Development of a novel clinical assay for measuring everolimus levels

SUMMARY/ABSTRACT In 2017 nearly 35,000 solid organ transplants were performed in the United States, an increase of 27% over the past ten years. Organ transplantation requires lifelong immunosuppression to prevent rejection of the transplant. Many new classes of drugs have been developed to combat the side effects and interactions common to immunosuppressants, with several agents (sirolimus, everolimus, and belatacept) gaining momentum in immunosuppressive treatment regimens. Many of the immunosuppressants, including everolimus (EVR), have a narrow therapeutic range and warrant therapeutic drug monitoring (TDM), as sub-therapeutic levels increase the risk for rejection while supra-therapeutic levels are associated with significant toxicity. In addition to significant pharmacokinetic variability, andgt;25% of patients are non-adherent to their immunosuppressant regimen, leading to unnecessarily high graft failure rates and a staggering financial burden on the healthcare system. Current methods to measure EVR are costly and time consuming, or fraught with metabolite interference and significant variability. Because transplant recipients must take their immunosuppressant for the life of the graft, there is an urgent and unmet need to develop a simple, cost effective, and precise way of monitoring EVR levels.Liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) is the current gold standard for EVR TDM, but it is costly, requires extensive technical expertise, has long turnaround times and low throughput, and large inter-lab variations (i.e. poor harmonization). Immunoassays represent a more affordable, high-throughput, and simple platform for TDM. However, the only immunoassay on the market is a competitive assay that indirectly measures EVR in patient samples and suffers from cross-reactivity with metabolites and a related immunosuppressant. Additionally, use of this approved immunoassay on different analyzer platforms and differences in calibration methods between labs has led to inconsistencies and large variability from lab to lab. Because supra-therapeutic dosing of EVR is associated with adverse events and patient discontinuation of EVR, accurate monitoring of EVR is critical for rapid detection of patient non-compliance and preservation of the graft. Affinergy has developed highly sensitive capture and detection reagents for a one-step sandwich-type assay that will enable frequent, simple, and affordable monitoring of EVR in whole blood. In this Direct- to-Phase II application, we will optimize our previously identified capture and detection reagents and adapt them to an existing automated clinical analyzer platform used in clinical chemistry labs. At the conclusion of this application, we expect to have a fully optimized assay for EVR TDM.PROJECT NARRATIVE Organ transplant recipients receive immunosuppressants to prevent graft rejection, but many of these drugs have a narrow therapeutic index and require routine drug monitoring to ensure patients are receiving an appropriate dose and to monitor patient compliance. Current technologies to measure the immunosuppressant everolimus are either costly, time-consuming, and require highly skilled operators, or are plagued by cross- reactivity issues that overestimate the amount of everolimus present. In this application, we propose to further develop a novel assay to facilitate rapid and affordable measurement of everolimus in whole blood.