title: Label-free epitope binning assays of monoclonal antibodies enable the identification of antigen heterogeneity.
abstract: Label-free biosensors are often used in the discovery of therapeutic antibodies to characterize the epitope binding regions of a panel of monoclonal antibodies that target a specific antigen, thus facilitating their organization into epitope groups or "bins". When tested in a pairwise combinatorial manner, two antibodies that compete with one another for binding to a specific antigen may be grouped into the same epitope bin - that is, they recognize similar or overlapping epitopes - whereas two antibodies that bind simultaneously to the antigen are placed into different epitope bins. However, depending on the assay format used, results from such experiments can sometimes contradict one another. Here, we provide two examples that illustrate how antigen heterogeneity, either inherent in an antigen sample, or induced by the assay conditions, can confound the interpretation of epitope binning results and, in some cases, lead to erroneous conclusions. We highlight why assays that employ solution antigen are often more reliable than those that employ immobilized antigen and, by corroborating our binning results with assays that utilize native antigen, we determine which subpopulations of our heterogeneous antigen samples are biologically relevant and thus improve the correlation between epitope bins and functional activity. Furthermore, we provide recommendations for performing definitive binning assays and a diagnostic assay procedure that can be followed when antigen heterogeneity is suspected.
authors.affiliation: Rinat-Pfizer Inc, South San Francisco, CA 94080, USA. Yasmina.Abdiche@pfizer.com
authors.forename: Yasmina N
authors.initials: YN
authors.lastname: Abdiche
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Lindquist
authors.forename: Donna M
authors.initials: DM
authors.lastname: Stone
authors.forename: Arvind
authors.initials: A
authors.lastname: Rajpal
authors.forename: Jaume
authors.initials: J
authors.lastname: Pons
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000235:genetics* / Q000276:immunology; D015374:Biosensing Techniques; D000939:Epitopes / Q000276:immunology*; D050556:Fatty Acid-Binding Proteins / Q000032:analysis / Q000276:immunology; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology; D008969:Molecular Sequence Data; D000077153:Progranulins
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; C493410:FABP4 protein, human; D050556:Fatty Acid-Binding Proteins; C494993:GFRA3 protein, human; C492314:GRN protein, human; D051097:Glial Cell Line-Derived Neurotrophic Factor Receptors; D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins