EP1481009A2

EP1481009A2 - Peptides for use in antitumor immunotherapy

Info

Publication number: EP1481009A2
Application number: EP03743400A
Authority: EP
Inventors: Boris Linard; Francine Jotereau; Houssem Benlalam; Elizabeth Diez; Yannick Guilloux; Nathalie Labarriere; Nadine Gervois; Laurent Derre
Original assignee: Universite de Nantes; Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Universite de Nantes; Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2002-03-04
Filing date: 2003-03-04
Publication date: 2004-12-01
Also published as: WO2003074565A2; CA2477762A1; US20060122119A1; FR2836684B1; WO2003074565A3; JP2005533487A; AU2003232280A8; AU2003232280A1; FR2836684A1

Abstract

The invention concerns the use of immunogenic peptides representing T epitopes presented by the MHC I, derived from Melan-A, MAGE-A6, gp 100, tyrosinase and NY-ESO-1 antigens for the diagnosis or treatment of melanomas in HLA-B35 subjects.

Description

PEPTIDES FOR USE IN ANTI-TUMOR IMMUNOTHERAPY

The present invention relates to peptides representing epitopes shared by tumor antigens, and to their use in immunotherapy. Vaccination or peptide immunotherapy is a therapeutic approach which is currently the subject of great interest in the context of the prevention or treatment of cancers. Its principle is based on immunization with peptides reproducing T epitopes of tumor-associated antigens (TAA) recognized by cytotoxic T lymphocytes (CTL), which play a major role in the elimination of cancer cells expressing these antigens at their area.

It will be recalled that the CTLs do not recognize the entire protein antigens, but peptide fragments thereof, presented by the molecules of the major histocompatibility complex (MHC) expressed on the surface of different cells. It is these peptide fragments which constitute the T epitopes. The peptides presented by the major histocompatibility class I complex (MHC I) generally have 8 to 11 amino acids, and are recognized by the CD8 + T cells, which represent the major component. of the cytotoxic response. The peptides presented by the major class II histocompatibility complex (MHC II) generally have 13 to 18 amino acids and are recognized by CD4 + T cells. The identification of these epitopes, and in particular of those presented by the MHC I (taking into account the essential role of the CD8 + response in cytotoxicity), therefore constitutes an essential step for the development of anti-tumor immunotherapy compositions. In the case of melanomas, two main classes of melanoma-associated antigens (MAA) have been identified: melanoma-specific antigens, little or not expressed in normal tissues, and melanocyte differentiation antigens, which are also expressed by melanocytes (for review, see CASTELLI et al., 2000, J Cell Physiol, 182, 323-31; KIRKIN et al, 2002, Cancer Invest, 20, 222-36). Melanocyte differentiation antigens, such as Melan-A / MART-1, gp-100 and tyrosinase, are expressed by a significant proportion of melanoma-type tumors. In addition, these antigens are effectively recognized both by the CTLs of healthy subjects and by those of melanoma patients (BENLALAM et al, 2001, Ewr JImrnunol, 31, 2007-15; KAWAKAMI et al., 2000, J Immunother, 23, 17-27; LABARRIΕRΕ et al., 1998, Int J Cancer, 78, 209-15; PITTΕT et al, 1999, J Exp Med, 190, 705-15; VALMORI et al., 2002, Cancer Res, 62 , 1743-50). We currently know more than thirty epitopes of these antigens recognized by the CTLs, of which almost two thirds are presented in the context HLA-A * 0201.

Melanoma-specific antigens include families of antigens known as “shared by cancer and testes”: MAGE, GAGE, BAGE and LAGE. These antigens, which are expressed by different tumors, generate CTL epitopes presented in a wide variety of HLA contexts, including HLA-B and C (KIRKIN et al, 2002). With the exception of NY-ESO-1 (JAGER et al, 1998, JExp Med, 187, 265-70), and unlike melanocyte differentiation antigens, antigens shared by cancer and testes are rarely recognized by tumor infiltrating lymphocytes (TILs). The identification of most of the epitopes present in these antigens was carried out using CTLs generated ex-vivo by repeated stimulation of peripheral blood lymphocytes (PBL) by presenting cells (CPA) loaded with antigens (SCHULTZ et al, 2001, Tissue Antigens, 57, 103-9), with the exception of the epitopes of MAGE-A6 and MAGE-A12, since they have been identified with CTL clones derived from TILs (PANELLI et al, 2000, J Immunol, 164, 4382-92; ZOON & HERCEND, 1999, EwrJ Immunol, 29, 602-7).

Until now, most of the peptide immunotherapy trials on melanomas have been carried out in the HLA-A * 0201 or HLA-A * 0101 context and using only a limited number of TAA epitopes (LAU et al, 2001, J Immunother, 24, 66-78; MACKΕNSΕN et al, 2000, Int J Cancer, 86, 385-92; MARCHAND et al, 1999, Int J Cancer, 80, 219-30; PANΕLLI et al, 2000, J Immunother, 23, 487-98; ROSΕNBΕRG et al, 1998, JNatl Cancer Inst, 90, 1894-900; SALGALLΕR et al, 1996, Cancer Res, 56, 4749-57). However, the under-expression of HLA alleles, and the appearance of TAA variants having lost the epitopes recognized by the CTLs could constitute mechanisms allowing the escape of tumor cells, with respect to the immune system (FΕRRONΕ & MARINCOLA, 1995, Immunol Today, 16, 487-94; MAΕURΕR et al, 1996, Clin Cancer Res, 2, 641-52), which could explain the low efficacy of vaccines using peptides. To overcome this drawback, it has been proposed to use polyvalent vaccines composed of a multitude of peptides from different TAA and presented in various HLA contexts. However, this requires the identification of new peptide / HLA complexes which are efficiently presented by tumor cells. In addition, the identification of peptides presented in different HLA contexts also makes it possible to develop tools making it possible to measure the T cell response in immunized patients. Indeed, if the identification of new antigenic peptides is essential to increase the availability and effectiveness of vaccines, it is also essential to improve the monitoring of the CTL response of patients who have been immunized with peptides or other forms of antigens, such as complete recombinant proteins or recombinant viruses.

Since HLA-B35 is one of the most frequent HLA-B alleles, present in approximately 20% of the Caucasian population (60% of which corresponds to allele B * 3501; MORI et al, 1997, Transplantation, 64, 1017-27), the identification of new peptides antigens presented in the context HLA-B35 is very desirable for the development of cancer immunotherapy.

