Cell Penetrating Peptides and Complexes Comprising the Same

Abstract

The present invention provides a cell penetrating peptide derived from ZEBRA, which is optionally linked to a cargo molecule, such as at least one antigen or antigenic epitope. The present invention also provides a complex comprising the cell penetrating peptide and the cargo molecule. In particular, compositions, such as a pharmaceutical compositions and vaccines are provided, which may be useful for example in the prevention and/or treatment of a diseases and/or a disorder including cancer, hematological disorders, infectious diseases, autoimmunity disorders and transplant rejections.

Claims

1. A cell penetrating peptide comprising an amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1, wherein the amino acid sequence sharing at least 80% sequence identity with SEQ ID NO: 1: provides cell penetrating functionality; has a serine at position 12; and has a length of at least 36 amino acids in total.

2.-4. (canceled)

5. The cell penetrating peptide according to claim 1, wherein the cell penetrating peptide consists of an amino acid sequence according to SEQ ID NO: 1.

6. A complex comprising the cell penetrating peptide according to claim 1 and a cargo molecule, wherein the cell penetrating peptide according to claim 1 and the cargo molecule are preferably covalently linked.

7. The complex according to claim 6, wherein the cargo molecule is selected from the group consisting of: (i) a peptide, a polypeptide, or a protein; (ii) a polysaccharide; (iii) a lipid; (iv) a lipoprotein; (v) a glycolipid; (vi) a nucleic acid; (vii) a small molecule drug or toxin; and (viii) an imaging or contrast agent.

8. (canceled)

9. The complex according to claim 6, wherein the cargo molecule is at least one antigen or antigenic epitope.

10. The complex according to claim 9, wherein the at least one antigen or antigenic epitope comprises or consists of at least one pathogen epitope and/or at least one tumor epitope, preferably the at least one antigen or antigenic epitope comprises or consists of at least one tumor epitope.

11. (canceled)

12. The complex according to claim 9, wherein the complex comprises more than one antigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenic epitopes.

13. (canceled)

14. (canceled)

15. The complex according to claim 9, wherein the complex further comprises a TLR peptide agonist.

16. The complex according to claim 15, wherein the TLR peptide agonist is a TLR2, TLR4 and/or TLR5 peptide agonist, preferably a TLR2 peptide agonist and/or a TLR4 peptide agonist.

17. (canceled)

18. The complex according to claim 16, wherein the TLR peptide agonist comprises or consists of an amino acid sequence according to SEQ ID NO: 5 or a functional sequence variant thereof.

19. (canceled)

20. (canceled)

21. The complex according to claim 15, wherein the complex is a recombinant polypeptide or a recombinant protein and the cell penetrating peptide, the at least one antigen or antigenic epitope and the TLR peptide agonist are positioned in N-terminal.fwdarw.C-terminal direction of the main chain of said complex in the order: (a) cell penetrating peptideat least one antigen or antigenic epitopeTLR peptide agonist; or (b) TLR peptide agonistcell penetrating peptideat least one antigen or antigenic epitope, wherein the cell penetrating peptide, the at least one antigen or antigenic epitope and the TLR peptide agonist may be optionally linked by a further component, in particular by a linker or a spacer.

22. (canceled)

23. A nucleic acid encoding the cell penetrating peptide according to claim 1.

24. A vector comprising the nucleic acid according to claim 23.

25. A host cell comprising the vector according to claim 24.

26. (canceled)

27. A cell loaded the complex according to claim 6.

28. The cell according to claim 27, wherein said cell is an antigen presenting cell, preferably a dendritic cell.

29. (canceled)

30. (canceled)

31. A pharmaceutical composition comprising at least one complex according to claim 6 and a pharmaceutically acceptable carrier.

32.-37. (canceled)

38. A method of preventing and/or treating of a diseases and/or a disorder including cancer, hematological disorders, infectious diseases, autoimmunity disorders and transplant rejections in a subject, the method comprising administering to the subject a complex according to claim 6.

39. The method of claim 38, wherein the disease to be prevented and/or treated is cancer and/or a hematological disorder, preferably a malignant neoplasm of the brain or a malignant neoplasm of lymphoid, hematopoietic and related tissue, most preferably glioblastoma.

40. (canceled)

41. The nucleic acid according to claim 23 encoding a complex comprising the cell penetrating peptide and a cargo molecule, wherein the complex is a recombinant peptide, a recombinant polypeptide or a recombinant protein.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0333] 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.

[0334] FIG. 1 shows a schematic overview (A) and the amino acid sequences (B) for the different ZEBRA CPP truncations (Z12, Z13, Z14, Z15, Z16, Z17, Z18, Z19 and Z20), which were designed and synthesized as described in Example 1. Numbers at the beginning and at the end of each ZEBRA truncation refer to the corresponding amino acid position in ZEBRA.

[0335] FIG. 2 shows for Example 2 the identification of the best Zebra CPP truncations in vitro (proliferation assay) and in vivo (CD8 T cell immune response after vaccination). (A) BMDCs were loaded 4 h with 300 nM of each Zebra CPP truncation (Z13 to Z20) conjugated to OVACD8 epitope, then washed and incubated o.n. with 100 ng/ml of LPS. Efficient MHC class I-restricted processing and presentation was monitored after 4 days with CFSE-labeled na?ve OVA.sub.257-264-specific CD8.sup.+ T cells from OT-1 T cell receptor (TCR) transgenic mice. Data from one experiment representative of two independent experiments. (B) Mice were vaccinated by s.c. injection (right flank) at wk0, and wk2 with 10 nmoles of each Zebra CPP truncation (Z12 to Z20) conjugated to OVACD8 epitope with 100 pg of anti-CD40. Mice were also injected with 50 ?g of Hiltonol i.m. (right hind leg). Mice were bled 1 wk after the 2.sup.nd vaccination for assessing OVA.sub.257-264-specific CD8 T cells.

