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

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.

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life

“…Accordingly, UOX-PAS 100 exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Similar results have been obtained by the previous studies performed on other PASylated proteins such as Certolizumab Fab, Tα1, leptin, and IFN-β1b . UOX-PAS 100 retained 89.0% of original activity after 72 h incubation in rabbit plasma in 37 °C.…”

“…The high hydrophilicity of PAS polypeptide helps form a watery cloud around the therapeutic molecule and hence reduces the interaction with proteolytic enzymes and neutralizing antibodies in plasma. , PASylated biotherapeutics show better stability and function, decreased immunogenicity and antigenicity and offer new hope of improved clinical outcomes . There are successful examples of PASylation in production of biopharmaceuticals, e.g., ferritin, Leptin, IFN-β1b, Tα1, GH, Interleukin-1­(IL-1) and Coversin . In 2018, a recombinant PASylated UOX was developed by Horizon Pharma plc and XL-protein GmbH for the treatment of people with uncontrolled gout.…”

“…PASylation is a cost-effective technology that improves plasma half-life, solubility, stability, and potency of biopharmaceutical, making them suitable for administration. 16 So far, PASylation has been utilized for a series of protein biopharmaceuticals, i.e., interferon alpha (IFN-α2α), 17 growth hormone (GH), 18 interferon-β1b (IFN-β1b), 19 Thymosin α1 (Tα1), 20 Certolizumab Fab, 21 Adnectin C, 22 and leptin. 23 In 2018, a recombinant PASylated UOX was developed by Horizon Pharma plc and XL-protein GmbH for the treatment of people with uncontrolled gout.…”

Current Use of Asparaginase in Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma

“…Alternative strategies are being explored including a method termed PASylation in which the therapeutic protein is fused with a conformationally disordered 600 residue polypeptide rich in proline/alanine +/- serine ( 120 ). PASylation has been used to extend the half-life of a number of compounds ( 121 ) including immunostimulatory peptides ( 122 ), interferons ( 123 ), and urate oxidase ( 124 ). A PASylated Erwinia asparaginase is currently in the pre-clinical pipeline for Jazz pharmaceuticals 3 , offering hope for a long-acting Erwinia asparaginase in the future.…”

Peptides for Health Benefits 2020

“…In the study of Bryzek et al [ 4 ], arginine residues in human cathelicidin LL-37 are substituted by homoarginine citrulline to produce a new generation of agents to treat bacterial infections and other inflammatory diseases associated with peptidyl-arginine deiminases activity. Binder and Skerra [ 5 ] combine PASylation ® technology with enzymatic in situ N-acetylation by RimJ to obtain a long-acting version of immunostimulatory peptide thymosin α1 in Escherichia coli at high yield. The findings of this study provide the basis for the therapeutic development of a next generation thymosin α1 with prolonged plasma circulation that promotes its benefits against hepatitis B and C viruses, among other infections.…”