GPC3 chimeric antigen receptor (CAR)-NK cells combined with Enoblituzumab enhance the anti-tumor efficacy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Although CAR-NK cell therapies have shown promise in hematologic malignancies, their efficacy in solid tumors like HCC is often limited by antigen escape. Here, we investigated a dual-targeting strategy combining GPC3 CAR-NK cells with the B7H3-blocking monoclonal antibody Enoblituzumab. We confirmed GPC3 and B7H3 co-expression in HCC tissues and cell lines via database analysis and flow cytometry. GPC3 CAR-NK cells were generated from expanded human PBMC-derived NK cells. In vitro cytotoxicity against HCC cells was assessed by LDH release and real-time cell analysis, while IFN-γ secretion and CD107a degranulation were measured by ELISA and flow cytometry. In vivo antitumor efficacy was evaluated in NCG mice bearing subcutaneous Huh7 tumors. High GPC3 and B7H3 co-expression correlated with poor patient prognosis. GPC3 CAR-NK cells exhibited potent cytotoxicity against HCC cells in vitro. Enoblituzumab triggered strong killing effects against B7H3-positive tumors. Notably, the combination synergistically enhanced cytotoxicity, IFN-γ production, and CD107a degranulation compared to either monotherapy. In vivo, combination treatment significantly suppressed tumor growth without inducing weight loss or splenomegaly, demonstrating favorable efficacy and safety. Our findings demonstrate that dual targeting of GPC3 and B7H3 effectively enhances NK cell-mediated antitumor activity. This strategy leverages both CAR-mediated specificity and NK cell-intrinsic mechanisms, providing a strong rationale for developing dual-target immunotherapies to improve outcomes in HCC patients.