title: Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach.
abstract: The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), 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 V(H)s with good antigen binding activity and solubility. The engineered human V(H) 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.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada Ottawa, Ontario, Canada K1A 0R6. jamshid.tanha@nrc-cnrc.gc.ca
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
authors.forename: Thanh-Dung
authors.initials: TD
authors.lastname: Nguyen
authors.forename: Andy
authors.initials: A
authors.lastname: Ng
authors.forename: Shannon
authors.initials: S
authors.lastname: Ryan
authors.forename: Feng
authors.initials: F
authors.lastname: Ni
authors.forename: Roger
authors.initials: R
authors.lastname: Mackenzie
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012995:Solubility; D013816:Thermodynamics
chemical_list: D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D005340:Fibrinogen