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.

Overcoming Drug Resistance with Alginate Oligosaccharides Able To Potentiate the Action of Selected Antibiotics

“…This inhibition was evident in the studies on bacterial biofilms, where OligoG not only inhibited biofilm formation and resulted in disruption of established 24-h biofilms but also potentiated antibiotic treatment. This observation was at first counterintuitive, as alginate per se is an important component of the EPS of certain bacterial biofilms and therapies targeting alginate, e.g., alginate lyases are currently being developed as antibiofilm therapies (23). However, unlike alginates derived from algae, bacterial alginates from most bacteria, such as P. aeruginosa, differ considerably structurally, having an absence of G blocks (homopolymeric regions of poly-L-guluronate) (31), which may be a contributing factor in these observed antimicrobial effects.…”

Alginate Lyase Exhibits Catalysis-Independent Biofilm Dispersion and Antibiotic Synergy

“…In particular, biocatalytic degradation of mucoid P. aeruginosa biofilms using alginate lyase enzymes (EC 4.2.2.3) has been the subject of more than 20 years of research. Alginate lyase treatment has been shown to reduce viscosity in cultures of clinical isolates and in CF sputum (9,10); it strips biofilms from abiotic surfaces (11,12), it enhances phagocytosis and killing of P. aeruginosa by human immune cells (13)(14)(15), and it improves the efficacy of various antipseudomonal antibiotics (14,(16)(17)(18)(19). In aggregate, these reports make a rather convincing case for the use of inhaled alginate lyases for treating chronic P. aeruginosa infections of the CF lung, although no clinical trials have been conducted to date.…”

“…Sphingomonas sp. A1 alginate lyase (A1-III) has been shown previously to exhibit high levels of activity toward bacterial alginate (20) and has also been subjected to molecular engineering with an eye toward therapeutic applications (12,21). P. aeruginosa alginate lyase (AlgL) is produced by the CF-associated pathogen itself, and its confirmed role in bacterial alginate biosynthesis (22,23) suggests the enzyme is an obvious choice for developing alginate-degrading CF biotherapies.…”

Nanomedicine and Drug Delivery Strategies for Treatment of Genetic Diseases

“…Classical examples of the first approach have been applied to the treatment of lysosomal storage disorders (LSDs), mostly caused by genetic defects affecting enzymes involved in lysosomal degradation of varied substrates (Burrow, et al, 2007), neuropathic phenylketonuria (PKU) as a consequence of a defect in phenylalanine hydroxylase (PAH) , Harding & Blau, 2010, or prolidase deficiency affecting collagen metabolism (Colonna, et al, 2008b), among many others. The second approach is illustrated in examples pertaining, for instance, administration of alginate lyase to degrade components of infectious biofilms in CF (Lamppa, et al, 2011) or delivery of uricase for gout treatment (Sherman, et al, 2008). However, effective delivery of enzymes often suffers from the impediments stated above for small molecule therapies, with the added disadvantages that arise from using proteins as therapeutic agents: susceptibility to proteases, high potential for immunogenicity, and even more reduced penetration within tissues and cells in the body.…”

“…Some preliminary attempts in this direction include the case of delivery of PEG-modified dextranase, which achieved prolonged activity by bypassing immunorecognition in a mouse model of LSD mimicked by lysosomal accumulation of dextran (Mumtaz & Bachhawat, 1994). Similar strategies of PEGylation have been useful for delivery of uricase for gout treatment in hyperuricemia (Sherman, et al, 2008), non-mammalian phenylalanine ammonia lyase for treatment of PKU (Ikeda, et al, 2005), or alginate lyase delivery targeted to CF infections (Lamppa, et al, 2011). In addition to protecting from inactivation and immunogenic responses, by masking the enzymes from the immune and clearance systems, it is expected that these strategies involving incorporation of PEG in the formulation will also provide prolonged circulation, hence enhancing the chances to reach tissues and cell targets in the body, a capacity otherwise considerably restricted for large molecules such as these enzymes.…”