Thayer School of Engineering
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.
Lamppa JW, Ackerman ME, Lai JI, Scanlon TC, Griswold KE. 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. PMID: 21340021; PMCID: PMC3038863.
Dartmouth Digital Commons Citation
Lamppa, John W.; Ackerman, Margaret E.; Lai, Jennifer I.; Scanlon, Thomas C.; and Griswold, Karl E., "Genetically Engineered Alginate Lyase-PEG Conjugates Exhibit Enhanced Catalytic Function and Reduced Immunoreactivity" (2011). Dartmouth Scholarship. 2736.