A Reactive Antibody Platform for One-Step Production of Antibody–Drug Conjugates through a Diels–Alder Reaction with Maleimide

The normal electron-demand Diels–Alder (DA) cycloaddition is a classic transformation routinely used in synthesis; however, applications in biological systems are limited. Here, we report a spiro[2.4]­hepta-4,6-diene-containing noncanonical amino acid (SCpHK) capable of efficient incorporation into antibodies and subsequent coupling with maleimide via a DA reaction. SCpHK was stable throughout protein expression in mammalian cells and enabled covalent attachment of maleimide drug-linkers yielding DA antibody–drug conjugates (DA-ADCs) with nearly quantitative conversion in a one-step process. The uncatalyzed DA reaction between SCpHK and maleimide in aqueous buffer was rapid (1.8–5.4 M–1 s–1), and the antibody–drug adduct was stable in rat serum for at least 1 week at 37 °C. Anti-EphA2 DA-ADCs containing AZ1508 or SG3249 maleimide drug-linkers were potent inhibitors of tumor growth in PC3 tumor models in vivo. The DA bioconjugation strategy described here represents a simple method to produce site-specific and stable ADCs with maleimide drug-linkers.