WO2003084990A1

WO2003084990A1 - Bage genes, proteins coded by the same, and their uses for the treatment or the diagnosis of melanomas

Info

Publication number: WO2003084990A1
Application number: PCT/EP2002/003811
Authority: WO
Inventors: Albertina De Sario; Myriam Ruault
Original assignee: Centre National De La Recherche Scientifique
Priority date: 2002-04-05
Filing date: 2002-04-05
Publication date: 2003-10-16
Also published as: AU2002317715A1

Abstract

The invention relates to new BAGE genes, tumor antigen proteins encoded by the same, or derived proteins, or fragments thereof, said proteins of the BAGE family comprising the following SEQ ID NO: 1 sequence: MAAGX1VFLALSAQLLQARLMKEESPVVSWX2LEPEDGTALX3[X4X5F]n in which n represents 0 or 1, X1 represents V or A, V2 represents R or W, X3 represents Z, D or C, X4 represents V or F, X5 represents H or I. The invention also relates the uses of said proteins or corresponding nucleotide sequences for the treatment or the diagnosis of pathologies wherein fragments of said tumor antigens are expressed at the cell surface, such as melanomas.

Description

BAGE GENES, PROTEINS CODED BY THE SAME, AND THEIR USES FOR THE TREATMENT OR THE DIAGNOSIS OF MELANOMAS

The invention relates to new BAGE genes, tumor antigen proteins encoded by the same, or derived proteins, or fragments thereof, and to their uses for the treatment or the diagnosis of pathologies wherein fragments of said tumor antigens are expressed at the cell surface, such as melanomas.

Tumour antigens are of particular interest for i munotherapy of cancer patients. Naccines containing antigenic peptides or antigenic proteins are already employed in clinical trials for the iirimunization of melanomas, a tumour type that is particularly sensitive to immune attacks (Marchand et al., 1995; Thurner et al., 1999).

Several tumour-specific antigens were identified during the past decade such as MAGE (van der Bruggen et al., 1991), BAGE (Boel et al., 1995), GAGE (De Backer et al., 1999). These antigenic peptides are presented at the cell surface by the class I Major Histocompatibility Complex (MHC) and are recognized by cytolytic T lymphocytes (CTL).

Genes encoding these tumour antigens are expressed in tumour cells and silent in normal tissues with the exception of testes. However, expression in these tissues does not result in antigemc presentation, because germ cells do not express class I MHC molecules (Haas et al, 1988).

MAGE, the most extensively studied gene family, consists of five gene clusters mapping to independent loci in chromosome X (Rogner et al., 1995; Chomez et al, 2001). The GAGE family consists of at least eight genes mapping to chromosome X (De Backer et al., 1999).

A mRJΝfA encoding the BAGE antigen was isolated from a melanoma cell line (Boel et al., 1995). BAGE was expressed in 22% of melanomas, 30% of infiltrating bladder carcinomas, 10% of mammary carcinomas, 8% of head and neck squamous cell carcinomas and 6% of non-small cell lung carcinomas. During annotation of genomic sequences of human chromosome 21 (Hattori et al.,

2000), the Inventors have identified DΝA stretches having 92-99% nucleotide identity with the BAGE RΝA and have concluded that chromosome 21 contains E-4GE-related sequences. The E_4GE-related sequences are located less than 1 Mb away from the centromere: two map to 21p and one maps to 21q. Hence, the present invention relies on the demonstration made by the Inventors that BAGE is- a gene family comprising expressed genes that map to the juxtacentromeric regions of chromosomes 13 and 21 and unexpressed gene fragments that are scattered in the juxtacentromeric regions of acrocentrics and of chromosomes 9 and 18. The invention relates to proteins of the BAGE family comprising the following SEQ ID NO : 1 sequence:

MAAGX₁NFLALSAQLLQARLMKEESPVNSWX₂LEPEDGTALX₃[X₄X₅F]_n in which: - n represents 0 or 1 ,

- X. represents N or A,

- X₂ represents R or W,

- X₃ represents Z, D or C,

- X₄ represents N or F, - X₅ represents H or I, or fragments of these proteins comprising at least about 5 amino acids, or at least 9 amino acids, or peptide sequences derived from the above-mentioned proteins or fragments, by substitution, deletion or addition of one or more amino acids of the SEQ ID NO : 1 sequence, or of a fragment of this sequence, said fragments or derived sequences having the property, when forming complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence, provided that :

- the BAGE 1 protein represented by the following SEQ ID NO : 20 sequence: MAARAVFLALSAQLLQARLMKEESPNVSWRLEPEDGTALCFIFZ,

- and the BAGE 1 fragments, or of derived sequences from these fragments, represented by the following sequences:

. SEQ ID NO : 21: MAARAVFLALSAQLLQARLMKE . SEQ ID NO : 22: MAARAVFLALSAQLLQ . SEQ ID NO : 23: AARAVFLAL . SEQ ID NO : 24: ARAVFLALF . SEQ ID NO : 25 : MAARAVFLA being excluded.

The invention relates more particularly to proteins as defined above, having the formula of the following SEQ ID NO : 2 sequence:

MAAGVNFLALSAQLLQA-^M-^ESPNNSWRLEPEDGTALDVHFVSTLEPLSNAN -3-ΝNPRCπ-LNLQEPTXgFMSNTSSCFNQ in which X₆ represents A or P, and X₇ represents R or Q.

The invention concerns more particularly the proteins of the SEQ -D NO : 2 sequence as mentioned above, chosen among : - the BAGE 2 protein represented by the following SEQ ID NO : 4 sequence: MAAGVNFLALSAQLLQA-n--v-KEESPVNSWRLEPEDGTALDNHFNSTLEPLSNAN KRΝWRCmLNLQEPTAFMSNTSSCFNQΝTLT-π-LKDRR lQTNQCATARETS

- the BAGE 3 protein represented by the following SEQ ID NO : 6 sequence: MAAGVNFLALSAQLLQAP M-^ESPNNSWPLEPEDGTALDNHFNSTLEPLSNAV -a WRCII-LNLQEPTPFMSNTSSCFNQNTLTM.L-π RRKMQTVQCATAQETS

- or the BAGE 6 protein represented by the SEQ ID NO : 8 sequence which is identical to SEQ ID NO : 4.

The invention also concerns fragments of proteins having the formula of the SEQ ID NO : 2 sequence mentioned above, or the formula of sequences derived from the latter such as defined above, said fragments comprising at least about 5 amino acids, or at least 9 amino acids from said sequence.

The invention concerns more particularly fragments of proteins as mentioned above, said fragments having the following SEQ ID NO : 9 sequence: NSTLEPLSNAN]πm RC-mNLQEPTX₆FMSNTSSCFNQOT NQCATAX₇ETS in which X₆ represents A or P, and X₇ represents R or Q, or peptide sequences derived from the above-mentioned fragments by substitution, deletion or addition of one or more a ino acids of the SEQ ID NO : 9 sequence, said fragments or derived sequences having the property, when forming complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigen derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence. The invention relates more particularly to fragments of proteins as mentioned above, chosen among:

- the following peptide sequence SEQ ID NO : 11 : NSTLEPLSNAW-RJ>^RCmLNLQEPTAFMSNTSSCT^ NQCATARETS - the following peptide sequence SEQ ID NO : 13 :

NSTLEPLSNAVK--υSTvTRCπ-LNLQEPTPF-^ QCATAQETS

The invention concerns more particularly fragments comprising at least about 5 amino acids, or at least 9 amino acids, derived from the SEQ ID NO : 9, 11 or 13 sequences, or from sequences derived from the latter such as defined above.

The invention also relates to proteins of SEQ ID NO : 1 mentioned above, chosen among the following:

- the BAGE 4 protein represented by the following SEQ ID NO : 15 sequence: MAAGAWLALSAQLLQA-^-V--K-EESPNNSVv^TWLEPEDGTAL - the BAGE 5 protein represented by the following SEQ ID NO : 17 sequence: MAAGAWLALSAQLLQARLMKEESP SV -EPEDGTALCFIF

- the BAGE 7 protein represented by the following SEQ ID NO : 19 sequence: MAAGAVFLALSAQLLQARLMKEESPNNS VRLEPEDGTAL The invention relates more particularly to fragments of the BAGE 4 and BAGE 5 proteins mentioned above, or of sequences derived from the latter such as defined above, said fragments comprising at least about 5 amino acids, or at least 9 amino acids from the SEQ ID NO : 15, SEQ ID NO : 17, or SEQ ID NO : 19 sequences.

The invention also concerns nucleotide sequences coding for a protein of the BAGE family, or a fragment or a derived protein such as defined above, or nucleotide sequences derived from the latter by degeneration of the genetic code.

The invention relates more particularly to nucleotide sequences as defined above, coding for from tumor rejection antigen precursors of the BAGE family, said sequences being chosen among the following: - the sequence SEQ LO NO : 3, and more particularly part of SEQ ID NO : 3 delimited by the nucleotides located at the position 209 and the position 535 coding for the peptide sequence SEQ ID NO : 4, or any other possible open reading frame of SEQ ID NO : 3 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 5, and more particularly part of SEQ ID NO : 5 delimited by the nucleotides located at the position 209 and the position 535 coding for the peptide sequence SEQ ID NO : 6, or any other possible open reading frame of SEQ ID NO : 5 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 7, and more particularly part of SEQ ID NO : 7 delimited by the nucleotides located at the position 209 and the position 535 coding for the peptide sequence SEQ ED NO : 8, or any other possible open reading frame of SEQ ID NO : 7 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 10, and more particularly part of SEQ ID NO : 10 delimited by the nucleotides located at the position 13 and the position 210 coding for the peptide sequence SEQ ID NO : 11, or any other possible open reading frame of SEQ ID NO : 10 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 12, and more particularly part of SEQ LD NO : 12 delimited by the nucleotides located at the position 13 and the position 210 coding for the peptide sequence SEQ ID NO : 13, or any other possible open reading frame of SEQ ID NO : 12 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 14, and more particularly part of SEQ ID NO : 14 delimited by the nucleotides located at the position 189 and the position 305 coding for the peptide sequence SEQ ID NO : 15, or any other possible open reading frame of SEQ ID NO : 14 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 16, and more particularly part of SEQ ID NO : 16 delimited by the nucleotides located at the position 189 and the position 317 coding for the peptide sequence SEQ ID NO : 17, or any other possible open reading frame of SEQ ED NO : 16 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 18, and more particularly part of SEQ ID NO : 18 delimited by the nucleotides located at the position 189 and the position 305 coding for the peptide sequence SEQ ID NO : 19, or any other possible open reading frame of SEQ ED NO : 18 encoding a tumor rejection antigen precursor.

