A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence–Quencher Probe as a Tool for Functional Antibody Screening. (August 2015)

• Record Type: Journal Article
• Authors: Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D.; Hiraga, Kaori
• Is Part Of: Journal of biomolecular screening. Volume 20:Number 7(2015)
• Page Start: 869
• Page End: 875
• Publication Date: 2015-08
• Subjects: internalization -- degradation -- antibody labeling -- fluorescent dye -- quencher dye -- flow cytometry -- antibody screening -- cell-based assay; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; 572.36
• DOI: 10.1177/1087057115588511
• Languages: English
• ISSNs: 1087-0571

Abstract

For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody–drug conjugates. Here we describe a novel activatable fluorescence–quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process.