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.
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