Construction Of Chimeric EGFRvⅢ ScFv-CD137-CD3ζ And Selective Killing Effect Of Its Modified T Cells

Backgroud Based on immune effector cells, adoptive immunotherapy is one of the importantmethods of tumor biological treatment, which showed a certain amount of anti-tumor effect from thelaboratory to the clinic. More and more people pay attention to it. However, for most of the tumor, AITdid not show the satisfactory curative effect. It only played the auxiliary therapeutic action in clinic. Tcell which is genetically modified with chimeric antigen receptor is a new targeted immunotherapystrategy in recent years. It enhanced T cell proliferation, survival and targeted killing activity. Thismethod made T cells can resist the immunosuppressive microenvironment in tumor tissue and break thehost state of immune tolerance. It gave the tumor adoptive immunity treatment new vitality.Epidermal growth factor receptor mutant Ⅲ has a high expression rate in glioblastoma and othermalignant tumors, and is closely related with the genesis and progression of tumor. Therefore it is a idealtarget for targeted therapy. We designed the second generation of chimeric antigen receptor（EGFRvⅢscFv-CD137-CD3ζ）which used EGFRvⅢ scFv to target tumor antigen, costimulatory molecule CD137to enhance T cell activity, CD3ζ to activate the T cell toxicity reaction. Lentiviral vector system was usedto genetically modify T cells with CAR, so that the T cell could be redirected against EGFRvⅢexpressed at the surface of glioma cell. CAR modified T lymphocytes showed the selective killing effectboth in vitro and in vivo. It explored a new method for glioma and other malignancies immunotherapy.Objective To investigate the selective killing effect of chimeric EGFRvⅢ scFv-CD137-CD3ζmodified T cells to the epidermal growth factor type Ⅲ mutant-positive glioblastoma cells.Methods Hinge-TM-CD137-CD3ζ was obtained using gene synthesis method and inserted into theEcoRI and BamHI sites of lentivirus transfer vector pCDH-CMV-MCS by molecular cloning techniques.The new vector was named as pCDH-CD137-CD3ζ. EGFRvⅢ scFv gene sequence was amplified frompCR2.1TOPO-EGFRvⅢ scFv using PCR reaction, and it was cloned into the EcoRI site ofpCDH-CD137-CD3ζ. The connection direction of the gene sequences was confirmed with colony PCRidentification. After DNA sequence analysis. The new plasmid was named as pCDH-CAR. With thecalcium phosphate precipitation,293T cells were co-transfected by the packaging plasmid pDel8.9, the envelope protein plasmid pVSVG and transfer plasmid containing the objective gene pCDH-CAR. Themedium which contained virus was collected and concentrated with sucrose ultracentrifugation andprecipitation method. The virus was named as LV-EGFRvⅢ/CAR, and its titer was determinated bylentivirus titer kit（lentivirus-associated HIV p24）. Human peripheral blood CD3⁺T lymphocytes wereseparated and activated by immunomagnetic bead, and then they were infected by lentivirus carryingEGFRvⅢ/CAR. The expression of CAR in T cells was tested using flow cytometry and Western blot.Cytotoxicity of CAR-modified T lymphocyte was detected by⁵¹Cr release assay, and cytokines IFN_-γsecretion was measured by ELISA assay. The tumor inhibition targeting EGFRvⅢ⁺U87cell was testedin nude mouse.Results DNA sequencing analysis showed that each part of chimeric antigen receptor includingEGFRvⅢscFv, hinge region, transmembrane region, CD137intracellular domain and CD3ζ was correct.The titer of lentivirus LV-EGFRvⅢ/CAR is about1.8x10⁶TU/ml, and the transfection efficiency oflentivirus is68.73%. CAR modified T lymphocytes demonstrated antigen-dependent activation byEGFRvⅢ⁺U87glioma cells and specific killing effect. Significant IFN_-γ secretion could be detected inthe co-culture supernatant. CAR⁺T cells showed specific tumor inhibition through transplantation tumornude mice model.Conclusion EGFRvⅢ/CAR⁺T lymphocytes showed specific cytotoxicity both in vitro and in vivo,and established the foundation for adoptive cancer target immunotherapy.