DEVELOPMENT OF A RETROVIRUS-BASED COMPLEMENTARY-DNA EXPRESSION SYSTEMFOR THE CLONING OF TUMOR-ANTIGENS

A new retroviral system has been developed for the generation of a cDN A library and the functional cloning of tumor antigens, These retrovir al vectors contain a cytomegalovirus promoter in the 5' long terminal repeat, an extended packaging signal for rapid production of high-tite r retroviral particles, and many convenient cloning sites for cDNA lib rary construction. The vesicular stomatitis virus G protein has been u sed to generate pseudotype retroviral particles to enable efficient vi ral infection. Using this system, viral titers in the range of 10(6) c olony-forming units/ml could be generated routinely, and a high transd uction efficiency in human primary cells, including fibroblasts, was a chieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA library without a functional selection. The uti lity of this system was demonstrated by constructing a retrovirus-base d cDNA Library and re-isolating the NY-ESO-I tumor antigen from a cDNA library using an antigen-specific CTL. This approach can facilitate t he identification of novel tumor antigens recognized by T cells withou t knowledge of MHC class I restriction elements and is generally appli cable for the isolation of any gene as long as a biological assay is a vailable.