PO.ET02.05 · 实验与分子治疗
用于定量评估抗体-药物偶联物内化的实时成像
Real-time imaging for quantitative assessment of antibody-drug conjugate internalization
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
抗体-药物偶联物（ADC）代表了癌症治疗的重大进步，其最佳疗效依赖于高效的靶点介导内化。这凸显了对精确、便捷且经济高效的方法的需求，以监测抗体内化，用于候选物筛选、作用机制研究和安全性评估。我们提出了一种稳健的实时定量检测方法，结合使用Agilent xCELLigence RTCA eSight系统与CypHer5E-NHS-酯标记。CypHer5E是一种pH敏感的花青染料，在酸性环境中发出荧光，可实现对抗体转运进入胞内区室的动态追踪。采用这一方法，我们证明了CypHer5E标记的trastuzumab通过受体介导的摄取在HER2阳性细胞系中的选择性内化。初级CypHer5E标记和次级Fab-CypHer5E偶联均能够清晰可视化内化事件。此外，次级Fab-CypHer5E标记便于对多种HER2靶向治疗性抗体进行比较分析。这一集成平台为机制研究和药物筛选实验提供了强大工具，为在生物制药开发中优化抗体疗效、安全性和药代动力学提供了关键洞见。
查看英文原文 English abstract
Antibody-drug conjugates (ADCs) represent a major advancement in cancer therapy, relying on efficient target-mediated internalization for optimal efficacy. This underscores the need for precise, convenient, and cost-effective methods to monitor antibody internalization for candidate selection, mechanism-of-action studies, and safety evaluation. We present a robust, real-time quantitative assay using the Agilent xCELLigence RTCA eSight system combined with CypHer5E-NHS-Ester labeling. CypHer5E is a pH-sensitive cyanine dye that fluoresces in acidic environments, enabling dynamic tracking of antibody trafficking into intracellular compartments. Using this approach, we demonstrated selective internalization of CypHer5E-labeled trastuzumab in HER2-positive cell lines via receptor-mediated uptake. Both primary CypHer5E labeling and secondary Fab-CypHer5E conjugation enabled clear visualization of internalization events. Furthermore, secondary Fab-CypHer5E labeling facilitated comparative analysis of multiple HER2-targeting therapeutic antibodies. This integrated platform provides a powerful tool for mechanistic studies and drug-screening assays, offering critical insights to optimize antibody efficacy, safety, and pharmacokinetics in biopharmaceutical development.
利益披露 Disclosure
T. Wang, None..
Y. Pu, None..
R. Raver, None..
X. Zhang, None..
P. Ye, None..
N. Li, None.