Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

被引:113
作者
Hussack, Greg [1 ,2 ]
Hirama, Tomoko [1 ]
Ding, Wen [1 ]
MacKenzie, Roger [1 ,3 ]
Tanha, Jamshid [1 ,2 ,3 ]
机构
[1] Natl Res Council Canada, Inst Biol Sci, Ottawa, ON K1A 0R6, Canada
[2] Univ Ottawa, Fac Med, Dept Biochem Microbiol & Immunol, Ottawa, ON, Canada
[3] Univ Guelph, Sch Environm Sci, Guelph, ON N1G 2W1, Canada
关键词
MILK IMMUNOGLOBULIN CONCENTRATE; SITE-DIRECTED MUTAGENESIS; MONOCLONAL-ANTIBODIES; IN-VITRO; V-H; DISULFIDE BOND; III SECRETION; FRAGMENTS; PROTEINS; PHAGE;
D O I
10.1371/journal.pone.0028218
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 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 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 VHH trypsin resistance was similar to that of wild-type V(H)Hs, 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.
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页数:15
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