Modified Actriib-Fc Fusion Protein (ACE-536) Mitigates Sickling and Red Cell Pathology in a Murine Model of Sickle Cell Disease (2014)

Abstract Sickle cell disease (SCD) is a debilitating hereditary disorder caused by a single point mutation in the β-globin gene resulting in the production of sickle hemoglobin variant (HbS). In the deoxygenated state, HbS is labile and undergoes auto-oxidation and polymerizes to generate rigid and irreversibly sickled erythrocytes. The pathophysiology of SCD include increased red cell hemolysis, reactive oxygen species and phosphatidylserine (PS) exposure on RBC membranes which leads to a very short red cell half-life, increased reticulocytosis and splenomegaly. Sickled RBCs show enhanced adherence to activated endothelium causing chronic inflammation leading to frequent and acute painful vaso-occlusive crises in SCD patients. Hydroxyurea (HU) augments fetal hemoglobin production, decreases irreversible sickle cells and painful events, and is the only approved therapy for SCD patients. However, recent studies have shown dose limiting myelosupression with HU treatment. Approximately one-third of patients do not respond to HU therapy thereby highlighting the need for alternative treatment strategies. ACE-536 is a modified type IIB activin receptor-Fc fusion protein (ACE-536)1 which functions as a ligand trap for certain members of the TGFβ superfamily. In a murine model of β-thalassemia, RAP-536 (murine ortholog of ACE-536) treatment reduced hemichromes on RBC membranes, decreased reactive oxygen species, reduced hemolysis, improved red cell half-life and thus corrected anemia and mitigated disease complications of β-thalassemia syndrome2. In the present study, we evaluated RAP-536 as a monotherapy and combination therapy with HU in the murine model of sickle cell disease (βS/βS)3. SCD mice were dosed with RAP-536 (1 mg/kg, twice weekly, s.c.) or TBS vehicle (VEH) control (N=5/group) for 3 months. A combination treatment with HU (100mg/kg, i.p.) and RAP-536 (10mg/kg, s.c) twice weekly for 2 months was performed and compared with vehicle or HU monotherapy treated SCD mice. Non-symptomatic compound heterozygote (β/βS) littermates were treated similarly (N=5/group) and used as controls to confirm disease in SCD (βS/βS) mice. At study baseline, SCD mice had reduced RBC number (-28%, P&lt;0.01) and hemoglobin (-14.5%, P&lt;0.05) and increased reticulocytes (+50%, P&lt;0.001) compared to compound heterozygote mice. Following one month of treatment, RAP-536 (1mg/kg) significantly reduced spleen weight (-20.5%, P&lt;0.05), decreased serum bilirubin content (-17%, P&lt;0.01) and cell free hemoglobin (-30.7%, P=0.06) compared to vehicle treated mice indicating decreased hemolysis. Most remarkably, blood smears from RAP-536 treated SCD mice displayed a decrease in number of irreversibly sickled erythrocytes (-66.5%, P&lt;0.001) as well as reduced annexin V/PS exposure (-18.75%, N.S), suggesting improved membrane phospholipid asymmetry. RAP-536 treatment showed increased RBC number (+15.2%, P&lt;0.01) and hemoglobin (+9.28%, P&lt;0.05) compared to VEH treatment with concomitant decrease in reticulocytes (-13.5%, P&lt; 0.05), suggestive of an increase in red cell half-life. Furthermore, histopathological analysis of spleen, kidneys and heart revealed a trend toward reduced intravascular congestion in RAP-536 treated SCD mice. Preliminary data from the combination treatment of HU and RAP-536 in SCD mice displayed additive beneficial effects as compared to HU alone. The combination of RAP-536 and HU produced a greater reduction in annexin V/PS exposure on peripheral blood cells than did HU alone compared to vehicle treatment (-35.6%, P&lt;0.001 vs. -22.2%, N.S, respectively). Similarly, HU+RAP-536 showed a greater reduction in spleen size than HU alone (-50.7%, P&lt;0.05 vs. -20.2%, N.S) respectively, compared to vehicle treated SCD mice. Additional analyses are in progress. Taken together, these data demonstrates that RAP-536 reduces the RBC sickling and red blood cell pathology in SCD and also shows its utility as monotherapy and in combination with HU to further mitigate the disease severity. ACE-536 is currently being tested in Phase 2 clinical trials in MDS and β-thalassemia patients, and merits evaluation as a therapy for SCD patients. References: Suragani RN et.al; Nature Medicine 2014; 20: 408-14Suragani RN et.al; Blood 2014; 123: 3864-72Wu LC etal; Blood. 2006; 108:1183-8. Disclosures Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Cawley:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.