The inventors have identified new epitopes, which are derived from melanocytic antigens or from antigens shared by cancers and testes, and which are presented in the HLA-B35 context.

The peptides reproducing these epitopes can be used for diagnostic or therapeutic purposes, in the context of the prevention or treatment of melanomas in patients expressing an HLA-B35 allele, in particular HLA-B * 3501 or HLA-B * 3503. The subject of the present invention is the use of at least one immunogenic peptide representing a T epitope presented by the MHC I, chosen from: a) a peptide comprising the sequence EXιAGIGILX ₂ (SEQ ID NO: 1) in which Xj represents A or P, and X ₂ represents T or Y, capable of inducing a cytotoxic response directed against the Melan-A antigen; b) a peptide comprising the sequence ENDPIGHNY (SEQ ID ΝO: 2), capable of inducing a cytotoxic T response directed against the MAGE-A6 antigen; c) a peptide comprising the sequence NPLDCNLYR (SEQ ID ΝO: 3), capable of inducing a cytotoxic response directed against the gplOO antigen; d) a peptide comprising the sequence TPRLPSSADNEF (SEQ ID ΝO: 4) capable of inducing a cytotoxic response directed against the tyrosinase antigen; e) a peptide comprising the sequence MPFATPMEA (SEQ ID ΝO: 5), capable of inducing a cytotoxic response directed against the ΝY-ESO-1 antigen; for obtaining a drug intended for anti-tumor immunotherapy in an HLA-B35 patient. Advantageously, said medicament is intended for the treatment of melanomas. Advantageously, one can use:

- at least one peptide a) corresponding to a sequence chosen from: TAEEAAGIGILTV (SEQ ID NO: 6), EAAGIGILTVIL (SEQ ID NO: 7), EAAGIGILTV (SEQ ID NO: 8), EAAGIGILTY (SEQ ID NO: 9), EAAGIGILY (SEQ ID NO: 10), EPAGIGILTY (SEQ ID NO: 11), EPAGIGILTV (SEQ ID NO: 12); - a peptide b) corresponding to the sequence ENDPIGHNY (SEQ ID ΝO: 2);

- at least one peptide c) corresponding to a sequence chosen from NPLDCNLYR (SEQ ID ΝO: 3) and NPLDCNLYRY (SEQ ID ΝO: 13);

- at least one peptide d) corresponding to a sequence chosen from TPRLPSSADNEFCL (SEQ ID ΝO: 14) and TPRLPSSADNEF (SEQ ID ΝO: 4); - at least one peptide e) corresponding to a sequence chosen from: LAMPFATPMEAEL (SEQ ID ΝO: 15), LAMPFATPMEAE (SEQ ID ΝO: 16), MPFATPMEAEL (SEQ ID ΝO: 17), MPFATPMEAE (SEQ ID ΝO: 18), and MPFATPMEA (SEQ ID ΝO: 5). According to a preferred embodiment of the present invention, a combination is used comprising at least two peptides of two different categories among the categories a), b), c), d) or e) defined above, in order to d '' be able to induce a cytotoxic response against at least two tumor antigens. The present invention also relates to a multi-epitopic composition associating at least two peptides of two different categories among the categories a) b) c) d) or e) above. Advantageously, these compositions comprise at least one peptide from each of these categories a) b) c) d) or e). Multi-epitopic compositions in accordance with the invention may also comprise one or more other immunogenic peptides, derived from the antigens mentioned above, or from different antigens. These peptides can represent epitopes derived from the same antigen, or from two or more different antigens. By way of example, the peptide ENDPIGHLY (SEQ ID ΝO: 19) will be cited, which represents an epitope of MAGE-A3, already known as being able to be presented in a HLA-B35 context (PCT application WO 01/53833).

These compositions can also comprise, to be more widely usable in a population whose individuals carry different HLA alleles, one or more peptides presented by MHC I molecules other than HLA-B35. By way of example, mention will be made of the peptide PLDCNLYRY (SEQ ID ΝO: 20), described below. Multi-epitopic compositions in accordance with the invention may in particular be in the form of a chimeric polypeptide comprising one or more copies of each of the epitopes chosen.

Chimeric polypeptides in accordance with the invention can be easily obtained by methods known in themselves, and in particular by conventional techniques of recombinant DNA.

The present invention also relates to a polynucleotide coding for a chimeric polypeptide according to the invention, as well as a nucleic acid vector containing said polynucleotide. The present invention also encompasses the use of said polynucleotide or said nucleic acid vector in tumor immunotherapy.

Nonlimiting examples of implementation of the present invention are indicated below.

It is possible, for example, to inject the patient to be treated with a peptide, a chimeric polypeptide, or a multi-epitopic composition as defined above, optionally combined with an appropriate adjuvant.

One can also use one of the peptides defined above to charge in vitro cells presenting the professional HLA-B35 antigen, in particular dendritic cells, in order to induce the proliferation of anti-tumor CTLs, as described by example by BAKKER et al. (Cancer Res., 55, 5330-5334, 1995) or NAN ELSAS et al. (Eur. J. Immunol, 26, 1683-1689, 1996).

The HLA-B35 antigen presenting cells charged in this way are also part of the object of the present invention. The polynucleotides according to the invention preferably integrated into nucleic acid vectors, in particular viral vectors such as adenoviruses, can also be administered by injection to the patient to be treated.

A polynucleotide comprising a sequence coding for a peptide defined by one of the sequences SEQ ID NO: 1 to 18 above, and in particular a polynucleotide coding for a chimeric polypeptide according to the invention, can also be used to transfect in vitro professional HLA-B35 antigen presenting cells, in particular dendritic cells, which are then injected into the patient, as described for example by KAPLAN et al (J. Immunol., 163 (2), 699-707, 1999) or KIM et al. (Armais of Surgical Oncology, 5 (1), 64-76, 1998). These transfected antigen presenting cells are also part of the object of the present invention.

The present invention also encompasses the use of the peptides defined above, for the in vitro detection of CTLs directed against one or more of the antigens Melan-A, MAGE-A6, gplOO, tyrosinase, and NY-ESO-1, in a biological sample obtained from an HLA-B35 subject. These peptides can also be used to carry out the specific sorting of these CTLs.

The CTLs thus isolated can then be amplified in vitro and reinjected in large numbers (of the order of a billion) to the patient.

The present invention also relates to therapeutic compositions comprising, as active principle, a mutated ras peptide, a multi-epitopic composition, a chimeric polypeptide, a polynucleotide, or an antigen presenting cell according to the invention.

