The Antitumor Experiment Research Of Chimeric EGFRvⅢ ScFv-ICOS-CD3ξ Modified T Cells

Backgroud and ObjectiveThe development of antitutrmor immune therapy is a challenging work, the immunogenicity of many specific antigen which on the surface of the tumor is low. Plus the immune escape mechanisms on tumor formation, so it can be protected from attack by the immune system, mainly including:Decreased processing power of antigen, processing power of antigen expression of major histocompatibility complex, immunosuppressive molecules and cytokine decrease. In order to overcome the difficulties, researchers continue to try various methods to activate antitumor immune system. These include:the use of cancer vaccines, polyclonal antibody fusion and the adjustment factor. The most promising is the autologous tumor activated T lymphocytes, adoptive immunotherapy for in vitro amplification to a certain number. The application of this technology has achieved good results. However, because this treatment requires a certain number of tumor specific T cells, making this technology now applies only to melanoma. Some T cells are few in number, only through genetic modification methods can help them achieve the expected number. Two kinds of methods have now reported, one is polyclonal T cells can be targeted tumor antigen, the antigen by genetic modification with T cell receptor specificity and affinity. Another is the modification of T cells, the expression of a chimeric antigen receptor to recognize the original antigen.This paper on the basis of previous studies,chimeric EGFRvIII scFv-ICOS-CD3ζ was constructed using molecular cloning techniques,293T cells were co-transfected with three-plasmid lentiviral system to prepare the lentivirus LV-EGFRvⅢ-ICOS/CAR Human peripheral blood T lymphocytes were infected by lentivirus carrying LV-EGFRvⅢ-ICOS/CAR. To investigate and compared the selective killing effect of chimeric EGFRvⅢ scFv-ICOS-CD3ζ modified T cells to the epidermal growth factor type Ⅲ mutant (EGFRvⅢ)-positive glioblastoma cells, and carry out the further explore for cancer target immunotherapy.Methods1. Using molecular cloning techniques constructed chimeric EGFRvⅢscFv-ICOS-CD3ζ2.293T cells were co-transfected with three-plasmid lentiviral system to prepare the lentivirus LV-EGFRvⅢ-ICOS/CAR. Human peripheral blood T lymphocytes were infected by lentivirus carrying LV-EGFRvⅢ-ICOS/CAR3.the expression of CAR in T cells were tested using flow cytometry and Western blot4.Cytotoxicity of ICOS/CAR+T-modified T lymphocyte was detected by51Cr release assay5. cytokines IFN-γsecretion was measured by ELISA assayResults1.pCDH-ICOS/CAR were obtained,confirmed by PCR, restriction enzyme digestion and nucleotide sequencing analysis2.three-plasmid lentiviral system to prepare the lentivirus,and the titer of lentivirus LV-EGFRvⅢ-ICOS/CAR are about1.8x106TU/ml3.After human peripheral blood T lymphocytes were infected by lentivirus carrying LV-EGFRvⅢ-ICOS/CAR,using flow cytometry tested the transfection efficiency of lentivirus is ICOS/CAR73.65%4. Western-Blot analysis showed a specific band at the57kD5.ICOS/CAR+T modified T lymphocytes demonstrated antigen-dependent activation by EGFRvⅢ+U87glioma cells and specific killing effect. 6.ELISA tested the cytokines secretion of ICOS/CAR+T cells is respectively as high as (2103.6±97.58)pg/ml. Confirmed IFN-y secretion of lymphocyte is EGFRvIII tumor antigen specific after transfection.ConclusionEGFRvIII/CAR+T lymphocytes showed specific cytotoxicity in vitro.this study provides experiment basis for glioma cell targeting therapy,and established the foundation for cancer immunotherapy early applied to clinical.