Disulfide linkage engineering for improving biophysical properties of human VH domains

To enhance their therapeutic potential, human antibody heavy chain variable domains (VHs) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of VH domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (VHHs). Using four model domains we demonstrate that this strategy is also applicable to human VH domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of VHs by 14–18°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced VH aggregation in domains with the Cys54–Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the VHs to protein A. This indicates that it may be prudent to use the synthetic VH libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders.