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. 2016 Oct 12;6:35072.
doi: 10.1038/srep35072.

A Long-Acting GH Receptor Antagonist Through Fusion to GH Binding Protein

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Free PMC article

A Long-Acting GH Receptor Antagonist Through Fusion to GH Binding Protein

Ian R Wilkinson et al. Sci Rep. .
Free PMC article

Abstract

Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days.

In conclusion: we provide proof of concept that a fusion of GHR antagonist to its binding protein generates a long acting GHR antagonist and we confirmed that introducing the W104A amino acid change in the GH binding domain enhances antagonist activity.

Conflict of interest statement

JRS and RJR hold equity in Asterion Ltd.

Figures

Figure 1
Figure 1. Overview of molecular interactions of GH and GH-Fusions.
(a) Schematic showing the binding of GH to growth hormone binding protein (GHBP) (1).GH binding is via two binding sites: site 1 (high affinity) and site 2 (low affinity) (2). GH binds to preformed GH receptor (GHR) dimers at the cell surface (3). (b) Detailed view of the GH/GHR interfaces, GH (red) is bound to two GHR molecules at site 1 (GHR1) and site 2 (GHR2). A G120R amino acid change (red spheres) abolishes binding at site 2; and the GHR W104A amino acid change (pink and purple spheres) abolishes binding to GH. (c) Possible conformations of the GH ligand-receptor fusions. The GH domain could associate via intramolecular interactions with GHBP to form an inactive closed monomer conformation, exist as an open monomeric conformation or it could form a reciprocal head-to-tail dimer. (d) GHA1-3 antagonist molecules consisting of GHR antagonist linked directly to the N-terminus of GHBP. GHA molecules contain combinations of GH site 1 amino acid changes - H18D, H21N, R167N, K168A, D171S, K172R, E174S and I179T (purple), GH site 2 (G120R) amino acid change (red), and the GHR W104A amino acid change (yellow).
Figure 2
Figure 2. Pharmacokinetic studies in rats and rabbits.
(a) Pharmacokinetics in rats. A single bolus intravenous injection of 1 nmole/kg was given to Crl:CD(SD) male rats (n = 6 animals per group). Sera was taken at specified time points and assayed for the presence of GHA. Data is displayed as a ratio of C/Cmax (+/− SEM). (b) Pharmacokinetics in rabbits. A single subcutaneous injection of 2 mg/kg was given to New Zealand White male rabbits (n = 3 animals per group). Sera was taken at specified time points and assayed for the presence of GHA. Data is displayed as a ratio of C/Cmax (+/− SEM).
Figure 3
Figure 3. Pharmacodynamics of GHA3 in New Zealand White Male Rabbits.
A single subcutaneous injection of 2 mg/kg was given to New Zealand White male rabbits (n = 3 animals per group). A buffer only vehicle control was also tested (n = 3 animals per group).The pharmacodynamic effects of GHA3 were followed over 196 hours (7 days). (a) Concentrations of IGF-I from rabbit serum samples challenged with GHA3 (+/− SEM). Samples were analysed using an automated iDS human IGF-I ELISA. (b) Body weight measurements were obtained daily over the study period.

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