Real-time imaging for quantitative assessment of antibody-drug conjugate internalization

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.