Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogs exhibit short plasma circulation times. To improve clinical viability, between 4-6 molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the 9 amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper catalyzed click reaction yielded high purity, site-specific conjugates with *

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.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036–S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036–S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036–S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.

Decades of published research supports a role for growth hormone (GH) in cancer. Accordingly, there is increasing interest in targeting GH in oncology, with GH antagonists exhibiting efficacy in xenograft studies as single agents and in combination with anticancer therapy or radiation. Here we discuss challenges associated with using growth hormone receptor (GHR) antagonists in preclinical models and considerations for translation, such as the identification of predictive biomarkers for selecting patients and for monitoring drug efficacy. Ongoing research will determine whether suppressing GH signalling pharmacologically will also reduce the risk of developing cancer. An increase in GH-targeted drugs in preclinical development will ultimately provide researchers with new tools to test anti-cancer efficacy of blocking the GH signalling pathway in preclinical models.

Recombinant human growth hormone (rhGH) and GH receptor antagonists (GHAs) are used clinically to treat a range of disorders associated with GH deficiency or hypersecretion, respectively. However, these biotherapeutics can be difficult and expensive to manufacture with multiple challenges from recombinant protein generation through to development of long‐acting formulations required to improve the circulating half‐life of the drug. In this review, we summarise methodologies and approaches used for making and purifying recombinant GH and GHA protein, and strategies to improve pharmacokinetic and pharmacodynamic properties, including PEGylation and fusion proteins. Therapeutics that are in clinical use or are currently under development are also discussed.This article is protected by copyright. All rights reserved.