E-Poster 63rd Endocrine Society of Australia Annual Scientific Meeting 2020

Development of long-acting human growth hormone antagonists by solid-phase site-specific PEGylation (#57)

Yue Wang 1 , Ries Langley 2 , Kyle Tomshen 3 , Julia Harms 1 4 , Heather Maynard 3 , Stephen Jamieson 4 , Jo Perry 1
  1. Liggins Institution, The University of Auckland, Auckland
  2. Department of Molecular Medicine and Pathology, The University of Auckland, Auckland
  3. Department of Chemistry and Biochemistry, University of California, Los Angeles
  4. Auckland Cancer Society Research Centre, The University of Auckland, Auckland

Growth hormone (GH) mediates actions through binding to the GH receptor (GHR), activating key signalling pathways including the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway 1. Excessive GH secretion leads to acromegaly and tumoral expression has been implicated in cancer progression 2,3. GHR antagonist B2036 effectively inhibits GH signalling. B2036 is a biological agent based on GH; a single mutation in binding site 2 of the hormone converts it from an agonist into an antagonist. Conjugation of 4-6 of 5 kDa polyethylene glycol (PEG) to B2036 generates the clinically used agent, pegvisomant 4. PEGylation considerably extends the serum half-life of the antagonist but inevitable leads to a loss in bioactivity. In addition, the Ghr from rodents has very low affinity for pegvisomant and as a consequence very high doses are required for cancer xenograft studies (60-250 mg/kg/day) 5. This drug is therefore not suitable for routine preclinical studies. To generate an antagonist for in vivo study, B2036 was site-specifically conjugated to 20, 30, or 40 kDa PEG maleimide through an introduced cysteine at amino acid 144 (S144C). A codon optimised B2036-S144C was generated by gene synthesis and recombinantly engineered by gene fusion with thioredoxin. Recombinant B2036-S144C was produced from E. coli and was PEGylated using cysteine-specific conjugation chemistry. In vitro bioactivity of these conjugates was significantly improved compared with amine PEGylated B2036. The circulating half-life of the 20, 30, and 40 kDa PEG conjugates was 16.4, 18.6 and 58.3 h in mice, respectively. Administration of 40 kDa PEG conjugates (10 mg/kg/day) reduced serum IGF-I concentrations by 50.6% in mice. 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. Future studies will investigate their efficacy in cancer xenograft studies.

  1. Lu, M.; Flanagan, J. U.; Langley, R. J.; Hay, M. P.; Perry, J. K. Targeting Growth Hormone Function: Strategies and Therapeutic Applications. Signal Transduct. Target. Ther. 2019, 4 (1), 3. https://doi.org/10.1038/s41392-019-0036-y.
  2. Wang, Y.; Langley, R. J.; Tamshen, K.; Jamieson, S. M.; Lu, M.; Maynard, H. D.; Perry, J. K. Long-Acting Human Growth Hormone Receptor Antagonists Produced in E. Coli and Conjugated with Polyethylene Glycol. Bioconjug. Chem. 2020, 31 (6), 1651–1660. https://doi.org/10.1021/acs.bioconjchem.0c00208.