Expression Optimization Of Targeting CD138 Chimeric Antigen Receptor And Its Cytotoxic Effect On RPMI8226 Cells In Multiple Myeloma

Background:Multiple Myeloma (MM) is the second most common haematological malignancy. Clonal plasma cells accumulate in the bone marrow or extramedullary sites and eventually give rise to renal failure, immunosuppression with repeated infections, anaemia and bone lesions. Despite encouraging therapeutic advances, MM remains an incurable malignancy. Chimeric antigen receptors modified T cells (CAR-T) have become a hot research field for tumor immunotherapy and targeting treatment, especially the exciting results of CAR-based immunotherapy in CD 19+ B-cell malignancies. Research on CARs technology in the field of MM treatment may bring new hope for patients with MM.Objective:This study chose CD 138 as the target to design corresponding CAR structure. We optimized the vectors carrying CAR gene by replacing with a promoter of higher protein expression activity to increase the CARs’expression level on the cell surface and the killing activity of CAR-T cells to CD138 positive MM cell line RPMI8226 was compared before and after optimization.Methods:(1) Construction of vectors containing CAR gene and different promoters; (2) To verify the expression activity of different promoters to downstream CAR gene; (3) Preparation of the two group CD138 targeting CAR-T cells before and after optimization and flow cytometry detection; (4) CD138 targeting CAR-T cells co-cultured with RPMI8226 cells and cytotoxicity detection.Results:(1) The gene vectors needed for this experiment were successfully constructed; (2) Optimized gene vector has higher expression activity; (3) Two groups of CD 138 targeting CAR-T cells were prepared and the positive rates were 13.32% and 10.16%, respectively; (4) In the case of different effector-to-target ratios, the killing activity of both CAR-T groups were higher than CIK group, and P<.05, with significant difference; at different effector-to-target ratios, the killing activity of PWPXL-CAR-T group were higher than that of PWPT-CAR-T group, T test P>0.05, the difference was not significant.Conclusions:(1) The promoter EF-1 has higher expression activity than EF-1-short, which can be used to increase the expression level of CARs on the surface of CAR-T cells; (2) In this study, two groups of CD 138 targeting CAR-T cells were successfully prepared; (3) Compared with CIK cells without gene modification, the CD 138 targeting CAR-T cells have enhanced killing activity on CD 138 positive RPMI8226 cells; (4) Increasing the expression level of CARs on the surface of CD138 targeting CAR-T cells may increase its killing activity on CD 138 positive RPMI8226 cells.