The invention also relates to nucleotide sequences hybridizing with the nucleotide sequences such as defined above, under stringent conditions (e.g. O.lxSSPE 65°C), provided that the nucleotide sequences coding for the BAGE 1 protein represented by

SEQ ID NO : 20, or for the fragments of BAGE 1, or the derived sequences, represented by SEQ ID NO : 21 to 25, being excluded.

The invention relates more particularly to nucleotide sequences mentioned above, coding for polypeptide sequences having the property, when forming complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigen derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence.

The invention also concerns recombinant nucleotide sequences comprising a nucleotide sequence as mentioned above, under the control of a transcription promoter and a transcription terminator.

The invention also relates to expression vector, characterized in that it contains a recombinant sequence as mentioned above.

The invention also concerns host cells transformed by a nucleotide sequence as mentioned above, if necessary by means of a vector as defined above.

The invention concerns more particularly the host cells as mentioned above, characterized in that they express HLA molecules, or are transformed in such a way that they express HLA molecules.

The invention also relates to cytolytic T cells recognizing specifically the complexes between HLA molecules and the proteins of the BAGE family, or the fragments or derived sequences, such as defined above, said cytolytic T cells being such as obtained by contacting T cells from an individual with said complexes, or with host cells as mentioned above.

The invention also concerns polyclonal or monoclonal antibodies directed against the proteins of the BAGE family, or against the fragments or derived proteins, such as defined above, or directed against complexes of these proteins, fragments or derived sequences, with HLA molecules, said antibodies being such as obtained by immunizing an animal with said sequences, fragments or derived sequence, or with said complexes. The invention concerns more particularly antibodies as mentioned above, characterized in that they are coupled to antitumor agents.

The invention also concerns the use of the proteins of the BAGE family, or of fragments or sequences derived from the latter, or of host cells, or of cytolytic cells, or of antibodies, as mentioned above, for the manufacture of a medicament intended for the treatment of pathologies linked to the expression, at the surface of the cells of the organism, of complexes between HLA molecules and peptides corresponding to fragments from said proteins SEQ ED NO : 2, SEQ ED NO : 4, SEQ ED NO : 6, SEQ ED NO : 8, SEQ ED NO : 15, SEQ ED NO : 17, and SEQ ID NO : 19. The invention relates more particularly to the use mentioned above, in the scope of the treatment of tumours such as melanomas.

The invention also relates to a pharmaceutical composition characterized in that it comprises at least a protein of the BAGE family, and/or a fragment, and/or a derived sequence, or host cells, or cytolytic cells, or antibodies, as defined above, in association with a pharmaceutically acceptable vehicle.

The invention relates more particularly to a pharmaceutical composition as mentioned above, characterized in that it also comprises at least a protein, and/or a fragment, and/or a derived sequence, from the MAGE, GAGE family, and/or the BAGE 1 protein, or the fragments of the BAGE 1 protein such as defined above. The invention also concerns a method for treating a subject with a disorder linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigens corresponding to fragments of proteins SEQ ID NO : 2, SEQ ED NO : 4, SEQ ED NO : 6, SEQ ED NO : 8, SEQ ED NO : 15, SEQ ED NO : 17, and SEQ ID NO : 19, comprising the administration to said subject of a pharmaceutical composition as mentioned above, in a sufficient amount for alleviating said pathologies.

The invention relates more particularly to a method as mentioned above, for treating a subject with a tumor such as a melanoma. '

The invention also concerns the use of nucleotide sequences, or of host cells, or of cytolytic cells recognizing specifically the complexes between HLA molecules and the proteins of the BAGE family, or the fragments or derived sequences, such as defined above, or of antibodies directed against the proteins of the BAGE family, or against the fragments or derived proteins, such as defined above, for the implementation of methods for the in vitro diagnosis of disorders linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigens coiresponding to fragments of proteins SEQ ID NO : 2, SEQ ED NO : 4, SEQ ED NO : 6, SEQ ED NO : 8, SEQ ID NO : 15, SEQ ID NO : 17, and SEQ ID NO : 19, more particularly of tumors such as melanomas. The invention also relates to a method for the in vitro diagnosis of a disorder linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigen corresponding to fragments of proteins SEQ ID NO : 2, SEQ ID NO : 4, SEQ ED NO : 6, SEQ ID NO : 8, SEQ ED NO : 15, SEQ ID NO : 17, and SEQ ED NO : 19, more particularly of tumors such as melanomas, said method comprising contacting a biological sample from a subject with an agent specific for proteins of the BAGE family, or for fragments or derived proteins, such as defined above, or specific for the nucleotide sequences coding for a protein of the BAGE family, or a fragment or a derived protein such as defined above, and determining interaction between said agent with the above-mentioned proteins of the BAGE family, or with the complexes between said tumor antigens and the HLA molecules on cell membranes, or with the nucleotide sequences coding for the above-mentioned proteins of the BAGE family, as a determination of said disorder. The invention relates more particularly to a method for in vitro diagnosis as mentioned above, characterized in that the specific agent of the proteins of the BAGE family, or of the fragments or derived proteins, such as defined above, are T cells recognizing specifically the complexes between HLA molecules and the proteins of the BAGE family, or the fragments or derived sequences, such as defined above, or antibodies directed against the proteins of the BAGE family, or against the fragments or derived proteins, such as defined above.

The invention concerns more particularly the method for in vitro diagnosis as defined above, characterized in that the specific agent of the nucleotide sequences coding for a protein of the BAGE family, or a fragment or a derived protein such as defined above, are nucleotide sequences complementary to the latter, and which are used as probes or primers in PCR reactions.

The invention will be further illustrated with the following detailed description of the obtaining of the new BAGE proteins according to the invention.

I) Results

1) BAGE transcripts

We cloned and characterized different transcripts belonging to the BAGE family.

The 1,004-bp BAGE mRNA (gbU19180) (hereafter called BAGElά) was previously isolated by Pierre van der Bruggen (LICR, Brussels) from a melanoma cell line library (MZ2-Mel 43) (Boel et al., 1995). Five additional clones (BAGElb, BAGElc, BAGEld, BAGEle, and BAGElf) were isolated by Pierre van der Bruggen (LICR, Brussels) through hybridization of the MZ2-Mel 43 melanoma cDNA library with probes corresponding to the 5' and 3' regions of BAGEla. Sequencing confirmed that they were alternatively spliced n RNA variants of a single gene: BAGE Their sizes ranged from 1 to 2 kb.

To isolate transcripts expressed by new independent BAGE genes, we combined RACE with PCR experiments on cDNA libraries. 5' and 3 ' RACE were done on a testis Marathon cDNA library (Clontech) using primers designed to the BAGE1 sequence. 5' RACE subclones had 97-99% nucleotide identity with the 5' end ofBAGEl, whereas 3' RACE subclones differed in sequence and length from BAGE1 niRNA (Boel et ah, 1995). We derived primers from the ends of the RACE products by taking advantage of conserved sequences among different subclones and we amplified full length cDNAs from two testis and one melanoma cDNA libraries. The PCR products were cloned in a plasmid and 10 colonies per each amplification were sequenced. We isolated three 1.9- kb transcripts that were named BAGE2 (gbAF218570), BAGE3 (gbAF339514), and E-4GE5 (gbAF339516).

BAGE transcripts have open reading frames (ORF) of 132 or 330 bp encoding predicted proteins of 43 or 109 amino acids, respectively.

2) BAGE genes and gene fragments

In a search for BAGE genomic sequences, we retrieved from databases 14 sequences that show a significant nucleotide identity (85-100% P<10-4) with transcripts of the BAGE family (Table 1). Transcripts were tentatively assigned to a specific locus if they had >99.5% nucleotide identity with a given genomic sequence. We chose 99.5% as a threshold to distinguish nucleotide variations corresponding to independent loci from allelic variations, the frequency of which is estimated 0.1-0.3% in the human genome (Hattori et al, 2000). BAGE genomic sequences were classified as follows: gene, when a transcript could be assigned to the genomic sequence; predicted gene, when no transcript could be assigned to the genomic sequence, but the predicted ORF was intact; gene fragment, when the gene was truncated and the predicted ORF was disrupted by deleterious mutations (deletions and nucleotide changes) that introduced stop codons and/or erase the initiation codon (Table 1).

The genomic organization of BAGE 1 was obtained through alignment of the six transcripts (BAGEla, BAGElb, BAGElc, BAGEld, BAGEIQ, and BAGElf) with the genomic sequences AC064811 and 22AH8 (see material and method section) (Table 2). BAGE1 comprises seven exons and expresses an extremely complex and wide variety of alternatively spliced RNAs. This wide variation results from the presence of alternative splicing sites that produce exons of different lengths. Exon 3 is an Alu sequence included in the 3' untranslated region of two BAGE1 mRNA variants (BAGEla and BAGEle). All the splicing sites conform the consensus gt/ag sequence. The genomic organization of BAGE2 and BAGE 5 was obtained through alignment of the transcripts with the genomic sequences AL163201 and AL161418, respectively. These two genes span 76 kb and comprise 9 and 10 exons, respectively (Table 2). No genomic sequence could be assigned to BAGE3. Two BAGE genes were predicted in the genomic sequences AL158811 and AL049849, and named BAGE 6 and BAGE 7 respectively. The remaining sequences correspond to gene fragments (2 to 8 kb) and comprise only exons 1 and 2. No transcripts expressed by these truncated loci were identified.