[0336] FIG. 3 shows for Example 3 the comparison of the transduction capacity of (A) Z13, (B) Z14, (C) Z15 and (D) Z18 Zebra CPP truncations. Transduction was assessed in cells with high phagocytosis capacity (dendritic cells of human and mice origin) and in cells with poor phagocytosis capacity (T cells from human, K562, or mouse, EL4, origin). Cells were incubated for 30 minutes, 2 h or 4 h with the fluorescein-conjugated constructs (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM or Z18OVACD8FAM) then subjected to a 30s wash with an acidic buffer to remove membrane bound peptide before FACS analysis.

[0337] FIG. 4 shows for Example 4 the in vitro epitope presentation (MHC I and MHC II) assay. BMDCs were loaded 4 h with 300nM of the different Zebra CPP truncation conjugated to both ovalbumin CD8 and CD4 epitopes (Z13OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4), then washed and incubated o.n. with 100 ng/ml of LPS. Efficient MHC class I or class II-restricted presentation was monitored after 4 days with CFSE-labeled na?ve OVA.sub.257-264-specific CD8.sup.+T cells from OT-1 T cell receptor (TCR) transgenic mice OT1 cells (A) or CFSE-labeled na?ve OVA.sub.323-339-specific CD.sup.+T cells from OT-2 T cell TCR transgenic mice respectively. As positive control, BMDCs were pulsed for 1 h with 5 uM peptide. Data from one experiment representative of three independent experiments.

[0338] FIG. 5 shows for Example 5 the in vitro epitope presentation (MHC I) assay by human dendritic cells. Human monocyte-derived DCs were loaded with 1 ?M of scramble-MART1, Z13-MART1, Z14-MART1, Z15-MART1 or Z18-MART1 for 4 h, wash and then incubated o.n. at 37? C. Specific TCR-transfected T cells were then added to DCs and incubated for 5 h with monensin and brefeldinA before intracellular FACS staining for CD107, IFN-? and TNF-?.

[0339] FIG. 6 shows for Example 6 CD8 and CD4 T cell immune responses elicited by vaccination with Zebra CPP truncations combined to TLR3 agonist (Hiltonol). Mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmoles of OVACD8CD4 (the cargo without Zebra CPP truncation), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 and i.m. injection of 50 ?g of Hiltonol (right hind leg). One week after the last vaccination, Elispot assay was performed on spleen cells for detecting IFN-y-producing OVA.sub.257-264-specific CD8 T cells (A) and OVA.sub.323-339-specific CD4 T cells (B). *, p<0.05; **, p<0.01.

[0340] FIG. 7 shows for Example 6 CD8 and CD4 T cell immune responses elicited by vaccination with Zebra CPP truncations combined to TLR2 agonist (Pam3CSK4). Mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmoles of OVACD8CD4 (the cargo without Zebra CPP truncation), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 or Z18OVACD8CD4 and 20 ?g of Pam3CSK4. One week after the last vaccination, Elispot assay was performed on spleen cells for detecting IFN-?-producing OVA.sub.257-264-specific CD8 T cells (A) and OVA.sub.323-339-specific CD4 T cells (B). *, p<0.05; **, p<0.01.

[0341] FIG. 8 shows for Example 6 CD8 and CD4 T cell immune responses elicited by vaccination with Zebra CPP truncations combined to TLR4 agonist (MPLA). Mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmoles of Z13OVACD8CD4, Z15OVACD8CD4 or Z18OVACD8CD4 and 20 ?g of MPLA. One week after the last vaccination, Elispot assay was performed on spleen cells for detecting IFN-?-producing OVA.sub.257-264-specific CD8 T cells (A) and OVA.sub.323-339-specific CD4 T cells (B). *, p<0.05; **, p<0.01.

[0342] FIG. 9 shows for Example 6 that Z18 did not elicit self antigen-specific CD8 T cell responses, whereas Z13 and Z14 were able to promote high self antigen-specific CD8 immune responses. The constructs were tested in combination with Hiltonol (A) and MPLA (B). Briefly, Mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmol of Z13OVACD4gp1 00CD8, Z14OVACD4gp1 00CD8, Z18OVACD4gp100CD8 and i.m. injection of 50 pg of Hiltonol (right hind leg) or s.c. injection of 20 ?g of MPLA. One week after the last vaccination, spleen cells were restimulated in vitro for 7 days with gp100CD8 peptide and stained as described in Example 6.

[0343] FIG. 10 shows for Example 7 the therapeutic effect of Zebra CPP truncations on tumor growth. C57BL/6 mice were implanted s.c. with 3?10.sup.5 EG7-OVA tumor cells in the left flank and vaccinated three times (d5, d13 and d21) by s.c. injection of 10nmoles of Z-truncOVACD8CD4 peptides and 20 ?g of MPLA in the right flank. Tumor size was measured with a caliper. A, C and E: tumor growth (mean of 7 mice per group?SEM). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (2-way Anova test at the day when tumor size of all control mice reach a size superior to 1000 mm.sup.3). B, D and F: survival curve of 7 mice per group. Median survival is indicated on the graph (m.s.). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (Log-Rang test).

[0344] FIG. 11: shows for Example 8 the effect of complexes having different CPPs on CD8 T cells in spleen (A), draining lymph nodes (B) and bone marrow (C). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. (right flank) with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. Nine days after the 5.sup.th vaccination, mice were euthanized, organs recovered and multimer staining was performed.

