Antitumor Activity Of NK92 Cells Modified By A Soluble MICA-responsive Artificial Chimeric Receptor

BackgroundImmune escape is one of the most important characteristics of cancer.What methods can be used to restart the immune response blocked by tumor immune escape and mobilize the active killing of immune cells to tumor cells.Those have become the key problem to cure tumor.Based on this concept,tumor immunotherapy represented by chimeric antigen receptor modified T/NK cells(CAR-T/NK)has made a new breakthrough in tumor therapy.However,more prognostic recurrence cases have recently been found in patients treated with CAR-T,which is related to immune escape caused by loss of surface antigen of tumor cells.Therefore,it has become an urgent problem in tumor immunotherapy,especially in the design of new generation CAR.ObjectiveBy analyzing the protein structure of the complex of NKG2 D and ligand,we design and construct a chimeric antigen receptor that has both membrane-bound MICA and soluble MICA(sMICA)targeting recognition and response capabilities.Next,we explore the ability of the receptor to respond to sMICA and provide theoretical basis for enhancing the anti-tumor activity of NK92 cells.MethodsUsing NKG2 D,the main activated receptor of NK cells,as the construction element of CAR,the 3D structure prediction tools of NKG2D-CAR chimeric protein were used for structure modeling,and the design of trimer structure formed by binding with soluble MICA was obtained.The designed NKG2D-CAR was synthesized and inserted into the modified pCDH-CMV-MCS-copGFP-Puro lentivirus vector.In this study,NK92 cellswere transfected with lentivirus and enriched with puromycin resistance.NKG2D-CAR-NK92 cell line,which can stably and highly express NKG2D-CAR,was obtained.The expression level of NKG2D-CAR was identified by qPCR,flow cytometry and WB.The release of IFN-? from NKG2D-CAR was measured by ELISA after incubation with different concentrations of MICA protein.Flow cytometry was used to detect the phenotype of NKG2D-CAR-NK92 cells and the expression of MICA/B in various cancer cells.The cytotoxic effects of NK92,pCDH-NK92 and NKG2D-CAR-NK92 on SGC-7901 and SNU-1 cells,A2058 cells and Raji cells were compared.Results1.NKG2D-CAR with MICA response potential was successfully designed by three-level structure modeling of the transmembrane protein.2.The NKG2D-CAR expression vector was successfully constructed.The NK92 cell lines with stable and high expression NKG2D-CAR genes were obtained by lentivirus transfection.3.The release of IFN-? factors in NKG2D-CAR-NK92 cells was higher than that in NK92 cells after incubation with different concentrations of MICA.The results showed a trend of first increasing and then decreasing,which was consistent with the results of biological modeling.4.Cytotoxicity experiments verified that NKG2D-CAR-NK92 cells could effectively identify and significantly enhance the killing of MICA/B positive cancer cells.ConclusionThis project successfully constructed a chimeric receptor modified NK92 cell line(i.e.,NKG2D-CAR-NK92)that responds to soluble MICA.Those cells could significantly enhance the killing of MICA/B positive cancer cells and curb the immunosuppression caused by soluble MICA.