NEW VACCINAL STRATEGY

Abstract

The present invention relates to the prevention and treatment of disease like cancer. The inventors have previously characterized MELOE-1 antigen as an IRES dependent, melanoma specific translation product from a lncRNA mainly transcribed in the melanocytic lineage. MELOE-1 contains numerous class II epitopes and one HLA-A*0201-restricted CD8 epitope eliciting a frequent repertoire of high avidity T cells. They designed various synthetic long peptide (SLPs) comprising a CD4 epitope coupled to the CD8 epitope by a serie of linkers of 4 to 6 aa and studied the efficacy of T cell clone activation by SLP-loaded DC in vitro. Particularly, they evaluated the ability of a few selected SLPs to stimulate specific T cells proliferation of PBL from healthy donors in vitro and finally, they explored the vaccination potential of their best SLP candidate in vivo in an HLA*A0201/HLA-DRB0101 transgenic mouse. Thus, the present invention relates a SLP comprising a CD4 class II peptide linked to a CD8 class I peptide by a specific linker and its use in the treatment of disease like cancers.

Claims

1. A peptidic linker comprising the amino acids sequence: Xaa1-LSV-Xaa5-Xaa6 (SEQ ID NO:1) wherein Xaa1 is an amino acid selected from the group consisting of alanine (A), leucine (L), valine (V), serine (S) or Glycine (G) and Xaa5 and Xaa6 are each independently present or absent and if present are an amino acid selected from the group consisting of alanine (A), leucine (L), valine (V), and Glycine (G).

2. The peptidic linker according to claim 1 wherein the peptidic linker is selected from the group consisting of the amino acid sequences LLSV (SEQ ID NO:4), VLSV (SEQ ID NO:5), SLSV (SEQ ID NO:6), GLSV (SEQ ID NO:7), LLSVG (SEQ ID NO:8), LLSVGG (SEQ ID NO:9), VLSVG (SEQ ID NO:10), VLSVGG (SEQ ID NO:11) GLSVGG (SEQ ID NO:176), GLSVVV (SEQ ID NO:177), SLSVAA (SEQ ID NO:178), SLSVGG (SEQ ID NO:179), ALSVGG (SEQ ID NO:180) and LLSVGA (SEQ ID NO:191).

3. A synthetic long peptide (SLP) comprising a CD4 class II peptide linked to a CD8 class I peptide by the peptidic linker of claim 1, wherein the CD4 class II peptide is linked at its C-terminal position to the peptidic linker and the CD8 class I peptide is linked at its N-terminal position to the peptidic linker.

4. The SLP according to claim 3 wherein the CD4 class II peptide and the CD8 class I peptide are peptides derived from the proteins MELOE-1, MELOE-2, Melan-A, HTERT, NY-ESO-1 and GP100.

5. The SLP according to claim 3 wherein the CD4 class II peptide and/or the CD8 class I peptide has an amino acid sequence selected from the group consisting of: SEQ ID NO:15 to 64, SEQ ID NO:121 to 144 and SEQ ID NO:148 to 158.

6. The SLP according to claim 3, wherein the SLP has an amino acid sequence selected from the group consisting of: SEQ ID NO:65 to 120, SEQ ID NO:145 to 147, SEQ ID NO:159, SEQ ID NO:168 and SEQ ID NO:181 to 189.

7. A nucleic acid sequence encoding a linker according to claim 1 or an SLP comprising the linker.

8. (canceled)

9. (canceled)

10. (canceled)

11. A vaccine composition comprising an SLP according to claim 3.

12. (canceled)

13. A T lymphocyte that recognizes specifically a SLP according to claim 3.

14. A method for treating or preventing cancer, infectious diseases, inflammatory diseases or auto-immune diseases in a patient in need thereof comprising, administrating to the patient a SLP according to claim 3 or a nucleic acid encoding the SLP.

15. The method of claim 14, wherein the SLP is administered in a vaccine composition.

16. The method of claim 14, wherein the cancer is melanoma.

Description

FIGURES

[0136] According to the FIGS. 2 to 7 of the patent application, all the number of the peptidic sequences indicated in these figures correspond to the number of the peptidic sequences of the linker tested.

[0137] FIG. 1. Panel A. Representation of MELOE-1 antigen. MELOE-1 is a 46 aa antigen containing multiple class II epitopes presented in various HLA context and a HLA A*0201 restricted class I epitope. Depicted here are the epitopes used in this study. Panel B. Schematic representation of the Synthetic Long Peptide (SLP) used in this study. The designed SLP comprises a class II epitope in N-ter fused to a class I epitope via a cathepsin-sensitive linker. The SLP ranges from 30-35 amino acid length.

[0138] FIG. 2. Panel A. Titration curve of TNF-α production (intra-cellular staining and flow cytometry analysis) by a MELOE-1, HLA DRb1*11 restricted CD4 T cell clone activated by mature MO-DC loaded with relevant SLP (TSMELOE-1.sub.24-37 xxxxMELOE-1.sub.36-44 SL). Panel B. Titration curve of TNF-α production (intra-cellular staining and flow cytometry analysis) by a MELOE-1, HLA A*0201 restricted CD8 T cell clone activated by mature MO-DC loaded with relevant SLP (TSMELOE-1.sub.24-37 xxxxMELOE-1.sub.36-44 SL).

