A Novel Anti-Glypican-3 Monoclonal Antibody G₃Mab-25 for Flow Cytometry, Western Blotting, and Immunohistochemistry

Glypican-3 (GPC3) belongs to the glypican family of heparan sulfate proteoglycans and is fre-quently overexpressed in hepatocellular carcinoma (HCC). The overexpression of GPC3 is associ-ated 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, more than 80 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 and HuH-7 were determined to be 1.8 × 10⁻8 M and 3.9 × 10⁻9 M, respectively. Moreover, G3Mab-25 detects the N-terminal fragment of GPC3 in west-ern 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.