Construction And Functional Evaluation Of CD19/CD22 Dual-Target Tandem-CAR T Cells

Objective: Chimeric antigen receptor engineered-T(CAR-T)cell therapy has shown great therapeutic effects on the treatment of hematological malignancies,such as acute B lymphocytic leukemia and B cell lymphoma,on both a specific tumor antigen CD19 is highly expressed.Although many patients have achieved remission and recovery by the treatment of CD19-engineered CAR T cells,relapse and refractory still occurred in some cases.CD19-negative relapse was found the major mechanism of treatment failure.So,it is necessary for us to find a novel therapeutic strategy.We designed a bivalent chimeric antigen receptor(CD22×19-CAR)targeting both CD19 and CD22 on the surface of B lymphocytes as well as prepare CD22×19CAR-T cells.The cytotoxicity effect of CD22×19 CAR-T cells on B-cell malignant tumors and the therapeutic effect on a leukemia mouse model were detected.Methods: The CD22×19-CAR molecule was designed by stringing CD19-sc Fv and CD22-sc Fv together and loading them into a lentiviral vector.293 T cells were used to package lentivirus and infect healthy human T cells to prepare the CD22×19 CAR-T cells.The single target cells CD19 CAR-T and CD22 CAR-T were set as the control group.Cell lines K562(CD19-/CD22-),CD19-transfected K562(K562-CD19),CD22-transfected K562(K562-CD22),and Nalm6(CD19+/CD22+)were used as target cells for co-incubated with CD22×19 CAR-T cells,CD19 CAR-T cells,and CD22 CAR-T cells.Cytotoxicity as well as interleukin 2 and interferon gamma release were detected.The leukemia mouse model was established by Nalm6 cell injection,and the anti-tumor effects of CD22×19 CAR-T,CD19 CAR-T,CD22 CAR-T,and Loop CAR6(a previously reported CD19/CD22 dual-target CAR-T cell)cells were observed by performing survival curve analysis and bioluminescence imaging.Results: The second generation CD22×19-CAR was successfully constructed and stably expressed on T cells.CD22×19 CAR-T cells were prepared and compared with CD19 CAR-T and CD22 CAR-T cells,each of them showed significant cytotoxicity effects and higher cytokine secretion when co-cultured with specific target cells in vitro but no obvious effect on CD19/CD22-negative K562 cells,and sometimes CD22×19 CAR-T cells can even show better effects than CD19/CD22 single target CAR-T cells.In vivo experiments showed that CD22×19 CAR-T cells could effectively remove Nalm6 tumor cells,which were same as CD19 CAR-T,CD22 CAR-T,and Loop CAR6 cells.More importantly,compared to Loop CAR6 cells,CD22×19CAR-T cells could prolong the median survival in a leukemia mouse model.Conclusion: We successfully designed and prepared CD22×19 CAR-T cells that could double target CD19 and CD22 as well as demonstrated their feasibility and effectiveness using both in vitro and in vivo experiments.