title: Genetically engineered alginate lyase-PEG conjugates exhibit enhanced catalytic function and reduced immunoreactivity.
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.
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.
authors.forename: John W
authors.initials: JW
authors.lastname: Lamppa
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Ackerman
authors.forename: Jennifer I
authors.initials: JI
authors.lastname: Lai
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Scanlon
authors.forename: Karl E
authors.initials: KE
authors.lastname: Griswold
mesh_terms: D000818:Animals; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005818:Genetic Engineering; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects; D011092:Polyethylene Glycols / Q000378:metabolism* / Q000494:pharmacology; D011133:Polysaccharide-Lyases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000502:physiology; D015202:Protein Engineering; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase