Engineering of immunoglobulin domains

The present invention provides a single domain antibody (sdAb) scaffold comprising one or more than one non-canonical disulfide bond in the framework region (FR). The one or more than one non-canonical disulfide bond may be formed between cysteines introduced by mutations in FR2 and FR3. In the case where the sdAb scaffold is a V H， the Cys may be introduced at any one of positions 47-49 and any one of positions 67-71， based on Kabat numbering; in one example， the Cys may be introduced at positions 49 and 69， based on Kabat numbering. In the case where the sdAb scaffold is a VL， the Cys residues may be introduced at any one of positions 46-49 and any one of positions 62-66， based on Kabat numbering; in one example， the Cys residues may be introduced at positions 48 and 64， based on Kabat numbering.

本发明提供了一种骨架区（framework region, FR）包含一个或多个非经典二硫键的单域抗体（single domain antibody, sdAb）骨架。该一个或多个非经典二硫键可由骨架区2（FR2）与骨架区3（FR3）中经突变引入的半胱氨酸残基之间形成。当该单域抗体骨架为重链可变区（variable heavy chain, VH）时，基于Kabat编号系统，可在第47至49位的任意位置及第67至71位的任意位置引入半胱氨酸残基；在一个实施例中，可基于Kabat编号在第49位与第69位引入半胱氨酸残基。当该单域抗体骨架为轻链可变区（variable light chain, VL）时，基于Kabat编号系统，可在第46至49位的任意位置及第62至66位的任意位置引入半胱氨酸残基；在一个实施例中，可基于Kabat编号在第48位与第64位引入半胱氨酸残基。