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Background Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer-related mortality worldwide and current immunotherapies often fail due to insufficient tumour specificity and systemic toxicity. Although chimeric antigen receptor (CAR) T-cell approaches have transformed the treatment of haematologic malignancies, their application to solid tumours such as HCC is restricted by constitutive activation and off-target effects. This study aimed to establish an anti-glypican-3 (GPC3) CAR-T platform incorporating synthetic inducible gene circuits that enable antigen-gated, tumour-restricted activation to enhance efficacy while minimising off-tumour toxicity.Methods Human primary T cells were lentivirally transduced with a second-generation anti-GPC3 CAR construct. Cytotoxicity was evaluated against GPC3-positive HepG2 cells in two-dimensional co-cultures and immunodeficient xenograft models. Inducible control was achieved using a synthetic Notch (synNotch) receptor platform constructed in Jurkat T-cell reporter lines and indicated using an inducible mCherry. Multiple anti-GPC3 single-chain variable fragments (scFvs) were screened; receptor engineering included negative regulatory region (NRR) deletion and synthetic intramembrane proteolysis receptor (SNIPR) architecture. Functional validation was performed upon co-culture with GPC3-positive target cells.Results Anti-GPC3 CAR-T cells achieved satisfactory transduction efficiency and demonstrated potent cytotoxicity against HepG2 cells both in vitro and in vivo ( IDDF2026-ABS-0400 Figure 1; Production and characterization of anti-GPC3 CAR T cells). The synNotch system enabled inducible mCherry expression in Jurkat cells upon antigen stimulation; however, the initial anti-GPC3 version remained suboptimal (IDDF2026-ABS-0400 Figure 2; synNotch system allows inducible gene expression in Jurkat T cells but α-GPC3 synNotch remains suboptimal). Optimisation through scFv screening identified the A04 variant with the highest GPC3 binding index, superior induction fold-change, and minimal basal expression. Further engineering via NRR deletion and SNIPR architecture markedly enhanced receptor responsiveness, yielding robust, HCC-specific transgene induction exclusively upon co-culture with GPC3-positive HCC tumor cells (IDDF2026-ABS-0400 Figure 3; Optimization yielded an α-GPC3 receptor with robust, HCC-specific induction).Conclusions This modular inducible anti-GPC3 CAR-T platform establishes tumour-triggered gene expression with high specificity and low leakage. By enforcing spatial control, the approach simultaneously augments anti-tumour activity and mitigates systemic toxicity, providing a promising strategy to overcome current limitations of CAR-T therapy in HCC.Abstract IDDF2026-ABS-0400 Figure 1Abstract IDDF2026-ABS-0400 Figure 2Abstract IDDF2026-ABS-0400 Figure 3