Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcc receptors (Fc gamma Rs), Fc gamma RIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of Fc gamma RIIa to mAb-mediated cytotoxicity have been reported. Fc gamma RIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via Fc gamma RIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human Fc gamma RIIa-expressing reporter cell line. The Fc gamma RIIa reporter cell assay was able to estimate the activation of Fc gamma RIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate Fc gamma RIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the Fc gamma RIIa activation by EGFR-bound cetuximab. These results suggest that our Fc gamma RIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.