title: Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another.
abstract: Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another's binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen's unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.
authors.affiliation: Department of Protein Engineering, Rinat, Oncology Research and Development, Pfizer Inc., South San Francisco, California, United States of America.
authors.forename: Yasmina Noubia
authors.initials: YN
authors.lastname: Abdiche
authors.affiliation: Department of Protein Engineering, Rinat, Oncology Research and Development, Pfizer Inc., South San Francisco, California, United States of America.
authors.forename: Andy Yik
authors.initials: AY
authors.lastname: Yeung
authors.affiliation: Department of Protein Engineering, Rinat, Oncology Research and Development, Pfizer Inc., South San Francisco, California, United States of America.
authors.forename: Irene
authors.initials: I
authors.lastname: Ni
authors.affiliation: Department of Protein Engineering, Rinat, Oncology Research and Development, Pfizer Inc., South San Francisco, California, United States of America.
authors.forename: Donna
authors.initials: D
authors.lastname: Stone
authors.affiliation: Wasatch Microfluidics, Salt Lake City, Utah, United States of America.
authors.forename: Adam
authors.initials: A
authors.lastname: Miles
authors.affiliation: Department of Protein Engineering, Bristol-Myers Squibb, Redwood City, California, United States of America.
authors.forename: Winse
authors.initials: W
authors.lastname: Morishige
authors.affiliation: Compugen USA Inc., South San Francisco, California, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Rossi
authors.affiliation: Department of Protein Engineering, Bristol-Myers Squibb, Redwood City, California, United States of America.
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000941:Antigens / Q000276:immunology; D015374:Biosensing Techniques; D016000:Cluster Analysis; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D057166:High-Throughput Screening Assays; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes