| Research Abstract |
MAGE-1 gene was isolated from the genomic DNA of MZ2-MEL melanoma subclone by transfection of its cosmid library into E-antigen-loss variant MZ-2MEL.2.2 followed by screening with the susceptibility to CD8^+ CTL clones (Van der Bruggen et al., 1991). The MAGE multigene family includes the MAGE-1 and -3 henes that encode tumor-rejection antigens on HLA-A1 recognized by cytotoxic T-lymphocytes (CTL). MAGE genes seem to be a multigene family, the members of which share a high degree of homology. However, little is known about the MAGE-4, -41 and -6 genes. Therefore, we initialy amplified 1040-bp (MAGE-1), 1061-bp (MAGE-3 and -6) and 1064-bp (MAGE-4 and -41) cDNA fragments from cancer cells, including the entire coding sequences (927 to 951 bp), using the reverse transcription-polymerase chain reaction (RT-PCR) method followed by nucleotide (nt) sequencing. One member had greater than 80 or 66% homology with the other members at the nt or deduced amino acid (aa) levels, respectively. Highe
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r homology was found between MAGE-3 and-6 (98% at the nt level) and also between MAGE-4 and -41 (98%). The results of this investigation demonstrated the high homology, as well as the clear differences between the members of the MAGE family at the coding sequence level.
MAGE-1, -2, -3, -4a, -6, and -12 genes are investigated at the mRNA level in many different cancers including, colon cancers, esophageal cancers, and lung cancers. At least one of these genes were expressed approximately 35 to 50% of these cancers. For example, MAGE-1, -2, -31-6, and -4 genes were respectively expressed at the mRNA level on 6,7,20, and 7 of 53 lung cancers (50 non-small-cell-lung cancers and 3 small-cell-lung cancers). In contrast, neither normal cells nor normal tissues other than testis and placenta express the MAGE gene at the mRNA level. These results suggest that the MAGE gene products are appropriate target molecules for spcific immunotherapy of human cancers.
However, there was no available quantitative method to measure cellular MAGE protein expressed on human cancers. Therefore, an enzyme-linked immunosorbent assay (ELISA) was established for measuring cellular MAGE-4 protein (MAGE-4a and/or -4b) expressed on human tumor cells using the monoclonal antibody (mAb) and the polyclonal Ab to recombinant MAGE-4b protein. This ELISA also showed no apparent cross-reactivity with the other MAGE gene products (MAGE-1, -2, -3, -6, and -12). The minimum detectable level of MAGE-4 protein was determined to be 10 pg/well (100pg/ml).The results suggest that this ELISA is a reliable and quantitative method to measure cellular MAGE-4 protein that is a potential target molecule for specific immunotherapy of human cancers. Less
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