Construction Of EGFR Specific Chimeric Antigen Receptor And Establishment Of Luciferase Double Labeled Target Cells

Objective:Specific anti-human epidermal growth factor receptor?EGFR?single-chain antibody?scFv?was prepared and its biological activity was identified.A lentiviral vector targeting human EGFR was constructed and their viral fluid was prepared by transfecting 293T cells.The titer of the virus was determined and the expression was identified by infection within 293FT cells.The construction of human lung cancer cell lines stably expressing firefly luciferase?Fluc?and red fluorescent protein?RFP?provides premiss for the establishment of in vivo imaging of human lung cancer xenograft model in nude mice and further research on single-chain antibody-based immunotherapy.Methods:Following extraction of total RNA from hybridoma cell lines secreting anti-human EGFR monoclonal antibody,amplification of light and heavy chain variable region?V_L,V_H?genes using 5'RACE technology and construction of single-chain antibody genes of V_L and V_H genes by a linker,the constructed single-chain antibody cDNA was then cloned into eukaryotic vector pcDNA3.1 for their expressing determination.ELISA to identify the specificity of single-chain antibodies against antigens;Fortebio was used to detect the affinity between antigens and antibodies,and flow cytometry was used to measure the functional activity of single-chain antibodies in combination with native EGFR in lung cancer cell lines.The scFv-based CAR structure targeting EGFR was designed and the whole sequence gene was cloned into a lentiviral vector and transfected into 293T cells.The virus fluid was collected and the titer of the virus was determined by qRT-PCR.A lentiviral vector of luciferase and red fluorescent protein,pHBLV-Fluc-RFP,was transfected into 293T cells for viral packaging.The complete virus infects human lung cancer cell lines A549,H1975 and human B cell lymphoma cell line K562 with puromycin resistance.Stable cell lines were obtained by clonal screening,and the expression of red fluorescent protein and luciferase were confirmed by fluorescence microscopy and quantitative PCR,respectively.Results:The unique light and heavy chain variable region sequences V_L and V_H were obtained,and an EGFR-scFv was successfully constructed.The specificity was verified by its combination with native EGFR protein with an affinity of 3.22×10^-9 mol/L.A lentiviral vector containing EGFR-scFv was constructed and the constructed lentiviral vector was effective transfected into 293T cells.The collected virus concentrate was diluted to infect 293T cells and the titer of the virus was successfully determined by qRT-PCR.Infection of 293T cells with lentivirus showed that the virus could be normally expressed.The pHBLV-Fluc-RFP lentiviral vector was successfully constructed,and A549,H1975 and K562 cells stably expressing luciferase and red fluorescent protein were obtained.Conclusion:An anti-human EGFR single-chain antibody was successfully constructed and a lentiviral expression vector for EGFR-scFv-CD28-CD137-CD3 was finally constructed.Lentiviral particles were packaged by transfecting 293T cells.The virus fluid was obtained and virus titer was measured.After infection of the target cells,the expression of the target gene?EGFR-ScFv?was identified by flow cytometry,by which laid a foundation for the immuno-guided treatment of lung cancer study.Meantime,the obtained red fluorescent protein and luciferase dual expression human lung cancer cell lines A549,H1975 and human B cell lymphoma cell line K562 provided a new fluorescence color matching cell model for in vivo imaging of a human lung cancer cell immunodeficient mouse model.