A Dual-Ligand PDC Co-Targeting FOLR1 and c-Met Enhances Tumor Accumulation and Antitumor Efficacy in Ovarian Cancer.

Background: Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy. Single-targeted ADCs/PDCs are limited by "on-target, off-tumor" toxicity. We developed FMM-66, a dual-ligand peptide-drug conjugate targeting FOLR1 and c-Met, to improve tumor-selective drug delivery. Methods: FMM-66 was rationally designed by integrating FOLR1- and c-Met-targeting peptide ligands into a single PDC architecture. Binding and cellular internalization assays were performed in ovarian cancer cell lines with different FOLR1/c-Met expression profiles, as well as in engineered overexpression cell models, to comprehensively evaluate the targeting properties of FMM-66. Antitumor efficacy, intratumoral pharmacokinetics, and biodistribution were evaluated in female BALB/c nude mice bearing Caov3 xenografts, with the single-ligand PDCs FA-MMAE and GE137-MMAE as comparators. Normal-tissue biodistribution and systemic safety were further assessed in female Sprague-Dawley rats. Results: FMM-66 demonstrated enhanced binding affinity and significantly increased cellular internalization compared with single-ligand PDCs in vitro. Receptor-blocking experiments confirmed contributions from both FOLR1 and c-Met, whereas internalization was driven predominantly by FOLR1. In vivo studies revealed potent tumor growth inhibition and superior tumor accumulation in FOLR1/c-Met double-positive ovarian tumors than the single-ligand comparators. Furthermore, FMM-66 exhibited rapid systemic clearance while released MMAE showed prolonged intratumoral exposure. At the therapeutic-equivalent dose in rats, no significant biochemical abnormalities or body-weight loss were observed; transient ALT, AST, creatinine, and urea elevations at the higher dose largely resolved by Day 14. Conclusions: FMM-66 demonstrated enhanced tumor accumulation and potent antitumor efficacy, with preliminary tolerability at the tested dose levels. These findings support dual-ligand targeting as a promising strategy for improving tumor-selective payload delivery and warrant further preclinical evaluation of FMM-66.