Background: Chimeric antigen receptor engineered T cells (CAR-T) have demonstrated extraordinary efficacy in B cell malignancy therapy and have been approved by the US Food and Drug Administration for diffuse large B cell lymphoma and acute B lymphocytic leukemia treatment. However, treatment of T cell malignancies using CART cells remains limited due to the shared antigens between malignant T cells and normal T cells. CD5 is considered one of the important characteristic markers of malignant T cells and is expressed on almost all normal T cells but not on NK-92 cells. Recently, NK-92 cells have been utilized as CAR-modified immune cells. However, in preclinical models, CART cells seem to be superior to CAR-NK-92 cells. Therefore, we speculate that in addition to the short lifespan of NK-92 cells in mice, the costimulatory domain used in CAR constructs might not be suitable for CAR-NK-92 cell engineering. Methods: Two second-generation anti-CD5 CAR plasmids with different costimulatory domains were constructed, one using the T-cell-associated activating receptor-4-1BB (BB.z) and the other using a NK-cell-associated activating receptor-2B4 (2B4.z). Subsequently, BB.z-NK and 2B4.z-NK were generated. Specific cytotoxicity against CD5 + malignant cell lines, primary CD5 + malignant cells, and normal T cells was evaluated in vitro. Moreover, a CD5 + T cell acute lymphoblastic leukemia (T-ALL) mouse model was established and used to assess the efficacy of CD5-CAR NK immunotherapy in vivo. Results: Both BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5 + malignant cells in vitro and prolonged the survival of TALL xenograft mice. Encouragingly, 2B4.z-NK cells displayed greater anti-CD5 + malignancy capacity than that of BB.z-NK, accompanied by a greater direct lytic side effect versus BB.z-NK. Conclusions: Anti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cellassociated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.

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CD19 chimeric antigen receptor (CAR) T cells have shown robust efficacy in relapsed and refractory acute lymphoblastic leukemia (R/R ALL), but compromising result in chronic lymphoblastic leukemia (CLL) and non-Hodgkin's lymphoma (NHL). CD19− relapse and the lack of CAR-T cell persistence which result in treatment failure are considerable obstacles to overcome.CAR-T targeting CD20 is an option for salvaging CD19 CAR-T failure. Previous studies have established variant structures of bispecific CAR-T which could avoid antigen-loss and immune escape. Here, we constructed tandem and loop CAR structures targeting both CD19 and CD20 antigen. Bispecific CAR-T cells could eliminate either CD19 or CD20 negative lymphoma cells, suggesting they exhibited dual antigen targeting of CD19 and CD20. By comparing the efficiency of four bispecific CAR modified T cells, it was found that loop2019 CAR was the best structure among them to eradicate lymphoma cell lines and patients' primary lymphoma or CLL cells in a very low dose in vitro and prolong the survival time dramatically in lymphoma xenograft mice model. These data highlighted the potential of loop2019 CAR-T in clinical treatment.