Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity

PLoS One. 2011 Feb 14;6(2):e17042. doi: 10.1371/journal.pone.0017042.

Abstract

Alginate lyase enzymes represent prospective biotherapeutic agents for treating bacterial infections, particularly in the cystic fibrosis airway. To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented. Rationally designed, site-specific PEGylation variants were constructed by orthogonal maleimide-thiol coupling chemistry. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produced a conjugate that maintained wild type levels of activity towards a model substrate. Significantly, the PEGylated variant exhibited enhanced solution phase kinetics with bacterial alginate, the ultimate therapeutic target. The immunoreactivity of the PEGylated enzyme was compared to a wild type control using in vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer. In both cases, the PEGylated enzyme was found to be substantially less immunoreactive. Underscoring the enzyme's potential for practical utility, >90% of adherent, mucoid, Pseudomonas aeruginosa biofilms were removed from abiotic surfaces following a one hour treatment with the PEGylated variant, whereas the wild type enzyme removed only 75% of biofilms in parallel studies. In aggregate, these results demonstrate that site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yielded an enzyme with enhanced performance relative to therapeutically relevant metrics.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Catalysis
  • Enzyme Activation / genetics
  • Genetic Engineering*
  • Humans
  • Immunity, Innate / drug effects
  • Polyethylene Glycols / metabolism*
  • Polyethylene Glycols / pharmacology
  • Polysaccharide-Lyases / genetics
  • Polysaccharide-Lyases / immunology*
  • Polysaccharide-Lyases / metabolism*
  • Polysaccharide-Lyases / physiology
  • Protein Engineering
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology

Substances

  • Recombinant Proteins
  • Polyethylene Glycols
  • Polysaccharide-Lyases
  • poly(beta-D-mannuronate) lyase