Impact of Maleimide Disubstitution on Chemical and Biological Characteristics of HER2 Antibody–Drug Conjugates

Abstract

Antibody−drug conjugates (ADCs) are the spearhead of targeted therapies. According to the technology used, the conjugation of a cytotoxic drug to an antibody can produce suboptimal heterogeneous species, impacting the overall efficacy. Herein, we describe the synthesis of HER2targeting ADCs with three disulfide rebridging heads, allowing homogeneous and site-specific bioconjugation: dibromomaleimide (DBM), dithiomaleimide (DTM), and hybrid thiobromomaleimide (TBM) chemical bricks to combine the properties of both previously used heads. The primary purpose of this study was to compare the reactivity of these three chemical bricks in the bioconjugation process. Then, the resulting ADCs were evaluated in terms of physicochemical stability, binding, and biological activity. We have demonstrated that the higher percentage of a drug-to-antibody ratio of 4 was obtained with TBM. Additionally, the reaction time was drastically reduced with TBM in comparison to DTM. The three ADCs showed good binding to HER2 and in vitro cytotoxicity, which validate the TBM structure as an attractive alternative scaffold for rebridging bioconjugation.

Therapeutic Potential of MF-TTZ-MMAE, a Site-Specifically Conjugated Antibody-Drug Conjugate, for the Treatment of HER2-Overexpressing Breast Cancer

“…16,31 B HIC Analysis. Samples of 50 μg were analyzed at 280 nm as previously described 36 on a MAbPac HIC-butyl, 5 μm, 4.6 × 100 mm column (ThermoScientific), and eluted with a step gradient over 30 min at 1 mL/min, at 25 °C.…”

“…E SEC Analysis. Samples of 50 μg were analyzed at 280 nm as previously described, 36 on an AdvanceBio SEC 2.7 μm, 7.8 × 300 mm column (Agilent Technologies), and eluted with an isocratic gradient (PBS pH 7.0, 0.02% sodium azide) over 24 min at 1 mL/min, at 25 °C.…”

Site-selective modification strategies in antibody–drug conjugates

“…135 Furthermore, Feuillâtre et al reported the use of hybrid thiobromomaleimides (TBMs) which were shown to produce homogenous ADCs with a marginally narrower DAR distribution compared to dibromomaleimide and dithiophenolmaleimide reagents. 136 NGMs have repeatedly demonstrated the ability to generate ADCs with high levels of homogeneity. Optimisation of the ring hydrolysis has significantly improved the utility of this method and allowed for the synthesis of ADCs with excellent plasma stability.…”

The Chemistry Behind ADCs

“…They lead to the substitution with the two nucleophilic thiol groups and enable better stability compared to classic maleimides, which are prone to retro-Michael reaction. Recently it was reported the synthesis of hybrid thiobromomaleimide (TBM) combining the properties of dibromo- and dithiomaleimide [ 112 ]. This TBM reagent conjugates faster and demonstrates higher percentage of DAR 4 probably due to the bromide that reduces steric hindrance and represents a better living group than the thiophenolate.…”