Experimental Study On Antitumor Activity Of T Cells Targeting EGFRvⅢ Chimeric Antigen Receptor

Background : Chimeric antige receptor-modified T cell is a new strategy for cancer immunotherapy. CAR endows T cells new properties of tumor targeting, proliferation and persistence, which therefore can overcome tumor immunomicro environment and status of immune suppressive host. Epidermal growth factor receptor variant III(EGFRvⅢ) is a tumor specific antigen highly expressed in glioma and many types of cancers. Its expression is closely related with oncogenesis of many human cancers. Due to the tumor specificity, EGFRvⅢ received much attention as an ideal molecular marker for cancer target immunotherapy.Based on previously generated anti-EGFRvⅢ CAR of third generation, lentiviral EGFRvⅢ/3G-CAR were created and then infect CD3~+T cells, which redirect T cells orientation toward EGFRvⅢ. The purpose is to provide the experimental data for immunotherapy of glioma and human cancers.Objective CD3~+T cells modified by lentiviral EGFRvⅢ/3G-CAR, redirect its specificity for killing of EGFRvⅢ+ glioma cells, to explore the cytotoxicity of CAR engineered T cells.Methods(1)Lentiviral EGFRvⅢ/3G-CAR were generated by calcium phosphate co-transfection of 293 T cells with three plasmids(pVSVS,pDel8.9 and pCDH-EGFRvⅢ/3G-CAR), condensed by sucrose gradient ultracentrifugation, titrated by serial dilution.(2) CD3~+T cells were isolated from healthy human donors, activated by CD3/CD28 immune beads, and then expanded.(3)The expression of EGFRvⅢ/3G-CAR on CD3~+T cells were checked by flow cytometry and Western blot.(4) The secretion of IFN-γ of EGFRvⅢ/3G-CAR+T cells were examined by ELISA, specific cytotoxicity was done by 51 Cr release experiment.Results(1)The titer of Lenti EGFRvⅢ/3G-CAR could reach 1.2x108TU/ml, the transfection efficiency was about 73.65%.(2) In the supernatant of co-culture of EGFRvⅢ/3G-CAR+T cells and EGFRvⅢ+U87 cells, the level of IFN-γ could be(1902.7±96.23)pg/ml(P﹤0.01). EGFRvⅢ/3G-CAR+T cells demonstrated specific cytotoxicity toward EGFRvⅢ+U87 cells and the killing activity was elevated with the increase of Effector/Target ratio.Compared with the controls(EGFRvⅢ/3G-CAR-T, GFP+T), the specific cytotoxicity of EGFRvⅢ/3G-CAR+T cells had statistical significance.Conclusion Lentivirus encoding EGFRvⅢ/3G-CAR were generated successfully, which can efficiently express on the surface of T cells.The EGFRvⅢ/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvⅢ specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvⅢ-directed T cells lays a foundation to employ such approach in future cancer treatment.