Engineered single-domain antibodies with high protease resistance and thermal stability.
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 (V(H)Hs) were...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3227653?pdf=render |
| _version_ | 1811264659665190912 |
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| author | Greg Hussack Tomoko Hirama Wen Ding Roger Mackenzie Jamshid Tanha |
| author_facet | Greg Hussack Tomoko Hirama Wen Ding Roger Mackenzie Jamshid Tanha |
| author_sort | Greg Hussack |
| collection | DOAJ |
| description | 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 (V(H)Hs) 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 V(H)Hs 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 V(H)H 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 V(H)Hs 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 V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H 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. |
| first_indexed | 2024-04-12T20:08:22Z |
| format | Article |
| id | doaj.art-b24e4679d3a64b85b9233518ddbe5e16 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-12T20:08:22Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-b24e4679d3a64b85b9233518ddbe5e162022-12-22T03:18:20ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-01611e2821810.1371/journal.pone.0028218Engineered single-domain antibodies with high protease resistance and thermal stability.Greg HussackTomoko HiramaWen DingRoger MackenzieJamshid TanhaThe 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 (V(H)Hs) 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 V(H)Hs 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 V(H)H 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 V(H)Hs 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 V(H)H trypsin resistance was similar to that of wild-type V(H)Hs, although the trypsin resistance of one V(H)H mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases V(H)H thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase V(H)H 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.http://europepmc.org/articles/PMC3227653?pdf=render |
| spellingShingle | Greg Hussack Tomoko Hirama Wen Ding Roger Mackenzie Jamshid Tanha Engineered single-domain antibodies with high protease resistance and thermal stability. PLoS ONE |
| title | Engineered single-domain antibodies with high protease resistance and thermal stability. |
| title_full | Engineered single-domain antibodies with high protease resistance and thermal stability. |
| title_fullStr | Engineered single-domain antibodies with high protease resistance and thermal stability. |
| title_full_unstemmed | Engineered single-domain antibodies with high protease resistance and thermal stability. |
| title_short | Engineered single-domain antibodies with high protease resistance and thermal stability. |
| title_sort | engineered single domain antibodies with high protease resistance and thermal stability |
| url | http://europepmc.org/articles/PMC3227653?pdf=render |
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