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• W2082091678 abstract "Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLWe have developed a bioluminescent cell-based reporter bioassay with reduced variability and complexity compared with traditional antibody-dependent cell-mediated cytotoxicity (ADCC) bioassays, in order that more accurate and precise quantification of therapeutic antibody Fc functionality can be achieved.Traditional bioassays to quantify ADCC are labor intensive, have high inherent assay variability, and routinely use primary NK cells from blood donors. Our bioluminescent cell-based reporter bioassay is based on the same NFAT pathway-based activation of gene transcription which occurs in NK cells after ligation of FcγRIIIA with target cell-bound antibody in a traditional ADCC assay system. In NK cells this leads to activation of cytokine and FasL gene transcription; FasL participates in lysis of target cells. We generated Jurkat T-cell lines which stably express both human FcγRIIIA and an NFAT-luciferase reporter. These cell lines were evaluated as effector cell replacements of primary NK effector cells in bioassays. The resultant reporter bioassay signal was robust and exhibited appropriate specificity, meaning robust signal above background and appropriate dose-response were achieved and were absolutely dependent on specific target and effector cells, and specific antibody. A “best” effector cell clone was selected based on bioassay performance and stability of the clone.Two types of target systems were evaluated: suspension CD20+ B cell targets with rituximab (anti-CD20) and adherent Her2+ cell targets with trastuzumab (anti-Her2). Our reporter bioassay performed well with both target test systems to quantify biological activity of the therapeutic antibodies.With a goal toward obtaining high inter-assay precision of the bioassay, we evaluated growing and freezing cells under specific conditions to identify those which allowed the cells to be ready for bioassay with minimal handling after thaw and we were able to identify such conditions. Additionally, we identified passage range suitable for banking vials of cells to which we can return time after time, obviating the variability that can arise when using continuously cultured cells for bioassay.We further optimized our bioassay using design of experiments (DoE) and established range of effector to target cell ratios over which the assay gave suitable bioassay performance in test systems. A qualification study was performed for characterization of precision, accuracy, linearity across potency range and stability-indicating ability.The bioluminescent cell-based reporter bioassay provides a robust, low variability and easier-to-perform alternative to traditional ADCC bioassays for quantification of Fc effector functionality of therapeutic antibody candidates.Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2011-1768" @default.
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• W2082091678 date "2011-04-15" @default.
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• W2082091678 title "Abstract 1768: A bioluminescent cell-based reporter bioassay alternative to ADCC bioassay" @default.
• W2082091678 doi "https://doi.org/10.1158/1538-7445.am2011-1768" @default.
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