Bioconjugation is an important chemical biology research focus, especially in the development of methods to produce pharmaceutical bioconjugates and antibody-drug conjugates (ADCs). In this report, an enzyme-catalyzed conjugation method combined with a chemical reaction was used to modify a native antibody under mild reaction conditions. Our investigation revealed that lipoic-acid ligase (LplA) modifies native IgG1 with biased site-specificity. An intact mass analysis revealed that 98.3% of IgG1 was modified by LplA and possessed at least one molecule of octanocic acid. The average number of modifications per antibody was calculated to be 4.6. Peptide mapping analysis revealed that the modified residues were K225, K249 and K363 in the Fc region, and K30, K76 and K136 in the heavy chain and K39/K42, K169, K188 and K190 in the light chain of the Fab region. Careful evaluation including solvent exposed amino acid analysis suggested that these conjugate sites were not only solvent exposed but also biased by the site-specificity of LplA. Furthermore, antibody fragment conjugation may be able to take advantage of this enzymatic approach. This feasibility study serves as a demonstration for preparing enzymatically modified antibodies with conjugation site analysis.

生物偶联是重要的化学生物学研究重点，特别是在开发生产药物生物偶联物和抗体-药物偶联物 (ADC) 的方法方面。在本报告中，在温和的反应条件下，结合化学反应的酶催化偶联方法对天然抗体进行了修饰。我们的调查显示，硫辛酸连接酶 (LplA) 会修饰具有偏向位点特异性的天然 IgG1。完整的质量分析显示 98.3% 的 IgG1 被 LplA 修饰并具有至少一个辛酸分子。每个抗体的平均修饰数计算为 4.6。肽图分析显示，修饰残基为 Fc 区的 K225、K249 和 K363，重链的 K30、K76 和 K136 以及 K39 / K42、K169，Fab 区轻链中的 K188 和 K190。包括溶剂暴露氨基酸分析在内的仔细评估表明，这些缀合物位点不仅暴露于溶剂，而且还受到 LplA 的位点特异性的影响。此外，抗体片段偶联可能能够利用这种酶促方法。该可行性研究可作为通过结合位点分析制备酶修饰抗体的示范。