title: Validation of a flow cytometry-based assay to assess C5aR receptor occupancy on neutrophils and monocytes for use in drug development.
abstract: The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial.
authors.affiliation: Research and Development Department, ICON Laboratory Services, Farmingdale, New York.
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Quadrini
authors.affiliation: Department of Development Bioanalysis, Novo Nordisk A/S, DK-2760, Maaloev, Denmark.
authors.forename: Anne Charlotte
authors.initials: AC
authors.lastname: Hegelund
authors.affiliation: Research and Development Department, ICON Laboratory Services, Farmingdale, New York.
authors.forename: Kasia E
authors.initials: KE
authors.lastname: Cortes
authors.affiliation: Research and Development Department, ICON Laboratory Services, Farmingdale, New York.
authors.forename: Chengsen
authors.initials: C
authors.lastname: Xue
authors.affiliation: Cellular Immunology, ICON Laboratory Services, Leopardstown, Dublin, Ireland.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Kennelly
authors.affiliation: Department of PharmacoDynamics, Novo Nordisk A/S, DK-2760, Maaloev, Denmark.
authors.forename: Hong
authors.initials: H
authors.lastname: Ji
authors.affiliation: Department of PharmacoDynamics, Novo Nordisk A/S, DK-2760, Maaloev, Denmark.
authors.forename: Carl-Magnus
authors.initials: CM
authors.lastname: Högerkorp
authors.affiliation: Research and Development Department, ICON Laboratory Services, Farmingdale, New York.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Mc Closkey
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology; D044087:Receptor, Anaphylatoxin C5a / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C488033:C5AR1 protein, human; C000612951:NNC0215-0384; D044087:Receptor, Anaphylatoxin C5a
keywords: biomarker; clinical trial; drug development; flow cytometry; receptor occupancy; validation