[0345] FIG. 12: shows for Example 8 the effect of complexes having different CPPs on T cells in spleen (CD8 T cell response (A) and CD4 T cell response (B)). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. (right flank) with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. (A) nine days after the 5.sup.th vaccination, Elispot assay was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide. (B) nine days after the 5.sup.th vaccination, Elispot assay was performed on spleen cells stimulated with OVACD4 peptide.

[0346] FIG. 13: shows for Example 8 the effect of complexes having different CPPs on CD8 T cell effector function. C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. (right flank) with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. Nine days after the 5.sup.th vaccination, intracellular staining was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide.

[0347] FIG. 14: shows for Example 9 the effect of complexes having different CPPs on tumor growth (A) and survival rates (B). C57BL/6 mice were implanted s.c. with 3?10.sup.5 EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank. (A) Tumor growth of 7 mice per group (mean?SEM); *, p<0.05; ****, p<0.0001 (2-way Anova test at day 28). (B) Survival curve of 7 mice per group. Median survival is indicated on the graph (m.s.). *, p<0.05; **, p<0.01; ***,p<0.001 (Log-rank test).

EXAMPLES

[0348] 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.

Example 1

Design and Synthesis of Different ZEBRA CPP Truncations

[0349] Different ZEBRA CPP truncations were designed and synthesized as shown in FIG. 1. In particular, peptides were synthesized on an ABI 433 synthesizer customized to perform Boc chemistry with in situ neutralization as already described (Hartley (2004), Proc. Natl. Acad.

[0350] Sci. 101, 16460-16465). Purity and integrity of each peptide were routinely verified by HPLC and mass spectrometry. The amino acid sequences of the different ZEBRA CPP truncations are shown in the following:

TABLE-US-00009 Z12: (SEQ ID NO: 20) KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLK Z13: (SEQ ID NO: 1) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLK Z14: (SEQ ID NO: 21) KRYKNRVASRKSRAKFKQLLQHYREVAAAK Z15: (SEQ ID NO: 22) KRYKNRVASRKSRAKFK Z16: (SEQ ID NO: 23) QHYREVAAAKSSEND Z17: (SEQ ID NO: 24) QLLQHYREVAAAK Z18: (SEQ ID NO: 25) REVAAAKSSENDRLRLLLK Z19: (SEQ ID NO: 26) KRYKNRVA Z20: (SEQ ID NO: 27) VASRKSRAKFK

Example 2

Identification of the Best ZEBRA CPP Truncations In Vitro and In Vivo

[0351] The objective of this study was to select the best ZEBRA CPP truncations in vitro and in vivo from the different ZEBRA CPP truncations designed and synthesized in Example 1.

[0352] To this end, each Zebra CPP truncation (Z12 to Z20) was conjugated to the OVACD8 epitope. Accordingly, the following fusion peptides/fusion proteins were synthesized as described in Example 1:

TABLE-US-00010 Z12OVACD8 Sequence: [SEQ ID NO: 28] KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKEQLESIIN FEKLTEWT Z13OVACD8 Sequence: [SEQ ID NO: 29] KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKEQLESIIN FEKLTEWT Z14OVACD8 Sequence: [SEQ ID NO: 30] KRYKNRVASRKSRAKFKQLLQHYREVAAAKEQLESIINFEKLTEWT Z15OVACD8 Sequence: [SEQ ID NO: 31] KRYKNRVASRKSRAKFKEQLESIINFEKLTEWT Z16OVACD8 Sequence: [SEQ ID NO: 32] QHYREVAAAKSSENDEQLESIINFEKLTEWT Z17OVACD8 Sequence: [SEQ ID NO: 33] QLLQHYREVAAAKEQLESIINFEKLTEWT Z18OVACD8 Sequence: [SEQ ID NO: 34] REVAAAKSSENDRLRLLLKEQLESIINFEKLTEWT Z19OVACD8 Sequence: [SEQ ID NO: 35] KRYKNRVAEQLESIINFEKLTEWT Z20OVACD8 Sequence: [SEQ ID NO: 36] VASRKSRAKFKEQLESIINFEKLTEWT

[0353] For comparison, the following OVACD8 peptide (without any CPP) was also synthesized:

TABLE-US-00011 OVACD8 Sequence: [SEQ ID NO: 37] EQLESIINFEKLTEWT

[0354] The functionality of the different ZEBRA CPP truncations was validated both in vitro and in vivo, based on the capacity to stimulate MHC class-I restricted CD8 T cells specific for a model antigen OVA (OVA.sub.257-264-specific CD8.sup.+ T cells from OT-1 T cell receptor (TCR) transgenic mice). This capacity should reflect transport of the OVA antigen cargo into APCs. The functional read-out of these experiments was proliferation of OVA-specific CD8 T cells in vitro (FIG. 2A), and for the in vivo studies, the induction of OVA-specific CD8 T cells in the blood of mice vaccinated with the different CPP-OVA conjugates together with adjuvant (FIG. 2B).

[0355] To assess the proliferation of OVA-specific CD8 T cells in vitro, bone marrow derived dendritic cells (BMDCs) were prepared from C57BL/6 mice as previously described (Santiago-Raber (2003), J. Exp. Med. 197, 777-788), and used at day 9-10 of culture.

