Melanoma antigen peptide and uses thereof

Abstract

The present invention relates to a melanoma antigen peptide comprising the amino acids sequence selected in the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 or a function-conservative variant thereof. Moreover the invention also relates to a melanoma antigen peptide according to the invention for use in the prevention or the treatment of melanoma in patient.

Claims

1. A melanoma antigen peptide having less than 40 amino acids comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; or a fusion peptide comprising said amino acid sequence and a melanoma antigen peptide comprising the amino acids motif: TX2NDECWPX9(SEQ ID NO: 23), wherein X2 is leucine, methionine, valine, isoleucine or glutamine and X9 is alanine, valine or leucine; or a composition comprising said melanoma antigen peptide or said fusion peptide.

2. A method of treating melanoma in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent selected from the group consisting of: i) a melanoma antigen peptide according to claim 1, ii) a fusion protein according to claim 1, or iii) a composition according to claim 1.

3. The method according to claim 2 wherein the patient is genotyped with HLA-DQ1*0201 or HLA-DQ1*0202 alleles.

Description

FIGURES

(1) FIG. 1: (A) Percentages of TNF- producing CD4 T cells among positive microcultures. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2-46), microcultures were re-stimulated with each indicated peptide during 5 hours. TNF- production was then assessed by a double labeling TNF--CD4. Results were analyzed with a non-parametric test (Kruskal-Wallis) followed by a Dunns post-test. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides (assessed by TNF- intracellular staining after re-stimulation with the 4 indicated peptides). 624 microcultures (from 7 healthy donors) were analyzed by a contingency table followed by a Fisher exact test.

(2) FIG. 2: (A) Percentages of cytokine producing CD4 T cells among positive microcultures. Fourteen days after PBMC stimulation with MELOE-1 whole polypeptide (2-46), microcultures were re-stimulated with each indicated peptide during 5 hours. IFN- (left panel) and IL4 (right panel) production was then assessed by a triple labeling IFN--IL4-CD4. Results were analyzed with a non-parametric test (Kruskal-Wallis) followed by a Dunns post-test. (B) Frequency of microcultures containing CD4 T cells specific for MELOE-1 peptides (assessed by IFN- or IL4 intracellular staining after re-stimulation with the 3 indicated peptides). 576 microcultures (from 10 melanoma patients) were analyzed by a contingency table followed by a Fisher exact test.

(3) FIG. 3: HLA-restricting element of MELOE-1 specific T cell clones and reactivity against HLA-matched melanoma cell lines. The HLA restriction of MELOE-1 specific T cell clones was first assessed using anti-HLA blocking antibodies (upper panel). T cell clones were stimulated for 5 hours in the presence of brefeldin A (10 g/mL) either with peptide alone (10 M) in an autopresentation assay and in presence or not of blocking antibodies at a concentration of 12.5 g/mL. HLA restriction was confirmed with HLA-matched B-EBV cell lines pulsed 2 hrs with the cognate peptide, at a ratio 1:2 (middle panel). Reactivity of each T cell clone against HLA-class II expressing melanoma cells was assessed, in presence or not of exogeneous peptide (lower panel). After 5 hours of stimulation, cells were then stained with APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-TNF- mAb and analyzed by flow cytometry.

(4) FIG. 4: Class II epitopes are naturally processed from MELOE-1 whole antigen. Autologous DC, were loaded (before or after fixation) with MELOE-1.sub.2-46 (1 M) or, as a negative control, with Melan-A.sub.16-40L peptide (1 M), and matured. T cell clones were then stimulated with DC at a ratio 1:1, during 5 h in presence of Brefeldin A, then stained with APC-conjugated anti-CD3 mAb, fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-TNF- mAb and analyzed by flow cytometry. Histograms illustrated the % of TNF- producing cells among CD3 positive T cells.

