Nadal-Bufi Ferran, Salomon Paulin L, de Moliner Fabio, Sarris Kathy A, Wang Zhi, Wills Rachel D, Marin Violeta L, Shi Xiaona, Zhou Kuo, Wang Zhongyuan, Xu Zhou, McPherson Michael J, Marvin Christopher C, Hobson Adrian D, Vendrell Marc
Centre for Inflammation Research, The University of Edinburgh, Edinburgh EH16 4UU, U.K.
IRR Chemistry Hub, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh EH16 4UU, U.K.
J Am Chem Soc. 2025 Mar 5;147(9):7578-7587. doi: 10.1021/jacs.4c16842. Epub 2025 Feb 18.
Antibody-drug conjugates (ADCs) represent promising therapeutic constructs to enhance the selective delivery of drugs to target cells; however, attaining precise control over the timing and location of payload release remains challenging due to the complex intracellular processes that define ADC internalization, trafficking, and linker cleavage. In this study, we present novel real-time fluorogenic probes to monitor both subcellular dynamics of ADC trafficking and payload release. We optimized a tandem molecular design of sequential pH- and enzyme-activatable naphthalimide fluorophores to (1) track their subcellular localization along the endolysosomal pathway and (2) monitor linker cleavage with OFF-to-ON fluorescence switches. Live-cell imaging microscopy revealed that fluorogenic ADCs can traffic to the lysosomes and yet require residence time in these subcellular compartments for efficient linker cleavage. Notably, the compact size of fluorogenic naphthalimides did not impair the recognition of target cell surface reporters or the kinetics of payload release. This modular platform is applicable to many ADCs and holds promise to inform their rational design for optimal release profiles and therapeutic efficacy.
抗体药物偶联物（ADCs）是一种很有前景的治疗性构建体，可增强药物向靶细胞的选择性递送；然而，由于定义ADC内化、运输和连接子裂解的复杂细胞内过程，要实现对有效载荷释放的时间和位置的精确控制仍然具有挑战性。在本研究中，我们展示了新型实时荧光探针，用于监测ADC运输的亚细胞动力学和有效载荷释放。我们优化了串联分子设计，采用顺序pH和酶激活的萘二甲酰亚胺荧光团，以（1）追踪它们沿内溶酶体途径的亚细胞定位，以及（2）通过从关闭到开启的荧光开关监测连接子裂解。活细胞成像显微镜显示，荧光ADC可以运输到溶酶体，但需要在这些亚细胞区室中停留一段时间以实现有效的连接子裂解。值得注意的是，荧光萘二甲酰亚胺的紧凑尺寸并不影响对靶细胞表面报告分子的识别或有效载荷释放的动力学。这个模块化平台适用于许多ADC，并有望为其合理设计提供依据，以实现最佳的释放曲线和治疗效果。
