Long-Acting Human Growth Hormone Receptor Antagonists Produced in E. coli and Conjugated with Polyethylene Glycol

Abstract

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 °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.

Enhanced Bioactivity of a Human GHR Antagonist Generated by Solid-Phase Site-Specific PEGylation

“…As shown in Figure A, monomeric B2036–S144C inhibited Ba/F3-GHR cell growth in a concentration-dependent manner with an IC 50 value of 10.1 ± 2.5 nM. This value is higher than that we have previously reported for unmodified B2036 (3.4 ± 0.1 nM) . Interestingly, the B2036–S144C dimer also inhibited Ba/F3-GHR cell growth with an IC 50 value of 4.9 ± 1.0 nM (Figure A).…”

“…The Ba/F3-GHR cell line has been modified to stably express the human GHR . Ba/F3-GHR cells were serum-starved and treated with the GHR antagonists for 20 min, followed by treatment with 20 ng/mL hGH and 48 h culture, as previously described . As shown in Figure A, monomeric B2036–S144C inhibited Ba/F3-GHR cell growth in a concentration-dependent manner with an IC 50 value of 10.1 ± 2.5 nM.…”

“…Recombinant proteins were essentially produced as previously described with minor modifications. 17 Detailed methodology for expression and purification is provided in the Supporting Information.…”

“…As B2036 contains eight lysine residues and an N-terminal amine, this method creates a heterogeneous mixture of B2036 conjugates with 4–6 PEG moieties attached. PEGylation dramatically reduces the inhibitory activity of the antagonist; studies from our laboratory have demonstrated that amine-PEGylated B2036 possesses possesses approximately 140-fold lower in vitro bioactivity compared with unmodified B2036 . However, this is compensated for in vivo , as pegvisomant has a significantly improved circulating half-life of 72 h in humans and effectively reduces circulating IGF-I …”

“…PEGylation dramatically reduces the inhibitory activity of the antagonist; studies from our laboratory have demonstrated that amine-PEGylated B2036 possesses possesses approximately 140-fold lower in vitro bioactivity compared with unmodified B2036. 17 However, this is compensated for in vivo, as pegvisomant has a significantly improved circulating half-life of 72 h in humans and effectively reduces circulating IGF-I. 16 Conducting in vivo preclinical studies into the use of GHR antagonism in cancer is challenging because amine PEGylation of B2036 (pegvisomant) dramatically reduces the in vivo bioactivity in rodents, requiring the use of very high doses in mice (60−250 mg/kg/day).…”