T-cell Receptor-like Nanobody Chimeric Antigen Receptor T-cells Directed Towards Tumor Antigen

Background:CAR-T cell therapy for cancer research is one of the research hotspots in the field of malignant tumor treatment in recent years.Most of the CAR-T cells currently are constructed to target tumor extracellular antigens in order to produce an anti-tumor effect.However,the number of extracellular antigens in tumors is limited,and most tumor antigens are antigens in tumor cells.It is of great significance to develop TCR-like antibody CAR-T cells to target tumor cell antigens for anti-tumor effects.The extracellular antigen binding regions used in conventional CAR-T cells are single-chain antibodies.A single-chain antibody is composed of an antibody heavy chain variable region,a hinge region,and a light chain variable region.The activity and application of traditional CAR-T is limited due to the ease of mismatching of the heavy chain variable region and the light chain variable region or the complexity of the hinge region optimization process.Nanobodies can overcome the shortcomings of traditional single-chain antibodies,so exploring the anti-tumor effect of building TCR-like Nanobody CAR-T cells will create new breakthroughs in the field of CAR-T cell therapy research.Objective:To prepare nano-antibody-based WT1 Nb-CAR T cells(WT1 Nb-CAR)by using nano-antibodies to target the WT1 peptide antigen complex,and to verify its anti-tumor effect by in vitro and in vivo experiments.TCR-like antibody CAR-T cell anti-tumor research provides new technical means and new ideas.Method:1.The human peripheral blood T lymphocytes were isolated and cultured,and the construction of WTI Nb-CAR T cells was completed by lentivirus transfection.After the completion of the construction,the GFP expression of WT1 Nb-CAR T cells was observed under a fluorescence microscope;The ratio of GFP expression on the surface of WT1 Nb-CAR T cells was examined.2.Cell proliferation assay was used to detect the proliferation of T lymphocytes in Utd group,Mock group,GPC3 Nb-CAR group and WT1 Nb-CAR group after co-incubation with OVCAR3 cells.3.Cytotoxic killing method was ued for the detection of killing of target cells after co-incubation of the T lymphocytes in Utd group,Mock group,GPC3 Nb-CAR group and WT1 Nb-CAR group with OVCAR3 cells,Naml6 cells,K562 cells,MCF7 cells,empty T2 cells and T2 cells of each peptide plused.4.The content of the cytokines(IFN-γ,TNF-α,IL-2,IL-10)in the supernatant of the Utd group,Mock group,GPC3 Nb-CAR group and WT1 Nb-CAR group co-incubated with OVCAR3 cells were detected by ELISA.5.Flow cytometry analysis was used to detect the expression of effector cell surface activation molecules CD25,CD69,memory molecule CD62L and the lysosomal protein CD 107a of T cells in Utd group,Mock group,GPC3 Nb-CAR group and WT1 Nb-CAR group after co-incubation with OVCAR3 cells.6.The OVCAR3 cell mouse subcutaneous xenograft model was constructed and randomly divided into PBS,Utd,Mock,GPC3 Nb-CAR and WT1 Nb-CAR groups.After injecting each group of CAR-T cells and untransfected T cells into the tail vein,the effects of WT1 Nb-CAR T cells on tumor volume,tumor weight and survival time of mice were observed.Result:1.After transfecting Lentivirus into T cells,GFP fluorescence was observed under fluorescence microscope.Flow cytometry showed that GFP expression of WT1 Nb-CAR T cells was(34±1.54)%,indicating that WT1 Nb-CAR T cells The preparation was successful.2.Proliferation experiments showed that WT1 Nb-CAR T cells proliferated more than the other control groups after stimulation with HLA-A2± WT1+ target cells(OVCAR3 cells).3.The results of cytotoxic killing experiments showed that the killing efficiency of WT1 Nb-CAR cells against HLA-A2+ WT1+ target cells(ovCAR3 cells)was higher than that of other control groups.4.The secretion of cytokines(IFN-γ,TNF-α,IL-2)was significantly higher in WT1 Nb-CAR T cells and HLA-A2+ WTI+ target cells(OVCAR3 cells)than in other control groups.There was no significant difference between the groups of the secretion of IL-10.5.The expression levels of surface activating molecules(CD25,CD69),memory molecules(CD62L)and lysosomal protein CD 107a in WT1 Nb-CAR cells stimulated by HLA-A2+ WT1+ target cells(OVCAR3 cells)were higher than those in the other control groups.6.In vivo experiments showed that WT1 Nb-CAR treatment can inhibit tumor growth in mice bearing OVCAR3 tumors and prolong the survival time of mice.Conclusion:This study successfully constructed a nanobody-based WT1 Nb-CAR T cell.The constructed WT1 Nb-CAR T cells can specifically target HLA-A2+ WT1+ target cells in vitro,and have good anti-tumor effect in vivo.It provides new technical means and new research direction for anti-tumor research of TCR-like antibody CAR-T cells.