Use\nof a Cyclic α‑Alkylidene-β-Diketone\nas a Cleavable Linker Strategy for Antibody-Drug Conjugates

In\nthe fast-evolving landscape of targeted cancer therapies, the\nrevolutionary class of biotherapeutics known as antibody-drug conjugates\n(ADCs) are taking center stage. Most clinically approved ADCs utilize\ncleavable linkers to temporarily attach potent cytotoxic payloads\nto antibodies, allowing selective payload release under tumor-specific\nconditions. In this study, we explored the utilization of 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl\n(Dde), a cyclic β-diketone featuring an active alkylidene group,\nto develop a novel chemically labile linker. This linker was designed\nto exploit the difference in reduction potential between the intracellular\ncompartment and plasma. Upon reduction of an azido trigger strategically\ninstalled neighboring the cyclic β-diketone, the resulting nucleophilic\nprimary amine reacts with the alkylidene group facilitated by a favorable\nring closure reaction in accordance with Baldwin’s rules. Consequently,\nthis reaction enables the simultaneous release of the attached cytotoxic\npayload. The therapeutic utility of this novel linker strategy was\ndemonstrated by separate conjugation of the linker to two epidermal\ngrowth factor receptor (EGFR)-targeting ligands to afford a peptide-drug\nconjugate and an ADC. This work comprises a significant contribution\nto the bioconjugation field by introducing the alkylidene cyclic β-diketone\nas a tunable scaffold used for the temporary conjugation of therapeutic\nagents to peptides and proteins.