In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16432-7. doi: 10.1073/pnas.1006044107. Epub 2010 Sep 1.

Abstract

This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R(h)): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R(h): 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Female
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Green Fluorescent Proteins / pharmacokinetics
  • Inteins / genetics
  • Mice
  • Mice, Nude
  • Molecular Structure
  • Neoplasms, Experimental / metabolism
  • Polyethylene Glycols / chemistry
  • Polyethylene Glycols / metabolism*
  • Polyethylene Glycols / pharmacokinetics
  • Protein Engineering
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Recombinant Fusion Proteins / pharmacokinetics

Substances

  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Polyethylene Glycols