Advances on The Relationship Between Stability and Structure of Nanobody

Camel nanobody has a wide range of application prospects due to its simple structure,easymodification,and the characteristics of low immunogenicity,high stability,high specificity as well as highaffinity.It is reported that nanobody with high stability could be stored and transported in more easily waycompared with conventional antibodies.It can evenly refold and recover its antigen binding affinity after beingdenatured under polar conditions such as high temperature,high concentration organic solvents,high pressure,chemical reagents,extreme acid-base environments,and proteases.This review summarized the stabilitycharacterization of nanobody under these conditions.It is shown that nanobody can not only expand itsapplication in the detection of targets under complex polar conditions but also enrich multiple routes of drugdelivery in medical therapy,including intravenous and subcutaneous injection,nasal inhalation,and oraladministration.Emphasis was made on describing the relationship between amino acid sequence,number andposition of disulfide bonds,structural domains,and stability,which revealed that highly stable nanobody havecommon structural features such as higher net charge surfaces,disulfide bonds that limit conformationalmigration,framework regions with more hydrophilic amino acid substitutions,conserved hydrophobic pockets,and highly interacting structural domains.Based on these structural features,several strategies for stabilitystructure optimization of nanobody were also discussed in this paper,including shared sequence-driven sequencerepair,the substitution of easily modified amino acids,alteration of net protein charge,the introduction ofunnatural disulfide bonds,and transposition of CDRs.It is expected to provide theoretical guidance on thestability regulation of nanobody to expand their wide applications as therapeutic drugs,diagnostic reagents,and biosensors