[0139] FIG. 3. Titration curve of TNF-α production (intra-cellular staining and flow cytometry analysis) by a MELOE-1, HLA A*0201 restricted CD8 T cell clone activated by mature MO-DC loaded with relevant SLP (TSMELOE-1.sub.11-23xxxxMELOE-1.sub.36-44 SL).

[0140] FIG. 4. Titration curve of TNF-α production (intra-cellular staining and flow cytometry analysis) by a MelanA, HLA A*0201 restricted CD8 T cell clone activated by mature MO-DC loaded with relevant SLP (TSMELOE-1.sub.13-27xxxxMelanA.sub.26-35A27LIL).

[0141] FIG. 5. Titration curve of TNF-α production (intra-cellular staining and flow cytometry analysis) by a MELOE-1, HLA DRB1*01 restricted CD4 T cell clone activated by mature MO-DC loaded with relevant SLP (TSMELOE-1.sub.13-27xxxxMELOE-1.sub.36-44 SL).

[0142] FIG. 6. Panel A. in vitro PBMC stimulation with MELOE-1 SLP (TSMELOE-1.sub.13-27xxxxMELOE-1.sub.36-44 SL). Panel B. in vitro PBMC stimulation with MELOE-1 SLP (MELOE-1.sub.13-27xxxxMELOE-1.sub.36-44) containing various linkers or the native MELOE-1.sub.11-46 Assessment of CD8 responses (number of positive microcultures and frequency of positive tetramer+/CD8+ lymphocytes per well) on a representative healthy donor (HD1). PBMC were stimulated in 96 well plates with cytokines cocktail and SLP (5 μM) for 21 days. Microcultures were screened by tetramer and CD8 double staining (cut off 0.5% of total). Panel C. Comparison of in vitro stimulation with the same aSLP as in panel B containing either the linker GGGG or the linker LLSVGG with PBMC from six healthy donors (black circle) and one melanoma patient (open triangle). **p=0.004, paired t-test. Panel D. in vitro PBMC stimulation with MelanA SLP (TSMELOE-1.sub.13-27xxxxMelanA.sub.26-35A27LIL). Assessment of CD8 responses. PBMC from a healthy donor were stimulated and analysed as described above.

[0143] FIG. 7. Immunisation of HLA DRB1*01xHLA A*0201 transgenic mice with 2 SLP (TSMELOE-1.sub.13-27xxxxMELOE-1.sub.36-44 SL). Mice received a prime injection of 100 μg of SLP followed by 2 boosts of 50 μg. SLP were emulsified in Incomplete Freund Adjuvant and administered with 50 μg of PolyI:C. Panel A. linker GGGG (SEQ ID NO:160). Panel B and C. linker LLSVGG (SEQ ID NO:9). At day 28 splenocytes were harvested, CD8+ T cells (panel A and B) or CD4+ T cells (panel C) were sorted and stimulated ex vivo with (black bars) or without (white bars) MELOE-136-44 peptide (panel A and B) or MELOE-113-27 (panel C). IFN-g production was assessed by ELISPOT.

[0144] FIG. 8. Panel A. Comparison of tumor sizes at day 36 in the three groups of mice, vaccinated with PBS, or adjuvant alone or aSLP containing the LLSVGG linker. Bars indicate median of each group. Panel B and C. Monitoring of the growth of subcutaneous SARC-A2-MELO-1 cells (2×105 cells) in PBS-treated mice (panel B) or vaccinated mice (panel C). Prime vaccination and boost are indicated by arrows.

[0145]

TABLE-US-00010 TABLE 1 Evaluation of different linkers to promote cross-presentation of the SLP MELOE-1.sub.13-27 xxxx MELOE-1.sub.36-44 Sequence Fold Codes linkers Linkers Codes SLP change.sup.a N.sup.b MELOE-1.sub.13-27 xxxx MELOE-1.sub.36-44 SEQ ID NO: 160 GGGG SEQ ID NO 173 <0, 1 5 SEQ ID NO: 4 LLSV SEQ ID NO: 64  6-14 4 SEQ ID NO: 10 VLSVG SEQ ID NO: 71  3-13 2 SEQ ID NO: 8 LLSVG SEQ ID NO: 89 10-20 3 SEQ ID NO: 175 PLSVII SEQ ID NO: 79 2.3-10  2 SEQ ID NO: 9 LLSVGG SEQ ID NO: 87 12-56 3 SEQ ID NO: 11 VLSVGG SEQ ID NO: 69 1.5-2   2 SEQ ID NO: 176 GLSVGG SEQ ID NO: 81 1 SEQ ID NO: 177 GLSVVV SEQ ID NO: 85 1 SEQ ID NO: 178 SLSVAA SEQ ID NO: 73 7-8 SEQ ID NO: 179 SLSVGG SEQ ID NO: 75 1-8 SEQ ID NO: 180 ALSVGG SEQ ID NO: 77 1-3 .sup.aFold change is calculated as EC50 (M) of MELOE-1.sub.11-46/EC50 (M) SLP. EC50 were determined by the TNF-a response curves of a MELOE-1.sub.11-46 specific CD8 T cell clone to SLP-loaded DC. .sup.bnumber of independent experiments