(5) FIG. 5: Minimal peptides recognized by MELOE-1 specific CD4 T cell clones. MELOE-1 specific CD4 T cell clones were incubated with various concentrations of the indicated peptides during 5 hours in presence of Brefeldin A. TNF- production was assessed by intracellular labeling with an anti-TNF- specific antibody. The core peptide sequence is indicated in bold on each figure panel, and black circles illustrate the best fitting peptide.

(6) TABLE-US-00004 TABLEI MELOE-1andMELOE-1derivedpeptidesequences. Peptide Sequences SeqIDNo MELOE-1 MSCVGYPDEATSREQFLPSEGAACPPWHPSERISSTLNDECWPASL 20 MELOE-1.sub.2-21 SCVGYPDEATSREQFLPSEG 1 MELOE-1.sub.11-30 TSREQFLPSEGAACPPWHPS 10 MELOE-1.sub.18-37 PSEGAACPPWHPSERISSTL 16 MELOE-1.sub.26-46 PWHPSERISS SL 6 MELOE-1.sub.22-46 AACPPWHPSERISS SL 17

(7) All the peptides were purchased from Millegen company (France), with a purity >85%. In bold are indicated the DR-11 (SEQ ID NO:18) and the DQ-6 (SEQ ID NO:8) overlapping epitopes already described, and in italics is indicated the HLA-A2 restricted class I epitope (TLNDECWPA, SEQ ID NO:23)).

(8) TABLE-US-00005 TABLE II Assessment of MELOE-1 CD4 T cell responses in PBMC from healthy donors. Microcultures containing MELOE-1 specific CD4+ T cells (TNF- production) Donor MELOE-1.sub.2-21 MELOE-1.sub.11-30 MELOE-1.sub.18-37 MELOE-1.sub.26-46 Class II HLA HD9 9/96 13/96 6/96 5/96 DP1*0902/1501 (1.4% 0.6) (2.4% 1.5) (1.4% 0.6) (1.5% 0.5) DQ1*0301 DR1*1104/1201 HD17 6/96 26/96 7/96 10/96 DP1*0401/0301 (3.2% 1.9) (8.2% 6.5) (3% 0.7) (5.2% 4.1) DQ1*0301/0603 DR1*1101/1301 HD22 1/96 2/96 0/96 2/96 DP1*0401/1101 (1%) (3.9% 3.3) (1% 0.1) DQ1*0202 DR1*0701 HD24 3/96 46/96 0/96 0/96 DP1*0401/0101 (1.3% 0.8) (2% 1.4) DQ1*0501/0602 DR1*0101/1501 HD25 3/96 3/96 0/96 15/96 DP1*0401/0402 (0.6% 0.1) (1.2% 0.7) (1.1% 0.9) DQ1*0201 DR1*0301 HD27 0/96 5/96 0/96 0/96 DP1 NA (5% 3.4) DQ1*501/0201 DR1*0101/0301 HD28 30/48 14/48 0/48 0/96 DP1 NA (2.7% 1.2) (2.4% 0.-9) DQNA DR1*0301

(9) PBMC from healthy donors were stimulated with 10 M of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by restimulating cells with MELOE-1.sub.2-21, MELOE-1.sub.11-30, MELOE-1.sub.18-37 and MELOE-1.sub.26-46 peptides, followed by CD4/TNF- double staining and flow cytometry analysis. Between brackets is indicated the mean % of TNF- producing CD4 T cells, in positive microcultures. NA: not available.

