Growth hormone receptor antagonists, GHA2 and GHA3, with dual activity against the human and mouse receptor.

Aberrant human growth hormone (GH) expression has been increasingly linked to poor outcomes in certain cancers and reduced health span. GH receptor (GHR) signal transduction activates signaling pathways that promote cell proliferation and survival, contributing to tumor progression. Consequently, there has been growing interest in creating antagonists that target GH signaling for anticancer applications. Pegvisomant is the only clinically available GHR antagonist (GHA). It is a protein antagonist that consists of a mutated GH protein (B2036) conjugated to polyethylene glycol (PEG). While pegvisomant is a potent human GHR antagonist, it has lower efficacy in mice, necessitating the use of higher doses in preclinical models. Here, we describe two antagonists, GHA2 and GHA3, based on an earlier reported antagonist, B2024, that have improved efficacy in mice. Recombinant GHA2 and GHA3 were produced in E. coli and then conjugated to multiple 5 kDa amine-reactive mPEG (GHA2-PEG5K) or site-specifically PEGylated with 40 kDa mPEG at amino acid site 144 (GHA3-PEG40K). When compared with B2036, GHA2 and GHA3 had significantly improved in vitro activity against the mouse GHR in a Ba/F3 cell viability assay, and GHA2 more effectively reduced GH-dependent signal transduction in the mouse melanoma cell line, B16-F10. A single dose of 30 mg/kg of amine-PEGylated GHA2-PEG5K or site-specific conjugate, GHA3-PEG40K, reduced serum IGF-1 in mice at 24 h by 40.7% and 45.8%, respectively. In contrast, amine-PEGylated B2036-PEG5K did not significantly reduce serum IGF-I levels. In conclusion, we demonstrate here that GHA2-PEG5K and GHA3-PEG40K are more potent inhibitors than B2036-PEG5K in mouse models.