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.

阐明单克隆抗体 (mAb) 的受体占据 (RO) 是表征 mAb 治疗功效的关键步骤。然而， RO的体内评估，特别是在外周组织内，受到当前技术的极大限制。在本研究中，我们开发了一种基于生物发光共振能量转移 (BRET) 的系统，该系统利用大信噪比和严格的能量供体-受体距离依赖性以高度选择性和时间方式测量抗体 RO。这种多功能和微创系统能够对体内进行纵向监测抗体受体参与数天。作为原理证明，我们使用该系统量化了西妥昔单抗-表皮生长因子受体结合动力学，并在肿瘤异种移植模型中评估了西妥昔单抗 RO。即使在 50 mg/kg 的超治疗剂量下，也观察到不完全的 RO，表明实现了部分目标可及性。基于 BRET 的成像方法能够对体内RO抗体进行量化，并提供优化治疗性 mAb 功效所需的关键信息。