Abstract 3740: Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors
Authors: Zhi-Jie Jey Cheng, Denise Garvin, Aileen Paguio, Rich Moravec, Frank Fan, Teresa Surowy
Journal: Cancer Research (2014)

Abstract Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to clinical efficacy of a broad range of therapeutic antibodies. FcγRIIIa polymorphisms of individual cancer patients are correlated with clinical efficacy of several antibody drugs. An understanding of in vitro antibody activity via both Fc receptor variants is the key to ultimately understanding drug efficacy in vivo. Yet, with classic ADCC assays that rely on primary effector cells which are highly heterogeneous and variable, it remains challenging to quantitatively measure antibody ADCC activity and evaluate the impact of FcγRIIIa polymorphisms by in vitro ADCC assays. To address this problem, we developed a pair of reporter-based ADCC assays, where two engineered effector cell lines were generated in Jurkat T-cells which stably express a NFAT-RE driven luciferase reporter and either FcγRIIIa/V158 or FcγRIIIa/F158 polymorphism variant. The engineered Jurkat effector cells were further developed in frozen, thaw-and-use format to reduce handling time and minimize assay variability. We compared the V variant ADCC reporter assay with the classic ADCC assay using PBMCs from homozygous 158VV donors, by testing a panel of glyco-modified trastuzumab mixtures. The results demonstrate that the reporter-based ADCC assays using engineered Jurkat effector cells provide ADCC biological activity ranking equivalent to that obtained using classic PBMC-based ADCC assay. When tested side by side, both V variant and F variant ADCC reporter assays appropriately measure the biological activities in ADCC pathway activation of various human and mouse antibody isotypes of rituximab. The two ADCC reporter assays are also able to measure antibody potencies for multiple therapeutic antibodies, in various native target cell systems including suspension and adherent cell lines, and also genetically engineered cell lines such as a membrane-bound TNFα cell line. When tested in the same antibody/target cell system, the V variant ADCC assay showed higher antibody biological activity in ADCC reporter response than the F variant ADCC assay. This result appropriately reflects the reported impact of FcγRIIIa polymorphism on antibody binding and ADCC activity. In summary, the pair of V and F variant ADCC reporter assays provides a valuable approach to quantitatively measure the potency of therapeutic antibodies in ADCC and evaluate the impact of FcγRIIIa polymorphism in drug discovery and development for antibody therapeutics. Citation Format: Zhi-Jie Jey Cheng, Denise Garvin, Aileen Paguio, Rich Moravec, Frank Fan, Teresa Surowy. Paired ADCC reporter bioassays enable quantification and differentiation of antibody Fc-effector activities via V158 and F158 variant FcγRIIIa receptors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2014-3740