Construction And Identification Of PNE Peptide-regulated Universal CAR-T Cells

BackgroundChimeric antigen receptor T cells(CAR-T)immunotherapy has achieved exciting results in the treatment of hematological tumors.However,side effects such as severe tumor lysis syndrome(TLS),fatal cytokine release syndrome(CRS),CAR-T-related encephalopathy syndrome(CRES)and off-target effects caused by CAR-T immunotherapy should not be ignored.Hence,how to improve the controllability and safety of CAR-T has become the most important part of CAR-T immunotherapy.The design of molecular switches may be an effective strategy to alleviate adverse effects of drug administration,antigen loss,recurrent mutations,and heterogeneous tumors.Peptide neo-epitope(PNE)is derived from the 14-amino acid peptide of yeast transcription factor GCN4(NYHLENEVARLKKL).Although PNE is an exogenous peptide,it has been confirmed to have low immunogenicity in vivo and weak ability to induce neutralizing antibodies.PNE can bind with high affinity to the single-chain variable fragment(scFv)composed of Anti-PNE monoclonal antibody(52SR4)at the front end of CAR.It can guide and bridge the binding of CAR-T to target cells by linking PNE with antibodies or affinity peptides that recognize Tumor-associated antigen(TAA)and can achieve the killing of tumor cells.ObjectTo construct a lentiviral vector overexpressing Anti-PNE CAR to infect human activated T cells,so as to construct universal CAR-T cells regulated by PNE and realize the generalization and modularization of CAR-T cells,laying the foundation for the personalized clinical application and precise effect of universal CAR-T cells.MethodsThe humanized Anti-PNE CAR sequence(Anti-PNE scFv-IgG4m-CD8-CD137CD3ζ)was amplified by PCR and cloned into plasmid GV401.After positive clone screening and sequencing validation,plasmid extraction was performed to obtain the recombinant plasmid Anti-PNE GV401.The three plasmid systems(Anti-PNE CAR GV401,pHelper1.0 and pHelper2.0)were co-transfected into HEK293T cells to package lentiviral vectors.The lentiviral vector was determined by gradient dilution method.The target gene expression of the plasmid was verified by Real-time quantitative polymerase chain reaction(RT-qPCR).Peripheral blood mononuclear cells were isolated by Ficoll-Paque density centrifugation.The proliferation ability and activity of T cells were detected by CCK-8 reagent.The dynamic changes in the proportion of activated T cells and the expression of CD25 and CD69 in the activated phenotype were detected by flow cytometry.The activated T cells were infected with the constructed lentiviral vectors,and the transfection efficiency was observed by fluorescence microscopy.The relative expression of CAR mRNA in Anti-PNE CART cells was detected by RT-qPCR.ResultsThe sequencing results of the recombinant plasmid were consistent with the target sequence,indicating that the recombinant plasmid vector Anti-PNE CAR GV401 was successfully constructed.HEK 293T cells transfected with the plasmid had high intensity green fluorescence under the microscope,and RT-qPCR showed that the relative expression of CAR mRNA was significantly higher than that of the control group,indicating that the expression of the target gene of the plasmid was normal,and the lentiviral vector overexpressing Anti-PNE CAR was successfully packaged.T cells had good proliferation ability and activity under the experimental conditions.T cells overexpressing Anti-PNE CAR had high intensity green fluorescence,and RT-qPCR assay showed that the relative expression of CAR mRNA was 1947.87 ± 89.14 times higher than that of the control(P<0.05).The relative expression level of CAR mRNA increased after PNE peptide intervention.ConclusionIn this paper,a kind of universal CAR-T cells overexpressing Anti-PNE CAR was successfully constructed.This modular design of Anti-PNE CAR-T cells can be used as a new tool for immunotherapy,laying the foundation for universal CAR-T cells and their safe application.