A Homogenous PBMC ADCC Bioassay Enables Bridging Studies with ADCC Reporter Bioassays in Immunotherapy Monoclonal Antibody Development
Stecha, P. et al., Promega Corporation
AACR 2020 Abstract #5658
Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for characterizing antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, these assays are relatively low throughput, present a biosafety risk due to radioactivity, and are prone to donor variability and weak assay signal. Therefore, these assays are not well-suited for implementation in quality-controlled drug development environments.
Previously, we developed a mechanism of action (MOA)-based ADCC reporter bioassay using genetically engineered Jurkat effector cells. When co-cultured with antigen-expressing target cells and a relevant antigen-specific antibody, engagement of FcγRIIIa expressed on the effector cells leads to NFAT-mediated expression of luciferase and a bioluminescent signal. The effector cells are optimized for use in thaw-and-use format and can accurately measure antibody potency for a wide range of therapeutic antibodies targeted to hematopoietic or solid tumors. The bioassay can distinguish effector functions for different antibody IgG isotypes, is stability indicating, and prequalified according to ICH guidelines (precision, accuracy, linearity, and robustness). The ADCC reporter bioassay can be developed as a QC lot release assay for antibody therapeutics that function via an ADCC MOA. However, prior to implementation, a bridging study with a primary cell-based cytotoxicity assay must be performed.
To enable this analysis, we developed a PBMC-based ADCC assay that overcomes limitations of traditional primary cell ADCC assays. First, we established a workflow to prequalify donor PBMCs for ADCC activity, significantly reducing the level of variability introduced with random donor PBMCs. We then developed a platform to genetically engineer target cells with a cytosolic protein (HaloTag) fused to HiBiT, an 11 amino acid protein tag. When cultured with a relevant antibody and ADCC-qualified PBMCs, the target cells are killed and HaloTag-HiBiT is released into the extracellular medium containing the protein LgBiT. Binding of HiBit and LgBiT reconstitutes a luciferase enzyme and produces a bioluminescent signal. The use of HaloTag as a binding partner offers the additional opportunity to image the target cells using fluorescent HaloTag ligands. This improved PBMC ADCC assay is simple, homogenous, highly sensitive, and yields a robust assay window. Importantly, we have demonstrated that this assay accurately measures the potency of a range of antibody therapeutics including rituximab, cetuximab, and trastuzumab, and correlates with the surrogate ADCC reporter bioassay.
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