Mouse Anti-Human GHR Hybridoma [IHI3.7.2]

Growth hormone (GH) is a peptide hormone that mediates actions through binding to a cell surface GH receptor (GHR). The GHR antagonist, B2036, combines an amino acid substitution at 120 that confers GHR antagonist activity, with eight additional amino acid substitutions. Conjugation to polyethylene glycol (PEG) increases the serum half-life of these proteins due to reduced renal clearance. Recombinant forms of GH and its antagonists are mainly produced in prokaryotic expression systems, such as E. coli. However, efficient production in E. coli is problematic, as these proteins form aggregates as inclusion bodies resulting in poor solubility. In the present study, we demonstrate that N-terminal fusion to a thioredoxin (Trx) fusion partner improves soluble expression of codon-optimized B2036 in E. coli when expressed at 18 degrees C. Expression, purification and PEGylation protocols were established for three GHR antagonists: B2036, B20, and G120Rv. Following purification, these antagonists inhibited the proliferation of Ba/F3-GHR cells in a concentration-dependent manner. PEGylation with amine-reactive 5 kDa methoxy PEG succinimidyl propionate yielded a heterogeneous mixture of conjugates containing four to seven PEG moieties. PEGylation significantly reduced in vitro bioactivity of the conjugates. However, substitution of lysine to arginine at amino acid residue 120 in B2036 improved the in vitro activity of the PEGylated protein when compared to unmodified PEGylated B2036. Pharmacokinetic analysis demonstrated that the circulating half-life of PEGylated B20 was 15.2 h in mice. Taken together, we describe an effective strategy to produce biologically active PEGylated human GHR antagonists.

Background: We proposed to verify the role of growth hormone receptor gene expression in growth failure of children with Crohn's disease (CD). Methods: We measured serum levels of growth hormone binding protein (GHBP) and insulin-like growth factor-I (IGF-I), and growth hormone receptor (GHR) gene expression in peripheral blood mononuclear cells of 21 patients with CD (before and after therapy) and in 27 age-sex-matched controls. Results: At diagnosis, significantly lower insulin-like growth factor-I and growth hormone binding protein levels were found in the CD group compared to the controls. Growth hormone receptor mRNA expression was lower in patients at diagnosis compared to the controls, even though the difference did not reach statistical significance, and significantly increased in patients in the following year. Insulin-like growth factor-I levels showed significant improvements 1 year after diagnosis compared to basal levels. On the contrary, growth hormone binding protein values had not significantly changed after 1 year of therapy. Conclusion: Our study raises the hypothesis of another mechanism through which cytokines interact with the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis.