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

• Main Authors: Uli Binder, Arne Skerra
• Format: Article
• Language: English
• Published: MDPI AG 2021-12-01
• Series: International Journal of Molecular Sciences
• Subjects: biobetter; cancer; COVID-19; drug delivery; half-life extension; immunostimulatory peptide
• Online Access: https://www.mdpi.com/1422-0067/22/1/124
• ISSN: 1661-6596; 1422-0067

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 <i>Escherichia coli</i> 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.