Serum Hepcidin Immunoassay: Laboratory to Marketplace

描述（由申请人提供）：铁代谢紊乱是人类最常见的疾病之一。在过去的十年中，在理解铁稳态及其紊乱的分子基础方面取得了快速进展。肽激素铁调素已经成为铁代谢的主要调节剂。铁调素的失调是大多数系统性铁障碍的主要或促成因素。在人类中，由于铁调素转录的负调节因子膜蛋白基质蛋白酶2（TMPRSS 6）的遗传缺陷导致铁调素的原发性过度产生，导致铁难治性缺铁性贫血（IRIDA）。IRIDA是一种铁调素合成相对于血浆铁过量的疾病，可以通过测量铁调素水平来诊断，而不是通过DNA测序，目前的确诊。对于用于血清铁调素的稳健且广泛可用的临床测定存在公认的未满足的需求，但由于铁调素的免疫原性差，该目标的进展已经放缓。Intrinsic Life Sciences（ILS）开发并验证了世界上第一个RUO血清铁调素免疫测定法（竞争性ELISA，C-ELISA），该方法依赖于有限的高质量兔多克隆抗体供应。该第一代铁调素测定有助于显示IRIDA表型是由与血浆铁水平相关的不适当升高的铁调素引起的，并且在初步数据分析后，我们已经证明该测定可以以高灵敏度和特异性明确区分由于TMPRSS 6突变而患有IRIDA的个体与1）正常对照群体的儿童和成人，2）患有单纯性铁缺乏（ID）的儿童和成人，和3）患有对口服铁治疗有抗性的ID的高度选择的个体群体，其被称为“排除”IRIDA。在第二阶段的研究中，ILS发现了铁调素的关键单克隆抗体，并开发了两种强有力的C-ELISA.这些原型新一代“湿”测定具有优异的动态范围，具有理想的精密度和线性特征，并显示LLOQ、LLOD和EC 50值以及平行于或超过我们目前的多克隆RUO C-ELISA的测定内和测定间CV。去识别的IRIDA患者样品的初步分析已经证明，MAb 583和NT-生物素化的铁调素示踪剂产生与我们的多克隆RUO C-ELISA平行的优异的灵敏度、特异性和AUROC。我们建议继续开发Hepcidin-IRidA Compete”IVD作为干燥微孔板试验，并证明其在我们将招募的贫血患者中诊断IRIDA的临床实用性。根据ISO/GMP监管指南，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，最终确定器械主记录，并向FDA提交IDE前材料。我们将生产合规批次，进行持续的FDA合规生产性能和质量研究。为了证明其临床用途，我们将寻求FDA的IDE前指导，最终确定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在患有IRIDA的TMPRSS 6（-/-）患者和临床上无法区分的贫血患者之间的诊断区分。

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.