FR2889959A1 - Use of peptides derived from the melanoma antigen MAGE-A1, or their derivatives, as vaccines and reagents for prevention, treatment and diagnosis of MAGE-associated cancers - Google Patents

Abstract

Use of a peptide (I), derived from the MAGE-A1 antigen, for preparing (a) a vaccine for prevention and treatment of MAGE-associated cancer in an individual carrying a DP4 allele and (b) a diagnostic reagent for the immune status of such an individual with respect to the cancer, is new. Use of a peptide (I), derived from the MAGE-A1 antigen, for preparing (a) a vaccine for prevention and treatment of MAGE-associated cancer in an individual carrying a DP4 allele and (b) a diagnostic reagent for the immune status of such an individual with respect to the cancer, is new. (I) (i) contains a T CD4 +>epitope that can be presented by HLA-DP4; (ii) can induce T lymphocytes specific for at least one MAGE antigen expressed by tumor cells and (iii) consists of (a) fragments containing 13-50, preferably 13-25, consecutive amino acids (aa) of the MAGE-A1 antigen, containing at least one of the sequences between aa 93-101, 271-279, 68-76, 107-115, 123-131, 139-147, 150-158, 215-223 and 274-282 or (b) variants of (a) of the same size and with binding activity for HLA-DP4 not over 1000 nM, able to induce CD4 +>T cells that recognize the epitopes defined in (a). Independent claims are included for the following: (1) similar use of a polyepitopic fragment (II) consisting of a concatenation of at least two epitopes (same or different) of at least one (I); (2) similar use of a fusion protein (III) containing a protein (or its fragment) fused to (I); (3) similar use of a lipopeptide (IV) prepared by adding a lipid to the alpha -amino group or reactive sidechain in (I); (4) similar use of an expression vector that encodes (I)-(IV); (5) immunogenic or vaccinating compositions that contain at least one of (I)-(IV) as antigen, plus a vehicle, carrier or adjuvant; (6) diagnostic reagent that contains the antigen of (5); (7) method for diagnosing the immune status of an individual with respect to a MAGE-related cancer by contact with (I) and detecting specific CD4 +>lymphocytes, in vitro; (8) method for selecting CD4 +>T cells that are specific for MAGE expressed in tumor cells; (9) (I) as new peptides (Ia) except for MAGE-A2 127-139; A3 111-125, 111-126, 113-126, 113-127, 113-128, 114-126, 114-127, 114-128, 127-142, 146-160, 156-170 and 281-295; A6 121-144, 140-158, 140-170, 150-165, 155-170 and 219-236; (10) new polyepitopic fragments (IIa) that include at least one (Ia); (11) new fusion proteins (IIIa) that include (Ia) or (IIa); (12) new lipopeptide (IVa) that includes (Ia) or (IIa); (13) new polynucleotides (NA) that encode (Ia), (IIa) or (IIIa); (14) expression vectors containing NA and regulatory sequences; and (15) host cells that contain NA or the vector of (15). ACTIVITY : Cytostatic. No details of tests for cytostatic activity are given. MECHANISM OF ACTION : Vaccine.

Description

CD4 + EPITOPES OF MAGE-A ANTIGENS

  HLA-DP4 RESTRICTIONS AND THEIR APPLICATIONS The present invention relates to CD4 + T epitopes of tumor-specific MAGE antigens restricted to HLA-DP4, and to their vaccine and diagnostic applications.

  Tumor cells express antigens that are recognized by CD4 + and CD8 + T cells and that are tumor specific (for review see Kirkin et al., Cancer Investigation, 2002, 20, 222-236). These tumor-specific antigens, called CT (Cancer Testis) antigens, are also expressed in certain healthy tissues (testes, placenta, ovaries) but are not recognized by T cells because these tissues lack conventional HLA molecules. The MAGE-A, MAGE-B and MAGE-C antigens, which are expressed in many tumors, represent particularly interesting targets for anti-tumor vaccination. These antigens belong to the MAGE antigen superfamily which also includes ubiquitous antigens (MAGE-D) or specifically expressed in the brain (necdine, MAGEL2). MAGE antigens comprise 3 domains (d1, d2 and d3); the d2 domains show strong homology (> 60%) whereas a lower homology is observed for some of the d1 domains, in particular those of the tumor-specific MAGE antigens and in particular those of the MAGE-A and MAGE-B antigens. MAGE-A antigens are the best known and most documented. They group together 12 different antigens, the MAGE-A 1 antigen (or MAGE-1) being the first tumor antigen identified. The expression MAGE-AI, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A 12 is found in many tumors, whereas that of MAGE-A5, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-Al 1 is found in only a few and MAGE-A7 has never been detected. Except for leukemic cells, lymphomas and carcinomas of the kidney, MAGE-AI and MAGE-A3 antigens are found in 15 to 50% of cancers such as melanomas, sarcomas and cancers of the lung, breast, breast cancer. stomach and prostate; the MAGE-Al antigen alone is especially expressed in 80% of liver tumors. The MAGE-A4 antigen has a different expression profile from other MAGE-A antigens and is frequently expressed in lymphomas.

  CD4 + T lymphocytes have been shown to play a vital role in tumor control. Indeed, the induction of cytotoxic T lymphocytes (CTL) is dependent on the activation of CD4 + T lymphocytes which intervene in particular in the recruitment and maintenance of CTL. Thus, immunization with CD8 + T epitopes alone, induces cytotoxic T lymphocytes specific for the tumor antigen, with a very low frequency (of the order of 10-4 to 10 'of CD8 + cells; Zhang et al., Eur J. Immunol., 2005, 35 :). Through cellular contacts and the secretion of many cytokines, CD4 + T cells induce the activation of antigen presenting cells (APCs) which in turn recruit tumor-specific CD8 + T cells. They also intervene in the maturation of effector cells that are cytotoxic T lymphocytes. In addition, the results observed in mice not expressing major class 1 histocompatibility complex molecules, also indicate that CD4 + T cells exert tumor control via CTL independent mechanisms probably via activation. of macrophages. Finally, CD4 + T lymphocytes may themselves be cytotoxic.

  CD4 + T cells recognize the tumor peptides presented to them by the class II major histocompatibility complex molecules; in humans, they are called HLA II molecules for Human Leucocyte Antigen class H. Recognition can take place directly (ie the tumor itself presents these peptides to T cells) but it is likely that the main route of activation is through the dendritic cells. These cells are in fact the main antigen presenting cells capable of recruiting naive T lymphocytes in vivo. These antigenic peptides, termed T epitopes, result from the proteolytic degradation of the antigens by the antigen presenting cells. They have variable lengths, generally from 13 to 25 amino acids and have a sequence that makes them capable of binding to HLA II molecules. It is well known that, like the native antigen, a peptide comprising a CD4 + T epitope is capable of stimulating specific CD4 + T cells in vitro or recruiting them in vivo. It is therefore sufficient to induce a CD4 + T response.

  These observations are in favor of the use of such antigenic peptides specific for MAGE antigens expressed by the tumor cells, such as the MAGE-A, MAGE-B or MAGE-C antigens, and capable of stimulating a strong CD4 + T response, for the preparation of an anti-tumor vaccine composition in humans, especially in combination with CD8 + T epitopes specific for these tumor-specific MAGE antigens.

  In addition, such peptides which are recognized by CD4 + T lymphocytes specific for tumor antigens are also useful as reagents in a test for diagnosis or monitoring treatment of a cancer in humans, based on the detection of said T cells. CD4 +, either directly, in particular by flow cytometry in the presence of multimers of complex class II molecules / peptide, or indirectly, in particular by a lymphocyte proliferation assay, or an assay of antibodies or cytokines.

  However, while numerous sequences of CD8 + T epitopes capable of being presented by molecules of the major histocompatibility complex of class I, in humans (HLA I molecules or Human Leucocyte Antigen type 1), have been identified in the MAGE antigens (US Patent 6,063,900; PCT Application WO 2004/052917; http: //www.cancerimmunity.org/peptidateatabase/ tumorspecific.htm, the known number of CD4 + T epitopes is smaller.

  One of the major problems limiting the use of these peptides as an antigen is the identification of CD4 + T epitopes, since their sequence varies from one individual to another due to the polymorphism of HLA II molecules. Indeed, the HLA II molecules are heterodimers consisting of a polymorphic chain alpha (a) and chain beta 03). There are four types of HLA II molecules per individual (2 HLA-DR, 1 HLADQ and 1 HLA-DP), named according to the allele encoding the beta chain which is the most polymorphic. The HLA-DR molecule is very polymorphic. Indeed, while its alpha chain has only 3 alleles, the beta chain (13) encoded by the DRB1 gene, which is the most expressed, currently has 458 alleles. For the HLA-DQ and HLA-DP molecules, the two chains (a and (3) that constitute them are polymorphic but have fewer alleles.There were 28 DQA1 alleles (HLA-DQ a-chain), 60 alleles DQB1 (HLA-DQ R chain), 22 DPA 1 alleles (HLA-DP a chain) and 116 DPB 1 alleles (HLA-DP 13 chain).

  However, the combination of the two α and β chains encoded by these alleles gives rise to numerous molecules FILA-DO and HLA-DP.

