Optimization of Targeted Erythropoietin for Treatment of Anemia

The design of cell-targeted protein therapeutics can be inspired by natural protein interactions that use cooperative contacts to achieve cell type specificity. We previously applied this approach to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors on red blood cell (RBC) precursors and prevent interaction with nonerythroid cells, such as platelet precursors. We described an engineered EPO molecule that was mutated to weaken its affinity for EPO-R, with its avidity for RBC precursors rescued by tethering to an antibody fragment that binds the human RBC marker glycophorin A (Burrill et al., PNAS 2016, 113(19):5245-50). This molecule stimulates red blood cell formation but not platelet formation in mice, as predicted. Surprisingly, we found that mice treated with the antibody-EPO fusion protein showed accelerated blood clotting relative to saline-treated mice or even mice treated with normal EPO (which also show somewhat accelerated blood clotting). The acceleration of clotting was associated with the glycophorin-binding antibody element (specifically, the 10F7 antibody). We further found that the 10F7 antibody-EPO fusion protein stimulated phosphorylation of Tyr9 in the red blood cell protein Band 3. This protein is known to heterodimerize with glycophorin A in a signaling complex to mediate H2O2 production, RBC stiffening, and ATP release – all indicators of pro-inflammatory signaling. We recently identified and alternative antibody fragment that is predicted not to cause inflammatory signaling, fused it to a mutated EPO, and found that the resulting fusion protein is 10- to 100-fold more active in vitro and in vivo.

Supported by: Wyss Institute for Biologically Inspired Engineering, and NIH- U54HL119145 (Boston Biomedical Innovation Center; B-BIC)