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

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.

Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

“…Similarly, the majority (Ͼ98%) of bovine-derived anti-C. difficile immunoglobulins were previously found to be degraded in the human gastrointestinal tract when administered orally (46). Engineering of the VHH fragments for improved proteolytic resistance has previously been shown to significantly improve stability and could therefore be a promising approach for further development of the VHH fragments (21,22). Since the increased neutralization of IgY antibody could be conferred both by a higher proteolytic stability and by its bivalency, we hypothesized that VHH antibody fragments could be more effective if produced continuously and displayed on the surfaces of lactobacilli.…”

“…They are well expressed in bacteria, and their excellent physicochemical stability combined with the possibility to be engineered for improved protease stability makes them ideal choices for passive immunity in the gastrointestinal tract (21,22).…”

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

“…The introduction of an additional disulfide bond at Ala49 and Ile69 by protein engineering (9, 10) is becoming a general approach to increase the equilibrium thermodynamic stability of VHHs (11). This mutation also increased resistance against pepsin (12,39) and was applicable to VH domains from other species than camelid (40,41). In spite of the efficiency of this mutation to stabilize VHHs and VHs thermodynamically, our data suggest caution when the mutated protein will be used in conditions that accelerate the chemical modification of cystine.…”

Affinity maturation of single-chain variable fragment specific for aflatoxin B1 using yeast surface display

“…In particular, scFv has been useful for AFB 1 detection, because of feasible antibody engineering tools for affinity or specificity, economical production using microorganisms such as Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae), broad applicability for diverse detection modules. Antibody engineering technologies have been continuously advanced to improve the clinical or diagnostic functions of recombinant antibodies, such as an affinity enhancement via yeast surface display technology (Lee et al, 2010) or phage display (Hu et al, 2015), avidity increase by dior polymerization (Zhu et al, 2010), thermal stability using nano-flow reversed-phase LC-MS (Hussack, Hirama, Ding, MacKenzie, & Tanha, 2011) or antibody solubility increase (Perchiacca, Ladiwala, Bhattacharya, & Tessier, 2012), prolonged half-life by PEGylation or increasing the affinity of fragment crystallizable region (Fc) for the Fc neonatal receptor (Patel et al, 2011). To obtain tailor-made antibodies, several technologies such as phage, ribosome, bacteria and yeast display have been developed and applied.…”