Abstract
The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics.
Highlights
The gastrointestinal (GI) tract is the site of numerous microbial infections caused by a range of pathogens, including: Helicobacter pylori, Salmonella Typhi, Vibrio cholerae, Escherichia coli, Campylobacter jejuni, and C. difficile
Soluble VHHs were extracted from the periplasm of TG1 E. coli and purified by immobilized-metal affinity chromatography (IMAC) with purified yields ranging from 3–12 mg/L of bacterial culture
Inhibition of toxins and colonization factors within the GI tract may be an effective means of disease control
Summary
The gastrointestinal (GI) tract is the site of numerous microbial infections caused by a range of pathogens, including: Helicobacter pylori, Salmonella Typhi, Vibrio cholerae, Escherichia coli, Campylobacter jejuni, and C. difficile. Disease-causing pathogens of the GI tract rely on a myriad of virulence factors for colonization, adherence, motility, cellular entry, and pathogenesis. These include, but are not limited to: surface-layer proteins, adhesins, invasins, flagella, highmolecular weight toxins, and quorum sensing molecules. Inhibition of bacterial virulence factors that are essential for disease pathogenesis represents a novel, non-antibiotic based strategy to treat infectious diseases, while reducing the risk of microbial resistance and maintaining commensal gut populations [1,2,3]. There are major limitations facing orally administered immunotherapeutics, including the susceptibility of antibodies to proteolytic degradation, instability at low pH, high dosing requirements and cost [29]
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