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Making Meaningful Measurements - Understanding Flow Cytometry-based Receptor Occupancy Assays [VIDEO]

Posted on: March 14, 2018

Many therapeutic drugs and biologics under development target the immune system to mediate their effects. These molecules may only be needed at very low concentrations to trigger the desired response, and understanding the pharmacokinetic and pharmacodynamic properties of an experimental biologic or drug is instrumental to determining the appropriate dose range to use for clinical trials.

receptor occupancy (RO) assay measures the binding of an experimental therapeutic molecule to its target on a cell’s surface and provides quantitative data that can be used to generate a pharmacodynamic profile. Flow cytometry is a widely used approach for RO assays, and flow cytometry-based assays can be adapted to measure RO of a biologic or drug with any type of target cell. Consider these three elements as you develop a flow cytometry-based RO assay.

 Flow cytometry is a widely used approach for RO assays, and flow cytometry-based assays can be adapted to measure receptor occupancy of a biologic or drug with any type of target cell.

1. Is A Flow Cytometry-Based RO Assay Relevant To Your Experimental Biologic or Drug?

A receptor occupancy assay measures the binding of a molecule to a target on the surface of a cell. RO assays are well-suited to experimental molecules like therapeutic antibodies that bind to specific cell surface targets, and are not as well suited to therapeutic molecules that enter into the cell and work via intracellular mechanisms or to soluble targets like cytokines.

2. Are Your Reagents Well-Suited For A Flow Cytometry-Based RO Assay?

Your experimental molecule will have to be labeled with a fluorochrome or must be detected using a secondary fluorochrome-labeled antibody in order to be measured by flow cytometry. You typically need to use multiple antibodies in an RO assay, including antibodies that compete with the target molecule and non-competing antibodies that measure the total target cell frequency, so multiple fluorochromes are necessary.

3. How Do You Want To Measure RO?

RO can be measured in different ways depending on your reagents, as well as the characteristics of the experimental biologic or drug and its target. You can measure the frequency of free target using a fluorochrome-conjugated antibody that only binds to unoccupied targets. Alternatively, occupied targets can be measured using a secondary antibody that binds to the target-bound molecule of interest. In addition, the total number of targets can be measured using a fluorescent antibody that binds to targets but does not compete with the binding of the experimental molecule. These different assay formats are appropriate for different types of molecules, and pilot experiments are critical to determining the format that suits your experimental needs.

Flow cytometry-based assays for clinical trials also need to fulfill regulatory requirements in order to assure the assays are validated and the measurements are accurate and reliable. Consider working with an experienced contract research organization as you pursue the development of an RO assay. Expert guidance and support can assure that your assay will provide meaningful data and help guide your preclinical and clinical research decisions.

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