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 analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(
ethylene glycol) (PEG) are nonspecifically conjugated to the nine
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 >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to
pegvisomant, with an inverse relationship between
polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to
pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.