2B4 Co-Stimulation and Dasatinib Modulation Enhance Anti-CD19 CAR-NK Cell Cytotoxicity

Chimeric Antigen Receptor (CAR)-cell-based therapies have shown remarkable achievements in cancer treatment, especially for hematological malignancies. In CAR-T cells, the costimulatory[P1] domain is a key factor that significantly affects their therapeutic success. However, the effects of different costimulatory domains on the activation and performance of CAR-natural killer (CAR-NK) cells have not been sufficiently studied. We designed novel CAR constructs incorporating NK-specific costimulatory domains, such as 2B4 (CD244) and DAP12, to enhance CAR NK functionality. We evaluated their efficacy using in vitro cytotoxicity assays, cytokine profiling, and in vivo tumor models. We further investigated dasatinib, a tyrosine kinase inhibitor, as a pharmacological modulator of CAR NK cell activity. Short-term dasatinib exposure temporarily and reversibly suppressed CAR-NK activity yet enhanced function upon withdrawal. Accordingly, 2B4-DAP12 CAR-NK cells demonstrated superior tumor control and survival compared to conventional 4-1BB? CAR-NK cells in vivo following dasatinib pretreatment. Our findings highlight that NK-optimized co-stimulation (2B4-DAP12) with reversible dasatinib pharmacological control synergistically enhanced CAR-NK cytotoxic function, providing an innovative strategy for next-generation cellular therapies. Overall design: The cells were then co-culture against CD19+positive Nalm-6 cells for 4 hours. After co-culture, cells were magnetically separated and the RNA of CAR-NK cells were purified for differencial gene expression analisys.