The therapeutic compositions in accordance with the invention may also comprise the usual excipients, as well as adjuvants usually used in immunotherapy, and making it possible, for example, to favor the administration of the active principle, to stabilize it, to increase its immunogenicity, etc.

Among the peptides of categories a), b), c), d), and e) mentioned above, some were already known to be capable of inducing a CTL response, but in contexts other than HLA-B35.

Others had never been described as epitopes capable of inducing a cytotoxic T response and had therefore not been isolated. These are the following peptides:

- the peptides EAAGIGILTY (SEQ ID NO: 9), EAAGIGILY (SEQ ID NO: 10), EPAGIGILTY (SEQ ID NO: 11), EPAGIGILTV (SEQ ID NO: 12);

- the peptides NPLDCNLYR (SEQ ID ΝO: 3) and NPLDCNLYRY (SEQ ID ΝO: 13); - the TPRLPSSADNEFCL peptides (SEQ ID ΝO: 14) and TPRLPSSADNEF (SEQ ID ΝO: 4).

The present invention also encompasses these particular peptides, as well as any multi-epitopic composition comprising at least one of these peptides. This notably includes chimeric polypeptides containing at least one of these peptides. The polynucleotides encoding these chimeric polypeptides, and the nucleic acid vectors containing these polynucleotides are also part of the subject of the present invention. The inventors have also found that the peptide NPLDCVLYRY (SEQ ID ΝO: 13) derived from the gplOO antigen, was also recognized, in the HLA * A0101 context, by a CD8 T clone (M199.6.12) from a population TILs of melanoma. From this peptide, they identified a shorter peptide, (PLDCNLYRY, SEQ ID ΝO: 20), which is not recognized in the HLA-B35 context, but is very effectively recognized in the A * 0101 context. The peptide SEQ ID ΝO: 20 also forms part of the subject of the present invention, as well as the chimeric polypeptides containing at least this peptide. The polynucleotides encoding these chimeric polypeptides, and the nucleic acid vectors containing these polynucleotides are also part of the subject of the present invention. This peptide, the chimeric polypeptides containing it, as well as the polynucleotides coding for these chimeric polypeptides can be used in the context of the detection or treatment of melanomas in HLA-A1 subjects, and in particular HLA * A0101, according to the same techniques as those indicated above for the peptides recognized in the HLA-B35 context.

On the other hand, studies carried out on animal models have made it possible to identify other tumor rejection antigens, many of which are mutated oncogenic proteins (PREHN et al, J. Natl. Cancer Inst, 18, 769, 1998; DE PLAEN et al, PNAS, 85, 2274, 1988; DUBEY et al, J. Exp. Med., 185, 695, 1997). The presentation by MHC molecules of the mutated fragments of these proteins on the surface of tumor cells induces the specific destruction of these by CTLs and rejection of the tumor. In humans, mutated oncogenic proteins also appear to be particularly attractive targets for anti-tumor immunotherapy. However, this involves identifying the epitopes presented on the surface of tumor cells and capable of inducing a cytotoxic T response.

Among the oncogenes most frequently involved in different types of tumors, mention will be made of ras oncogenes (K-ras, H-ras and N-ras) which result from point mutations (substitution of a single amino acid) of ras proto-oncogenes p21. These mutations occur mainly at codon 12, codon 13, or codon 61 (BOS, Cancer Res., 49, 4682, 1989; WEIJZEN et al, Leukemia, 13, 502, 1999). Due to the limited number of oncogenic substitutions that can intervene, ras mutations present in many identical tumors can thus generate shared tumor epitopes, presented by a significant fraction of human tumors (WEIJZEN et al, Leukemia, 13, 502, 1999).

It has thus been proposed to use anti-tumor immunotherapy for synthetic peptides reproducing ras mutated epitopes. Thus, PCT application WO 92/14756 proposes the use of peptides reproducing ras epitopes mutated at codon 12 or codon 61. However, these epitopes are presented by MHC II (DQ and DR), and therefore induce a CD4 + response. However, although it may be advantageous to induce this type of response, insofar as the CD4 + helper lymphocytes make it possible to increase the cytotoxic response (WALTER et al, N. Engl. J. Medicine, 333, 1038, 1995 ), the CD8 + response remains the essential player in cytotoxicity. In addition, recent studies carried out in mice suggest that immunization with peptides presented by MHC II could induce stimulation of tumor growth instead of the expected protection (SIEGEL et al, J. Exp. Med., 191 , 1945, 2000). Several ras epitopes mutated at positions 12, 13 or 61 have been selected on the basis of their ability to anchor to MHC class I molecules (NAN ELSAS et al, Int. J. Cancer, 61, 389, 1995; BERGMANN and al, Cell Immunol., 187, 103, 1998; GOUTTEFANGEAS et al, Human Immunol, 55, 117, 1997). The peptides thus selected can stimulate the growth of specific CTLs from PBL in vitro. However, these CTLs weakly recognize mutated tumor cells, suggesting that the endogenous expression of these epitopes is limited (VAN ELSAS et al, Int. J. Cancer, 61, 389, 1995; ABRAMS et al, Cell Immunol, 182, 137, 1997; BERGMANN et al, Cell Immunol, 187, 103, 1998), and therefore do not allow efficient elimination of tumor cells by specific CTLs, which considerably limits their interest in immunotherapy.

It therefore appears necessary to have other mutated ras epitopes restricted to MHC I and presented effectively by a significant fraction of human tumors. The inventors have now identified a ras epitope mutated in position 61 by substitution of a glutamine by an arginine (Q61R), and restricted to MHC I. This epitope, hereinafter called 55-64 ^{Q 1R} is presented effectively by several lines of HLA-A * 0101 + melanoma expressing a ras oncogene carrying the Q61R mutation. It is capable of specifically inducing the expansion of clones of tumor infiltrating lymphocytes (TIL) obtained from these melanomas. In addition, the dendritic cells loaded with this peptide efficiently stimulate specific CTLs from peripheral blood lymphocytes (PBL) from healthy donors HLA-A * 0101, and these CTLs recognize all of the HLA-A * 0101 melanoma lines. expressing the ras Q61R oncogene, and do not recognize cells expressing the non-mutated ras protein. The peptide 55-64 ^Q61R does not have a higher anchoring capacity than that of the corresponding wild-type peptide. The HLA-A * 0101 binding affinity of the wild type peptide is similar to that of peptide 55-64 ^Q61R . However, even at high concentrations, this wild-type peptide cannot induce the expansion of specific TILs or of specific CTLs. It therefore appears that the substitution of a glutamine by an arginine at position 61 creates a new epitope presented by HLA-A * 0101. In addition, an analysis carried out on the BIMAS database (http://bimas.dcrt.nih.gov/molbio/hla_bind; PARKER et al, J. Immunol. 152, 163, 1994;), shows that this peptide has a Identical binding score for HLA-A * 0101 and HLA-B * 1501. Consequently, the present invention also relates to the use of an immunogenic ras mutated peptide of sequence ILDTAGREEY (SEQ ID NO: 35) for obtaining a medicament intended for the immunotherapeutic treatment of tumors in a HLA-A * 0101 or HLA-B * 1501 patient,: Advantageously, said medicament can be used for the treatment of tumors expressing a K-ras, H-ras or N protein -rαs mutated by substitution of glutamine in position 61 with an arginine.