  Because of this polymorphism, these isoforms have different binding properties to each other, which implies that they can bind different peptides of the same antigen. Thus, the mode of binding of the peptides to the HLA II molecules is more complex than that of the HLA I molecules and there are no reliable programs for predicting peptides capable of binding to the HLA II molecules. Indeed, the HLA molecules have a very close three-dimensional structure but the binding site of the FILA II molecules is an open groove at both extremities. For this reason, peptides restricted to HLA II molecules accept peptides of variable size, generally between 13 and 25 amino acids. This throat is characterized by five pockets of specificity (PI, P4, P6, P7 and P9 corresponding to the positions of the residues of the peptide that they accommodate) which harbor polymorphic residues. Since the sequence differences between the different HLA II molecules are essentially located in the pockets of the peptide binding site, they intervene directly in the repertoire of peptides that bind to each molecule.

  Thus, each individual recognizes in an antigen a set of peptides whose nature depends on the HLA II molecules that characterize it. Since there are a large number of HLA II alleles, there is therefore in a given sequence an important repertoire of T epitopes of very different sequences, each specific for a different allele. A peptide capable of stimulating a CD4 + T-specific response of a tumor antigen in a few individuals may therefore be inactive in the majority of other individuals, because the latter do not recognize the tumor antigen by the same epitopes.

  Most CD4 + T epitopes of MAGE antigens are restricted to a FILA DR molecule and are derived from the MAGE-A3 antigen [http: // www. cancerimmunity.org/peptidedatabase/tumorspecific.htm; MAGE-A3 267-282 restricted to DRI (PCT International Application WO 02/095051); MAGE-A3 149160 restricted to DR4 and DR7 (Kobayashi et al., Cancer Research, 2001, 61, 4773-4778); MAGE-A3 191-205 and 281-295 restricted to DR11 (Consogno et al., Blood, 2003, 101, 1038-1044, Manici et al., J. Exp.Med., 1999, 189, 871-876) and MAGE-A3 121-134 restricted to DR13 (US Patent 6,716,809); MAGE-A1 281-292 restricted to DR15 (PCT International Application WO 00/78806); DR4-restricted MAGE-A6 102-116, 121-144, 140-170, 145-160, 150-165 and 246-263 (Tatsumi et al., Clinical Cancer Research, 2003, 9, 947954) MAGE-A 1,281 -292 restricted to DR 15 (PCT International Application WO 00/78806); MAGE-A6 102-116, 121-144, 140-170, 145-160, 150-165 and 246263 restricted to DR4 (Tatsumi et al., Clinical Cancer Research, 2003, 9, 947-954)].

  However, for the HLA-DR molecules, a dozen alleles are necessary to cover more than 60% of the gene frequency found in the Caucasian population and therefore affect more than 85% of this population. Thus, a peptide carrying an HLA-DR restricted epitope is generally not recognized by all predominant HLA-DR molecules in the population. Consequently, each peptide carrying an HLA-DR restricted epitope must be tested against a dozen HLA-DR molecules in order to select the peptides recognized by the majority of these molecules, and it is generally necessary to use a mixture of at least two or three different peptides to obtain an effective vaccine in the majority of individuals in the population to be vaccinated.

  Two HLA-DP4 molecules (DP401 and DP402) which have only 3 amino acids of difference have the particularity of being very frequent, especially in the Caucasian population. These two DP4 molecules alone cover the majority of the HLA-DP allelic frequency in the Caucasian population (around 50% in Europe and 80% in North America) and are also present at significant frequencies. in other populations (allelic frequency in the order of 60% in South America, 60% in India, 25% in Africa and 15% in Japan). Each of these DP4 molecules comprises an alpha chain coded either by the DPAI * 0103 allele which is the most frequent (78.2%) or by the DPAI * 0201 allele (20.2%) and a beta chain. coded by the allele DPB 1 * 0401 (molecule HLA-DP401) or DPB 1 * 0402 (molecule HLA-DP402). The HLA-DP4 molecules are perfectly capable of presenting peptides to T lymphocytes since many T clones have been isolated from a wide variety of antigens such as tetanus toxin, hepatitis B virus and allergen. major of Dermatophagoides pteronissimus.

  However, only one HLA-DP restricted (HLA-DP4) and HLA-DQ (HLA-DQ6) CD4 + T epitope was identified, only in MAGE-A3 antigen (MAGE-A3 243-258; US Patent 6,716,809; Schultz et al., J. Immunol., 2004, 172: 13041310), indicating that these two HLA II molecules would apparently be only weakly used for the recognition of tumors.

  The inventors have identified other CD4 + T epitopes restricted to HLADP4, in the MAGE-A 1 antigen; these epitopes, which are different from the known epitope of the MAGE-A3 antigen, are also present in other tumor-specific MAGE antigens, in particular in the MAGE-A2, MAGE-A3, MAGE-A4 and MAGE-A9 antigens, MAGE-Al 0, MAGE-Al 1 and MAGE-Al 2. Furthermore, these peptides, which are capable of being presented by an HLA-DP4 molecule and of inducing specific CD4 + T lymphocytes in naive individuals, are capable of inducing, by themselves, an antigen-specific CD4 + T response (s). MAGE expressed by tumor cells, in the majority of vaccinated individuals.

  The subject of the present invention is therefore the use of a peptide derived from the MAGE-A 1 antigen comprising a CD4 + T epitope capable of being presented by an HLA-DP4 molecule and to induce T lymphocytes specific for at least a MAGE antigen expressed by the tumor cells, which peptide is selected from the group consisting of: a) the 13 to 50 consecutive amino acid fragments of the MAGEAI antigen, preferably 13 to 25 amino acids, comprising at least the sequence located between the following positions: - positions 68-76 (SEQ ID NO: 1); these fragments include MAGE epitope I, positions 93-101 (SEQ ID NO: 2); these fragments include MAGE epitope II, positions 107-1 (SEQ ID NO: 3); these fragments include MAGE epitope III, positions 123-131 (SEQ ID NO: 4); these fragments include the MAGE epitope IV, positions 139-147 (SEQ ID NO: 5); these fragments include the MAGE epitope V, positions 150-158 (SEQ ID NO: 6); these fragments include the MAGE epitope VI, positions 215-223 (SEQ ID NO: 7); these fragments include MAGE epitope VII, positions 271-279 (SEQ ID NO: 8); these fragments include MAGE epitope VIII, positions 274-282 (SEQ ID NO: 9); these fragments comprise the epitope IX of MAGE, b) the variant peptides of the peptides defined in a), from 13 to 50 amino acids, preferably from 13 to 25 amino acids, which exhibit an activity of binding to an HLA-DP4 molecule less than or equal to 1000 nM and are capable of inducing CD4 + T cells recognizing one of the MAGE epitopes defined in a), for the preparation of a vaccine intended for the prevention or treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent for diagnosing the immune status of said individual with respect to this cancer.

  The term "individual carrying a DP4 allele" means an individual carrying an allele coding for the beta chain of a DP4 molecule, preferably the DPB 1 * 0401 or DPB 1 * 0402 allele.

  The peptides according to the invention have the following properties: HLA-DP4 binding activity: the peptides according to the invention have a high affinity for HLA-DP4 (binding activity <1000 nM); the binding activity can be measured by a HLADP4 / peptide binding assay, in competition, with immunoenzymatic revelation, according to the principle described in PCT International Application WO 03/040299, immunogenicity: the peptides according to the invention are recognized by CD4 + T cells in the majority of individuals because of the preponderance of the HLA-DP4 molecule in the Caucasian population but also in the populations of South America and India, and to a lesser extent in those from Africa and Japan. These peptides are also capable of inducing CD4 + T cells specific for a MAGE antigen expressed by tumor cells from the precursors present in the majority of naive individuals or to stimulate such cells in the majority of individuals suffering from cancer associated with the expression of a MAGE antigen. In addition, the CD4 + T cells induced by these peptides recognize the epitope of the native antigen naturally present by the dendritic cells. Consequently, such peptides represent potential candidates for prophylactic or therapeutic vaccination against cancers associated with the expression of a MAGE antigen, which are particularly interesting because they can induce a CD4 + T response specific for a MAGE antigen in the majority of individuals in the population. In addition, peptides whose sequence is conserved in the various MAGE antigens or which cross-react with CD4 + T epitopes present in other MAGE antigens are capable of inducing a CD4 + T response towards several antigens. MAGE expressed by tumor cells. The immunogenicity of the peptides can be determined, in particular from peripheral blood mononuclear cells (PBMCs), by any appropriate assay known to those skilled in the art, for example: a cell proliferation assay, an ELISPOT assay (cell assay cytokine producers) or an intracellular cytokine assay (IFN-γ. IL-2, IL-4 and IL-10).

  The sequence of the MAGE-Al antigen is available in the databases and corresponds to the access number SWISSPROT P43355.

  The peptides according to the invention comprise a CD4 + T epitope consisting of a sequence of 9 to 12 amino acids as defined above, flanked at one of its ends, preferably at both ends, with at least 2 amino acids, preferably 3 amino acids. The sequence of 9 to 12 amino acids, generally 9 amino acids, includes residues P1, P4, P6 and P9, anchoring to an HLA-DP4 molecule and residues in position 1, 4, 6 and 9 of said sequence correspond respectively to residues P1, P4, P6 and P9. For example, in the peptides comprising the epitope II which includes the sequence ILESLFRAV (SEQ ID NO: 2), the anchoring residues are I (P1), S (P4), F (P6) and V (P9).

