Generation of Long-acting Growth Hormone Receptor Antagonists

Abstract

Growth hormone (GH) has been implicated in cancer progression and is a potential target for anticancer therapy. The GH receptor (GHR) antagonist, B2036, effectively inhibits GH signalling. B2036 is a biological agent based on GH; a single mutation in binding site 2 of the hormone converts it from an agonist into an antagonist. Conjugation of four to six 5 kDa polyethylene glycol (PEG) moieties to B2036 generates the clinically used agent, pegvisomant. PEGylation considerably extends the serum half-life of the antagonist but inevitable leads to a loss in bioactivity. In addition, the Ghr from rodents has very low affinity for pegvisomant and as a consequence very high doses are required for cancer xenograft studies (60-250 mg/kg/day). This drug is therefore not suitable for routine preclinical studies. The work in this thesis attempted different approaches to generate long-acting GHR antagonists. All GHR antagonists were expressed in E.coli and purified using a series of chromatographic methods. N-terminal fusion to Thioredoxin (Trx)-His tag improved soluble protein expression in E. coli when expressed at low temperature. Protein expression and purification protocols were established for a series of GHR antagonists, including B2036, B20, G120Rv, B2036-S144C and B24. PEGylation was employed to extend their circulating half-life of the proteins. PEGylation of B2036, B20 and G120Rv with amine-reactive 5 kDa PEG yielded a heterogeneous mixture of conjugates containing four to seven PEG moieties. This PEGylation significantly reduced their in vitro bioactivity, but prolonged circulating half-life would be expected to compensate for this. Substitution of lysine to arginine at amino acid residue 120 in B2036 improved the in vitro activity compared to unmodified PEGylated B2036. To further improve bioactivity of the antagonist, B2036 was site-specifically conjugated to 20, 30, or 40 kDa PEG maleimide through an introduced cysteine at amino acid 144 (S144C). In vitro bioactivity of these conjugates was significantly improved compared with amine PEGylated B2036. The circulating half-life of the 20, 30, and 40 kDa PEG conjugates was 16.4, 18.6 and 58.3 h in mice, respectively. Administration of 40 kDa PEG conjugates (10 mg/kg/day) reduced serum Insulin-like growth factor-I (IGF-I) concentrations by 50.6% in mice. Finally, another GH variant, B24, was found to have improved activity as a mouse Ghr antagonist compared to B2036. Following amine PEGylation, B24 effectively reduced circulating IGF-I in mice (55.7% reduction). Site-specific PEGylation of this variant should improve the in vivo bioactivity of this protein further. In conclusion, this thesis reports effective strategies to produce biologically active PEGylated GHR antagonists. Future studies will investigate site-specific attachment of PEG to B24 and will investigate efficacy in cancer xenograft studies.