A novel peptide assay for hepcidin clinical monitoring

摘要/摘要 这一第二阶段SBIR计划的目标是推进血清学诊断测试的商业化 对于海普西丁，铁代谢的荷尔蒙主控调节剂，现在是几种治疗方法的靶点 临床试验的高级阶段。海普西丁是在全身铁水平升高时产生的 随后会阻碍饮食中铁的吸收。荷尔蒙触发铁转运蛋白的泛素化 蛋白质铁转运蛋白，从而减少细胞对铁的摄取和外流，并鼓励其全身 清除，直到达到健康的平衡。海普西丁的功能处于遗传性铁代谢的中心 包括血色沉着症和地中海贫血在内的疾病，使其成为耐人寻味的新药靶点 发展努力。针对海普西丁调节因子的反义寡核苷酸、siRNAs和小分子 目前正在进行临床试验，以调节海普西丁的表达，以改善铁超载和贫血 各种临床适应症。因此，出现了对海普西丁诊断测试的迫切需求，并且一旦 可能会被临床医生用作下一代的辅助诊断 治疗，但也监测工具铁失调和造血系统疾病的患者。 目前还没有FDA批准的用于量化患者中庚西丁的诊断测试，这主要是由于 荷尔蒙的抗原性低，以及存在多种异构体，其中许多是不活跃的，因此 临床上无关紧要。AffinEnergy已经开发出一种独特的基于多肽的三明治检测方法，将使 对血浆中生物活性海普西丁-25水平的频繁、简单和负担得起的监测。通过一个 之前资助的第一阶段计划和进一步的内部发展，AffinEnergy利用了其核心能力， 噬菌体展示生物扫描，以鉴定两个序列中唯一的专有海普西丁结合肽(HBPs) 和结构，专门识别海普西丁。进一步的噬菌体展示生物扫描鉴定出一种噬菌体 它在纳摩尔浓度下唯一识别HBP-海普西丁-25复合体。平板法 使用这些试剂，检测下限为4 NM，与正常海普西丁的低端一致 一般报告为15毫微克/毫升或5毫微米。结果表明，该方法具有较高的灵敏度和准确性。 与基于质谱学的海普西丁-25检测有很强的相关性。根据这些数据，阶段 II计划将专注于优化基于多肽的分析，扩大这些专利的生产 试剂，测试污染物(药物、常见毒物等)的影响在化验性能方面， 以及在组装新的510(K)上市前许可申请之前验证该化验 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.