PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins

M Schlapschy,A Skerra,S Kisling,I Theobald,C Borger,U Binder,K Wachinger,D Haller

doi:10.1093/protein/gzt023

Abstract

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

Highlights

Short plasma half-life in humans of just a few hours after intravenous (i.v.) bolus injection is a typical feature of almost all biopharmaceuticals (Kontermann, 2012), or corresponding drug candidates, with renal clearance providing the predominant route of fast elimination
PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to polyethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension
Our concept for the design of polypeptides with PEG-like properties such as, in particular, large hydrodynamic volume, high solubility, and lack of charges was based on a hypothetical L-a-amino acid sequence whose backbone should adopt robust random chain conformation in aqueous solution and at ambient or body temperature and whose side chains have low tendency to form intermolecular interactions other than with solvent molecules

Summary

Introduction

Short plasma half-life in humans of just a few hours after intravenous (i.v.) bolus injection is a typical feature of almost all biopharmaceuticals (Kontermann, 2012), or corresponding drug candidates, with renal clearance providing the predominant route of fast elimination. A remarkable exception are intact IgG antibodies, which show a half-life of 1–3 weeks in humans (Lobo et al, 2004) and 6–8 days in mice (Vieira and Rajewsky, 1988; Milenic et al, 2010), owing to their large size as well as FcRn-mediated endosomal recycling (Roopenian and Akilesh, 2007). PEG forms a highly solvated, structurally disordered random chain in aqueous solution which, if conjugated, leads to an expanded hydrodynamic volume for the pharmaceutically active biomolecule and, to strongly retarded glomerular filtration. This phenomenon depends on the length of the PEG chain and is essentially based on a pure biophysical size effect (Pasut and Veronese, 2007)

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Conclusion