Antibodies Against the Erythroferrone N-Terminal Domain Prevent Hepcidin Suppression and Ameliorate Murine Thalassemia

Erythroferrone (Erfe) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating Erfe levels in conditions of stress erythropoiesis, such as in patients with β-thalassemia, promote the tissue iron accumulation that decisively contributes to morbidity in these patients. Here we developed neutralizing antibodies against Erfe to prevent hepcidin suppression and correct the iron loading phenotype in a mouse model of β-thalassemia (Hbb Th3/+ mice) and used these antibodies as tools to further characterize Erfe's mechanism of action. We demonstrate that Erfe binds to BMP6 with low nanomolar affinity, but also binds BMP2 and BMP4 with lower affinities. We further show that BMP6 binds the N-terminal domain of ERFE. This domain in itself was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in vivo in wildtype mice. Concurrently, anti-Erfe antibodies targeting the N-terminal domain prevented hepcidin suppression in Erfe-treated Huh7 cells and in EPO-treated mice. Crystal structure of the antibodies in contact with an N-terminal peptide of Erfe demonstrated critical contacts in the Erfe N-terminal domain imparting antibody selectivity to human and murine protein. Finally, we tested these antibodies in vivo in a mouse model of thalassemia. We observed a decrease in serum and liver iron in antibody-treated Hbb Th3/+ mice. In addition, treatment with anti-Erfe antibodies increased the number of red blood cells, hemoglobin concentration and hematocrit, while decreasing the number of reticulocytes and the red cell distribution width. These changes were more pronounced when mice are treated for eight weeks. Anti-Erfe treatment caused an increase in hepatic hepcidin mRNA expression, red blood cells, hemoglobin and hematocrit, while reticulocytes levels were lower and peripheral red cell lifespan was increased. In summary, we demonstrate that antibodies targeting the N-terminal domain of Erfe constitute a potential therapeutic tool for iron-loading anemias.