BAGE sequences (genes and gene fragments) share extensive regions of high nucleotide identity: nucleotide identity is higher among genes (97-99%) than between genes and gene fragments (95-96%). Nucleotide identity among different BAGE genes is as high in coding as in non-coding sequences.

3) Chromosome mapping To map BAGE genomic sequences as a whole, we hybridized a 1.8-kb genomic probe to human metaphase chromosomes. The probe was amplified by PCR from the 5' region that is common to all the BAGE sequences. BAGE sequences mapped to the juxtacentromeric regions of the acrocentrics and of chromosomes 9 and 18. Both the p and the q arms of chromosome 9 hybridized with the probe. Hybridization to chromosomes 14 and 15 was observed only in some metaphases suggesting that these BAGE sequences were more divergent.

We then amplified exon 1, which is common to all the BAGE sequences, on a panel of monochromosome somatic cell lines (NIGMS mapping panel 2; Dubois and Naylor, 1993). Specific amplifications were obtained with chromosomes 9, 13, 15, 18, 21, and 22. No amplification was obtained with chromosome 14, but this result can be due to nucleotide divergence between the primers used and the target sequence.

To map individual BAGE loci, we analysed the localization of the genomic sequences retrieved from databases (Table 1). Assignment of' genomic sequences to chromosomes 9, 13, 18, and 21 was consistent with our mapping results; by contrast, assignment to chromosomes 4 and 5 was at variance with both in situ hybridization and somatic hybrid analysis. This is not surprising because in databases 50% of the genomic clones derived from the juxtacentromeric regions are likely to be misassigned (Bailey et al, 2001). To ascertain the actual localization of BAGE 1, which has 100% nucleotide identity with the genomic clone (AC064811) assigned to chromosome 4, we cloned the BAGE sequences amplified from somatic hybrid cell lines and sequenced 10 colonies per each amplification reaction. A genomic sequence matching (100% nucleotide identity) the BAGE1 transcripts was isolated from chromosome 13 and from no other chromosome. In conclusion, BAGE sequences map to the juxtacentromeric regions of different human chromosomes and each chromosome contains more than one locus: BAGE genes map to chromosomes 13 and 21, whereas BAGE gene fragments map to acrocentrics and to chromosomes 9 and 18.

4) Predicted BAGE proteins

Multialignment of BAGE predicted proteins shows that amino acid sequence identity ranges from 88% to 98%. Although BAGE2 and BAGE3 differ only in two amino acids, transcripts encoding these proteins are unlikely to be allelic because they have 1.2% nucleotide divergence.

The nonapeptide AARANFLAL (amino acids 2 to 10 of the BAGE1 predicted protein; boxed in Fig. 2) is the sequence of the BAGE antigen recognized by a CTL (Boel et al, 1995). BAGE predicted proteins have two amino acid changes with respect to the sequence of the BAGE1 antigenic peptide: (R->G)3 and (A->N)4. After databases searches, we concluded that BAGE predicted proteins have no significant identity/similarity to any known protein.

5) Expression analysis

We analyzed the expression of BAGE genes in 215 tumour samples of various histological types. To distinguish individual genes, we took advantage of the few nucleotide variations that characterize different transcripts and we designed a strategy based upon PCR followed by restriction enzyme digestion.

A first set of primers (bagel4/bagel9) amplified a PCR product of 490 bp corresponding to BAGE1 and BAGE5 transcripts. Fifteen percent (15/103) of melanomas scored positive, whereas all the normal tissues were negative, with the exception of testis (Table 3). In a previous study with a different set of primers that amplified the same transcripts, the percentage of positive melanomas was comparable (22%) (Boel et al., 1995). '

A second set of primers (bage20/bage21) was also used to amplify a PCR product of 368 bp corresponding to BAGE2, BAGE3, and BAGE5 transcripts. Thirty-four percent (35/103) of melanomas scored positive, indicating that a significant number of tumours expressed BAGE2 or BAGE3 without expressing the other BAGE genes, in particular in primary melanomas (Table 3). Here again, all the normal tissues were negative, with the exception of testes. PCR product were then digested with restriction enzymes BssKI, AM, BstNI, to identify individual transcripts for BAGE1IBAGE5, BAGE2 or BAGE3, respectively (Fig. 3). In melanomas, BAGE2 (23%) was more frequently expressed than BAGE1 (14%), BAGE3 (14%) and BAGE5 (9%) (Table 3). Individual tumours generally expressed more than one BAGE gene simultaneously. In accordance to a previous analysis (Boel et al., 1995), our results confirmed that BAGE genes are expressed in bladder and lung carcinomas and in a few tumours of other histological types. Leukemias, colorectal carcinomas, and renal carcinomas scored negative. Here, no head and neck tumours expressed BAGE, whereas 8 % were found positive in the previous study. This could be explained by the small number of samples analyzed.

II) Conclusion

We show that the BAGE gene family comprises genes that are transcribed and translated (as suggested by the antigenic properties of BAGE1) and gene fragments that are not expressed. Genes and predicted genes map to the juxtacentromeric regions of human chromosomes 13 and 21, whereas gene fragments map to the juxtacentromeric regions of acrocentrics and of chromosomes 9 and 18.

Since human juxtacentromeric regions are currently believed to be devoid of genes and are enriched with pseudogenes (Ruault et al., 1999), a gene family which maps to these regions, is a novelty.

Because of their complex structure (presence of repetitive sequences along with inter- and intrachromosome duplications), human juxtacentromeric regions mostly correspond to the unsequenced portion (roughly 5%) of the human genome (Bailey et al., 2001), so the list of BAGE loci (genes and gene fragments) reported in this work is far from being complete.

BAGE genes and gene fragments share extensive regions of high nucleotide identity (up to 99%) which may be accounted for by concerted evolution. Concerted evolution was suggested to be the molecular mechanism responsible for sequence conservation among human ribosomal genes. Ribosomal genes map to the short arms of the five acrocentric chromosomes and participate in the formation of a common nucleolus. In the human germline cells, acrocentrics undergo frequent interchromosome DNA exchanges (Ferguson-Smith, 1964; Marre et al, 1980). 'E_4GE sequences may therefore undergo similar interchromosome exchanges that promote sequence homogeneity. In addition, it was shown that sequence identity is maximal between chromosomes 13 and 21 that, besides ribosomal DNA, also share a common centromeric α satellite (Van Camp et al, 1992; our unpublished results). Consistently, we observed that BAGE genes, which map to chromosomes 13 and 21, have a higher nucleotide identity than BAGE gene fragments, which are scattered in different acrocentrics and in chromosomes 9 and 18, the latter two chromosomes being devoid of ribosomal DNA.

Alternatively, extensive regions of sequence homology in BAGE genes may be due to functional constraints that prevent the fixation of deleterious mutations, (and by concerted evolution that simply promote sequence homogeneity among different loci) However, the percentages of nucleotide identity among BAGE genes is as high in coding as in non-coding sequences. Moreover, whether BAGE proteins have a function and which role they play still remain open questions.

To determine whether either concerted evolution or functional constrains account for sequence conservation, we are currently investigating the evolution of the BAGE gene family.

In this work, we confirmed that BAGE genes are expressed in different cancer cells and silent in normal tissues other than testes and we showed that BAGE2 has the largest expression profile compared to other members of the BAGE family. Similar to BAGE genes, TPTE (Transmembrane Phosphatase with Tensin homology), a gene mapping 30 kb distal to BAGE2 (Chen et al., 1999; Guipponi et al.,

2000), and CT2 (Creatin Transporter 2), a gene mapping next to the centromere of chromosome 16 (Iyer et al., 1996), are exclusively expressed in testes. These results lead us to suggest that the restricted pattern of expression may be a general feature of the few genes located within a heterochromatic environment. Chromatin compaction and DNA methylation may account for gene silencing in this chromosome domain.

DNA methylation was shown to be the primary mechanism responsible for the silencing of the genes encoding the MAGE antigens (De Smet et al. 1996; 1999). However, given their localization in the Xq27-q28 region, methylation of MAGE genes is not due to centromeric heterochromatin.

Table 1. BAGE gene structure

BAGEl

AC064811 ( -xons (2) size (bp > 5' splice site 3' splice site i ntrons size (kb)

(1) 1 >237 GGAGCG/gtaag I 1.5

(1) 2a 170 tgtag/GTTTT GGAGG/gtaa II nd

(1) 2b 190 tgtag/GTTTT AAGTG/gtagga

(1) 2c 1856 tgtag/GTTTT t

133,676 - 133,58 3 91 gtag/AGATG TACAG/gtgtg III 1.4

132,178 - 132,01 4a 163 ttcag/CGATG AAGAT/gtaagt

132,215 - 132,01 4b 200 tcaag/GTTTT AAGAT/gtaagt

132,178 - 131,65 4c 528 ttcag/CGATG t

132,178 - 131,09 4d 1080 ttcag/CGATG t

120,733 - 120,38 5 (3) 351 aaag/GATGA t IV nd

120,345 - 118,66 6 (3) 1680 gtcag/TAATA t V 0.03

5,420 - 4,887 7 (3) 533 acag/GGTCT t VI nd

BAGE2

HS21C001 exons size (bp i 5' splice site 3' splice site i introns size (kb)

-191,680 1 >223 GGAGTG/gtaa I 1.1

190,623 - 190,52 2 101 ttgtag/GTTTT AGCTCT/gtga II 38.8

151,755 - 151,59 3 163 tttcag/CGAT AAAGAT/gtaa III 8.5

143,054 - 142,88 4 172 ttatag/CGAA CTGCAA/gtaa IV 1.8

141,028 - 140914 5 115 ttttag/ACAA TGCAAA/gtaa V 8.1

132,862 - 132,16 6 702 atacag/TGAA AAACAG/gta VI 9.3

123,134 - 123,01 7 120 tcttag/GAGG AAAGTG/gtac VII 2.8

120,252 - 120,13 8 117 acatag/GGCC CCTGGG/gtga VIII 5.5

114,624 - 114,52 9 102 ttatag/GTGT AATCAG/gttt IX 0.2

114,330 - 10 >76 cttcag/GATATG

BAGE5

AL161418 exons size (bp » 5' splice site 3' splice site i [ntrons size (kb)

