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

Lamppa JW, Ackerman ME, Lai JI, Scanlon TC, Griswold KE - PLoS ONE (2011)

Bottom Line: To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented.In both cases, the PEGylated enzyme was found to be substantially less immunoreactive.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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

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.

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Disruption of mucoid P. aeruginosa biofilms.Adherent biofilms of a mucoid clinical isolate were established in                            96-well plates and subsequently treated with 1 mg/ml enzyme for 1 hour.                            Remaining biofilm was then quantified using an alginate-sensitive                            lectin-HRP conjugate and ABTS substrate. Signals were normalized to a                            buffer only treatment. Both enzymes removed a significant proportion of                            biofilm relative to the buffer control (p<0.01). Importantly,                            theA53C-his-PEG enzyme removed >15% more biofilm than the                            WT-his enzyme (p<0.025). Error bars represent standard deviation.
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pone-0017042-g006: Disruption of mucoid P. aeruginosa biofilms.Adherent biofilms of a mucoid clinical isolate were established in 96-well plates and subsequently treated with 1 mg/ml enzyme for 1 hour. Remaining biofilm was then quantified using an alginate-sensitive lectin-HRP conjugate and ABTS substrate. Signals were normalized to a buffer only treatment. Both enzymes removed a significant proportion of biofilm relative to the buffer control (p<0.01). Importantly, theA53C-his-PEG enzyme removed >15% more biofilm than the WT-his enzyme (p<0.025). Error bars represent standard deviation.

Mentions: There exists considerable evidence that P. aeruginosa grows in biofilm communities during CF lung infection [26], and it is likely that disrupting alginate biofilms represents a key challenge in the fight to eradicate CF-associated P. aeruginosa infections. To assess this therapeutically relevant aspect of enzyme function, biofilms of the alginate-producing P. aeruginosa strain Xen5, a derivative of clinical isolate ATCC 19660, were first established by growth in 96-well plates. Subsequently, adherent biofilms were treated for one hour with 1 mg/ml of WT-his or A53C-his-PEG and then washed to remove degraded biofilm. The remaining adherent alginate biofilm matrix was quantified using a ConA lectin-HRP conjugate that binds to mannuronate residues of alginate [27]. The percentage of biofilm removed by each enzyme was determined by comparison to wells receiving a buffer control treatment. Both the wild type and PEGylated enzymes were found to effectively remove the majority of established biofilm from the wells (Fig. 6). Consistent with its enhanced solution phase activity towards bacterial alginate, A53C-his-PEG exhibited a significant (pā€Š=ā€Š0.025) increase in mucoid biofilm disruption relative to the WT-his protein (94% vs. 75% biofilm removal, respectively). These results suggest that the enhanced catalytic performance of the genetically engineered A53C-his-PEG enzyme may have relevance to clinical applications.


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

Lamppa JW, Ackerman ME, Lai JI, Scanlon TC, Griswold KE - PLoS ONE (2011)

Disruption of mucoid P. aeruginosa biofilms.Adherent biofilms of a mucoid clinical isolate were established in                            96-well plates and subsequently treated with 1 mg/ml enzyme for 1 hour.                            Remaining biofilm was then quantified using an alginate-sensitive                            lectin-HRP conjugate and ABTS substrate. Signals were normalized to a                            buffer only treatment. Both enzymes removed a significant proportion of                            biofilm relative to the buffer control (p<0.01). Importantly,                            theA53C-his-PEG enzyme removed >15% more biofilm than the                            WT-his enzyme (p<0.025). Error bars represent standard deviation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3038863&req=5

pone-0017042-g006: Disruption of mucoid P. aeruginosa biofilms.Adherent biofilms of a mucoid clinical isolate were established in 96-well plates and subsequently treated with 1 mg/ml enzyme for 1 hour. Remaining biofilm was then quantified using an alginate-sensitive lectin-HRP conjugate and ABTS substrate. Signals were normalized to a buffer only treatment. Both enzymes removed a significant proportion of biofilm relative to the buffer control (p<0.01). Importantly, theA53C-his-PEG enzyme removed >15% more biofilm than the WT-his enzyme (p<0.025). Error bars represent standard deviation.
Mentions: There exists considerable evidence that P. aeruginosa grows in biofilm communities during CF lung infection [26], and it is likely that disrupting alginate biofilms represents a key challenge in the fight to eradicate CF-associated P. aeruginosa infections. To assess this therapeutically relevant aspect of enzyme function, biofilms of the alginate-producing P. aeruginosa strain Xen5, a derivative of clinical isolate ATCC 19660, were first established by growth in 96-well plates. Subsequently, adherent biofilms were treated for one hour with 1 mg/ml of WT-his or A53C-his-PEG and then washed to remove degraded biofilm. The remaining adherent alginate biofilm matrix was quantified using a ConA lectin-HRP conjugate that binds to mannuronate residues of alginate [27]. The percentage of biofilm removed by each enzyme was determined by comparison to wells receiving a buffer control treatment. Both the wild type and PEGylated enzymes were found to effectively remove the majority of established biofilm from the wells (Fig. 6). Consistent with its enhanced solution phase activity towards bacterial alginate, A53C-his-PEG exhibited a significant (pā€Š=ā€Š0.025) increase in mucoid biofilm disruption relative to the WT-his protein (94% vs. 75% biofilm removal, respectively). These results suggest that the enhanced catalytic performance of the genetically engineered A53C-his-PEG enzyme may have relevance to clinical applications.

Bottom Line: To effectively deimmunize one therapeutic candidate while maintaining high level catalytic proficiency, a combined genetic engineering-PEGylation strategy was implemented.In both cases, the PEGylated enzyme was found to be substantially less immunoreactive.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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire, United States of America.

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.

Show MeSH
Related in: MedlinePlus