Mention will in particular be made of melanomas, as well as of other tumors in which the mutations ras affecting the residue 61 are detected with a high frequency, such as congenital melanocytic nεevi (PAPP et al, Journal of Medical Genetics, 36, 610, 1999) , multiple myelomas (BEZIEAU et al, Hum. Mutât., 18, 281, 2001), and thyroid tumors (ESAPA et al, Cinical Endocrinology, 50, 529, 1999). Said peptide can be used in particular in the context of multi-epitopic compositions, and in particular of chimeric polypeptides, as mentioned above. A polynucleotide encoding such a chimeric polypeptide, as well as a nucleic acid vector containing said polynucleotide can also be used as mentioned above.

Said peptide or said polynucleotide can also be used respectively to load or transfect in vitro cells presenting the professional HLA-A * 0101 or HLA-B * 1501 antigen, in order to induce the proliferation of anti-tumor CTLs. The antigen presenting cells, HLA-A * 0101 or HLA-B * 1501 loaded or transfected in this way are also part of the object of the present invention. Said mutated ras peptide can also be used to detect in vitro CTLs directed against the mutated ras antigen from which it is derived, in a biological sample obtained from a subject HLA-A * 0101 or HLA-B * 1501. It can also be used to perform the specific sorting of these CTLs.

The present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples illustrating the properties of ras peptides. mutated according to the invention usable in immunotherapy, by implementing the method according to the invention.

EXAMPLE 1: EVIDENCE OF ANTIGENIC EPITOPES PRESENT IN THE HLA-B35 CONTEXT AND RECOGNIZED BY CTLS CLONES:

In a previous study (BENLALAM et al, 2001), the inventors obtained clones of CD8 + TILs restricted to HLA-B35, and recognizing respectively the antigens Tyrosinase (TIL M171), Melan-A (TIL M171 and M28), and gplOO (TIL M171, M28 and Ml 10). They completed this collection of clones with a clone recognizing MAGE-A3 / MAGE-A6 (TIL-M171) and a clone recognizing NY-ESO-1 (TIL Ml 18). The response of these different TIL clones to COS-7 cells co-transfected with the relevant HLA-B35 antigen and allele is detected by measuring their production of TNF. COS-7 cells, cultured in DMEM medium (Sigma) containing 10% fetal calf serum, antibiotics and L-glutamine, were transfected with cDNA coding for one of the HLA-B * alleles 3501, HLA-B * 3503, HLA-B * 3508, alone or in combination with a cDNA encoding one of the antigens MAGE-A3, MAGE-A6, tyrosinase, Melan- A / MART-1, gp-100, and NY-ESO1 / LAGE-2. The transfection was carried out according to the protocol described by DE PLAEN et al. (Methods, 12, 125-42, 1997). 16.5 10 ³ COS cells-

7 were transfected with 100 ng of plasmid pcDNA3 (Invitrogen, San Diego, CA), containing the cDNA of the HLA-B * 35 allele concerned, and, for co-transfection, 100 ng of plasmid containing the cDNA encoding the selected antigen.

These COS-7 cells were used 48 hours after transfection to stimulate the various TILs clones (2x10 ³ to 10 ⁴ ). The culture supernatants were removed 6 hours later and their TNF concentration was determined by measuring the cytotoxicity of these culture supernatants for the clone 13 WEHI 164, as described by DE PLAEN et al.

(1997, supra).

The results are illustrated in Figure 1. On the ordinate, the concentration of TNF in pg / ml

On the x-axis the different populations of TIL

D: Secretion of TNF by the TIL clones in the presence of COS-7 cells transfected only with a cDNA coding for a HLA-B35 molecule.

H: Secretion of TNF by the TILs clones in the presence of COS-7 cells co-transfected with a cDNA coding for a HLA-B35 molecule and a cDNA coding for an MAA.

Two clones (M171.95B and M28.10B) recognize a Melan-A / B * 3501 complex, one clone (M28.9B) recognizes a gpl00 / B * 3501 complex, one clone (M171.100B) a tyrosinase / B * 3501 complex, a clone (M171.8C) recognizes a MAGE-A3 and MAGE-A6 epitope in the context B * 3501 and a clone (Ml 18.45) recognizes a NY-ESO- epitope

I in the contexts B * 3501 and B * 3503.

EXAMPLE 2: IDENTIFICATION OF THE ANTIGENIC EPITOPES PRESENT IN THE HLA-B35 CONTEXT.

To identify the regions coding for the epitopes recognized by the TIL clones, the inventors constructed a series of fragments of the cDNAs of the various MAAs. The Melan-A / MART-1 and NY-ESO-1 cDNA fragments were obtained by exonuclease III digestion: the plasmids comprising the cDNA coding for Melan-A / MART-1 or NY-ESO-1 were opened by Xbal and Apal, or SpHI and Notl respectively. The fragments obtained were then digested with exonuclease III using the Erasea-base system (Promega, Madison, WI).

A restriction fragment of gp-100 (1156-1986) containing the nucleotides located between the Kpnl 1156 bp site downstream of the initiation codon, and the end of the coding sequence, was generated by enzymatic digestion with Kpnl.

The cDNA fragments corresponding to the fragments of the tyrosinase and gplOO antigens were obtained by PCR from the plasmids containing the complete sequence coding for each of these two antigens. The expression of these different fragments by the COS-7 cells is carried out as in Example 1. The responses of the different TIL clones towards these COS-7 cells co-transfected with the antigen fragments and the relevant HLA-B35 allele are measured as in Example 1. The results are illustrated in FIG. 2: The positions of the regions of the cDNAs coding for the potential epitopes are indicated in base pairs.