TABLE-US-00011 TABLE 2 Assessement of CD8 responses in 5 healthy donors after PBMC stimulation in vitro with the SLP MELOE-1.sub.13-27 xxxx MELOE-1.sub.36-44 Linkers Codes SLP HD1 HD2 HD3 HD4 HD5 GGGG SEQ ID 21/96  8/96 0/96 3/96 (SEQ ID NO: 173 NO: 160) LLSV SEQ ID  38/96*  27/96** 4/96 5/96 (SEQ ID NO: 64 NO: 4) LLSVGG SEQ ID 16/96 (SEQ ID NO: 87 NO: 9) Meloe-1.sub.11-46 / 18/96  8/96

TABLE-US-00012 TABLE 3 CD8 responses in HLA-DRB1*0101/HLA-A*0201 transgenic mice following immunization with SLP MELOE- 1.sub.13-27 xxxx MELOE-1.sub.36-44. Codes SLP Exp#1 Exp#2 Exp#3 MELOE-1.sub.11-46 / 0/5 Linker GGGG SEQ ID 1/3 3/5 (SEQ ID NO: 173 NO: 160 Linker LLSV SEQ ID 2/3 (SEQ ID NO: 64 NO: 4) Linker LLSVG SEQ ID 2/5 (SEQ ID NO: 89 NO: 8) Linker LLSVGG SEQ ID 5/5 (SEQ ID NO: 87 NO: 9)

[0146] CD8+ splenocytes from immunized mice were tested by INFg-ELlspot following restimulation with the short epitope MELOE-1.sub.36-44. Mice were considered positive if the number of spots after restimulation were more than twice the background level and above ten spots.

Example

[0147] Material & Methods

[0148] Peptide Synthesis

[0149] Designed peptides were purchased at >90% purity (Protogenix, Genecust). Lyophilised powder was resuspended at 10 mMstock in DMSO and stored at −80° C. Purity was assessed by HPLC and precise quantification was calculated based on protein content analysis.

[0150] Mice

[0151] HLA-DRB1*0101/HLA-A*0201 transgenic mice (A2/DR1 mice) have been previously described (Pajot et al Eur. J. Immunol 2004, Dosset et al Clin Cancer Res, 2012, Rangan et al Oncotarget, 2017). Mice were bred and housed at Animalerie centrale UFC/UFR “SMP” Besançon. Female mice 6-10 weeks old were used in the experiments. All experiments were performed according to the good laboratory practices after agreement #58 by the local ethical committee.

[0152] Tumor Cell Line

[0153] The SARC-L1 cell line which spontaneously occurred in A2/DR1 mice was genetically modified to over-express HLA-A*0201 as previously described (Rangan et al Oncotarget, 2017). SARC-A2 cells were then transduced with a gammaretroviral dicistronic vector encoding the whole MELOE-1 antigen and, downstream of an IRES element, a puromycin N-acetyltransferase allowing selection of the transduced cells with puromycin (5 μg/mL). HLA-A*0201 expression was checked by flow cytometry. The capacity of the MELOE-1-transduced SARC-A2 (SARC-A2-MELOE-1) cells to present the A2-restricted MELOE-1 epitope was confirmed by their recognition by an HLA-A*0201-restricted T cell clone (data not shown).

[0154] Monocyte—Derived Dendritic Cells (MO-DC) Generation

[0155] Monocytes were purified from PBMC of HLA-A*0201 and/or HLA-DRB1*0101 healthy donors (Etablissement Français du Sang, Nantes, France) by negative selection using magnetic sorting (Stem Cell). Immature dendritic cells were generated by culturing monocytes in RPMI supplemented with 2% albumin, 1000 IU/mL of GM-CSF and 200 IU/mL of IL-4 (Cellgenix, Freiburg, Germany) for 5 days. Then, DC were pulsed with a concentration range of the various SLP tested and matured with 20 ng/mL of TNF-α and 50 μg/mL of PolyI:C (Sigma-Aldrich, France) for 16 h at 37° C.

[0156] T Cell Clone Assay

[0157] Once loaded and matured, MO-DC were washed twice in RPMI-2% albumin before being co-cultivated with T cell clones at a 1:1 ratio for 5 h in presence of brefeldinA (Sigma-Aldrich, 10 μg/ml). Intra-cellular TNF production was assessed by flow cytometry after 4% paraformaldehyde fixation and 0,1% saponin permeabilisation (clone Mab11, 5 μg/ml, Biolegend, San Diego, USA). The percentage of TNF positive cells was assessed amongst CD8+ (clone RPA-T8, BioLegend) or CD4+ cells (clone RPA-T4, BioLegend). Curve fitting and EC50 analysis were performed with GraphPad Prism v6.01 (log (agonist) vs. response, 4 parameters).

[0158] In Vitro Stimulation

[0159] At day 0, PBMCs were plated in 96 wells at 2×10.sup.5 cells/wells in RMPI 1640 medium containing 8% human serum, 501 U/mL 11-2 (Proleukin, Novartis) and stimulated with 5-10 μM of various SLP tested. Medium was supplemented following the acDC protocol (Martinuzzi et al., 2011) by addition of 1000 U/mL of GM-CSF (CellGenix) and 500 UI/mL of II-4 (CellGenix). After 24 hours TNF-α(10001 U/mL), IL-113 (10 ng/mL) and prostaglandin E.sub.2 (1 μM) (R&D Systems, Minneapolis, USA) were added as DC maturation agents. After 21 days, the percentage of microcultures containing specific CD8+ T cells was evaluated by surface staining with anti-CD8 mAb and tetramer staining (HLA A*0201-MELOE-1.sub.36-44 (SEQ ID NO:31) or MelanA.sub.A27L26-35, SEQ ID NO:64) (recombinant protein facility, P2R, Nantes). Comparison of a number of positive microcultures after stimulation with aSLP in each subject was evaluated by Fisher's exact test and as a group using paired t-test (α=0.5%).

