Expression of human FcγRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human Fc gamma RIIIa, the key receptor for ADCC, an attractive alternative method of assessment. Here, we describe the development of cell lines and assays for this purpose. The transmembrane receptor, Fc gamma RIIIa, requires co-expression with signal transducing subunits to prevent its degradation, unlike the homologous receptor Fc gamma RIIIb that is expressed as a GPI-anchored molecule. Therefore, to simplify the production of cell lines as reliable assay components, we expressed Fc gamma RIIIa as a GPI-anchored molecule. Separate, stable CHO cell lines that express either the 158F or the higher-affinity 158V allotype of Fc gamma RIIIa were isolated using fluorescence-activated cell sorting. The identities of the expressed receptors were confirmed using a panel of monoclonal antibodies that distinguish between subclasses and allotypes of Fc gamma RIII and the cell lines were shown to have slightly higher levels of receptor than Fc gamma RIII-positive peripheral blood mononuclear cells. Because the affinity of Fc gamma RIIIa for IgG is intermediate amongst the receptors that bind IgG, we were able to use these cell lines to develop flow cytometric assays to measure the binding of both complexed and monomeric immunoglobulin. Thus, by choosing the appropriate method, weakly- or strongly-binding IgG can be efficiently compared. We have quantified the difference in the binding of wildtype IgG1 and IgG3 molecules to the two functional allotypes of the receptor and report that the Fc gamma RIIIa-158V-antibody interaction is 3- to 4-fold stronger that the interaction with Fc gamma RIIIa-158F. Overall, these robust assays should be valuable for batch-testing clinical material as well as providing tools for improving the design of therapeutic IgG. (C) 2010 Elsevier B.V. All rights reserved.