects of a combined administration of a STING agonist and a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa), antigen-specific CD8 T cell responses were assessed in the TC-1 cold tumor model.

[0574] To this end, 10.sup.5 TC-1 tumor cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated with two administrations of (i) a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa); (ii) a STING agonist; or (iii) a combination thereof at one week interval, essentially as described in Example 1. One week after the last treatment, mice were sacrificed, tumor harvested, and CD8 T cells presence and phenotype was analyzed by FACS staining.

[0575] TC-1 tumor cells being lowly immunogenic, very low proportion and number of circulating HPV-specific CD8 T cells were found in vehicle treated mice (FIG. 3). Similarly to the observation in tumor-free mice, vaccination with a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist significantly increases peripheral HPV-specific response, and while STINGa monotherapy had no effect, combination treatment increases antigen-specific CD8 T cells number.

[0576] Next, the ability of HPV-specific CD8 T cells to infiltrate TC-1 tumors was investigated. TC-1 being a well-known cold tumor model, very few total or HPV-specific CD8 T cells were found within control tumors, either taking into account proportion—they represent less than 1% of tumor infiltrating leukocytes—or total number (FIG. 4A-D). Vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist induced a significant increase of CD8 T cells tumor infiltration, of which over 50% were HPV-specific. Of note, HPV-specific CD8 T cells are massively present within the tumor despite the rather low percentage in the blood, suggesting that measurement of peripheral responses can only partially predict the intra-tumoral outcome (FIG. 3A-B). STINGa monotherapy did not modulate CD8 T cells tumor infiltration nor the proportion of HPV-specific, thus differing from the observation in tumor-free mouse spleen (FIG. 1). Interestingly, combination of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with a STING agonist showed a synergic effect, increasing both CD8 T cells infiltration and HPV-specific proportion. This confirms that systemic administration of a STING agonist can modulate the intra-tumoral effect of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist.

[0577] In addition, the functionality of tumor-infiltrating HPV-specific CD8 T cells was monitored by measuring IFNγ, TNFα and degranulating marker CD107α expression after ex vivo stimulation with HPV peptide-loaded bone marrow derived dendritic cells (BMDCs), and a significant increase of HPV-specific cytokine-producing and degranulating CD8 T cells was found in mice treated with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist compared to control or STINGa monotherapy group (FIG. 5). Interestingly, vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist particularly increased the proportion of multifunctional CD8 T cells, able to simultaneously produce IFNγ, TNFα and/or CD107α. Combination with STINGa further increased CD8 T cells functionality, and importantly the frequency of multifunctional cells.

[0578] To further characterize tumor infiltrating T cells functionality, also intracellular production of Granzyme B (GzB) was measured, following brief ex vivo TILs culture in presence of Golgi inhibitor, as GzB is one of the main weapons used by CD8 T cells to eliminate cancer cells. Significantly higher frequency and number of GzB-producing total or HPV-specific CD8 T cells were found in mice vaccinated with a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, compared to vehicle or STINGa treated (FIG. 6). Despite not changing the frequency of GzB-positive among HPV-specific CD8 T cells, combination with STINGa further increased their total number. Importantly, contrarily to the intra-tumoral compartment, very low frequency of cytokine- or GzB-producing splenic HPV-specific CD8 T cells was observed in all the different treatments (FIG. 7), demonstrating that CD8 T cells are not systemically activated.

[0579] The efficacy of cancer-specific T cells is often limited by tumor induced exhaustion. Therefore, the expression of activation and exhaustion markers on intra-tumoral and peripheral CD8 T cells was analyzed next. T cells exhaustion is a gradual process eventually resulting in a loss of cell functionality, which can be monitored by the progressive expression of exhaustion markers. While most of tumor infiltrating CD8 T cells in control and STINGa single treated mice expressed only PD-1 or no exhaustion marker at all, in mice vaccinated with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist or in combination treated mice the majority of CD8 T cells, and in particular HPV-specific cells, expressed exhaustion markers PD-1 and Tim-3 (FIG. 8A-B). Interestingly, in the combination group a lower proportion of CD8 T cells co-express PD-1 and Tim-3, suggesting a less exhausted phenotype, which correlate with the higher proportion of cytokine-secreting cells. In addition, a higher proportion of mice vaccinated with complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist showed CD8 T cells, which also expressed CD38 (FIG. 8C-D), NKg2a (FIG. 8E-F), and TCF-1 (FIG. 8G-H), which are other markers associated with reduced T cell functionality. FIG. 81 shows the co-expression of PD1, Granzyme B and TCF-1 on HPV-specific CD8 T cells.

[0580] Similarly to functionality analysis, peripheral CD8 T cells showed a less-exhausted phenotype, with the majority of cells that expressed only PD-1 and some cells still expressing the early activation marker KLRG1 (FIG. 9), suggesting that exhaustion is acquired within the TME.