[0160] Mice and Immunization

[0161] HLA-DRB1*0101/HLA-A*0201 transgenic mice (A2/DR1 mice) were used to evaluate immunogenicity of peptides. Group of 5 female A2/DR1 mice (6 to 10 weeks) were immunized subcutaneously with 100 μg of each peptides emulsified in incomplete Freund adjuvant (v/v) plus 50 μg of polyI:C. At day 14 and day 28 boost vaccinations were performed with 50 μg of each peptide along with the same adjuvants. All experiments were carried out according to the good laboratory practices defined by the animal experimentation Rules in France.

[0162] ELISpot

[0163] Specific immune responses were evaluated 10 days after the last boost by ELISpot-IFN-g (Diaclone, France). Briefly, freshly isolated splenocytes or CD8 positive T cells from spleen (CD8+ T Cell Isolation Kit, Miltenyi Biotec) were incubated (2.105 cells per well) during 15-20 hours in ELISpot plate in the presence of the class I MELOE-1 epitope (5 μg/ml final) or medium (X-vivo, as negative control) or PMA-ionomycin (positive control). Ex vivo CD4 T cell responses were evaluated on the CD8 negative fraction as above with the HLA-DRB1*0101-restricted epitope MELOE-1.sub.13-27. Spots were revealed according to the supplier's recommendations. Spot-forming cells were counted using the «C.T.L. Immunospot» system (Cellular Technology Ltd). Results were considered positive when the ELISPOT counts were >10 spots and above 2× the background (medium). (Dosset et al Clin Cancer Res, 2012)

[0164] Anti-Tumor Vaccination

[0165] A2/DR1 mice were subcutaneously injected with 2×105 SARC-A2-MELOE-1 cells in 100 μl in the right flank. The first vaccination with aSLP (100 μg in IFA, as above) or adjuvant alone or PBS started when tumor became detectable (around 10 mm2). A boost vaccination with 50 μg of aSLP was performed at day 20. Tumor growth was evaluated twice a week using a caliper and mice were euthanized when their tumor exceeded 300 mm.sup.2. The efficiency of anti-tumor vaccination was evaluated by Fisher's exact test (α=0.5%).

[0166] Results

[0167] Design of SLP

[0168] Our aim was to design SLP of 25-35aa long that could be efficiently processed by DC to generate both defined class I and class II restricted T cell epitopes. The rationale for designing our SLPs was as follows: we decided to place the class II-restricted epitope first, then the protease sensitive linker and then the class I-restricted epitope (FIG. 1B). We figured that with this design, even if protease cleavage generated a class I epitope elongated at the N-terminus, the trimming necessary for loading into HLA class I molecules would be performed by the physiological ERAD system in DC (Serwold, Nature 2002). On the other hand, the production of a slightly elongated class II-restricted epitope should not prevent its loading into class II molecules since class II HLA are much more permissive in terms of epitope length than class I HLA (O'Brien, Immunome Res 2008ref). The next critical choice which constitutes the core of this work was the amino-sequence of the linker peptide. Considering that SLP are internalized and processed by DC through the endosomal route ( ) the main enzymes involved in initial processing of SLP are cathepsins. They represent a large family with multiple cathepsins involved in antigen processing by DC among which the main endopeptidases are cathepsins S, L and D (for review, Chapman, 2006). Using the MEROPS on-line data base (merops@ebi.ac.uk, Rawlings Nucleic Acids Res 2016 ref), we designed our linker sequences so that they could presumably be cut by at least one of these three cathepsins.

[0169] Separating Overlapping Epitopes Increases DC Presentation

[0170] The first situation that we wanted to explore was the case of an overlap between a class II epitope and a class I epitope i.e. where the processing of the class II epitope should result in the destruction of the class I epitope. In our MELOE-1 antigen, this is precisely the case between the DRB01*11-restricted epitope CPPWHPSERISSTL (SEQ ID NO:26) and the HLA-A*0201-restricted TLNDECWPA (SEQ ID NO:31) (FIG. 1A). We figured that the competition for processing between the two epitopes could possibly be alleviated by separating these two epitopes in a synthetic long peptide. We designed two such SLP (31aa) where the two epitopes where linked either with a control GGGG linker (SEQ ID NO:160) or with a potential cathepsin-sensitive linker LVGS (SEQ ID NO:161) (SLP control: AACPPWHPSERISSTLGGGGTLNDECWPASL (SEQ ID NO:171).

[0171] We then assessed the efficiency of DC processing and presentation of these SLP at various concentrations by measuring the dose-dependent activation of a DRB01*11-restricted specific T cell clone through classical presentation (FIG. 2A) and the dose-dependent activation of a CD8 HLA-A*0201-restricted specific T cell clone (FIG. 2B) through cross-presentation. This was compared to DC loaded with either full length MELOE-1 (46 aa) or a 31 aa-long peptide that contains the native overlapping epitopes (data not shown).

[0172] For the presentation of the DRB01*11-restricted epitope, there was no difference of efficacy between the full length MELOE-1 and the 31aa-long native long peptide (EC50 around 7×10.sup.−8M for both). The presentation was improved when the two epitopes were separated by the GGGG linker (SEQ ID NO:160) (EC50=2.6×10.sup.−8M) and further improved with the LVGS linker (SEQ ID NO:161) (1.4×10.sup.−8M) (FIG. 2A).

