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By: Jennifer J. Stewart

Jennifer Stewart - Final Estimating receptor occupancy (RO) requires an accurate measuring technique, and flow cytometry methods can fill this need. However, RO assays are among the most challenging flow cytometric methods to develop, validate, and implement.

For development of therapeutics in humans, rational dose selection and clinical trial design is critical. The primary challenges for such approaches are to develop a robust biomarker for demonstrating pharmacodynamic (PD) effect of the therapeutic in relevant animal species, and to construct a pharmacokinetic (PK)/PD model for effectively integrating PK and PD information from animal studies for human dose prediction. First-in-human (FIH) dose selection traditionally relies on a no-observed-adverse-effect level (NOAEL) identified from nonclinical toxicity studies in order to identify a safe starting dose and a dose-escalation scheme.

Severe adverse events observed in a FIH clinical trial of TGN1412, an anti-CD28 superagonist monoclonal antibody, highlighted the importance of selecting safe starting doses in FIH trials. Due to the difference in pharmacology in monkeys and humans for TGN1412, the NOAEL obtained from monkeys was shown to not perfectly correlate to humans. As a result, the human equivalent dose corresponding to the NOAEL in monkeys was more than 30,000 times higher than the dose that was predicted to lead to 10% receptor occupancy (RO) in humans. The actual administered starting dose thus led to greater than 90% RO and caused a life-threatening cytokine release syndrome in healthy volunteers. Target engagement PD biomarkers, such as receptor occupancy, can be used as a parameter to define safe starting doses for FIH clinical trials, and the TGN1412 case further demonstrated the utility of RO estimation in rational dose selection.

The journal Cytometry Part B: Clinical Cytometry has just published the special issue Receptor Occupancy Assays, which includes a recommendation paper on the development and validation of RO assays, authored by members of the Flow Cytometry Action Program Committee, a subgroup of the AAPS Ligand Binding Assay Bioanalytical focus group.

In addition, the workshop Detective Workshop: How to Solve the Mystery of Challenging Receptor Occupancy Assays will be held at the Cyto 2016 Conference in Seattle on June 13, 2016. With a target audience of flow cytometry users from industry, academia, contract research organizations, and government agencies, this workshop will be an in-depth discussion of the most challenging aspects of RO that were not fully explored in the recommendation paper. We look forward to seeing you there!

Jennifer J. Stewart, Ph.D., is laboratory director at Flow Contract Site Laboratory, LLC. She has been a member of the AAPS Flow Cytometry Action Program Committee since 2009.