- 76,589 1 203 GGAGCG/gtaa I 1.5

78,261 - 78,450 2 190 ttgtag/GTTTT CAAGTG/gtag II >38.6

132,037 - 132,19 3 163 tttcag/CGAT AAAGAT/gtaa III 8.5

140,734 - 140,90 4 172 ttatag/CGAA CTGCAA/gtaa IV 1.8

142,760 - 142,87 5 115 ttttag/ACAA TGCAAA/gtaa V 8

150,921 - 151,62 6 702 atacag/TGAA AAACAG/gta VI 11,9

163,514 - 163,63 7 117 acatag/GGCC CCTGGG/gtga VII >5,6

169,281 - 169,38 8 102 ttatag/GTGT AATCAG/gttt VIII 0,2

169,575 - 169,65 9 >76 cttcag/GATATG IX

(1) The gene structure was determined on the paitial sequence of cosmid 22H8

(2) a, b, c are alternative exons of different length

(3) order of exons 5-7 is not known because they are terminal exons and the genomic sequence (AC064811 is unordered t = terminal exon and nd = not determined Table 2. BAGE genomic sequences

Genbank gene type chromosome positions

AC064811(i; ) BAGE1 gene 13 (2)

AL163201 BAGE 2 gene 21p 191,902-114,255

AL161418 BAGE 5 gene 13 76,589-169,650

AL158811 BAGE6 predicted ge 13 4,541-147,162

AL049849 BAGE7 predicted ge 21p 23,819-27,258

AL163203 gene fragment 21q 66,6624-68,522

AC008443 (Ξ » gene fragment nd 18,576-16,465

AC008454 (2 . gene fragment nd 32,862-22,256

AL161615 gene fragment 13 210,981-213,643

AL163539 gene fragment 9 99,935-101,853

AL356136 gene fragment 9 6,126-4,209

AL359312 gene fragment 9 22,315-20,417

AC068255 gene fragment 18 76,105-78,004

AP001896 gene fragment 18q22 (2)

(1) genomic clone misassigned to chromosome 4

(2) unordered genomic sequence

(3) genomic clone misassigned to chromosome 5 nd = not determined

Table 3. Expression of BAGE genes in normal tissues and tumour samples

Histological type umber Number of BAGE -positive samples tested

PCR PCR-restriction enzyme

1 a b | BssKI + Alul + BstNI + BssKI- 1

BAGE1 BAGE1 BAGE2 BAGE3 BAGE5 ^'

BAGE2

BAGE3

BAGE5 BAGE5

Tumour samples

Melanoma 103 15 35 14 24 14 9 primary lesion 26 3 10 2 4 4 2 π-etastatic lesion 77 12 25 12 20 10 7

Bladder carcinoma 20 4 6

Lung carcinoma NSC 13 0 3

Marrtmary carcinoma 16 0 1 Sarcoma 10 0 1

Prostatic carcinoma 6 0 1

Neuroblastoma 1 1 1

Head & neck tumour 11 0 0

Leukemia 10 0 0

Renal carcinoma 10 0 0

Colorectal carcinoma 9 0 0

Uterus 2 0 0

Brain tumour 2 0 0

2 0 0

Normal tissues testis 1 1 1 placenta 1 0 0 heart 1 0 0 brain 1 0 0 lung 1 0 0 liver 1 0 0 skeletal muscle 1 0 . 0 kidney 1 0 0 pancreas 1 0 0

(a) RT-PCR with primers bagel4/bage

(b) RT-PCR with primers bage20/bag II) References

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IN) Materials and methods

1) 5' and 3' RACE and cDΝA cloning

Five μl of Marathon Ready human testes cDΝA (#7414-1, Clontech) was amplified in a 30-cycle RACE PCR. We used the Expand PCR kit (Boehringer) with specific primers in conjunction with the API adaptor-specific primer (Clontech). Each PCR mix was diluted 1/50 and 5 μl was used in a second 20-cycle PCR using the nested specific primers in conjunction with the nested adaptor-specific primer AP2 (Clontech). Primers bage5R (5'-GCCATCTCTGTTGCCACCACCATCC-3') and nested bage5ΝR (5'-TGAGACGAGCCACGGTGTTACAGGC-3') were used for 5' RACE experiments. Primers bage3R (5'-TGTCTGCCCAGCTGCTCCAAGCCAG-3') and nested bageSNR (5'-GAAGGAGGAGTCCCCTGTGGTGAGC-3') were used for 3' RACE experiments. 5' and 3'RACE products were purified using the Wizard kit (Promega) and cloned using the pGem-T PCR cloning kit (Promega). Afterwards, primers bagel4 (5'- GTCTCTGGTATCTCCCGCTGAGCTG-3') and ah.33 (5'-

ACTGACCACACTGAGAACAGGCAAG-3') were designed to conserved regions of the 5' and 3' end sequences and were used to reamplify three cDNA libraries: two home-made testis and melanoma (MZ2-Mel43) cDNA libraries (Boel et al, 1995) and a purchased testis cDNA library (Clontech #7414-1). The obtained 2 kb-products were gel purified and cloned using the pGem-T PCR cloning kit (Promega).

2) BAGE1 genomic structure

To determine the exon/intron structure of the 5' region of BAGE 1, for which no genomic sequences were retrieved f om databases, we partially sequenced cosmid 22H8, a genomic clone that we isolated through the screening oi. a genomic library with a E-4GE-⁷ probe.

3) Sequencing

Sequencing was done with an Applied Biosystems 373XL sequencer.

4) Computer analysis Nucleotide sequences and predicted amino acid sequences were analyzed by

BLAST (Altschul et al., 1990) to do searches on public databases.

Sequences were aligned by CLUSTALW at http://www2.ebi.ac.uk/clustalw 5)Chromosome mapping

In situ hybridization.

A 1.8-kb genomic fragment spanning from exon 1 to exon 2 was amplified from the 5' region that is common to all the BAGE sequences using the primers bagel (5'- GGGTCTCCGGTATCTCCCGC-3') and bage2 (5'-ATTGCTCCTGTTGAGCTGCCG- 3') and the BAG HSB1L1C6 (AL049849) as DNA matrix. The obtained PCR product was labelled by nick translation with Bio-16-dUTP (Boehringer) and hybridized to normal human metaphase chromosomes as described in Fantes et al. (1993). Hybridization signals were detected with successive layers of avidin FITC, biotinylated anti-avidin, and avidin FITC. The chromosomes were counterstained with DAPI (lmg/ml in Vectashield). Hybridization signals were visualised using a Zeiss Axioplan epifluorescence microscope. Images were captured using Digital Scientific Smartcapture software.

PCR. Primers bagel and bage2 were designed to exonl, which is common to all the

BAGE sequences, and used to amplify DNA from the monochromosome somatic hybrid cell lines (NIGMS mapping panel 2; Dubois and Naylor, 1993). To localize BAGE sequences on chromosomes 9 and 18, we used primers bage3 (5'- CTGCCATCACTGTTGCCACC-3') and bage4 (5'-TGTTCCCGGCTTAGAGGACC- 3').

PCR products were cloned in pGem-T vector (Promega). Ten colonies per cloning reaction were sequenced with universal primers SP6 and T7.

6) Expression analysis Total RNA was extracted by the guanidine-isothiocyanate procedure as described

(Davis and al, 1986). Reverse transcription was done on 2 μg of total RNA 2 niM oligo(dT)₁₅ primer and and 200 U of MoMLV reverse transcriptase (GIBCO BRL). One twentieth of the cDNA product was used for the PCR assay using Taq DNA polymerase (Promega) and the following program (1 rnin at 94°C, 2 min at 60°C, and 2 min at 72°C for 30 cycles). The quality of the RNA preparation was tested by PCR amplification of human β-actin cDNA with primers (5'-GGCATCGTGATGGACTCCG-3') and (5'- GTCGGAAGGTGGACAGCGA-3') for 21 cycles of 1 min at 94°C, 2 min at 68°C, and 2 min at 72°C by Amp-i-Tαg DNA polymerase (Perkin-Elmer Cetus, Norwalk, Connecticut). Primers used for the expression analysis were located in different exons. A first set of primers, bagel4 (5'-GTCTCTGGTATCTCCCGCTGAGCTG-3') and bagel9 (5'-TTGCTCCTGTTGAGCTGCCGTCTCC-3'), was used to amplify a PCR product of 490 bp corresponding to BAGE1 and BAGE5 transcripts. A second set of primers, bage20 (5'-CATTTTGTAAGCACTTTGGAGCCAC-3') and bage21 (5'-CACTATCAGCCTCATTAGAAATCTG-3'), was used to amplify a 368-bp PCR product corresponding to BAGE2, BAGE3, and BAGE5 transcripts.

