Fluorogenic Platform for Real-Time Imaging of Subcellular Payload Release in Antibody-Drug Conjugates

Area of Science:

• Bioconjugate Chemistry
• Cellular Biology
• Drug Delivery Systems

Background:

• Antibody-drug conjugates (ADCs) offer targeted drug delivery but face challenges in controlling payload release timing and location.
• Understanding ADC intracellular trafficking and linker cleavage is crucial for optimizing therapeutic efficacy.

Purpose of the Study:

• To develop novel real-time fluorogenic probes for monitoring ADC subcellular dynamics and payload release.
• To investigate the kinetics of ADC trafficking and linker cleavage within live cells.

Main Methods:

• Design and optimization of sequential pH- and enzyme-activatable naphthalimide fluorophores.
• Live-cell imaging microscopy to track fluorogenic ADC localization and fluorescence activation.
• Assessment of probe impact on ADC recognition and payload release kinetics.

Main Results:

• Fluorogenic probes successfully tracked ADC trafficking along the endolysosomal pathway.
• Linker cleavage was monitored in real-time via OFF-to-ON fluorescence switches.
• ADCs require residence time in lysosomes for efficient linker cleavage, despite successful trafficking.
• The compact fluorogenic probes did not impede ADC targeting or payload release.

Conclusions:

• The developed fluorogenic probes provide a powerful tool for real-time monitoring of ADC intracellular processes.
• This platform enables the study of ADC release kinetics and informs rational ADC design for improved therapeutic outcomes.