[0581] In summary, vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist highly increases HPV-specific CD8 T cells tumor infiltration and functionality, and while STINGa monotherapy has no effect, and combination treatment further enhances the efficacy of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist. Nevertheless, intra-tumoral CD8 T cells have a partially exhausted phenotype, which is less advanced in combination treated mice compared to mice vaccinated with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist alone.

Example 4: Combinations of STING Agonists and Vaccine Complexes Modulate Intra-Tumoral CD4 T Cell Responses

[0582] Immunotherapy research has widely focused on CD8 T cells, neglecting CD4 T cells, which are often only considered as immune-suppressives due to the regulatory T cells (Treg). However, recent studies highlight the importance of CD4 T cells, in particular the Th1 and Th17 polarized, for the development of a proper anti-tumoral CD8 T cells response (Meissen, M. and C. L. Slingluff, Jr. (2017). “Vaccines targeting helper T cells for cancer immunotherapy.” Curr Opin Immunol 47: 85-92; Muranski, P., et al. (2008). “Tumor-specific Th17-polarized cells eradicate large established melanoma.” Blood 112(2): 362-373).

[0583] In view thereof, intra-tumoral CD4 T cells were monitored next. To this end, the TC-1 tumor model, as described in Example 3, was used.

[0584] Results are shown in FIG. 10. The data show a significantly increased tumor infiltration by CD4 T cells in mice treated with a combination of complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa) and a STING agonist (STINGa), as compared to all other groups (FIG. 10A-B). Differently form the observation in the spleen, STINGa monotherapy had no effect on intra-tumoral CD4 T cells recruitment. Interestingly, this increased CD4 T cell infiltration was not led by Treg, as their percentage was highly reduced in combination treated mice, but rather by effector CD4 T cells (FIGS. 10C, D and G). The ratio between intra-tumoral CD8 and total or regulatory CD4 T cells is often used as a predictive value for the immunological state of TME. Vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist induced a higher CD8/CD4 T cells ratio compared to vehicle or STINGa treated groups (FIG. 10E). While combination treatment resulted in a CD8/CD4 T cells ratio similar to vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, it induced a significantly higher CD8/Treg ratio (FIG. 10F), highlighting a less immunosuppressive TME. Further analysis revealed that in combination treated mice most of intra-tumoral CD4 T cells are T-bet+ Th1, while only a minimal part are GATA-3+Th2 cells, resulting in a positive Th1/Th2 ratio (FIG. 10H-J). Contrarily from the spleen, a slight decrease in intra-tumoral Th17 CD4 T cells was observed in combination treated mice (FIG. 10K). As Th1 CD4 T cells are usually characterized by the production of IFN-γ and TNF-α production of cytokines was measured by flow cytometry after ex vivo restimulation with HPV peptide-loaded BMDCs. However, contrarily to CD8 T cells, neither IFN-γ nor TNF-α production by intra-tumoral CD4 T cells (FIG. 10L) was detected.

Example 5: Combinations of STING Agonists and Vaccine Complexes Modulate Tumor Microenvironment (TME)

[0585] Despite T cells being the principal target of immunotherapy, due to their ability to directly kill cancer cells, the TME is a very complex network constituted by different immune cell types able to promote or inhibit cancer growth. In view thereof, the composition of TME was deeply dissected in order to obtain a complete overview of its immunological status. To this end, the TC-1 tumor model was used, as described above.

[0586] Results are shown in FIG. 11. As previously mentioned, TC-1 is a cold tumor model, characterized by very low CD4 and CD8 T cells infiltration that combined represent less than 2% of tumor infiltrating CD45+ cells in vehicle treated mice (FIG. 11A). The most prominent cell type are tumor associated macrophages (TAM), representing up to 75% of the infiltrate, and in particular the immunosuppressive TAM2, which have been associated to promotion of tumor growth in different cancer types. Myeloid derived suppressor cells (MDSC)—whose role is less clear and have been associated to tumor promotion or control depending on cancer type—represent another 15%, with the monocytic type (mMDSC) being prevalent. Other cell types found with lower frequency were dendritic cells (DCs, 7%), B cells (2%), NK and NKT cells (1.5%) and neutrophils (1%). Vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist induced a profound modification of the TME, characterized by a strong increase in CD8 T cells and DCs frequency and the appearance of non-Treg CD4 T cells. Interestingly, the increase of DCs infiltration is also characterized by an increase of monocytic DCs (moDCs) proportion (FIG. 11B), a particular DC phenotype which has been described to differentiate only in inflammatory conditions and has been shown to activate anti-tumoral T cell responses (Kuhn, S., et al. (2015). “Monocyte-Derived Dendritic Cells Are Essential for CD8(+) T Cell Activation and Antitumor Responses After Local Immunotherapy.” Front Immunol 6: 584). While the TAM1 compartment remains mostly unaltered, TAM2 frequency is strongly decreased resulting in a higher TAM1/TAM2 ratio (FIG. 11C-E). Contrarily, the frequency of mMDSC is increased by vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, while granulocytic MDSC remains mostly unchanged (FIG. 11F-G). The inverse effect on TAM2 and mMDSC, suggests a possible cell re-polarization, as both population of monocytic origins are known for their plasticity and ability to change differentiation status depending on the environment.

