title: IL-1beta epitope mapping using site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry analysis.
abstract: Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus, and rabbit IL-1beta but only weakly binds to mouse and rat IL-1beta. Biacore experiments demonstrated that Hu007 and the type-I IL-1 receptor competed for binding to IL-1beta. Increasing salt concentrations decrease the association rate with only moderate effects on the dissociation rate, suggesting that long-range electrostatics are critical for formation of the initial complex. To understand the ligand-binding specificity of Hu007, we have mapped the critical residues involved in the recognition of IL-1beta. Selected residues in cynomolgus IL-1beta were mutated to the corresponding residues in mouse IL-1beta, and the effects of the changes on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically, substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobic interactions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N- and C-terminal regions of cIL-1beta with those found in mouse IL-1beta (V3I/S5Q/F150S) decreased the binding affinity of Hu007 to IL-1beta by about 1000-fold. Conversely, mutating the corresponding residues in mouse IL-1beta to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1beta were protected from exchange because of antibody binding. The results from this study demonstrate that Hu007 binds to a region located in the open end of the beta-barrel structure of IL-1beta and blocks binding of IL-1beta to its receptor.
authors.affiliation: Biotechnology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. Lu_Jirong@lilly.com
authors.forename: Jirong
authors.initials: J
authors.lastname: Lu
authors.forename: Derrick R
authors.initials: DR
authors.lastname: Witcher
authors.forename: Melissa A
authors.initials: MA
authors.lastname: White
authors.forename: Xiliang
authors.initials: X
authors.lastname: Wang
authors.forename: Lihua
authors.initials: L
authors.lastname: Huang
authors.forename: Radhakrishnan
authors.initials: R
authors.lastname: Rathnachalam
authors.forename: John M
authors.initials: JM
authors.lastname: Beals
authors.forename: Stuart
authors.initials: S
authors.lastname: Kuhstoss
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D001666:Binding Sites, Antibody / Q000502:physiology; D003903:Deuterium / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods; D006801:Humans; D007375:Interleukin-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008252:Macaca fascicularis; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D007375:Interleukin-1; D003903:Deuterium