Humanized antibody Fab-TCR T cells targeting GPC2 effectively regress neuroblastoma via enhanced TCR signaling and sustained cytotoxicity

Background and Significance: Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of hematologic cancers but remains less effective against solid tumors, where heterogeneous antigen expression poses a major barrier to efficacy. Neuroblastoma, a pediatric solid tumor with poor survival rates in high-risk patients, often expresses glypican-2 (GPC2), a promising yet variably expressed surface antigen. To overcome these challenges, we engineered antibody-T cell receptors (AbTCRs) that combine antibody-based antigen recognition with the signaling machinery of gamma/delta T cell receptors, aiming to promote more physiological activation and sustained antitumor activity in GPC2-positive neuroblastoma. Methods: We developed AbTCRs containing anti-GPC2 Fab fragments (humanized CT3 or murine CT3) fused to gamma/delta TCR constant regions and a co-stimulatory domain, CD30. Primary human T cells were transduced with these constructs and tested through in vitro tumor-killing assays, repeated cytotoxicity tests, western blotting, and in vivo xenograft models bearing GPC2 neuroblastoma (IMR5, NBEB, LAN1, and SH-SY5Y) tumors. The LAN1 and SH-SY5Y models have low GPC2 antigen density, enabling evaluation of efficacy under stringent antigen conditions. Results: We found strong anti-tumor effects in humanized CT3 AbTCR-T cells, including significant tumor reduction and complete responses in multiple xenograft models. Tumors from mice administered with humanized CT3 AbTCR-T cells showed notably augmented infiltration of CD8⁺ and CD8⁺CD27⁺ T cells, consistent with enhanced effector persistence. Humanized CT3 AbTCR-T cells demonstrated downregulation of exhaustion markers PD1, LAG3, and TIM3 and an enriched less-differentiated Tscm subset, as assessed by peripheral blood testing. These cells maintained lower PD1 levels after prolonged coculture with tumor cells, and retained cytotoxic activity in second- and third-round killing assays. Mechanistically, upon tumor engagement, hCT3 AbTCRs elicited more robust TCR signaling, evidenced by higher NFAT nuclear translocation and greater phosphorylation of key TCR pathway proteins. Conclusion: Humanized CT3 AbTCR T cells exhibit durable and potent antitumor effects against low-GPC2 neuroblastoma by combining antibody specificity with physiological TCR signaling. Their enhanced TCR signaling, reduced exhaustion, and sustained cytotoxic ability suggest that AbTCR T cells represent a promising next-generation cellular therapy for solid tumors with heterogeneous or low antigen expression.