Kocheise, L.; Bacil, G.; Bhimalli, P.; Benmebarek, M.-R.; Li, D.; Huang, P.; Ma, C.; Muralidaran, V.; Hernandez-Felix, J.; Bugliarelli, J. R.; Chari, R.; Bauer, K.; Myojin, Y.; Firdaus, S.; Zhu, X. B.; Morris, C.; Korangy, F.; Kroemer, A.; Ho, M.; Greten, T. F.

Background & Aims: Liver transplantation improves outcomes in hepatocellular carcinoma (HCC), yet treatment options for patients with tumor recurrence remain limited to tyrosine kinase inhibitors. Glypican-3 (GPC3)-targeted CAR T cells offer a tumor-directed immune-based therapeutic strategy, but their efficacy may be limited by post-transplant immunosuppression. We developed a CAR T cell platform combining CRISPR/Cas9-mediated FKBP1A disruption to confer resistance to FKBP12-dependent immunosuppressive agents, including tacrolimus, everolimus, and sirolimus, with TRAC knockout to eliminate endogenous T cell receptor expression and reduce alloreactivity. Methods: Human T cells were edited using Cas9 ribonucleoprotein complexes targeting FKBP1A and TRAC, expanded, and transduced with an anti-GPC3 CAR construct. Cytokine production and cytotoxicity were assessed in vitro. Antitumor activity under tacrolimus treatment was evaluated in a Hep G2 xenograft model, and xenoreactivity was assessed in a graft-versus-host disease model. FKBP1A/TRAC double-knockout T cells were enriched using mTOR inhibitor selection combined with CD3-based MACS depletion. PBMCs from liver transplant recipients were used to evaluate feasibility for clinical translation during the early post-transplant period. Results: Tacrolimus suppressed wild-type CAR T cell function but not FKBP1A/TRAC double-knockout CAR T cells, which retained cytokine production, cytotoxicity, and in vivo antitumor activity. Cyclosporine A remained suppressive, enabling its potential use as a pharmacologic control strategy. TRAC disruption reduced xenoreactivity. CD3-based MACS depletion and mTOR inhibition achieved functional double-knockout efficiencies greater than 98%, without compromising cell viability. Functional FKBP1A/TRAC knockout CAR T cells were generated from patient PBMC samples 30 days post-transplant. Conclusions: Dual-edited GPC3 CAR T cells resist tacrolimus-based immunosuppression while limiting alloreactivity, supporting their use for recurrent HCC after liver transplantation. Sequential, high-viability selection in a modular cellular engineering framework enables adaptation to alternative tumor targets and next-generation CAR T cell designs.