Rational payload selection enables high antitumoral efficacy of an anti-EGFR antibody-drug conjugate against ovarian tumors

The epidermal growth factor receptor (EGFR) is frequently expressed in ovarian cancer, yet its potential as a therapeutic target remains underexplored. We investigated EGFR expression and its therapeutic exploitation using antibody-drug conjugates (ADCs) based on the clinical anti-EGFR antibody cetuximab. EGFR protein and mRNA levels were evaluated in patient-derived tumors and ovarian cancer cell lines by western blotting and qPCR, and cell surface localization was analyzed by flow cytometry. Several cetuximab-based ADCs were generated using clinically validated cytotoxics: DM1, deruxtecan (DXd), and monomethyl auristatin F (MMAF), with cleavable or non-cleavable linkers. Their structural integrity, antiproliferative activity, internalization dynamics, and mechanisms of action were examined in vitro, and in vivo efficacy and pharmacokinetics were assessed in SKOV3 and OVCAR8 xenograft models. All ADCs showed successful conjugation and preserved antibody integrity. MMAF-conjugated ADCs displayed superior antiproliferative effects, particularly the cleavable cetuximab-vc-MMAF, while A2780 and PEO4, which showed low EGFR expression, were less responsive. Cetuximab alone or free MMAF were markedly less active. Mechanistic studies revealed rapid internalization and lysosomal trafficking of cetuximab-vc-MMAF, leading to G2/M arrest, increased mitotic markers, spindle defects, DNA damage, and apoptosis. In vivo, cetuximab-vc-MMAF significantly inhibited tumor growth and prolonged survival without systemic toxicity. Pharmacokinetic analyses confirmed selective intratumoral accumulation of the ADC and its payload with minimal off-target distribution. Treated tumors exhibited reduced proliferation and increased markers of mitotic arrest, DNA damage, and apoptosis. These results demonstrate that cetuximab-based EGFR-targeting ADCs exert potent and selective antitumor activity in ovarian cancer, supporting their further preclinical and clinical development.