Expression System for the Cloning of Tumor Antigens Development of a Retrovirus-based Complementary DNA

A new retroviral system has been developed for the generation of a cDNA library and the functional cloning of tumor antigens. These retroviral vectors contain a cytomegalovirus promoter in the 5' long terminal repeat, an extended packaging signal for rapid production of high-titer retroviral particles, and many convenient cloning sites for cDNA library construction. The vesicular stomatitis virus G protein has been used to generate pseudotype retroviral particles to enable efficient viral infection. Using this system, viral titers in the range of 10'' colony-forming units/ml could be generated routinely, and a high transduction efficiency in human primary cells, including fibroblasts, was achieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA li brary without a functional selection. The utility of this system was dem onstrated by constructing a retrovirus-based cDNA library and re-isolat ing the NY-ESO-1 tumor antigen from a cDNA library using an antigenspecific CTL. This approach can facilitate the identification of novel tumor antigens recognized by T cells without knowledge of MHC class I restriction elements and is generally applicable for the isolation of any gene as long as a biological assay is available.