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Published on: October 15, 2016
A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for
Yan Li1, Peter Corbett Liu2, Yang Shen2
1Antibody Technology, Eli Lilly and Company, New York, NY, USA yan.li@lilly.com.
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.
Insights
A new assay uses a novel activatable fluorescence-quencher pair to measure antibody internalization and degradation. This method allows for rapid screening of anti-cancer antibody drugs, improving antibody discovery.
Area of Science:
- Biotechnology
- Immunology
- Drug Discovery
Background:
- Therapeutically potent anti-cancer antibody drugs require efficient cellular internalization for efficacy.
- Antibody internalization mediates receptor downregulation and is crucial for antibody-drug conjugate development.
- Current methods for assessing antibody internalization can be cumbersome and require purified antibodies.
Purpose of the Study:
- To develop a novel assay for quantifying antibody internalization and degradation in target cells.
- To enable rapid, high-throughput screening of candidate antibodies for efficient internalization.
- To facilitate early-stage discovery of potent anti-cancer antibody drugs.
Main Methods:
- Development of an activatable fluorescence-quencher pair assay.
- Labeling candidate antibodies with a fluorescent dye and a quencher.
- Utilizing an activatable secondary antibody for screening purified antibodies or those in culture supernatants.
Main Results:
- The assay activates fluorescence upon antibody endocytosis and degradation within cells.
- Demonstrated a process for rapid characterization of candidate antibodies.
- Enabled screening without the need for antibody purification, identifying antibodies with efficient internalization.
Conclusions:
- The novel assay effectively quantifies antibody internalization and degradation.
- This method offers a direct, functional screen for identifying internalizing antibodies early in the drug discovery process.
- The assay supports high-throughput screening, accelerating the development of anti-cancer antibody therapeutics.
