Directed Evolution of Human Heavy Chain Variable Domain (V_H) Using <i>In Vivo</i> Protein Fitness Filter

<div><p>Human immunoglobulin heavy chain variable domains (V_H) are promising scaffolds for antigen binding. However, V_H is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the V_H domain, we performed <i>in vivo</i> protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of <i>E. coli</i>. After screening a human germ-line V_H library, 95% of the V_H proteins obtained were identified as V_H3 family members; one V_H protein, MG2x1, stood out among separate clones expressing individual V_H variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in V_H. Comparison of the crystal structures of the V_H variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the V_H interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered V_H acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the V_H variant as a scaffold was qualified as most of V_H clones selected randomly were expressed as soluble form in <i>E. coli</i> regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected V_H conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.</p>