Genetic Code Expansion Enables Site-Specific PEGylation of a Human Growth Hormone Receptor Antagonist through Click Chemistry

Abstract

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 *

Polyglycerol for Half-Life Extension of Proteins—Alternative to PEGylation?

“…PEGylation was first introduced by Abuchowski and co-workers in 1977 and describes the covalent conjugation of one or multiple poly­(ethylene glycol) (PEG) chains to a protein in a random or site-specific manner, − being already exploited to a large number of biomolecules. , The resulting PEG-protein conjugates exhibit longer circulation time, mainly due to diminished renal clearance and shielding of immunogenic protein epitopes, preventing fast depletion from the bloodstream. Within the last few years, PEG’s image as being a safe and nonimmunogenic polymer, however, has been challenged by numerous reports on the occurrence of anti-PEG antibodies ,, in clinic, diminishing the benefits of PEGylation, thereby compromising its therapeutic efficacy. , Therefore, manifold efforts to find alternative polymer platforms for protein conjugation, covering synthetic as well as biodegradable, natural polymer systems, have been made. ,− …”

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

“…Conjugation of a 20 or 40 kDa PEG to the N-terminal of B2036 resulted in approximately 2- and 17-fold reduction in receptor binding activity, respectively . In addition, we have previously incorporated an unnatural amino acid into B2036 at amino acid Y35 for site-specific PEGylation using copper-catalyzed click chemistry and reported that 20 kDa PEG-conjugated B2036 exhibited 5.8-fold reduction in bioactivity compared to a 72.8-fold reduction observed for pegvisomant . Using this approach led to a conjugation efficiency of 90%.…”

“…37 In addition, we have previously incorporated an unnatural amino acid into B2036 at amino acid Y35 for site-specific PEGylation using coppercatalyzed click chemistry and reported that 20 kDa PEGconjugated B2036 exhibited 5.8-fold reduction in bioactivity compared to a 72.8-fold reduction observed for pegvisomant. 25 Using this approach led to a conjugation efficiency of 90%. However, the incorporation of an unnatural amino acid into B2036 reduced the protein expression yield and a byproduct (truncated protein) caused by the amber code requires additional purification steps to remove.…”

“…The preparation and evaluation of mono PEGylated GH have been reported, including one conjugate that is used clinically. − However, only two publications describe site-specific PEGylation of a GHR antagonist, , and information regarding the in vivo behavior is limited. The aim of this study was to generate a long-acting GHR antagonist with improved in vivo bioactivity in rodents.…”

Bioorthogonal Chemistry and Bioconjugation: Synergistic Tools for Biology and Biomedicine

“…Enzymatic 9 and affinity-guided tagging 10 provide effective selectivity, while genetic code expansion offers the unique opportunity to incorporate the orthogonal handles during protein biosynthesis. 11 The use of bioorthogonal catalysis coupled with DNA nanostructures is a particularly intriguing new direction to site-selectively modify proteins. 12 Bioorthogonal functionalization is an enabling technology for a broad range of applications.…”