In Vitro Study Of Chimeric Antigen Receptor Modified T Cells Targeting NKG2D Ligands Against Non-small Cell Lung Cancer

Objective: Lung cancer is one of the most common malignant tumors in the chest and the most deadly cancer in the world.The early symptoms of lung cancer patients are not obvious,about three-quarters of patients are late in their discovery,and most of them are non-small cell lung cancer(NSCLC).The traditional surgical,radiotherapy,and chemotherapy treatments are not clinically satisfactory and the patient's five-year survival rate did not improve significantly.Although the emerging biological therapies such as tyrosine kinase inhibitors,immunodetection inhibitors,etc.,have been widely used in clinical practice,there are still some deficiencies.The purpose of this experiment is to prepare a minicircle DNA(mcDNA)vector containing NKG2 D chimeric antigen receptor(CAR),transduce into autologou T cells by using electroporation,to produce third-generation CAR-T effectors,and evaluate the transduction efficiency of mcDNA and the biological function of NKG2D-CAR-T effectors.Methods: The NKG2D-CAR-T cells were constructed by transfecting mcDNA plasmids containing NKG2 D target fragments into T lymphocytes by electroporation.To observe the effects of transfection of mcDNA on T cells and transfection efficiency,the effect of electrotransfection on the survival rate of T cells,the effect of microcircle DNA and electrotransfection on the phenotype of T cells,and the detection of NKG2D-CAR by flow cytometry The expression of NKG2D-CAR protein in T cells was detected by Western blot.NKG2D-CAR-T cells were co-cultured with HCC827 cell line with low expression of NKG2 D ligands and PC-9 cell line with high expression of natural NKG2 D ligands.CCK8 assay was used to detect the in vitro selective killing activity of CAR-T cells,and ELISA was used to detect cytokine IL-2 and IFN-? secretion.Results:1.The third-generation NKG2D-CAR-T cells were successfully constructed by electroporation.The expression of NKG2D-CAR protein in T cells was detected by Western blot and flow cytometry.2.Methods of electroporation and microcircle DNA vectors have no effect on the surface molecular phenotype of T cells.3.The inhibition of PC-9 in non-small cell lung cancer cells with high expression of NKG2 D ligands by NKG2D-CAR-T cells was higher than that of non-small cell lung cancer cells HCC827 with low expression of NKG2 D ligands.4.Compared with HCC827,a non-small cell lung cancer cell with low expression of NKG2 D ligands,NKG2 D ligands highly expressed PC-12 in non-small cell lung cancer cells can promote IL-2 and IFN? secretion from NKG2D-CAR-T cells.Conclusions:1.T lymphocytes can be successfully transfected by electroporation to generate NKG2D-CAR-T cells and express NKG2D-CAR protein.2.NKG2D-CAR-T cells inhibit the growth of non-small cell lung cancer cells expressing NKG2 D ligands in vitro,and secrete IL-2 and IFN?.