In-Depth Structure and Function Characterization of Heterogeneous Interchain Cysteine-Conjugated Antibody–Drug Conjugates

Abstract

Antibody−drug conjugates (ADCs), integrating high specificity of antigen-targeting antibodies and high potency of cellkilling chemical drugs, have become one of the most rapidly expanding therapeutic biologics in oncology. Although ADCs were widely studied from multiple aspects, overall structural elucidation with comprehensive understanding of variants is scarcely reported. Here, for the first time, we present a holistic and in-depth characterization of an interchain cysteine-conjugated ADC, focusing on conjugation and charge heterogeneity, and in vitro biological activities. Conjugation mapping utilized a bottom-up approach, unraveled positional isomer composition, provided insights into the conjugation process, and elucidated how conjugation affects the physicochemical and biological properties of an ADC. Charge profiling combined bottom-up and top-down approaches to interrogate the origin of charge heterogeneity, its impact on function, and best practice for characterization. Specifically, we pioneered the utilization of capillary isoelectric focusing-mass spectrometry to decode not only critical post-translational modifications but also drug load and positional isomer distribution. The study design provides general guidance for in-depth characterization of ADCs, and the analytical findings in turn benefit the discovery and development of future ADCs.

“Build Your Own” ADC Mimics: Identification of Nontoxic Linker/Payload Mimics for HIC-Based DAR Determination, High-Throughput Optimization, and Continuous Flow Conjugation

“…Other maleimides show similar results: Reactions with both MAL-83 and MAL-75 also provide HIC chromatograms with baseline-separated peaks (Figure B/D). However, both chromatograms also show an additional peak right next to the DAR4 peak, which could be attributed to another DAR4 positional isomer (see Figure ); however, no further identification of these peaks was pursued. Another notable outcome is the HIC chromatogram obtained with MAL-81: even though the peaks are not baseline-separated, each peak is clearly visible (Figure C).…”

Recent Technological and Intellectual Property Trends in Antibody–Drug Conjugate Research

“…Site-specific conjugation methods present a groundbreaking approach in ADC development, aiming to precisely attach the payload at specific locations and overcome challenges associated with traditional coupling methods. For example, the introduction of engineered reactive cysteine residues selectively inserted at specific positions enables precise conjugation at that site, enhancing the homogeneity of ADCs and providing tunable reactivity through the alteration of the modification site [ 32 , 33 , 34 ]. In enzymatic conjugation methods, a variety of enzymes, such as bacterial-derived formyl glycine-generating enzymes, transglutaminases, glycotransferases, and sortases, have been utilized for tag-free antibody modification techniques [ 35 , 36 , 37 , 38 , 39 ].…”