[0587] Similarly to the observation on CD8 T cells infiltration and phenotype, systemic administration of STINGa alone does not affect the composition of TME, which is essentially identical to vehicle treated mice. However, in combination treatment, the STING agonist showed a synergic effect with vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, further expanding CD8 and non-Treg CD4 T cells infiltration by 2.5 fold, while decreasing TAM2 frequency, thus resulting in an even more inflammatory environment.

Example 6: Combinations of STING Agonists and Vaccine Complexes Modulate Intra-Tumoral Expression of PD-L1 and MHC

[0588] The PD-1/PD-L1 axis is the major pathway leading to T cells exhaustion, thus inhibiting the anti-tumoral effect of antigen-specific CD8 T cells, and PD-L1 expression has been shown to be up-regulated on tumoral cells upon intra-tumoral treatment with STING agonist. Furthermore, down-regulation of MHC-I expression on tumor cells is one of the main mechanism of immune evasion. Therefore, the intra-tumoral expression of PD-L1 and MHC-I as well as MHC-II was also monitored in the TC-1 tumor model as described above.

[0589] Results are shown in FIG. 12. Increased PD-L1 expression was found upon treatment with the complex (Z13Mad25Anaxa) comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist alone or in combination with the STINGa, as compared to vehicle or STINGa s.c. monotherapy (FIG. 12A-B). PD-L1 expression was increased on both CD45− and CD45+ cell compartments, highlighting that both, tumoral and immune cells, could promote T cell exhaustion. The main immune cell population to express PD-L1 was identified as TAMs of both types (FIG. 12C-D). Furthermore, with regard to MHC-I expression on tumor cells, both, H2-Kb and H2-db alleles expression, was up-regulated by tumor cells upon treatment with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist alone or in combination with the STINGa, as compared to both vehicle and STINGa monotherapy (FIG. 12E-F), excluding this mechanism of immune evasion and suggesting that vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist could even promote epitope presentation by tumor cells. At the same time, vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist alone or in combination with the STINGa also increased MHC-II expression on CD11b+ cells (FIG. 12G-H) as compared to both vehicle and STINGa monotherapy, thus promoting the presentation of epitopes to CD4 T cells.

[0590] Altogether, these results highlight the profound modulation of TME induced by vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, which is able to turn a cold tumor into hot, and the synergistic effect of STING agonist treatment combined with the vaccine complex, which further increases intra-tumoral immunogenicity, although no effect was observed with STING agonist monotherapy.

Example 7: Antitumoral Effects of the Combination of STING Agonists and Vaccine Complexes

[0591] Next, the anti-tumoral effect of therapeutic of the combination of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with a STING agonist was evaluated in the TC-1 tumor model.

[0592] 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice. When tumors were visible, mice were treated twice at one-week interval as described above in therapeutic settings of the TC-1 tumor model with (i) the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa); (ii) the STING agonist; or (iii) a combination of both.

[0593] Results are shown in FIG. 13. In the TC-1 model, two vaccinations with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist resulted in a significant delay of tumor development and an increased median survival, (FIG. 13A-B). While STINGa monotherapy had only a small effect on tumor growth, its combination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist considerably increased this effect, thereby significantly delaying tumor development and enhancing median survival.

Example 8: Antitumoral Effects of the Combination of the Vaccine Complex with a Distinct STING Agonist

[0594] Next, the anti-tumoral effect of therapeutic of the combination of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide (Z13Mad25Anaxa) agonist with a distinct STING agonist was evaluated in the TC-1 tumor model. Instead of STING agonist ADU-S100 (Aduro), as used in the experiments described above, STING agonist STINGa 2 was used.

[0595] Similarly as described above, 10.sup.5 TC-1 cells were implanted on the back of C57BL/6 mice and assigned to distinct groups (control (no treatment); complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa); STING agonist STINGa 2; and a combination thereof). For the treatment with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa), mice were vaccinated by s.c. injection of 10 nmoles of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad25Anaxa) at day 6 (when tumors were visible) and day 13 post tumor implantation. For the treatment with the STING agonist STINGa 2, mice received 10 μg of STING agonist STINGa 2 administered systemically (s.c.) at days 6, 10, 13 and 17. Whole blood was collected at day 20 and used for antigen-specific CD8 T cells measurement by multimer flow cytometry staining.

[0596] As shown in FIG. 14, monotherapy with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist increased survival and reduced tumor growth, while STING agonist monotherapy only provided a slight improvement. However, combination of both showed a synergistic effect with considerably increased survival and reduced tumor growth. This confirms the results described in Example 7 above with a systemic administration of a distinct STING agonist.

[0597] As shown in FIG. 15, vaccination with the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist is able to elicit circulating HPV-specific CD8 T cells. However, combination with STING agonist treatment further increased the frequency of antigen-specific CD8 T cells, thereby confirming the results described in Example 1 for a distinct STING agonist.

