Fig. 4

CAR-MS10 structural optimization and cytokine coexpression enhance CAR-NK cytotoxicity and functionality. a Four CAR constructs were designed to evaluate combinations of transmembrane (CD8, NKG2D), costimulatory (4-1BB, OX40, DAP10, and 2B4), and signaling (CD3ζ, DAP12) domains. b Cytotoxicity of linCAR-NK cells at 12 h and 24 h post coculture with Capan-2 cells determined via a luciferase-based assay (n = 3). c CD107a degranulation in CD56⁺ linCAR-NK cells after coculture with Capan-2 cells was analyzed by flow cytometry (n = 4). d IFN-γ secretion after 24 h of coculture (n = 3). e, f Metabolic activity [oxygen consumption rate (OCR)] after 2 h of stimulation on matMSLN-coated plates (20 µg/mL) (n = 3). g Intracellular signaling pathway activation by the lead construct (linCAR-MS10-OX40ζ), quantified by normalizing the levels of phosphorylated PLCγ and ERK to their respective total forms (n = 3). h–j Functional assessment of primary NK cells cotransfected with linCAR-MS10-OX40ζ and cytokine-encoding linRNAs (IL-2, IL-15, or IL-21). Luciferase-based cytotoxicity (i) and CD107a degranulation (j) were measured in NK cells cotransfected with single or combined cytokines (n = 3). The data are shown as the means ± s.d. One-way ANOVA was used in (b−d, f, g, i, j); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001