A Novel Anti-Glypican-3 Monoclonal Antibody G₃Mab-25 for Versatile Applications

Glypican-3 (GPC3) belongs to the glypican family of heparan sulfate proteoglycans and is frequently overexpressed in hepatocellular carcinoma (HCC). The overexpression of GPC3 is associated with the poor clinical outcomes, suggesting its potential as a clinically relevant biomarker and therapeutic target. Therefore, anti-GPC3 monoclonal antibodies (mAbs) have been developed in various modalities for tumor diagnosis and therapy. In this study, 88 clones of novel anti-GPC3 mAbs were established using a flow cytometry-based high-throughput screening, the Cell-Based Immunization and Screening (CBIS) method. Among them, a clone G₃Mab-25 (IgG₁, κ) recognized GPC3-overexpressed Chinese hamster ovary-K1 (CHO/GPC3) but not parental CHO-K1 in flow cytometry. Furthermore, G₃Mab-25 recognizes endogenous GPC3 in GPC3-expressing HCC cell lines, including HepG2, HuH-7, and JHH-5. G₃Mab-25 specifically recognized only CHO/GPC3, but not other GPC family-overexpressed CHO-K1. The dissociation constant values of G₃Mab-25 for CHO/GPC3, HepG2, HuH-7, and JHH-5 were determined to be 1.8 × 10^-8 M, 7.3 × 10^-9 M, 3.9 × 10^-9 M, and 1.4 × 10^-9 M, respectively. Moreover, G₃Mab-25 detects the N-terminal fragment of GPC3 in western blotting. In immunohistochemistry, G₃Mab-25 showed a diverse staining pattern for GPC3 in HCC tissues. G₃Mab-25, established by the CBIS method, is a versatile mAb for basic research and is expected to contribute to tumor diagnosis and therapy.