Example 9: ATP128 Immunogenicity in Mouse; the Combination of STINGa and ATP128 Induces a CEA-Specific CD8 T Cells Response

[0598] Female C57BL/6J mice were implanted with 5*10.sup.5 MC38-CEA tumor cells subcutaneously on the back of the mouse. At day 6 and day 13 post tumor implantation, mice were vaccinated with 10 nmol of ATP128, 25 μg of a STING agonist (ADU-S100) or a combination of the two. Both the ATP128 vaccine and the STING agonist were injected subcutaneously at the base of the tail. One week after the second vaccination, mouse blood was collected from the tail vein and the frequency and the total number of CEA-specific CD8 T cells was analyzed by flow cytometry using a custom designed multimer.

[0599] Results are shown in FIG. 16. An IFN-g Elispot assay was carried out one week after the third vaccination with ATP128. ATP128 vaccination elicits CEA-specific CD8 T cells, which can be monitored by multimer staining (FIG. 16A). Multimer staining was performed one week after the second vaccination. The data show that the addition of the STING agonist to ATP128 enhances CEA-specific CD8 T cell responses (FIG. 16 B,C).

Example 10: Combination of STING Agonist and Vaccine Complex Enhances Functionality of CD8 T and CD4 T Cell Peripheral Responses in Tumor-Free Mice

[0600] Similarly to Example 1, the immunogenicity of the combination was evaluated in tumor-free C57BL/6 mice, but using a different complex (Z13Mad39Anaxa; SEQ ID NO: 58). Briefly, tumor-free C57BL/6 mice were vaccinated twice at one week interval (at day 0 and 7) by s.c. injection of 10 nmoles of Z13Mad39Anaxa (SEQ ID NO: 58; an exemplified a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist, which contains CD4 and CD8 epitopes derived from ovalbumin (OVA; SEQ ID NOs 59 and 57, respectively). At about the same time of vaccination, mice received 25 μg of STING agonist ADU-S100 administered via 2×50 μl s.c. injections in each side of the low back. Serum was collected 4 and 24 hours after the first vaccination and IFN-α concentration was measured by ELISA. Whole blood was collected at day 14 and used for antigen-specific CD8 T cells measurement by multimer flow cytometry staining. At the same time, spleens were harvested and splenocytes used for ex vivo stimulation and intracellular cytokine production was analyzed by flow cytometry. Alternatively, splenocytes were used for TCR avidity assay.

[0601] The results are shown in FIG. 17. As shown in FIG. 17A, one week after the second vaccination, circulating (left) and splenic (right) SIINFEKL-specific CD8 T cells were measured by multimer staining. FIG. 17B shows SIINFEKL-specific CD8 T cell TCR avidity measured by ex vivo ELIspot (upper panel) and antigen-specific cytokine production by CD8 T cells measured by intracellular staining after ex vivo stimulation with SIINFEKL peptide (lower panel). FIG. 17C shows the frequency of Treg (FoxP3.sup.+), Th1 (T-bet.sup.+), and Th2 (GATA-3.sup.+) among splenic CD4 T cells as well as the Th1/Th2 ratio (measured by flow cytometry one week after the second vaccination). In addition, antigen-specific cytokine production by CD4 T cells is shown, which was measured by intracellular staining after ex vivo stimulation with ISQAVHAAHAEINEAGR (OVA-CD4) peptide (SEQ ID NO: 59).

[0602] In summary, similar modulation of CD8 and CD4 T cell response was observed as in Example 1 using a different complex (Z13Mad39Anaxa containing CD4 and CD8 epitopes derived from ovalbumin (OVA) in the present case vs. HPV-E7-epitope containing Z13Mad25Anaxa in Example 1). This confirms that the modulation of the T cell response does not depend on the antigenic cargo. Z13Mad39Anaxa vaccination elicited polyfunctional CD8 and CD4 antigen-specific T cells, which produced IFNγ and TNFα following ex vivo stimulation with the specific peptide (fig. S2). Altogether, addition of the STING agonist to a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist profoundly impact frequency and quality of CD8 T cell response along with polarization of CD4 T cell toward Th1.

Example 11: Combination of STING Agonist and Vaccine Complex Inhibits B16-OVA Tumor Growth

[0603] The anti-tumoral efficacy of the combination of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide (Z13Mad39Anaxa) agonist with the STING agonist (STINGa) was then evaluated in the B16-OVA pulmonary metastases tumor model.

[0604] Briefly, 10.sup.5 B16-OVA cells were injected intravenously into C57BL/6 mice. Starting three days post tumor cell intravenous injection, mice were vaccinated twice at one-week interval with an exemplified a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad39Anaxa; SEQ ID NO: 58), STING agonist (STINGa) or a combination of the two. At day 20 (10 days after the last vaccination), lungs were perfused to eliminate blood, the number of pulmonary metastasis was counted, and lung infiltrating lymphocytes (LILs) were analyzed.