[0356] BMDCs were loaded with 0.3 ?M of each Zebra CPP truncation (Z13 to Z20) conjugated to OVACD8 epitope as described above for 4 hours at 37? C., washed 3 times, then matured overnight at 37? C. with 100 ng/ml LPS (from Salmonella abortus, equi S-form, Enzo Life Sciences). Antigen-loaded mature BMDC were then mixed at a ratio of 1:10 with OT-1 T cell receptor (TCR) transgenic mice splenocytes that had been stained with 10 ?M 5-(and 6) Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies). After 4 days co-incubation of spleen cells and BMDCs, antigen-specific proliferation was assessed by flow cytometry, measuring CFSE dilution on CD8 T cells and live-gated cells. OT-1 mice express TCR specific for MHC class I restricted OVA.sub.257-264. Results are shown in FIG. 2A.

[0357] To assess the induction of OVA-specific CD8 T cells in vivo, C57/BL6 mice were assigned to nine different groups (Z12 to Z20, respectively). Each group of mice was vaccinated by subcutaneous injection of 10 nmol of one selected Zebra CPP truncation (one selected from Z12 to Z20) conjugated to OVACD8 epitope as described above with 100 ?g of anti-CD40 at week0 and week2. Mice were also injected with 50 ?g of Hiltonol i.m. (right hind leg). Mice were bled 1 wk after the 2.sup.nd vaccination in order to assess OVA.sub.257-264-specific CD8 T cells. Results are shown in FIG. 2B.

[0358] Taken together, the data show that truncated CPPs generally conserved their immunogenicity. In general, the in vivo vaccination experiment (FIG. 2B) showed more pronounced differences than the in vitro experiment (FIG. 2A). In summary, the data indicate that Z13, Z14, Z15 and Z18 were the best truncations for promoting cross-presentation and were therefore selected for further study.

Example 3

In Vitro Transduction with ZEBRA CPP Truncations

[0359] In order to more directly assess the transduction capacities of the selected ZEBRA CPP truncations Z13, Z14, Z15 and Z18 fluorescein-conjugated constructs (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM and Z18OVACD8FAM) were synthesized. To this end, 5-(and-6)-carboxyfluorescein (mixed isomers; e.g. ThermoFisher Scientific catalogue no. C194) was added to each of Z13OVACD8 (SEQ ID NO: 29), Z14OVACD8 (SEQ ID NO: 30), Z15OVACD8 (SEQ ID NO: 31) and Z18OVACD8 (SEQ ID NO: 34).

[0360] These fluorescein-conjugated constructs (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM and Z18OVACD8FAM) were tested for their ability to transduce different human and mouse cell types, in particular (i) cells with high phagocytosis capacity, namely dendritic cells (DCs) of human (BMDCs) and mice origin (mo-DCs), and (ii) cells with poor phagocytosis capacity, namely T cells from human, K562, or mouse, EL4, origin). The EL-4 thymoma cell line was maintained in complete RPMI 1640 medium. The K562 cell line was maintained in complete Iscove's modified Dulbecco's medium. BMDC, mo-DCs, EL-4 or K562 cells were incubated for 4 h at 37? C. with the fluorescein-conjugated constructs as described above (Z13OVACD8FAM, Z14OVACD8FAM, Z15OVACD8FAM or Z18OVACD8FAM). For removal of membrane bound peptide prior to cell transduction, a 30s wash with an acidic buffer (0.2 M Glycine, 0.15 M NaCl, pH 3) was performed. Cells were then analyzed by flow cytometry (FACS). Results are shown in FIG. 3.

[0361] Transduction efficacy was different depending on the cell type. Z13 and Z14 were rapidly entering in the majority of the cells. In contrast, the kinetic of transduction for Z15 and Z18 was slower. Overall, transduction efficacy was higher in human DCs, which might be correlated to phagocytic activity. However, high transduction was also observed in EL4 cells. All the peptides showed a modest transduction in dendritic cells of murine origin. For all cell types, Z18 exhibited however a lower transduction efficacy compared to other ZEBRA CPP truncations.

Example 4

In vitrocapacity of ZEBRA CPP Truncations to Promote Epitope Presentation on MHC class I and MHC class II

[0362] In order to assess the capacity of the ZEBRA CPP truncations to stimulate an integrated immune response including CD4 T cells, antigenic cargoes were produced that included both CD8 and CD4 T cell epitopes from OVA, and conjugated to Z13, Z15 and Z18. Accordingly, the following constructs (Z13OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4) were synthesized:

TABLE-US-00012 Z13OVACD8CD4: Sequence: [SEQ ID NO: 42] KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKEQLESIIN FEKLTEWTESLKISQAVHAAHAEINEAGREVVG Z15OVACD8CD4 Sequence: [SEQ ID NO: 43] KRYKNRVASRKSRAKFKEQLESIINFEKLTEWTESLKISQAVHAAHAEIN EAGREVVG Z18OVACD8CD4 Sequence: [SEQ ID NO: 44] REVAAAKSSENDRLRLLLKEQLESIINFEKLTEWTESLKISQAVHAAHAE INEAGREVVG

[0363] Processing and presentation of the CD8 and CD4 T cell epitopes were monitored, as described in Example 2 for CD8 T cell epitopes, by measuring the in vitro proliferation of na?ve OT-1 T cells (CD8 T cell epitopes; FIG. 4A) and OVA.sub.323-339-specific CD4.sup.+ T cells from OT-2 T cell TCR transgenic mice, respectively (FIG. 4B). Briefly, OT-2 T cell TCR transgenic mice express TCR specific for MHC class II restricted OVA.sub.323-339. Results are shown in FIG. 4A (CD8) and 4B (CD4).

[0364] The data indicate that proliferation of both CD8 and CD4 T cells occurred in vitro with all tested ZEBRA CPP truncations (Z13, Z15 and Z18), confirming an efficient transduction of the DCs and delivery of the cargo into antigen processing pathways for both MHC class I and MHC class II. However, Z18 was less efficient that Z13 or Z15.

