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Decreased hepcidin mobilizes iron, which facilitates erythropoiesis, but excess iron is pathogenic in beta-thalassemia and other iron-loading anaemias. Erythropoietin (EPO) enhances erythroferrone (ERFE) synthesis by erythroblasts, and ERFE suppresses hepatic hepcidin production, through an unknown mechanism. The BMP/SMAD pathway in the liver is critical for control of hepcidin, and we show that EPO suppressed hepcidin and other BMP target genes in vivo in a partially ERFE-dependent manner. Furthermore, recombinant ERFE suppressed the hepatic BMP/SMAD pathway independently of changes in serum and liver iron, and in vitro, ERFE decreased SMAD 1/5/8 phosphorylation and inhibited expression of BMP target genes in hepatoma cells. ERFE specifically abrogated the induction of hepcidin by BMP5, BMP6 and BMP7, but had no or very little effect on hepcidin induction by BMP2, 4, 9 or Activin B. A neutralising anti-ERFE antibody prevented the ability of ERFE to inhibit hepcidin induction by BMP5, 6 and 7. Cell-free Homogeneous Time Resolved Fluorescence assays showed that BMP5, BMP6 and BMP7 competed with anti-ERFE for binding to ERFE. Biacore analysis showed that ERFE binds to BMP6 with a higher affinity compared to its binding to BMP2, BMP4 or Activin B, and does not bind to GDF15. We propose that ERFE suppresses hepcidin by inhibiting hepatic BMP/SMAD signaling via preferentially binding and impairing the function of an evolutionarily closely related BMP sub-group consisting of BMP5, BMP6 and BMP7. These findings indicate that ERFE can act as a natural ligand trap generated by stimulated erythropoiesis in order to regulate availability of iron. Disclosures Arezes: Pfizer: Research Funding. Foy:Pfizer: Employment. McHugh:Pfizer: Research Funding. Sawant:Pfizer: Employment. Benard:Pfizer: Employment. Quinkert:Pfizer: Research Funding. Terraube:Pfizer: Employment. Brinth:Pfizer: Employment. Tam:Pfizer: Employment. LaVallie:Pfizer: Employment. Cunningham:Pfizer: Employment. Lambert:Pfizer: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer: Employment. Drakesmith:La Jolla Pharmaceutical Company: Research Funding; Pfizer: Research Funding; Alnylam: Consultancy; Kymab: Membership on an entity's Board of Directors or advisory committees.

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. Disclosures Arezes: UCB: Employment. Foy:Pfizer Inc.: Employment. Benard:pfizer: Employment. Sawant:Pfizer Inc.: Employment. Tam:Pfizer Inc.: Employment. Maben:Pfizer Inc.: Employment. LaVallie:Pfizer Inc.: Employment. Cunningham:Pfizer Inc.: Employment. Lambert:Pfizer Inc.: Employment. Pittman:Pfizer Inc.: Employment. Murphy:Pfizer Inc.: Employment. Draper:Pfizer: Research Funding. Jasuja:Pfizer Inc.: Employment. Drakesmith:Pfizer: Consultancy, Research Funding; Kymab: Other: Scientific Advistory; La Jolla Pharmaceutical: Research Funding.

Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies of the intrinsic coagulation plasma proteins Factor VIII (FVIII) or Factor IX (FIX), respectively. An approach to achieving hemostatic pharmacology in hemophilia is to augment the extrinsic cascade is to neutralize Tissue Factor Pathway Inhibitor (TFPI). TFPI is a multi-Kunitz (K) domain inhibitor which binds to and inhibits Factor Xa via the K2 domain and Factor VIIa/Tissue Factor activity (K1 domain). Marstacimab is a fully human monoclonal antibody that binds to and neutralizes TPFI activity and is under development for treatment of hemophilia. To support lead identification during late discovery, five anti-tissue factor pathway inhibitor (TFPI) antibodies (EC50 Disclosures Parng: Pfizer: Employment. Jin:Pfizer: Employment. Holsti:Pfizer: Employment. Benard:pfizer: Employment. Rakhe:Pfizer Inc.: Employment. Patel-Hett:Pfizer: Employment. Joyce:Pfizer: Employment. Webster:pfizer: Employment. Pittman:Pfizer Inc.: Employment.