Receptor
Occupancy Assays
by Flow Cytometry
RO assays, or receptor occupancy assays by flow cytometry, are commonly employed to produce data on pharmacodynamic biomarkers. The data can then be correlated with pharmacokinetic profiles to establish PK/PD relationships.
These assays have a broad range of uses in the pre-clinical and early stages of clinical trials, including helping in the selection of compounds, verifying target engagement in disease models, and determining the initial dose for further trials. In later stages of drug development, RO assays can be combined with PK profiles to make decisions on dosing and administering schedules.
At Precision for Medicine, we can provide flow cytometry services from multiple labs in the US and Europe. In addition, our ability to develop customized kitting for sample collection, global network of sample processing labs, and logistics services helps to ensure reliable and robust assay results.
Understand how biotherapeutics engage their targets
- Simultaneous detection of RO and other surface PD markers by multiparametric flow cytometry
- Optimal matrix selection: whole blood or fixed whole blood
- Reagent specificity determined by extensive characterization during assay development
- Assays are validated for intended use suitable for proof of concept, exploratory, secondary, or primary clinical trial endpoints
Customized to your objective
Considerations for assay development include choice of appropriate antibody clones and fluorochromes, quality control measures, receptor stability, and appropriate assay reportables. At Precision, we can help to tailor an RO strategy to your specific therapeutic development needs.
5 Common RO Assay Formats
Flow Cytometry Resources
Resources
Webinar: Flow Cytometry for Clinical Applications: Recommendations on Multi-site Assay Transfer and Method Validation
Deborah Phippard, PhD, and Laura Lozza, PhD. July 2022.
Poster: An Assay to Monitor the Engagement and Modulation of CD6 on T cells as a Clinical Biomarker of Treatment With Itolizumab
Angelina Bisconte, et al. AAI Immunology, 2021.
Case Study: Developing and Validating Clinical Receptor Occupancy Pharmacodynamic Biomarker Assays
Precision for Medicine. 2022.
Article: Advancing Clinical Development of Immuno-modulatory Therapies With Flow Cytometry-Based Receptor Occupancy Assays
Laura Lozza, PhD, and Deborah Phippard, PhD. 2022.
Frequently Asked Questions
Pre-clinical 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 I: 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 II: 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 III: Population PK for long-term safety and efficacy studies.
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.
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 non-competing antibodies to the target
- Modulation of the receptor after treatment
Values of Precision Flow Cytometry
Established experience running complex flow cytometry in gene therapy, cell therapy, and immuno-oncology therapies, validating up to CLIA
Sample testing by senior staff with >5 years of experience who understand the disease biology and gating strategy
Real-time quality control during the run to ensure sample integrity
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