ADCC Bioassay Effector Cells, Propagation Model, allows for propagation and banking of the engineered Jurkat effector cells developed for the ADCC Reporter Bioassay line of products.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of action (MOA) of antibodies through which virus-infected or other diseased cells are targeted for destruction by components of the cell-mediated immune system. The ADCC Reporter Bioassay is a bioluminescent assay for quantifying Fc effector function of therapeutic antibodies as measured by activation of NFAT signaling pathway (Figure 1). The assay includes effector cel...
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ADCC Bioassay Effector Cells, Propagation Model, allows for propagation and banking of the engineered Jurkat effector cells developed for the ADCC Reporter Bioassay line of products.
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism of action (MOA) of antibodies through which virus-infected or other diseased cells are targeted for destruction by components of the cell-mediated immune system. The ADCC Reporter Bioassay is a bioluminescent assay for quantifying Fc effector function of therapeutic antibodies as measured by activation of NFAT signaling pathway (Figure 1). The assay includes effector cells only; user needs to supply the target cells, reference and test antibodies and all other reagents. The performance characteristics of the ADCC Bioassay Effector Cells make them suitable for application across antibody drug research, development and manufactured lot release. The engineered Jurkat effector cells are generated under highly controlled conditions.
ADCC is a desirable mechanism for killing target cancer cells using antibody-based drugs. The antibody binds to target antigens on the cell surface. When the Fc effector portion of target-bound antibodies also binds to FcγRIIIa receptors on the cell surface of effector cells (natural killer cells predominantly), multiple cross-linking of the two cell types occurs, leading to pathway activation of ADCC MOA. Killing of target cells is an endpoint of this pathway activation and is used in classic ADCC bioassays, which use donor peripheral blood mononuclear cells (PBMCs) or the natural killer (NK) cell subpopulation as effector cells. These cells can be highly variable in response, are tedious to prepare and can result in high background readings.
ADCC Bioassay Effector Cells, which comprise engineered Jurkat cells, use an alternative readout at an earlier point in ADCC MOA pathway activation: the activation of gene transcription through the NFAT (nuclear factor of activated T-cells) pathway in the effector cell. In addition, the ADCC Reporter Bioassay uses engineered Jurkat cells stably expressing the FcγRIIIa receptor, V158 (high affinity) variant, and an NFAT response element driving expression of firefly luciferase as effector cells. Antibody biological activity in ADCC MOA is quantified through the luciferase produced as a result of NFAT pathway activation; luciferase activity in the effector cell is quantified with luminescence readout (Figure 2). Signal is high, and assay background is low.
The ADCC Bioassay Effector Cells exhibit the clear specificity desired for a bioassay, as shown in Figure 3. A good assay response is only obtained when target cells with the correct surface antigen, the correct specific antibody, and effector cells expressing FcγRIIIa are present. If any one of these is missing, there is no response.
The ADCC Bioassay Effector Cells possess performance characteristics suitable for many applications of a bioassay used across antibody drug discovery, development and manufacture; it is stability-indicating and has the precision and accuracy suitable for a lot-release bioassay (Figure 4). Additionally the ADCC Bioassay Effector Cells can be used to quantify effects of glycosylation differences on Fc effector function of antibodies in ADCC MOA (Figure 5), which is useful for ADCC efficiency variant analysis, for example.
Features - Benefits
- Simple, Easy and Homogeneous Assay: Reduced assay-to-assay variability.
- Bioluminescent Reporter Bioassay: Sensitive with excellent signal-to-noise ratios.
- ADCC MOA-Based: Correlates with and suitable replacement for cytotoxic ADCC assays.
- Scalable: Adaptable to 384-well format.
- Tested with FDA-Approved Antibodies.
- Suitable for QC Lot Release: Stability-indicating, excellent linearity, accuracy and precision.
Applications
- Use in bioluminescent, reporter-based ADCC applications (with signal detection in the effector cells).
- Demonstrate ADCC MOA (or lack of).
- Discriminate levels of glycosylation and afucosylation.
- QC lot release bioassay.
- Antibody screening.
Notes
ADCC Bioassay Effector Cells, Propagation Model, allow propagation and banking of the engineered Jurkat effector cells developed for the ADCC Reporter Bioassay line of products. These cells can only be used in bioluminescent, reporter-based ADCC applications (with signal detection in the effector cells). These cells do not possess inherent or added cytotoxicity factors to lyse target cells.
Bio-Glo™ Luciferase Assay System is the required reagent for use with ADCC Bioassay Effector Cells, Propagation Model.
Product includes two vials at 2 × 107 cells/ml and 0.65ml/vial. One vial should be thawed, propagated and cells frozen to create a cell bank. The remaining vial should be reserved as backup.
References
- Hogarth, P.M. and Pietersz, G.A. (2012) Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nature Reviews Drug Discovery 11, 311–31.
- Chung, S. et al. (2012) Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities. mAbs 4, 1–15.
- Parekh, B.S. et al. (2012) Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay. mAbs 4, 1–9.
- Surowy, T. et al. (2012) Low variability ADCC bioassay. Genetic Engineering News 32.
- Cheng, Z.J. et al. (2012) Development of a bioluminescent cell-based bioassay to measure Fc receptor functionality in antibody-dependent cell-mediated cytotoxicity. American Association of Cancer Research (AACR) Annual Meeting, poster #2840.
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