  The invention also encompasses natural or synthetic variants obtained from the preceding sequences, by mutation (insertion, deletion, substitution) of one or more amino acids in the peptide sequence, since said variant retains a high affinity for HLA-DP4 (binding activity <1000 nM) and is immunogenic.

  The natural variants correspond to the homologous peptides derived from other MAGE antigens expressed by the tumor cells and in particular MAGE-A2, MAGE-A3 antigens. MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A1, MAGE-A1 and MAGE-A 2. The sequences of the other MAGE antigens are available in the databanks; the access numbers of the other human MAGE-A antigens in the SWISSPROT base are as follows: MAGE-A2: P43356; MAGE-A3: P43357; MAGE-A4: P43358; MAGE-A5: P43359; MAGE-A6: P43360; MAGE-A8: P43361; MAGE-A9: P43362; MAGE-AlO: P43363; MAGE-A1: P43364; MAGE-Al2: P43365. The sequence of these variants can be deduced easily after optimal alignment of the MAGE antigen sequences expressed by the tumor cells. using appropriate software known to those skilled in the art such as CLUSTAL W (Thompson et al., 1994, N.A.R., 22: 4673-4680).

  In addition, other variants can be easily constructed since the amino acid residues involved in HLA-DP4 binding (PI, P4, P6 and P9 anchor residues) and the effect of modifications of these residues on the binding to HLA-DP4 are known to those skilled in the art; PCT International Application WO 03/040299 teaches in particular that the residue in P6 must be aromatic or hydrophobic or consist of a cysteine residue (C), and at least one of the residues P1, P9 is such that PI is aromatic or hydrophobic and / or P9 is aromatic or hydrophobic or is a C, D, Q, S, T or E residue, while the residue at P4 can be any amino acid residue.

  The invention also encompasses modified peptides derived from the foregoing by introduction of any modification at amino acid residue (s), peptide bond or peptide ends, as long as said modified peptide retains a high affinity for HLA. DP4 (binding activity <1000 nM) and is immunogenic. These modifications, which are introduced into the peptides by conventional methods known to those skilled in the art, include, without limitation: the substitution of an amino acid by a nonproteinogenic amino acid (amino acid D or the like of an acid amine); adding a chemical group (lipid, oligo or polysaccharide) at a reactive function, in particular the side chain R; modification of the peptide bond (CO-NH-), in particular by a retro-type or retro-inverso bond (-NH-CO-) or a bond different from the peptide bond; cyclization; the fusion of a peptide (epitope of interest for vaccination, label useful for the purification of the peptide, especially in cleavable form with a protease); the fusion of the sequence of said peptide with that of a protein, in particular an α chain or of an HLA-DP4 molecule or the extracellular domain of said chain, or else a targeting sequence in the endosome derived in particular from the invariable chain II or LAMP-1 protein; coupling to a suitable molecule, in particular a marker, for example a fluorochrome. These modifications are intended in particular to increase the stability and more particularly the resistance to proteolysis, as well as the solubility, the immunogenicity or to facilitate the purification or the detection, either of the peptide according to the invention, or of specific CD4 + cells of said peptide.

  According to an advantageous embodiment of said use, the peptide defined in a) is a peptide of the MAGE-A1 antigen selected from the group consisting of: SCILESLFRAVITK (positions 90-104, SEQ ID NO: 10, epitope II); PRALAETSYVKVLEY (positions 268-282: SEQ ID NO: 11, epitope VIII); 65-79: SEQ ID NO: 12; epitope I); 104-118: SEQ ID NO: 13; epitope III), 120-134: SEQ ID NO: 14; epitope IV), 136-150: SEQ ID NO: 15; epitope V), 147-161: SEQ ID NO: 16; epitope VI); PTTINFTRQRQPNEG (positions KKVADLVGFLLLKYR (positions REPVTKAEMLESVIK (positions YKHCFPEIFGKASES (positions ASESLQLVFGIDVKE (positions EEIWEEISVMEVYDG (positions 212-226: SEQ ID NO: 17, epitope VII) and LAETSYVKVLEYVIK (positions 271-285: SEQ ID NO: 18, epitope IX).

  Preferably, said peptide is selected from the group consisting of SEQ ID NO: 10, 11, 13, 14 and 17.

  According to another advantageous embodiment of said use, the variant peptide in b) is a fragment of another MAGE antigen expressed by the tumor cells, whose sequence is homologous to that of the fragment in a), preferably said homologous fragment. is derived from a MAGE-A antigen selected from: MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A1 0, MAGE-A1 1 and MAGE-A 12.

  According to an advantageous arrangement of this embodiment, said variant peptide is selected from the group consisting of: the fragments of 13 to 50 consecutive amino acids of the MAGE-A9 antigen, preferably from 13 to 25 amino acids, comprising at least minus the sequence VYYTLWSQF (positions 71-79: SEQ ID NO: 19); the peptides including this sequence correspond to the epitope I of MAGE, the fragments from 13 to 50 consecutive amino acids of the MAGE-A2, MAGE-A3 or MAGE-Al2 antigens, preferably from 13 to 25 amino acids, comprising at least the A / VELVHFLLL sequence (positions 114-122: SEQ ID NO: 20); the peptides including this sequence correspond to the MAGE epitope III, the 13 to 50 consecutive amino acid fragments of the MAGE-A2, MAGE-A3, MAGEA4 antigens. MAGE-A9, MAGE-A10, MAGE-A1 or MAGE-Al2, preferably from 13 to 25 amino acids, comprising at least the sequence XiTKAEX2LX3X4 in which XI represents F, V or I; X2 is M or I; X3 is G or E and X4 is S or R (SEQ ID NO: 21). The sequence SEQ ID NO: 21 corresponds to the following positions: 130-138 of MAGE-A2, MAGE-A3 and MAGE-A1 2, 131-139 of MAGE-A4, 129-138 of MAGE-A9, 155-163 of MAGE-A2. 10, 133-141 of MAGE-A 11. The peptides including this sequence correspond to the MAGE epitope IV, the 13 to 50 consecutive amino acid fragments of the MAGE-A2 antigen, preferably from 13 to 25 amino acids, comprising at least the sequence FFPVIFSKA (positions 146-154: SEQ ID NO: 22); the peptides including this sequence correspond to the epitope V of MAGE, the fragments of 13 to 50 consecutive amino acids of the MAGE-A9 antigen, preferably from 13 to 25 amino acids, comprising at least the sequence FMQVIFGTD (positions 156-164: SEQ ID NO: 23); the peptides including this sequence correspond to the epitope VI of MAGE, the fragments from 13 to 50 consecutive amino acids of the MAGE-A2 and MAGE-A10 antigens, preferably from 13 to 25 amino acids, comprising at least the sequence WEXILX2SMX3X4X5 ( SEQ ID NO: 24) wherein X1 is E or A, X2 is S or N, X3 is L or M, X4 is E or G and X5 is V or L. The sequence SEQ ID NO: 24 corresponds to the following positions: 222 to 230 of MAGE-A2 or 247 to 253 of MAGE-AlO; the peptides including this sequence correspond to the epitope VII of MAGE, and the fragments of 13 to 50 consecutive amino acids of the MAGE-A10 antigen, preferably from 13 to 25 amino acids, comprising at least the sequence IRKMSLLKF (positions 306-314: SEQ ID NO: 25); the peptides including this sequence correspond to the IX epitope of MAGE.

  The positions are indicated relative to the sequence of each of the different MAGE-A antigens, as specified in the Table below: Table I: Position of Epitopes I to IX in the MAGE Antigen Sequence HLA-DP4-restricted CD4 Epitope T Epitope T *

