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 provided in frozen, thaw-and-use format and reagents and an optimized protocol to provide a bioassay that has low variability and high accuracy. Moreover the bioassay can be performed in a single day. These performance characteristics make the bioassay suitable for application across antibody drug research, development and manufactured lot release. The thaw-and-use cells provided in the ADCC Reporter Bioassay kits are generated under highly controlled conditions that drive low assay variability run to run, while providing the convenience of an assay reagent that eliminates the need to propagate and prepare cells each time.
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.
The ADCC Reporter Bioassay uses 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 Reporter Bioassay exhibits 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 Reporter Bioassay has 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 assay 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
• Cells in Frozen, Thaw-and-Use Format: No propagation and cell culture fuss
• 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
• Demonstrate ADCC MOA (or lack of)
• Discriminate levels of glycosylation and afucosylation
• QC lot release bioassay
• Antibody screening