development of a cell-based assay measuring the activation of fcγriia for the characterization of therapeutic monoclonal antibodies推荐.pdf

Development of a Cell-Based Assay Measuring the Activation of FccRIIa for the Characterization of Therapeutic Monoclonal Antibodies Minoru Tada, Akiko Ishii-Watabe*, Takuo Suzuki, Nana Kawasaki Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, Japan Abstract 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 (FccRs), FccRIIIa 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 FccRIIa to mAb-mediated cytotoxicity have been reported. FccRIIa 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 FccRIIa-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 FccRIIa- expressing reporter cell line. The FccRIIa reporter cell assay was able to estimate the activation of FccRIIa 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 FccRIIa 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 FccR