title: Robust Antibody-Antigen Complexes Prediction Generated by Combining Sequence Analyses, Mutagenesis, In Vitro Evolution, X-ray Crystallography and In Silico Docking.
abstract: Hu 15C1 is a potent anti-human Toll-like receptor 4 (TLR4) neutralizing antibody. To better understand the molecular basis of its biological activity, we used a multidisciplinary approach to generate an accurate model of the Hu 15C1-TLR4 complex. By combining site-directed mutagenesis, in vitro antibody evolution, affinity measurements and X-ray crystallography of Fab fragments, we identified key interactions across the Hu 15C1-TLR4 interface. These contact points were used as restraints to predict the structure of the Fab region of Hu 15C1 bound to TLR4 using computational molecular docking. This model was further evaluated and validated by additional site-directed mutagenesis studies. The predicted structure of the Hu 15C1-TLR4 complex indicates that the antibody antagonizes the receptor dimerization necessary for its activation. This study exemplifies how iterative cycles of antibody engineering can facilitate the discovery of components of antibody-target interactions.
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Loyau
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Gérard
authors.initials: G
authors.lastname: Didelot
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Pauline
authors.initials: P
authors.lastname: Malinge
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Ulla
authors.initials: U
authors.lastname: Ravn
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Magistrelli
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Depoisier
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Guillemette
authors.initials: G
authors.lastname: Pontini
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Yves
authors.initials: Y
authors.lastname: Poitevin
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Marie
authors.initials: M
authors.lastname: Kosco-Vilbois
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Fischer
authors.affiliation: Department of Molecular Biology, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva, Switzerland.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Thore
authors.affiliation: Novimmune SA, Chemin des Aulx 14, 1228 Plan-les-Ouates, Switzerland. Electronic address: frousseau@novimmune.com.
authors.forename: François
authors.initials: F
authors.lastname: Rousseau
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology / Q000648:ultrastructure*; D001666:Binding Sites, Antibody / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D061505:Cell Surface Display Techniques; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000648:ultrastructure*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013045:Species Specificity; D020349:Surface Plasmon Resonance; D051197:Toll-Like Receptor 4 / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4
keywords: Fab; TLR4; crystallography; docking; in vitro evolution