Design Of A Universal Chimeric Antigen Receptor For Enhanced Cytotoxicity Of NK92 Cells Based On The CID System

BackgroundIn recent years,chimeric antigen receptor（CAR）modified immune cell therapy has shone brilliantly in the treatment of malignant tumors,but it also shows a variety of side effects.Safety and effectiveness are two major problems in the clinical application of CAR therapy.In recent years,based on the structure of traditional CAR,a universal CAR is constructed by separating the tumor antigen recognition domain from CAR through modular design,which is a major breakthrough in immune cell therapy.In addition,CAR modified natural killer（NK）cells show the potential for allogeneic application.Combined with the engineering concept of synthetic biology,this paper proposes to use chemically induced dimerization（CID）elements to construct universal CAR and modify NK92 cells,in order to be able to use universal CAR-NK92 cells to treat all kinds of tumor patients.ObjectiveBased on the engineering concept of synthetic biology,a universal chimeric antigen receptor（CAR）which can be regulated by switch molecules was constructed by using chemically induced dimerization elements,and the allogeneic NK92 cells were engineered to construct a safe and effective universal CAR-NK92 cells.MethodsBased on the chemical induced dimerization element FKBP12 mutant FKBP12F36V（hereinafter referred to as m FKBP）and the mutant FRBT2098 L of FRB（hereinafter referred to as m FRB）,they were combined with CAR elements respectively,that is,m FRB was used as the extracellular domain of car,and m FKBP was fused with antibodies to form a complete structure of CAR molecule mediated m FKBP-m FRB dimerization by small molecule rapamycin（RAPA）.On the one hand,the molecular module m FRB-CAR was obtained by whole gene synthesis,and then constructed into lentiviral vector by restriction endonuclease ligation technique.The NK92 cell line with high expression of target module m FRB-CAR,namely m FRB-CAR-NK92,was obtained by stable transfection of lentivirus and enrichment of puromycin.The expression and phenotype of m FRB-CAR were detected by flow cytometry,and the proliferation of m FRB-CAR-NK92 was detected by CCK-8.On the other hand,using the corresponding antibodies of different antigen targets,the whole gene was synthesized and the element m FKBP was used to construct the protein expression vector p ET28 a.The antibody protein was purified and concentrated after protein expression in E.coli.The obtained protein antibody,RAPA and m FRB-CAR-NK92 were used to compare the killing effect of NK92,p CDH-NK92 and m FRB-CAR-NK92 on tumor cells by lactate dehydrogenase（LDH）,and the release of cytokines was detected by ELISA.Results1.Based on chemical induced dimerization elements m FKBP and m FRB,m FRB-CAR and m FKBP-sc Fv（m FKBP-La G16）.2.Construct of m FRB-CAR expression vector and preparation of m FRB-CAR-expressing NK92 cells.3.Construct m FKBP-La G16 protein expression vector and obtain target protein with higher purity.4.Through cytotoxicity test and cytokine detection test,under the premise of combining protein and small molecule drugs,COMPARED with NK92 cells and p CDH-NK92 cells,CID（m FRB）-CAR-NK92 cells can effectively recognize and act on target cells,and the expression of granulase B,perforin,IFN-γ,TNF-α has been significantly improved.ConclusionIn this study,a universal CID（m FRB）-CAR system,namely CID（m FRB）-CAR and m FKBP-nanobody fusion protein,was successfully designed based on chemically induced dimerization elements using the concept of synthetic biology engineering.It was verified by cytotoxicity experiments and cytokine detection that m FRB-CAR-NK92 cells combined with m FKBP-sc Fv exhibited significant and controllable in vitro killing activity against tumor cells expressing the corresponding targets under the induction of small molecule drug rapamycin.