title: A high-performance, non-radioactive potency assay for measuring cytotoxicity: A full substitute of the chromium-release assay targeting the regulatory-compliance objective.
abstract: Standardized and biologically relevant potency assays are required by the regulatory authorities for the characterization and quality control of therapeutic antibodies. As critical mechanisms of action (MoA) of antibodies, the antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) must be characterized by appropriate potency assays. The current reference method for measuring cytotoxicity is the51Cr-release method. However, radioactivity handling is difficult to implement in an industrial context because of environmental and operator protection constraints. Alternative non-radioactive methods suffer from poor validation performances and surrogate assays that measure FcγR-dependent functions do not comply with the regulatory requirement of biological relevance. Starting from these observations, we developed a non-radioactive luminescent method that is specific for target cell cytolysis. In adherent and non-adherent target cell models, the ADCC (using standardized effector cells) or CDC activities of rituximab, trastuzumab and adalimumab were compared in parallel using the51Cr or luminescent methods. We demonstrated that the latter method is highly sensitive, with validation performances similar or better than the51Cr method. This method also detected apoptosis following induction by a chemical agent or exposure to ultraviolet light. Moreover, it is more accurate, precise and specific than the concurrent non-radioactive calcein- and TR-FRET-based methods. The method is easy to use, versatile, standardized, biologically relevant and cost effective for measuring cytotoxicity. It is an ideal candidate for developing regulatory-compliant cytotoxicity assays for the characterization of the ADCC, CDC or apoptosis activities from the early stages of development to lot release.
authors.affiliation: a Clean Cells , Boufféré , France.
authors.forename: Alexis
authors.initials: A
authors.lastname: Rossignol
authors.affiliation: a Clean Cells , Boufféré , France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Bonnaudet
authors.affiliation: b Centre Hospitalier Universitaire de Nantes , Nantes cedex 01 , France.|c UMR INSERM U892 , Nantes Cedex , France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Clémenceau
authors.affiliation: c UMR INSERM U892 , Nantes Cedex , France.
authors.forename: Henri
authors.initials: H
authors.lastname: Vié
authors.affiliation: a Clean Cells , Boufféré , France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Bretaudeau
mesh_terms: D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D006801:Humans; D008156:Luciferases; D008163:Luminescent Measurements / Q000379:methods*
chemical_list: D008156:Luciferases
keywords: ADCC; CDC; apoptosis; cell-based assay; cytotoxicity; potency; regulatory compliance; therapeutic antibody