Research paper One-step assay for quantification of neutralizing antibodies to biopharmaceuticals Christophe Lallemand a , Jean-Francois Meritet b , Brigitte Blanchard a , Pierre Lebon b , Michael G. Tovey a, ⁎ a Laboratory of Viral Oncology, CNRS FRE 2937, Institut André Lwoff, Villejuif, France b Laboratory of Virology, Groupe Hospitalier Cochin-Saint-Vincent-de-Paul, Université René Descartes, Paris, France article info abstract Article history: Received 7 January 2010 Received in revised form 24 February 2010 Accepted 2 March 2010 Available online 15 March 2010 Assessment of immunogenicity is an important part of biopharmaceutical drug safety evaluation and a prerequisite for the development of less immunogenic and safer biopharmaceuticals since anti-drug antibodies can impair the activity and compromise the safety of biopharmaceuticals. Although regulatory authorities recommend cell-based assays for detection of neutralizing antibodies (NAbs), such assays are difficult to standardize, and ill adapted to high-throughput analysis. These limitations have been overcome by the development of a unique one-step cell-based assay that allows both drug activity and drug NAbs to be quantified rapidly and with a high degree of precision simply be adding reporter cells to a sample. The reporter cells have been engineered to express firefly luciferase (FL) under the control of a drug-responsive promoter, and to express the drug of interest, the production of which is normalized relative to the expression of Renilla luciferase (RL) transcribed from a common doxycycline-inducible promoter. Residual drug levels present in a sample are first quantified by determination of FL expression, autocrine drug synthesis is then induced, and NAb activity is quantified from the difference in the ratio of FL/RL expression in the presence or absence of the sample. Since assay results are normalized relative to the expression of an internal standard, results are independent of cell number or differences in cell viability thus affording a high degree of assay precision and reducing serum matrix effects to a minimum. This unique assay platform is ideally suited for high-throughput analysis, is applicable to most biopharmaceuticals, and will facilitate standardization and comparison of immunogenicity data. The performance of the one-step assay is illustrated for interferon alpha2 (IFNα2) used widely to treated chronic hepatitis C (HCV) infection and neoplastic disease. © 2010 Elsevier B.V. All rights reserved. Keywords: Cytokines Cell-based assays Interferons Immunogenicity Neutralizing antibodies Reporter gene 1. Introduction Recombinant biopharmaceuticals are an important class of therapeutic agents the safety and efficacy of which can be severely impaired by their immunogenicity. Anti-drug anti- bodies (ADAs) can adversely affect drug pharmacokinetics or even neutralize drug activity resulting in a reduced clinical response or treatment failure (Baert et al., 2003; Kappos et al., 2005; Aarden et al., 2008; Radstake et al., 2009; Bertolotto, 2009). ADAs can also induce hypersensitivity, or potentially life-threatening autoimmunity if they cross-react with a non- redundant endogenous protein such as erythropoietin (EPO) (Casadevall et al., 2002) or megakaryocyte growth and development factor, MGDF (Neumann and Foote, 2000). Assessment of immunogenicity is therefore an important component of drug safety evaluation (Gupta et al., 2007; Baumann, 2009) in both pre-clinical and clinical studies and is a prerequisite for the development of less immunogenic and safer biopharmaceuticals (Koren et al., 2008). Monitoring patients for the presence of ADAs capable of neutralizing drug activity requires the use of cell-based assays that are difficult Journal of Immunological Methods 356 (2010) 18–28 ⁎ Corresponding author. E-mail address: tovey@vjf.cnrs.fr (M.G. Tovey). 0022-1759/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2010.03.003 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim