Evaluation of a noncanonical Cys40-Cys55 disulfide linkage for stabilization of single-domain antibodies.

Incorporation of noncanonical disulfide linkages into single-domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here， we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T s)， reversibility of thermal unfolding， solubility， and antigen-binding affinities of three types of sdAbs (V H， V ， and V domains). The Cys40-Cys55 disulfide linkage was tolerated by 9/9 V Hs， 12/12 V s， and 2/11 V s tested and its formation was confirmed by mass spectrometry. Using circular dichroism， we found that the Cys40-Cys55 disulfide linkage increased sdAb T by an average of 10.0°C (range: 0-21.8°C). However， enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as， for some sdAbs， significantly decreased solubility and antigen-binding affinity. Thus， Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains， although its effects should be tested empirically for individual sdAbs.