PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

Int J Mol Sci. 2020 Dec 24;22(1):124. doi: 10.3390/ijms22010124.

Abstract

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Keywords: COVID-19; PASylation; biobetter; cancer; drug delivery; half-life extension; immunostimulatory peptide; pharmacokinetics; thymosin alpha 1; viral disease.

MeSH terms

  • Acetylation
  • Acetyltransferases / metabolism
  • Adjuvants, Immunologic / genetics
  • Adjuvants, Immunologic / pharmacokinetics*
  • Adjuvants, Immunologic / pharmacology
  • Animals
  • COVID-19 / drug therapy
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / metabolism
  • Female
  • Half-Life
  • Mass Spectrometry
  • Microscopy, Electron, Scanning
  • Neoplasms / drug therapy
  • Peptides / chemistry
  • Proteolysis
  • Rats
  • Rats, Wistar
  • Recombinant Fusion Proteins / blood
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / pharmacokinetics
  • Recombinant Fusion Proteins / ultrastructure
  • Ribosomal Proteins / metabolism
  • Thymalfasin / blood
  • Thymalfasin / chemistry
  • Thymalfasin / genetics
  • Thymalfasin / pharmacokinetics*
  • Virus Diseases / drug therapy

Substances

  • Adjuvants, Immunologic
  • Escherichia coli Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Ribosomal Proteins
  • RimJ protein, E coli
  • Acetyltransferases
  • Thymalfasin