Disulfide Bond Influence of Single Domain Antibody (sdAb) Stability
sdAbs, including the variable domain of camelid heavy-chain antibody (VHH) and the variable domain of the new antigen receptor (VNAR) in sharks, are known to be remarkably stable. For example, sdAb can be engineered to have high thermodynamic stability, stability against unfolding with chemical denaturants, or protease resistance. The high stability of sdAb is mainly attributed to its efficient refolding after chemical or thermal denaturation and to a lesser extent because of increased resistance against denaturation. The excellent biochemical and biophysical properties (e.g., high chemical and physical stability, thermal tolerance, and proteolytic resistance) lead to the successful application of sdAbs for diagnostic. Moreover, the exceptional stability of sdAb opens the possibility for nonconventional therapeutic applications and alternative routes of administration, such as oral administration.
Disulfide Bond Influence
Compared to the conventional antibody, additional disulfide bonds are naturally present in sdAbs from both camelid and shark in most cases. sdAb from camelid often contains one noncanonical Cys pair, located in CDR1-CDR3 from camel, FR2-CDR3 from alpaca and llama. sdAb from shark usually has one or several noncanonical Cys pairs located in FR2-FR4, within CDR3, or in CDR1-CDR3. Apparently, the insertion of the naturally occurring cystine consistently increases the sdAb stability and resistance against aggregation without any adverse effect on expression yield or antigen affinity. These features show that sdAb is an ideal candidate for developing viable treatment strategies in harsh environments.
Fig.1 Introduction of disulfide bond for sdAb stability. (Goldman, 2017)
Disulfide linkage involving pairs of Cys residues is a significant stabilizing force in sdAb. Thus, the introduction of non-canonical disulfide bonds can improve sdAb stability, including thermodynamic stability and protease resistance. It is based on two amino acid substitutions within the framework to form an additional intra-domain disulfide bond while having minimal effects on antigen affinity. Through this strategy, stabilized sdAb without significantly compromising its affinity can be developed that retains its binding ability under extreme conditions. sdAb with increased stability will provide a superior reagent for a myriad of biotechnology, detection, and diagnostic applications, especially promising for the novel administration (e.g., oral administration).
However, for pharmaceutical purposes, sdAb clones without the additional disulfide bond might be preferable to avoid the potential risk of disulfide bond mismatch during the downstream manufacture. Probably, inappropriately positioned unpaired cysteines may lead to folding problems in the expression system, resulting in reduced expression yields. Moreover, the additional disulfide bond may also affect the degree of humanization.
References
- Saerens, D.; et al. Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains. Journal of molecular biology. 2008, 377(2): pp.478-488.
- Goldman, E.R.; et al. Enhancing stability of camelid and shark single domain antibodies: an overview. Frontiers in immunology. 2017, 8: p.865.
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