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.

PUBLIC HEALTH RELEVANCE: The founders of Intrinsic Life Sciences (ILS) pioneered discovery of hepcidin, the master iron regulatory hormone in humans, and developed the world's first validated serum hepcidin immunoassay, as well as described its key role regulating dietary iron absorption and iron recycling. With NIH Phase II funding our team discovered specific monoclonal antibodies (MAb) to hepcidin and used one, MAb 583 to make two prototype serum hepcidin diagnostic tests that are suitable for FDA-approval. With NIH Phase II-B funding, ILS will complete pre-commercialization R&D and select one prototype assay for cGMP/ISO manufacturing of the Hepcidin-IRidA Compete" IVD, perform a prospective clinical trial in collaboration with Boston Children's Hospital, and clinically validate our diagnostic testfor definitive differential diagnosis of a rare genetic iron disorder, Iron Refractory Iron Deficiency Anemia (IRIDA) that has clinical characteristics common in anemic pediatric/adolescent patients without the disorder: FDA approval of the Intrinsic Hepcidin-IRidA Compete" IVD will enable rapid diagnosis of IRIDA without expensive genetic testing or extensive clinical work-ups.