Figure 4.

Split anti-hGPC3 CAR-T cells have potent and specific cytotoxicity against hGPC3⁺ HCC cell lines. 293T, HepG2, and Huh7 cells were cocultured with Mock-T, conventional anti-hGPC3 CAR-T, and SpyCatcher CAR-T cells with or without 10 nM anti-hGPC3 scFv-SpyTag in 96-well plates at different E:T ratios for 24 h. (a) Pictures were taken under an inverted optical microscope at 200x magnification field of view. Scale bar, 100 μm. (b) Culture supernatants were collected, and the killing effect of each well was calculated using a LDH-based cytotoxicity assay kit according to the manufacturer’s instruction. (c) HepG2 and Huh7 cells were cultured alone or cocultured with SpyCatcher CAR-T cells at an E:T ratio of 9:1 followed by the addition of a serial diluted anti-hGPC3 scFv-SpyTag. After 24 h coculture, supernatants were collected and cytotoxicities determined by LDH-based assays. Experiments were repeated twice with three replicates each. Repeated-measures ANOVA was used to compare different groups.

ANOVA, analysis of variance; CAR-T, chimeric antigen receptor T; E:T, effector:target; HCC, hepatocellular carcinoma; hGPC3, human glypican-3; LDH, lactose dehydrogenase; SD, standard deviation.