[0173] For the cross-presentation of the HLA-A0201-epitope, differences between SLP were more striking. In fact, full length MELOE-1 and the natural long peptide did not differ significantly in terms of cross-presentation (7.9×10.sup.−6M vs 5.9×10.sup.−6M respectively) while separating the two epitopes with the GGGG linker (SEQ ID NO:160) improved cross presentation (1.9×10.sup.−6M). The linker LVGS (SEQ ID NO:161), designed as a target for cathepsins, markedly improved cross-presentation (1.1×10.sup.−7M) (FIG. 2B). This observation prompted us to test a variety of linkers for their ability to favor cross-presentation.

[0174] Strong Influence of the Linker on Cross Presentation of the HLA Class I Epitope

[0175] To assess the influence of the linker sequence on cross-presentation, we designed another serie of SLP containing the previously described DRB1*0101-restricted epitope MELOE-1.sub.11-23 (SEQ ID NO:24) linked to the HLA-A*0201-restricted MELOE-1 epitope. The choice of a DRB1*0101-restricted epitope was motivated by the fact that this HLA haplotype is frequent in the population and DRB1*0101 transgenic mice are available for in vivo studies. As presented in FIG. 3, we first focused on cross-presentation and observed major differences in the presentation of the HLA-A*0201-restricted MELOE-1 epitope depending on the linker used. In three independent experiments, using full length MELOE-1 as reference (EC50=2.5×10.sup.−7M), we observed that some linkers induced poorer cross-presentation (GGGG (SEQ ID NO:160), 1.2×10.sup.−6M and GSGS (SEQ ID NO:162), 4.3×10.sup.−7M), ten fold better presentation (ASLG (SEQ ID NO:163), 1.2×10.sup.−8M and the previously tested LVGS (SEQ ID NO:161), 3.4×10.sup.−8M) or hundred fold better (PIVLG (SEQ ID NO:164), 3.2×10.sup.−9M; LLSV (SEQ ID NO:4), 2.8×10.sup.−9M; VLSVG (SEQ ID NO:10), 2.1×10.sup.−9M) (SLP control: AATSREQFLPSEGAACPPWGGGGTLNDECWPA (SEQ ID NO:172)).

[0176] In the next serie of experiments, we designed SLP with a newly identified DRB1*0101-restricted epitope, MELOE-1.sub.13-27 (SEQ ID NO:21) (FIG. 1A), obtained after in vitro T cell stimulation and cloning from a DRB1*0101 blood donor (data not shown). We wanted to assess the effect of changing the C-terminus of the class II epitope on SLP cross presentation i.e. how it may affect processing at the level of the linker. According to the MEROPS data base, the C-terminus of MELOE-1.sub.13-27 (PPW, SEQ ID NO:165) should be less favorable for cutting at the linker level than the C-terminus of MELOE-1.sub.11-23 (AAC, SEQ ID NO:166). Indeed, when we tested the two linkers previously identified as the most favorable for cross presentation (LLSV (SEQ ID NO:4) and VLSVG (SEQ ID NO:10)) with this new epitope MELOE-1.sub.13-27 (SEQ ID NO:21), they were still better than full length MELOE-1 but only ten fold so instead of hundred fold (Table 1). This suggested than the aminoacid sequence contributed by the C terminus of the class II epitope could affect processing efficiency at the linker level. Considering this observation, we decided to lengthen the linker to 6 aa and explored variations in the aa sequence around the LSV (SEQ ID NO:167) core that was favorable for processing. SLP containing the linker LLSVGG (SEQ ID NO:9) was the most efficiently processed and cross-presented in this experiment (SLP control: TSREQFLPSEGAACPPWGGGGTLNDECWPASL (SEQ ID NO:173)). The different SLP designed have a sequence of SEQ ID NO:65 to SEQ ID NO:120).

[0177] In the same line of thought, we wanted to check whether the linkers defined as favorable for cross presentation of the HLA-A*0201-restricted MELOE-1 would remain so if we changed the class I epitope. We replace the MELOE-1.sub.36-44 (SEQ ID NO:31) epitope by the HLA-A*0201-restricted Melan-A A27L epitope (ELAGIGILTV, SEQ ID NO:64), thus changing the aa downstream of the linker from TLND (SEQ ID NO:169) to ELAG (SEQ ID NO:171) and assessed DC cross presentation to a Melan-A/A*0201 specific T cell clone. The long peptide Melan-A.sub.16-40 A27L that can crosspresent the HLA-A*0201-restricted Melan-A A27L epitope (SEQ ID NO:64) was used as reference in these experiments. As shown on FIG. 4, this epitope replacement resulted in dramatic changes in terms of linker efficiency to promote cross-presentation. SLP containing the linker LLSV (SEQ ID NO:4) became one of the worst in terms of cross presentation (EC50=2.0×10.sup.−5M) suggesting that cleavage downstream of the linker was favored and thus destroyed the Melan-A epitope. With an additional G, the linker LLSVG (SEQ ID NO:8) did slightly better (8.1×10.sup.−6M) but was less efficient than the native Melan-A sequence (2.5×10.sup.−6M). Finally, with the longer linker LLSVGG (SEQ ID NO:9), very efficient cross presentation was restored (1.5×10.sup.−7M) suggesting that with this longer sequence, cleavage was favored within the linker (SLP control REQFLPSEGAACPPWGGGGELAGIGILTV (SEQ ID NO:174)).

