Directed Evolution of Human Heavy Chain Variable Domain (V(H)) Using In Vivo Protein Fitness Filter

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 in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line V(H) library, 95% of the V(H) proteins obtained were identified as V(H)3 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 E. coli 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.