Example 5

In Vitro Capacity of ZEBRA CPP Truncations to Promote Epitope Presentation on MHC Class I by Human DCs

[0365] In order to assess the capacity of the ZEBRA CPP truncations to promote epitope presentation by human DCs, the following constructs (Z13-MART1, Z14-MART1, Z15-MART1 and Z18-MART1, with each of the Z13, Z14, Z15 and Z18 CPP conjugated to the HLA-A2 epitope of the tumor associated antigen (TAA) MART1)) were synthesized:

TABLE-US-00013 Z13-MART1 Sequence: [SEQ ID NO: 45] KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKTTAEELAG IGILTVILGV Z14-MART1 Sequence: [SEQ ID NO: 46] KRYKNRVASRKSRAKFKQLLQHYREVAAAKTTAEELAGIGILTVILGV Z15-MART1 Sequence: [SEQ ID NO: 47] KRYKNRVASRKSRAKFKTTAEELAGIGILTVILGV Z18-MART1 Sequence: [SEQ ID NO: 48] REVAAAKSSENDRLRLLLKTTAEELAGIGILTVILGV As control, scramble-MART1 was used: [Sequence ID NO: 38] ISQAVHAAHAEINEAGRTTAEELAGIGILTVILGV

[0366] These constructs (Z13, Z14, Z15, and Z18 CPPs conjugated to the HLA-A2 epitope of the tumor associated antigen (TAA) MART1) were assessed for their capacity to promote epitope presentation on MHC class I by human DCs. To this purpose, DCs from an HLA-A2+ donor were prepared from elutriated monocytes, cultured in CelIGro DC medium (CellGenix, Freiburg, Germany) with granulocyte-macrophage colony-stimulating factor (GM-CSF; 2500 U/ml) (Leucomax; Schering-Plough, Kenilworth, N.J.) and interleukin-4 (IL-4; 1000 U/ml) (CellGenix). The immature DCs were matured with cytokines: IL-1? (10 ng/ml), IL-6 (1000 U/ml), tumor necrosis factor ? (TNF?; 10 ng/ml) (all from CellGenix) and prostaglandin E.sub.2 (1 ?g/ml) (Sigma-Aldrich). DCs were rested in CellGro DC medium for 2 hours and loaded with 1 ?M Scramble-MART1, Z13-MART1, Z14-MART1, Z15-MART-1 or Z18-MART1 for 4 hrs at 37? C. before being washed once and plated at 300 000 cells per well in round-bottomed 96-well plates. Non-peptide loaded DCs were used as a negative control. T cells were expanded from peripheral blood mononuclear cells using Dynabeads ClinExVivo CD3/CD28 (Life Technologies, Oslo, Norway). Expanded T cells (day 10 post activation) were transfected with the DMFS TCR specific for MART1 by mRNA electroporation. T cells were co-incubated with DCs at a ratio of 1:2 for 5 hours in the presence of GolgiPlug and GolgiStop (both BD Biosciences) before intracellular staining was performed using the following antibodies after FcR blocking; CD4 (RPAT4), CD8 (RPAT8), CD107a (H4A3) (BD Biosciences), IFN-? (4S.B3) and TNF-? (MAb11) (BD Biosciences), all from eBioscience except where noted. Fixation and permabilization was performed using the PerFix kit from Beckman Coulter according to manufacturer's instructions. Cells were analysed using an LSR II flow cytometer (BD Biosciences) and results were processed with Kaluza (Beckman Coulter) software. All conditions were tested in duplicate, and the experiment was repeated up to three times.

[0367] These experiments showed an efficient stimulation of human MART1-specific CD8 T cells that was CPP dependent (FIG. 5), and particularly efficacious using Z13, Z14 and Z15 CPPs. Z18 CPP was identified as markedly less efficient.

Example 6

Capacity of ZEBRA CPP Truncation-Based Vaccines to Elicit CD8 and CD4 T Cell Immune Responses

[0368] To extend the previous findings to in vivo vaccination, Z13, Z14, Z15 and Z18 conjugated to antigenic cargo containing both CD8 and CD4 T cell epitopes from OVA (as described in Example 4 for Z13, Z15 and Z18) was synthesized. For Z14 the construct was as follows:

TABLE-US-00014 Z14OVACD8CD4 Sequence: [SEQ ID NO: 49] KRYKNRVASRKSRAKFKQLLQHYREVAAAKEQLESIINFEKLTEWTESLK ISQAVHAAHAEINEAGREVVG

[0369] The following construct without ZEBRA CPP was used as control:

TABLE-US-00015 OVACD8CD4 Sequence: [SEQ ID NO: 50] EQLESIINFEKLTEWTESLKISQAVHAAHAEINEAGREVVG

[0370] Firstly, those constructs were tested with Hiltonol adjuvant (a TLR3 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and i.m. injection of 50 ?g of Hiltonol. One week after the last vaccination, mice were bled for assessing OVA.sub.257-264-specific CD8 T cells by FACS MHC-peptide multimer staining and elispot analysis. The enzyme-linked immunospot (ELISPOT) assay for detection of peptide-specific gamma interferon (IFN-?)-secreting T cells was performed essentially as described previously (Miyahira (1995), J. Immunol. Methods, 181, 45-54) For analysis of ex vivo cytokine secretion, splenocytes were incubated o.n. in ELISPOT plates in the presence or absence of 5 ?M of OVA.sub.257-264 or OVA.sub.323-339. The number of peptide-specific IFN-?-producing cells was calculated by subtracting the number of IFN-?-secreting cells cultured without peptide to that obtained with cells cultured with peptide. Results are shown in FIG. 6B.

