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 Fcγ receptors (FcγRs), FcγRIIIa 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 FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa 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 FcγRIIa-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 FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa 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 FcγRIIa 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 FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.
Highlights
Antibody-dependent cellular cytotoxicity (ADCC) plays an important role in anti-tumor activity of therapeutic monoclonal antibodies targeting tumor cells.[1,2,3] To date, many mAbs exhibiting ADCC activity have been approved and have contributed to anticancer therapy (e.g., the anti-EGFR mAb cetuximab for colorectal cancers, the anti-CD20 mAb rituximab for B-cell lymphomas, and the antiHER2 mAb trastuzumab for metastatic breast cancers)
With the addition of rituximab, the calcein/calcein-violet double-positive population was significantly increased (Fig. 1C), indicating that the Jurkat/Fcc receptors (FccRs) cells recognized rituximab bound to CD20 at the surface of the Daudi cells; that is, Daudi and Jurkat/FccR cells were bridged by rituximab. These results suggest that the Jurkat/FccRIIa and Jurkat/FccRIIIa cell lines we developed can function as reporter cells mimicking FccRexpressing immune cells such as natural killer (NK) cells and macrophages
We found that the luciferase activity of Jurkat/FccRIIa/NFATLuc cells as well as Jurkat/FccRIIIa/NFAT-Luc cells was dosedependently increased by rituximab in co-culture with Daudi cells (Fig. 2B), suggesting that the activation of FccRIIa by the bridging of target and effector cells via mAbs can be assessed by using Jurkat/FccRIIa/NFAT-Luc cells
Summary
Antibody-dependent cellular cytotoxicity (ADCC) plays an important role in anti-tumor activity of therapeutic monoclonal antibodies (mAbs) targeting tumor cells.[1,2,3] To date, many mAbs exhibiting ADCC activity have been approved and have contributed to anticancer therapy (e.g., the anti-EGFR (epidermal growth factor receptor) mAb cetuximab for colorectal cancers, the anti-CD20 mAb rituximab for B-cell lymphomas, and the antiHER2 mAb trastuzumab for metastatic breast cancers). There are four types of activating FccRs: FccRI, FccRIIa, FccRIIIa and FccRIIIb.[17] FccRIIIa is the only activating FccR expressed on NK cells, and it is thought to play a pivotal role in ADCC induced by IgG1 subclass mAbs. Its significance in clinical efficacy is supported by reports of correlations between FccRIIIa polymorphism (F158V) and response to rituximab [18,19] or other mAbs. Much of the Fc engineering in tumor-targeting mAbs has increased the affinity to FccRIIIa, resulting in enhanced ADCC activity via NK cells
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