One-Step Assembly of Homogeneous DAR4 ADCs via a Traceless Fc Ligand-Directed Acylation Strategy.

Site-specific antibody-drug conjugates (ADCs) represent a promising class of biotherapeutics with enhanced pharmacological profiles. we herein report a novel one-step strategy for preparing homogeneous ADCs with a drug-to-antibody ratio (DAR) of 4. This approach leverages a Fc ligand-directed thioester-based acylating reagent combined with a β-glutamic acid-based branching linker to enable precise conjugation of four MMAE molecules per antibody. Through systematic optimization of buffer composition and pH, we successfully mitigated the hydrophobicity-driven aggregation typically associated with K248-linked DAR4 ADCs, while maintaining exceptional conjugation efficiency. The method demonstrates excellent tolerance to protein concentration and is applicable to multiple IgG subtypes, including those targeting HER2, cMet, ROR1, and FRα. All resulting ADC products exhibited high homogeneity. Notably, ADC-4, functionalized with a VK(SO3H)-modified linker, showed enhanced aggregation stability, potent tumor suppression, and a favorable safety profile, highlighting its promising therapeutic potential.