Tuning the Diels–Alder Reaction for Bioconjugation to Maleimide Drug-Linkers

The thiol–maleimide linkage is widely used for antibody–drug conjugate (ADC) production; however, conjugation of maleimide–drugs could be improved by simplified procedures and reliable conjugate stability. Here, we report the evaluation of electron-rich and cyclic dienes that can be appended to antibodies and reacted with maleimide-containing drugs through the Diels–Alder (DA) reaction. Drug conjugation is fast and quantitative due to reaction acceleration in water, and the linkage is more stable in serum than in the corresponding thiol–maleimide adduct with the same drug. ADCs produced using the DA reaction (DAADCs) are effective in vitro and in vivo, demonstrating the utility of this reaction in producing effective biotherapeutics. Given the large number of commercially available maleimide compounds, this conjugation approach could be readily applied to the production of a wide range of antibody (or protein) conjugates.