Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada.
PLoS One. 2011;6(11):e28218. doi: 10.1371/journal.pone.0028218. Epub 2011 Nov 30.
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.
上消化道的极端 pH 值和富含蛋白酶的环境是口服蛋白治疗药物（包括抗体）面临的主要障碍。通过蛋白质工程，通过引入 Ala/Gly54Cys 和 Ile78Cys 突变，表达了几种具有额外二硫键的艰难梭菌毒素 A 特异性重链抗体可变区（V(H)H）。与野生型相比，突变抗体在表达产量、非聚集状态、对毒素 A 的亲和力、圆二色性（CD）结构特征、热稳定性、蛋白酶抗性和毒素 A 中和能力方面进行了比较。突变的 V(H)H 表达良好，尽管产量低于野生型，是非聚集的单体，保留了对毒素 A 的低 nM 亲和力，尽管大多数与野生型相比亲和力略有降低，并且能够在细胞基础测定中进行体外毒素 A 中和。远紫外和近紫外 CD 光谱一致表明，野生型和突变 V(H)H 对的峰强度和选择性峰最小值发生了变化；然而，整体 CD 谱仍然非常相似。在中性和酸性 pH 下，所有突变体的热变性中点温度都显著升高。在生物相关浓度下，用主要的胃肠道蛋白酶消化 V(H)H 时，所有突变体的胃蛋白酶抗性显著增加，大多数突变体的胰凝乳蛋白酶抗性增加。突变体 V(H)H 的胰蛋白酶抗性与野生型 V(H)H 相似，尽管一种 V(H)H 突变体的胰蛋白酶抗性显著降低。因此，在疏水性核心中引入第二个二硫键不仅如前所述增加了 V(H)H 在中性 pH 下的热稳定性，而且还代表了一种增加 V(H)H 在低 pH 下稳定性并赋予蛋白酶抗性的通用策略，对靶标结合亲和力只有微小的干扰。这些都是蛋白质口服治疗药物设计的理想特征。
