Optimization of an Enzymatic Antibody–Drug Conjugation Approach Based on Coenzyme A Analogs

作者：Jan Grünewald, Yunho Jin, Julie Vance, Jessica Read, Xing Wang, Yongqin Wan, Huanfang Zhou, Weijia Ou, Heath E. Klock, Eric C. Peters, Tetsuo Uno, Ansgar Brock, Bernhard H. Geierstanger · 发表于：Bioconjugate Chemistry · 年份：2017 · DOI：10.1021/acs.bioconjchem.7b00236 · 被引用次数：20 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Biochemical and Structural Characterization、Peptidase Inhibition and Analysis

Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.