MAGE I II III IV V VI VII VIII IX

  Al 68-76 93-101 107-115 123-131 139-147 150-158 215-223 271-279 274-282 A2 75-83 100-108 114-122 130-138 146-154 157-165 222-230 278-286 281-289 A3 75-83 100-108 114-122 130-138 146-154 157-165 222-230 278-286 281-289 A4 76-84 101-109 115-123 131-139 147-155 158-166 223-231 279-287 282-290 A5 75-83 100-108 114-122 - - - - _ _ A6 75-83 100-108 114-122 130-138 146154 157-165 222-230 - - A8 75-83 100-111 117-125 133-141 149-157 160-168 225-233 - A9 71-79 96-107 113-121 129-138 145-153 156-164 221-229 277285 280-288 A10 97-105 122-133 139-147 155-163 171-179 182-190 247-253 303-311 306-314 A11 75-83 100-111 114-122 133-141 149-157 160-168 225233 281-289 284-292 Al2 75-83 100-108 114-122 130-138 146-154 157-165 222230 278-286 281-289 killed by: - the sequence SISVYYTLWSQFDEG of the MAGE-A9 antigen (positions 68-82; SEQ ID NO: 26) corresponding to the epitope I, the sequence RKMVELVHFLLLKYR of the MAGE-A2 antigen (positions 111-125: SEQ ID NO: 27) corresponding to the epitope III, the sequestr RKVAELVHFLLLKYR antigen of the MAGE-A3 antigen (positions 111-125: SEQ ID NO: 28) corresponding to the epitope III, - the RKMAELVHFLLLKYR sequence of the MAGE-Al2 antigen, (positions 111-125: SEQ ID NO: 29) corresponding to the epitope III, the positions are those of the anchoring residues P1 and P9 except for the 12 amino acid sequence (). Preferably, said fragment is selected from the group consisting of: MAGE-A2 antigen (positions 127-141: SEQ ID NO: 30) corresponding to the IV epitope, - - the KELVTKAEMLERVIK sequence of the MAGE-A4 antigen (positions 128-142: SEQ ID NO: 31) corresponding to the IV epitope, the KEPVTKAEMLESVIK sequence of the MAGE-A9 antigen (positions 126-140: SEQ ID NO: 32) corresponding to the IV epitope, - the KEPITKAEILESVIK sequence of the MAGE-AlO antigen (positions 152- 166: SEQ ID NO: 33) corresponding to the epitope IV, the KGLITKAEMLGSVIK sequence of the MAGE-A1110 antigen (positions 130-144: SEQ ID NO: 34) correspo ndant to the epitope IV, - the REPFTKAEMLGSVIR sequence of the MAGE-Al2 antigen (positions 127-141: SEQ ID NO: 35) corresponding to the IV epitope, - the CQDFFPVIFSKASEY sequence of the MAGE-A2 antigen (positions 143-157: SEQ ID NO: 36) corresponding to the V epitope, the ASEFMQVIFGTDVKE sequence of the MAGE-A9 antigen (positions 153-167: SEQ ID NO: 37) corresponding to the VI epitope, the sequence EKIWEELSMLEVFEG of the MAGE-A2 antigen (positions 219-233: SEQ ID NO: 38) corresponding to the epitope VII, the sequence EVIWEALNMMGLYDG of the MAGE-A10 antigen (positions 244-258; SEQ ID NO: 39) corresponding to the epitope VII, and the HAEIRKMSLLKFLAK sequence of the MAGE-AlO antigen (positions 303-317: SEQ ID NO: 40) corresponding to the IX epitope.

  According to another advantageous embodiment of said use, the peptide variant in b) is a synthetic variant obtained by the substitution of at least one of the residues P1, P6 and / or P9 by an aromatic or hydrophobic amino acid, of the residue P6. by cysteine (C), residue P9 by C, D, Q, S, T or E and / or residue P4 by another natural or synthetic amino acid.

  The term natural or synthetic amino acid is understood to mean the naturally occurring α-amino acids commonly found in the proteins (A, R, N, D, C, Q, E, G, H, I, L, K, M, F). P, S, T, W, Y and V), some amino acids rarely encountered in proteins (hydroxyproline, hydroxylysine, methyllysine, dimethyllysine ..), amino acids that do not exist in proteins such as (3- alanine, y-amino-butyric acid, homocysteine, ornithine, citrulline, canavanine, norleucine, cyclohexylalanine, and the enantiomers and diastereoisomers of the foregoing amino acids.

  The term hydrophobic amino acid is understood to mean an amino acid selected from (code to a letter): A, V, I, P, W, F and M. The term aromatic amino acid means an amino acid selected from (code to a letter): F, W and Y. According to another advantageous embodiment of said use, the peptide has a sequence of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 acids. 10 amines.

  According to another advantageous embodiment of said use, the peptide is labeled or complexed; it may in particular be complexed with labeled HLA-DP4 molecules, for example biotinylated, to form HLADP4 / peptide complexes, in particular multimeric complexes such as tetramers.

  According to yet another advantageous embodiment of said use, the DP4 molecule comprises a chain (3 coded by the DPB1 * 0401 allele (HLA-DP401 molecule) or DPBI * 0402 (HLA-DP402 molecule), the oc chain being coded by the DPA l * 0103 or DPA 1 * 0201 allele.

  The present invention also relates to the use of a polyepitopic fragment comprising the concatenation of at least two identical or different epitopes of which at least one is a CD4 + T epitope of a MAGE antigen included in a peptide as defined above, for the preparation of a vaccine for the prevention or treatment of cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a diagnostic reagent for the immune status of said individual with respect to this cancer.

  Preferably, said polyepitopic fragment has a length of 20 to 1000 amino acids, preferably 20 to 100 amino acids.

  Said polyepitopic fragment advantageously comprises a label fused at one of its ends for the purification or detection of said fragment. The tag, particularly a polyhistidine sequence or an epitope B of an antigen, is preferably separated from the polyepitopic sequence by a protease cleavage site so as to isolate the polyepitopic sequence from the fusion.

  According to an advantageous embodiment of said use, the polyepitopic fragment comprises the concatenation of at least one CD4 + T epitope of a MAGE antigen included in a peptide as defined above and of at least one epitope selected from the group consisting of: - a CDS + T epitope of a MAGE antigen expressed by the tumor cells, presented by an HLA 1 molecule and recognized by cytotoxic T lymphocytes specific for said antigens; such CD8 + T epitopes are described in particular at http: //www.cancerimmunity. org / peptidedatabase / tumorspecific.htm; preferably, the CD8 + T epitope is chosen from MAGE-Al 96-104, MAGE-A3 168-176, MAGE-Al 0 254-262 and MAGE-A 12 170-178, a universal natural or synthetic CD4 + T epitope, such as tetanus toxin peptide TT 830-846 (O'SULLIVAN et al., J. Immunol., 1991, 147, 2663-2669), haemagglutinin peptide of HA 307-319 influenza virus (O Sullivan et al, supra), PADRE peptide (KXVAAWTLKAA, Alexander et al., Immunity, 1994, 1, 751-761) and peptides derived from Plasmodium falciparum antigens such as CS.T3 peptide (SINIGAGLIA et al. , Nature, 1988, 336, 778-780) and the peptides CSP, SSP2, LSA-1 and EXP-1 (DOOLAN et al., J. Immunol., 2000, 165, 1123-1137), an epitope B consisting of a sugar (ALEXANDER et al., supra), said epitope B being preferably in the form of a glycopeptide, and a B epitope of a MAGE antigen expressed by the tumor cells specifically recognized by antibodies directed against said antigen. tumor.

  The combination of the CD4 + T epitope with at least one of the epitopes as defined above advantageously makes it possible to trigger or modulate an anti-tumor immune response.

  The present invention also relates to the use of a lipopeptide comprising a peptide or a polyepitopic fragment as defined above, for the preparation of a vaccine intended for the prevention or treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent for diagnosing the immune status of said individual with respect to this cancer.

  Said lipopeptide is especially obtained by adding a lipid on an α-amino function or on a reactive function of the side chain of an amino acid of said peptide or polyepitopic fragment; it may comprise one or more chains derived from C4-20 fatty acids, optionally branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-amino hexadecanoic acid, pimelautide, trimetauxide) or a derivative of a steroid. The preferred lipid portion is especially represented by a N-acetyl-lysine Nc (palmitoyl) group, also called Ac-K (Pam).

  The present invention also relates to the use of a fusion protein consisting of a protein or a fragment of protein, fused with a peptide or a polyepitopic fragment as defined above, for the preparation of a vaccine intended for the prevention or treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent for diagnosing the immune status of said individual vis-à-vis -vis of this cancer.

  The peptide or polyepitope fragment may be fused with the NH 2 or COOH end of said protein or inserted into the sequence of said protein.

  According to an advantageous embodiment of said use, the fusion protein consists of a MAGE peptide as defined above, fused with a targeting sequence in the endosome, preferably derived from a human invariable chain II or LAMP-1 protein. The targeting sequences in the endosome and their use for the targeting of antigens in the endosome are in particular described in Sanderson et al. (P.N.A.S., 1995, 92: 7217-7222), Wu et al. (P.N.A.S., 1995, 92: 11671-11675) and Thompson et al. (J. Virol., 1998, 72: 2246-2252).

  According to another advantageous embodiment of said use, the fusion protein consists of a MAGE peptide as defined above, fused with one of the chains of an HLA-DP4 molecule, preferably the beta chain, or with a fragment thereof corresponding to a soluble HLA-DP4 molecule, in particular a fragment corresponding to the extracellular domain preceded by the homologous signal peptide or a heterologous signal peptide. Said MAGE peptide is advantageously inserted between the signal peptide and the NH 2 end of the extracellular domain of the p chain, as described for the HLADR molecule (Kolzin et al., PNAS, 2000, 97, 291-296).

  Alternatively, said MAGE peptide or said polyepitopic fragment are fused with a protein facilitating their purification or their detection, known to those skilled in the art such as in particular Glutathione STransferase (GST) and fluorescent proteins (GFP and derivatives). In this case, the sequence of the peptide or polyepitopic fragment of interest is preferably separated from the rest of the protein by a protease cleavage site, to facilitate the purification of said peptide or said polyepitopic fragment.

  The present invention also relates to the use of an expression vector comprising a polynucleotide encoding a peptide, a polyepitopic fragment or a fusion protein as defined above, under the control of appropriate regulatory sequences, transcription and possibly for translation, for the preparation of a vaccine intended for the prevention or treatment of cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation a reagent for diagnosing the immune status of said individual with respect to this cancer.

