CAR-T Cell Therapy Of Multiple Myeloma Targeting Cancer Antigen BCMA And CD138

Background:Multiple myeloma(MM)is a malignant plasma cell disease that requires constant exploration of new treatments.Immunotherapy is one of the treatments and its mechanism is different from that of chemotherapy drugs.The chimeric antigen receptor(CAR)fuses proteins such as the antigen recognition domain and the T cell signaling domain.The successful application of CAR-T cells in leukemia and lymphoma has promoted the use of CAR-T in the treatment of MM.The design of CAR-T requires a suitable target antigen.The ideal antigen is expressed in myeloma cells,but not in normal cells.Such CAR-T cells can kill tumors well and have fewer side effects.Based on the above conditions,we chose human BCMA and human GD138 as target antigens.The purpose of selecting two target antigens is to lay the foundation for the subsequent design of a CAR-That can recognize two target antigens simultaneously.With the increase of CAR-T clinical studies,the tumors are down-regulated or even do not express target antigens during the course of treatment.However,the specific mechanism is not yet fully understood.There are currently mature mouse myeloma models,which are very close to the mechanism of human MM and are good model of tumor escape and treatment strategies,but there are no CAR-T cells for mouse myeloma yet,so we have designed a CAR-T that can target mouse CD138 that could be used in animal models to lay the foundation for the tumor escape mechanism.As the CAR-T study progresses,in order to optimize the therapeutic effect,CAR assumes more and more functions,making the fragment length longer,resulting in a significant decrease in transfection efficiency.Therefore,the purification and enrichment of CAR-T cells is required for better treatment effect.Methods:1、CAR-targeting BCMA:anti-BCMA-CAR was successfully constructed in this study and cloned into retrovirus MSCV.The virus was packaged by using 293T cells and transfected into mouse T cells.Flow cytometry was used to detect the effciency.The anti-BCMA-MSCV CAR-T cells were co-cultured with tumor cells to detect the secretion of cytokines by CAR-T cells,the proliferation of CAR-T cells,the activation of CAR-T cells,and the killing of tumor cells.It was found that anti-BCMA-MSCV CAR-T can be specifically activated by MM cells,secrete cytokines,proliferate,and specifically kill MM cells.In the next step,we cloned anti-BCMA-CAR into lentivirus PWPT,transfected human T cells,and co-cultured with tumor cells to detect CAR-T killing function.2.This study successfully constructed two CARs targeting CD138.They are anti-CD138(46F2)CAR and anti-CD138(062)CAR.The anti-CD138(46F2)CAR recognizes murine and human CD 138 antigens.Anti-CD138(062)CAR can recognize human CD138 antigen.Both of them were cloned into retrovirus MSCV to make virus.T cells were transfected and its killing function was detected.3.NOG mice were injected subcutaneously with tumor to establish a tumor-bearing mouse model.Anti-CD 138-CAR and anti-BCMA-CAR was injected to observe the changes of tumor CD138 and BCMA expression.CAR-T retention in mice was also detected.4.This study used two methods to purify CAR-T cells.A Strep tag was added to the CAR fragment,meaning that the anti-BCMA-3ST CAR-T cells express the strep protein.Because strep binds specifically to streptavidin,CAR-T cells can be purified by using streptavidin beads.The second method is using biotin-labeled F(ab)2 antibody,and biotin can specifically bind to streptavidin magmetic beads and thus the CAR-T cell could be enriched or purified.Results:1、Anti-BCMA-CAR and two anti-CD 13 8-CARs were successfully constructed.2、In vitro experiments anti-BCMA-CAR-T,anti-CD 13 8-CAR-T cells can be specifically activated by MM tumor cells,proliferate,secrete cytokines and kill tumor cells.3、Animal experiments showed that anti-BCMA-CAR-T and anti-CD 13 8-CAR-T significantly decreased the number of tumor cells and CAR-T cell existed in mice for a long time.4、The addition of Strep tag method and biotin-labeled F(ab)2 antibody method successfully enriched CAR-T cells with a purity of more than 85%.Conclusion:Anti-BCMA-CAR-T and anti-CD 13 8-CAR-T can effectively kill MM cells.The CAR-T cells were successfully enriched using strep tag method and biotin-labeled F(ab)2 antibody method.