[0605] Results are shown in FIGS. 18 and 19. FIG. 18A shows the experimental schedule. The number of metastatic nodules per lung shown in FIG. 18B demonstrate that Z13Mad39Anaxa vaccination resulted in a significant reduction of the number of metastasis and while STINGa monotherapy had no effect, in combination with KISIMA it significantly further lowered the number of metastasis. In addition, the presence and functionality of lung infiltrating lymphocytes (LILs) was analyzed by flow cytometry. The vaccination induced polyfunctional OVA-specific CD8 T cells infiltration, characterized by the expression of granzyme B (GzB), IFNγ and TNFα (FIG. 18C), which were significantly increased with STINGa combination. Similar increase in T cells phenotype and functionality was observed in the periphery (blood and spleen) with a lower magnitude, suggesting that antigen-specific T cells are prevalently recruited to the tumor site, as shown in FIGS. 19A and B. As observed in tumor-free mice (Example 10), the combination of the complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist (Z13Mad39Anaxa; SEQ ID NO: 58) and the STING agonist (STINGa) modulated the polarization of intratumoral CD4 T cells, decreasing the presence of Tregs while increasing the Th1/Th2 ratio (FIG. 18D). Ex vivo stimulation with OVA peptide highlighted the presence of functional antigen-specific CD4 T cells in the spleen but not in the lungs, suggesting that the helping to CD8 T cell response is prevalently happening in the secondary lymphoid organ (FIGS. 18D and 19C).

[0606] Taken together these results show that combination treatment of a complex comprising a cell penetrating peptide, at least one antigen or antigenic epitope, and a TLR peptide agonist with a STING agonist promotes both intratumoral infiltration of antigen-specific effector CD8 T cells and the functionality of peripheral CD4 T cells, resulting in the inhibition of B16-OVA tumor growth.