D: Fragment recognized by specific CTLs clones (significant TNF secretion). 0: Fragment not recognized by specific CTLs clones (non-significant TNF secretion).

It appears that the different TIL populations recognize epitopes which are encoded respectively by the Melan-A cDNA fragment extending from nucleotides 95 to 119

(amino acids 32 to 39), the cDNA fragment gplOO 1200 to 1601 (amino acids 400 to 533), the cDNA fragment tyrosinase 937 to 975 (amino acids 313 to 325) and the cDNA fragment NY-ESO -1 259 to 339 (amino acids 87 to 113).

EXAMPLE 3: IDENTIFICATION OF THE ANTIGENIC PEPTIDES PRESENT IN THE HLA-B35 CONTEXT.

The wild type and modified peptides whose sequences are indicated in Table I below were obtained from EPYTOP (Nîmes, France). Purity (> 70%) is controlled by high yield reverse phase liquid chromatography (HPLC). The peptides are lyophilized, then dissolved in DMSO at 10 mg / ml and stored at -80 ° C.

The response of the various TIL clones was evaluated, by a TNF release test, after

6 hours of co-culture with EBN cells expressing an HLA-B * 3501 molecule loaded with 10 μM of the peptide of interest. The results are collated in Table I.

Table I

On the other hand, the antigenicity of the peptides was tested by evaluating the capacity of the TIL clones to lyse BM36.1 cells (KELLY et al, 1992, Nature, 355, 641-4), which are deficient in the TAP transporter, and which naturally express HLA-B * 3501 and HLA-A * 0101. BM36.1 cells are labeled for 1 h at 37 ° C. with 100 Ci of ⁵¹ Cr (Na2 ⁵¹ CrO4, ORIS, Gif-sur-Yvette, France). The cells are then pulsed for 20 minutes with the various synthetic peptides. 10 ³ BM36.1 cells thus treated are incubated with 10 ⁴ T cells of the clone (Effector: Target ratio of 10: 1) for 4 hours. The culture supernatants are recovered and the amount of ⁵¹ Cr released is evaluated using a ^β plate counter (EG&G Wallac, Evry, France). For each TIL clone, a negative control is carried out with an irrelevant peptide. The amount of peptide necessary to obtain 50% of the maximum lysis (EC50) has been determined. On the other hand, the affinity and the stability of the peptides for HLA-B35 were measured as described by TOURDOT et al, Eur. J. Immunol., 30: 3411-3421, 2000). To measure the affinity of the peptides, the BM36.1 cells are incubated for 18 h with a range of concentrations of each peptide. BM36.1 cells are simultaneously incubated with a range of a reference peptide, which binds to HLA-B35 (peptide 37F, TAKAMIYA et al, Int. Immunol, 6: 255-261, 1994; SCHÔNBACH et al, J. Immunol, 154: 5951-5958, 1995). The relative amount of fixed peptide is then estimated at each concentration, for each of the peptides, by measuring the stabilization of HLA-B35 on the cell surface. This measurement is carried out by flow cytometry using an anti HLA-B / C antibody (which in the case of BM36.1 cells recognizes only HLA-B * 3501, these cells spontaneously expressing no molecule HLA-C). The% of binding to B * 35 is then calculated for each concentration, by fixing the 100% of binding for 100 μM of each peptide. The relative affinity (RA) is then determined as follows: RA = [concentration of peptide] for 20% of binding / [concentration of peptide 37F] for 20% of binding.

To measure the stability of the peptides, the BM36.1 cells are incubated for 18 h with 100 μM of each of the peptides. They are then incubated in the presence of BFA (10 μg / ml) for one hour in order to block the transport of newly synthesized HLA molecules on the cell surface. BM36.1 cells are washed in PBS and taken up in culture medium containing 5% SNF and 0.5 μg / ml of BFA, which constitutes time 0. Cells are then removed, after 30 minutes, 1 h, 2 h, 4 h and 6 hours of incubation. The relative amount of fixed peptide is then estimated at each time, for each of the peptides, by the measurement of stabilization of HLA-B35 on the cell surface. This measurement is carried out in flow cytometry using an anti-HLA-B / C antibody. The time indicated in Table II corresponds to the time of 1/2 life of the peptide on HLA-B * 35. The results are illustrated in Figure 3, and Table II below. Table II

Figure 3 legend: On the ordinate, the percentage of cell lysis obtained. On the abscissa the peptide concentration (in nM). The results of Tables I and II and of Figure 3 show that: * Three overlapping Melan-A peptides are recognized by the clone M28.10B. The most effectively recognized peptide is decapeptide 26-35 (EAAGIGILTV, SEQ ID NO: 8,). This Melan-A 26-35 peptide probably corresponds to the peptide naturally presented, in the context B * 3501, by the melanoma cells and recognized by the TILs of the M28 patient. It is known that this same Melan-A 26-35 peptide is presented by HLA molecules, but only in the context of HLA-A * 0201 (KAWAKAMI et al, 1994, J Exp. Med, 180, 347-52; VALMORI and al, 1998, J Immunol, 160, 1750-8). A cross presentation of the same epitope by different isotypes of HLA class I molecules is relatively uncommon. * The specific clone of gplOO (M28.9B) recognizes, in the HLA-B * 3501 context, three overlapping peptides (two decameras and one nonamer) located between amino acids 470 and 480. However, significant concentrations of the peptide QVPLDCVLYR ( SEQ ID NO: 24) are necessary to induce a response from the specific T clone (EC50 at 20 μM). The affinity and stability measurement results for this peptide show that it is not able to attach to HLA B * 35. The weak response obtained by the clone can be explained by contamination of this peptide (prepared at 70% purity) with other peptides recognized by this clone T.

Several epitopes derived from gplOO presented in the HLA-A or C contexts are known (CASTELLI et al, 1999, J Immunol, 162, 1739-48; KAWAKAMI et al, 1998; TSAI et al, 1997, J Immunol, 158, 1796 -802). In contrast, gplOO epitopes presented in the HLA-B context have not yet been known.

