title: 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.
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).
authors.affiliation: Department of Chemistry, Washington University in St. Louis , St. Louis, Missouri 63130-4889, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Biologics Development, Bristol-Myers Squibb , 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
authors.forename: Hui
authors.initials: H
authors.lastname: Wei
authors.affiliation: Molecular Structure & Design, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Stanley R
authors.initials: SR
authors.lastname: Krystek
authors.affiliation: Process Development, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Derek
authors.initials: D
authors.lastname: Bond
authors.affiliation: Discovery Biology, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Ty M
authors.initials: TM
authors.lastname: Brender
authors.affiliation: Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Cohen
authors.affiliation: Applied Genomics, Bristol-Myers Squibb , 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534, United States.
authors.forename: Jena
authors.initials: J
authors.lastname: Feiner
authors.affiliation: Molecular Structure & Design, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Nels
authors.initials: N
authors.lastname: Hamacher
authors.affiliation: Molecular Structure & Design, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Johanna
authors.initials: J
authors.lastname: Harshman
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Richard Y-C
authors.initials: RY
authors.lastname: Huang
authors.affiliation: Protein Engineering, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Susan H
authors.initials: SH
authors.lastname: Julien
authors.affiliation: Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Kristina
authors.initials: K
authors.lastname: Moore
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Luciano
authors.initials: L
authors.lastname: Mueller
authors.affiliation: Protein Engineering, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Claire
authors.initials: C
authors.lastname: Noriega
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Preeti
authors.initials: P
authors.lastname: Sejwal
authors.affiliation: Protein Engineering, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Paul
authors.initials: P
authors.lastname: Sheppard
authors.affiliation: Protein Engineering, Bristol-Myers Squibb , 1201 Eastlake Ave E., Seattle Washington 98102, United States.
authors.forename: Brenda
authors.initials: B
authors.lastname: Stevens
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Guodong
authors.initials: G
authors.lastname: Chen
authors.affiliation: Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Adrienne A
authors.initials: AA
authors.lastname: Tymiak
authors.affiliation: Department of Chemistry, Washington University in St. Louis , St. Louis, Missouri 63130-4889, United States.
authors.forename: Michael L
authors.identifier: 0000-0003-1159-4636
authors.initials: ML
authors.lastname: Gross
authors.affiliation: Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.
authors.forename: Lumelle A
authors.identifier: 0000-0003-3226-0774
authors.initials: LA
authors.lastname: Schneeweis
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D003001:Cloning, Molecular; D041961:Deuterium Exchange Measurement; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D053759:Interleukin-23 / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D053759:Interleukin-23; D000409:Alanine