CAR-T cell therapy has demonstrated significant clinical success in treating B cell–derived hematologic malignancies. However, its efficacy in solid tumors remains limited due to the heterogeneous antigen expression across tumor and normal tissues. Glypican-3 (GPC3), a heparan sulfate proteoglycan involved in key oncogenic signaling pathways, is highly expressed in the placenta and fetal liver but minimally detected in most adult tissues. Importantly, GPC3 is highly expressed in hepatocellular carcinoma (HCC), making it a promising therapeutic target. Recent clinical trials of GPC3-targeted CAR-T therapy in advanced HCC patients have demonstrated encouraging efficacy and safety profiles.
In this study, we developed and evaluated a novel GPC3 CAR construct. Specifically, we used the GPC3-specific antibody clone 2B8, developed through mouse immunization and the hybridoma technique, to design a second-generation CAR construct. The variable heavy (VH) and light (VL) chains of 2B8 were engineered into a single-chain variable fragment (scFv) and incorporated into a lentiviral vector harboring CD28 and CD3ζ intracellular domains. Lentiviral transduction of this construct successfully generated GPC3 CAR-T cells with preserved antigen-binding capacity. Co-culture of these CAR-T cells with the HCC cell line HepG2 confirmed reactivity, evidenced by the expression of effector cytokines IL-2, IFN-γ, and TNFα. In a xenograft mouse model bearing HepG2 cells expressing CD19, GPC3 CAR-T cells demonstrated potent anti-tumor activity comparable to CD19 CAR-T cells expressing a second-generation CAR construct with CD28 or 4-1BB co-stimulatory domain. To enhance the clinical applicability of this CAR construct, we humanized the VH and VL domains, resulting in 16 humanized GPC3 antibody candidates. Following comprehensive characterization of specificity, stability, and binding affinity, we designed and tested two high-affinity and two low-affinity GPC3 CAR variants in a xenograft mouse model.
This study shows the potential of GPC3-targeted CAR-T therapy as a treatment for refractory HCC and provides valuable insights for advancing CAR-T therapies and cancer immunotherapy.