title: A quantitative flow cytometric assay for determining binding characteristics of chimeric, humanized and human antibodies in whole blood: proof of principle with rituximab and ofatumumab.
abstract: Clinical successes of antibody-based drugs has led to extensive (pre-) clinical development of human(ized) monoclonal antibodies in a great number of diseases. The high specificity of targeted therapy with antibodies makes it ideally suited for personalized medicine approaches in which treatments needs are tailored to individual patients. One aspect of patient stratification pertains to the accurate determination of target occupancy and target expression to determine individual pharmacodynamic properties as well as the therapeutic window. The availability of reliable tools to measure target occupancy and expression on diseased and normal cells is therefore essential. Here, we evaluate a novel human antibody detection assay (Human-IgG Calibrator assay), which allows the flow cytometric quantification of therapeutic antibodies bound to the surface of cells circulating in whole blood. This assay not only permits the determination of the number of specific antibody bound per cell (sABC), but, when combined with quantification of exogenously added mouse antibody, also provides information on binding kinetics and antigen modulation. Our data indicate that the calibrator assay has all properties required for a pharmacodynamic tool to quantify target occupancy of chimeric, humanized and human therapeutic antibodies during therapy, as well as to collect valuable information on both antibody and antigen kinetics.
authors.affiliation: Genmab, Utrecht, The Netherlands. p.engelberts@genmab.com
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Engelberts
authors.forename: Carole
authors.initials: C
authors.lastname: Badoil
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Beurskens
authors.forename: Danièle
authors.initials: D
authors.lastname: Boulay-Moine
authors.forename: Karine
authors.initials: K
authors.lastname: Grivel
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: Maxime
authors.initials: M
authors.lastname: Moulard
mesh_terms: D000911:Antibodies, Monoclonal / Q000097:blood / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived / Q000097:blood / Q000494:pharmacology*; D018951:Antigens, CD20 / Q000276:immunology*; D001666:Binding Sites, Antibody; D001769:Blood / Q000187:drug effects / Q000276:immunology*; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000069283:Rituximab; C527517:ofatumumab