A novel peptide assay for hepcidin clinical monitoring

总结/摘要 SBIR计划的第二阶段目标是推进血清学诊断测试的商业化 铁调素是铁代谢的激素主要调节剂，现在是几种治疗药物的靶点。 临床试验的高级阶段。铁调素响应于全身铁水平升高而产生， 随后阻止膳食铁吸收。这种激素触发了铁转运蛋白的泛素化 铁转运蛋白，从而减少细胞吸收和铁的流出，并促进其系统性 直到达到健康的平衡。铁调素的功能是在遗传性铁代谢的中心 包括血色素沉着症和地中海贫血在内的疾病，使其成为新药的一个有趣的目标 发展努力。靶向铁调素调节剂的反义寡核苷酸、siRNA和小分子 目前在临床试验中调节铁调素的表达，以改善铁过载和贫血。 各种临床适应症。因此，出现了对铁调素诊断测试的迫切需要，并且一旦 可用，可能会被临床医生使用，不仅作为这些下一代的伴随诊断 它不仅是治疗药物，也是铁失调和造血系统疾病患者的监测工具。 目前，没有FDA批准的用于定量患者中的铁调素的诊断测试，这主要是由于 激素的低抗原性和多种亚型的存在，其中许多是无活性的，因此 临床上无关。Affinergy开发了一种独特的基于肽的夹心测定法， 频繁、简单和负担得起地监测血浆中的生物活性铁调素-25水平。通过 先前资助的第一阶段计划和进一步的内部发展，Affinergy利用其核心竞争力， 噬菌体展示生物淘选，以鉴定专有的铁调素结合肽（HBPs），在两个序列中都是独特的 和结构，特异性识别hepcidin。进一步的噬菌体展示生物淘选已经鉴定了噬菌体 其唯一识别纳摩尔浓度的HBP-hepcidin-25复合物。平板检测 使用这些试剂显示检测下限为4 nM，与正常铁调素的下限一致 通常报告为15 ng/mL或5 nM。结果表明，该方法具有较高的灵敏度和准确度 与铁调素-25的基于质谱的检测强相关。根据这些数据，阶段 II计划将专注于优化基于肽的测定，扩大这些专利产品的生产规模， 试剂、测试污染物的影响（药物、常见毒物等）在测定性能上， 并在组装重新分类510（k）上市前许可申请之前验证检测试剂盒， 提交给FDA。

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.