The emergence of immuno-modulatory therapies has sparked increased interest in measuring cell-based receptor engagement and modulation in patients who have been treated with these novel medicines. Measuring receptor occupancy (RO) can be challenging and developing reproducible RO assays that can be used in clinical trials requires careful planning.
When an assay is reproducible, the result obtained by one lab can reliably be replicated by other labs. Reproducibility is a fundamental requirement for assays used:
Using a reproducible assay accommodates complex sample logistics, allows analysis of time-sensitive marker(s), and minimizes the risk of false data interpretation or loss of a signal in “noisy” data.
Reproducibility is achieved through a process called standardization, which focuses on eliminating any source of variability, from sample preparation and instrument setup to standard operating procedure (SOP) generation and operator training. With flow cytometry-based assays, standardization may be challenging for several reasons, including:
RO is a flow cytometry-based pharmacodynamic (PD) that quantifies the binding of a drug to a specific target on the cell surface. Often, RO needs to be tested on fresh whole blood samples because over time ex-vivo, the receptor may be downregulated/internalized or the target cells may undergo apoptosis. Thus, when developing an RO assay, it is important to clarify the stability of receptor occupancy and to identify labs in close proximity to the sites where samples are collected unless stabilization of the signal is possible.
RO assays need to be customized to the drug of interest, so it is critical to define the best approaches for designing and developing these assays. There are three main formats that can be used to develop an RO assay:
The optimal format will depend on the aim of the study, the biology of the receptor, and the type of drug and perhaps most crucially what reagents are available or can be derived.
Careful assay design and reagent selection is crucial for the development of a reproducible RO assay that can be transferred from one lab to another. When designing an RO assay, it is critical to:
Transitioning from design to development of an RO assay, it is essential to develop a comprehensive understanding of the biology of the RO. Mechanism of action, target cell population, receptor expression, and specimen stability should all be analyzed when performing in vitro testing of drug activity. All these factors contribute to the reliability of the calibration curve.
For flow cytometry-based assays, including appropriate controls is paramount. Marker expression, measured as mean fluorescent intensity (MFI), can be determined by tracking the antibody and ensuring that all the markers to be detected during flow cytometry analysis are saturated. The compensation matrix and the acceptable level of spillover can be set by standardizing the voltage of the instrument. The gating strategy, which defines positive or negative populations for a specific marker, can be determined using isotype or fluorescence minus one (FMO) controls. Finally, it is important to conduct experimental controls that ensure the assay performs as expected for the defined characteristics of the clinical sample or testing conditions.
Once an RO assay has been developed, it needs to be validated. The validation process—along with the parameters and acceptance criteria—should be customized to the intent of the study. It is important to consider the study phase, the endpoint, and how the data will be reported. Flow cytometry assays are rarely used for a primary endpoint. For exploratory and secondary endpoints, Precision for Medicine has developed general guidelines on method validation for clinical studies, based on the intent of the data and with a focus on robustness (see Figure 3).
For real-time assays, we generally recommend training at least three operators to ensure full coverage over the life of a study. Validating post-staining and sample stability is important for clarifying the test window and time sensitivity of the assay. Verifying the assay in the disease state is also useful for understanding the true frequency of the cells in the expected clinical study population. For RO assays that will be used as a secondary endpoint, method validation will need to be more rigorous because the data acquired will be reported to regulatory agencies. In such cases, validation may also include linearity and sensitivity to clarify the performance of the assay and increase confidence in the clinical data.
Assay transfer is most seamless when each lab involved in the clinical study uses the same flow cytometry machine with the same configuration, as the reference values measured by the lab that developed and validated the assay can be transferred across all labs. What will be needed for the transfer are:
Utilizing reagents from the same lots may also be useful. If this is not possible, a bridging study will need to be run to minimize lot-to-lot variability. For RO assays, it will be necessary to test at least three different concentrations of the drug to validate reproducibility within the defined acceptance criteria. To complete assay validation and transfer, it will also be important to train multiple operators and test multiple samples to further minimize variability.
The ability to simultaneously evaluate receptor expression and drug occupancy on specific cell subsets is critical throughout the development of immuno-modulatory therapies. Developing, optimizing, and validating a robust, reproducible RO assay facilitates dose selection, safety, and pharmacological monitoring in clinical studies, supporting development of safe, effective therapeutics.
Pharma industry veteran and expert at biomarker-driven clinical trial design and execution. Leader of biomarker and drug development programs for pharmaceutical and diagnostics companies, as well as the National Institutes of Health. Spearheaded the discovery of pharmacodynamic biomarkers and novel targets for inflammatory disease therapy.
Dr. Laura Lozza is Senior Director for Cell Biology at Precision for Medicine. She has long-standing expertise in immune monitoring and immune assay development with a particular focus on flow cytometry assays. Her focused on human immunology, including characterizing the regulatory activities of CD4 T cells, as well as analyzing the response of human dendritic cells in tuberculosis disease and BCG vaccination. She was a leader in establishing Precision’s Cell Biology Lab in Berlin which supports assay development and validation utilizing flow cytometry as well as other cell biology technologies.
Precision for Medicine is part of the Precision Medicine Group, an integrated team of experts that extends Precision for Medicine’s therapeutic development capabilities beyond approval and into launch strategies, marketing communication, and payer insights. As one company, the Precision Medicine Group helps pharmaceutical and life-sciences clients conquer product development and commercialization challenges in a rapidly evolving environment.
