Abstract
BackgroundChimeric 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 CAR-T 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, CAR-T 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.MethodsTwo 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.ResultsBoth BB.z-NK and 2B4.z-NK exhibited specific cytotoxicity against CD5+ malignant cells in vitro and prolonged the survival of T-ALL 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.ConclusionsAnti-CD5 CAR-NK cells, particularly those constructed with the intracellular domain of NK-cell-associated activating receptor 2B4, may be a promising strategy for T cell malignancy treatment.
Highlights
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
After 6 h, cells were harvested and stained with PE-conjugated anti-human CD69 antibody, APC-Cy7-conjugated anti-human HLA-DR antibody, and PE-conjugated anti-human NKG2D antibody (Biolegend, USA) for 30 min at 4 °C, and washed and Construction of CD5 chimeric antigen receptor (CAR) and preparation of CAR-natural killer (NK) cells To improve the cytotoxicity of CAR-NK cells against CD5+ hematologic malignant cells, two second-generation CARs with different costimulatory domains were generated, one with T-cell-associated costimulatory domain 4-1BB, referred to as BB.z, and the other with NK-cell-associated costimulatory domain 2B4, referred to as 2B4.z (Fig. 1a)
NK-92 cells were infected with CAR structures lentivirus carrying CAR DNAs to generate CAR-NK cells
Summary
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. Chimeric antigen receptor (CAR)-modified immune cells have shown outstanding efficacy for the treatment of B cell malignancies [5, 6]. This indicated that using similar concepts to develop CAR-modified immune cells may help fight against T cell malignancies. The relapsed leukemia cells were CD19 negative, but anti-CD19 CAR was aberrantly expressed. When targeting T-malignant cells, it is necessary to try other types of effector cells to circumvent the shortcomings of CAR-T cells
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