Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies

ABSTRACT

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

KEYWORDS:

• Alanine scan
• cross-linking
• Epitope mapping
• Hydrogen deuterium exchange
• hydroxyl radical footprinting
• monoclonal antibodies

Acknowledgments

Ryan Wenzel from CovalX (Saugus, MA, USA), John Schenkel and Mark Chance from NEO Proteomics, Inc. (Cleveland, OH, USA) and Jennifer Pawlowski of Merck & Co., Inc., Kenilworth, NJ, USA for editorial support.

Disclosure statement

D.L. Hulsik and J. Kreijtz are employed by, and may own stock in, Aduro Biotech Europe who partially provided funding for this study and own stock in Chinook Therapeutics (KDNY). A. Beebe, X. Dang, M. Beaumont, E. Hsieh, G. Ermacov, T. Fischmann, and V. Juan, are employed by, and may own stock in, Merck & Co., Inc., Rahway, NJ, USA, who partially provided funding for this study. A. Nazabal is employed by, and may own stock in, CovalX. Inc, who provided chemical cross-linking data for this study. L. Guelen and H.v. Eenennaam hold patents in Merck Sharpe & Dohme Corp and own Chinook stock.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2285285

Additional information

Funding

Merck Sharp & Dohme Corp. LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and Aduro Biotech Europe B.V.