TABLE-US-00042 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks SEQ ID NO: 1 RQIKIYFQNRRMKWKK CPP: Penetratin SEQ ID NO: 2 YGRKKRRQRRR CPP: TAT minimal domain SEQ ID NO: 3 MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLG ZEBRA amino acid GPSQAPLPCVLWPVLPEPLPQGQLTAYHVSTAPTGSWF sequence (natural SAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGE sequence from APQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQ Epstein-Barr virus PAPVAAPARRTRKPQQPESLEECDSELEIKRYKNRVASRK (EBV)) (YP_401673) CRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDV DSIIPRTPDVLHEDLLNF SEQ ID NO: 4 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLR CPP1 (Z11) LLLKQMC SEQ ID NO: 5 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLR CPP2 (Z12) LLLK SEQ ID NO: 6 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRL CPP3 (Z13) LLK SEQ ID NO: 7 KRYKNRVASRKSRAKFKQLLQHYREVAAAK CPP4 (Z14) SEQ ID NO: 8 KRYKNRVASRKSRAKFK CPP5 (Z15) SEQ ID NO: 9 QHYREVAAAKSSEND CPP6 (Z16) SEQ ID NO: 10 QLLQHYREVAAAK CPP7 (Z17) SEQ ID NO: 11 REVAAAKSSENDRLRLLLK CPP8 (Z18) SEQ ID NO: 12 KRYKNRVA CPP9 (Z19) SEQ ID NO: 13 VASRKSRAKFK CPP10 (Z20) SEQ ID NO: 14 MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEA MAGE-A3 ASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNY PLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAEL VHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKAF SSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQI MPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGRED SILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGP RALVETSYVKVLHHMVKISGGPHISYPPLHEWVLREGEE SEQ ID NO: 15 MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAG mesothelin ETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLST ERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALP LDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPER QRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFV AESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYG PPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQR SSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDES LIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLK HKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLE TLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRG QLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQ DLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLG GAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEV QKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLG LQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLL ASTLA SEQ ID NO: 16 MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPER survivin MAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEE HKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKE TNNKKKEFEETAKKVRRAIEQLAAMD SEQ ID NO: 17 RISTFKNWPF survivin epitope SEQ ID NO: 18 APTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEELT survivin fragment LGEFLKLDRER SEQ ID NO: 19 MQAEGRGTGGSTGDADGPGGPGIPDGPGGNAGGPGE NY-ESO-1 AGATGGRGPRGAGAARASGPGGGAPRGPHGGAASGL NGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLA QDAPPLPVPGVLLKEFTVSGNILTIRLTAADHRQLQLSISS CLQQLSLLMWITQCFLPVFLAQPPSGQRR SEQ ID NO: 20 MERRRLWGSIQSRYISMSVWTSPRRLVELAGQSLLKDE PRAME ALAIAALELLPRELFPPLFMAAFDGRHSQTLKAMVQAW PFTCLPLGVLMKGQHLHLETFKAVLDGLDVLLAQEVRPR RWKLQVLDLRKNSHQDFWTVWSGNRASLYSFPEPEAA QPMTKKRKVDGLSTEAEQPFIPVEVLVDLFLKEGACDEL FSYLIEKVKRKKNVLRLCCKKLKIFAMPMQDIKMILKMV QLDSIEDLEVTCTWKLPTLAKFSPYLGQMINLRRLLLSHIH ASSYISPEKEEQYIAQFTSQFLSLQCLQALYVDSLFFLRGRL DQLLRHVMNPLETLSITNCRLSEGDVMHLSQSPSVSQLS VLSLSGVMLTDVSPEPLQALLERASATLQDLVFDECGITD DQLLALLPSLSHCSQLTTLSFYGNSISISALQSLLQHLIGLS NLTHVLYPVPLESYEDIHGTLHLERLAYLHARLRELLCELG RPSMVWLSANPCPHCGDRTFYDPEPILCPCFMPN SEQ ID NO: 21 MDGGTLPRSAPPAPPVPVGCAARRRPASPELLRCSRRR ASCL2 RPATAETGGGAAAVARRNERERNRVKLVNLGFQALRQ HVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRN ALAGGLRPQAVRPSAPRGPPGTTPVAASPSRASSSPGR GGSSEPGSPRSAYSSDDSGCEGALSPAERELLDFSSWLG GY SEQ ID NO: 22 SAVEYIRALQ ASCL2 epitope SEQ ID NO: 23 ERELLDFSSW ASCL2 epitope SEQ ID NO: 24 AAVARRNERERNRVKLVNLGFQALRQHVPHGGASKKLS ASCL2 fragment KVETLRSAVEYIRALQRLLAEHDAVRNALAGGLRPQAVR PSAPRGPSEGALSPAERELLDFSSWLGGY SEQ ID NO: 25 MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSAT MUC-1 QRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVT LAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHD VTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST APPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA PGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTR PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAP PAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGST APPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPA PGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTR PAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPD TRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVT SAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHG VTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAP PVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPF SIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTS PQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDIS EMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINV HDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSG AGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYG QLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEK VSAGNGGSSLSYTNPAVAATSANL SEQ ID NO: 26 GSTAPPVHN MUC-1 epitope SEQ ID NO: 27 TAPPAHGVTS MUC-1 epitope SEQ ID NO: 28 MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIV TGFPR2 TDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSIC EKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTS NPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS TVVETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNT ELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFP YEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQY WLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHL HSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLS LRLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENV ESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSK VREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVC ETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSE EKIPEDGSLNTTK SEQ ID NO: 29 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIE CEA STPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNR QIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTG FYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVED KDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNG NRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLY GPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVN GTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTV TTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTY LWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYE CGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVN LSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNS GLYTCQANNSASGHSRTIVKTITVSAELPKPSISSNNSKP VEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQL SNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTL DVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSW RINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNN SIVKSITVSASGTSPGLSAGATVGIMIGVLVGVAL SEQ ID NO: 30 YLSGANLNLS CEA epitope SEQ ID NO: 31 SWRINGIPQQ CEA epitope SEQ ID NO: 32 NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLP CEA fragment DSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIA KITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGL SA SEQ ID NO: 33 MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQA P53 MDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAP APAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFL HSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPP PGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLA PPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSD CTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGR NSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKR ALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNE ALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLM FKTEGPDSD SEQ ID NO: 34 MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDS KRas YRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEG FLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGN KCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAF YTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM SEQ ID NO: 35 VVVGAGGVG KRas epitope SEQ ID NO: 36 MASSVGNVADSTEPTKRMLSFQGLAELAHREYQAGDF OGT EAAERHCMQLWRQEPDNTGVLLLLSSIHFQCRRLDRSA HFSTLAIKQNPLLAEAYSNLGNVYKERGQLQEAIEHYRH ALRLKPDFIDGYINLAAALVAAGDMEGAVQAYVSALQY NPDLYCVRSDLGNLLKALGRLEEAKACYLKAIETQPNFAV AWSNLGCVFNAQGEIWLAIHHFEKAVTLDPNFLDAYINL GNVLKEARIFDRAVAAYLRALSLSPNHAVVHGNLACVYY EQGLIDLAIDTYRRAIELQPHFPDAYCNLANALKEKGSVA EAEDCYNTALRLCPTHADSLNNLANIKREQGNIEEAVRL YRKALEVFPEFAAAHSNLASVLQQQGKLQEALMHYKEAI RISPTFADAYSNMGNTLKEMQDVQGALQCYTRAIQINP AFADAHSNLASIHKDSGNIPEAIASYRTALKLKPDFPDAY CNLAHCLQIVCDWTDYDERMKKLVSIVADQLEKNRLPS VHPHHSMLYPLSHGFRKAIAERHGNLCLDKINVLHKPPY EHPKDLKLSDGRLRVGYVSSDFGNHPTSHLMQSIPGMH NPDKFEVFCYALSPDDGTNFRVKVMAEANHFIDLSQIPC NGKAADRIHQDGIHILVNMNGYTKGARNELFALRPAPI QAMWLGYPGTSGALFMDYIITDQETSPAEVAEQYSEKL AYMPHTFFIGDHANMFPHLKKKAVIDFKSNGHIYDNRIV LNGIDLKAFLDSLPDVKIVKMKCPDGGDNADSSNTALN MPVIPMNTIAEAVIEMINRGQIQITINGFSISNGLATTQI NNKAATGEEVPRTIIVTTRSQYGLPEDAIVYCNFNQLYKI DPSTLQMWANILKRVPNSVLWLLRFPAVGEPNIQQYA QNMGLPQNRIIFSPVAPKEEHVRRGQLADVCLDTPLCN GHTTGMDVLWAGTPMVTMPGETLASRVAASQLTCLG CLELIAKNRQEYEDIAVKLGTDLEYLKKVRGKVWKQRISS PLFNTKQYTMELERLYLQMWEHYAAGNKPDHMIKPVE VTESA SEQ ID NO: 37 MAEDSGKKKRRKNFEAMFKGILQSGLDNFVINHMLKN CASP5 NVAGQTSIQTLVPNTDQKSTSVKKDNHKKKTVKMLEYL GKDVLHGVFNYLAKHDVLTLKEEEKKKYYDTKIEDKALIL VDSLRKNRVAHQMFTQTLLNMDQKITSVKPLLQIEAGP PESAESTNILKLCPREEFLRLCKKNHDEIYPIKKREDRRRLA LIICNTKFDHLPARNGAHYDIVGMKRLLQGLGYTVVDEK NLTARDMESVLRAFAARPEHKSSDSTFLVLMSHGILEGIC GTAHKKKKPDVLLYDTIFQIFNNRNCLSLKDKPKVIIVQA CRGEKHGELWVRDSPASLALISSQSSENLEADSVCKIHEE KDFIAFCSSTPHNVSWRDRTRGSIFITELITCFQKYSCCCH LMEIFRKVQKSFEVPQAKAQMPTIERATLTRDFYLFPGN SEQ ID NO: 38 MSSPLASLSKTRKVPLPSEPMNPGRRGIRIYGDEDEVDM COA-1 LSDGCGSEEKISVPSCYGGIGAPVSRQVPASHDSELMAF MTRKLWDLEQQVKAQTDEILSKDQKIAALEDLVQTLRP HPAEATLQRQEELETMCVQLQRQVREMERFLSDYGLQ WVGEPMDQEDSESKTVSEHGERDWMTAKKFWKPGD SLAPPEVDFDRLLASLQDLSELVVEGDTQVTPVPGGARL RTLEPIPLKLYRNGIMMFDGPFQPFYDPSTQRCLRDILDG FFPSELQRLYPNGVPFKVSDLRNQVYLEDGLDPFPGEGR VVGRQLMHKALDRVEEHPGSRMTAEKFLNRLPKFVIRQ GEVIDIRGPIRDTLQNCCPLPARIQEIVVETPTLAAERERS QESPNTPAPPLSMLRIKSENGEQAFLLMMQPDNTIGDV RALLAQARVMDASAFEIFSTFPPTLYQDDTLTLQAAGLV PKAALLLRARRAPKSSLKFSPGPCPGPGPGPSPGPGPGP SPGPGPGPSPCPGPSPSPQ SEQ ID NO: 39 MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIV IL13Ralpha2 DPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSET WKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEV QSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCS WKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQ NIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQN IVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIR EDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCS DDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIF VTGLLLRKPNTYPKMIPEFFCDT SEQ ID NO: 40 LPFGFIL IL13Ralpha2 epitope SEQ ID NO: 41 MNKLYIGNLSENAAPSDLESIFKDAKIPVSGPFLVKTGYA KOC1 FVDCPDESWALKAIEALSGKIELHGKPIEVEHSVPKRQRI RKLQIRNIPPHLQWEVLDSLLVQYGVVESCEQVNTDSET AVVNVTYSSKDQARQALDKLNGFQLENFTLKVAYIPDE MAAQQNPLQQPRGRRGLGQRGSSRQGSPGSVSKQKP CDLPLRLLVPTQFVGAIIGKEGATIRNITKQTQSKIDVHRK ENAGAAEKSITILSTPEGTSAACKSILEIMHKEAQDIKFTE EIPLKILAHNNFVGRLIGKEGRNLKKIEQDTDTKITISPLQE LTLYNPERTITVKGNVETCAKAEEEIMKKIRESYENDIAS MNLQAHLIPGLNLNALGLFPPTSGMPPPTSGPPSAMTP PYPQFEQSETETVHLFIPALSVGAIIGKQGQHIKQLSRFA GASIKIAPAEAPDAKVRMVIITGPPEAQFKAQGRIYGKIK EENFVSPKEEVKLEAHIRVPSFAAGRVIGKGGKTVNELQ NLSSAEVVVPRDQTPDENDQVVVKITGHFYACQVAQRK IQEILTQVKQHQQQKALQSGPPQSRRK SEQ ID NO: 42 MAPKFPDSVEELRAAGNESFRNGQYAEASALYGRALRV TOMM34 LQAQGSSDPEEESVLYSNRAACHLKDGNCRDCIKDCTSA LALVPFSIKPLLRRASAYEALEKYPMAYVDYKTVLQIDDN VTSAVEGINRMTRALMDSLGPEWRLKLPSIPLVPVSAQK RWNSLPSENHKEMAKSKSKETTATKNRVPSAGDVEKAR VLKEEGNELVKKGNHKKAIEKYSESLLCSNLESATYSNRAL CYLVLKQYTEAVKDCTEALKLDGKNVKAFYRRAQAHKAL KDYKSSFADISNLLQIEPRNGPAQKLRQEVKQNLH SEQ ID NO: 43 MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAV RN F-43 ESERSAEQKAIIRVIPLKMDPTGKLNLTLEGVFAGVAEITP