Construction Of NK92 Cells Modified By Dual Targeting Chimeric Receptors PD1-DAP10&NKG2D And Its Anti-cancer Effect

Background In recent years,tumor immunotherapy has ushered in a new era of cancer therapy.Immunotherapy,represented by chimeric antigen receptor T cell therapy(CAR-T),has demonstrated great promise in hematological malignancies therapy.However,CAR-T immunotherapy has limited effectiveness in the treatment of solid tumors and the risks such as cytokine release syndrome in clinical treatment.Nowadays,mounting research suggests that natural killer(NK)cells have many unique immune characteristics and advantages when compared with T cells,and various methods have been used to enhance their killing ability.Therefore,NK cells are promising in solid tumor treatment.Objective A novel dual-targeted chimeric receptor of PD-L1 and MICA/B was constructed to enhance the specific recognition and killing ability of NK92 cells to gastric cancer cells.Methods A High Activition Dual Targeting Chimeric Receptor(Ha-DTCR)was obtained by whole-genome synthesis and constructed on the lentiviral vector p CDH.The target gene was stably transfected into NK92 cell line by slow-transfection method,referred to as Ha-DTCR.After successful transfection,the target gene successfully transfected was identified by q PCR at the m RNA level,and flow cytometry was also performed.The successful expression of the target gene was identified in the protein expression level,and the various phenotypes of the modified NK92 were analyzed and verified.The effect of transfection on cell proliferation was detected by CCK-8 proliferation assay.In vitro,the killing effect of NK92 and Ha-DTCR on gastric cancer cell SGC-7901 was compared by luciferase assay.In vivo,the anti-tumor effect of Ha-DTCR on gastric cancer cell SGC-7901 was verified by NOG mouse xenograft model.Results1.Successfully synthesize a dual targeting transduction receptor encoding gene and construct a recombinant expression vector,stably transfect NK92 cells and express the above dual targeting receptor.2.The expression of the target gene and the validation of related phenotypes were confirmed by q PCR and flow cytometry,respectively.PD1 expression was 40% and NKG2 D expression was 95%.The proliferation level of Ha-DTCR cells and untransfected NK92 cells was not significantly different by CCK-8 proliferation assay.3.In vitro experiments,the killing effect was significantly higher in the Ha-DTCR group than in the NK92 group and the PCDH group(**P<0.01)compared with the NK92 group and the PCDH group.There was no significant difference between the NK92 group and the PCDH group.4.In the in vivo NOG mouse test,Ha-DTCR compared with the parental NK92 cells in the killing effect of gastric cancer cell SGC-7901,in the tumor volume,Ha-DTCR has extremely significant tumor inhibition compared with the PBS group.Effect(***P<0.001).In the comparison between tumor weight,Ha-DTCR was significantly different from the PBS group(**P<0.01).Conclusions Successfully constructed a NK92 cell(Ha-DTCR cell)expressing the highly active dual-targeting chimeric receptor PD1-DAP10&NKG2D,and demonstrated that Ha-DTCR cells showed up on gastric cancer cell SGC-7901 by in vitro and in vivo experiments.Significant killing effect and antitumor effect.