Abstract
Background: Unlike T cells, natural killer (NK) cells lack a dominant activating receptor analogous to the T cell receptor (TCR) that governs their activation. Whether chimeric antigen receptor (CAR) constructs engineered specifically for T cells can effectively drive NK cell activation remains unresolved. NK cells inherently possess non-specific recognition capacities and exert broad-spectrum cytotoxicity against diverse tumor targets. However, the complexity of receptor-ligand interactions between CAR NK cells and susceptible target cells has impeded efforts to delineate the specific functional contributions of individual CAR constructs. Methods: CAR NK cells were generated via electroporation. The murine B16 melanoma cell line was modified to express various target proteins using lentiviral transduction. In vitro functional assays, including conjugate formation, granule polarization, degranulation, cytotoxicity, and cytokine production, were employed to assess CAR NK cell efficacy. Recombinant protein-coated beads were used to investigate downstream activation signaling pathways. The in vivo antitumor activity of CAR NK cells was evaluated using NPG mouse xenograft models. Results: B16 cell line was first validated to be a suitable model for specifically assessing CAR construct function in CAR NK cells. Among nine distinct CAR molecules generated, the construct incorporating the NKG2DTM-2B4-FCER1G exhibited the most potent capacity to enhance NK cell-mediated functionalities. Consistent with these functional improvements, this CAR construct induced robust phosphorylation of key activation pathways, including AKT, VAV1, ERK, PLCγ1, and NF-κB. Conclusions: The CAR construct incorporating the NKG2DTM-2B4-FCER1G is demonstrated to be the most effective in enhancing NK cell functionality.