Hepcidin-Ferroportin axis dictates optimal absorption of dietary iron as well as systemic iron levels. This is crucial for providing sufficient iron needed for cellular functions while also preventing iron toxicity. PTG-300 (currently in a Phase 2 clinical study for beta-thalassemia) is a peptide mimetic of natural hepcidin that targets the major iron transporter, ferroportin, and causes its internalization & subsequent degradation. The pharmacodynamic effects of PTG-300 are the reductions in serum iron and transferrin-saturation (TSAT) due to reduced ferroportin expression on cells that store or recycle iron. We chose to demonstrate in two mouse models with iron dysregulation, that our hepcidin mimetics improve disease parameters by correcting dysregulated iron homeostasis. Beta-thalassemia is characterized by an imbalance in alpha-beta globin ratio in erythrocytes due to underlying beta-globin gene mutations. The excess alpha-globin, along with associated heme and iron, form "hemichrome" aggregates that integrate into the membranes of RBCs. The labile iron in these hemichromes generate ROS and are toxic to the cells, causing premature hemolysis of circulating RBCs and reduction in their lifespan. In a mouse model for beta-thalassemia, Hbbth3/+, we investigated the efficacy of a hepcidin mimetic in reducing hemichrome aggregation by limiting iron in the erythroid progenitors, and thereby reducing iron toxicity in RBCs. Subcutaneous injections of 1 mg/kg PN-8772 (analog of PTG-300 which has similar in vitro and in vivo potency) were administered every other day (Q2D) for a period of 4 weeks. At the end of the study, hemichrome aggregates were extracted from RBC membranes, and then analyzed on a TAU gel to quantify the cytoskeleton α-globin band intensities (Casu et al, Blood 2016). Hemichrome aggregates were reduced in groups treated with PN-8772 as compared to untreated controls, with concurrent improvements in hemoglobin and reductions in reticulocytes. Treatment with oral chelator Deferasirox (200 mg/kg; daily) did not show reduction in hemichrome aggregation, while it significantly lowered liver iron-overload. RBCs in Hbbth3/+ mice express aberrant morphologies due to the underlying hemichrome toxicity, similar to the phenotypes expressed in human beta-thalassemia. Chronic treatment with PN-8772 (as described above) also resulted in a significant reduction in aberrant morphologies that are indicative of hemolysis, viz. spherocytes & schistocytes. In a separate study, flow cytometry was used to monitor the survival of RBCs in Hbbth3/+ mice. At the end of 4 weeks of PTG-300 treatment (1 mg/kg, Q2D) the RBCs were marked by an in-life biotinylation method (Schmidt et al, Blood 2013) and subsequently followed over 49 days with continued treatment. There was a significant increase in survival of RBCs as compared to untreated controls. In summary, we demonstrate that by limiting iron in the developing erythroblasts and iron toxicity in RBCs, PTG-300 therapy has the potential to improve the quality of the RBCs and their oxygen carrying capacity, thereby ameliorating anemia. In beta-thalassemia, the clinical presentation includes secondary iron overload in various organs because of hyperabsorption of dietary iron, exacerbated by frequent blood transfusions that are required for management of anemia. Similarly, in hereditary hemochromatosis (HH) there is hyperabsorption of dietary iron leading to primary iron overload. We used a hemochromatosis mouse model (HFE) to demonstrate the effectiveness of PTG-300 therapy in limiting systemic iron toxicity by regulating TSAT and in preventing hyper-iron absorption. The model is characterized by homozygous deletion of HFE with severely low hepcidin levels and consequently very high TSAT (~100%). In this model, a single dose of PTG-300 at 2.5mg/kg reduced TSAT by ~60% at 10-hour post-dose, as compared to untreated controls. Sustained TSAT reduction by chronic treatment will therefore mitigate toxic effects of labile iron. Two weeks of chronic treatment with PTG-300 (2.5 mg/kg, Q2D) effectively prevented iron deposition in the liver. Overall our data suggests that PTG-300 has the potential to be an effective treatment in hemoglobinopathies, like beta-thalassemia, and Hereditary Hemochromatosis, by reducing systemic labile iron toxicity by limiting TSAT, preventing organ iron deposition & improving anemia (in case of thalassemia). Disclosures Taranath: Protagonist Therapeutics: Employment. Bourne:Protagonist Therapeutics: Employment. Zhao:Protagonist Therapeutics: Employment. Frederick:Protagonist Therapeutics: Employment. King:Protagonist Therapeutics: Employment. Liu:Protagonist Therapeutics: Employment.