To identify individual genes, 15 μl of the PCR product was digested with restriction enzymes in a total volume of 20 μl and the products of digestion were size fractionated on a 2 % agarose gel. Digestion of the 495-bp PCR product (bage20- bage21) with either AM or BstNI allowed us to identify BAGE2 and BAGE3 cDNAs, respectively. Digestion of the 368-bp PCR product (bagel4-bagel9) with BssKI allowed us to identify BAGE1 (presence of the restriction site) and BAGE5 (absence of the restriction site).

nucleotide sequences cited above

- SEQ ID NO : 3 gtctctGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGTTGGAGGCTGGAGTCTGTAACACCGTGGCTCGTCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGCGCAGCTGGAGTGTTAGGAG GGCGGCCTGAGCGGTAGGAGTGGGGCTGGAGCAGTAAGATGGCGGCCGGA GCAGTAAGATGGCGGCTGGAGTGGTTTTTCTGGCATTGTCTGCCCAGCTG CTCCAAGCCAGGCTGATGAAGGAGGAGTCCCCTGTGGTGAGCTGGAGGTT GGAGCCTGAAGATGGCACAGCTCTCGATGTGCATTTTGTAAGCACTTTGG AGCCACTATCAAATGCTGTGAAGAGAAATGTACCCAGATGTATCATTATC CTTGTGCTGCAGGAGCCGACAGCTTTCAGGATTTCAGTCACATCTTCCTG CTTTGTCCAGAACACATTGACCAAGCTCCTGAAAGATCGAAGGAAGATGC AAACTGTGCAGTGTGCGACAGCCCGGGAGACCTCTTAGATCAGTTCTTTT GTACTACTTGTGGTCAGCACTATCATGGAATGTGCCTGGATATAGCGGTT ACTCCATTAAAACGTGCAGGTTGGCAATGTCCTGAGTGCAAAGTGTGCCA GAACTGCAAACAATCGGGAGAAGATAGCAAGATGCTAGTGTGTGATACGT GTGACAAAGGGTATCATACTTTTTGTCTTCAACGAATTATGAAATCTGTA CCAACCAATGGCTGGA-ATGCAAATGAATACCGAATTGGAAAAACAGATT TCTAATGAGGCTGATAGTGAAGAAATGAAAATGTCTTCTGAAGTGAAGCA TATTTGTGGCGAAGATCAAATTGAAGATAAAATGGAAGTGACAGAAAACA TTGAAGTCGTTACACACCAGATCACTGTGCAGCAAGAGCAACTGCAGTTG TTAGAGGAACCTAAAACAGTGGTATCCAAAGAAGAATCAAGGCCTCCAAA ATTTGTCATTGAATCTGTCACTCTTCCACTAGAAACCTTAGTGTCCCCAC ATGAGGAAAGCACTTCATTATGTCCTGAGGAACAGTTGGTTATAGAAAGG CTACAAGGAGAAAAGGAACAGAAAGAAGATTCTGAACTTTCTACTGGATT GATGGACTCTGAAATGACTCCTACAATTGAGGGTTGTGTGAAAGATGTTT CATACCAAGGAGGCAAATCTATAAAGTTATCATCTGAGACAGAGTCATCA TTTTCATTATCAGCAGACATAAGCAAGGCAGATGTGTCTTCCTCCCCAAC ACCTTCTTCAGACTTGCCTTCGCATGACATGCTGCGTAATTACCCTTCAG CTCTTAGTTCCTCTGCTGGAAACATCATGCCAACAACTTACATCTCAGTC ACTCCAAAAAACTGGCATGGGTAAACCAGCTATTACTAAGAGAAAATTTT CTCCTGGTAGACCTCGGTCCAAACAGGAGGCTTGGAGTACCCATAATACA GTGAGCCCACCCTCGTGGTCCCCAGACATTTCAGGAGGTCGGGAAATTTT TAAACCCAGGCAGCTTCCTGGCAGTGCCATTTGGAGCATCAAAGTGGGCC ATGGGTCTGGATTTCCAAGAAAGCGGAGACCTCGAGGTGCAGGACTGTCG GGGCGAGGTGGCCGAGGCAGGTCAAAGCTGAAAAGTGGAATCGGAGCTGT TGTATTGCCTGGGGTGTCTACTGCAGATATTTCATCAAATAAGGATGATG AAGAAAACTCTATGCACACTACGGTTGTGTTGTTTTCTAGCAGTGACAAG TTCACTTTGAATCAGGATATGTGTGTAGTTTGTGGCAGTTTTGGCCAAGG AGCAGAAGGAAGACTACTTGCCTGTTCTCAGTGTGGTCAGT the nucleotides encoding SEQ ID NO : 4 being situated at positions 209 to 535 of SEQ ID NO : 3 - SEQ ID NO : 5

GTCTCTGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGTTGGAGGCTGGAGCCTGTAACACCGTGGCTCGTCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGCGCAGCTGGAGTGTTAGGAG GGCGGCCTGAGCGGTAGGAGTGGGGCTGGAGCAGTAAGATGGCGGCCGGA GCAGTAAGATGGCGGCTGGAGTGGTTTTTCTGGCATTGTCTGCCCAGCTG CTCCAAGCCAGGCTGATGAAGGAGGAGTCCCCTGTGGTGAGCTGGAGGTT GGAGCCTGAAGATGGCACAGCCCTCGATGTGCATTTTGTAAGCACTTTGG AGCCACTATCAAATGCTGTGAAGAGAAATGTACCCAGATGTATCATTATC CTTGTGCTGCAGGAGCCGACACCTTTCAGGATTTCAGTCACATCTTCCTG CTTTGTCCAGAACACATTGACCAAGCTCCTGAAAGATCGAAGGAAGATGC AAACTGTGCAGTGTGCAACAGCCCAGGAGACCTCTTAGATCAGTTCTTTT GTACTACTTGTGGTCAGCACTATCATGGAATGTGCCTGGATATAGCGGTT ACTCCATTAAAACGTGCAGGTTGGCAATGTCCTGAGTGCAAAGTGTGCCA GAACTGCAAACAATCGGGAGAAGATAGCAAGATGCTAGTGTGTGATACGT GTGACAAAGGGTATCATACTTTTTGTCTTCAACGAATTATGAAATCTGTA CCAACCAACGGCTGGAAATGCAAATGAATACCGAATTGGAAAAACAGATT TCTAATGAGGCTGATAGTGAAGAAATGAAAATGTCTTCTGAAGTGAAGCA TATTTGTGGCGAAGATCAAATTGAAGATGAAATGGAAGTGACAGAAAACA TTGAAGTCGTTACACACCAGATCACTGTGCAGCAAGAGCAACTGCAGTTG TTAGAGGAACCTAAAACAGTGGTATCCAAAGAAGAATCAAGGCCTCCAAA ATTTGTCATTGAATCTGTCACTCTTCCACTAGAAACCTTAGTGTCCCCAC AGGAGGAAAGCTCTTCATTATGTCCTGAGGAACAGTTGGTTATAGAAAGG CTACAAGGAGAAAAGGAACAGAAAGAAGATTCTGAACTTTCTACTGGATT GATGGACTCTGAAATGACTCCTACAATTGAGGGTTGTGTGAAAGATGTTT CATACCAAGGAGGCAAATCTATAAAGTTATCATCTGAGACAGAGTCGTCA TTTTCATTATCAGCAGACATAAGCAAGGCAGATGTGTCTTCCTCCCCAAC ACCTTCTTCAGACTTGCCTTCGCATGACATGCTGCATAATTACCCTTCAG CTCTTAGTTCCTCTGCTGGAAACATCATGCCAACAACTTAACATCTCAGT CACTCCAAAAATTGGCATGGGTAAACCAGCTATTACTAAGAGAAAATTTT CTCCTGGTAGACCTCGGTCCAAACAGGAGGCTTGGAGTACCCATAATACA GTGAGCCCACCCTCGTGGTCCCCAGACATTTCAGGAGGTCGGGAAATTTT TAAACCCAGGCAGCTTCCTGGCAGTGCCATTTGGAGCACAAAAGTGGGCC GTGGGTCTGGATTTCCAAGAAAGC^'GGAGACCTCGAGGTGCAGGACTGTCG GGGTGAGGTGGCCGAGGCAGGTCAAAGCTGAAAAGTGGAATTGGAGCTGT TGTATTGCCTGGGGTGTCTACTGCAGATATTTCATCAAATAAGGATGATG AAGAAAACTCTATGCTCGATATGGTTGTGTTGGTTTCTAGCAGTGACAAG

TTCACTTTGAATCAGGATATGTGTGTAGt11gtggcagt111ggccaagg agcagaaggaggattacttgcctgttctcagtgtggtcagt the nucleotides encoding SEQ ID NO : 6 being situated at positions 209 to 535 of SEQ ID NO : 5 - SEQ ID NO : 7

GTCTCTGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGTTGGAGGCTGGAGCCTGTAACACCGTGGCTCGTCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGCGCAGCTGGAGTGTTAGGAG GGCGGCCTGAGCGGTAGGAGTGGGGCTGGAGCAGTAAGATGGCGGCCGGA GCAGTAAGATGGCGGCTGGAGTGGTTTTTCTGGCATTGTCTGCCCAGCTG CTCCAAGCCAGGCTGATGAAGGAGGAGTCCCCTGTGGTGAGCTGGAGGTT GGAGCCTGAAGATGGCACAGCTCTCGATGTGCATTTTGTAAGCACTTTGG AGCCACTATCAAATGCTGTGAAGAGAAATGTACCCAGATGTATCATTATC CTTGTGCTGCAGGAGCCGACACCTTTCAGGATTTCAGTCACATCTTCCTG CTTTGTCCAGAACACATTGACCAAGCTCCTGAAAGATCGAAGGAAGATGC AAACTGTGCAGTGTGCGACAGCCCGGGAGACCTCTTAGATCAGTTCTTTT GTACTACTTGTGGTCAGCACTATCATGGAATGTGCCTGGATACAGCGGTT ACTCCATTAAAACGTGCAGGTTGGCAATGTCCTGAGTGCAAAGTGTGCCA GAACTGCAAACAATCGGGAGAAGATAGCAAGATGCTAGTGTGTGATACGT GTGACAAAGGGTATCATACTTTTTGTCTTCAACGAATTATGAAATCTGTA CCAACCAACGGCTGGAAATGCAAATGAATACCGAATTGGAAAAACAGATT TCTAATGAGGCTGATAGTGAAGAAATGAAAATGTCTTCTGAAGTGAAGCA TATTTGTGGCGAAGATCAAATTGAAGATAAAATGGAAGTGACAGAAAACA TTGAAGTCGTTACACACCAGATCACTGTGCAGCAAGAGCAACTGCAGTTG TTAGAGGAACCTAAAACAGTGGTATCCAAAGAAGAATCAAGGCCTCCAAA ATTTGTCATTGAATCTGTCACTCTTCCACTAGAAACCTTAGTGTCCCCAC AGGAGGAAAGCTCTTCATTATGTCCTGAGGAACAGTTGGTTATAGAAAGG CTACAAGGAGAAAAGGAACAGAAAGAAGATTCTGAACTTTCTACTGGATT GATGGACTCTGAAATGACTCCTACAATTGAGGGTTGTGTGAAAGATGTTT CATACCAAGGAGGCAAATCTATAAAGTTATCATTTGAGACAGAGTCGTCA TTTTCATTATCAGCAGACATAAGCAAGGCAGATGTGTCTTCCTCCCCAAC ACCTTCTTCAGACTTGCCTTCGCATGACATGCTGCATAATTACCCTTCAG CTCTTAGTTCCTCTGCTGGAAACATCATGCCAACAACTTAACATCTCAGT CACTCCAAAAATTGGCATGGGTAAACCAGCTATTACTAAGAGAAAATTTT CTCCTGGTAGACCTCGGTCCAAACAGGAGGCTTGGAGTACCCATAATACA GTGAGCCCACCCTCGTGGTCCCCAGACATTTCAGGAGGTCGGGAAATTTT TAAACCCAGGCAGCTTCCTGGCAGTGCCATTTGGAGCATCAAAGTGGGCC ATGGGTCTGGATTTCCAAGAAAGCGGAGACCTCGAGGTGCAGGACTGTCG GGGCGAGGTGGCCGAGGCAGGTCAAAGCTGAAAAGTGGAATCGGAGCTGT TGTATTGCCTGGGGTGTCTACTGCAGATATTTCATCAAATAAGGATGATG AAGAAAACTCTATGCACACTACGGTTGTGTTGTTTTCTAGCAGTGACAAG TTCACTTTGAATCAGGATATGTGTGTAGttt tggcagttttggccaagg agcagaaggaagactacttgcctgttctcagtgtggtcagt the nucleotides encoding SEQ ID NO : 8 being situated at positions 209 to 535 of SEQ ID NO ^': 7 - SEQ ID NO : 10

GATGTGCATTTTGTAAGCACTTTGGAGCCACTATCAAATGCTGTGAAGAG AAATGTACCCAGATGTATCATTATCCTTGTGCTGCAGGAGCCGACAGCTT TCAGGATTTCAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACCAAG CTCCTGAAAGATCGAAGGAAGATGCAAACTGTGCAGTGTGCGACAGCCCG GGAGACCTCTTAGATCAGTTCTTTTGTACTACTTGTGGTCAGCACTATCA TGGAATGTGCCTGGATATAGCGGTTACTCCATTAAAACGTGCAGGTTGGC AATGTCCTGAGTGCAAAGTGTGCCAGAACTGCAAACAATCGGGAGAAGAT AGCAAGATGCTAGTGTGTGATACGTGTGACAAAGGGTATCATACTTTTTG TCTTCAACGAATTATGAAATCTGTACCAACCAATGGCTGGAAATGCAAAT GAATACCGAATTGGAAAAACAGATTTCTAATGAGGCTGATAGTGAAGAAA TGAAAATGTCTTCTGAAGTGAAGCATATTTGTGGCGAAGATCAAATTGAA GATAAAATGGAAGTGACAGAAAACATTGAAGTCGTTACACACCAGATCAC TGTGCAGCAAGAGCAACTGCAGTTGTTAGAGGAACCTAAAACAGTGGTAT CCAAAGAAGAATCAAGGCCTCCAAAATTTGTCATTGAATCTGTCACTCTT CCACTAGAAACCTTAGTGTCCCCACATGAGGAAAGCACTTCATTATGTCC TGAGGAACAGTTGGTTATAGAAAGGCTACAAGGAGAAAAGGAACAGAAAG AAGATTCTGAACTTTCTACTGGATTGATGGACTCTGAAATGACTCCTACA ATTGAGGGTTGTGTGAAAGATGTTTCATACCAAGGAGGCAAATCTATAAA GTTATCATCTGAGACAGAGTCATCATTTTCATTATCAGCAGACATAAGCA AGGCAGATGTGTCTTCCTCCCCAACACCTTCTTCAGACTTGCCTTCGCAT GACATGCTGCGTAATTACCCTTCAGCTCTTAGTTCCTCTGCTGGAAACAT CATGCCAACAACTTACATCTCAGTCACTCCAAAAAACTGGCATGGGTAAA CCAGCTATTACTAAGAGAAAATTTTCTCCTGGTAGACCTCGGTCCAAACA GGAGGCTTGGAGTACCCATAATACAGTGAGCCCACCCTCGTGGTCCCCAG ACATTTCAGGAGGTCGGGAAATTTTTAAACCCAGGCAGCTTCCTGGCAGT GCCATTTGGAGCATCAAAGTGGGCCATGGGTCTGGATTTCCAAGAAAGCG GAGACCTCGAGGTGCAGGACTGTCGGGGCGAGGTGGCCGAGGCAGGTCAA AGCTGAAAAGTGGAATCGGAGCTGTTGTATTGCCTGGGGTGTCTACTGCA GATATTTCATCAAATAAGGATGATGAAGAAAACTCTATGCACACTACGGT TGTGTTGTTTTCTAGCAGTGACAAGTTCACTTTGAATCAGGATATGTGTG TAGTTTGTGGCAGTTTTGGCCAAGGAGCAGAAGGAAGACTACTTGCCTGT TCTCAGTGTGGTCAGT the nucleotides encoding SEQ ID NO : 11 being situated at positions 13 to 210 of SEQ ID NO : 10 - SEQ ID NO : 12

GATGTGCATTTTGTAAGCACTTTGGAGCCACTATCAAATGCTGTGAAGAG AAATGTACCCAGATGTATCATTATCCTTGTGCTGCAGGAGCCGACACCTT TCAGGATTTCAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACCAAG CTCCTGAAAGATCGAAGGAAGATGCAAACTGTGCAGTGTGCAACAGCCCA GGAGACCTCTTAGATCAGTTCTTTTGTACTACTTGTGGTCAGCACTATCA TGGAATGTGCCTGGATATAGCGGTTACTCCATTAAAACGTGCAGGTTGGC AATGTCCTGAGTGCAAAGTGTGCCAGAACTGCAAACAATCGGGAGAAGAT AGCAAGATGCTAGTGTGTGATACGTGTGACAAAGGGTATCATACTTTTTG TCTTCAACGAATTATGAAATCTGTACCAACCAACGGCTGGAAATGCAAAT GAATACCGAATTGGAAAAACAGATTTCTAATGAGGCTGATAGTGAAGAAA TGAAAATGTCTTCTGAAGTGAAGCATATTTGTGGCGAAGATCAAATTGAA GATGAAATGGAAGTGACAGAAAACATTGAAGTCGTTACACACCAGATCAC TGTGCAGCAAGAGCAACTGCAGTTGTTAGAGGAACCTAAAACAGTGGTAT CCAAAGAAGAATCAAGGCCTCCAAAATTTGTCATTGAATCTGTCACTCTT CCACTAGAAACCTTAGTGTCCCCACAGGAGGAAAGCTCTTCATTATGTCC TGAGGAACAGTTGGTTATAGAAAGGCTACAAGGAGAAAAGGAACAGAAAG AAGATTCTGAACTTTCTACTGGATTGATGGACTCTGAAATGACTCCTACA ATTGAGGGTTGTGTGAAAGATGTTTCATACCAAGGAGGCAAATCTATAAA GTTATCATCTGAGACAGAGTCGTCATTTTCATTATCAGCAGACATAAGCA AGGCAGATGTGTCTTCCTCCCCAACACCTTCTTCAGACTTGCCTTCGCAT GACATGCTGCATAATTACCCTTCAGCTCTTAGTTCCTCTGCTGGAAACAT CATGCCAACAACTTAACATCTCAGTCACTCCAAAAATTGGCATGGGTAAA CCAGCTATTACTAAGAGAAAATTTTCTCCTGGTAGACCTCGGTCCAAACA GGAGGCTTGGAGTACCCATAATACAGTGAGCCCACCCTCGTGGTCCCCAG ACATTTCAGGAGGTCGGGAAATTTTTAAACCCAGGCAGCTTCCTGGCAGT GCCATTTGGAGCACAAAAGTGGGCCGTGGGTCTGGATTTCCAAGAAAGCG GAGACCTCGAGGTGCAGGACTGTCGGGGTGAGGTGGCCGAGGCAGGTCAA AGCTGAAAAGTGGAATTGGAGCTGTTGTATTGCCTGGGGTGTCTACTGCA GATATTTCATCAAATAAGGATGATGAAGAAAACTCTATGCTCGATATGGT TGTGTTGGTTTCTAGCAGTGACAAGTTCACTTTGAATCAGGATATGTGTG TAGTTTGTGGCAGTTTTGGCCAAGGAGCAGAAGGAGGATTACTTGCCTGT TCTCAGTGTGGTCAGT the nucleotides encoding SEQ ID NO : 13 being situated at positions 13 to 210 of SEQ ID NO : 12

