Study The Effects Of CAR-T Cells On Anti-multiple Myeloma

Multiple myeloma is a malignant cell proliferative disease that mainly affects the elderly population.Due to it’s high aggressiveness,metastasis and drug resistance of myeloma cells,all patients will relapse or resist eventually.So myeloma is considered an incurable disease and new treatment is urgently needed.With the in-depth understanding of the pathogenesis with myeloma,immunotherapy of myeloma has made great progress.For the disease,the preferred treatment is still the transplantation of hematopoietic stem cell.But the method would cause graft versus host reaction easily,resulting in high morbidity and mortality.The main problems with myeloma immunotherapy,including the lack of specific expression of the target molecules on the surface of myeloma cells and the local bone marrow microenvironment is prone to immunosuppression and immune escape as well as immune tolerance.Chimeric antigen receptor T cell therapy(CART)has brought new hope for the treatment of myeloma.Due to the structural features of CAR molecule,it can target the targeted molecule and activate T cells simultaneously.and activate T cells continuously.The clinical trials of CD 19-CART cells showed surprising and lasting results.With the success of CART therapy in the treatment of blood diseases,more and more attention are turned to other diseases.Currently,there are three clinical trials about myeloma,which two targeted B-cell maturation antigen(BCMA)and one targeted CD138 molecules.In this study,we selected CD 13 8,BCMA,CD38,CD40 molecules that were highly expressed on the surface of myeloma cells and CD 19 molecules specifically expressed on the surface of B-cell malignancies as targets for research.The scFv segments of the targets were searched by the freepatent online website and then PCR amplification and agarose gel electrophoresis were done to identify it.After identification,the recombinant plasmids were formed with scFv segments and tool plasmids digested with EcoRI and Xbal.The results showed that the bands of scFv fragments,tool plasmids and recombinant plasmids were in accordance with the expected results.The DNA sequencing results of the recombinant plasmid also confirmed that the recombination was successful.Each recombinant plasmids transfected into 293T cells for 24 hours and infected for 48 hours respectively.After the concentration and purification of each targeted lentivirus,we obtained the average titer of virus was 10 ^9 IU/ml.The expression of each antigen on K562 cells was higher than 96%.CAR molecules transduced into T cells and efficiency of BCMA-CAR above 64%.The killing experiments used LDH method showed that the killing effect is obvious and the highest effect reached 65%.We also have succeeded in establishing the correlation between the killing results and the release of IFN-γ,IL-2 and TNF cytokines.This study can provide an important basis for the selection of clinical treatment program and determination of cellular dosimetry and prediction clinical treatment results.