Study On The Treatment Of Advanced Non-small Cell Lung Cancer By CAR-T Therapy Targeting MUC1 And Combining PD-1 Knockout

BackgroundGlobally,the incidence of lung cancer(LC)has been increasing.Lung cancer is already the most common and deadly cancer in the world.The main treatments for lung cancer include surgery,chemotherapy and radiation.In recent years,the chimeric antigen receptor(CAR)T lymphocyte therapy,as an immunotherapy method,has made encouraging progress in the treatment of blood system malignant tumors.Studies on the application of CAR-T cell therapy in solid tumors are also under way.MUC1 is a transmembrane glycoprotein with large molecular weight(>200k D),whose epitopes can be recognized by antibodies,and can be used as superantigen to bind to T cell receptor and activate T cells.When expressed in normal cells,the epitopes are covered by the peripheral carbohydrate chain of MUC1 and cannot be recognized.On the surface of cancer cells,incomplete glycosylation is caused by the increased activity of glycosyltransferase,which in turn leads to the shortening of the side chains and the exposure of the core peptides.As a result,abnormal glycosyllables and peptide antigen epitopes were observed,which has become the ideal target of immunotherapy.MUC1 is overexpressed in a variety of epithelial cell derived tumors,including lung cancer.At present,MUC1 has become one of the popular immunotherapy targets.It has been proved by animal experiments that CAR-T therapy targeting MUC1 is effective in the treatment of non-small cell lung cancer.Programmed cell death receptor-1(PD-1),also known as B7-H1 or CD274,is the corresponding ligand of PD-L1 and is widely expressed on the surface of activated T cells,B cells,macrophages,dendritic cells and tumor cells.Activating the PD-1 / PD-L1 signaling pathway leads to the formation of immunosuppressive tumor microenvironment,enabling tumor cells to escape from immune surveillance and attack.This is one of the mechanisms by which tumor cells escape attack by immune cells.Studies have shown that blocking PD-1 / PD-L1 can improve T-cell-mediated cytotoxicity and enhance its killing effect.PD-1 is also one of the most popular immune checkpoints in recent years.We have carried out research on CAR-T targeting MUC1 combined with PD-1 knockout technology in the treatment of non-small cell lung cancer(NSCLC),which is expected to enhance the effect of CAR-T targeting MUC1 cell therapy while resisting the immunosuppressive microenvironment,and provide a new treatment method for NSCLC.Methods1.CAR sequences are synthesized by universal biosynthesis.Use lentivirus to infect T cells.Cas RNP was imported into CAR-T cells by means of electroporation.Subsequently,PD-1 gene was knocked out.2.Flow cytometry was used to detect the positive rate of CAR-T cells and the knockout efficiency of PD-1 gene.3.The killing effect of CAR-T cells on tumor cells was detected by flow cytometry.4.The expression of MUC1 in tumor tissues of NSCLC was detected by immunohistochemistry.5.Ten enrolled patients with advanced NSCLC received CAR-T cell therapy in the form of dose climbing.6.Observe and record the adverse reactions carefully during the treatment.Evaluate the safety of CAR-T cells in the treatment of advanced NSCLC.7.Imaging and serum tumor-related tests were used to evaluate the efficacy of CAR-T cells in the treatment of advanced NSCLC.8.Polymerase chain reaction(PCR)was used to detect the copy number of CAR-T DNA in the subjects’ peripheral blood.Results1.We successfully prepared CAR-T cells targeting MUC1 combined with PD-1 knockout.2.The positive rate of CAR-T cells and the knockout efficiency of PD-1 gene could be stably detected by flow cytometry.3.CAR-T cells have a stronger killing effect on tumor cells than ordinary T cells.4.None of the subjects had serious adverse reactions after the infusion of CART cells,which demonstrated the safety of CAR-T cells in the treatment of NSCLC.5.In some patients(4/10),the tumor was well controlled after CAR-T cell infusion,which proved that CAR T was effective in the treatment of advanced NSCLC.6.In some patients,CAR T cells can survive in the body for a period of time and were amplified.ConclusionCAR-T therapy targeting MUC1 combined with PD-1 knockout is safe and effective in the treatment of advanced NSCLC.