(10) TABLE-US-00006 TABLE III Assessment of MELOE-1 CD4 T cell responses in PBMC from melanoma patients. Microcultures containing MELOE-1 specific CD4+ T cells Th1 responses (IFN- positive Th2 responses (IL4 positive microcultures) microcultures) MELOE- MELOE- MELOE- MELOE- MELOE- MELOE- 1.sub.2-21 1.sub.11-30 1.sub.22-46 1.sub.2-21 1.sub.11-30 1.sub.22-46 Pt 1 1/48 10/48 11/48 0/48 1/48 4/48 (0.7%) (1.4% 0.7) (4.8% 7.2) (1%) (0.8% 0.3) Pt 2 4/96 16/96 2/96 0/96 0/96 0/96 (0.8% 0.2) (1.6% 1.2) (0.6% 0.02) Pt 3 10/96 5/96 4/96 0/96 0/96 0/96 DP1*0201/ (1.7% 2.9) (16.2% 33.2) (1.9% 1.3) 2001 DQ1*0303/ 0501 DR1*0101/ 0701 Pt 4 0/48 9/48 0/48 1/48 0/48 0/48 (0.9% 0.5) (0.7%) Pt 5 1/48 6/48 0/48 2/48 0/48 1/48 (0.6%) (1.5% 1.1) (0.7% 0.1) (0.8%) Pt 6 0/48 5/48 0/48 0/48 0/48 0/48 (1.5% 1.9) Pt 7 0/48 0/48 28/48 0/48 0/48 7/48 (2.9% 3.5) (1.5% 0.7) Pt 8 0/48 2/48 1/48 0/48 0/48 1/48 (2.9% 0.04 ) (0.5%) (0.6%) Pt 9 0/48 0/48 0/48 1/48 9/48 3/48 (0.6%) (0.6% 0.09) (0.7% 0.15)

(11) PBMC from melanoma patients were stimulated with 10 M of MELOE-1. After 14 days, the presence of CD4 T cells specific for the different regions of MELOE-1 was assessed by restimulating cells with MELOE-1.sub.2-21, MELOE-1.sub.11-30 and MELOE-1.sub.22-46 peptides, followed by CD4/IFN- double staining for the detection of Th1 responses, and by CD4/IL4 double staining for Th2 responses. Between brackets is indicated the mean % of cytokine producing CD4 T cells, in positive microcultures.

(12) TABLE-US-00007 TABLEIV TCRcharacterizationandcytokineprofileof MELOE-1specificCD4Tcellclones CDR3beta Cytokine chain profile MELOE-1.sub.2-21specificCD4Tcellclone (9C12-DQ1*0202) Vbetachain V2.1 TNF.sup.high CDR3beta CSASPDTHWGTDTQYFG IFN.sup.high Jbetachain J 2.3 IL2.sup.high GM-CSF.sup.high IL4.sup.high IL5.sup.low IL13.sup.high IL10.sup.neg MELOE-1.sub.11-30specificCD4Tcellclone (1A5-DR1*1101) Vbetachain ND TNF.sup.high CDR3beta ND IFN.sup.high Jbetachain ND IL2.sup.high GM-CSF.sup.high IL4.sup.low IL5.sup.neg IL13.sup.neg IL10.sup.neg MELOE-1.sub.11-30specificCD4Tcellclone (5F9-DR1*0101) Vbetachain ND TNF.sup.high CDR3beta ND IFN.sup.high Jbetachain ND IL2.sup.low GM-CSF.sup.high IL4.sup.high IL5.sup.neg IL13.sup.high IL10.sup.neg MELOE-1.sub.26-46specificCD4Tcellclone (4E2-DQ1*0201) Vbetachain V2.1 TNF.sup.high CDR3beta CSASGRRKFYEQYFG IFN.sup.high Jbetachain J 2.7 IL2.sup.high GM-CSF.sup.high IL4.sup.high IL5.sup.low IL13.sup.high IL10.sup.neg CD4 T cell clones were stimulated for 5 hours in the presence of brefeldin A (10 g/mL) either with the cognate peptide (10 M) in an autopresentation assay. After 5 hours of stimulation, cells were stained with APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE- conjugated anti-cytokine mAb and analyzed by flow cytometry.

EXAMPLE

(13) Material & Methods

(14) Cells

(15) Blood samples from healthy subjects and melanoma patients were respectively obtained from Etablissement Francais du Sang, Nantes, France and from the department of onco-dermatology, Nantes Hospital, France. Melanoma and B-EBV cell-lines were maintained in RPMI 1640 (GIBCO) containing 10% fetal calf serum (FCS). Lymphocytes were grown in RPMI 1640 8% human serum (HS) with 50 or 75 IU/ml of recombinant interleukin-2 (IL-2, Chiron, France) and 2 nM of L-Glutamin. For experiments using dendritic cells (DC), RPMI supplemented with 20 mg/mL of human albumin (LFB BIOMEDICAMENTS, France) was used to avoid peptide degradation by serum proteases.

