Huo, M.; Li, D.; Li, N.; Quan, A.; Liang, T.; Henderson, D.; Sagert, J.; Pharm, M.; Hanley, L.; Maeng, K.; Eule, M.; Ho, M.

Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy in solid tumors, in part due to variability in autologous T cells derived from heavily pretreated patients with advanced disease. To address these constraints, we developed an off-the-shelf allogeneic CAR-T platform using CRISPR-Cas9-mediated genome editing in T cells from healthy donors to enable targeted CAR insertion at the TRAC locus with concurrent disruption of B2M. Using adeno-associated virus (AAV) delivery, we designed CAR-T cells targeting glypican-2 (GPC2) and glypican-3 (GPC3), emerging antigens expressed in pediatric and adult solid tumors. Genome-edited allogeneic CAR-T cells exhibited potent, antigen-specific cytotoxicity across multiple tumor models. GPC2-directed allogeneic CAR-T cells demonstrated enhanced or comparable activity relative to conventional lentiviral CAR-T cells in neuroblastoma models and mediated tumor regression with prolonged survival in preclinical models. Notably, repeated dosing augmented antitumor efficacy without evidence of toxicity, supporting multi-dose regimens for solid tumors. Similarly, GPC3-targeted allogeneic CAR-T cells based on a single-domain antibody showed robust activity against hepatocellular carcinoma cells in vitro and in vivo. These findings establish a scalable, genome-engineered allogeneic CAR-T strategy with strong therapeutic potential and support the clinical development of off-the-shelf cell therapies for pediatric and adult solid tumors.