Study On The Preparation And Function Of PD-L1 Single-chain Antibody Secretory CAR-T

BackgroundIn recent years,the incidence of cancer has gradually increased,and cases tend to be younger.Cancer has the characteristics of high mortality rate,poor prognosis,and lack of effective clinical treatment methods.It has become a disease that seriously threatens the health of modern people.The current clinical treatment methods for cancer mainly include surgery,radiotherapy,chemotherapy and immunotherapy,but the first three methods are still very limited effects on cancer treatment,especially for advanced patients.Therefore,the immunotherapy has become the focus of research for it is more effective for advanced caccer.In recent years,chimeric antigen receptor T cell(CAR-T)therapy has attracted much attention for its great potential in tumor treatment.Although CAR-T therapy has shown gratifying results in clinical treatments,the complex tumor microenvironment and tumor immune escape mechanism severely limit the effect of CAR-T therapy.Especially for the PD-1/PD-L1 signaling pathway,tumor cells can inhibit cytotoxic T lymphocytes by overexpressing PD-L1 protein,which seriously affects the performance of CAR-T therapy.In addition,anti-PD-1 and anti-PD-L1 have shown great effect in the treatment of various tumors such as non-small cell lung cancer,melanoma,lymphoma.Therefore,we speculate that let CAR-T express PD-L1 single-chain antibody can greatly enhance the ability of CAR-T to slay the tumor cells and enhance CAR-T’s tumor suppressor ability.PurposeThis research is to prepare CAR-T cells expressing both CD19-CAR structure and PD-L1 antibody.Realizing the combined therapy of CAR-T cell therapy and PD-1/PD-L1 immunosuppressive.It provides a theoretical basis for further exploring in clinical application of CAR-T therapy and in the treatment of solid tumors.Method1.By molecular cloning to constructed the lentiviral expression plasmid which can express CD19-CAR structure and PD-L1 single-chain antibody.Infecting 293 T cell by use lentivirus,detecting the protein expression by western blot,cell Immunofluorescence technique and other methods.2.The human acute B lymphoblastic leukemia cell line nalm6 was infected with lentivirus to construct a cell line which can express the luciferase reporter gene named Luc-nalm6.3.Constructing CAR-T cells through lentivirus and cell transfection.Detecting the killing of CAR-T cells to tumor cells by bioluminescence,and the ELISA method was used to detect the release level of cytokines.4.Establishing a mice human acute B lymphoblastic leukemia model.The mice were divided into PBS group,T lymphocyte group,CAR-T group,scfv secretory CAR-T group and injected different therapeutic cells respectively.Testing the treatment effect of CAR-T cells which can secreting PD-L1 scfv on B lymphoblastic leukemia in mice in-vivo experiments.Result1.We successfully constructed the expression plasmid p CDH-CD19 and p CDH-CD19-a PDL1 and infect 293 T cells by lentivirus.By western blot and cell Immunofluorescence,we certificate that the 293 T cells which were infected can express PD-L1 scfv and CD19-CAR structure,and the PD-L1 scfv can be secreted out of the cell and bind to PD-L1 protein.2.Multifunctional enzyme marker certify that the Luc-nalm6 cells can express luciferase reporter gene,and the luminous intensity is positively correlated with the number of cells.3.Through mixing tumor cells with killer target cells in vitro,it is shown that T cells expressing CAR structure can kill tumor cells more effectively.4.In vivo experiments in mice shown that mice in the group was infected scfv secretory CAR-T had a longer average survival time and more obvious inhibition of tumor growth than the mice in the other three groups.ConclusionBy molecular cloning techniques,we successfully constructed CAR-T cells that can secrete PD-L1 single-chain antibody.And through experiments in mice,it is shown that sc Fv secretory CAR-T cells have a more significant effect in tumor treatment than traditional CAR-T cells.