(16) Reagents

(17) Antibodies were purchased from BD Biosciences-France or from Miltenyi Biotec, France. Purified cytokines were purchased from CellGenix, Germany. The different peptides (Millegen, France, purity >85%) used in this study are described in Table I. HLA-A*0201/MELOE-1.sub.36-44 monomers were generated by the recombinant protein facility of our institute (SFR 26).

(18) Dendritic Cells Generation and Loading Monocytes were purified from PBMC of healthy donors by a CD14-enrichment kit, according the recommendations of the supplier (Stem Cell, France). Immature dendritic cells (iDC) were generated by culturing monocytes in RPMI supplemented with 20 mg/mL of human albumin, 1000 IU/mL of GM-CSF and 200 IU/mL of IL-4 for 5 days. Then, iDC were pulsed with the whole MELOE-1 (1 M) protein or the modified Melan-A.sub.16-40 A27L as negative control (1 M) and matured with 20 ng/mL of TNF- and 50 g/mL of PolyI:C for 4 hours at 37 C. Finally they were fixed for 1 minute with PBS/0.015% glutaraldehyde. Alternatively, iDC were first matured, fixed and then pulsed with antigens at the same concentration.

(19) Stimulation of MELOE-1 Specific T Cells

(20) PBMC from healthy donors or melanoma patients (2.Math.10.sup.5 cells/well) were cultured for 14 days with 10 M of MELOE-1 whole antigen (46 amino acids) in RPMI medium supplemented with 8% HS, 50 IU/ml of rIL-2 (Chiron, France) and L-Glutamin, in 96-well multiplates. Micro cultures were then restimulated individually with each overlapping peptide (MELOE-1.sub.2-21, MELOE-1.sub.11-30, MELOE-1.sub.18-37, MELOE-1.sub.26-46 or MELOE-1.sub.22-46 for melanoma patients) in the presence of 10 g/mL brefeldin A for 5 hours and the percentage of CD4.sup.+ specific T cells was assessed by TNF-, IFN- or IL-4 intracellular staining A negative control without peptide was included in all experiments.

(21) Alternatively, MELOE-1 specific CD4+ T cell clones were stimulated by autologous MELOE-1 loaded and matured DC at a 1:1 ratio.

(22) T Cell Cloning and TCR Characterization

(23) Polyclonal cultures containing specific CD4+ T cells were cloned by limiting dilution as previously described (Gervois N. et al., 2000). After 2 weeks, each clone was checked for peptide specificity by TNF production assay. For TCR sequencing, RNA from 5.Math.10.sup.6 T cell clones was extracted with RNable reagent (Eurobio, France) according to the supplier's instructions. Reverse transcriptions, PCR amplifications and sequencing were performed as described (Davodeau F. et al, 2001). We used the TCR nomenclature established by Arden et al. (Arden et al., 1995).

(24) TNF Production Assay

(25) CD4.sup.+ T cell clones were cultured for 5 hours at 37 C. in the presence of the recognized 20-mer peptide. Culture supernatants were harvested and TNF was measured in a biological assay using cytotoxicity against WEHI 164 clone 13 (Espevik T. et al., 1986).

(26) Cytokine Intracellular Staining

(27) Lymphocytes were stimulated for 5 hours in the presence of brefeldin A (10 g/mL) either with peptide alone (10 M) in an autopresentation assay or with B-EBV or HLA-class II expressing melanoma cells pulsed 2 hours with the cognate peptide, at a ratio 1:2. In some experiments, blocking mAb against HLA-DP (clone B7.21 from Dr Charron, UMR940, Paris), HLA-DQ (clone SPVL3, Beckman Coulter) or HLA-DR (clone L243, BD Biosciences) were added at a concentration of 12.5 g/ml. Cells were then stained with APC-conjugated anti-CD4 mAb, fixed with 4% paraformaldehyde, labeled with PE-conjugated anti-cytokine mAb and analyzed by flow cytometry.