- SEQ ID NO : 14 GTCTCTGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGCTGGAGGCTGGAGCCTGTAACACCGTGGCTCGTCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGCGCAGCTGGAGTGTTAGGAG GGCAGCCTGAGCAGTAGGATTGGGGCTGGAGCAGTAAGATGGCAGCCGGA GCGGTTTTTCTGGCATTGTCTGCCCAGCTGCTCCAAGCCAGGCTGATGAA GGAGGAGTCCCCTGTGGTGAGCTGGTGGTTGGAGCCTGAAGACGGCACAG CTCTGTGATTCATCTTCTGAGGTTGTGGCAGCCACGGTGATGGAGACAGC AGCTCAACAGGAACAATAGGAGGGTACCCGTGGAGGCCAAGTGCGATGTG CATTTTGTAAGCACTTTGGAGCCACTATCAAATGCTGTGAAGAGAAATGT ACCCAGATGTATCATTATCCTTGTGCTGCAGGAGCCGGCTCCTTTCAGGA TTTCAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACCAAGCTCCAG AAAGATCGAAGGAAGATGCAAACTGTGCAGTGTGCGACAGCCCGGGAGAC CTCTTAGATCAGTTCTTTTGTACTACTTGTGGTCAGCACTATCATGGAAT GTGCCTGGATATAGCGGTTACTCCATTAAAACGTGCAGGTTGGCAATGTC CTGAGTGCAAAGTGTGCCAGAACTGCAAACAATCGGGAGAAGATAGCAAG ATGCTAGTGTGTGATACGTGTGACAAAGGGTATCATACTTTTTGTCTTCA ACGAATTATGAAATCTGTACCAACCAATGGCTGGAAATGCAAATGAATAC CGAATTGGAAAAACAGATTTCTAATGAGGCTGATAGTGAAGAAATGAAAA TGTCTTCTGAAGTGAAGCATATTTGTGGTGAAGATCAAATTGAAGATAAA ATGGAAGTGACAGAAAACATTGAAGTCGTTACACACCAGATCACTGTGCA GCAAGAGCAACTGCAGTTGTTAGAGGAACCTAAAACAGTGGTATCCAAAG AAGAATCAAGGCCTCCAAAATTTGTCATTGAATCTGTCACTCTTCCACTA GAAACCTTAGTGTCCCCACATGAGGAAAGCACTTCATTATGTCCTGAGGA ACAGTTGGTTATAGAAAGGCTACAAGGAGAAAAGGAACAGAAATAAGATT CTGAACTTTCTACTGGATTGATGGACTCTGAAATGACTCCTACAATTGAG GGTTGTGTGAAAGATGTTTCATACCAAGGAGGCAAATCTATAAAGTTATC ATCTGAGACAGAGTCATCATTTTCATTATCAGCAGACATAAGCAAGGCAG ATGTGTCTTCCTCCCCAACACCTTCTTCAGACTTGCCTTCGCATGACATG CTGCGTAATTACCCTTCAGCTCTTAGTTCGTCTGCTGGAAACATCATGCC AACAACTTACATCTCAGTCACTCCAAAAAACTGGCATGGGTAAACCAGCT ATTACTAAGAGAAAATTTTCTCCTGGTAGACCTCGGTCCAAACAGGGCCG TGGGTCTGGATTTCCAGGAAAGCGGAGACCTCGAGGTGCAGGACTGTCAG GGCGAGGTGGCCGAGGCAAGTCAAAGCTGAAAAGTGGAATCGGAGCTGTT GTATTGCCTGGGGTGTCTACTGCAGATATTTCATCAAATAAGGATGATGA AGAAAACTCTATGCACACTACGGTTGTGTTGTTTTCTAGCAGTGACAAGT TCACTTTGAATCAGGATATGTGTGTAGTTTGTGGCAGTTTTGGCCAAGGA GCAGAAGGAAGATTACTTGCCTGTTCTCAGTGTGGTCAGT the nucleotides encoding SEQ ID NO : 15 being situated at positions 189 to 305 of SEQ ID NO : 14

- SEQ ID NO 16

GTCTCTGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGCTGGAGGCTGGAGCCTGTAACACCGTGGCTCGTCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGCGCGGCTGGAGTGTTAGGAG GGTGGCCTGAGCAGTAGGATTGGGGCTGGAGCAGTAAGATGGCAGCCGGA GCGGTTTTTCTGGCATTGTCTGCCCAGCTGCTCCAAGCCAGACTGATGAA GGAGGAGTCCCCTGTGGTGAGCTGGAGGTTGGAGCCTGAAGACGGCACAG CTCTGTGCTTCATCTTCTGAGGTTGTGGCAGCCACGGTGATGGAGACGGC AGCTCAACAGGAGCAATAGGAGGGTACCCGTGGAGGCCAAGTGCGATGTG CATTTTGTAAGCACTTTGGAGCCACTATCAAATGCTGTGAAGAGAAATGT ACCCAGATGTATCATTATCCTTGTGCTGCAGGAGCCGACACCTTTCAGGA TTTCAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACCAAGCTCCTG AAAGATCGAAGGAAGATGCAAACTGTGCAGTGTGCGACAGCCCGGGAGAC CTCTTAGATCAGTTCTTTTGTACTACTTGTGGTCAGCACTATCATGGAAT GTGCCTGGATATAGCGGTTACTCCATTAAAACGTGCAGGTTGGCAATGTC CTGAGTGCAAAGTGTGCCAGAACTGCAAACAATCGGGAGAAGATAGCAAG ATGCTAGTGTGTGATACGTGTGACAAAGGGTATCATACTTTTTGTCTTCA ACGAATTATGAAATCTGTACCAACCAACGGCTGGAAATGCAAATGAATAC CGAATTGGAAAAACAGATTTCTAATGAGGCTGATAGTGAAGAAATGAAAA TGTCTTCTGAAGTGAAGCATATTTGTGGCGAAGATCAAATTGAAGATAAA ATGGAAGTGACAGAAAACATTGAAGTCGTTACACACCAGATCACTGTGCA GCAAGAGCAACTGCAGTTGTTAGAGGAACCTAAAACAGTGGTATCCAAAG AAGAATCAAGGCCΤCCAAAATTTGTCATTGAATCTGTCACTCTTCCACTA GAAACCTTAGTGTCCCCACAGGAGGAAAGCTCTTCATTATGTCCTGAGGA ACAGTTGGTTATAGAAAGGCTACAAGGAGAAAAGGAACAGAAAGAAGATT CTGAACTTTCTACTGGATTGATGGACTCTGAAATGACTCCTACAATTGAG GGTTGTGTGAAAGATGTTTCATACCAAGGAGGCAAATCTATAAAGTTATC ATCTGAGACAGAGTCGTCATTTTCATTATCAGCAGACATAAGCAAGGCAG ATGTGTCTTCCTCCCCAACACCTTCTTCAGACTTGCCTTCGCATGACATG CTGCATAATTACCCTTCAGCTCTTAGTTCCTCTGCTGGAAACATCATGCC AACAACTTAACATCTCAGTCACTCCAAAAATTGGCATGGGTAAACCAGCT ATTACTAAGAGAAAATTTTCTCCTGGTAGACCTCGGTCCAAACAGGGCCG TGGGTCTGGATTTCCAAGAAAGCGGAGACCTCGAGGTGCAGGACTGTCGG GGTGAGGTGGCCGAGGCAGGTCAAAGCTGAAAAGTGGAATTGGAGCTGTT GTATTGCCTGGGGTGTCTACTGCAGATATTTCATCAAATAAGGATGATGA AGAAAACTCTATGCACACTACGGTTGTGTTGTTTTCTAGCAGTGACAAAT TCACTTTGAATCAGGATATGTGTGTAGTTTGTGGCAGTTTTGGCCAAGGA GCAGAAGGAGGATTACTTGCCTGTTCTCAGTGTGGTCAGT the nucleotides encoding SEQ ID NO : 17 being situated at positions 189 to 317 of SEQ ID NO : 16

- SEQ ID NO : 18 GTCTCCGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACCAG GAGAAGGGGGAGCTGGAGGCTGGAGCCTGTAACACCGTGGCTCATCTCGC TCTGGATGGTGGTGGCAACAGAGATGGCAGTGCGGCTGGAGTGTTAGGAG AGTGGCCTGAGCAGTAGGATTGGGGCTGGAGCAGTAAGATGGCAGCCGGA GCGGTTTTTCTGGCATTGTCTGCCCAGCTGCTCCAAGCCAGACTGATGAA GGAGGAGTCCCCTGTGGTGAGCTGGAGGTTGGAGCCTGAAGATGGCACAG CTCTGTGATTCATCTTCTGCGGTTGTGGCAGCCACGGTGATGGAGACGGC AGCTCAACAGGAGCAATAGGAGGGTACCCATGGAGGCCAAGTG the nucleotides encoding SEQ ID NO : 19 being situated at positions 189 to 305 of SEQ ID NO : 18

Claims

1. Proteins of the BAGE family comprising the following SEQ ID NO : 1 sequence: MAAGX₁NFLALSAQLLQARLMKEESPNNSWX₂LEPEDGTALX₃[X₄X₅F]_n in which:

- n represents 0 or 1,

- X. represents V or A,

- X₂ represents R or W, - X₃ represents Z, D or C,

- X₄ represents N or F,

- X₅ represents H or I, or fragments of these proteins comprising at least about 5 amino acids, or at least 9 amino acids, or peptide sequences derived from the above-mentioned proteins or fragments, by substitution, deletion or addition of one or more amino acids of the SEQ ID NO : 1 sequence, or of a fragment of this sequence, said fragments or derived sequences having the property, when fonning complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence, provided that :

. SEQ ID NO : 21: MAARAVFLALSAQLLQARLMKE . SEQ J-D NO : 22: MAARAVFLALSAQLLQ . SEQ ID NO : 23 : AARAVFLAL

. SEQ ID NO : 24: ARAVFLALF . SEQ ID NO : 25: MAARANFLA being excluded.

2. Proteins according to claim 1, having the formula of the following SEQ ID

NO : 2 sequence:

MAAGNNFLALSAQLLQA-^M-^ESPVNSWRLEPEDGTALDVHFNSTLEPLSNAV KRj>WPRCmLVLQEPTX₆FMSNTSSCFNQNTLT]π,LKDRRKMQTNQCATAX₇ETS in which X₆ represents A or P, and X₇ represents R or Q.

