CD28 signaling domain boosts persistence and in vivo anti-tumor activity of stem cell-derived CD19-CAR-NK cells

Allogeneic natural killer (NK) cell-based therapies with an outstanding safety profile, are a compelling alternative to autologous T cell-based approaches for cancer immunotherapy, offering innate tumor-killing ability that can be further augmented via introduction of tumor antigen-specific chimeric antigen receptors (CARs). In this study, we genetically engineered primary human hematopoietic stem cells using an optimized lentiviral backbone carrying CD19 CAR cassettes with varied hinge, transmembrane, and signaling domains to evaluate their role in CAR-NK development and function. Our platform integrates early genetic modification with our unique expansion/differentiation system, enabling high CAR expression with low vector copy numbers. Notably, CARs incorporating CD28 transmembrane and signaling domains with CD3ζ, promoted enhanced tonic signaling, accelerated NK differentiation, enhanced antigen-specific kinome activation, and improved cytotoxicity and persistence both <i>in vitro</i> and <i>in vivo</i>. These findings offer a robust strategy for development of stem cell-based CAR-NK immunotherapies, combining potent innate, and antigen-specific antitumor responses.