Identification on a human sarcoma of two new genes with tumor-specific expression.

Genes MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 code for antigens that are recognized on melanoma cells by autologous CTLs. Because the pattern of expression of these genes results in the presence of antigens on many tumors of various histological types and not on normal tissues, these antigens qualify for cancer immunotherapy. To identify new genes with tumor-specific expression, we applied a cDNA subtraction approach, ie., representational difference analysis, to a human sarcoma cell line. We obtained two cDNA clones that appeared to be tumor specific. The corresponding genes were named SAGE and HAGE because they have the same pattern of expression as genes of the MAGE family. SAGE encodes a putative protein of 904 amino acids and shows no homology to any recorded gene. Like the MAGE-A genes, it is located in the q28 region of chromosome X. Expression of gene SAGE was observed mainly in bladder carcinoma, lung carcinoma, and head and neck carcinoma but not in normal tissues, with the exception of testis. Gene HAGE, which is located on chromosome 6, encodes a putative protein of 648 amino acids. This protein is a new member of the DEAD-box family of ATP-dependent RNA helicases. Gene HAGE is expressed in many tumors of various histological types at a level that is 100-fold higher than the level observed in normal tissues except testis. Because of this tumor-specific expression, genes SAGE and HAGE ought to encode antigens that could be useful for antitumoral therapeutic vaccination.

High frequency of autologous anti-melanoma CTL directed against an antigen generated by a point mutation in a new helicase gene.

We have identified an Ag recognized by autologous CTL on the melanoma cells of a patient who enjoyed an unusually favorable clinical evolution. The antigenic peptide, which is presented by HLA-A28 molecules, is encoded by a mutated sequence in a new gene. This gene, which was named MUM-3, is expressed ubiquitously and shows homology with the RNA helicase gene family. Limiting dilution analysis indicated that at least 0.15% of the blood CD8 T cells were tumor-specific CTL precursors. The MUM-3 Ag was recognized by 90% of these CTL, indicating that it is the dominant target Ag of the tumor-specific CTL response. The high frequency of anti-MUM-3 CTL was confirmed with tetramers of soluble HLA-A28 molecules loaded with the antigenic peptide. MUM-3 tetramers stained 1.2% of blood CD8 cells, a frequency that has never been reported for T cells directed against a strictly tumor-specific Ag. To confirm these results, the CD8 T cells that were clearly labeled with tetramers were restimulated in clonal conditions. About 90% of these cells proliferated, and all the resulting clones proved lytic and MUM-3 specific. By improving the conditions used for the in vitro restimulation of CTL precursors by the tumor cells, the same frequency could be obtained in limiting dilution analysis. These results show that some cancer patients have a high frequency of circulating CTL that are directed against a strictly tumor-specific Ag. These CTL are responsive to restimulation in vitro and are easily detected with tetramers. Such responses may therefore be an achievable goal for therapeutic vaccination with tumor-specific Ags.

DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter.

A subset of male germ line-specific genes, the MAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation. It was also shown that MAGE-A1 transcription was induced in cell cultures treated with demethylating agent 5'-aza-2'-deoxycytidine. We have now analyzed all of the CpG sites within the 5' region of MAGE-A1 and show that for all of them, demethylation correlates with the transcription of the gene. We also show that the induction of MAGE-A1 with 5'-aza-2'-deoxycytidine is stable and that in all the cell clones it correlates with demethylation, indicating that demethylation is necessary and sufficient to produce expression. Conversely, transfection experiments with in vitro-methylated MAGE-A1 sequences indicated that heavy methylation suffices to stably repress the gene in cells containing the transcription factors required for expression. Most MAGE-type genes were found to have promoters with a high CpG content. Remarkably, although CpG-rich promoters are classically unmethylated in all normal tissues, those of MAGE-A1 and LAGE-1 were highly methylated in somatic tissues. In contrast, they were largely unmethylated in male germ cells. We conclude that MAGE-type genes belong to a unique subset of germ line-specific genes that use DNA methylation as a primary silencing mechanism.