AEGKLMQSHPLYLCNASDDDNLEPGFISIVKLESPRRAPR PCLSLASKARAGERGASAVLFDITEDRAAAEQLQQPLGLT WPVVLIWGNDAEKLMEFVYKNQKAHVRIELKEPPAWP DYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQR TAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCA ICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLC MFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAHY HLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMGPRHHRF PRAAHPRAPGEQQRLAGAQHPYAQGWGLSHLQSTSQ HPAACPVPLRRARPPDSSGSGESYCTERSGYLADGPASD SSSGPCHGSSSDSVVNCTDISLQGVHGSSSTFCSSLSSDF DPLVYCSPKGDPQRVDMQPSVTSRPRSLDSVVPTGETQ VSSHVHYHRHRHHHYKKRFQWHGRKPGPETGVPQSRP PIPRTQPQPEPPSPDQQVTRSNSAAPSGRLSNPQCPRAL PEPAPGPVDASSICPSTSSLFNLQKSSLSARHPQRKRRGG PSEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHP LICGPPGLDKRLLPETPGPCYSNSQPVWLCLTPRQPLEPH PPGEGPSEWSSDTAEGRPCPYPHCQVLSAQPGSEEELEE LCEQAV SEQ ID NO: 44 MAPPQVLAFGLLLAAATATFAAAQEECVCENYKLAVNC EpCAM FVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNG SKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGT SMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKA REKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDL VQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDL TVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVI VVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRE LNA SEQ ID NO: 45 GLKAGVIAV EpCAM epitope SEQ ID NO: 46 MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPAS Her2/neu PETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDI QEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALA VLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLI QRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHP CSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLP TDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPAL VTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGS CTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGME HLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASN TAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNL QVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIH HNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVG EGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEE CRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEA DQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDE EGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVV GILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPL TPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGI WIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGV GSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGR LGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLV KSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALE SILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREI PDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFR ELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLE DDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHR HRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVF DGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSET DGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARP AGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQ GGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTF KGTPTAENPEYLGLDVPV SEQ ID NO: 47 MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAP WT1 VLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQE PSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFG PPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTV TFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLG EQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQ MTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEG QSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRR VPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQ MHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHT GVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCR WPSCQKKFARSDELVRHHNMHQRNMTKLQLAL SEQ ID NO: 48 NRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLDVLP antigenic cargo of DSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVLFIA ATP128 KITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGL SAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQFEE LTLGEFLKLDRERAAVARRNERERNRVKLVNLGFQALRQ HVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAVRN ALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSWLG GY SEQ ID NO: 49 STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE TLR2 peptide agonist Anaxa SEQ ID NO: 50 STVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE TLR peptide agonist ″Anaxa″ sequence variant SEQ ID NO: 51 MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVN TLR2 agonist mo- FSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREM HMGB1 KTYIPPKGETKKKFKDPNAPKRPPSAFFLFCSEYRPKIKGE HPGLSIGDVAKKLGEMWNNTAADDKQPYEKKAAKLKEK YEKDIAAYRAKGKPDAAKKGVVKAEKSKKKK SEQ ID NO: 52 NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPE EDA DGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDD MESQPLIGIQST SEQ ID NO: 53 DPNAPKRPPSAFFLFCSEKRYKNRVASRKSRAKFKQLLQH Hp91 YREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAG REVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANI AVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 54 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRL ATP128 LLKNRTLTLFNVTRNDARAYVSGIQNSVSANRSDPVTLD VLPDSSYLSGANLNLSCHSASPQYSWRINGIPQQHTQVL FIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSP GLSAAPTLPPAWQPFLKDHRISTFKNWPFLEGSAVKKQF EELTLGEFLKLDRERAAVARRNERERNRVKLVNLGFQAL RQHVPHGGASKKLSKVETLRSAVEYIRALQRLLAEHDAV RNALAGGLRPQAVRPSAPRGPSEGALSPAERELLDFSSW LGGYSTVHEILSKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 55 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRL Z13Mad25Anaxa LLKQAEPDRAHYNIVTFSSKSSTVHEILSKLSLEGDHSTPP SAYGSVKPYTNFDAE SEQ ID NO: 56 RAHYNIVTF HPV-E7 CD8 epitope SEQ ID NO: 57 SIINFEKL OVA CD8 epitope SEQ ID NO: 58 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRL Z13Mad39Anaxa LLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQD WLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIIN FEKLTEWTGSSTVHEILSKLSLEGDHSTPPSAYGSVKPYT NFDAE SEQ ID NO: 59 ISQAVHAAHAEINEAGR OVA CD4 epitope