Abstract
Lysine-conjugated antibody-drug conjugates (ADCs) are formed by attaching cytotoxic drugs to reactive lysine residues of monoclonal antibodies (mAbs) through chemical linkers. During production, the payloads are conjugated nonspecifically to lysine residues in mAbs, resulting in a heterogeneous mixture of ADCs with both different number and conjugation sites of drug payloads per mAb. On account of the drug conjugation sites and levels that both have significant influences on physical and pharmaceutical properties of ADCs, a reliable and straightforward approach for conjugation site analysis for ADCs is highly demanded. Herein, we used a lysine-conjugated ADC, Trastuzumab-MCC-DM1 (T-DM1), as a model ADC, and described an integrative strategy that combines the signature ion fingerprinting method for rapid and reliable filtering of DM1-conjugated peptides, and the normalized area quantitation approach for accurately gauging the conjugation levels for each identified site. This approach is believed to be readily applicable to other maytansinoid derivatives-modified ADCs, and more importantly, universally applicable to lysine-conjugated ADCs for both the recognition of conjugation sites and the measurement of conjugation levels.
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Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (No. 81872838), the Natural Science Foundation of Jiangsu Province (BK20180079), National Key R&D Program of China (2018YFD0901101), Double First-rate Project (CPU2018GY09, CPU2018GF09), the Project of State Key Laboratory of Natural Medicines in China Pharmaceutical University (SKLNMZZCX201817), and the Project for Major New Drugs Innovation and Development (2018ZX09711001-002-003). The authors also appreciated useful discussions with Prof. Lingjun Li at the University of Wisconsin-Madison.
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Sang, H., Wan, N., Lu, G., Tian, Y., Wang, G., Ye, H. (2020). Conjugation Site Analysis of Lysine-Conjugated ADCs. In: Tumey, L. (eds) Antibody-Drug Conjugates. Methods in Molecular Biology, vol 2078. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9929-3_16
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DOI: https://doi.org/10.1007/978-1-4939-9929-3_16
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