Chimeric Antigen Cytotoxic Receptors for In-Vivo Engineering of Tumor-targeting Natural Killer Cells

ABSTRACT Ex vivo chimeric antigen receptor (CAR) NK cells face challenges in manufacturing, and have limited tumor infiltration and in vivo persistency. A method leveraging mRNA-based delivery for in-vivo engineering of human NK cells could address these issues but has not been established. Here we developed an in-vivo NK cell engineering method by designing CARs that capitalize on inherent NK receptor biology for specific expression and function. These CARs utilize the Immunoreceptor Tyrosine-based Activation Motif (ITAM)-containing signaling adaptor in human NK cells for tumor destruction and cytokine response. We demonstrated that an NKp44-based CAR’s expression and function depend on the signaling adaptor DAP12. This approach enables precise mRNA-driven in-vivo NK cell programming against tumors, ensuring specificity and reducing off-target expression in non-immune healthy tissues.