CAR-T cells with GPC3-inducible gene circuits for targeting hepatocellular carcinoma

Despite the remarkable success of chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies, its application to solid tumors such as hepatocellular carcinoma (HCC) remains limited by insufficient tumor-specific activation and systemic toxicity from constitutive immunostimulation. To overcome these challenges, we engineered anti-glypican-3 (GPC3) CAR T cells incorporating inducible gene circuits that enable spatially and temporally controlled activation within the tumor microenvironment. Utilizing a lentiviral delivery system, we generated GPC3-specific CAR T cells exhibiting potent antigen-dependent cytotoxicity against HepG2 cells in vitro and significant tumor regression in immunodeficient xenograft models. To achieve precise antigen-gated regulation, we implemented a synthetic Notch (synNotch) receptor system and systematically characterized its induction dynamics and kinetics using a Jurkat T-cell reporter platform. Through single-chain variable fragment (scFv) screening, we identified an optimized anti-GPC3 scFv that conferred high sensitivity and specificity for HCC-associated GPC3. Subsequent engineering refinements, including deletion of the negative regulatory region (NRR) and incorporation of a SyNthetic Intramembrane Proteolysis Receptor (SNIPR) architecture, substantially enhanced receptor responsiveness, yielding robust, GPC3-dependent transgene expression with minimal basal leakage. Functional validation in both two-dimensional co-cultures and three-dimensional tumor spheroids confirmed that the optimized anti-GPC3 SNIPR system drives potent, target antigen-restricted transgene activation exclusively upon engagement with GPC3-positive tumor cells. Collectively, these findings establish a modular and tunable platform for inducible CAR T-cell therapy in HCC, demonstrating that synthetic gene circuits can enforce stringent spatial control over T-cell activation in the anti-GPC3 context. This approach holds significant promise for improving the therapeutic index of CAR T-cell therapy in HCC by simultaneously enhancing antitumor efficacy and mitigating off-tumor toxicity.