1986 年 106 巻 12 号 p. 1084-1091
The immunological and chemical properties of melanoma antigens have been analysed by using C57BL/6 derived B16 melanoma cells. Cytotoxic T lymphocytes or helper T cells involved in the positive immune responses were easily induced by the antigen on the cell surface, whereas soluble melanoma antigens secreted into the culture medium selectively stimulate and induce antigen-specific suppressor T cells. Furthermore, the soluble antigen works as an inhibitor for the blocking of the cytotoxic T cell activity. Therefore, the soluble melanoma antigen stimulates the negative immune response in order for tumor cells to escape from immune system. By using two syngeneic monoclonal antibodies against the soluble melanoma antigens (M2590 and M562), we found that the melanoma antigen was composed of GM3 (NeuAc) in association of proteins. M2590 recognized GM3 carbohydrate moiety with cross-species specificities, while M562 reacts with the protein with species specific melanoma antigenic determinant. M2590 can react with the extracted GM3 from normal cells, not with normal cells expressing GM3 on the cell surface. Thus, this suggests that the tertially structure of GM3 on melanoma cells is different from that of normal cells. Finally, we succeeded in cloning of the genomic deoxyribonucleic acid (DNA) which contains genes encoding melanoma antigen with species specific determinant (M562). The B16-melanoma DNA cosmid libraries were constructed with the shuttle vector pCV103. They were infected into ED8767 E. coli, and then transfected into the human melanoma cell line P-36 by protoplast fusion with polyethylene glycol. The transfectants were selected in the presence of mycophenolic acid, stained with FITC-labeled M562, and selected by FACS. Total DNA was isolated from the transfectants, and the B16 genomic DNA clones were rescued by the in vitro packaging with lysogenic bacterial extracts. pD2-7 (34.8kb) reproducibly expressed M562 determinants in the p-36 human melanoma cells.