[0371] Next, those constructs were tested with Pam3CSK4 adjuvant (a TLR2 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and of 20 ?g of Pam3CSK4. One week after the last vaccination, Elispot analysis was performed on spleen cells as described above. Results are shown in FIG. 7.

[0372] Thereafter, the constructs were tested with MPLA adjuvant (a TLR4 agonist). To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection of 10 nmol of OVACD8CD4 (the cargo without Zebra CPP truncation; as described above), Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4, Z18OVACD8CD4 (as described in Examples 4 and 6) and of 20 ?g of MPLA. One week after the last vaccination, Elispot analysis was performed on spleen cells as described above. Results are shown in FIG. 8.

[0373] Taken together, the data showed that: i) Z18 induced the highest CD8 and CD4 immune response when combined with Pam3CSK4 whereas ii) Z13 is the most efficacious when combined with MPLA; iii) in contrast, for any adjuvant, the Z15 CPP elicited low CD8 T cell immune responses.

[0374] To extend the previous findings to self antigen-specific immune response, Z13, Z14 and Z18 conjugated to antigenic cargo containing a self antigen (CD8 epitope from gp100) and CD4 T cell epitope from OVA were synthesized.

TABLE-US-00016 Z13OVACD4gp100CD8 Sequence: [SEQ ID NO: 39] KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKISQA VHAAHAEINEAGREVVGVGALKVPRNQDWLGVPR Z14OVACD4gp100CD8 Sequence: [SEQ ID NO: 40] KRYKNRVASRKSRAKFKQLLQHYREVAAAKiESLKISQAVHAAHAEINEA GREVVGVGALKVPRNQDWLGVPR Z18OVACD4gp100CD8 Sequence: [SEQ ID NO: 41] REVAAAKSSENDRLRLLLKiESLKISQAVHAAHAEINEAGREVVGVGALK VPRNQDWLGVPR

[0375] Firstly, those constructs were tested with Hiltonol or MPLA adjuvant. To this end, mice were vaccinated three times (wk0, wk2 and wk9) by s.c. injection (right flank) of 10 nmol of Z13OVACD4gp100CD8, Z14OVACD4gp100CD8, Z18OVACD4gp100CD8 and i.m. injection of 50 ?g of Hiltonol (right hind leg) or s.c. injection of 20 ?g of MPLA. One week after the last vaccination, spleen cells were restimulated in vitro for 7 days with gp100CD8 peptide and stained as follow. Briefly, for surface staining, after FcR blocking, the following mAb were used: CD4 (RMA4-4), CD8 (53-6.7), CD11b (M1/70), CD19 (6D5), all from BD Biosciences. Dead cells were identified with LIVE/DEAD yellow fluorescent reactive dye (L34959) from Life Technologies and were excluded from analyses. MHC-peptide multimers were from Immudex (Copenhagen, Denmark). Multimer gating strategy used a dump gate (CD4, CD11b, CD19) and excluded dead cells. Results are shown in FIG. 9.

[0376] The data indicated that: i) Z18 did not elicit self antigen-specific CD8 T cell responses; ii) Z13 and Z14 are able to promote high self antigen-specific CD8 immune response when combined with MPLA or Hiltonol.

Example 7

Therapeutic Effect of ZEBRA CPP Truncation-Based Vaccines on Tumor Growth

[0377] To investigate the effects of different ZEBRA CPP truncation-based vaccines on tumor growth and survival, the effects of the constructs Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4 (as described in Examples 4 and 6) were investigated in the EG.7-OVA s.c. model. On d0, C57BL/6 mice were implanted s.c. with 3?10.sup.5 EG7-OVA tumor cells in the left flank and assigned to five different groups (control, Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 and Z18OVACD8CD4). Mice were vaccinated three times (namely, at d5, d13 and d21 after tumor implantation) by s.c. injection of either 10 nmol of Z13OVACD8CD4, Z14OVACD8CD4, Z15OVACD8CD4 or Z18OVACD8CD4 and 20?g of MPLA in the right flank. Tumor size was measured with a caliper.

[0378] Results are shown in FIG. 10. These results show that vaccination with all of the constructs reduce tumor volume and increase survival time. However, those effects were only slight with constructs with Z15 and Z18, but much more pronounced and highly significant for constructs with Z13 and Z14. Thus, Z13 and Z14 are superior to Z15 and Z18.

Example 8

Comparison of T Cell Immune Responses with Complexes Having Different Cell Penetrating Peptides

[0379] Based on the results of Example 7, the best ZEBRA CPP truncations Z13 and Z14 were selected for further investigation, namely in a complex according to the present invention. To this end, two fusion proteins Z13Mad5Anaxa and Z14Mad5Anaxa were designed and synthesized. The two fusion proteins differed only in the cell penetrating peptides (Z13 or Z14). Both fusion proteins comprisein addition to the CPP (Z13 or Z14)-(i) the protein MAD5, which contains various epitopes of different antigens, namely OVACD4, gp100CD8, EalphaCD4 and OVACD8 epitopes, and (ii) the TLR2 peptide agonist Anaxa.

[0380] In the following, the amino acid sequences of Z13Mad5Anaxa and Z14Mad5Anaxa are shown:

TABLE-US-00017 Z13Mad5Anaxa Sequence: [SEQ ID NO: 19] MHHHHHHKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLR LLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINF EKLTEWTGSS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE Z14Mad5Anaxa Sequence: [SEQ ID NO: 51] MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKESL KISQAVHAAH AEINEAGREV VGVGALKVPR NQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWT GSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE

[0381] To investigate the CD8 T cell immune responses in more detail, C57BL/6 mice were assigned to three different groups (3-4 mice per group): naive, Z13Mad5Anaxa or Z14Mad5Anaxa.

