2B4 Co-Stimulation and Dasatinib Modulation Synergistically Enhance Anti-CD19 CAR-NK-92 Cell Cytotoxicity

Matheus Henrique Dos Santos¹Júlia Teixeira Cottas De Azevedo²Mara Elisama da Silva Januário¹Dayane de Fátima Schmidt¹Mariane Cariati Tirapelli¹Alisson Felipe Bordini Biggi¹Sima Ebrahimabadi¹Renata Nacasaki Silvestre¹Dimas Tadeu Covas¹Rodrigo T Calado¹Virginia Picanço-Castro^1*

• ¹Center for Cell-based Therapy CTC, Regional Blood Center of Ribeirão Preto, University of Sao Paulo, Ribeirao Preto, Brazil
• ²Department of Hemotherapy and Cellular Therapy, Hospital Israelita Albert Einstein, Sao Paulo, Brazil

Word count: 331 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. In vivo, 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 cellsChimeric Antigen Receptor (CAR)-cell-based therapies have shown remarkable achievements in cancer treatment, especially for hematological malignancies. In CAR-T cells, the co-stimulatory domain is a key factor that significantly affects their therapeutic success. However, the effects of different co-stimulatory 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 co-stimulatory 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 CAR19-NK-92 cells demonstrated superior tumor control and survival compared to conventional 4-1BBζ CAR19-NK-92 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.