3. Proteins according to claim 1 or 2, chosen among :

- the BAGE 2 protein represented by the following SEQ ID NO : 4 sequence: -V--AAGV LALSAQLLQA-RLMKEESPVNSWRLEPEDGTALDNHFNSTLEPLSNAN Ol-WPRCiπLNLQEPTAFMSNTSSCFVQΝTLT-^^

- the BAGE 3 protein represented by the following SEQ ID NO : 6 sequence: MAAGVNFLALSAQLLQA-^MKEESPNVSWRLEPEDGTALDNHFNSTLEPLSNAV Ki WRCIIILNLQEPTPFRISNTSSCFNQNTLTKLLKDRRKMQTNQCATAQETS - or the BAGE 6 protein represented by the SEQ ID NO : 8 sequence which is identical to SEQ ID NO : 4.

4. Fragments of proteins having the formula of the SEQ ID NO : 2 sequence according to claim 1 or 2, or of sequences derived from the latter such as defined in claim 1, said fragments comprising at least about 5 amino acids, or at least 9 amino acids from said sequence.

5. Fragments of proteins according to claim 4, said fragments having the following SEQ ID NO : 9 sequence: NSTLEPLSNAN-πWNPRCπiiNLQEPTX₆FMSNTSSCFNQNTLT]α-LKD]?-RKMQT VQCATAX₇ETS in which X₆ represents A or P, and X₇ represents R or Q, or peptide sequences derived from the above-mentioned fragments by substitution, deletion or addition of one or more amino acids of the SEQ ID NO : 9 sequence, said fragments or derived sequences having the property, when forming complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigen derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence.

6. Fragments of proteins according to claim 5, chosen among:

- the following peptide sequence SEQ ID NO : 11 : VSTLEPLSNAVia WRCπiLNLQEPTAFMSNTSSCFNQNTLT-π-L-πDRRKMQT VQCATARETS - the following peptide sequence SEQ ID NO : 13:

VSTLEPLSNANIOWWRC-I-LNLQEPTPFMSNTSSCFNQN^ QCATAQETS

7. Fragments comprising at least about 5 amino acids, or at least 9 amino acids, derived from the SEQ ID NO : 9, 11 or 13 sequences, or from sequences derived from the latter such as defined in claims 5 or 6.

8. Proteins according to claim 1, chosen among the following:

- the BAGE 4 protein represented by the following SEQ ID NO : 15 sequence: IV-AAGANFLALSAQLLQARLMKEESPNNSWWLEPEDGTAL

- the BAGE 5 protein represented by the following SEQ ID NO : 17 sequence: MAAGANFLALSAQLLQARLMKEESPNNSWRLEPEDGTALCFIF - the BAGE 7 protein represented by the following SEQ ID NO : 19 sequence:

MAAGA LALSAQLLQARLMKEESP SWRLEPEDGTAL

9. Fragments of the BAGE 4 and BAGE 5 proteins according to claim 8, or of sequences derived from the latter such as defined in claim 1, said fragments comprising at least about 5 amino acids, or at least 9 amino acids from the SEQ ID NO : 15, SEQ ID NO : 17, or SEQ ID NO : 19 sequences.

10. Nucleotide sequences coding for a protein of the BAGE family, or a fragment or a derived protein such as defined in one of the claims 1 to 9, or nucleotide sequences derived from the latter by degeneration of the genetic code.

11. Nucleotide sequences according to claim 10, chosen among the following:

- the sequence SEQ ID NO : 3, and more particularly part of SEQ ID NO : 3 delimited by the nucleotides located at the position 209 and the position 535 coding for the peptide sequence SEQ ID NO : 4, or any other possible open reading frame of SEQ ID NO : 3 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 7, and more particularly part of SEQ ID NO : 7 delimited by the nucleotides located at the position 209 and the position 535 coding for the peptide sequence SEQ ID NO : 8, or any other possible open reading frame of SEQ ID NO : 7 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 10, and more particularly part of SEQ ID NO : 10 delimited by the nucleotides located at the position 13 and the position 210 coding for the peptide sequence SEQ ID NO : 11, or any other possible open reading frame of SEQ ID NO : 10 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 12, and more particularly part of SEQ ID NO : 12 delimited by the nucleotides located at the position 13 and the position 210 coding for the peptide sequence SEQ ID NO : 13, or any other possible open reading frame of SEQ ID NO : 12 encoding a tumor rejection antigen precursor, - the sequence SEQ ID NO : 14, and more particularly part of SEQ JO NO : 14 delimited by the nucleotides located at the position 189 and the position 305 coding for the peptide sequence SEQ ID NO : 15, or any other possible open reading frame of SEQ J-D NO : 14 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 16, and more particularly part of SEQ ID NO : 16 delimited by the nucleotides located at the position 189 and the position 317 coding for the peptide sequence SEQ ID NO : 17, or any other possible open reading frame of SEQ JD NO : 16 encoding a tumor rejection antigen precursor,

- the sequence SEQ ID NO : 18, and more particularly part of SEQ ID NO : 18 delimited by the nucleotides located at the position 189 and the position 305 coding for the peptide sequence SEQ ID NO : 19, or any other possible open reading frame of SEQ ID NO : 18 encoding a tumor rejection antigen precursor.

12. Nucleotide sequences which hybridize with the nucleotide sequences such as defined in claims 10 and 11, under stringent conditions, provided that the nucleotide sequences coding for the BAGE 1 protein represented by SEQ ID NO : 20, or for the fragments of BAGE 1, or the derived sequences, represented by SEQ ID NO : 21 to 25, being excluded.

13. Nucleotide sequences according to claim 12, coding for polypeptide sequences having the property, when forming complexes with HLA molecules, of stimulating the cytolytic activity of T cells against the cells expressing at their surface complexes between HLA molecules and tumor antigen derived from tumor rejection antigen precursors having the SEQ ID NO : 1 sequence.

14. Recombinant nucleotide sequences comprising a nucleotide sequence according to anyone of claims 10 to 13, under the control of a transcription promoter and a transcription terminator.

15. Expression vector, characterized in that it contains a recombinant sequence according to claim 14.

16. Host cells transformed by a nucleotide sequence according to anyone of claims 10 to 13, if necessary by means of a vector according to claim 15.

17. Host cells according to claim 16, characterized in that they express HLA molecules, or are transformed in such a way that they express HLA molecules.

18. Cytolytic T cells recognizing specifically the complexes between HLA molecules and the proteins of the BAGE family, or the fragments or derived sequences, such as defined in anyone of claims 1 to 9, said cytoiytic T cells being such as obtained by contacting T cells from an individual with said complexes, or with host cells according to claim 16.

19. Polyclonal or monoclonal antibodies directed against the proteins of the

BAGE family, or against the fragments or derived proteins, such as defined in anyone of claims 1 to 9, or directed against complexes of these proteins, fragments or derived sequences, with HLA molecules, said antibodies being such as obtained by immunizing an animal with said sequences, fragments or derived sequence, or with said complexes.

20. Antibodies according to claim 19, characterized in that they are coupled to antitumor agents.

21. Use of the proteins of the BAGE family, or of fragments or sequences derived from the latter, according to anyone of the claims 1 to 9, or of host cells according to claim 15 or 16, or of cytolytic cells according to claim 17, or of antibodies according to claim 18 or 19, for the manufacture of a medicament intended for the treatment of pathologies linked to the expression, at the surface of the cells of the organism, of complexes between peptides corresponding to fragments from said proteins according to claims 1, 2, 3 and 8, and HLA molecules.

22. Use according to claim 21, in the scope of the treatment of tumors such as melanomas.

23. Pharmaceutical composition characterized in that it comprises at least a protein of the BAGE family, and/or a fragment, and/or a derived sequence according to one of the claims 1 to 9, or host cells according to claim 15 or 16, or cytolytic cells according to claim 17, or antibodies according to claim 18 or 19, in association with a pharmaceutically acceptable vehicle.

24. Pharmaceutical composition according to claim 23, characterized in that it also comprises at least a protein, and/or a fragment, and/or a derived sequence, from the MAGE, GAGE family, and/or the BAGE 1 protein, or the fragments of the BAGE 1 protein such as defined in claim 1.

25. Method for treating a subject with a disorder linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigens corresponding to fragments of proteins according to claims 1, 2, 3 and 8, comprising the administration to said subject of a pharmaceutical composition according to claim 23 or 24, in a sufficient amount for alleviating said pathologies.

26. Method according to claim 25, for treating a subject with a tumor such as a melanoma.

27. Use of nucleotide sequences according to anyone of claims 10 to 13, or of host cells according to claim 16 or 17, or of cytolytic cells according to claim 18, or of antibodies according to claim 19 or 20, for the implementation of methods for the in vitro diagnosis of disorders linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigens corresponding to fragments of proteins according to claims 1, 2, 3 and 8, more particularly of tumors such as melanomas.

28. Method for the in vitro diagnosis of a disorder linked to the expression at the surface of the cells of the organism, of complexes between HLA molecules and tumor antigens derived from tumor rejection antigen precursors, said tumor antigen corresponding to fragments of proteins according to claims 1, 2, 3 and 8, more particularly of tumors such as melanomas, said method comprising contacting a biological sample from a subject with an agent specific for proteins of the BAGE family, or for fragments or derived proteins, such as defined in one of the claims 1 to 9, or specific for the nucleotide sequences according to anyone of the claims 10 to 13, and determining interaction between said agent with the above-mentioned proteins of the BAGE family, or with the complexes between said tumor antigens and the HLA molecules on cell membranes, or with the nucleotide sequences coding for the above- mentioned proteins of the BAGE family, as a determination of said disorder.

29. Method for in vitro diagnosis according to claim 28, characterized in that the specific agent of the proteins of the BAGE family, or of the fragments or derived proteins, such as defined in anyone of claims 1 to 9, are T cells according to claim 18, or antibodies according to claim 19.

30. Method for in vitro diagnosis according to claim 28, characterized in that the specific agent of the nucleotide sequences according to anyone of claims 10 to 13, are nucleotide sequences complementary to the latter, and which are used as probes or primers in PCR reactions.