Construction And Application Of Anti-EGFRv? Modified Chimeric Antigen Receptor Jurkat T Cells

Objects:we construct a third-generation EGFRv?-sepcific CAR gene in lentiviral expression vector and packaging lentivirus in 293T cells.The recombinant lentivirus infected Jurkat T cells,and we obtained Jurkat Clonal cell lines of a stable expression CAR gene by screening.To validate the in vitro targeting of third-generation CAR-modified Jurkat T cells.Methods:EGFRvIII-CAR gene was obtained by overlapping PCR and EGFRv?-CAR gene were cloned into lentiviral expression vector.The recombinant plasmid was transiently transfected into 293T cells,then we extracted the cell protein and the positive expression of CAR gene was confirmed by western-blot.293T cells were co-transfected by the packaging plasmid psPAX2,the envelope protein plasmid pMD2G and the transfer plasmid containing the objective gene.The medium supernatant which contained virus was collected and concentrated by ultrafiltration tube.The virus titer was measured by the fluorescence counting method.293T cells were infected by lentivirus carrying EGFRvIII-CAR.The expression of CAR in the 293T cells was tested using western-blot.Jurkat T cells were infected by lentivirus carrying EGFRvIII-CAR.Jurkat monoclonal cell line were obtained by using puromycin to detect the Jurkat T cells.Jurkat monoclonal cell line which were stably expressing CAR gene were confirmed with PCR identification.The assays of CCK-8 and ELISA were used to detect the cell proliferation,IL-2 secretion,respectively.Results:1.we successfully constructed pCDH-EGFRv?scFv-CAR-copGFP-T2A-puro lentivirus expression and the recombinant plasmid transfected into 293T cells.The positive expression of CAR was confirmed by western-blot.2.293T cells were co-transfected with three plasmids psPAX2,pMD2G and recombinant plasmids containing the objective gene.The virus was successfully packed and concentrated.The titer of virus was about 4 × 106TU/mL.293T cellswere infected with the virus.The expression of CAR protein in the 293T cells was tested using western-blot.3.Jurkat T cells were infected with the virus,and Jurkat T cell lines which were stably expressing CAR gene were obtained.The genomic DNA of the selected cell line was verified by PCR.The CAR gene was integrated into Jurkat T cells.4.After Jurkat-CAR monoclonal cell EGFRv? intermolecular contacts found Jurkat-CAR cells were stimulated to proliferate.CAR-modified Jurkat monoclonal cells and U87 cells co-culture,by ELISA detection IL-2 secretion of CAR-modified Jurkat T monoclonal cells,The amount of IL-2 secretion of the EGFRv?-specific CAR-engineered Jurkat T cells was significantly higher than the negative control.Conclusions:The monoclonal cell of the third generation of EGFRv?-CAR was successfully screened,and the monoclonal antibody of this cell had obvious targeting effect on EGFRv?,which provided the theoretical basis for the subsequent clinical cell immunotherapy.