[0178] No Strong Influence of the Linker on CD4 Antigen Presentation

[0179] In parallel, SLP containing MELOE-1.sub.13-27 (SEQ ID NO:21) and MELOE-1.sub.36-44 (SEQ ID NO:31) with linkers LLSV (SEQ ID NO:4), LLSVG (SEQ ID NO:8) or LLSVGG (SEQ ID NO:9) were tested for presentation to a CD4 MELOE-1.sub.13-27-specific T cell clone to assess the influence of the linker sequence on HLA class II presentation by DC. MELOE-1.sub.11-46 native long peptide and SLP with the linker GGGG (SEQ ID NO:160) were used as controls. As shown on FIG. 5, the SLP with linker GGGG (SEQ ID NO:160) was poorly presented in comparison to the native long peptide (EC50: 3.5×10.sup.−8M vs 8.4×10.sup.−9M) whereas all the other linkers tested were as efficient (LLSVG (SEQ ID NO:8), 6.8×10.sup.−9M) or slightly better (LLSV (SEQ ID NO:4), 2.7×10.sup.−9M) than the native sequence in terms of processing for class II presentation.

[0180] T Cell Specific Expansion In Vitro is Increased

[0181] We next tested the ability of SLP containing MELOE-1.sub.13-27 (SEQ ID NO:21) and MELOE-1.sub.36-44 (SEQ ID NO:31) with linkers GGGG (SEQ ID NO:160), LLSV (SEQ ID NO:4), or LLSVGG (SEQ ID NO:9) to reactivate and expand CD8 specific T cells within PBMC from HLA-A*0201 healthy donors in vitro. For this purpose, PBMC were treated for 24 h with GMCSF and IL-4 to accelerate the differentiation of monocytes into DC together with SLP at 5 μM and then TNF-α, IL-1b and PGE.sub.2 were added as maturation agents as previously described (see M&M). After 21 days, the percentage of CD8 specific T cells in each microculture was assessed by tetramer staining. Results of a typical experiment is presented in FIGS. 6A and 6B showing the number of positive wells (threshold set at 0.5% of tetramer positive CD8 T cells) and the percentages of positive cells following stimulation with either the SLP containing the most efficient linker LLSVGG (SEQ ID NO:9), LLSV (SEQ ID NO:4, the linker GGGG (SEQ ID NO:160) or the native MELOE-1 sequence. Stimulation and in vitro expansion was more efficient with the SLP containing LLSVGG then with the native sequence (16/96 vs 8/96 positive wells, respectively). Stimulation and in vitro expansion of MELOE-1.sub.36-44-specific T cells with the aSLP containing LLSV was more efficient than with the aSLP containing the GGGG linker (27/96 vs 8/96, p<0.01) and also than the native sequence MELOE-1.sub.11-46 (27/96 vs 18/96) although not significantly so. The summary of all the experiments performed is presented on table 2. Two donors (HD3 and HD4) displayed few positive microcultures reflecting low frequencies of circulating CD8 MELOE-1 specific cells and thus differences in SLP cross-presentation according to the linker used could not be considered. With the three other healthy donors who showed higher frequencies of T cell responses, the optimal linker LLSV (SEQ ID NO:4) or the latest LLSVGG (SEQ ID NO:9) always increased cross-presentation when compared to either the control linker GGGG (SEQ ID NO:160) or the native sequence. We next assessed our optimal linker LLSVGG using PBMC from seven healthy donors and two melanoma patients. Within this group, one healthy donor and one patient were excluded from the analysis for lack of any T cell response against MELOE-1. With the remaining six donors and one patient, we showed that stimulation with aSLP containing the linker LLSVGG reactivated significantly more specific responses then aSLP containing the linker GGGG (p=0.004, paired t-test) (FIG. 6C). To further confirm this observation, we tested the cross presentation of the SLP containing Melan-A A27L (SEQ ID NO:64) epitope instead of MELOE-1.sub.36-44 (SEQ ID NO:31) with linkers GGGG (SEQ ID NO:160) or LLSVGG (SEQ ID NO:9). The linker LLSVGG (SEQ ID NO:9) significantly increased in vitro cross-presentation of the Melan-A epitope as compared to GGGG (SEQ ID NO:160) (47/96 vs 7/96 positive wells respectively, p<0.0001) (FIG. 6D).

[0182] In Vivo Immunogenicity is Increased

[0183] Finally, we explored the immunogenicity of our SLP in vivo in HLA-DRB1*0101/HLA-A*0201 transgenic mice. Mice were immunized subcutaneously with 100 μg of SLP in IFA and poly I:C and boosted at D14 and D28 with 50 μg of SLP with the same adjuvants (see M&M). We focused on cross-presentation and thus assessed CD8 T cell responses by ELISpot-IFN-g after re-stimulation with the short MELOE-11.sub.36-44 epitope (SEQ ID NO:31) or with medium alone. A mouse was considered responder when ELIspots were above 10 and at least twice about background. A typical experiment is presented in FIG. 7 comparing immunization with SLP containing MELOE-1.sub.13-27 (SEQ ID NO:21) and MELOE-1.sub.36-44 (SEQ ID NO:31) with linker GGGG (SEQ ID NO:160) or LLSVGG (SEQ ID NO:9). Immunization with SLP containing GGGG (SEQ ID NO:160) generated moderate CD8 responses in 3 out of five mice while immunization with SLP with LLSVGG (SEQ ID NO:9) was efficient in all 5 mice with two mice showing robust CD8 responses. Previous experiments performed with the shorter linkers LLSV (SEQ ID NO:4) or LLSVG (SEQ ID NO:8) also supported the hypothesis that our synthetic linkers favored cross-presentation when compared to either the linker GGGG or the native sequence (Table 4). In contrast, CD4 responses evaluated ex vivo towards the HLA-DRB1*0101 epitope were very low (FIG. 7C). This suggested that the mouse T cell repertoire against this epitope is scarce as compared to the human T cell repertoire and thus T cell help may not have contributed much to the CD8 responses in those mice.

[0184] Antitumor Effect In Vivo Triggered by aSLP Vaccination

[0185] To evaluate the anti-tumor potential of vaccination with aSLP, we used the previously described SARC-A2 cell line transduced with a cDNA encoding the whole MELOE-1 antigen. The ability of this transduced cell line to present the HLAA*0201-restricted MELOE-1.sub.36-44 epitope was confirmed in vitro by its ability to stimulate a MELOE-1 specific CD8 T cell clone (data not shown). Following tumor engraftment, mice were vaccinated at day 6 by a prime injection of 100 μg of aSLP with the LLSVGG linker plus adjuvant, or adjuvant alone or PBS, followed by a boost 14 days later. Mice were monitored until the end point at day 36. Comparison of median tumor sizes at endpoint in the three groups of mice is shown in FIG. 8A. At that time, setting the threshold at 50 mm.sup.2, all tumors were growing in the seven untreated mice despite variability in tumor size while tumor growth was inhibited in four out of eight mice (0/7 vs 4/8, p=0.0513, Fisher's exact test) (FIGS. 8B and 8C).

[0186] Conclusion:

[0187] To determine the optimal linker sequence for crosspresentation, we tested a number of linker sequences predicted to be recognized by the endocathepsins present in DC endosomes i.e. mainly cathepsins L, S and D. Our results showed an unexpected wide range in cross-presentation efficiency among them with some aSLP being 100 fold more efficient then the native antigen sequence. In addition, we demonstrated that the aa sequences provided by the class II and class I epitopes upstream and downstream of the linker could critically affect the processing. The most striking example was the abrogation of cross-presentation of the Melan-A.sub.A27L epitope when short linkers where used. We thus decided to extend the linker to a 6 aa length to favor cleavage within the linker sequence. A few selected aSLP were then further evaluated for their ability to expand CD8-specific T cells from healthy donors in vitro. It should be pointed out that in those experiments, aSLP were used at high concentrations (5 μM) and persisted in the culture for many days. Therefore, extracellular partial digestion of the aSLP by surface or secreted proteases may have occurred and somewhat blurred differences between the different linkers. Nonetheless, even in those conditions, we confirmed that aSLP containing linkers LLSV and LLSVGG were more efficient than the native MELOE-111-46 or the aSLP with a GGGG linker to expand MELOE-136-44 specific CD8 T lymphocytes. In addition, we confirmed in vivo in transgenic mice that vaccination with aSLP designed with cathepsin-sensitive linkers were the most efficient to trigger CD8+ specific T cell responses. However, our data showed that mouse CD4 T cell responses against the HLA-DRB1*0101-restricted MELOE-1 epitope were weak and thus probably provided little help to the CD8 T cell responses. Such differences in T cell repertoire between those HLA-DRB1*0101/HLA-A*0201 transgenic mice and humans have been previousl reported before by some of us: in fact among the four HLA-DRB1*0101-restricted epitopes derived from telomerase reverse transcriptase (hTERT) that are immunogenic in humans (coined UCP 1,2, 3 and 4), only UCP2 and UCP3 triggered significant CD4 T cell responses and provided help for the CD8 responses in those mice.13 Thus, we reckon that with our aSLPs in this mouse model, we mainly assessed the linker-dependent efficiency of in vivo cross-presentation of the class I epitope with little influence of the class II epitope. Finally, vaccination with our optimal aSLP inhibited the growth of transplanted SARC-A2 tumors expressing MELOE-1 antigen. Although vaccination inhibited tumor growth in only four out of eight mice, we believe that the vaccination protocol could be further improved in the future. Indeed, in the recent publication of Ott et al. (Ott et al (2017) Nature; 547:217-21) showing impressive clinical results in melanoma patients following vaccination with long peptides, priming consisted in five injections followed by two boosts while we only injected our mice twice. In conclusion, our data provide evidence that (i) designing aSLPs with defined class II and class I epitopes connected by a cathepsin-sensitive linker is a valid approach to allow presentation of both CD4 and CD8 epitopes by DCs and (ii) choosing the optimal linker can significantly enhance cross-presentation to CD8+ T cells and (iii) vaccination with such aSLP can trigger an anti-tumor response in vivo. This approach should now be evaluated in vaccination trials in cancer patients.

