Development of Glypican 3-Targeting Antibody-Drug Conjugates for Hepatocellular Carcinoma Therapy.

UNLABELLED: Hepatocellular carcinoma (HCC), a leading cause of global cancer mortality, is characterized by intratumoral heterogeneity, chemoresistance, and limited treatments. Glypican 3 (GPC3), an oncofetal glycoprotein overexpressed in ∼70% of HCC cases but absent in normal adult tissues, represents an attractive target for antibody-drug conjugates (ADC). To address this unmet need, three GPC3-specific antibodies were developed: a fully human Mab-A with moderate affinity (Kd = 45.7 nmol/L), recognizing a unique epitope distal to GPC3's membrane region; a humanized Mab-B with high affinity (Kd = 8.84 nmol/L), targeting an epitope similar to those of several known therapeutic antibodies; and a biparatopic BpAb-AB with the highest affinity (Kd = 0.383 nmol/L). Three payloads were used for conjugation: duocarmycin SA (DUBA), pyrrolobenzodiazepine (PBD) dimer, and deruxtecan (Dxd). In cell line-derived xenograft (CDX) models of HCC, DUBA conjugates demonstrated satisfactory efficacy but less potent than other conjugates, whereas PBD dimer conjugates showed potent tumor suppression with narrow therapeutic windows due to dose-limiting toxicity. Mab-B conjugates exhibited comparatively lower efficacy and suboptimal pharmacokinetics. In contrast, Mab-A-Dxd and BpAb-AB-Dxd ADCs produced sustained tumor regression and acceptable safety. Despite lower binding affinity and in vitro activities, Mab-A-Dxd outperformed BpAb-AB-Dxd in vivo, driven by superior tumor penetration, prolonged exposure, and reduced target-mediated drug disposition (TMDD). Efficacy was further validated in patient-derived xenograft (PDX) models. Collectively, the optimized Mab-A-Dxd ADC, engineered with high drug-to-antibody ratio (DAR∼10) and Fc-silencing mutations, achieves a favorable balance among tumor penetration, minimized TMDD, and effective GPC3-mediated delivery, positioning it as a promising therapeutic candidate for refractory HCC and other GPC3-expressing malignancies.

SIGNIFICANCE: We identified Mab-A, a moderate-affinity GPC3 antibody targeting a unique membrane-distal epitope. Combined with a high-DAR (10) Dxd payload and engineered Fc, Mab-A-Dxd shows superior efficacy in CDX/PDX models, balancing internalization, cytotoxicity, tumor penetration, exposure, and reduced TMDD, redefining ADC design principles and supporting therapeutic potential in HCC and beyond.