Serum Hepcidin Immunoassay: Laboratory to Marketplace

描述（申请人提供）：铁代谢紊乱是人类最常见的疾病之一。在过去的十年中，在了解铁稳态及其失调的分子基础方面取得了快速进展。肽激素hepcidin已成为铁代谢的主要调节因子。hepcidin的失调是大多数系统性铁紊乱的主要或促成因素。在人类中，由于hepcidin转录的负调节因子-膜蛋白基质酶2 （TMPRSS6）的遗传缺陷，hepcidin的原发性过量产生导致铁难逆性缺铁性贫血（IRIDA）。IRIDA是一种与血浆铁相关的hepcidin合成过量的疾病，可以通过测量hepcidin水平来诊断，而不是通过DNA测序，这是目前的确诊诊断。对于血清hepcidin的一种强大的、广泛可用的临床检测方法的需求是公认的，但由于hepcidin的免疫原性较差，这一目标的进展已被减缓。Intrinsic Life Sciences （ILS）开发并验证了世界上第一个RUO血清hepcidin免疫测定（竞争性ELISA， C-ELISA），该方法依赖于有限供应的高质量兔多克隆抗体。第一代hepcidin检测有助于证明IRIDA表型是由与血浆铁水平相关的hepcidin不适当升高引起的，经过初步数据分析，我们已经证明该检测可以明确地以高灵敏度和特异性区分由于TMPRSS6突变而患有IRIDA的个体1)正常对照人群儿童和成人，2)儿童和成人无复杂性铁缺乏症（ID）。3)高度选定的对口服铁治疗有耐药性的ID患者，他们被称为“排除”IRIDA。在II期研究中，ILS发现了hepcidin的关键单克隆抗体，并开发了两种强大的C- elisa。这些新一代的原型“湿”检测具有优异的动态范围，具有理想的精度和线性特性，并且显示LLOQ， LLOD和EC50值以及检测内和检测间CV，与我们目前的多克隆RUO C-ELISA相当或超过。对未鉴定的IRIDA患者样本的初步分析表明，MAb 583和nt生物素化hepcidin示踪剂具有优异的灵敏度、特异性和AUROC，与我们的多克隆RUO C-ELISA相似。我们建议继续开发Hepcidin-IRidA Compete IVD作为一种干燥微孔板试验，并证明其在我们将招募的贫血患者中诊断IRIDA的临床应用。在ISO/GMP法规指导下，我们将完成hepcidin IVD的生产前研发，最终确定设计特征并生产检测组分的测试批次，确认试剂性能，最终确定设备主记录，并向FDA提交ide前材料。我们将生产符合fda要求的批次，进行持续的符合fda要求的生产性能和质量研究。为了证明其临床应用，我们将寻求FDA的ide前指导，最终确定一项将在波士顿儿童医院进行的前瞻性临床试验的设计，并测试Hepcidin-IRidA Compete”IVD在使用IRIDA的TMPRSS6（-/-）患者和参加试验的临床无法区分的贫血患者之间的诊断区别。

DESCRIPTION (provided by applicant): Disorders of iron metabolism are among the most common human diseases. During the last decade, rapid progress has been made in understanding the molecular basis of iron homeostasis and its disorders. The peptide hormone hepcidin has emerged as the master regulator of iron metabolism. Dysregulation of hepcidin is the principal or contributing factor in most systemic iron disorders. In humans, primary overproduction of hepcidin due to genetic deficiency of a negative regulator of hepcidin transcription, the membrane protein matriptase 2 (TMPRSS6), leads to iron-refractory iron-deficiency anemia (IRIDA). IRIDA is a disorder of excessive hepcidin synthesis relative to plasma iron, and can be diagnosed by measuring hepcidin levels, rather than by DNA sequencing, the current confirmatory diagnosis. There is a well-recognized unmet need for a robust and widely available clinical assay for serum hepcidin but progress towards this goal has been slowed by the poor immunogenicity of hepcidin. Intrinsic Life Sciences (ILS) developed and validated the world's first RUO serum hepcidin immunoassay (competitive ELISA, C-ELISA), that relied on a finite supply of high quality rabbit polyclonal antibodies. This first generation hepcidin assay was instrumental in showing that the IRIDA phenotype is caused by inappropriately elevated hepcidin in relation to plasma iron levels and after preliminary data analysis we have demonstrated that this assay can definitively distinguish with high sensitivity and specificity, individuals with IRIDA due to TMPRSS6 mutations from 1) a normal control population of children and adults, 2) children and adults with uncomplicated iron deficiency (ID), and 3) a highly selected population of individuals who have ID that is resistant to oral iron therapy who have been referred to as "rule out" IRIDA. During Phase II research, ILS discovered key monoclonal antibodies to hepcidin and developed two robust C- ELISAs. These prototype new generation 'wet' assays have an excellent dynamic range, possess ideal precision and linearity characteristics, and display LLOQ, LLOD and EC50 values and intra- and inter-assay CV's that parallel or exceeds those of our current polyclonal RUO C-ELISA. Preliminary analysis of de- identified IRIDA patient samples have demonstrated that MAb 583 and the NT-biotinylated hepcidin tracer yield excellent sensitivity, specificity, and AUROC that parallel our polyclonal RUO C-ELISA. We propose to continue to develop the Hepcidin-IRidA Compete" IVD as a desiccated microplate assay and demonstrate its clinical utility for diagnosis of IRIDA in anemic patients we will enroll. Under ISO/GMP regulatory guidelines we will complete pre-manufacturing R&D on our hepcidin IVD, finalize design features and produce test lots of assay components, confirm reagent performance, finalize the Device Master Record, and file pre-IDE materials to the FDA. We will manufacture compliance lots, conduct continuous FDA-compliant manufacturing performance and quality studies. To demonstrate its clinical use, we will seek FDA pre-IDE guidance, finalize design of a prospective clinical trial to be performed at Boston Children's Hospital, and test the Hepcidin-IRidA Compete" IVD for diagnostic discrimination between TMPRSS6 (-/-) patients with IRIDA and clinically indistinguishable anemic patients enrolled in the trial.