Abstract 5523: Measuring the Fc effector function of therapeutic antibody in antibody-dependent cell-mediated cytotoxicity using a bioluminescent cell-based reporter bioassay.

Primary peripheral blood mononuclear cells (PBMCs) or the sub-population of natural killer (NK) cells are used to quantify antibody drug biological activity in antibody-dependent cell-mediated cytotoxicity (ADCC) in classic ADCC bioassays. However, cell acquisition and preparation is labor intensive and those ADCC bioassays have high inherent variability, resulting primarily from the primary effector cell source. This variability has been challenging for acceptance of classic ADCC bioassays for use in lot release potency determination and stability-testing for antibody drugs. Here, we developed a cell-based bioassay that can quantify potency of Fc effector function of target cell-bound monoclonal antibody in pathway activation in effector cells that leads to ADCC. For this, Jurkat T-cell lines that stably express NFAT-luciferase reporter and either human FcγRIIIa(V158) or FcγRIIIa (F158) were generated to replace primary PBMCs or NK cells as effector cells in ADCC bioassay while retaining measurement of pathway activation mediated by Fc effector functionality of antibody drugs. Cells were developed in thaw-and-use format, to minimize assay variability due to cell culture and handling. The resultant bioluminescent reporter-based bioassay outperforms classic ADCC bioassays in several key ways: it has good assay precision (low variability), high accuracy, and is simple, robust and specific. In addition, we have demonstrated that the ADCC reporter bioassay is able to quantify potency of rituximab, trastuzumab, and cetuximab, which are on-market monoclonal antibody drugs for cancer using a variety of suspension or adherent target cells, and that this bioassay is stability-indicating for these antibody drugs after heat-treatment. These performance characteristics demonstrate suitability of the ADCC reporter bioassay for lot release potency determination and stability monitoring of antibody drugs. Furthermore, new antibody drugs with enhanced binding affinity to Fc receptors and improved ADCC efficiency are being developed to address improvement of drug efficacy in patient populations. Many of these ‘enhanced ADCC’ antibodies differ structurally in the N-glycan moiety. By testing blended mixtures of intact and glycan-modified antibody drugs in the ADCC reporter bioassay, we have demonstrated the linear correlation between relative antibody biological activity and the percentage of Fc N-glycan modification. The ability of this bioassay to quantifiably differentiate such differences in Fc effector functionality due to N-glycan moiety structural differences demonstrates its suitability as a tool for biosuperior antibody drug research and development. Citation Format: Zhi-Jie Jey Cheng, Denise Garvin, Rich Moravec, Aileen Paguio, Frank Fan, Teresa Surowy. Measuring the Fc effector function of therapeutic antibody in antibody-dependent cell-mediated cytotoxicity using a bioluminescent cell-based reporter bioassay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5523. doi:10.1158/1538-7445.AM2013-5523