  According to the invention, the sequence of said polynucleotide is that of the cDNA encoding said peptide or polyepitopic fragment or said fusion protein. Said sequence may advantageously be modified in such a way that codon usage is optimal in the host in which it is expressed. In addition, said polynucleotide may be linked to at least one heterologous sequence.

  Within the meaning of the present invention, heterologous sequence is understood to mean a nucleic acid sequence coding for a MAGE antigen expressed by the tumor cells, any nucleic acid sequence other than those which, in nature, are immediately adjacent to said nucleic acid sequence encoding said peptide of the MAGE antigen.

  According to the invention said recombinant vector comprises an expression cassette including at least one polynucleotide as defined above, under the control of appropriate transcriptional regulatory and possibly translational sequences (promoter, activator, intron, codon of (ATG), stop codon, polyadenylation signal). Many vectors in which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known. in themselves; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form, or integration into the chromosomal material of the host), as well as the nature of the host cell. For example, it is possible to use, inter alia, viral vectors such as adenoviruses, retroviruses, lentiviruses, AAVs and baculoviruses, in which the sequence of interest has been inserted beforehand; said sequence (isolated or inserted in a plasmid vector) may also be associated with a substance enabling it to cross the membrane of the host cells, such as a transporter such as a nanotransporter or a preparation of liposomes, or of cationic polymers, or the to introduce into said host cell using physical methods such as electroporation or microinjection. In addition, these methods can advantageously be combined, for example by using electroporation associated with liposomes.

  The subject of the present invention is also a peptide as defined above, with the exception of the MAGE-A3 peptides 111-125, 111-126, 113-126, 113127, 113-128, 114-126, 114-127, 114 -128, 146-160, 281-295 and MAGE-A6 121144, 140-170, 150-165 and 219-236, as an antigen for prophylactic or therapeutic vaccination, diagnosis or therapeutic follow-up of a cancer associated with the expression of a MAGE antigen.

  The present invention also relates to a polyepitopic fragment, a fusion protein, a lipopeptide or an expression vector as defined above, excluding those derived from the MAGE-A3 peptides 111-125, 111-126. , 113-126, 113-127, 113-128, 114-126, 114-127, 114-128, 146-160, 281-295 and MAGE-A6 121-144, 140-170, 150-165 and 219- 236, as an antigen for prophylactic or therapeutic vaccination, diagnosis or therapeutic follow-up of a cancer associated with the expression of a MAGE antigen.

  The subject of the present invention is also an immunogenic or vaccinal composition, characterized in that it comprises at least one antigen as defined above and a pharmaceutically acceptable vehicle, a carrier substance or an adjuvant.

  The immunogenic composition according to the invention is in a dosage form suitable for parenteral (subcutaneous, intramuscular, intravenous), enteral (oral, sublingual) or local (rectal, vaginal) administration.

  Pharmaceutically acceptable carriers, carrier substances and adjuvants are those conventionally used.

  The adjuvants are advantageously chosen from the group consisting of: oily emulsions, mineral substances, bacterial extracts, saponin, alumina hydroxide, monophosphoryl lipid A and squalene.

  The carrier substances are advantageously selected from the group consisting of: unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, solid microspheres of saccharide nature (poly (lactideco-glycolide)) or auriferous, and nanoparticles.

  According to an advantageous embodiment of said composition, it comprises at least one MAGE peptide including a CD4 + T epitope restricted to HLADP4 as defined above and another MAGE peptide including a CD8 + T epitope as defined above, in the form of a mixture of peptides, a polyepitopic fragment and / or an expression vector encoding said peptides or said fragment.

  According to an advantageous arrangement of this embodiment of said composition, the CD8 + T epitope is chosen from MAGE-Al 96-104, MAGE-A3 168-176, MAGE-AlO 254-262 and MAGE-Al2 170-178.

  According to another advantageous embodiment of said composition, it comprises at least two MAGE peptides including a CD4 + T epitope restricted to HLA-DP4 as defined above, and in particular one of the following combinations: the MAGE-A1 peptides 90-104, MAGE-A2 111-125 and MAGE-127141, MAGE-A1 peptides 268-282, MAGE-A112 127-141, MAGE-A9 68-82 and MAGE-A9 153-167.

  Such combinations advantageously make it possible to induce CD4 + T lymphocytes in almost all vaccinated individuals.

  According to yet another advantageous embodiment of said composition, it comprises a peptide including a universal CD4 + T epitope, as defined above.

  The peptides according to the present invention and the derived products (polyepitopic fragment, lipopeptide, recombinant vector) can be used in immunotherapy in the treatment of tumors expressing a MAGE antigen. Said peptides or derivatives are used either as a vaccine or in cell therapy, or even by a combination of both approaches.

  Cellular therapy comprises, the preparation of antigen presenting cells (dendritic cells) by a conventional protocol comprising isolation of peripheral blood mononuclear cells (PBMC) from a patient to be treated and culturing of the dendritic cells in the presence of peptide (s). In a second step the antigen presenting cells loaded with the peptide are reinjected into the patient.

  The subject of the present invention is also a reagent for diagnosing the immune status of an individual with respect to a cancer associated with the expression of a MAGE antigen, characterized in that it comprises an antigen such as as defined above. Preferably, said reagent comprises a peptide or a fusion protein as defined above, optionally labeled or complexed, in particular complexed with labeled HLA-DP4 molecules, for example biotinylated, in the form of multimeric complexes such as tetramers.

  The present invention also relates to a method for diagnosing the immune status of an individual with respect to a cancer associated with the expression of a MAGE antigen, characterized in that it comprises: contacting a biological sample of said individual with a peptide as defined above, and the detection of CD4 + T lymphocytes specific for a MAGE antigen expressed by the tumor cells, in vitro, by any appropriate means.

  According to an advantageous embodiment of said method, said MAGE antigen is selected from the group consisting of: MAGE-AI, MAGEA2, MAGE-A3. MAGE-A4. MAGE-A6, MAGE-A9, MAGE-A1, MAGE-A11 and MAGE-Al2.

  The present invention also relates to a kit for detecting the immune status of an individual with respect to a cancer associated with the expression of a MAGE antigen, characterized in that it comprises at least one less a peptide as defined above, associated with a means of detecting antigen-specific CD4 + T cells expressed by the tumor cells.

  The method according to the invention makes it possible to follow the evolution of the CD4 + T response directed against a MAGE antigen expressed by tumor cells during a cancer or an antitumor treatment, in particular an antitumor immunotherapy.

  Detection is performed from a biological sample containing CD4 + T cells, including a sample of mononuclear cells isolated from a peripheral blood sample (PBMCs).

  The CD4 + T lymphocytes specific for a MAGE antigen expressed by the tumor cells are detected by any means, known per se. For example, it is possible to use direct means such as flow cytometry in the presence of multimeric complexes as defined above, or indirect means such as lymphocyte proliferation assays and cytokine assays such as IL-2, VIL-4, VIL-5, IL-10 and IFN-γ, in particular by immunoenzymatic techniques (ELISA, RIA, ELISPOT) or by flow cytometry (intracellular cytokine assay).

  More specifically: A suspension of cells (PBMC, PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with the peptides as defined above or cloned T lymphocytes) is cultured for 3 to 5 hours. days in the presence of said peptides and, if necessary, appropriate presenting cells, such as dendritic cells, autologous or heterologous PBMCs, lymphoblastoid cells such as those obtained after infection with EBV or genetically modified cells. The presence of CD4 + T cells specific for a MAGE antigen expressed by the tumor cells in the initial suspension is detected using the MAGE peptides, according to one of the following methods: Proliferation test: Proliferation of T cells CD4 + specific for a MAGE antigen expressed by the tumor cells is measured by incorporation of tritiated thymidine into the DNA of the cells.

  * ELISPOT test: The ELISPOT test reveals the presence of secretory cytokine T cells (IL-2, IL-4, IL-5, IL-10 and IFN-γ), which are specific for a peptide as defined above. The principle of this test is described in Czerkinsky et al., J. Immunol. Methods, 1983, 65, 109-121 and Schmittel et al., J. Immunol. Methods, 1997, 210, 167-174, and its implementation is illustrated in International Application WO 99/51630 or Gahéry-Ségard et al., J. Virol., 2000, 74, 1694-1703.

  cytokine detection: The presence of MAGE antigen-specific T cells expressed by tumor cells, secreting cytokines such as IL-2, FIL-4, IL-5, IL-10 and IFN-γ is detected, either by assaying cytokines present in the culture supernatant, by an immunoenzymatic test, in particular using a commercial kit, or by detecting intracellular cytokines in flow cytometry. The principle of detection of intracellular cytokines is described in Goulder et al. , J. Exp. Med., 2000, 192, 1819-1832 and Maecker et al., J. Immunol. Methods, 2001, 255, 27-40 and its implementation is illustrated in Draenert et al., J. Immunol. Methods, 2003, 275, 19-29.

  multimeric complexes - a biological sample, preferably peripheral blood mononuclear cells (PBMC), is brought into contact with marked multimeric complexes, in particular with a fluorochrome, formed by the binding between soluble HLA-DP4 molecules and MAGE peptides. as defined above, and the cells labeled with said multimeric complexes are analyzed, in particular by flow cytometry.

  Advantageously, prior to bringing the biological sample into contact with said complexes. it is enriched in CD4 + T cells, bringing it into contact with anti-CD4 antibodies.

