Site-Selective Antibody–Drug Conjugation by a Proximity-Driven S to N Acyl Transfer Reaction on a Therapeutic Antibody

Immunoglobulin Gs (IgGs) contain many Lys and Cys residues, which results in an unwanted complex product mixture with conventional drug conjugation methods. We selectively acylated the ε-NH₂ of K248 on trastuzumab using an IgG Fc-binding peptide (FcBP) equipped with a 5-norbornene-2-carboxylic acid thioester (AbClick-1). AbClick-1 locates its thioester close to the ε-NH₂ of K248 while binding to trastuzumab. Consequently, the thioester underwent proximity-driven selective acylation of ε-NH₂ through an S to N acyl transfer reaction. Furthermore, N-tert-butyl maleimide accelerated the cross-linking reaction with an approximately 95% yield of the desired product by scavenging the byproduct (FcBP-SH). Only K248 was modified selectively with the 5-norbornene-2-carbonyl group, which was further modified by click reaction to afford an antibody–drug conjugate (ADC) with two drugs per antibody. The resulting ADCs showed remarkable in vitro and in vivo anticancer activity. Our results demonstrate that a thioester is a promising chemical entity for proximity-driven site-selective conjugation of antibodies.