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

Abstract

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 <i>S</i> to <i>N</i> acyl transfer reaction. Furthermore, <i>N</i>-<i>tert</i>-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 <i>in vitro</i> and <i>in vivo</i> anticancer activity. Our results demonstrate that a thioester is a promising chemical entity for proximity-driven site-selective conjugation of antibodies.