The Study On Anti-tumor Effect And Mechanism Of A Novel Type CD105-targeted And CTLA-4-Secretiing CAR T Cells

Background At present,chimeric antigen receptor-engineered(CAR T)therapy is one of the hot spots for malignant,especially in the treatment of hematoma.While its efficacy against non-haematological malignancies has been limited because of tumor microenvironment in solid tumors.Abnormal increase of tumor neovascularization in tumor microenvironment closely correlates tumorigenesis.The expression of CD105(Endoglin)in tumor tissues,especially tumor neovascularization,is much higher than that in normal tissues and can be used as a specific molecular marker of tumor neovascularization.The immunosuppressive molecule CTLA-4(Cytoxic T-lymphocyte Associated protein-4)will cause T cell depletion by releasing inhibitory signals.It is of great significance to develop CAR T cells to target CD 105 and secrete CTLA-4 antibody for anti-tumor effects.However,the extracellular antigen binding regions and secreting antibodies used in conventional CAR T cells are single-chain antibodies.This type of antibody is consisted of heavy chain variable region,hinge region and light chain variable region.The activity and application of traditional CAR T cells 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.Nanobody,which can overcome the shortcomings of traditional single-chain antibodies,has the advantage of small molecular weight,simple structure,good thermal stability,water solubility,low immunogenicity,strong tissue permeability.Therefore,we engineered a novel CAR T cell to target CD 105 and produce CTLA-4Nb(here after referred to as CD105-CTLA-4/Nb CAR T)and explore its anti-tumor effect and action mechanism will create breakthroughs in the field of CAR-T cell therapy research.According to the existing literature research results,there is no similar research on the design of this new CAR T cell at home and abroad.Objective To develop a novel CAR T cell which can target to CD 105 and secret CTLA-4 Nb by using CD 105 nanobody and CTLA-4 nanobody.Then to verify its anti-tumor effect through in vitro and in vivo experiments and discuss its mechanism.This novel CAR T will provide new technical means and new ideas in the area of tumor therapy.Methods 1.The gene sequence of CD105-CTLA-4/Nb CAR was designed and synthesized.The double enzyme digestion scheme was used to cut GV400 lentivirus vector and complete genetic recombination.CD105-CTLA-4/Nb CAR lentivirus vector was packaged and concentrated.Lentivirus titer was measured by using fluorescence counting.2.The human peripheral blood T lymphocytes were isolated and cultured,and the construction of CD105-CTLA-4/Nb CAR T cells was generated by lentivirus transfection.The ELISA method was used to detect the secretion of CTLA-4 Nb.3.The flow cytometry analysis was used to detect the proliferation of T lymphocytes in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD105-CTLA-4/Nb CART group after co-incubation with Bel7404 cells.4.The flow cytometry analysis was used to detect the expression of effector cell surface activation molecules CD25 and CD69 of T cells in in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD105-CTLA-4/Nb CAR T group after co-incubation with Bel7404 cells.5.The flow cytometry analysis was used to detect the killing efficacy of target cells after co-incubation with T lymphocytes in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD105-CTLA-4/Nb CAR T group with 293T-CD105 cells。6.ELISA was used to detect cytokines(IFN-γ,TNF-α,IL-2,IL-10)secreted in the supernatant of T lymphocytes in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD105-CTLA-4/Nb CAR T group after co-incubation with Bel7404 cells.7.ELISPOT was used to detect the number of positive T cells secreting IFN-y in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD105-CTLA-4/Nb CAR T group after co-incubation with Bel7404 cells.8.The NOD/SCID mice subcutaneous xenograft model was constructed by using Bel7404 cells.The T cells in Utd group,Mock group,CD105/Nb CAR T group,CTLA-4/Nb CAR T group,irr-CTLA-4/Nb CAR T group,CD105/Nb CAR T+CTLA-4 Nb group and CD 105-CTLA-4/Nb CAR T group was injected through tail vein.Tumor volume and survival efficacy of mice were measured.Results 1.The cleavage of GV400 lentiviral vectors were completed by using double enzyme digestion scheme.The PCR method indicated that CD105-CTLA-4/Nb CAR genes can be successfully inserted into GV400 vector,the harvested lentivirus titer reached 2-5E+8 TU/mL.2.CD105-CTLA-4/Nb CAR T cells were generated successfully by using lentivirus technology.GFP expression of CD 105-CTLA-4/Nb CAR T cells was around 40%,ELISA results showed that CTLA-4 Nbs can secrete by CD105-CTLA-4/Nb CAR T cells.These results indicating that CD105-CTLA-4/Nb CAR T cells were generated successfully.3.The results of cell proliferation assay showed that CD105-CTLA-4/Nb CAR T cells proliferated more than the other control groups after stimulated by Bel7404 cells.4.The expression of CD25 and CD69(surface activating molecules)in CD105-CTLA-4/Nb CAR T cells were higher than that of other groups after stimulated by Bel7404 cells.5.The results of cytotoxic killing experiments showed that the killing efficiency of CD105-CTLA-4/Nb CAR T cells against CD105+cells was higher than that of other control groups.6.The results of ELISA showed that the secretion of IFN-γ,TNF-α,IL-2 was higher in CD105-CTLA-4/Nb CAR T cells were higher than that of other control groups after stimulated by CD105+cells.7.In vivo experiments showed that CD105-CTLA-4/Nb CAR T cells can inhibit tumor growth and prolong the survival time of tumor-bearing mice.Conclusion CD105-CTLA-4/Nb CAR T cells were generated successfully based on CTLA-4 Nb and CD105 Nb.The CD105-CTLA-4/Nb CAR T cells can target CD 105+target cells specifically in vitro and have anti-tumor effect in vivo.It can provide new technical means and new research direction for anti-tumor research of CAR T cells which can secret antibody.