* The specific tyrosinase clone (M171.100B) effectively recognizes three overlapping peptides: 14-mer 309-322, 13-mer 309-321, and 12-mer 309-320. 12-mer is the most effectively recognized peptide, with 50% of the maximum lysis obtained at 0.2 nM, against 2 nM and 12 nM for 14-mer and 13-mer respectively (Figure 3). The deletion of phenylalanine at the C-terminal end of 12-mer greatly reduces recognition by the CTLs. The peptide 312-320, which has been shown to be recognized by a CTL clone in the context of HLA-B * 3501 (MOREL et al, 1999, Int J Cancer, 83, 755-9), is not recognized by the clone TIL M171.100B. In addition, if examples of epitopes of type 11-mer cells (AARNOUDSE et al, 1999, Int. J Cancer, 82, 442-8; CHIARI et al, 1999, Cancer Res, 59, 5785-92; KAWAKAMI et al, 2001, J Immunol, 166, 2871-7) and 14-mer exists (PROBST-KEPPER et al, 2001; J Exp Med, 193, 1189-98), these are significantly larger epitopes to that of the epitopes usually presented by T cells.

* The clone M171.8C specific for MAGE-A3 / A6 recognizes the epitope EVDPIGHLY MAGE-A3 / B * 3501 previously described by SCHULTZ et al, (2001, cited above). As this TIL clone also reacts with MAGE-A6, this indicates that the peptide MAGE-A6 168-176 (ENDPIGHNY), which differs from the MAGE-A3 epitope by a single amino acid in position 8, constitutes another melanoma epitope restricted to context HLA-B * 3501.

* The specific clone of an ΝY-ESO-1 epitope in contexts B * 3501 / B * 3503 (Ml 18.45) effectively recognizes five overlapping peptides: 13-mer 92-104, 12-mer 92-103, 11 -mer 94-104, decamer 94-103 and nonamer 94-102. The nonamer 94-102 is the most effectively recognized peptide, with 50% of the maximum lysis obtained at 1 pM, against 0.05 nM for 11-mer, 0.1 nM for 13-mer, 1 nM for the decamer and 5 nM for 12-Wed.

It is known that the nonamers 94-102 and 92-100 are presented in the contexts HLA-B51 (JAGER et al, Cancer Immunity, 2002, vol2, page 12) and HLA-Cw3 (GNJATIC et al, 2000, Proc Natl Acad Sci USA, 97, 10917-22) respectively. However, their presentation in an HLA-B35 context had not been described before. EXAMPLE 4 MODIFICATION OF THE ANTIGENIC PEPTIDES PRESENT IN THE HLA-B35 CONTEXT

Some of the identified antigenic peptides do not have the appropriate anchoring residues for the HLA-B * 3501, e molecules. P, A or N in position 2, Y, F, M, L or I in position 9, for nonamers, and in position 10 for decamers (FALK et al, 1993, Immunogenetics, 38, 161-2 [erratum in Immunogenetics 1994; 39 (5): 379]). To try to increase the affinity of this peptide for the HLA-B * 3501 context and the T cell response, the inventors have introduced modified residues at positions 2 and / or 9, or 10, of the Melan-A / peptides. MARTl 26-34 and 26-35 respectively. The results obtained with these modified peptides are presented in FIG. 3 (Melan A). It is observed that the modified decapeptides EAAGIGILTY (SEQ ID ΝO: 9) and EPAGIGILTY (SEQ ID ΝO: 11) induce maximum lysis by the clone M28.10B at concentrations 10 to 50 times lower than the wild peptide (10 and 50 μM instead 500 pM). In addition, these peptides exhibit improved stability (Figure 3). These modifications therefore make it possible to increase the binding affinity of these peptides for HLA-B * 3501, but also to induce a higher reactivity of the specific TIL clone.

EXAMPLE 5 NATURAL PRESENTATION OF ANTIGENIC PEPTIDES IN THE HLA-B35 CONTEXT BY MELANOMA CELLS.

To test the interest, as targets for immunotherapy, of the different antigenic peptides identified in Examples 3 and 4, the inventors analyzed their presentation by a panel of melanoma cell lines expressing the different antigens, from which are derived. these peptides, and the HLA-B * 3501 molecules. To increase the expression on the surface of cells of HLA molecules, the melanoma cells were preincubated, for certain experiments, 48 hours in medium containing 500 U / ml of IFN-γ (Tebu, Paris, France).

The response of the various TIL clones with respect to these lines is detected by measuring their production of TNF as in Example 1 (Effector Ratio: Target 1: 3). Negative controls were performed using melanoma cell lines not expressing HLA molecules (Ml 13). The melanoma cell lines were established from fragments of metastatic tumors or of tumors having invaded the lymph nodes, and cultured in RPMI 1640 medium (Sigma, St Loins, USA) containing 10% fetal calf serum (Gibco-BRL, Cergy-Pontoise, France), penicillin (10 mg / ml), streptomycin (1 OU / ml) (Sigma) and L-glutamine (2nM) (Sigma, St Louis, USA) . The results are illustrated in FIG. 4: on the ordinate, the concentration of TNF in pg / ml; on the abscissa, the melanoma lines expressing HLA-B35 and the various antigens (M47, M125, M131, M140, and M147), or not expressing the HLA-B35 allele (Ml 13). g: melanoma cell lines pretreated with 500 U / ml INF-γ. D: melanoma cell lines not pretreated with INF-γ. The specific Melan-A, tyrosinase and MAGE-A3 clones recognize the melanoma cell lines M47, M131 and M147 independently of treatment with IFN-γ. However, these same clones do not recognize the M125 and M140 lines until after the expression of HLA-B35 has been induced by treatment with IFN-γ.

The specific clone of gplOO recognizes a melanoma line independently of treatment with IFN-γ (M147). This clone also recognizes the lines M125 and M140 after treatment with IFN-γ (weakly for Ml 25). The specific clone of NY-ESO-1 recognizes one of these lines spontaneously expressing this antigen (M47) and the other two lines after treatment with IFN-γ (M131 and M140, FIG. 4).

In the case of the M125 and M140 lines, the lack of recognition of these lines by the different TIL clones is probably linked to a limited number of MHC / peptide complexes present on the surface of these cells. Indeed, these two cell lines express the HLA-B35 molecule only after treatment with IFN-γ.

The spontaneous recognition of the melanoma lines by the different CD8 T clones, shows that the epitopes identified are naturally presented by these tumors.

