FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody–Drug Conjugates

Despite the recent success of antibody–drug
conjugates (ADCs)
in cancer therapy, a detailed understanding of their entry, trafficking,
and metabolism in cancer cells is limited. To gain further insight
into the activation mechanism of ADCs, we incorporated fluorescence
resonance energy transfer (FRET) reporter groups into the linker connecting
the antibody to the drug and studied various aspects of intracellular
ADC processing mechanisms. When comparing the trafficking of the antibody–FRET
drug conjugates in various different model cells, we found that the
cellular background plays an important role in how the antigen-mediated
antibody is processed. Certain tumor cells showed limited cytosolic
transport of the payload despite efficient linker cleavage. Our FRET
assay provides a facile and robust assessment of intracellular ADC
activation that may have significant implications for the future development
of ADCs.