(28) Statistical Analyses

(29) Statistical analyses were done with GraphPad Prism software. Bar graphs were used to compare frequencies of T cells specific for MELOE-1-derived peptides, in all donors and patients and were analyzed by a contingency table followed by a Fisher exact test. Scatter-dot graphs were made to compare the percentage of TNF positive cells among positive microcultures and were analyzed with a non-parametric test (Kruskal-Wallis followed by a Dunns post-test).

(30) Results

(31) Frequency and Distribution of MELOE-1 Specific CD4 responses in healthy donor's PBMC Stimulated with MELOE-1 antigen

(32) Our purpose was to look for the existence of class II helper epitopes all along MELOE-1 sequence (SEQ ID NO: 20), in order to document the immunogenicity of the different regions of this melanoma antigen. We stimulated 2.Math.10.sup.7 PBMC from seven healthy donors with MELOE-1 whole antigen and tested, after a 14-day culture period the presence of CD4 T cells specific for each region of the protein. Microcultures were screened for TNF production by CD4+ T cells, after restimulation with four MELOE-1 derived overlapping peptides (Table I), in an autopresentation assay. As shown in table II, all donors exhibited CD4 responses against at least 1 out of 4 overlapping peptides. Responses against the N-terminal region of MELOE-1 (2-21) were detected in 6/7 donors, with rather low frequencies (from 1 to 9% of positive microcultures containing between 0.6 to 5.6% of TNF producing CD4 T cells), unless in HD28 healthy donor, who exhibited 62% of positive microcultures. The region 11-30 appears especially immunogenic, with CD4 specific responses detected in each tested donor (from 2 to 48% of positive microcultures containing between 0.7 to 24% of TNF producing CD4 T cells), and with very high frequencies in three donors (HD17, HD24 and HD28). On the contrary, the central region 18-37, containing an already described DR11-restricted epitope (24-37) located just at the end of this 20-mer peptide (Rogel et al., 2011), induced specific responses in microcultures deriving from only 2/7 donors (HD9 and HD17, both expressing the DR11 element). In these two donors, we detected 6 and 7% of positive microcultures, containing between 0.6 to 3.7% of TNF producing CD4 T cells. Finally, the C-terminal region (26-46), containing an already described DQ6-restricted epitope (Rogel et al., 2011), was recognized by stimulated microcultures from 4 out of 7 donors (all do not expressing the DQ6 element), with frequencies ranging from 2 to 16% of microcultures containing between 0.5 and 16% of TNF producing CD4 T cells. Overall, the frequency of MELOE-1.sub.11-30 positive microcultures was significantly higher than frequency of microcultures specific for the three other regions of MELOE-1 (FIG. 1B) (p<0.0001). The two terminal regions (2-21 and 26-46) were equivalent in terms of frequencies of positive microcultures, and these two regions induced significantly more responses than the central region 18-37 (FIG. 1A). Nonetheless, the mean fractions of CD4 reactive T cells induced in positive microcultures were not significantly different from a region to another (FIG. 1B).

(33) Frequency, Distribution and Th Profile of MELOE-1 Specific CD4 Responses in Melanoma Patient's PBMC Stimulated with MELOE-1 Antigen

