A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity

Abstract

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 (EC 80 )] 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 dosedependent 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.

Sialic acid–binding immunoglobulin-like lectin (Siglec) 8 in patients with eosinophilic disorders: Receptor expression and targeting using chimeric antibodies

“…3 Chimeric 2E2 IgG 1 (c2E2 IgG 1 ) is a chimeric afucosylated IgG 1 antibody with the same specificity as c2E2 IgG 4 that is anticipated to enhance antibody-dependent cellular cytotoxicity (ADCC) through natural killer (NK) cell engagement of CD16, as described with benralizumab, an afucosylated IgG 1 anti-IL-5 receptor antibody approved for the treatment of asthma. 8,9 Although prior studies in small numbers of NDs and patients with HESs have demonstrated Siglec-8 expression on peripheral blood eosinophils 10 and detected soluble Siglec-8 (sSiglec-8) in sera from some patients with HESs, 11 little is known about the variability and regulation of Siglec-8 expression in vivo. A weak correlation was noted between serum sSiglec-8 levels and absolute eosinophil counts (AECs).…”

Selection of a Ligand-Binding Neutralizing Antibody Assay for Benralizumab: Comparison with an Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Cell-Based Assay

“…Methods for quality control preparation, screening cutoff determination, sensitivity, drug tolerance, selectivity, cell culture, and assay procedures for the ADCC cell-based NAb assay have been described in detail (15). In brief, the validated CBA is an ADCC assay that consists of target cells (a murine cytotoxic cell line CTLL-2 stably transfected with human IL-5R) and an effector cell line (human NK92 cells).…”

“…For the CLBA, the concentrations of the polyclonal anti-benralizumab idiotype antibody were 2.0, 1.5, 1.0, 0.75, 0.5, 0.25, 0.1, 0.05, 0.025, 0.0125, and 0 μg/mL; and the benralizumab concentrations were 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, and 0 μg/mL. For the CBA, the concentrations of the polyclonal anti-benralizumab idiotype antibodies were 10, 5, 2.5, 1.25, 0.63, 0.31, and 0 μg/mL, and the benralizumab concentrations were 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, and 0 μg/mL (15). For the binding ADA assay, the concentrations of the polyclonal anti-benralizumab idiotype antibody were 4.0, 2.0, 1.0, 0.5, 0.25, and 0 μg/mL, and the benralizumab concentrations were 500, 250, 100, 50, 25, and 0 μg/mL.…”

“…Two operators analyzed each sample in duplicate wells in four assays over 2 days. The data set consisting of 320 normalized responses (signal to background values) was evaluated by statistical methods for the determination of the cutoff factor with a 1% false-positive rate (15).…”

Drug Target Interference in Immunogenicity Assays: Recommendations and Mitigation Strategies

“…Nonspecific matrix interference in a cell-based NAb assay can be a limitation and can often be eliminated by sample dilution, provided that the assay sensitivity and drug tolerance limit are not adversely impacted. In a cell-based NAb assay, either the drug target (soluble shed receptor) or a competitive antagonist to the drug (ligand) may be present as potential specific interfering factors [33].…”