A Novel System for the Quantification of the ADCC Activity of Therapeutic Antibodies

Received May 23, 2017; Revised Jul 19, 2017; Accepted Aug 1, 2017

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1. Introduction

The activity of numerous therapeutic antibodies is mediated in part by immune-mediated effector cell function following binding of the variable regions of the antibody to a specific antigen on the surface of target cells and the interaction of the Fc moiety of the antibody with an Fc receptor on an immune effector cell [1]. This leads to clustering of the Fc receptors, tyrosine phosphorylation, calcium flux, and activation of several transcription factors in the immune effector cell(s). Antibody-mediated effector functions, in particular, antibody-dependent cellular cytotoxicity (ADCC), are considered to play a determining role in the action of a number of therapeutic antibodies including rituximab, trastuzumab, and cetuximab [2]. Traditional methods for quantifying ADCC activity are labor intensive and have a high level of inherent variability [3]. This is due to the use of primary human peripheral blood mononuclear cells (PBMC) or natural killer (NK) cells from different donors as the effector cells and the use of a complex endpoint which is difficult to standardize, namely, cytotoxicity. Although the traditional ⁵¹CR release assay has been largely replaced by alternative assays using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), calcein-acetoxymethyl, or lactate dehydrogenase-release assays or various flow cytometric assays using Annexin V, propidium iodide, or 7-amino-actinomycin D, all these assays are subject to poor reproducibility, low sensitivity, and high levels of spontaneous release [4]. These limitations have been overcome in part by the use of engineered effector cells expressing the low-affinity Fc receptor, FcγRIIIA (CD16a), that responds to ligation of the Fc moiety of an antibody bound to the specific antigen expressed on target cells by activation of a NFAT responsive reporter gene construct [5, 6]; there is a need, however, for a method for the evaluation of ADCC activity with improved sensitivity and specificity. We describe herein a method for the quantification of ADCC activity based on the use of...