ALK2 Inhibition and a Modified Activin Receptor Type Iia Ligand Trap Cotherapy Maximized Hematologic Improvements in a Mouse Model of Anemia of Inflammation

Introduction: Anemia of inflammation (AI) is commonly seen in patients with chronic diseases, including chronic kidney disease (CKD). Current therapeutic options to mitigate anemia in AI include treating the underlying inflammatory disease, oral or intravenous iron supplementation, erythropoietin, and blood transfusions. The persistent release of inflammatory cytokines in AI not only induces the iron regulator hepcidin to inhibit iron absorption and recycling, but also directly impacts the marrow to suppress erythropoiesis, further worsening the ineffective, iron-restricted erythropoiesis. Therefore, targeting both iron and erythropoiesis could comprehensively address anemia in AI.

We previously showed that ALK2 inhibition was sufficient to reduce serum hepcidin and improve hemoglobin in a CKD mouse model. We also demonstrated that a modified activin receptor IIA ligand trap RKER-050 increased hemoglobin and red blood cell production by promoting erythroid differentiation in mice. Here, we explored the potential of additive improvements in hematologic parameters that could be achieved with a combination of hepcidin suppression by ALK2 inhibition and increased hematopoiesis with RKER-050 in a mouse model of AI.

Methods: C57BL/6 mice were fed a control or 0.2% adenine with 40 ppm iron diet (Ade 40) for 5 weeks to induce CKD. After confirming anemia, CKD mice were subcutaneously treated with 3 mg/kg isotype control or KTI-m216, a neutralizing antibody against ALK2, twice a week for 4 weeks while continuing the Ade 40 diet or switching to a 0.2% adenine with 3 ppm low iron diet (Ade 3). Combination therapy was evaluated in CKD mice on Ade 40 diet receiving biweekly isotype or KTI-m216 and also received either weekly PBS or 7.5 mg/kg RKER-050 intraperitoneally for 4 weeks.

Results: CKD mice developed impaired kidney function and characteristics of AI, including increased serum IL-6 and hepcidin, hypoferremia, tissue iron retention, and anemia compared to control mice. Serum hepcidin was reduced >95% in KTI-m216 treated mice on both Ade diets compared to mice receiving isotype control (CKD control). Transferrin saturation (TSAT) increased 65% in KTI-m216 treated mice on Ade 40 diet, with a similar trend in mice on Ade 3 diet. When compared to CKD control of the same diet, KTI-m216 treated mice had similar hematologic improvements: hemoglobin (+2.2 g/dL), hematocrit (+5.4%), red blood cells (+1.1 M/μl), and reticulocyte hemoglobin (CHr, +1.8 pg). These data support that increased circulating iron by KTI-m216 mediated hepcidin suppression can increase erythropoiesis in CKD mice. Data also suggest that dietary iron contribution is negligible in supporting erythropoiesis in this context.

With combination therapy, mice receiving KTI-m216 alone (KTI-m216 and PBS) and a combined KTI-m216 and RKER-050 therapy had a similar reduction in serum hepcidin (>91%) and a similar increase in TSAT (>1.7-fold), compared to mice receiving vehicle (isotype and PBS) controls. Mice treated with KTI-m216 alone increased hemoglobin to 11.4 g/dL compared to vehicle treated mice with hemoglobin of 8.0 g/dL. Treatment with a combined KTI-m216 and RKER-050 therapy showed a further increase in hemoglobin to 13.5 g/dL. Additionally, red blood cell production was 6.4 M/μl in mice given vehicle controls and increased to 8.3 M/μl in KTI-m216 alone and 9.8 M/μl in combined KTI-m216 and RKER-050; hematocrit was 23.4% in vehicle control, 32.5% in KTI-m216 alone and 38.7% in combined KTI-m216 and RKER-050. Although hematocrit was not fully rescued, hemoglobin and red blood cells in mice receiving a combined KTI-m216 and RKER-050 therapy were not different from the non-CKD controls.

Conclusion: These data illustrate that inhibition of ALK2 with KTI-m216 could potentially release sufficient iron from the recycling pathway to ameliorate anemia, but not restore RBC parameters to normal levels. Combined treatment with activin receptor ligand trap, RKER-050 and KTI-m216 normalized the RBC production in this preclinical model. These data support that increasing erythropoiesis with RKER-050 combined with iron mobilization with KTI-m216 has the potential to achieve the maximal amelioration of anemia in AI.

Wang:Keros Therapeutics: Current Employment. Melgar-Bermudez:Keros Therapeutics: Current Employment. Welch:Keros Therapeutics: Current Employment. Tseng:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cadena:Keros Therapeutics: Current Employment. Lerner:Keros Therapeutics: Current Employment. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.