Development of a Long-Acting Parathyroid Hormone fo the Treatment of Hypoparathyroidism

Abstract

Background: Parathyroid hormone (PTH) is a peptide hormone consisting of 84 amino acids with residues 1-34 responsible for its biological activity. In Hypoparathyroidism, patients lack PTH due to loss or damage to the parathyroid glands leading to impaired mineral homeostasis. Hypoparathyroidism is commonly treated with oral calcium and active vitamin D and more recently with Natpara (PTH 1-84). However, because of a short serum half-life, PTH 1-84 requires daily injections and treatments are complicated by fluctuating calcium levels. An unmet need exists for a long-acting PTH that is effective, easy to administer and has realistic manufacturing costs. It is hypothesised that a fusion of PTH to a native binding protein, Growth Hormone Binding Protein (GHBP), will retain biological activity, and have a prolonged serum half-life through increased size and reduced proteolysis. This hypothesis has been tested in this thesis by studying three PTH fusion proteins: 14A7 (PTH linked to GHBP), 14A2c (PTH linked to the extracellular domain of PTH receptor and GHBP) and 14A8 as control for 14A2c wherein the PTH binding site on the receptor has been mutated to prevent PTH binding. Methods: Stable PTH fusions generated in CHO Flp-In cells were confirmed by RT-PCR and sequencing. Fusions were expressed in suspension culture in roller bottles as a secreted product and purified by ion exchange and affinity chromatography. Potency of Fusions was assessed using a Dual Luciferase Reporter Assay (DLRA) in the PTH responsive rat osteosarcoma cell line, UMR-106. EC50 values were obtained using GraphPad Prism software. Results: Protein expression for all PTH fusions was confirmed by western blotting. 14A7 separated as diffuse bands between 37 and 50 kDa and purified to 0.93 mg/mL. 14A2c and 14A8 separated as diffuse bands between 75 and 100 kDa and purified to 2.57 mg/mL and 2.29 mg/mL, respectively. During the DLRA validation process, pRL-TK was shown to be superior to pRL-CMV as an internal control. pRL-TK at 10 or 20 ng per transfection yielded more consistent results with no variation in Renilla expression with increasing concentrations of PTH 1-34 or PTH fusions. Using the validated DLRA, Mean ± SD EC50’s nM were: PTH 1-34 = 2.39 ± 0.58 (n = 8), 14A7 = 68.02 ± 4.01 (n = 4), 14A2c = 699.4 ± 55.6 (n = 3), and 14A8 = 253.8 ± 26.3 (n = 3). Conclusion: All three PTH fusions were successfully expressed and for 14A8 and 14A7 stable CHO Flp-In cell lines were created. Protein was obtained in the expression media and purified for all PTH fusions. All three fusion proteins showed biological activity although this was less than that for PTH 1-34. The addition of the extracellular PTH receptor to the fusion, as in 14A2c, reduced biological activity which was partially restored in 14A8, by mutating the PTH receptor moiety, suggesting that inter- or intra-molecular binding was taking place. In conclusion, it is possible to express PTH linked to a binding protein and retain biological activity providing potential for a long-acting PTH molecule.