Detection and Characterization of In Vitro Payload-Containing Catabolites of Noncleavable Antibody-Drug Conjugates by High-Resolution Mass Spectrometry and Multiple Data Mining Tools [Articles]

Abstract

The formation and accumulation of payload-containing catabolites (PCCs) from a noncleavable antibody-drug conjugate (ADC) in targeted and normal tissues are directly associated with the therapeutic effect and toxicity of the ADC, respectively. Understanding the PCC formation is important for supporting the payload design and facilitating preclinical evaluation of ADCs. However, detection and identification of PCCs of a noncleavable ADC are challenging due to their low concentrations and unknown structures. The main objective of this study was to develop and apply a generic liquid chromatography–high-resolution mass spectrometry (LC-HRMS) method for profiling PCCs in vitro. Noncleavable ADCs, ado-trastuzumab emtansine (T-DM1) and ADC-1, were incubated in liver lysosomes, liver S9, and/or cancer cells followed by data acquisition using LC-HRMS. Profiling PCCs mainly relied on processing LC-HRMS datasets using untargeted precise and thorough background subtraction (PATBS) processing and targeted product ion filtering (PIF). As a result, 12 PCCs of T-DM1 were detected and structurally characterized in human liver lysosomal incubation, a majority of which consisted of 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (MCC)-DM1 and a few amino acids. Additionally, the incubation of ADC-1 in human, rat, and monkey liver S9 and cancer cells generated one major and three very minor PCCs, verifying the payload design. The results demonstrate that PATBS enabled the comprehensive profiling of PCCs regardless of their molecular weights, charge states, and fragmentations. As a complementary tool, PIF detected specific PCCs with superior sensitivity. The combination of the in vitro metabolism systems and the LC-HRMS method is a useful approach to profiling in vitro PCCs of noncleavable ADCs in support of drug discovery programs.

SIGNIFICANCE STATEMENT Profiling in vitro payload-containing catabolites (PCCs) of a noncleavable antibody-drug conjugate (ADC) is important for optimization of the payload design and preclinical evaluation of ADC. However, currently used analytical approaches often fail to quickly provide reliable PCC profiling results. The work introduces a new liquid chromatography high resolution mass spectrometry method for comprehensive and rapid detection and characterization of PCCs released from a noncleavable ADC in liver lysosomes and S9 incubations.

FootnotesReceived September 27, 2022.Accepted January 20, 2023.

This study was funded by WuXi AppTec.

No author has an actual or perceived conflict of interest with the contents of this article.

1T.C. and L.S. contributed equally to this manuscript.

dx.doi.org/10.1124/dmd.122.001135.

Embedded ImageEmbedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics

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