EXAMPLE 6: ANTIGENICITY OF RAS MUTE PEPTIDE 55-64 ^Q61R

The wild type ras peptides 55-64 ^wτ (ILDTAGQEEY; SEQ ID NO: 34), the mutated decamer 55-64 ^Q61R , and the peptide MAGE-A3 (ENDPIGHLN; SEQ ID ΝO: 20) were obtained from SYΝT: EM (Nîmes, France). The purity (> 85%) is checked by high yield liquid chromatography in reverse phase. The peptides are lyophilized, then dissolved in DMSO at 10 mg / ml and stored at -80 ° C. The antigenicity of the peptide 55-64 ^Q61R and that of its wild type analogue (55-64 ^wτ ), are evaluated by testing the capacity of these peptides to induce the growth of specific cytotoxic T lymphocytes (CTL) by in vitro stimulation of peripheral blood mononuclear cells (PBMC) by dendritic cells (DC) pulsed with these peptides. CD8 + lymphocytes are obtained from the PBMCs of an HLA-A * 0101 donor by negative sorting of CD4 + T cells on magnetic beads (MILTENY BIOTECH, France).

The dendritic cells are prepared from adherent PBMCs cultured for 7 days in 6-well culture plates containing RPMI culture medium supplemented with 10% fetal calf serum, 50 ng / ml of GM-CSF (SIGMA) and 50 ng / ml of IL-4 (SIGMA). On D + 7, the maturation of the dendritic cells is induced for 2 days in an RPMI culture medium supplemented with 10% fetal calf serum, 10 ng / ml of TNF-α (SIGMA) and 100 μg / ml of Poly-IC (SIGMA). At D + 9, the mature dendritic cells are incubated for 2 hours with 5 μg / ml of peptide ras 55-64 ^Q61R or of peptide ras 55-64 ^τ ; they are then washed to remove the free peptides.

The dendritic cells pulsed with the peptide ras 55-64 ^Q61R or the peptide ras 55-64 ^wτ are used to stimulate CD8 + lymphocytes (3.10 ⁷ cells). 3 stimulations are performed one week apart.

Each culture well is tested for the presence of CTLs specific for the peptide. For this purpose, 7 days after the last stimulation, 2.10 ⁶ BM36.1 cells and expressing HLA-A * 0101 (KELLY et al. Nature. 355. 641. 1992) previously incubated at 37 ° C for 12 hours in RPMI supplemented with 100 μM of peptide N-rαs 55-64 ^wτ or 55-64 ^Q61R , 1 μM of β2 microglobulin, and washed in PBS, are added to each well.

The specific CTL response to stimulation by the wild-type or mutated peptide Nr <zs is measured by assaying for interferon γ (IFN-γ), as described by LAB ARRIERE et al. (Int. J. Cancer, 78, 209, 1998). Two of the five culture wells stimulated with the peptide ras show a proliferation of CTL specific for the peptide (0.3 and 0.5% of reactive cells per well), while no proliferation is observed in the wells stimulated with the peptide 55-64 ^wτ .

EXAMPLE 7 PROPERTIES OF A PEPTIDE-INDUCED T-LYMPHOCYTE CLONE 55-64 ^Q61R Lymphocyte clones were obtained by limiting dilution from the cells of the culture well containing 0.5% of reactive T cells.

The capacity of the T cells from one of these clones to lyse BM36.1 cells presenting the peptide 55-64 ^{Q IR} or the peptide 55-64 ^τ is evaluated according to a standard assay for the release of ⁵¹ Cr (HERIN et al. , Int. J. Cancer, 39, 390-396, 1987). BM36.1 cells labeled with ⁵¹ Cr (Na2 ⁵¹ CrO4, ORIS, Gif-sur-Yvette, France). The cells are then pulsed for 1 hour at 37 ° C. with 10 μM of peptide 55-64 ^ or peptide 55-64 ^Q61R , and washed. 10 ³ BM36.1 cells thus treated are incubated with 5.10 ³ T cells of the clone to be tested for 4 hours. The culture supernatants are recovered and the percentage of ⁵¹ Cr released is evaluated. We observe a significant lysis of BM36.1 cells pulsed with the mutated ras peptide 55-64 ^Q61R . In contrast, the lysis of BM36.1 cells pulsed with the wild type ras peptide is very low.

The specificity of this clone with respect to cells expressing HLA-A * 0101 and the wild-type protein or the N-ras protein carrying the Q61R mutation is evaluated on transfected COS cells, or on HLA-A * melanoma cells. 0101, expressing or not the Q61R mutation. For the transfection of COS cells, a 334 bp cDNA coding for a fragment of the wild-type N-ras protein is obtained by PCR amplification, from complete cDNA of the wild-type N-ras protein. A cDNA coding for a fragment of the protein Nr s mutated at position 61 by substitution of glutamine with an arginine is obtained by site-directed mutagenesis.

The wild-type or mutated cDNA is inserted into the vector pcDNA3 and amplified in the bacterial strain E. coli TOP 10 F '(INVITROGΕN, reference C2020-03). A cDNA coding for the molecule HLA-A * 0101 is introduced into the vector pcDNA 3.1 (INVITROGΕN, reference CV790-20). COS-7 cells are co-transfected with these constructs as described below:

COS-7 cells, (BRICHARD et al, J. Εxp. Med., 178, 489, 1993) are cultured in DMΕM medium (BIOWHITTAKΕR) containing 10% fetal calf serum, 100 U / ml of penicillin, 100 μg / ml of streptomycin (SIGMA, St Louis, USA) and 2 mM of glutamine-L (SIGMA, St Louis, USA). 16.5.10 ³ COS cells are co-transfected with 100 ng of a mixture of the pcDNA 3.1 vector expressing HLA-A * 0101 and a pcDNA3 vector expressing the wild-type or mutated N-rαs protein, by the chloroquine-dextran process DΕAΕ (BRICHARD et al, J. Εxp. Med., 178, 489, 1993; SΕΕD et al, PNAS, 84, 3365, 1987,). The details of this method are described by DE PLAEN et al. (Methods, 12, 125, 1997). The stimulation of T cells is measured by assaying TNF (DE PLAEN et al, Methods, 12, 125, 1997; LABARRIERE et al, Int. J. Cancer, 78, 209, 1998). 2.10 ³ to 10 ⁴ cells of the tested T clone are added to 3.10 ⁴ COS cells, 48 hours after transfection, or to 3.10 ⁴ melanoma cells. The culture supernatants are recovered 6 hours later and their TNF content is determined by measuring their cytotoxic effect on clone 13 of the WEHI 164 murine fibrosarcoma (BRICHARD et al, J. Exp. Med., 178, 489, 1993) by MTT colorimetric assay. The results obtained with the transfected COS cells are presented in FIG. 5a: on the abscissa, peptides ras of wild type or mutated at position 61 by substitution of glutamine with an arginine; ordinate, concentration of TNF in pg / ml The results show that the T cells of the clone are strongly stimulated by the cells expressing the protein Nr -s Q61R, whereas only a weak stimulation with the protein N- is observed wild ras.