  The HLA-DP4 / peptide multimeric complexes can be prepared from native molecules extracted from cells expressing HLA-DP4 or from recombinant molecules produced in appropriate host cells as specified, for example in NOVAK et al. (J. Clin. Investig., 1999, 104, R63-R67) or in KURODA et al. (J. Virol., 2000, 74, 18, 8751-8756). These HLA-DP4 molecules can in particular be truncated (deletion of the transmembrane domain) and their sequence can be modified in order to make them soluble or to facilitate the pairing of alpha and beta chains (Novak et al., Cited above).

  The loading of HLA-DP4 molecules with the peptide can be done by contacting a preparation of HLA-DP4 molecules as above, with the peptide. For example, soluble and biotinylated HLA-DP4 molecules are incubated, for 72 hours at 37 ° C., with a 10-fold excess of MAGE peptides as defined above, in a 10 mM phosphatecitrate buffer, 0.15 NaCl. M at a pH of between 4.5 and 7.

  Alternatively, the peptide sequence can be introduced into one of the chains of the DP4 molecule in the form of a fusion protein which allows the preparation of HLA-DP4 / peptide multimeric complexes from appropriate host cells expressing said protein of fusion. Said complexes can then be labeled, in particular with biotin.

  The multimeric complexes of the tetramer type are obtained in particular by adding, to the charged HLA-DP4 molecules, streptavidin labeled with a fluorochrome in a quantity four times lower (mole to mole) relative to the HLA-DP4 molecules, the whole being then incubated during sufficient time, for example one night at room temperature.

  The multimeric complexes can also be formed, either by incubation of HLA-DP4 monomers / peptides with streptavidin-coupled magnetic beads as described for the HLA I molecules (Bodinier et al., Nature, 2000, 6, 707-710), or by inserting HLA-DP4 / peptide monomers into lipid vesicles as described for murine class II MHC molecules (Prakken, Nature Medicine, 2000, 6, 1406-1410).

  In order to use these multimeric HLA-DP4 / peptide complexes, especially of the tetramer type, a cell suspension (PBMC, PBMC depleted in CD8 + cells, T cells, previously enriched by an in vitro culture step with MAGE peptides as defined above or cloned T lymphocytes) with HLADP4 / peptide multimeric complexes at a suitable concentration (for example of the order of 10 to 11 g / ml), for a time sufficient to allow the binding between the complexes and the CD4 + T lymphocytes. specific to the MAGE antigen expressed by the tumor cells (for example, of the order of 1 to 3 hours). After washing, the suspension is analyzed by flow cytometry: the labeling of the cells is visualized by the multimeric complexes which are fluorescent.

  Flow cytometry makes it possible to separate the cells labeled with the HLA-DP4 / peptide multimeric complexes from the unlabeled cells and thus perform cell sorting.

  The present invention thus also relates to a method for sorting CD4 + T lymphocytes specific for a MAGE antigen expressed by the tumor cells, characterized in that it comprises at least the following steps: contacting, in vitro, a cell sample with labeled FILA-DP4 / peptide multimeric complexes, in particular with a fluorochrome, said complexes being formed by the binding of soluble HLA-DP4 molecules with at least one MAGE peptide as defined above, and sorting the cells bound to said HLA-DP4 / peptide complexes, in particular in flow cytometry.

  The subject of the present invention is also a peptide as defined above, excluding the peptides MAGE-A2 127-139, MAGE-A3 111-125, 111126, 113-126, 113-127, 113-128, 114- 126, 114-127, 114-128, 127-142, 146160, 156-170 and 281-295, MAGE-A6 121-144, 140-158, 140-170, 150-165, 155170 and 219-236.

  The present invention further relates to a poly-epitopic fragment, a lipopeptide, a polynucleotide, an expression cassette, a recombinant vector and a modified prokaryotic or eukaryotic host cell derived from the foregoing peptides.

  The invention encompasses in particular: a) expression cassettes comprising at least one polynucleotide as defined above. under the control of appropriate transcriptional regulatory and optionally translational sequences (promoter, enhancer, intron, initiation codon (ATG), stop codon, polyadenylation signal), and b) recombinant vectors comprising a polynucleotide conforming to 'invention. Advantageously, these vectors are expression vectors comprising at least one expression cassette as defined above.

  The polynucleotides, the recombinant vectors and the transformed cells as defined above are particularly useful for the production of the peptides, polyepitopic fragments and fusion proteins according to the invention.

  The polynucleotides according to the invention are obtained by conventional methods, known per se, following the standard protocols as those described in Current Protocols in Molecular Biology (Frederick M AUSUBEL, 2000, Wiley and his Inc, Library of Congress, USA ). For example, they can be obtained by amplification of a nucleic sequence by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with a homologous probe, or by total or partial chemical synthesis. Recombinant vectors are constructed and introduced into host cells by conventional methods of recombinant DNA and genetic engineering, which are known per se.

  The peptides and their derivatives (variants, modified peptides, lipopeptides, polyepitopic fragments, fusion proteins) as defined above, are prepared by standard techniques known to those skilled in the art, in particular by solid or liquid phase synthesis or by expression. recombinant DNA in a suitable cellular system (eukaryotic or prokaryotic).

  More specifically, the peptides and their derivatives (variants, polyepitopic fragments) can be synthesized in solid phase, according to the Fmoc technique, originally described by Merrifield et al. (J. Am Chem Soc., 1964, 85: 2149-) and purified by reverse phase high performance liquid chromatography, the lipopeptides can be prepared in particular according to the method described in International Applications WO 99/40113 or WO 99 / 51630.

  peptides and derivatives such as variants, polyepitopic fragments and fusion proteins can also be produced from the corresponding cDNAs, obtained by any means known to those skilled in the art; the cDNA is cloned in a eukaryotic or prokaryotic expression vector and the protein or fragment produced in the cells modified with the recombinant vector are purified by any appropriate means, in particular by affinity chromatography.

  In addition to the foregoing, the invention further comprises other provisions, which will emerge from the following description, which refers to examples of implementation of the object of the present invention.

  EXAMPLE 1: BINDING ACTIVITY OF ANTIGEN PEPTIDES MAGE VIS-NOTICE OF MOLECULES HLA-DP401 and DP402 1) Materials and Methods a) Peptides Fifteen amino acid peptides (15-mer) representative of the MAGE-A1 antigen ( SWISSPROT P43355), as well as the natural variants derived from the MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9 MAGE-A10, MAGE-A1 I and MAGEAl2 antigens (SWISSPROT P43356, P43357, P43362, P43363, P43364, P43365 ) were selected according to the presence of several aromatic or hydrophobic residues which are the main anchoring residues for the HLA-DP molecules.

  The peptides were synthesized according to the Fmoc strategy in solid phase parallel synthesis, purified by HPLC and controlled by mass spectrometry (ES-MS). The selected sequences that have been synthesized are presented in Table III and the attached sequence listing.

  b) HLA-DP4 / peptide binding assay The binding assays to the HLADP4 molecules encoded by the DPBI * 0401 and DPB1 * 0402 alleles, designated DP401 and DP402, respectively, are binding tests in competition with an immunoenzymatic revelation described in FIG. PCT International Application WO 03/040299.

  More specifically, the peptide Oxy 271-287 (EKKYFAATQFEPLAARL, SEQ ID NO: 41) biotinylated at the NH2 terminal residue, according to the protocol described in Texier et al., J. Immunol., 2000, 164, 3177-3184. ), is used as a tracer peptide under the conditions as specified in the Table below.

  TABLE II: HLA-DP4 binding test conditions Alleles Tracers pH concentration Optimal incubation tracer time (nM) (h) DBP1 * 401 Oxy 271-287 10 5 24 DPB1 * 402 Oxy 271-287 10 5 24 2) Results The majority (18 out of 19) of the peptides bind with a good affinity (IC50 <1000 nM) to at least one of the DP4 molecules (Table III). In addition, the binding activity of some of the natural variants of the MAGE-A1 peptides conies to the MAGE-A2, -A3, -A4, -A9, -A10, A11 and -Al2 peptides which is comparable to that of the peptides. MAGE-A1, as illustrated by the example of MAGE-A1 120-134 peptide variants, indicates that there is a cross-reaction between the selected MAGE peptides; some of the selected MAGE-A peptides are capable of inducing CD4 + T cells capable of recognizing peptides derived from another MAGE-A MAGE 1120-134 antigen MAGE-MAGE3127-141 "REPVTK MAGE4128-142 "KELVTK MAGE9126-140" KEPVTK MAGE 10152-166 * KEPITKA *: variant of magel 104-118 ": varying from magel 120-134 the anchoring residues to DP4 (Pl, P4, P6 and P9) AEMLESVIK 3500 424 AEMLESVLR 1600 310 AEMLGSVVG 26458 4183 AEMLERVIK 1500 414 AEMLESVIK 1800 639 EILESVIK 1500 570 Table III: Binding activities of MAGE peptides with respect to HLA-DP4 molecules (IC50 in nM) Al.42 A3 A4 A5 A6 A7 A8 A9 DP401 DP402 TSCILESLFRAVITK 155 100 PRALAETSYVKVLEY 13 16 RKMVELVHFLLLKYR 6 4 CQDFFPVIFSKASEY 335 50 EKIWEELSMLEVFEG 43 37 MAGE3111-125 'RKVAELVHFLLLKYR 100 35 MAGE9 68-82 S 1 s VYYTLWS QFDEG 15 30 MAGE9153-167 ASEFMQVIFGTDVKE 40 21 MAGE10 244-258 EVIWEALNMMGLYDG 245 64 MAGE10 303-317 HAEIRKMSLLKFLAK 730 175 MAGE11130-144 * 'KGLITKAEMLGSVIK 260 184 MAGE12127-141 REPFTKAEMLGSVIR 120 47 MAGE 12111-125 * RKMAELVHFLLLKYR 32 18 Seven strong peptides affinity for DP401 and DP402 (MAGE-A1 90-104 and 268-282; MAGE-A2 11-125 and 219-233; MAGE-A9 68-82 and 153-167; MAGE-Al2 127-141) were chosen for the immunogenicity tests. EXAMPLE 2 IMMUNOGENICITY OF IN VITRO MAGE PEPTIDES The ability of peptides having a good affinity for HLA-DP4 molecules to induce in vitro stimulation of specific T lymphocytes was evaluated from blood samples from healthy (non-tumor-bearing) individuals. ). It is a question of evaluating the capacity to recruit CD4 + precursor lymphocytes whereas they are in a naïve individual at a very low frequency, that is to say to carry out an in vitro immunization using these peptides.

