Improving the solubility of single domain antibodies using <scp>VH</scp>‐like hallmark residues

Single domain antibodies (sdAbs) can be generated from variable regions of heavy-chain antibodies, which lack light chain and CH1 region. They have attracted attention due to their small size and molecular characteristics. Hydrophilic hallmark amino acids at framework region 2 (FR2) are key residues involved in the solubility of sdAbs. Nevertheless, previous studies reported that several sdAbs with human VH-like hydrophobic hallmark residues were soluble in a monomeric state and suggested that solubility also depends on the amino acid sequences in the complementarity-determining region. In this study, we obtained two sdAbs (sdAb A and B) with VH-like hallmark residues and low solubility from an alpaca immune library. We introduced VHH-like mutations (V37Y, G44E, L45R, W47L) into the hallmark residues in FR2 of both sdAb A and B. We were able to prepare sdAb A as a monomer without an additive in the buffer, but sdAb B was polydispersed when arginine was not added to the buffer. We also predicted the hydrophobicity of the sdAb B surface by spatial aggregation propensity calculations and identified W99 as the residue responsible for its low solubility. Subsequently, we obtained the sdAb B mutant as a monomer by introducing the W99A mutation. We characterized the engineered sdAbs using structural, physicochemical, and biophysical analyses and found that the solubility-improved sdAbs retained their functionality. Our findings can be applied to improving the solubility of sdAbs even in the absence of structural information.