Teresa Surowy, Jey Cheng, Rich Moravec, Denise Garvin, Aileen Paguio, Neal Cosby, Richard Somberg and Frank Fan
Promega Corporation, Madison, WI 53711
Primary peripheral blood mononuclear cells (PBMCs) or the sub-population of natural killer (NK) cells are used in traditional ADCC bioassays to quantify antibody drug biological activity. However, cell acquisition and preparation is labor intensive and the ADCC bioassay has high inherent variability, resulting primarily from the primary effector cell source. This variability has been challenging for acceptance of traditional ADCC bioassays for use in lot release potency and stability bioassays of antibody drugs.
We have developed an alternative cell-based bioassay that can quantify potency of Fc effector function of target cell-bound monoclonal antibodies in ADCC mechanism of action. For this, Jurkat T-cells that stably express NFAT-luciferase reporter and human FcgRIIIa were generated to replace primary PBMCs or NK cells as effector cells in ADCC bioassay while retaining measurement of pathway activation in ADDC MOA. Cells were developed in thaw-and-use format, for immediate use, to minimize assay variability due to cell culture and handling. Cell preparation, and bioassay protocol and reagents were optimized. The resultant bioluminescent ADCC reporter bioassay outperforms classic ADCC bioassays in several key ways: it has good assay precision (low variability), high accuracy, and is simple, robust and specific.
We have demonstrated that the bioassay is able to quantify potency of rituximab, trastuzumab, and cetuximab, which are on-market monoclonal antibody drugs for cancer, using a variety of different target cells. With some of these same target systems, we have also shown that the ADCC reporter bioassay is stability-indicating. These performance characteristics demonstrate suitability of the ADCC reporter bioassay for assay validation for lot release potency and stability of antibody drugs. Furthermore, new antibody drugs with enhanced ADCC efficiency are being developed to address improvement of drug efficacy in patient populations.
Many of these ‘enhanced ADCC’ antibodies have modifications in the N-glycan moiety. Using the ADCC reporter bioassay, demonstration of linear correlation between relative antibody biological activity and the percentage of Fc N-glycosylation or Fc fucosylation has been obtained for antibody drug preparations. The ability of the bioassay to quantifiably differentiate such differences in ADCC efficiencies demonstrates its capabilities and applicability to biosuperior antibody drug research and development.