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.