Elastin-based polypeptide–Fc fusion for extended subcutaneous exposure and sustained thrombopoietic activity

Immune thrombocytopenia is characterized by increased platelet destruction and impaired platelet production. Thrombopoietin receptor agonists (TPO-RAs) stimulate megakaryopoiesis, but the influence of elastin-based polypeptide (EBP) length on the hydrodynamic properties and platelet responses of Fc-fused TPO-RAs remains unclear. We generated three EBP–Fc fusion TPO-RAs—NT-202 G3, G6, and G12—and evaluated their physicochemical properties, biological activity, and platelet responses; G12 was also examined in an exploratory pharmacokinetic study. The variants, produced in Expi293 cells, differed only in EBP length; all remained soluble at 37°C in phosphate-buffered saline, and hydrodynamic size increased with EBP length. Binding was detected in BaF3/hMPL cells expressing the human thrombopoietin receptor but was minimal in parental BaF3 cells. The variants induced M-07e cell proliferation, JAK2–STAT signaling, and megakaryocytic differentiation of human CD34 + cells. After a single subcutaneous dose of 500 µg/kg in normal mice, platelet elevation persisted longest with NT-202 G12. At 2000 µg/kg, NT-202 G12 peaked at 72 h and remained quantifiable through 336 h. Because the pharmacokinetic study lacked an intravenous arm, used a different dose from the platelet-response studies, and relied on composite mean profiles, it was interpreted descriptively and could not distinguish absorption from elimination or establish an exposure–response relationship. In an anti-CD41 antibody-induced acute thrombocytopenia model, NT-202 G12 and romiplostim, an Fc–peptide TPO-RA, increased platelet counts without a significant between-group difference on day 9. Overall, this exploratory proof-of-concept study suggests that EBP block length is a genetically encoded design variable associated with hydrodynamic size and platelet-response duration.