Evaluation of assay interference and interpretation of CXCR4 receptor occupancy results in a preclinical study with MEDI3185, a fully human antibody to CXCR4

Abstract

BackgroundReceptor occupancy (RO) assays provide a means to measure the direct interaction of therapeutics with their cell surface targets. Free receptor assays quantify cell‐surface receptors not bound by a therapeutic while total receptor assays quantify the amount of target on the cell surface.MethodsWe developed both a flow cytometry‐based free RO assay to detect free surface CXCR4, and a total surface CXCR4 assay. In an effort to evaluate potential displacement interference, we performed in vitro experiments to compare on‐cell affinity with the IC50 values from in vitro and in vivo from the free CXCR4 assay. We determined free and total surface CXCR4 on circulating blood cells in cynomolgus monkeys dosed with MEDI3185, a fully human monoclonal antibody to CXCR4.ResultsWe devised an approach to evaluate displacement interference during assay development and showed that our free assay demonstrated little to no displacement interference. After dosing cynomolgus monkeys with MEDI3185, we observed dose‐dependence in the magnitude and duration of receptor occupancy and found CXCR4 to increase on lymphocytes, monocytes, and granulocytes. In a multiple dose study, we observed time points where surface CXCR4 appeared fully occupied but MEDI3185 was not detectable in serum. These paradoxical results represented a type of assay interference, and by comparing pharmacokinetic, ADA and total CXCR4 results, the most likely reason for the free CXCR4 results was the emergence of neutralizing anti‐drug antibodies (ADA). The total CXCR4 assay was unaffected by ADA and provided a reliable marker of target modulation in both in vivo studies. © 2015 The Authors Cytometry Part B: Clinical Cytometry Published byWiley Periodicals, Inc.

Receptor Occupancy Measurement of Anti-PD-1 Antibody Drugs in Support of Clinical Trials

“…A fluorescent-labeled drug was used to detect free receptor sites, if the amount of free receptors is underestimated in the presence of neutralizing ADAs (Nab). In contrast, the amount of free receptors is overestimated in the presence of non-neutralizing ADA [29,30]. We used the drug-based bound strategy to analyze JS001 RO in this study.…”

Flow cytometry and receptor occupancy in immune-oncology

“…The latter may cause the presence of interfering factors which could neutralize the detection reagent and cause underestimation of the free receptor. The latter can be observed in some pre-clinical studies where animals develop antibodies to a human therapeutic [2] but also in clinical trials, when the biotherapeutic agent is administered repeatedly [3].…”