GPC2-CARs lead to significant in vivo tumor regression in orthotopic tumor models via intravenous or intraventricular administration route and had equivalent activity to the B7-H3-CAR against D283 and enhanced activity than GD2-CAR in both models in vivo. T cell kinetic studies revealed that GPC2-CAR T cells home to the area of the primary tumor, expand, and upregulate genes critical for cytotoxicity and T cell homing. These results provide a preclinical rationale for including children with GPC2+ MB in our upcoming clinical GPC2-CAR T cell trial.