A novel peptide assay for hepcidin clinical monitoring

SUMMARY/ABSTRACT The goal of this Phase II SBIR program is to advance the commercialization of a serological diagnostic test for hepcidin, the hormone master regulator of iron metabolism that is now the target of several therapeutics in advanced stages of clinical testing. Hepcidin is produced in response to elevated systemic iron levels and subsequently blocks dietary iron absorption. The hormone triggers the ubiquitylation of the iron transporter protein ferroportin, thereby reducing both cellular uptake and efflux of iron and encouraging its systemic clearance until reaching a healthy equilibrium. Hepcidin's function is at the center of inherited iron metabolism disorders including hemochromatosis and -thalassemia, rendering it an intriguing target for new drug development efforts. Antisense oligonucleotides, siRNAs, and small molecules targeting hepcidin regulators are currently in clinical trials to modulate hepcidin expression to ameliorate both iron overload and anemia in various clinical indications. An acute need for hepcidin diagnostic tests has therefore emerged, and once available, would likely be used by clinicians as not only a companion diagnostic for these next generation therapeutics, but also monitoring tools for patients with iron dysregulation and hematopoietic disorders. Currently there is no FDA-approved diagnostic test for quantifying hepcidin in patients, largely due to the hormone's low antigenicity and the presence of multiple isoforms, many of which are inactive and therefore clinically irrelevant. Affinergy has developed a unique peptide-based sandwich assay that will enable frequent, simple, and affordable monitoring of bioactive hepcidin-25 levels in plasma. Through a previously funded Phase I program and further internal development, Affinergy has used its core competency, phage display biopanning, to identify proprietary hepcidin-binding peptides (HBPs), unique in both sequence and structure, that specifically recognize hepcidin. Further phage display biopanning has identified a phage which uniquely recognizes the HBP-hepcidin-25 complex at nanomolar concentrations. Plate-based assays using these reagents revealed a lower-limit of detection of 4 nM, consistent with the low end of normal hepcidin levels which is generally reported as 15 ng/mL or 5 nM. The sensitivity and accuracy of the assay was shown to strongly correlate with mass spectrometry-based detection of hepcidin-25. Based on these data, the Phase II program will focus on optimizing a peptide-based assay, scaling up manufacturing of these proprietary reagents, testing for the impact of contaminants (medications, common toxicants, etc.) on assay performance, and validating the assay prior to assembling a de novo 510(k) premarketing clearance application for submission to the FDA.