Tag‐Free Antibody Modification Mediated by Lipoic Acid Ligase A: Application to Antibody‐Drug Conjugates Production

Abstract Enzymatic modifications of proteins are of great interest in the pharmaceutical industry as it enables the production of homogeneous protein conjugates such as antibody‐drug conjugates, which are considered to be promising for cancer medicine. Several biotechnology companies have established proprietary conjugation technologies using specific enzymes. Most enzymatic modifications require the use of tags and enzyme‐recognition moieties. Therefore, site‐specific and “tag‐free” modifications are required to avoid the time‐consuming process of installing tags on proteins. Lipoic acid ligase A (LplA), is a recently reported enzyme having the potential to be used for the site‐specific modification of native antibodies without requiring a tag moiety. In this manuscript, investigations focused on the optimization of this enzymatic modification applied to antibody‐drug conjugates (ADCs) production. First, an analysis of the pH dependency was performed. Next, the effects of excess adenosine mono‐ and di‐phosphate were also examined to show that these by‐products from LplA modification did not have a significant impact on inhibiting the reaction efficiency. Furthermore, ADC syntheses by LplA‐mediated tag‐free modification followed by click reaction were achieved. To the best of our knowledge, this is the first example of actual ADC synthesis produced by LplA modification of tag‐free antibodies. A stability assessment of the resulting ADCs indicated that this enzymatic approach is a promising tool for installing biorthogonal groups to native antibodies for the next generation of ADC manufacturing.