Construction And Identification Of EGFRvⅢscFv- CD28-CD137-CD3 ξ Lentivirus Expression Vector

Backgrounds Adoptive immunotherapy (adoptive immunotherapy, AIT) is one of the most important means in tumor immunotherapy, showing certain anti-tumor effect. In recent years, the chimeric antigen receptor (chimeric antigen receptor, CAR) modification of T lymphocytes targeted immunotherapy of a new strategy gives T lymphocyte lasting vitality, more fertile, targeted killing activity, further improve the therapeutic efficacy of the AIT. Epidermal growth factor receptor mutation type III (EGFRvIII) expression rate is higher in glioma, and other malignant tumors. It is closely related to the occurrence and development of tumor, and is the ideal molecular target to targeted therapy cancer. This study design the third generation chimeric antigen receptors EGFRvIIIscFv-CD28-CD137-CD3 zeta, build lentivirus expression vector, which lays the condition for chimeric antigen receptor modified T lymphocyte immunotherapy of tumors.Objective Construct a chimeric antigen receptor EGFRvIIIscFv-CD28-CD137-CD3 zeta expression vector. Pack lentivirus and infect. 293T cells in vitro. Provide the experimental basis for improving the antitumor activity of adoptive immunotherapy.Methods The third generation chimeric antigen receptor targeting EGFRvIII was composed of EGFRvIIIscFv (726 bp), hinge region、transmembrane region (213 bp), CD28 (123 bp), CD137 intracellular signal region (126 bp) and CD3 zeta chain (336 bp). It was cloned into EcoRl and BamHl sites of vector pCDH-CMV-MCS-EF-copGFP. Synthesize gene of chimeric antigen receptor EGFRvIIIscFv-CD28-CD137-CD3 zeta successfully by Oligo chemosynthesis and PCR. The targen gene was colned into EcoRl and BamHl sites of vetor pCDH-CMV-MCS-EF-copGFP. After verifying right by double enzyme digestion and sequencing. The lentivirus was obtained by using pVSVG, pDel8.9 and pCDH-EGFRvⅢ/CAR to transfect 293T cells. Test the lentivirus titer. Lentivirus infect 293T cells. Western blot test the expression of purpose gene.Results EGFRvⅢscFv and gene synthesis of CD28-CD137-CD3 zeta fragments were spliced into EGFRvⅢscFv-CD28-CD137-CD3 zeta (EGFRvⅢ/CAR) by overlapping PCR. The purpose gene and expression vector were digested by EcoRl and BamHl and then were connected. The sequence of EGFRvⅢ/CAR was confirmed right by double enzyme digestion and sequencing. lentivirus was packaged successfully. The titer of lentivirus was 1.5x 108TU/ml. Infecting 293T cells, extracting protein and CD3 zeta antibody immunoblot confirmed EGFRvⅢ/CAR expression (molecular weight about 57 KDa).Conclusion The chimeric antigen receptor EGFRⅢ/CAR expression vector was successfully constructed. The lentivirus of expression EGFRⅢ/CAR was prepared successfully and can infect 293T cells efficiently. Result efficient EGFRⅢ/CAR expression, which lays the foundation for researching on lentivirus mediated the chimeric antigen receptor modified T lymphocyte targeting tumor.