The results obtained with the melanoma cells are presented in FIG. 5b: on the abscissa, melanoma cell lines expressing the ras Q61R mutation (M6, M90, MEL4) or not (M36, M105, M106, M122, M138, MVl); ordinate, concentration of TNF in pg / ml These results show that among the HLA-A * 0101 melanoma lines, only those expressing the Q61R mutation (lines M6, M90 and MEL4) stimulate a response of the T cells of the clone.

EXAMPLE 8 IDENTIFICATION OF AN ANTIGENIC PEPTIDE PRESENT IN THE CONTEXT HLA-A * 0101.

A CD8 + T clone from a TIL population of melanoma recognizes an undescribed peptide derived from the gplOO antigen, in the context A * 0101 (PLDCVLYRY, SEQ ID NO: 20). The antigenicity of this peptide was tested by evaluating the ability of the clone to lyse BM36.1 cells presenting the peptide of interest in the context of HLA-A * 0101. BM36.1 cells are labeled for 1 h at 37 ° C. with 100 Ci of ⁵¹ Cr (Na2 ⁵¹ CrO4, ORIS, Gif-sur-Yvette, France). The cells are then pulsed for 20 minutes with the various synthetic peptides. 10 ³ BM36.1 cells thus treated are incubated with 10 ⁴ T cells of the clone (Effector: Target ratio of 10: 1) for 4 hours. The culture supernatants are recovered and the amount of ⁵¹ Cr released is evaluated using a β plate counter (EG&G Wallac, Evry, France). A negative control is carried out with an irrelevant peptide.

The results are presented in FIG. 6: on the ordinate the percentage of cell lysis obtained, on the abscissa the concentration of peptide PLDCVLYRY (D), or VPLDCVLYRY (Φ). The LC50 for the PLDCVLYRY peptide is 0.6 μM, while the EC50 for the PLDCVLYRY peptide is 10 μM.

The sequence of this peptide is included in the sequence of the decamer recognized in the context B * 3501, but this peptide is not recognized by the clone M28.9B. On the other hand, the peptide VPLDCVLYRY (SEQ ID NO: 13) is also recognized in the context A * 0101 by the clone M199.6.12 (Table I and Figure 6). In addition, the spontaneous recognition by the clone Ml 99.6.12, of melanoma lines sharing HLA-A * 0101 shows that this epitope is effectively presented by these tumors.

Claims

1) Use of at least one immunogenic peptide representing a T epitope presented by MHC I, chosen from: a) a peptide comprising the sequence EX]AGIGILX ₂ (SEQ ID NO: 1) in which Xi represents A or P, and X ₂ represents T or Y, capable of inducing a cytotoxic response directed against the Melan-A antigen; b) a peptide comprising the sequence EVDPIGHVY

(SEQ ID NO: 2), capable of inducing a cytotoxic T response directed against the MAGE-A6 antigen; c) a peptide comprising the sequence VPLDCVLYR

(SEQ ID NO: 3), capable of inducing a cytotoxic response directed against the gplOO antigen; d) a peptide comprising the sequence TPRLPSSADVEF

(SEQ ID NO: 4), capable of inducing a cytotoxic response directed against the tyrosinase antigen; e) a peptide comprising the sequence MPFATPMEA

(SEQ ID NO: 5), capable of inducing a cytotoxic response directed against the NY-ESO-1 antigen; for obtaining a drug intended for anti-tumor immunotherapy in an HLA-B35 patient.

2) Use according to claim 1, characterized in that said peptide is chosen from: a) a peptide with a sequence chosen from: TAEEAAGIGILTV

(SEQ ID NO: 6), EAAGIGILTVIL

(SEQ ID NO: 7), EAAGIGILTV

(SEQ ID NO: 8), EAAGIGILTY

(SEQ ID NO: 9), EAAGIGILY (SEQ ID NO: 10), EPAGIGILTY (SEQ ID NO: 11), EPAGIGILTV (SEQ ID NO: 12); b) a peptide of sequence EVDPIGHVY (SEQ ID NO: 2); c) a peptide of sequence chosen from VPLDCVLYR (SEQ ID NO: 3) and VPLDCVLYRY (SEQ ID NO: 13); d) a peptide of sequence chosen from TPRLPSSADVEFCL (SEQ ID NO: 15) and TPRLPSSADVEF (SEQ ID NO: 4); e) a peptide of sequence chosen from: LAMPFATPMEAEL (SEQ ID NO: 16), LAMPFATPMEAE (SEQ ID NO: 17), MPFATPMEAEL (SEQ ID NO: 18), MPFATPMEAE (SEQ ID NO: 19) and MPFATPMEA (SEQ ID NO: 19) : 5).

3) Immunogenic peptide representing a T epitope presented by MHC I, chosen from:

- a peptide of sequence chosen from: EAAGIGILTY (SEQ ID NO: 9), EAAGIGILY (SEQ ID NO: 10), EPAGIGILTY (SEQ ID NO: 11), EPAGIGILTV (SEQ ID NO: 12);

- a peptide of sequence chosen from: VPLDCVLYR (SEQ ID NO: 3), VPLDCVLYRY (SEQ ID NO: 13) and PVPLDCVLYRY.(SEQ ID NO: 14);

- a peptide with a sequence chosen from: TPRLPSSADVEFCL (SEQ ID NO: 15) and TPRLPSSADVEF (SEQ ID NO: 4). 4) Multi-epitope composition comprising at least two peptides from two different categories among categories a) b) c) d) or e) as defined in claim 1 or 2.

5) Multi-epitope composition according to claim 4, characterized in that it comprises at least one peptide from each of these categories a) b) c) d) or e) as defined in claim 1 or 2.

6) Multi-epitope composition according to any one of claims 4 or 5, characterized in that it consists of a chimeric polypeptide comprising one or more copies of each of said peptides. 7) Polynucleotide encoding a chimeric polypeptide according to claim 6.

8) Antigen-presenting cell expressing an HLA-B35 allele of MHC I, characterized in that it is loaded with a peptide as defined in any one of claims 1 or 2.

9) Antigen-presenting cell expressing an HLA-B35 allele of MHC I, characterized in that it is transfected with a polynucleotide according to claim 7.

10) Use of at least one peptide as defined in any one of claims 1 to 3, for the in vitro detection of CTLs directed against one or more of the antigens Melan-A, MAGE-A6, gplOO, tyrosinase, and NY- ESO-1, in a biological sample obtained from an HLA-B35 subject.