Detection of mage‐4 protein in lung cancers

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Abstract
Expression of genes of the MAGE family, which encode tumor‐rejection antigens recognized on HLA‐AI and ‐Cw1601 by cytotoxic T lymphocytes (CTL), was investigated in lung cancers at the mRNA [MAGE‐1, ‐2, ‐3/‐6, and ‐4 (4a and/or 4b)] and protein (MAGE‐4) levels. MAGE‐1, ‐2, ‐3/‐6 and ‐4 genes were expressed, respectively, at the mRNA level in 6, 7, 20 and 7 of 53 lung cancers (50 non‐small‐cell lung cancers and 3 small‐cell lung cancers) by the reverse transcription‐polymer‐ase chain reaction (RT‐PCR) method. Polyclonal antibody (Ab) and monoclonal antibody (MAb) against recombinant MAGE‐4b protein were developed to detect MAGE‐4 protein. Both the polyclonal Ab and the R5 MAb recognized a 45‐kDa protein in extracts of MAGE‐4 mRNA‐positive lung cancers, and showed no apparent cross‐reactivity with the other MAGE gene products except with MAGE‐4a by immunoblot analyses and transfec‐tion experiments. MAGE‐4 protein was detected on 13 of 44 (30%) lung cancers (18 to 55,989 pg/mg) by ELISA with the polyclonal Ab and R5 MAb. These 13 lung cancers consisted of 6 of 6 MAGE‐4 mRNA‐detectable and 7 of 38 MAGE‐4 MRNA‐undetectable lung cancers. Histologically, these comprised 7 of 10 squamous‐cell carcinomas, 4 of 30 adenocarcinomas and 2 of 3 small‐cell lung cancers. The proportions of MAGE gene‐positive samples, at both the mRNA and protein levels, correlated with the size of the primary tumors and with regional node involvement. These results should provide important information on specific immunotherapy of lung cancers using MAGE gene products.