Polycythemia Vera (PV) is a rare blood disease where mutations in JAK2 kinase confer constitutive JAK2 activity leading to abnormally elevated erythropoiesis that is independent of erythropoietin. PV patients present with iron deficiency at diagnosis due to increased iron utilization for erythropoiesis (Ginzburg YZ, Leukemia 2018) which worsens after repeated therapeutic phlebotomy (TP) performed to maintain hematocrit below 45%. The resulting suppression of hepcidin, the body's main negative regulator of iron metabolism, fuels expanded erythropoiesis resulting in a continued need for TP and thereby exacerbating patients' iron deficiency. Rusfertide targets iron exporter membrane protein ferroportin to trigger its degradation, preventing iron export from cells responsible for dietary iron absorption and cells that store and recycle iron. The resulting pharmacodynamic effect of lowered serum iron has disease-modifying effects in PV (Ginzburg YZ, Leukemia 2018). Rusfertide essentially eliminated the need for therapeutic phlebotomy in all PV patients (Kremyanskaya M, Blood 2020 136 Suppl 1: 33). Rusfertide also reversed iron deficiency, as indicated by increased serum ferritin, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in these patients. We present results from studies in a mouse PV model with JAK2-V617F mutation as in human PV (Mullaly A, Cancer Cell 2010; JAX stock #031658). We show that rusfertide analog Peptide A is efficacious in lowering hematocrit (HCT) while modulating other hematological parameters. Further, we show redistribution of iron away from erythropoiesis and renormalization of iron homeostasis as evidenced by ferrokinetic parameters. PV mice were treated over 6-weeks (thrice per week) with Vehicle or Peptide A at 2.5 or 7.5 mg/kg. At the end of 6 weeks, hematology parameters HCT, hemoglobin, RBC counts, were elevated in the PV-Vehicle group as compared to wild type (WT-Vehicle) mice (Table 1). Hematology parameters in PV-2.5 mg/kg group were lowered to WT-Vehicle values. In PV-7.5 mg/kg group, these parameters were lower than WT-Vehicle values, indicating that excessive iron restriction (EIR) leads to the expected anemic conditions. MCH and mean corpuscular hemoglobin concentration (MCHC) in PV-Vehicle group and PV-2.5 mg/kg treated were comparable to WT-Vehicle, indicating a lack of EIR. For the PV-7.5 mg/kg treated group, MCH and MCHC were significantly lower than WT-Vehicle, suggesting EIR at a high dose impacts hemoglobin concentration of RBC. To investigate the impact of iron restriction with Peptide A on erythroblast precursor cells in bone marrow, we conducted flow-cytometry analysis by gating on CD71 and TER-119 expression, and measuring intracellular iron using Ferro Far Red (FFR) dye. The CD71 + early precursor cell population did not change with Peptide A treatment however, the CD71 -/TER-119 + late precursor cell population was significantly lowered (~4-fold and 7.5-fold, in 2.5 and 7.5 mg/kg Peptide A treated PV groups respectively). Iron levels of CD71 + cells were dose-dependently and statistically significantly reduced in the Peptide A treated groups as compared to PV-Vehicle group. Iron levels of CD71 - cells were marginally lowered only in the PV-7.5 mg/kg group. We investigated the nature of iron redistribution induced by Peptide A, by using flow assay to assess iron concentration in splenic macrophages (F4/80 +/CD11b +). Iron was ~2-fold higher in the PV-7.5 mg/kg group as compared to PV-vehicle, and marginally higher in PV-2.5 mg/kg group. Total tissue iron concentration in the spleen was elevated in a dose-related manner in Peptide A treated groups compared to PV-Vehicle group, and in commensuration serum ferritin was increased. Serum iron was ~2-fold lower in PV-Vehicle group as compared to WT-Vehicle indicating iron depletion due to increased iron utilization for erythropoiesis. Serum iron measured after clearance of Peptide A from circulation (48 hr post-dose), was marginally increased for both Peptide A treated groups compared to PV-Vehicle. These data demonstrate that treatment with rusfertide and analogs, restricts iron from erythropoiesis by sequestering it in macrophage storage compartments. These effects along with normalization of iron homeostasis contribute to usefulness of rusfertide dose titration treatment in maintaining HCT <45% and improving symptoms related to iron deficiency in human PV. Figure 1 Figure 1. Disclosures Taranath: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhao: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Vengalam: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tang: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dion: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Su: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tovera: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bhandari: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cheng: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Protagonist Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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In polycythemia vera (PV), point mutation in JAK2 kinase (V617F) confers constitutive intracellular activity to JAK2 leading to a condition with excessive erythropoiesis that is independent of erythropoietin. Elevated hematocrit (HCT) and hyper-viscosity in the blood are risk factors for thrombosis and other symptoms. There is an increased iron demand required to support excessive erythropoiesis in the bone marrow. Hence, regulation of systemic iron in PV provides a mechanism for erythropoiesis control with potential disease modifying effects (Ginzburg YZ, Leukemia 2018). PTG-300 is an injectable hepcidin peptidomimetic drug currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis. Pharmacodynamic (PD) effects of PTG-300 are reductions in serum iron and transferrin-saturation due to reduced expression on cells of ferroportin which exports iron into the peripheral circulation. Splenic red pulp macrophages that recycle iron from senescent RBCs are the primary source of serum iron for erythropoiesis. PTG-300 administration in cynomolgus monkey resulted in dose dependent reduction in serum iron (Fig. 1). Single subcutaneous (SC) dose of 0.3 mg/kg achieved a maximum of ~54.9% reduction in serum iron at 24 hr post-dose, serum iron returned to baseline in < 60 hr. Maximum PTG-300 concentration of 171.6±31.2 nM was observed at 8 hr, and concentration had reduced to 25.5±3.9 nM by 72 hr. SC dose of 1 mg/kg achieved ~75.8% reduction in serum iron at 24 hr post-dose (from baseline 31.3±2.3 µM to 7.6±0.9 µM). This dose exhibited a more sustained PD, with serum iron returning to baseline at ~108 hr. For 1 mg/kg, maximum PTG-300 concentration was 472.5±47.2 nM at 6 hr, and concentration reduced to 70.9±13.7 nM by 72 hr. At both doses a delay of 16-18 hr was observed between the maximum PTG-300 concentration and the maximum PD response. In a dose limiting GLP toxicity study in monkeys (over 13 weeks), weekly SC dosing of PTG-300 resulted in dose-dependent reductions in HCT (Fig. 2). In 2 mg/kg dose group, HCT reduction from baseline was 2.5% on Day 27 and 5.3% on Day 90 in males, and 3.5% on Day 27 and 6.8% on Day 90 in females. There were no decreases in mean corpuscular volume as is otherwise observed in phlebotomy dependent PV patients who are iron-deficient, indicating that PTG-300 may not create an iron deficient state but rather restricts iron availability to the bone marrow thereby reducing RBC levels. At higher dose of 6 mg/kg, HCT reduction was 14.2% at Day 27 and 19% at Day 90 in males, and 12.4% at Day 27 and 14.5% at Day 90 in females. In concurrence with HCT, dose dependent reductions were observed in hemoglobin (HGB). 0.6 mg/kg dose did not show significant reductions in HCT or HGB, albeit only marginal reductions at Day 90 (data not shown). HCT and HGB returned to baseline after a recovery period of 30 days post-last dose for all groups. In a mouse model for acquired secondary erythrocytosis, wild type mice were administered with daily injections of exogenous erythropoietin (EPO; 50 units/dose) for 7 days(Wang J, Haematologica 2018), resulting in elevated HCT. Concurrent treatment with peptide A (hepcidin mimetic with in vitro and in vivo potencies similar to PTG-300) on alternate days showed dose dependent reductions in HCT, RBCs and reticulocytes (Fig. 3). 5 mg/kg dose was able to normalize all three parameters to levels similar to control group that did not receive any EPO. Dose dependent reductions in spleen weight indicate that peptide A was effective in lowering extra medullary stress erythropoiesis in the spleen. The desired end points for PTG-300 as a therapy for PV are sustained reduction of HCT to circumvent the need for chronic phlebotomy and prevent the exacerbation of systemic iron deficiency. Our pre-clinical data support the efficacy of our hepcidin mimetics in limiting erythrocytosis with sustained control of hematocrit through iron redistribution. In addition to lowering hematocrit and reducing the need for phlebotomy treatment, we believe that PTG-300 has the potential to reduce debilitating symptoms associated with chronic iron deficiency by restoring tissue iron to meet the needs of critical/normal cellular functions (Krayenbuehl PA, Blood 2011). PTG-300 would potentially provide a novel mechanism for selective hematocrit control while maintaining adequate body iron levels in polycythemia vera and other congenital and acquired erythropoietic disorders. Disclosures Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in private company. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Zhao:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company.