[0382] C57BL/6 mice of the Z13Mad5Anaxa group and of the Z14Mad5Anaxa group were vaccinated five times (Week1, Week2, Week4, Week6 and Week8) s.c. with 2 nmol of either Z13Mad5Anaxa or Z14Mad5Anaxa. Nine days after the 5.sup.th vaccination, mice were euthanized, organs recovered and multimer staining was performed to identify the percentage of SIINFEKL-specific CD8 T cells in the spleen, bone marrow and draining lymph nodes (inguinal and axillary).

[0383] The results are shown in FIG. 11. Mice vaccinated with Z13Mad5Anaxa or with Z14Mad5Anaxa showed a similar increase in multimer-positive cells, in particular in the spleen and bone marrow as well as a slight increase in draining lymph nodes.

[0384] To further investigate the CD8 T cell effector function after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 37) nine days after the 5.sup.th vaccination in order to quantify IFN-? producing cells.

[0385] The results are shown in FIG. 12A. Mice vaccinated with Z13Mad5Anaxa showed a significant increase in IFN-? producing cells compared to na?ve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-? producing cells compared to na?ve mice, however, the increase was not significant.

[0386] To investigate the CD4 T cell responses after vaccination with complexes with different CPPs, in the same groups of mice as described above Elispot assay was performed on spleen cells stimulated with OVACD4 peptide (SEQ ID NO: 52) nine days after the 5th vaccination in order to quantify IFN-? producing cells.

TABLE-US-00018 OVACD4 Sequence: [SEQ ID NO: 52] ISQAVHAAHAEINEAGR

[0387] The results are shown in FIG. 12B. Mice vaccinated with Z13Mad5Anaxa showed a highly significant increase in IFN-? producing cells compared to na?ve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-? producing cells compared to na?ve mice, however, again the increase was not significant.

[0388] In addition, in the above described groups of mice, intracellular staining was performed on spleen cells stimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 37) to identify CD107a.sup.+IFN-?.sup.+TNF-?.sup.+ cells. Results are shown in FIG. 13. Mice vaccinated with Z13Mad5Anaxa or with Z14Mad5Anaxa showed a similar increase in CD107a.sup.+IFN-?.sup.+TNF-?.sup.+ cells.

Example 9

Comparison of the Effect of Complexes Having Different Cell Penetrating Peptides on Tumor Growth and Survival in the EG.7-OVA s.c. Model

[0389] To investigate the effects of complexes having different cell penetrating peptides on tumor growth and survival the EG.7-OVA s.c. model was used. On d0 C57BL/6 mice were implanted s.c. with 3?10.sup.5 EG7-OVA tumor cells in the left flank and assigned to three different groups (na?ve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinated twice at d5 and d13 after tumor implantation by s.c. injection of either 0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.

[0390] Results are shown in FIG. 14. Vaccination with Z13Mad5Anaxa or with Z14Mad5Anaxa resulted in significantly decreased tumor volumes compared to control mice (FIG. 14A) as well as to significantly increased survival rates compared to control mice (FIG. 14B). Those results indicate that both complexes, Z13Mad5Anaxa and Z14Mad5Anaxa, are able to significantly decrease tumor growth and to significantly prolong survival. However, the significance was considerably more pronounced with Z13Mad5Anaxa than with Z14Mad5Anaxa (cf. FIG. 14A and B). For example, with Z13Mad5Anaxa the median survival time was increased from 26 days (control) to 35 days, whereas with Z14Mad5Anaxa the median survival time was increased from 26 days (control) to 31 days only. Accordingly, the best results were achieved with Z13.

