Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development

Abstract

Receptor occupancy (RO) assays are designed to quantify the binding of therapeutics to their targets on the cell surface and are frequently used to generate pharmacodynamic (PD) biomarker data in nonclinical and clinical studies of biopharmaceuticals. When combined with the pharmacokinetic (PK) profile, RO data can establish PKPD relationships, which are crucial for informing dose decisions. RO is commonly measured by flow cytometry on fresh blood specimens and is subject to numerous technical and logistical challenges. To ensure that reliable and high quality results are generated from RO assays, careful assay design, key reagent characterization, data normalization/reporting, and thorough planning for implementation are of critical importance during development. In this article, the authors share their experiences and perspectives in these areas and discuss challenges and potential solutions when developing and implementing a flow cytometry‐based RO method in support of biopharmaceutical drug development. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

Multiplexing of receptor occupancy measurements for pharmacodynamic biomarker assessment of biopharmaceuticals

“…However, the targets need to be expressed on different cell populations for unambiguous measurement of RO. On the other hand, RO methods, with fluorescence-biopharmaceutical detection systems are prone to assay interference by neutralizing anti-drug antibodies (21). This interference could lead to misinterpretation of RO results and inaccurate RO assessment.…”

“…To minimize the effect of receptor internalization during receptor measurements, we performed staining reactions at low temperatures. Another way to mitigate impact of internalization on receptor measurement is to pretreat cells with sodium azide, a known inhibitor of endocytosis (21).…”

“…RO assays measure free receptors (unoccupied by drug and still available for signaling) or drug-bound receptors (6,12,21). These assays are typically performed in peripheral blood as a minimally invasive source of specimens.…”

“…However, when flow cytometry is applied to RO assessment, immunophenotyping methods are not directly translatable since RO is focused not on measurement of the receptor per se, but primarily on measurement of receptor binding. To this end, application of flow cytometry to RO measurement requires careful selection of assay reagents and determination of optimal assay conditions (21).…”

Recommendations for the development and validation of flow cytometry‐based receptor occupancy assays

“…Interference due to ADA can also be observed in some pre-clinical studies where animals develop antibodies to a human therapeutic (20). In clinical trials, this issue more prevalent when the biotherapeutic agent is administered repeatedly.…”