β-glucuronidase-triggered ortho-hydroxyl-mediated cyclization strategy for the efficient delivery of phenolic payloads in antibody-drug conjugates.

While the payload landscape of antibody-drug conjugates (ADCs) has diversified beyond amine-containing compounds, the application of potent cytotoxic agents possessing phenolic hydroxyl groups remains restricted. This is primarily due to the scarcity of linker technologies capable of balancing systemic stability with efficient release kinetics, as conventional strategies for phenolic conjugation often suffer from premature hydrolysis or sluggish activation. Herein, we report the design and evaluation of a novel self-immolative linker strategy based on an ortho-hydroxyaniline scaffold, which facilitates the efficient release of phenolic payloads via intramolecular cyclization. Triggered by β-glucuronidase, an enzyme overexpressed in the tumor microenvironment, this system utilizes a hydrophilic glucuronide moiety that not only ensures tumor-specific activation but also improves the hydrophilicity of ADCs compared to traditional hydrophobic linkers. We synthesized trastuzumab-based conjugates utilizing this novel strategy (Tras-GLU572 and Tras-GLU579) and compared them against a clinical benchmark ADC utilizing a conventional peptide linker (Tras-GGFG-DXd). In HER2-positive NCI-N87 xenograft models, the optimized conjugates, Tras-GLU572 and Tras-GLU579, exhibited superior antitumor efficacy compared to the benchmark Tras-GGFG-DXd. Notably, at equivalent doses, the lead candidate Tras-GLU579 achieved profound tumor regression with a T/C value of 2.33% on day 20, significantly outperforming the benchmark (T/C = 18.58%; p < 0.01), while maintaining a favorable safety profile. This study establishes a robust platform for overcoming the synthetic and kinetic hurdles associated with phenolic ADCs.