Construction Of Chimeric Antigen Receptors On Cell Surface And Study Of Immune Effects

Objective:We aimed to construct a lentivirus recombinant plasmid targeting the Epidermal growth factor receptor variant ?(EGFRv?)chimeric antigen receptor(CAR)and targeting the Epithelial cell adhesion molecule(EPCAM)chimeric co-stimulatory receptor(CCR),which were used to modify the Jurkat T cells,and to investigate the activation in vitro and the tumor immune effects in vivo of the EPCAM-CCR and EGFRvIII-CAR co-modified T cells.Methods:The EPCAM-CCR and EGFRvIII-CAR gene fragments were synthesized and cloned into lentiviral expression vectors by genetic engineering techniques to construct lentiviral recombinant plasmid.Lentiviruses were packaged in a three-plasmid packaging system by non-liposomal transfection,infected and screened for stable Jurkat T monoclonal cell lines.PCR,Western Blot and flow cytometry were performed to verify the expression of CCR and CAR in the Jurkat T monoclonal cell line.Elisa method was used to detect the affinity and activation of EPCAM ScFv and EGFRvIII ScFv on the surface of Jurkat T monoclonal cell line in vitro.The effect of CCR and CAR co-modified T cells on the tumorigenicity of nude mice was studied in a nude mice bearing tumor model.Result:1.CCR-T2A-CAR-PCDH lentivirus expression recombinant plasmid was constructed and transfected into 293T cells successfully.Western blot showed that both CCR and CAR genes were normally expressed.2.The recombinant plasmid lentivirus was successfully packaged and the titer of the virus measured after concentrated was 5 × 108 TU/mL.3.Jurkat T cells were successfully infected with lentivirus,and CCR+CAR+Jurkat T monoclonal cell lines stably expressing CCR and CAR was obtained and identified.4.The high affinity between ScFv and the corresponding antigenic protein on the surface of CCR+CAR+Jurkat T monoclonal cell line was detected by the Elisa.The co-culture with HepG2 and Caco2 cells showed that the CCR+CAR+Jurkat T monoclonal cell line could effectively activated and secretes IL-2 cytokines.5.The T cells co-modified by EPCAM-CCR and EGFRv?-CAR showed a certain degree of specific tumor immunosuppression through transplantation tumor nude mice model.Conclusion:The Jurkat T monoclonal cell line co-modified by EPCAM scFv-CD8a hinge-TM-CD28-CD137 and EGFRv? scFv-CD8a hinge-TM-CD3 was successfully screened,which have a effect targeting tumor cells in vitro and can be effectively activated in vitro.Animal experiments further proved that chimeric antigen receptor and chimeric co-stimulation receptor modified T lymphocytes have a certain degree of immunosuppressive effect on tumors in nude mice,which is important for anti-tumor immunity research.