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Assessment of Receptor Occupancy via Flow Cytometry: Benefits and Pitfalls of Two Common Approaches

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Assessment of Receptor Occupancy via Flow Cytometry: Benefits and Pitfalls of Two Common Approaches presented by Andrea Wong, Scientist, Flow Cytometry

Authors: A. S. Wong, M. Patel, and T. Rogers. Altasciences, Seattle, WA.

Receptor occupancy assays can be a powerful tool to determine drug dosage. Recently, flow cytometry has been the method of choice to assess receptor occupancy due to its ability to measure drug-target binding interactions on multiple cell populations simultaneously. Receptor occupancy assays using flow cytometry involve measurement of target receptors bound by the drug, number of receptors not bound to the drug, and the total number of receptors present. The methods for measuring these parameters, particularly for antibody-based therapeutics, can be categorized into two approaches based on the detection antibodies used. Here, we discuss the benefits and pitfalls to these methods. In one approach, fluorescently labelled competing and non-competing antibodies to the drug were used to detect free and total receptors respectively. In this approach, the competing antibody is often the labelled drug itself. One of the main obstacles in method development is to identify a non-competing antibody with comparable binding affinity to the target as the drug tested. We observed that when competing and non-competing antibodies display different affinities to the target, the results can be difficult to interpret. In addition, it is imperative that in a multiplex assay, competing and non-competing antibodies do not interfere with each other’s binding to the target. The other approach utilized a single, fluorescently labelled secondary antibody to detect both bound and total receptors. Samples from animals treated in vivo were first incubated with excess drug ex vivo to fully saturate all targets, then stained with secondary antibody to detect total receptor. Staining with a single-detection antibody ensured that a direct comparison could be made between bound and total receptors, and removed challenges related to different binding affinities between reagents. Identifying a suitable secondary antibody was crucial, especially when the targeted populations consist of sticky cells such as granulocytes and monocytes. In addition, we found that incubation of samples ex vivo with excess drug can lead to down regulation of the target, leading to underestimation of the total receptors and overestimation of receptor occupancy of the drug. The technical considerations presented will aid to determine the most suitable approach and avoid common pitfalls in assay designs when incorporated early in the RO design phase.


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