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 Cysresidues, which results in an unwanted complex product mixture withconventional drug conjugation methods. We selectively acylated the epsilon-NH2of 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 epsilon-NH2of K248 whilebinding to trastuzumab. Consequently, the thioester underwentproximity-driven selective acylation of epsilon-NH2through anStoNacyl transfer reaction. Furthermore,N-tert-butyl maleimide acceleratedthe cross-linking reaction with an approximately 95% yield of thedesired product by scavenging the byproduct (FcBP-SH). Only K248was 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. Theresulting ADCs showed remarkablein vitroandin vivoanticancer activity. Our results demonstrate that a thioester is a promising chemical entity for proximity-driven site-selective conjugation of antibodies

Automated summary

In this study, a new method for antibody conjugation was developed, which achieved site-selective modification using an Fc-binding peptide equipped with a 5-norbornene-2-carboxylic acid thioester. The resulting antibody-drug conjugates showed remarkable anticancer activity.