title: A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.
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.
authors.affiliation: Antibody Technology, Eli Lilly and Company, New York, NY, USA yan.li@lilly.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Antibody Technology, Eli Lilly and Company, New York, NY, USA.
authors.forename: Peter Corbett
authors.initials: PC
authors.lastname: Liu
authors.affiliation: Antibody Technology, Eli Lilly and Company, New York, NY, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Antibody Technology, Eli Lilly and Company, New York, NY, USA.
authors.forename: Marshall D
authors.initials: MD
authors.lastname: Snavely
authors.affiliation: Antibody Technology, Eli Lilly and Company, New York, NY, USA.
authors.forename: Kaori
authors.initials: K
authors.lastname: Hiraga
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays; D006801:Humans; D018796:Immunoconjugates / Q000494:pharmacology; D021381:Protein Transport; D059748:Proteolysis
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D018796:Immunoconjugates
keywords: antibody labeling; antibody screening; cell-based assay; degradation; flow cytometry; fluorescent dye; internalization; quencher dye