Receptor Occupancy Assays by Flow Cytometry

Understand how your biotherapeutics are engaging their targets

Receptor occupancy (RO) assays are commonly employed to measure pharmacodynamic (PD) data. These assays have a broad range of uses across preclinical and early stages of clinical trials, all the way through later stages. PD data can then be correlated with pharmacokinetic (PK) profiles to establish PK/PD relationships and guide decision-making in dosing.

Preclinical and discovery:

Optimal matrix selection: whole blood or fixed whole blood
Reagent specificity determined by extensive characterization during assay development
Guide selection of compounds

Early phase trials:

Measure pharmacodynamics (PD) of biomarkers of interest
Assess efficacy

Late phase trials:

Define the population PD characteristics of the biotherapeutic agent
Combine data with PK profiles to guide dosing and administering schedules

	Free Receptor Assays	Direct Assessment of Bound Receptor	Total Receptor Assay	Receptor Modulation Assay	Functional Receptor Occupancy Assay
Function	Measure unbound or unoccupied receptors on a cell using an unlabeled antibody and competing therapeutic antibody or drug-conjugated antibody	Measure therapeutic antibodies bound to the receptor with an anti-therapeutic antibody detection reagent	Measure binding of a therapeutic antibody to its receptor site and couple with binding of an alternative antibody that binds to a different site on the same receptor	Measure effect of the therapeutic antibody binding on the function of the target receptor	Measure biological effects of the therapeutic antibody binding to the target receptor
Clinical Significance	Typically used for antagonistic therapeutic drugs that block ligand binding to the receptor	Often applied when monoclonal antibodies to the receptor are not readily available or conjugation of the therapeutic itself may compromise binding to its receptor target	Useful where receptor level or cell numbers can change over time	Receptor shedding or inhibition/activation of receptor internalization over time or with increasing drug dose	Cell proliferation or cytokine production, which may have implications in drug safety/efficacy
Illustration

Free Receptor Assays

Direct Assessment of Bound Receptor

Total Receptor Assay

Receptor Modulation Assay

Functional Receptor Occupancy Assay

Function

Measure unbound or unoccupied receptors on a cell using an unlabeled antibody and competing therapeutic antibody or drug-conjugated antibody

Measure therapeutic antibodies bound to the receptor with an anti-therapeutic antibody detection reagent

Measure binding of a therapeutic antibody to its receptor site and couple with binding of an alternative antibody that binds to a different site on the same receptor

Measure effect of the therapeutic antibody binding on the function of the target receptor

Measure biological effects of the therapeutic antibody binding to the target receptor

Clinical Significance

Typically used for antagonistic therapeutic drugs that block ligand binding to the receptor

Often applied when monoclonal antibodies to the receptor are not readily available or conjugation of the therapeutic itself may compromise binding to its receptor target

Useful where receptor level or cell numbers can change over time

Receptor shedding or inhibition/activation of receptor internalization over time or with increasing drug dose

Cell proliferation or cytokine production, which may have implications in drug safety/efficacy

Illustration

Free receptor assay

Direce assessment

Total receptor occupancy

Frequently asked questions

Which clinical phase can benefit from receptor occupancy flow cytometry?

Preclinical target verification and MOA: Determining RO through development is critical because many of these biologics have long half-lives. Understanding their binding characteristics can have an impact on lead compound selection.

Phase 1: Typically, this class of drug is very potent, and improper dosing protocols can potentially result in severe side effects or even be lethal. The identification of minimal anticipated biological effect (MABEL) model starting doses or Pharmacologically Active Doses (PAD) may require RO assessments in conjunction with PD and PK in order to appropriately guide dosing protocols.

Phase 2: Efficacy of dosing and administration protocols to help predict the levels of RO and whether the receptor is modulated up or down on cells that are engaged by the biotherapeutic.

Phase 3: Population PD for long-term safety and efficacy studies.

What is the regulatory level requested for a clinical flow cytometry panel?

Flow cytometry assays are validated and used under a fit-for-purpose approach. The parameters included in the validation of an assay should always depend on the purpose of the assay.

What are some challenges for receptor occupancy assays?

Because RO assays are typically performed on whole blood in real time and can be performed on PBMCs, sample stability must be taken into consideration.

Stability considerations:

Shipment time of blood from site to lab: age of blood impacts quality of results
Where is the site located and how long will sample shipment take?
Use of stabilizing fixative collection tubes (Smart Tube, Transfix, CytoChex) can be considered
Is the target receptor compatible with the fixative?
How long are the fixed samples stable for testing?

Other considerations:

Availability of competing or noncompeting antibodies to the target
Modulation of the receptor after treatment

Explore flow cytometry publications and resources

Explore best-practice methods and find useful considerations and tips for experimental design, sample prep, and data analysis.

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Case Study
Developing and Validating Clinical Receptor Occupancy Pharmacodynamic Biomarker Assays
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Brochure
Multiparametric Flow Cytometry
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