Integrated strategy for biotransformation of antibody–drug conjugates and multidimensional interpretation via high-resolution mass spectrometry

作者：Ziyi Li, Jingxian Zhang, Yue Wu, Fan Wang, Tingting Cai · 发表于：Drug Metabolism and Disposition · 年份：2025 · DOI：10.1016/j.dmd.2025.100081 · 被引用次数：5 · 研究领域：Monoclonal and Polyclonal Antibodies Research、HER2/EGFR in Cancer Research、Radiopharmaceutical Chemistry and Applications

The characterization of the release mechanism and stability in circulation for novel antibody-drug conjugates (ADCs) has become essential to address the complex variables (linker or payload selection, antibody, conjugating site). Understanding the integrated biotransformation of released catabolites and intact ADCs is necessary to elucidate the mechanism of action and mitigate the premature payload release and formation of inactive ADCs during circulation, which can lead to pharmacokinetic/pharmacodynamic disconnection. Herein, we present a comprehensive strategy for the biotransformation of ADCs from both small- and large-molecule perspectives. Two ADCs with common cleavable linkers were investigated: ADC-1 (maleimidocaproyl glycine-glycine-phenylalanine-glycine deruxtecan) and ADC-2 (maleimidocaproyl valine-citrulline-p-aminobenzyl carbamate monomethyl auristatin E). First, the payload-related catabolites released from lysosome, S9, and tumor cells were identified by sensitive data mining based on high-resolution mass spectrometry to reveal the pharmacologically active components. Second, we demonstrated the biotransformation occurring in intact ADCs using middle-down and bottom-up approaches, which particularly contributed to their instability in incubation. The combined immune capture with subunit or peptide analysis enables a comprehensive evaluation of the structural integrity of ADCs, whereas solely quantitative payload release is insufficient to determine the ADCs' st...