Background: The UK acromegaly register reported that <60% of acromegalics on medical therapy had controlled disease (1). This is because many patients do not respond to somatostatin therapy. Pegvisomant, a growth hormone antagonist (GHA), controls disease in >95% cases, but is not cost-effective and requires high dose daily injections (2). There is therefore an unmet need for a cost-effective GHA. We have developed a fusion technology for making a cost-effective long-acting GH molecule (3), and generated a GHA by linking mutated growth hormone to its binding protein (GHBP).
Design of GHA: Growth hormone (GH) contains two binding sites. A mutation (G120R) within site two produces a receptor antagonist and mutations in site 1 enhance binding creating a super antagonist. Linking to GHBP delays clearance but has the problem that site 1 in GH binds to GHBP reducing activity. We hypothesised that the addition of a W104A mutation in GHBP would prevent intramolecular binding and generate a potent antagonist.
Methods: Four target molecules were gene synthesised to include either site 2 mutation (GHA1), site 1 and 2 mutations (GHA2), site 2+W104A mutations (GHA3) and site 1 and 2+W104A mutations (GHA4). Proteins were expressed in CHO cells and purified using antibody based affinity chromatography. Analysis by SDSPAGE confirmed integrity and purity of protein. GH antagonist potency was tested using a GH-specific in vitro dual luciferase reporter assay.
Results: Median IC50s of 45 nM (GHA1); 133 nM (GHA2); 40 nM (GHA3) and 16 nM (GHA4) were obtained. Proteins were judged to be stable over an 8 day period when incubated at 4 °C, room temperature and multiple freeze/thaw cycles at −80 °C.
Conclusions: Site 1 mutations designed to enhance binding (GHA2) decreased bioactivity. However, the inclusion of a W104A mutation (GHA4) increased bioactivity. The results supported our hypothesis that the W104A mutation reduced intra and intermolecular binding. GHA4 has the potential to be a long-acting potent GHA and with no requirement for post-translational modification (e.g. pegylation) is likely to be a cost-effective treatment for acromegaly.
1. Howlett et al. Clinical Endocrinology. 2013 79(5) 68999.
2. Moore et al. BMC Endocr Disord. 2009; 9: 20.
3. Wilkinson et al. Nat Med. 2007; 13(9): 110813.