Improving solubility and refolding efficiency of human VHs by a novel mutational approach

• Published in: Protein engineering, design and selection Vol. 19; no. 11; pp. 503 - 509
• Main Authors: Tanha, Jamshid, Nguyen, Thanh-Dung, Ng, Andy, Ryan, Shannon, Ni, Feng, MacKenzie, Roger
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.11.2006; Oxford Publishing Limited (England)
• Subjects: human VH; immunogenicity; mutation; phage display library; solubility and refolding; mutation; solubility and refolding; phage display library; immunogenicity; human V
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzl037

The antibody VH domains of camelids tend to be soluble and to resist aggregation, in contrast to human VH domains. For immunotherapy, attempts have therefore been made to improve the properties of human VHs by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama VH domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human VH, derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human VHs with good antigen binding activity and solubility. The engineered human VH domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.