NQO1-responsive Trimethyl lock benzoquinone: A cleavable linker strategy for antibody-drug conjugates

Antibody-drug conjugate (ADC) efficacy is hindered by premature systemic payload release and insufficient tumor activation. Here, an NQO1-responsive linker utilizing trimethyl lock benzoquinone is developed to create NRTL-24, a glioma-targeting ADC. In vitro studies showed NRTL-24 maintained plasma stability while achieving NQO1-dependent MMAE release. It demonstrated potent cytotoxicity in EGFR/NQO1-high glioma cells (U251 IC₅₀ = 0.03 nM; T98G IC₅₀ = 0.06 nM), comparable to free MMAE (U251 IC₅₀ = 0.01 nM; T98G IC₅₀ = 0.02 nM). Notably, toxicity dropped significantly in EGFR/NQO1-low normal cells (HMC3 IC₅₀ = 0.58 mM), yielding >10,000 selectivity index. In vivo, NRTL-24 demonstrated superior tumor-targeting ability, significantly prolonging median survival compared to untreated and temozolomide-treated groups while maintaining favorable safety. This NQO1-responsive linker technology enables precise payload activation in tumor while minimizing off-target effects, establishing NRTL-24 as a promising therapeutic candidate. The strategy provides a blueprint for enzyme-targeted ADC development through tumor- selective linker design.