Systematic Evaluation of Maleimide Spacer Impact on Drug-Linker Deconjugation in Antibody-Drug Conjugates Ex Vivo and...

Antibody-drug conjugates (ADCs) are an important class of targeted therapeutics that require comprehensive stability characterization with robust analytical methods for their proper design and development. To date, most ADCs in the clinic or in preclinical development possess some degree of instability; in the case of thiol-maleimide ADCs, drug-linker deconjugation can happen through the retro-Michael reaction; simultaneously, maleimide hydrolysis can prevent further deconjugation, as the ring-opened derivative is more resistant to elimination. Here, we describe the development of a novel generic immunocapture method to recover ADCs from complex biological matrices prior to characterization by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS), focusing primarily on deconjugation and succinimide ring-opening quantification. Employing this method, we systematically evaluated 22 thiol-maleimide ADCs in ex vivo and in vivo settings across rat, mouse, and monkey sera, with ADCs featuring either maleimidocaproyl (MC) or maleimido-triethylene glycol (MT) spacer units, with diversity in antibody, drug loading, cleavable linker, and payload composition, including clinically validated drug-linkers such as MC-GGFG-DXd and MC-VCit-MMAE. We observed that ADCs bearing a hydrophilic MT spacer achieved higher levels of ring-opening and thioether adduct stabilization, which led to lower levels of deconjugation compared to hydrophobic MC spacer ADCs, which showed moderate ring-opening and substantial deconjugation in vivo. These trends were generally observed regardless of antibody backbone and remaining drug-linker structure, reflecting the influence of spacer structure on water accessibility to the maleimide ring. Our immunoprecipitation-LC-MS results were compared to orthogonal total antibody (T_ab) and ADC (conjugated antibody) ELISA pharmacokinetic (PK) profiles. While deconjugation profiles were generally consistent across methods, IP-MS provided more quantitative measurements and was more sensitive to subtle differences in deconjugation. Our findings shed light on the importance of the spacer structure on thiol-maleimide ADC stability.