title: Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.
abstract: Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs.
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States. Electronic address: zhengh@amgen.com.
authors: Jenny
authors: J
authors: Hu
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Iwona
authors: I
authors: Wala
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Hong
authors: H
authors: Han
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Janice
authors: J
authors: Nagatani
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Troy
authors: T
authors: Barger
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Francesca
authors: F
authors: Civoli
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Arunan
authors: A
authors: Kaliyaperumal
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States.
authors: Yao
authors: Y
authors: Zhuang
authors: Clinical Immunology, Amgen Inc., Thousand Oaks, CA 91320, United States. Electronic address: shalinig@amgen.com.
authors: Shalini
authors: S
authors: Gupta
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects / Q000276:immunology; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004921:Erythropoietin / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007158:Immunologic Techniques / Q000379:methods*; D015847:Interleukin-4 / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D011485:Protein Binding / Q000187:drug effects / Q000276:immunology; D017467:Receptors, Erythropoietin / Q000378:metabolism; D019948:Receptors, Interleukin-4 / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: C549611:AMG 317; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D008024:Ligands; D017467:Receptors, Erythropoietin; D019948:Receptors, Interleukin-4; D004921:Erythropoietin; D015847:Interleukin-4
keywords: Cell-based assay; Competitive ligand binding assay; Immunogenicity; NAb assay; Neutralizing antibodies; Non-cell-based assay