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 *

Vaults" are ubiquitously expressed endogenous ribonucleoprotein nanoparticles with potential utility for targeted drug delivery. Here, we show that recombinant human vault nanoparticles are readily engulfed by certain key human peripheral blood mononuclear cells (PBMC), predominately dendritic cells, monocytes/macrophages, and activated T cells. As these cell types are the primary targets for human immunodeficiency virus type 1 (HIV-1) infection, we examined the utility of recombinant human vaults for targeted delivery of antiretroviral drugs. We chemically modified three different antiretroviral drugs, zidovudine, tenofovir, and elvitegravir, for direct conjugation to vaults. Tested in infection assays, drug-conjugated vaults inhibited HIV-1 infection of PBMC with equivalent activity to free drugs, indicating vault delivery and drug release in the cytoplasm of HIV-1-susceptible cells. The ability to deliver functional drugs via vault nanoparticle conjugates suggests their potential utility for targeted drug delivery against HIV-1.

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.

Poly(ethylene glycols) (PEGs) with protein-reactive end-groups are widely utilized in bioconjugation reactions. Herein, we describe the use of ring-opening metathesis polymerization (ROMP) to synthesize unsaturated protein-reactive PEG analogs. These ROMP PEGs (rPEGs) contained terminal aldehyde functionality and ranged in molecular weight from 6 to 20 kDa. The polymers were readily conjugated to free amines on the protein hen egg-white lysozyme (Lyz). Biocompatibility of the unsaturated PEGs was assessed in vitro, revealing the polymers to be nontoxic up to concentrations of at least 1 mg/mL in human dermal fibroblasts (HDFs). The resulting unsaturated rPEG-lysozyme conjugates underwent metathesis-based depolymerization, resulting in decreased molecular weight of the conjugate.