Serum Hepcidin Immunoassay: Laboratory to Marketplace

描述(申请人提供)：铁代谢障碍是最常见的人类疾病之一。在过去的十年里，在理解铁稳态及其紊乱的分子基础方面取得了快速的进展。多肽荷尔蒙海普西丁已成为铁代谢的主要调节剂。海普西丁的失调是大多数全身性铁紊乱的主要或致病因素。在人类中，由于海普西丁转录的负调控因子膜蛋白基质裂解酶2(TMPRSS6)的遗传缺陷，导致海普西丁的原发过量生产，导致铁耐受性缺铁性贫血(IRIDA)。IRIDA是一种相对于血浆铁的海普西丁合成过多的疾病，可以通过测量海普西丁水平来诊断，而不是通过DNA测序，目前的确证诊断。有一个公认的尚未得到满足的需求，即一种强大的和广泛可用的血清海普西丁临床检测方法，但由于海普西丁的免疫原性较差，这一目标的进展已经放缓。内在生命科学(ILS)开发并验证了世界上第一个依赖于有限供应的高质量兔多克隆抗体的RuO血清海普西丁免疫分析(竞争性ELISAC-EL ISA)。第一代海普西丁检测有助于证明IRIDA表型是由与血浆铁水平有关的海普西丁水平异常升高引起的，经过初步数据分析，我们证明该检测方法能够以高敏感性和特异性明确区分TMPRSS6突变导致的IRIDA个体，这些个体来自1)正常对照人群儿童和成人，2)儿童和成人单纯缺铁(ID)，以及3)高度挑选的对口服铁疗法耐药的IRIDA个体被称为IRIDA。在第二阶段的研究中，ILS发现了针对海普西丁的关键单抗，并开发了两种强大的C-ELISA。这些新一代“湿”检测的原型具有极佳的动态范围，具有理想的精密度和线性特性，并可显示LLOQ、LLOD和EC50值，以及平行或超过我们目前的多克隆Ruo C-ELISA的批内和批间变异系数。对未鉴定的IRIDA患者样本的初步分析表明，MAb 583和NT-生物素标记的海普西丁示踪剂产生了出色的灵敏度、特异性和AUROC，与我们的多克隆Ruo C-ELISA平行。我们建议继续开发海普西丁-IRIDA竞争IVD作为一种干燥的微板法，并展示其在贫血患者IRIDA诊断中的临床应用价值。根据ISO/GMP法规指南，我们将完成海普西丁IVD的生产前研发，最终确定设计特征并生产大量测试组件，确认试剂性能，最终确定设备主记录，并向FDA提交IDE前材料。我们将生产符合标准的批次，进行持续的符合FDA标准的制造性能和质量研究。为了证明其临床应用，我们将寻求FDA的IDE前指导，最终确定将在波士顿儿童医院进行的前瞻性临床试验的设计，并在试验中测试海普西丁-IRidA竞争“IVD”对患有IRIDA的TMPRSS6(-/-)患者和临床上无法区分的贫血患者的诊断区分。

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.