Project Summary/Abstract Erythroferrone (ERFE), the erythroid regulator of hepcidin has recently been discovered and characterized as a hormone expressed in erythroblasts in the marrow. ERFE responds to erythropoietin (EPO) signaling from the EPO receptors in the marrow that stimulate ERFE synthesis and release into the plasma. ERFE then acts as a suppressor of hepcidin expression in the liver. Hepcidin is the master regulator of iron absorption from the diet and iron recycling of red blood cells (RBCs) by macrophages. Thus, ERFE as a suppressor of hepcidin, acts to increase iron absorption from the diet and plasma iron concentrations by freeing iron from stores in hepatocytes. ERFE was discovered in phlebotomized mice as a hormone involved with stress erythropoiesis following hemorrhage and was shown to be highly elevated from near undetectable levels at baseline in mouse models of β-thalassemia and ineffective erythropoiesis. Intrinsic LifeSciences licensed patent-pending ERFE technology from UCLA and has subsequently developed a small suite of monoclonal antibodies (mAbs) directed at ERFE. Using a pair of these mAbs we produced a mAb sandwich research use only (RUO) ELISA and began technical validation and clinical assessment of the kit. This proposal presents Preliminary Data supporting the technical quality of the ERFE test as well as its potential clinical utility. We recapitulated the mouse phlebotomy experiments by sampling serum ERFE before and after plasma apheresis in blood donors and established the preliminary normal range for ERFE in humans in serum samples from first-time blood donors. We also established a preliminary lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) of 0.1 and 0.15 ng/ml. The median value of the normal range was observed to be 0.8 ng/ml. This suggests that the ERFE sandwich ELSA is sensitive and suitable for commercial development. We confirmed clinical significance of the ERFE sandwich ELISA with de-identified samples from β-thalassemia and X-linked sideroblastic anemia (XLSA) patients. The PI is requesting Direct to Phase II SBIR funding under PAR-14-088 to continue our exciting research and development efforts on the ERFE monoclonal sandwich ELISA over the next two years. Our experience to date with the ERFE assay indicates that our research strategy is feasible and we expect the outcome of our Phase II R&D studies will be a fully automated clinical ERFE laboratory developed test (LDT) validated under Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP) guidelines. The CLIA/CAP-validated LDT would be available to healthcare providers in the US and would guide development and validation of a RUO kit for basic biology studies of ERFE in various disease states. An additional deliverable will be a set of Design Controls necessary to manufacture a monoclonal sandwich IVD for ERFE that is suitable for FDA-clearance. We believe that our ERFE IVD will gain widespread clinical use for detection of ineffective erythropoiesis and as an important clinical diagnostic test for β-thalassemia syndromes.

Project Narrative The hallmark of β-thalassemia and other common genetic and acquired iron-loading anemias is ineffective erythropoiesis (IE; impaired blood production) that leads to debilitating anemia in the presence of iron overload. Recently discovered erythroferrone (ERFE) is a hormone produced in developing red blood cells (RBCs) and has been shown to be a negative regulator of hepcidin, the master hormone regulating plasma iron levels, and to be highly elevated in β-thalassemia patients. Intrinsic LifeSciences is requesting Direct to Phase II SBIR funding to optimize an existing dual monoclonal sandwich ELISA as a kit suitable for FDA 510K clearance, as well as a validated laboratory developed test (LDT) that will be offered in our accredited clinical laboratory, IntrinsicDx, to U.S. clinicians for diagnosis and management of ineffective erythropoiesis.