Fluorogenic Imaging Enables the Identification of Phosphoramidate Antibody–Drug Conjugate Linkers

Modular, tunable linker chemistries that are stable in circulation yet selectively cleaved are needed to realize the therapeutic potential of antibody–drug conjugates (ADCs). Near-infrared (NIR) fluorogenic imaging using norcyanine carbamate (CyBam) probes can quantitatively compare ADC linkers across in vitro and in vivo settings. A series of substituted CyBams modified with phosphoramidates, peptides, and control triggers were conjugated to EGFR-targeting monoclonal antibody (mAb) panitumumab. Imaging in cellular and in vivo settings reveals the potential for novel phosphoramidate linkers, showing selective cellular activation, excellent tumor localization, and reduced liver signal compared to conventional proteolytic linkers. Guided by these imaging results, monomethyl auristatin E (MMAE) conjugates bearing the new linkers were prepared and displayed picomolar potency, receptor-dependent activity, and promising in vivo tumor growth inhibition. In total, these studies demonstrate that quantitative fluorogenic imaging can enable the discovery and prioritization of new ADC linkers.