Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis

  • Anal Chem. 2017 Feb 21;89(4):2250-2258. doi: 10.1021/acs.analchem.6b03058.
Jing Li  1 ,  Hui Wei  2 ,  Stanley R Krystek Jr  3 ,  Derek Bond  4 ,  Ty M Brender  5 ,  Daniel Cohen  6 ,  Jena Feiner  7 ,  Nels Hamacher  3 ,  Johanna Harshman  3 ,  Richard Y-C Huang  8 ,  Susan H Julien  9 ,  Zheng Lin  6 ,  Kristina Moore  6 ,  Luciano Mueller  8 ,  Claire Noriega  9 ,  Preeti Sejwal  8 ,  Paul Sheppard  9 ,  Brenda Stevens  9 ,  Guodong Chen  8 ,  Adrienne A Tymiak  8 ,  Michael L Gross  1 ,  Lumelle A Schneeweis  6
Affiliations
  • 1. Department of Chemistry, Washington University in St. Louis , St. Louis, Missouri 63130-4889, United States.
  • 2. Biologics Development, Bristol-Myers Squibb , 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
  • 3. Molecular Structure & Design, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
  • 4. Process Development, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
  • 5. Discovery Biology, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
  • 6. Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
  • 7. Applied Genomics, Bristol-Myers Squibb , 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
  • 8. Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
  • 9. Protein Engineering, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
PMID: 28193005 DOI: 10.1021/acs.analchem.6b03058
Abstract

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).

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