Improving the solubility of single domain antibodies using VH‐like hallmark residues

Abstract 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.