Fig. 3

mRNA-based screening identifies CLMS10 as a potent shedding-resistant CAR construct in NK cells. a Generation of linear mRNA-based CAR (linCAR)-NK cells via electroporation (EP) of human primary NK cells. b, c Establishment of a rapid and cost-effective screening platform for evaluating CAR-NK cytotoxicity against Capan-2 cells. b Visualization of CAR-NK-mediated cancer cell killing. Capan-2 cells were stained with CellTrace Violet Cell (blue) and live-cell Caspase-3/7 dye (red), and cocultured with CAR-NK cells (green). c Quantification of apoptotic cancer cells based on Caspase-3/7 activation (n = 3). d Shedding inhibition assay for determining CAR-NK cytotoxicity in the presence of increasing concentrations of solMSLN (residues 296–592); dashed lines indicate the baseline cytotoxicity of allogeneic NK cells. e CD107a activation in CAR-NK cells stimulated with matMSLN-coated plates (20 µg/mL) in the presence or absence of solMSLN (50 µg/mL) was analyzed by flow cytometry (n = 3). f Cell–cell avidity between linCAR-NK and Capan-2 cells was quantified via acoustic force-based microfluidic microscopy (n = 5). g Luciferase-based cytotoxicity of lentivirus (LV)-generated CAR(_LVCAR)-NK cells against Capan-2-Luc and SK-OV-3-Luc cells (n = 3). h Predicted docking model of CLMS10 scFv in complex with matMSLN using Schrödinger software. Residues with side chains within 2.5 Å of the binding interface are highlighted, revealing key antigen-antibody interactions. The soluble MSLN regions are indicated in marine blue, and the membrane-bound matMSLN regions are indicated in dark blue. The data are presented as the means ± s.d. Statistical analyses: one-way ANOVA in (c) and (g) and two-way ANOVA in (f); two-tailed Student’s t tests in (d) and (e); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001