title: A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.
abstract: Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA. wuyu@medimmune.com.
authors.forename: Yuling
authors.identifier: http://orcid.org/0000-0002-6922-7452
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Jia J
authors.initials: JJ
authors.lastname: Li
authors.affiliation: Analytical Biotechnology Development, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Hyun Jun
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Weiyi
authors.initials: W
authors.lastname: Liu
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Akhgar
authors.affiliation: Antibody Development and Protein Engineering, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bowen
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Spitz
authors.affiliation: Analytical Biotechnology Development, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Xu-Rong
authors.initials: XR
authors.lastname: Jiang
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Lorin K
authors.initials: LK
authors.lastname: Roskos
authors.affiliation: Clinical Pharmacology and DMPK, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, USA.
authors.forename: Wendy I
authors.initials: WI
authors.lastname: White
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized / Q000097:blood* / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000097:blood*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects* / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007694:Killer Cells, Natural / Q000187:drug effects / Q000378:metabolism; D051379:Mice
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; C571386:benralizumab
keywords: ADCC; benralizumab; cell-based assay; mechanism of action; neutralizing antibody