REFERENCES

[0188] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0189] 1. Melief C J M, van Hall T, Arens R, Ossendorp F, van der Burg S H. Therapeutic cancer vaccines. J Clin Invest 2015; 125:3401-12. [0190] 2. Pol J, Bloy N, Buqué A, Eggermont A, Cremer I, Sautes-Fridman C, Galon J, Tartour E, Zitvogel L, Kroemer G, et al. Trial Watch: Peptide-based anticancer vaccines. Oncoimmunology 2015; 4:e974411-3. [0191] 3. Bijker M S, van den Eeden S J F, Franken K L, Melief C J M, Offringa R, van der Burg S H. CD8+ CTL priming by exact peptide epitopes in incomplete Freund's adjuvant induces a vanishing CTL response, whereas long peptides induce sustained CTL reactivity. J Immunol 2007; 179:5033-40. [0192] 4. Rosalia R A, Quakkelaar E D, Redeker A, Khan S, Camps M, Drijfhout J W, Silva A L, Jiskoot W, van Hall T, van Veelen P A, et al. Dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and T-cell activation. Eur J Immunol 2013; 43:2554-65. [0193] 5. Wada H, Isobe M, Kakimi K, Mizote Y, Eikawa S, Sato E, Takigawa N, Kiura K, Tsuji K, Iwatsuki K, et al. Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen. J Immunother 2014; 37:84-92. [0194] 6. Kenter G G, Welters M J P, Valentijn A R P M, Lowik M J G, Berends-van der Meer D M A, Vloon A P G, Essahsah F, Fathers L M, Offringa R, Drijfhout J W, et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med 2009; 361:1838-47. [0195] 7. Ott P A, Hu Z, Keskin D B, Shukla S A, Sun J, Bozym D J, Zhang W, Luoma A, Giobbie-Hurder A, Peter L, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 2017; 547:217-21. [0196] 8. Masuko K, Wakita D, Togashi Y, Kita T, Kitamura H, Nishimura T. Artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP): Preparation and immunological analysis of vaccine efficacy. Immunology Letters 2015; 163:102-12. [0197] 9. Daftarian P, Mansour M, Benoit A C, Pohajdak B, Hoskin D W, Brown R G, Kast W M. Eradication of established HPV 16-expressing tumors by a single administration of a vaccine composed of a liposome-encapsulated CTL-T helper fusion peptide in a water-in-oil emulsion. Vaccine 2006; 24:5235-44. [0198] 10. Dosset M, Godet Y, Vauchy C, Beziaud L, Lone Y C, Sedlik C, Liard C, Levionnois E, Clerc B, Sandoval F, et al. Universal Cancer Peptide-Based Therapeutic Vaccine Breaks Tolerance against Telomerase and Eradicates Established Tumor. Clinical Cancer Research 2012; 18:6284-95. [0199] 11. Godet Y, Moreau-Aubry A, Guilloux Y, Vignard V, Khammari A, Dreno B, Jotereau F, Labarriere N. MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency. J Exp Med 2008; 205:2673-82. [0200] 12. Bobinet M, Vignard V, Florenceau L, Lang F, Labarriere N, Moreau-Aubry A. Overexpression of Meloe Gene in Melanomas Is Controlled Both by Specific Transcription Factors and Hypomethylation. PLoS ONE 2013; 8:e75421. [0201] 13. Carbonnelle D, Vignard V, Sehedic D, Moreau-Aubry A, Florenceau L, Charpentier M, Mikulits W, Labarriere N, Lang F. The Melanoma Antigens MELOE-1 and MELOE-2 Are Translated from a Bona Fide Polycistronic mRNA Containing Functional IRES Sequences. PLoS ONE 2013; 8. [0202] 14. Rogel A, Vignard V, Bobinet M, Labarriere N, Lang F. A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination. Cancer Immunol Immunother 2011; 60:327-37. [0203] 15. Bobinet M, Vignard V, Rogel A, Khammari A, Dreno B, Lang F, Labarriere N. MELOE-1 antigen contains multiple HLA class II T cell epitopes recognized by Th1 CD4+ T cells from melanoma patients. PLoS ONE 2012; 7:e51716. [0204] 16. Godet Y, Desfrancois J, Vignard V, Schadendorf D, Khammari A, Dreno B, Jotereau F, Labarriere N. Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapy. Eur J Immunol 2010; 40:1786-94. [0205] 17. Ohtake J, Ohkuri T, Togashi Y, Kitamura H, Okuno K, Nishimura T. Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine. Immunology Letters 2014; 161:20-30. [0206] 18. Pajot A, Michel M-L, Fazilleau N, Pancré V, Auriault C, Ojcius D M, Lemonnier F A, Lone Y-C. A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Eur J Immunol 2004; 34:3060-9. [0207] Serwold, T., Gonzalez, F., Kim, J., Jacob, R. & Shastri, N. ERAAP customizes peptides for MHC class I molecules in the endoplasmic reticulum. Nature 419, 480-483 (2002). [0208] O'Brien, C., Flower, D. R. & Feighery, C. Peptide length significantly influences in vitro affinity for MHC class II molecules. Immunome Res 4, 6-7 (2008). [0209] Rawlings, N. D., Barrett, A. J. & Finn, R. Twenty years of the MEROPSdatabase of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 44, D343-D350 (2016). [0210] Rangan L, Galaine J, Boidot R, Hamieh M, Dosset M, Francoual J, Beziaud L, Pallandre J-R, Joseph E L M, Asgarova A, et al. Identification of a novel PD-L1 positive solid tumor transplantable in HLA-A*0201/DRB1*0101 transgenic mice. Oncotarget 8:48959-48971 (2017).