  Peptide MAGE1 90-104 MAGE1 268-282 MAGE2 111-125 MAGE2 143-157 MAGE2 219233 are indicated in bold 1) Materials and methods a) Individuals tested Blood samples were taken from five healthy individuals expressing HLA-DP4. Alleles of the DPB1 gene of these individuals are shown in Table IV.

  Table IV: DPB1 Alleles of Individuals Tested Individual TYPAGE P78 DPB1 * 0402 DPB1 * 1301 P121 DPB1 * 0401 DPB1 * 0402 P122 DPB1 * 0401 P126 DPB1 * 0401 P129 DPB1 * 0401 DPB1 * 0402 P156 DPB1 * 0401 P158 DPB1 * 0402 b) How to Obtain CD4 + T cell lines specific for MAGE-A antigen and restricted to HLA-DP4. Peripheral blood mononuclear cells (PBMC) were separated on a Ficoll gradient. The PBMCs were then cultured in AIM V medium (LIFE TECHNOLOGIES) and incubated in flasks in an oven at 37 ° C. in the presence of 5% CO 2. After overnight incubation, the non-adherent cells were recovered, deposited on an LS + column (MYLTENYI), and the purified CD4 + T cells were frozen. The adherent cells were incubated for 5 days, in AIM V medium containing 1000 U / ml of GM-CSF and 1000 U / ml of IL-4, then the cells differentiated into dendritic cells were then cultured for 2 days, in the presence of 1 g / ml of LPS, 1000 U / mI of IL-4 and 1000 U / ml of GM-CSF, so as to induce their maturation.

  The mature dendritic cells (100,000 cells / well) were then incubated with a mixture of peptides (10 g of each peptide), for 4 hours at 37 ° C. The mature dendritic cells were then washed and then incubated, in the presence of the lymphocytes. CD4 + T (100,000 cells / well) previously thawed, in medium containing 1000 U / ml of IL-6 and 10 ng / ml of IL-12. After 7 days, the culture was restimulated a first time, using mature dendritic cells previously thawed and loaded with the peptide mixture, in medium containing IL2 (10 U / ml) and FIL-7 (5). ng / ml). After two additional stimulations, under the above conditions, the cells were tested in ELISPOT. e) Analysis of the Specificity of the ELISPOT Lines In order to verify their specificity with respect to the HLA-DP4 molecule, the T lymphocytes were tested in the presence of HLA-DP4 positive cells, namely an L-line of transfected fibroblasts. by the cDNA coding for a DP402 molecule (Yu et al., Hum Immunol., 1990, 27, 132-135) or the HOM2 line of human B lymphocytes transformed with the Epstein Barr virus (DPA1 * 0103 / DPB1 * 0401, EACC).

  More specifically, anti-IFN-γ (1-DIK, MABTECH) antibodies diluted to 1 g / ml in PBS buffer were adsorbed onto nitrocellulose (MILLIPORE) plates for 1 hour at 37 ° C. Plates were then washed with PBS and then saturated with ISCOVE medium containing 10% human serum AB group (100 l / well), for 2 h at 37 C. DP4 positive cells (10,000 cells / well) and 5000 lymphocytes To be tested were then added to the plates and incubated for 24 h at 37 ° C. in the presence or absence of a single peptide or a mixture of peptides. After washing with 0.05% PBS Tween buffer and then with PBS alone, 100 μl of biotin-conjugated anti-IFN-γ secondary antibody (7-B6-1-biotin, MABTECH), diluted to 0.25 g. / ml in PBS containing 1% BSA, was added to each well. After one hour of incubation, the plates were washed again and incubated with 100 l / well of Extravidin-AKP (E-2636, SIGMA), diluted 1/6000. After washing the plates in PBS buffer, 100 l of NBTBCIP substrate (B-5655, SIGMA) diluted in water (1 tablet in 10 ml of water) was dispensed into each well. Enzyme immunoenzymation was stopped after about 10 minutes, by extensive rinsing of the plates in water, and the colored spots were counted using a PLC (AID). The lines are considered positive when the number of spots is greater than three times that obtained with the negative control (control without peptides) with a minimum of 50 spots. The control without presenting cells makes it possible to check the specificity of the response for HLA-DP4 (restriction control).

  2) Results 28 lines specific for a MAGE-A antigen and restricted to HLADP4 were identified: these lines are stimulated by the mixture of peptides (mix) presented by HLA-DP4 but not in the absence of the mixture (control negative) or HLA-DP4 (restriction control, Table V).

  Table V: Immunogenicity of the peptides (induction with the mixture of the seven peptides) Number of individual spots Number Control Control mixture Mage-2 Mage-9 Mage-1 Mage-9 Mage-12 Mage-1 Mage-2 Negative 111.125 153-167 90-104 68-82 127-141 268-282 219-233 line restriction peptides P78 13 25 6 88 49 13 92 12 10 9 11 22 37 10 92 65 34 177 16 35 27 24 32 1 4 117 0 0 185 0 0 0 0 P121 6 2 0 92 9 2 123 3 3 2 2 9 3 0 392 2 3 366 0 4 5 1 19 4 0 216 5 3 153 3 280 8 9 21 2 0 106 4 2 147 6 1 0 1 24 19 1 140 23 23 208 21 15 34 19 5 2 171 4 7 329 12 7 10 10 34 5 1 36 23 4 73 9 4 5 3 38 18 0 68 15 13 104 35 8 17 5 1 0 208 4 3,250 6 6 5 2 44 5 1 62 15 8 63 6 110 8 7 48 6 0 225 6 5 253 7 8 9 6 49 2 0 87 5 2 122 2 3 2 1 P122 12 9 2 173 2 6 178 18 12 7 6 26 1 3 12 11 10 65 8 8 5 8 27 5 0 37 4 2 70 1 1 0 0 28 1 0 31 4 3 85 1 0 0 0 29 3 1 85 7 6 196 2 2 1 3 P129 7 5 1 97 3 2 1 2 307 22 6 12 1 1 39 2 1 112 0 2 1 3 14 0 1 23 1 1 97 1 3 2 2 24 2 0 6 21 1 70 0 4 1 1 0 1 28 0 1 86 8 6 7 5 3 4 3 2 22 115 19 5 2 3 4 1 37 4 0 19 1 2 51 3 3 3 7 39 7 1 23 4 7 93 5 5 2 3 Each line corresponds to at least one of the peptides of the mixture. The peptide MAGE-A1 (MAGE-1) is highly immunogenic since it induces 26 out of 28 lines. When the mixture of the seven peptides for the induction of CD4 + T lymphocytes is used, the induced MAGE-Al2 peptide 127-141 3 lines, the peptide MAGE-A2 111-125 induced a line and the other four peptides do not induce lineage.

  Given the preponderance of the response for the MAGE-Al 90104 peptide, an induction experiment was performed with a mixture containing only the other six peptides (Table VI).

  Table VI: Immunogenicity of the peptides (induction with the mixture without the MAGE-Al 90-104 peptide) Number of individual spots Number Mix control indicator Mage-2 Mage-9 Mage-9 Mage-12 Mage-1 Mage-2 negative restriction of 111-125 153-167 68-82 127-141 268-282 219-233 peptide line P126 21 12 6 85 37 30 27 38 72 38 31 4 5 68 3 5 3 65 14 10 36 11 5 55 9 4 5 6 62 7 42 7 3 61 11 2 3 6 67 10 54 12 13 81 58 21 51 60 45 22 23 23 100 39 107 22 47 30 48 57 30 9 155 33 26 16 20 137 34 59 6 65 121 3 3 In this context, it is observed that the MAGE-Al 268-282, MAGE-A9 968-82 and 153-167 peptides are also immunogenic since they induce, respectively, four, two and one of the eight lines. tested.

