A Bioluminescence Resonance Energy Transfer-Based Approach for Determining Antibody-Receptor Occupancy In Vivo

Elucidating receptor occupancy (RO) of monoclonal antibodies (mAbs) is a crucial step in characterizing the therapeutic efficacy of mAbs. However, the in vivo assessment of RO, particularly within peripheral tissues, is greatly limited by current technologies. In the present study, we developed a bioluminescence resonance energy transfer (BRET)-based system that leverages the large signal:noise ratio and stringent energy donor-acceptor distance dependency to measure antibody RO in a highly selective and temporal fashion. This versatile and minimally invasive system enables longitudinal monitoring of the in vivo antibody-receptor engagement over several days. As a proof of principle, we quantified cetuximab-epidermal growth factor receptor binding kinetics using this system and assessed cetuximab RO in a tumor xenograft model. Incomplete ROs were observed, even at a supratherapeutic dose of 50 mg/kg, indicating that fractional target accessibility is achieved. The BRET-based imaging approach enables quantification of antibody in vivo RO and provides critical information required to optimize therapeutic mAb efficacy.