(34) In order to confirm the immunogenicity of each MELOE-1 regions in melanoma patients, we stimulated melanoma patients PBMC with the MELOE-1 whole protein, and tested the reactivity of stimulated lymphocytes towards the three most immunogenic regions: 2-21, 11-30, and 22-46. For this study, instead of challenging microcultures with the 18-37 peptide, that appeared poorly immunogenic, we extended the C-terminus region from 26-46 to 22-46, in order to also detect responses to our previously described HLA-DR11-restricted epitope (24-37). Indeed, the location of this epitope just at the end of the 18-37 peptide could be deleterious for the detection of specific responses in additional DR contexts, and we previously showed that CD4 T cells specific for MELOE-1.sub.24-36 epitope were efficiently induced by 22-46 peptide stimulation (Rogel et al., 2011). We tested the induction of CD4 specific responses from MELOE-1 stimulated PBMC of 10 melanoma patients. We documented CD4 responses specific for the central region of MELOE-1 (11-30) for 7/9 patients, whereas responses specific for MELOE-1.sub.2-21 and MELOE-1.sub.22-46 were respectively detected in 4/9 and 5/9 patients (Table III). These responses were mainly Th1 responses (IFN-g production) while less frequent Th2 responses specific for the three regions of MELOE-1 were detected in 3/9 patients for MELOE-1.sub.2-21, 2/9 patients for MELOE-1.sub.11-30 and 5/9 patients for MELOE-1.sub.22-46 (Table III). Considering the various regions of MELOE-1, Th1 responses specific for the N-term region of MELOE-1 (2-21) were significantly less frequent than those specific for the central region (p<0.0001) and the C-term region (p=0.0001), with respectively 3.3%, 10.8% and 9.6% of 576 tested microculutures (FIG. 2A). Concerning Th2 responses, much less frequent, the C-term region appeared to induce more frequently the growth of IL-4 producing CD4 T cells than the two other regions (FIG. 2A). As observed for healthy donors, even if the frequencies were different, the mean fraction of reactive T cells (Th1 and Th2) induced in positive microcultures were not significantly different from a region to another (FIG. 2B). In summary, stimulation of patient's PBMC with MELOE-1 induced Th1 responses specific for diverse epitopes located all along the protein sequence, and among the different regions, the central region (11-30) and the C-term region (22-46) appeared to be especially immunogenic in term of frequency of responses.

(35) Production and Characterization of CD4 T Cell Clones Specific for the Different Regions of MELOE-1

(36) In order to formally characterize the recognized epitopes, we derived CD4 T cell clones specific for each region of MELOE-1 by limiting dilution, from microcultures of healthy donors or melanoma patients, containing at least 0.5% of specific CD4 T cells. We succeeded to derive CD4 specific T cell clones from HD17, HD22, HD25 and Pt3 microcultures, which were reactive against MELOE-1.sub.2-21 (HD22), MELOE-1.sub.11-30 (HD17 and Pt3) and MELOE-1.sub.26-46 (HD25). From each cloning experiment, we obtained between one and ten reactive CD4 T cell clones, that turned out to be the same clonotype after CDR3B sequencing (Table IV). A single CD4 T cell clone for each specificity was used for further experiments. The HLA-restriction was determined for each T cell clone, first by using HLA-class II blocking monoclonal antibodies (FIG. 3, upper panel), and further by testing the recognition of HLA-matched B-EBV cell lines loaded with each recognized long peptide (FIG. 3 middle panel). The two T cell clones named 9C12 and 4E2, derived from HD22 and HD25 and specific for MELOE-1.sub.2-21 and MELOE-1.sub.26-46 were restricted by the HLA-DQ1*0202 and the DQ1*0201 molecules respectively. As, these two donors were homozygous for the HLA-DQ locus, a single HLA-DQ matched B-EBV cell line was tested to confirm the HLA restriction of these two CD4 T cell clones. As shown on FIG. 3 (upper panel), the two other T cell clones 1A5 and 5F9 recognized the 11-30 region of MELOE-1, in a HLA-DR context. The use of HLA-matched B-EBV cell lines allowed to precise that 1A5 T cell clone was restricted by the HLA-DR1*1101 molecule and the 5F9 T cell clone by the HLA-DR1*0101 molecule.

