Abstract

Antibody-drug conjugates (ADCs) are constrained by limited tumor penetration of IgG formats and heterogeneity from stochastic conjugation. We addressed these challenges by engineering a compact trastuzumab Fab-based ADC with site-specific installation of DM1 using the peptide ligase butelase-1 and by extending serum persistence via genetic fusion of an albumin-binding domain (ABD). Butelase-1 ligation proceeded rapidly under mild conditions to afford a homogeneous product with a defined drug-to-antibody ratio. The conjugate selectively killed HER2-positive cells in vitro while sparing HER2-negative controls. ABD fusion increased serum albumin binding and improved in vivo exposure; the resulting T-Fab-ABD(H + L)-DM1 produced superior tumor growth inhibition in HER2-positive xenografts compared with its non-ABD analogue. The ADC showed no significant body weight loss or organ damage in mice, indicating good tolerability. These findings validate a modular strategy combining a Fab scaffold, enzymatic site-specific conjugation, and ABD-mediated half-life extension to generate potent, homogeneous ADCs and nominate T-Fab-ABD(H + L)-DM1 as a promising candidate for HER2-positive cancer therapy.