title: Disulfide linkage engineering for improving biophysical properties of human VH domains.
abstract: To enhance their therapeutic potential, human antibody heavy chain variable domains (V(H)s) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of V(H) domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (V(H)Hs). Using four model domains we demonstrate that this strategy is also applicable to human V(H) domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of V(H)s by 14-18°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced V(H) aggregation in domains with the Cys54-Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the V(H)s to protein A. This indicates that it may be prudent to use the synthetic V(H) libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders.
authors.affiliation: Human Health Therapeutics,  National Research Council Canada, Ottawa, ON,  Canada K1A 0R6.
authors.forename: Dae Young
authors.initials: DY
authors.lastname: Kim
authors.forename: Hiba
authors.initials: H
authors.lastname: Kandalaft
authors.forename: Wen
authors.initials: W
authors.lastname: Ding
authors.forename: Shannon
authors.initials: S
authors.lastname: Ryan
authors.forename: Henk
authors.initials: H
authors.lastname: van Faassen
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hirama
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Foote
authors.forename: Roger
authors.initials: R
authors.lastname: MacKenzie
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D055550:Protein Stability; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region