Abstract

Antibody−drug conjugates (ADCs) are important cancer therapeutics, yet efficacy in solid tumors is limited by poor penetration: the large size (∼150 kDa) restricts vascular extravasation and stromal diffusion, and strong bivalent IgG−antigen binding creates a binding-site barrier. Antibody fragment−drug conjugates (FDCs) penetrate better but clear rapidly, compromising retention. We developed a programmable Antibody Fragment-Cleavable PEGylated Drug Conjugate (AFCDC) platform enabling site-specific dual conjugation through genetically encoded bifunctional noncanonical amino acids, pTAF to attach cytotoxic payloads, and MMP2-cleavable PEG chains. PEGylation extends the systemic half-life, while tumor proteases remove PEG to yield smaller fragment−payload conjugates with improved tissue penetration. AFCDC improves intratumoral penetration, pharmacokinetics, and efficacy in HER2-positive tumor models. Under the evaluated regimen, AFCDC demonstrated enhanced activity over T-DXd in the NCI-N87 xenografts. Overall, AFCDC resolves the antibody fragment trade-off, providing a strategy compatible with multiple formats and payloads to enhance the ADC efficacy in solid tumors.