TABLE-US-00019 TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NO Sequence Remarks SEQ ID NO: 1 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSEN CPP: Z13 DRLRLLLK SEQ ID NO: 2 MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRV ZEBRA amino acid YQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAY sequence (natural HVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPV sequence from SDITQNQQTNQAGGEAPQPGDNSTVQTAAAV Epstein - Barr virus VFACPGANQGQQLADIGVPQPAPVAAPARRTR (EBV)) (YP_401673) KPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQ LLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDS IIPRTPDVLHEDLLNF SEQ ID NO: 3 ESLKISQAVHAAHAEINEAGREVVGVGALKVPR MAD5 cargo NQDWLGVPRFAKFASFEAQGALANIAVDKANL DVEQLESIINFEKLTEWTGS SEQ ID NO: 4 MHHHHHHKRYKNRVASRKSRAKFKQLLQHYRE Z13Mad5 VAAAKSSENDRLRLLLKESLKISQAVHAAHAEINE AGREVVGVGALKVPRNQDWLGVPRFAKFASFE AQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 5 STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE TLR2 peptide agonist Anaxa SEQ ID NO: 6 NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYR TLR4 peptide agonist VTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSE EDA YTVSVVALHDDMESQPLIGIQST SEQ ID NO: 7 DDDK enterokinase target site SEQ ID NO: 8 IEDGR factor Xa target site SEQ ID NO: 9 LVPRGS thrombin target site SEQ ID NO: 10 ENLYFQG protease TEV target SEQ ID NO: 11 LEVLFQGP PreScission protease SEQ ID NO: 12 RX(R/K)R furin target site SEQ ID NO: 13 GGGGG peptidic linker SEQ ID NO: 14 GGGG peptidic linker SEQ ID NO: 15 EQLE peptidic linker SEQ ID NO: 16 TEWT peptidic linker SEQ ID NO: 17 MHHHHHHNIDRPKGLAFTDVDVDSIKIAWESP EDAZ13Mad5 QGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAEL QGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRY KNRVASRKSRAKFKQLLQHYREVAAAKSSENDRL RLLLKESLKISQAVHAAHAEINEAGREVVGVGAL KVPRNQDWLGVPRFAKFASFEAQGALANIAVD KANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 18 MHHHHHHSTVHEILCKLSLEGDHSTPPSAYGSV AnaxaZ13Mad5 KPYTNEDAEKRYKNRVASRKSRAKFKQLLQHYRE VAAAKSSENDRLRLLLKESLKISQAVHAAHAEINE AGREVVGVGALKVPRNQDWLGVPRFAKFASFE AQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 19 MHHHHHHKRYKNRVASRKSRAKFKQLLQHYR Z13Mad5Anaxa EVAAAKSSENDRLRLLLKESLKISQAVHAAHAEIN EAGREVVGVGALKVPRNQDWLGVPRFAKFASFE AQGALANIAVDKANLDVEQLESIINFEKLTEWTG SSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 20 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSE CPP: Z12 NDRLRLLLK SEQ ID NO: 21 KRYKNRVASRKSRAKFKQLLQHYREVAAK CPP: Z14 SEQ ID NO: 22 KRYKNRVASRKSRAKFK CPP: Z15 SEQ ID NO: 23 QHYREVAAAKSSEND CPP: Z16 SEQ ID NO: 24 QLLQHYREVAAAK CPP: Z17 SEQ ID NO: 25 REVAAAKSSENDRLRLLLK CPP: Z18 SEQ ID NO: 26 KRYKNRVA CPP: Z19 SEQ ID NO: 27 VASRKSRAKFK CPP: Z20 SEQ ID NO: 28 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSE Z12OVACD8 NDRLRLLLKEQLESIINFEKLTEWT SEQ ID NO: 29 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSE Z13OVACD8 NDRLRLLLKEQLESIINFEKLTEWT SEQ ID NO: 30 KRYKNRVASRKSRAKFKQLLQHYREVAAAKEQL Z14OVACD8 ESIINFEKLTEWT SEQ ID NO: 31 KRYKNRVASRKSRAKFKEQLESIINFEKLTEWT Z15OVACD8 SEQ ID NO: 32 QHYREVAAAKSSENDEQLESIINFEKLTEWT Z16OVACD8 SEQ ID NO: 33 QLLQHYREVAAAKEQLESIINFEKLTEWT Z17OVACD8 SEQ ID NO: 34 REVAAAKSSENDRLRLLLKEQLESIINFEKLTEWT Z18OVACD8 SEQ ID NO: 35 KRYKNRVAEQLESIINFEKLTEWT Z19OVACD8 SEQ ID NO: 36 VASRKSRAKFKEQLESIINFEKLTEWT Z20OVACD8 SEQ ID NO: 37 EQLESIINFEKLTEWT OVACD8 peptide SEQ ID NO: 38 ISQAVHAAHAEINEAGRTTAEELAGIGILTVILGV Scramble-MART1 SEQ ID NO: 39 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSE Z13OVACD4gp100CD8 NDRLRLLLKESLKISQAVHAAHAEINEAGREVVG VGALKVPRNQDWLGVPR SEQ ID NO: 40 KRYKNRVASRKSRAKFKQLLQHYREVAAAKiESL Z14OVACD4gp100CD8 KISQAVHAAHAEINEAGREVVGVGALKVPRNQ DWLGVPR SEQ ID NO: 41 REVAAAKSSENDRLRLLLKiESLKISQAVHAAHAEI Z18OVACD4gp100CD8 NEAGREVVGVGALKVPRNQDWLGVPR SEQ ID NO: 42 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSE Z13OVACD8CD4 NDRLRLLLKEQLESIINFEKLTEWTESLKISQAVHA AHAEINEAGREVVG SEQ ID NO: 43 KRYKNRVASRKSRAKFKEQLESIINFEKLTEWTESL Z15OVACD8CD4 KISQAVHAAHAEINEAGREVVG SEQ ID NO: 44 REVAAAKSSENDRLRLLLKEQLESIINFEKLTEWTE Z18OVACD8CD4 SLKISQAVHAAHAEINEAGREVVG SEQ ID NO: 45 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSEN Z13-MART1 DRLRLLLKTTAEELAGIGILTVILGV SEQ ID NO: 46 KRYKNRVASRKSRAKFKQLLQHYREVAAAKTTAE Z14-MART1 ELAGIGILTVILGV SEQ ID NO: 47 KRYKNRVASRKSRAKFKTTAEELAGIGILTVILGV Z15-MART1 SEQ ID NO: 48 REVAAAKSSENDRLRLLLKTTAEELAGIGILTVILGV Z18-MART1 SEQ ID NO: 49 KRYKNRVASRKSRAKFKQLLQHYREVAAAKEQL Z14OVACD8CD4 ESIINFEKLTEWTESLKISQAVHAAHAEINEAGREV VG SEQ ID NO: 50 EQLESIINFEKLTEWTESLKISQAVHAAHAEINEAG OVACD8CD4 REVVG SEQ ID NO: 51 MHHHHHHKRY KNRVASRKSR AKFKQLLQHY Z14Mad5Anaxa REVAAAKESLKISQAVHAAHAEINEAGREVVGG ALKVPRNQDWLGVPRFAKFASFEAQGALANIAV DKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLS LEGDHST PPSAYGSVKP YTNFDAE SEQ ID NO: 52 ISQAVHAAHAEINEAGR OVACD4 peptide SEQ ID NO: 53 GGGGS (G4S)n linker repeated sequence