(37) We further tested the reactivity of these CD4 T cell clones against HLA-matched melanoma cell lines positive for meloe expression, by qPCR analysis. All the melanoma cell lines tested expressed HLA-DQ and DR at the cell surface. All the T cell clones were reactive against HLA-matched melanoma cell lines when loaded with the cognate peptide (FIG. 3, lower panel, black bars). The two DQ2-restricted T cell clones were reactive against peptide-loaded DQ1*0201 and 0202 melanoma cell lines. In absence of peptide, only the 4E2 DQ1*0201-restricted T cell clone was able to recognize unloaded M77 melanoma cell line (also DQ1*0201), but not the DQ1*0202 melanoma cell line, M88. Similarly, the DR1-restricted T cell clone 5F9 also recognized one of the DR1*0101 melanoma cell line, in the absence of exogenous peptide (M101).

(38) We also documented the T helper profile of each T cell clone, by stimulating the CD4 T cell clones with the cognate peptide, and analyzing cytokine production. All the clones expressed Th1 cytokines (TNF, IFN, IL2 and GM-CSF). On the contrary, these clones differ in their expression of Th2 cytokines. Indeed, the two DQ2 restricted T cell clones (9C12 and 4E2) and the DR1 restricted one (5F9) also strongly express two Th2 cytokines (IL4, and IL13), whereas the DR11-restricted T cell clone only weakly expressed IL4 (Table IV). None of the CD4 T cell expresses IL10 or IL5 at a significant level.

(39) Processing of the Recognized Epitopes from Autologous DC Loaded with MELOE-1 Antigen

(40) Initial PBMC stimulation was carried out with MELOE-1 whole antigen, and thus we assume that CD4 T cell responses were generated against peptides naturally processed by monocytes. Nonetheless, we could not formally exclude that the 14-day culture period artificially generated shorter class II epitopes that elicited CD4 T cell responses. Thus, it remained crucial to assess that all these new epitopes were naturally processed by autologous dendritic cells loaded with MELOE-1 whole protein, in serum-free medium. To this end, we loaded autologous iDC with 1 M of MELOE-1 antigen in serum-free medium, in presence of maturating agents, and fixed these DC before stimulation of the CD4 specific T cell clones. In these conditions, the four T cell clones were reactive against MELOE-1 loaded autologous DC (FIG. 4, left panel), whereas we could not detect any reactivity of CD4 T cell clones to DC loaded with an irrelevant long peptide, synthesized in the same conditions (Melan-A.sub.16-40L).

(41) As an additional control, we loaded autologous DC with MELOE-1 after DC fixation, and we could observe only a weak recognition by specific CD4 T cell clones, indicating that only a small fraction of the protein had been externally degraded into shorter peptides (FIG. 4, right panel). Thus, the four new epitopes identified by PBMC stimulation, are naturally processed from MELOE-1 antigen.

(42) Characterization of the Minimal Recognized Epitopes

(43) Our T cell clones were reactive against 20-mer peptides that are probably not the exact peptides naturally processed. In order to formally identify the minimal recognized epitopes, we tested shorter peptides derived from each of the MELOE-1 recognized regions, chosen on the basis of the core peptide sequence supposed to be recognized by the T cell clones (indicated in bold on FIG. 5).

(44) Three shorter peptides were better recognized by the DQ2-restricted 9C12 T cell clone, the shortest one being MELOE-1.sub.7-19 (13-mer), recognized with an EC50 of 100 nM. The deletion of the two amino acids in C-term strongly reduces T cell clone recognition. The other DQ2-restricted T cell clone (4E2) better recognized a 14-mer peptide (31-44) also with an EC50 of 100 nM, and also recognized to a lower extent the 32-44 epitope (FIG. 5) previously described in the HLA-DQ1*0603 context (Rogel et al., 2011). Concerning the DR-restricted T cell clones, optimal shorter peptides were 13-mer peptides, MELOE-1.sub.15-27 for the DR1*1101 restricted T cell clone 1A5 (EC50=100 nM), and MELOE-1.sub.11-23 or MELOE-1.sub.12-24 for the DR1*0101 restricted one (FIG. 5). Nonetheless all of these clones recognized a series of shortened peptides, thus we cannot formally assess that the shortest ones will be the exact peptides naturally presented on class II molecules.

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