The Study Of Glycosyl-phosphatidylinositol-B7-1 Anchored Into Tumor Cell Membranes Enhancing CTL's Ablity To Kill Urinary Tumor Cells

This project is to modify tumor cell membranes with costimulatory adhesion molecules B7-1 through protein transfer in order to develop therapeutic cancer vaccines for use in human immunotherapy. We demonstrated that protein transfer of glycosyl-phosphatidylinositol(GPI) anchored costimulatory molecule into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. The B7-1 recombinant gene was produced via chimerization of the extracellular coding sequence of B 7-1 with alternative GPI-modification signal sequences from decay-accelerating factor(DAF),and was directed into expressive plasmid pCDNA 3.the recombinant was identified by restrictive endonuclases and PCR. GPI ? B7-1 was expressed by CHO/DHFR~- cells transfected with pCDNA3 vector encoding this chimeric protein and plasmid pSV2-dhfr, identified by direct immunofluorescence (flow-cytometric) and direct immunofluorescence analyses. GPI anchorage was verified by demonstrating phosphatidylinositol-specific phosphalipase C(PIPLC),sensitivity of the chimeric polypeptide in flow-cytometry. the fusion protein isolated from the cell surface of CHO/DHFR" could be exogenously incorporated into the isolated tumor cell membranes stably. We Immunized C57BL/6 mice with the modified tumor membranes, then one big group mice's spleens were harvested for T cell proliferation and CTL assays. The other were injected RM-1 cells for tumor challenge experiments. Immunization of C57BL/6 mice with RM-1 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition,...