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

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.