Optimization of thrombopoietin mimetic peptide fusion proteins with albumin-binding domain for enhanced bioactivity and extended half-life

Primary immune thrombocytopenia is an autoimmune disorder marked by accelerated platelet destruction and reduced production, posing risks of severe bleeding and mortality. Thrombopoietin (TPO) mimetic peptide (TMP) stimulates platelet generation via TPO receptor activation but is hindered by rapid clearance and short half-life. The primary objective of this study is to optimize TMP fusion proteins with the albumin-binding domain (ABD) for enhanced expression in Escherichia coli (E. coli), extended half-life via indirect FcRn-mediated recycling, and improved thrombopoietic activity in cellular assays and murine models. TMP dimers were fused to the C-terminus of ABD. GS peptides with either Cys or Ala were fused to the N-terminus of ABD (yielding C-ABD-2TMP and A-ABD-2TMP fusion proteins). The fusion proteins were expressed in E. coli BL21 (DE3) with Trx-tags for solubility, purified by Ni-NTA and ion exchange chromatography, and dimerized via disulfide bonds (2C-ABD-2TMP). Thermal stability was assessed by circular dichroism; HSA affinity by ELISA; bioactivity by MO7e cell proliferation assay; and in vivo pharmacokinetics and platelet stimulation in C57BL/6 mice. The fusion proteins were successfully expressed in E. coli. Following purification via Ni²⁺-NTA, tag cleavage, ion-exchange chromatography, and disulfide bond formation, high-purity fusion proteins were obtained: disulfide-bonded 2C-ABD-2TMP and A-ABD-TMP lacking disulfide bonds. Fusion proteins displayed highly stability up to 70 °C. 2C-ABD-2TMP exhibited an apparent EC₅₀ for HSA of 2.1 ± 0.6 nM (vs. 5.5 ± 1.1 nM for A-ABD-2TMP). In MO7e cells, 2C-ABD-2TMP promoted dose-dependent proliferation, exceeding 2TMP at 25 nM (p < 0.01) and A-ABD-2TMP (p < 0.05). In mice, 150 nmol/kg 2C-ABD-2TMP increased platelets to 3.1 × 10⁹/mL by day 12, sustained exceeding 2.4 × 10⁹/mL at day 18 (AUC p < 0.01 vs. controls). The 2C-ABD-2TMP fusion protein displayed a half-life of 17.6 ± 2.9 h, while the A-ABD-2TMP exhibited a half-life of 13.2 ± 1.6 h. significantly longer than 2TMP alone (~ 1 h). ABD fusion enhanced the pharmacokinetics and thrombopoietic activity of 2TMP by enabling binding to HSA while retaining the ability to activate the TPO receptor.