  As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; it encompasses all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (36)

  1) Use of a peptide derived from the MAGE-A1 antigen comprising a CD4 + T epitope capable of being presented by an HLA-DP4 molecule and of inducing T lymphocytes specific for at least one MAGE antigen expressed by the tumor cells, wherein the peptide is selected from the group consisting of: a) the 13 to 50 consecutive amino acid fragments of the MAGE-AI antigen, preferably 13 to 25 amino acids, comprising at least the sequence between the following positions: at 101, 271 to 279, 68 to 76, 107 to 115, 123 to 131, 139 to 147, 150 to 158, 215 to 223 and 274 to 282, and b) the variants of the peptides defined in a), from 13 to 50 amino acids, preferably from 13 to 25 amino acids, which exhibit an HLA-DP4 binding activity of less than or equal to 1000 nM and are capable of inducing CD4 + T cells recognizing one of the epitopes defined in a ), for the preparation of a vaccine for prevention or in the treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent for diagnosing the immune status of said individual with respect to of this cancer.   2) Use according to claim 1, characterized in that the peptide is selected from the group consisting of the sequences located between the following positions of the MAGE-A1 antigen: 90 to 104, 268 to 282, 65 to 79, 104 to 118, 120 to 134, 136 to 150, 147 to 161, 212 to 226 and 271 to 285.   3) The use according to claim 1, characterized in that the variant peptide is a fragment of another MAGE antigen expressed by the tumor cells whose sequence is homologous to that of the fragment in a).   4) Use according to claim 3, characterized in that said fragment is a fragment of an antigen MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A9, MAGE-A 10, MAGE-A 11 or MAGE-A 12 comprising a sequence selected from the group consisting of SEQ ID NO: 19 to 25.   5) Use according to claim 4, characterized in that said fragment is selected from the group consisting of SEQ ID NO: 26 to 40 sequences.   6) Use according to claim 1, characterized in that the peptide variant in b) is a synthetic variant obtained by the substitution of at least one of the residues P1, P6 and / or P9 by an aromatic or hydrophobic amino acid, of the residue. P6 with a cysteine (C), the residue of the residue P9 with C, D, Q, S, T or E and / or residue P4 with another natural or synthetic amino acid.   7) Use according to any one of claims 1 to 6, characterized in that the peptide has a sequence of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids.   8) Use according to any one of claims 1 to 7, characterized in that the peptide is labeled or complexed.   9) Use according to claim 8, characterized in that said peptide is complexed with labeled HLA-DP4 molecules.   10) Use according to any one of claims 1 to 9, characterized in that said molecule DP4 comprises a chain (3 encoded by the allele DPB 1 * 0401 or DPB l * 0402.   11) Use of a polyepitopic fragment comprising the concatenation of at least two identical or different epitopes, at least one of which is a peptide as defined in any one of Claims 1 to 10, for the preparation of a vaccine for the prevention or treatment of cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a diagnostic reagent for the immune status of said individual to this cancer.   12) Use according to claim 11, characterized in that the polyepitopic fragment comprises the concatenation of at least one peptide as defined in any one of claims 1 to 10 and at least one CD8 + T epitope of an antigen MAGE expressed by the tumor cells and / or a universal CD4 + T epitope.   13) The use according to claim 12, characterized the CD8 + T epitope is selected from: MAGE-Al 96-104, MAGE-A3 168-176, MAGE-AlO 254-30 262 and MAGE-Al2 170-178.   14) Use of a fusion protein consisting of a protein or protein fragment, fused to a peptide as defined in any one of claims 1 to 10 or a polyepitopic fragment as defined in claim 12 or claim 13, for the preparation of a vaccine for the prevention or treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent of diagnosis of the immune status of said individual vis-à-vis this cancer.   15) Use according to claim 14, characterized in that said protein or said fragment is selected from the group consisting of an alpha or beta chain of an HLA-DP4 molecule, a fragment of said chain corresponding to a soluble HLA-DP4 molecule and a targeting sequence in the endosome.   16) Use according to claim 15, characterized in that said targeting sequence is derived from the invariable chain Ii or LAMP-I protein. 17) Use of a lipopeptide obtained by addition of a lipid on an α-amino function or a reactive function of the side chain of a peptide as defined in any one of claims 1 to 10 or a fragment polyepitope as defined in any one of claims 11 to 13, for the preparation of a vaccine for the prevention or treatment of cancer associated with the expression of a MAGE antigen. in an individual carrying a DP4 allele, or for the preparation of a diagnostic reagent for the immune status of said individual with respect to this cancer.   18) Use of an expression vector comprising a polynucleotide encoding a peptide as defined in any one of claims 1 to 10, a polyepitopic fragment as defined in any one of claims 11 to 13 or a fusion protein as defined in any one of claims 14 to 16, under the control of appropriate regulatory sequences, for the preparation of a vaccine for the prevention or treatment of a cancer associated with the expression of a MAGE antigen, in an individual carrying a DP4 allele, or for the preparation of a reagent for diagnosing the immune status of said individual with respect to this cancer.   19) A peptide as defined in any one of claims 1 to 10, excluding the MAGE-A3 peptides 111-125, 111-126, 113-126, 113-127, 113-128, 114-126, 114-127, 114-128, 146-160, 281-295 and MAGE-A6 121-144, 140-170, 150-165 and 219-236 as antigen for prophylactic or therapeutic vaccination, diagnosis or therapeutic follow-up. cancer associated with the expression of a MAGE antigen.   20) polyepitopic fragment, fusion protein, lipopeptide or expression vector as defined in any one of claims 11 to 17, excluding those derived from peptides MAGE-A3 111-125, 111-126, 113 -126, 113-127, 113-128, 114-126, 114-127, 114-128, 146-160, 281-295 and MAGE-A6 121-144, 140-170, 150-165 and 219-236, as an antigen for prophylactic or therapeutic vaccination, diagnosis or therapeutic follow-up of a cancer associated with the expression of a MAGE antigen.   21) Immunogenic or vaccine composition, characterized in that it comprises at least one antigen as defined in claim 19 or claim 20 and a pharmaceutically acceptable vehicle, a carrier substance or an adjuvant.   22) Immunogenic or vaccine composition according to claim 21, characterized in that it comprises at least one peptide as defined in claim 19 and a peptide including a CD8 + T epitope of a MAGE antigen, in the form of a mixture peptides, a polyepitopic fragment and / or an expression vector encoding said peptides or said fragment.   23) immunogenic or vaccine composition according to claim 22, characterized in that said CD8 + T epitope is selected from MAGE-Al 96104, MAGE-A3 168-176, MAGE-A 254-262 and MAGE-Al2 170-178.   24) Immunogenic or vaccine composition according to any one of claims 21 to 23, characterized in that it comprises a mixture of peptides selected from the group consisting of the mixture of peptides MAGE-Al 90-104, MAGE-A2 111- 125 and MAGE-12 127-141 and the mixture of peptides MAGE-Al 268-282, MAGE-Al2 127-141, MAGE-A9 68-82 and MAGE-A9 153167.   25) immunogenic composition according to any one of revendi-cations 21 to 24, characterized in that it comprises a peptide including a universal CD4 + T epitope.   26) A diagnostic reagent for the immune status of an individual with respect to a cancer associated with the expression of a MAGE antigen, characterized in that it comprises an antigen as defined in claim 19 or claim 20.   27) A method for diagnosing the immune status of an individual with respect to a cancer associated with the expression of a MAGE antigen, characterized in that it comprises: the contacting of a biological sample of said individual with a peptide as defined in any one of claims 1 to 10, and - the detection of CD4 + T lymphocytes specific for a MAGE antigen expressed by the tumor cells, in vitro, by any appropriate means.   28) Method according to claim 27, characterized in that said MAGE antigen is selected from the group consisting of: MAGE-A1, MAGEA2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE -Al 1 and MAGE-Al2.   29) A method for sorting CD4 + T lymphocytes specific for a MAGE antigen expressed by the tumor cells, characterized in that it comprises at least the following steps: - bringing into contact, in vitro, a cell sample with labeled HLA-DP4 / peptide multimeric complexes, formed by the binding of soluble HLA-DP4 molecules with at least one peptide as defined in one   any of claims 1 to 10, and   sorting the cells bound to said HLA-DP4 / peptide complexes.   30) Peptide as defined in any one of claims 1 to 10, excluding peptides MAGE-A2 127-139, MAGE-A3 111-125, 111-126, 113126, 113-127, 113-128 , 114-126, 114-127, 114-128, 127-142, 146-160, 156170 and 281-295, MAGE-A6 121-144, 140-158, 140-170, 150-165, 155-170 and 219-236.   31) A polyepitopic fragment comprising the concatenation of at least two identical or different epitopes of which at least one is a peptide according to claim 30.   32) fusion protein consisting of a protein or protein fragment fused to a peptide according to claim 30 or a polyepitopic fragment according to claim 31.   33) A lipopeptide obtained by addition of a lipid to a higher amine function or a reactive side chain function of a peptide according to claim 30 or a polepitopic fragment according to claim 31.   34) A polynucleotide encoding a peptide according to claim 30, a polyepitopic fragment according to claim 31 or a fusion protein according to claim 32.   An expression vector comprising a polynucleotide according to claim 34 under the control of appropriate regulatory sequences, transcription and optionally translation.   36) A polynucleotide-modified host cell according to claim 34 or an expression vector according to claim 35.