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.

Biological Evaluation of Antibody-Drug Conjugates Produced by Tag-Free Lipoate Ligase A Modification

“…All analytical results have been reported in previous studies. 12 In our ADC synthesis, azido-octanoic acid was introduced into trastuzumab through LplA enzymatic modification. This was followed by conjugation with DBCO-VC-PAB-MMAE, which served as the payload linker.…”

“…The ADC synthesis was conducted using LplA. This resulted in a DAR that was verified by nonreducing Q-TOF and RP-HPLC, two established analytical methods (Figures b and S1 in Supporting Information). All analytical results have been reported in previous studies .…”

“…Our research group also successfully synthesized the first ADCs using tag-free LplA techniques (Figures b and S1 in Supporting Information). The resulting ADCs displayed notable stability under prolonged storage conditions, thus bolstering the reliability and efficiency of LplA-mediated ADC production. Furthermore, meticulous peptide mapping analyses have suggested that LplA-induced modifications target specific lysines, particularly the light chain Lys188/190 site .…”

Lipoic Acid Ligase A‐Mediated Ligation: Mechanism, Applications, and Emerging Innovations in Bioconjugation

“…Notably, LplA showed preferential specificity for modifying Lys residues on the mAb surface, suggesting room for improvement of site specificity through reaction fine‐tuning. Furthermore, the same group generated four ADCs from an azide‐incorporated antibody modified with LplA and DBCO drug linkers [103] . The resulting conjugates were identified using reversed‐phase high‐performance liquid chromatography without prior treatment [104] .…”

“…Furthermore, the same group generated four ADCs from an azide-incorporated antibody modified with LplA and DBCO drug linkers. [103] The resulting conjugates were identified using reversed-phase high-performance liquid chromatography without prior treatment. [104] They demonstrated stability for over one month at 4 °C, confirming the potential of LplA as a strategic tool for incorporating biorthogonal groups into native antibodies for ADC production.…”