Construction And Functional Evaluation Of Universal CAR-T Cells Targeting To Multiple Myeloma

Multiple myeloma is the most common hematologic malignancy,with a higher incidence than acute lymphoblastic leukemia.Due to the high mutation and drug resistance of tumor cells,as well as the defects of existing treatment methods,patients still face the risk of failure and recurrence after receiving the existing treatment.However,chimeric antigen receptor T cell immunotherapy(CAR-T)has brought new hope to patients with multiple myeloma and shown great potential and advantages in clinical studies.However,the current CAR-T was mainly from each patient,the T cells usually are deficient both in quantity and quality,which can’t meet the needs of the clinical requirement.On the contrary,the Universal CAR-T comes from healthy donors,which are prepared with lower cost,shorter time.In addition,CAR-T cells from healthy dornors showed a better anti-tumor activity,which can provide faster and more efficient treatment for patients.Therefore,to successfully construct the Universal CAR-T for treating multiple myeloma,we intended to use human scFv fragments targeting BCMA and CD38molecules on the surface of myeloma cells from our lab,and the gene of scFv were synthesized to get recombinant plasmids.The recombinant plasmid with inserted scFv segments were digested with EcoR I and Xbal I.After sequencing,the results showed that the recombination was successful.Then the recombinant plasmids and package plasmid were transfected into 293T cells to get the lentivirus with the titer of 2×10~5TU/mL.T cells from healthy dornors were infected with concentrated virus,and the infection effciencies were up to 55%measured by FACS.After co-coulturing the CAR-T cells with target cells,we selected two optimal CAR sequences:anti-BCMA-2077-CAR and anti-CD38-2430-CAR by comparing the anti-tumor effects through the release of TNF-αand IFN-γas well as in vivo experiments.On the basis of this study,we used gene editing technology to knock out the TRAC and B2M in anti-2077-CAR-T cells to construct UCAR-T cells targeting BCMA.Then,we verify the activity of anti-tumor in vivo,which demonstrated that anti-2077-UCAR-T cells also had good anti-tumor effects comparing with CAR-T cells from Bluebird.It proved that after gene editing,CAR-T cells can still maintain strong killing ability of tumor cells.Our datas suggest an effective strategy for multiple myeloma in clinical practice by UCAR-T.