Abstract
<jats:p>Antibody-drug conjugates (ADCs) have become a cornerstone of targeted cancer therapy, with more than a dozen FDA-approved agents demonstrating the power of combining antibody specificity with potent small-molecule/heterobifunctional payloads. Despite this success, the chemical methods used to attach payloads to antibodies, particularly cysteine conjugation, still rely heavily on maleimides, whose thiosuccinimide linkages are prone to retro-Michael elimination and thiol exchange in plasma, compromising stability and therapeutic index. Alternative cysteine-reactive electrophiles have historically suffered from slow kinetics, incomplete labeling, or the need for excess reagent and basic conditions that can damage antibodies. Here we introduce a Kinetics-based Structure Activity Relationship (kSAR) approach that systematically maps the reactivity of vinyl heteroarenes and identifies 2-vinyl-4-aminopyrimidines (VAPs) as an optimum scaffold for ADC conjugation. Through a combination of rate constant measurements, Hammett analysis, pH-rate profiling, and DFT calculations, we establish that these linkers operate via a concerted but asynchronous proton-coupled thia-Michael addition that simultaneously activates the electrophile and generates the nucleophilic thiolate under the mildly acidic conditions (pH 6–7), which mechanistically departs from the canonical thiolate-driven paradigm and is preferred for antibody conjugation. The optimized VAP platform delivers second-order rate constants up to 5.2 M–1 s–1, enabling near-stoichiometric conjugation (1.25 equiv per cysteine) to trastuzumab affording homogeneous DAR up to 7.6 ADCs with <10% aggregation when appropriate PEG solubilizers are incorporated. The resulting conjugates exhibit exceptional plasma stability, no detectable thiol exchange over 96 hours, efficient papain-mediated payload release, and potent in vitro and in vivo activity. In HER2-positive breast cancer models, trastuzumab–VAP ADCs bearing deruxtecan matched the benchmark trastuzumab deruxtecan (EnhertuTM), while exatecan variants showed up to six-fold greater cytotoxicity and superior tumor-growth inhibition. This work provides a modular, tunable, and practical framework for cysteine-based ADC assembly that addresses long-standing limitations in stability, stoichiometry, and manufacturability.</jats:p>