A new family of mouse genes homologous to the human MAGE genes.

The human MAGE genes are expressed in a wide variety of tumors but not in normal cells, with the exception of the male germ cells, placenta, and, possibly, cells of the developing embryo. These genes encode tumor-specific antigens recognized by cytolytic T lymphocytes. The MAGE genes are located on the X chromosome, in three clusters denoted MAGE-A, B, and C, mapping at q28, p21.3, and q26, respectively. The function of these genes remains unknown. Because mice offer many advantages for the study of genes that may be involved in embryonic development, we looked for the murine equivalents of the 12 human MAGE-A genes. Using a MAGE-A probe, we isolated 8 new murine genes that are homologous to the MAGE genes. On average, the open reading frames (ORFs) of these 8 closely related genes display a slightly higher degree of nucleotide identity with the MAGE-A ORFs than with the MAGE-B or MAGE-C ORFs. Furthermore, like MAGE-A genes, they encode acidic proteins, whereas the MAGE-B genes encode basic proteins. Accordingly, these 8 murine genes were named Mage-a1 to 8 (approved symbols Magea1 to 8). Mage-a genes were mapped in two different loci on the mouse X chromosome. Mage-a4 and Mage-a7 are located in a region that is syntenic to either Xp21 or Xq28. The 6 other genes are arranged in a cluster located in a region syntenic to Xp22. Like their human counterparts, Mage-a genes were found to be transcribed in adult testis, but not in other tissues. Expression of some Mage-a genes was also detected in tumor cell lines. Two Mage-a genes were found to be expressed in blastocysts.

Identification of human testis-specific transcripts and analysis of their expression in tumor cells.

Tumor-specific antigens recognized by autologous T lymphocytes are encoded by genes, including those of the MAGE, BAGE, and GAGE gene families, that are expressed in a significant fraction of tumors of various types, but not in normal adult tissues, except for testis where they appear to be expressed in germ cells. Because male germ cells are known to express many genes that are not expressed in other normal adult tissues, we wished to determine whether most of these genes are occasionally activated in tumor cells. Representational difference analysis was used to obtain testis-specific transcripts. The expression of 15 testis-specific cDNA sequences was tested by RT-PCR in a series of tumor cell lines. Only one cDNA sequence showed a significant level of expression in some tumor cell lines. Remarkably, this cDNA clone proved to be a new gene of the MAGE family. These results suggest that MAGE, BAGE, and GAGE genes belong to a minor subset of testis-specific genes that is often activated in tumors of various types, whereas most testis-specific genes are either never or very rarely activated in tumors.

Two members of the human MAGEB gene family located in Xp21.3 are expressed in tumors of various histological origins.

Genes of the MAGE family direct the expression of tumor antigens recognized on a human melanoma by autologous cytolytic T lymphocytes. Twelve closely related MAGE genes are located in the Xq28 region. These genes share 60-98% nucleotide identity in their coding region. The presence of homologous genes in a region of Xp21.3 has been reported previously. We obtained the complete sequence of a 42-kb stretch of this region. It contains four MAGE-related genes, which we propose to name MAGE-B1, B2, B3, and B4 (HGMW-approved symbols MAGEB1, MAGEB2, MAGEB3, and MAGEB4). The coding regions of these genes share 66-81% nucleotide identity and show 45-63% identity with those of the MAGE genes located in Xq28. Like the MAGE genes located in Xq28, the MAGE-B genes are silent in normal tissues with the exception of testis. Like MAGE-1, 2, 3, 4, 6 and 12 (HGMW-approved symbols MAGEA1, 2, 3, 4, 6, and 12), genes MAGE-B1 and MAGE-B2 are expressed in a significant fraction of tumors of various histological types. The transcription of MAGE-B1 and MAGE-B2 can be induced by 5-aza-2'-deoxycytidine, suggesting that the activation of these genes in tumors results from a demethylation process.