2B4 co-stimulation and dasatinib modulation enhance anti-CD19 CAR-NK-92 cell cytotoxicity

These results highlight the importance of selecting NK-specific co-stimulatory domains and leveraging reversible Src-family kinase inhibition to optimize CAR-NK performance. The use of NK-92 cells enabled controlled mechanistic dissection of CAR signaling and pharmacologic effects, providing insights with translational relevance for engineering next-generation CAR-NK therapies in primary NK cells.Chimeric Antigen Receptor (CAR)-based therapies have transformed cancer treatment, especially for hematological malignancies. While the choice of co-stimulatory domains is a well-established determinant of CAR-T success, the optimal signaling modules for CAR-natural killer (CAR-NK) cells remain less defined. In this proof-of-concept study, we used the NK-92 cell line as a controlled experimental platform to evaluate CAR constructs incorporating NK-specific co-stimulatory domains, including 2B4 (CD244) and DAP12. Functional assays and transcriptomic profiling demonstrated that 2B4- and 2B4-DAP12-based CARs promoted NK cytotoxic programming. We further explored transient pharmacologic modulation with dasatinib, showing that short-term exposure reversibly suppressed CAR-NK activity but enhanced function upon withdrawal. <i>In vivo</i>, 2B4-DAP12 CAR19-NK-92 cells pretreated with dasatinib achieved superior tumor control compared to conventional 4-1BBζ CAR19-NK-92 cells. These findings underscore the value of different settings of co-stimulatory domains and reversible kinase inhibition as strategies to optimize CAR design. Importantly, by employing NK-92 cells as a proof-of-concept system, this work provides mechanistic insights that will guide the development of next-generation CAR-NK therapies in primary NK cells.