Comparative in vitro evaluation of fourth-generation anti-GPC3 CAR T-cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and remains difficult to treat in advanced stages because of tumor heterogeneity and an immunosuppressive microenvironment. Chimeric antigen receptor (CAR) T-cell therapy is an emerging strategy for solid tumors, but optimal CAR architecture for HCC remains under investigation. This study compared second-generation (anti-GPC3 CAR2), third-generation (anti-GPC3 CAR3), and fourth-generation (anti-GPC3 CAR4) CAR T-cells targeting glypican-3 (GPC3). Immunohistochemistry of liver biopsy specimens from 195 HCC patients showed that 82.6% were GPC3-positive. Anti-GPC3 CAR4 T-cells showed the strongest cytolytic activity against GPC3-positive target cells in both 2D monolayer and 3D spheroid models. At an effector-to-target ratio of 10:1, anti-GPC3 CAR4 showed significantly higher cytolytic activity against HepG2 cells (52.55 ± 10.04%, p = 0.003) and HeLa-GPC3 cells (64.45 ± 7.81%, p = 0.0002) than against GPC3-negative controls. In 3D spheroids, anti-GPC3 CAR4 also showed greater activity against GPC3-positive HepG2 (4.54 ± 0.48, p < 0.05) and HeLa-GPC3 spheroids (4.823 ± 0.18, p < 0.001). Anti-GPC3 CAR4 T-cells retained antigen-dependent proliferation with a moderated acute cytokine profile. After acute antigen exposure, anti-GPC3 CAR4 T-cells showed lower PD-1 and higher TIM-3 than anti-GPC3 CAR2 T-cells. Collectively, anti-GPC3 CAR4 T-cells were the most active construct in vitro; in vivo durability, biodistribution, and safety require validation.