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 m s), reversibility of thermal unfolding, solubility, and antigen-binding affinities of three types of sdAbs (VH H, VH , and VL domains). The Cys40-Cys55 disulfide linkage was tolerated by 9/9 VH Hs, 12/12 VH s, and 2/11 VL s tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40-Cys55 disulfide linkage increased sdAb T m 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.