title: Characterization of single-domain antibodies with an engineered disulfide bond.
abstract: Camelidae single-domain antibodies (VHHs) represent a unique class of emerging therapeutics. Similar to other recombinant antibody fragments (e.g., Fabs, scFvs), VHHs are amenable to library screening and selection, but benefit from superior intrinsic biophysical properties such as high refolding efficiency, high solubility, no tendency for aggregation, resistance to proteases and chemical denaturants, and high expression, making them ideal agents for antibody-based drug design. Despite these favorable biophysical characteristics, further improvements to VHH stability are desirable when considering applications in adverse environments like high heat, low humidity, pH extremes, and the acidic, protease-rich gastrointestinal tract. Recently, the introduction of a disulfide bond into the hydrophobic core of camelid VHHs increased antibody thermal and conformational stability. Here, we present additional protocols for characterizing the effects of the introduced disulfide bond on a panel of llama VHHs. Specifically, we employ mass spectrometry fingerprinting analysis of VHH peptides to confirm the presence of the introduced disulfide bond, size exclusion chromatography, and surface plasmon resonance to examine the effects on aggregation state and target affinity, and circular dichroism spectroscopy and protease digestion assays to assess the effects on thermal and proteolytic stability. The disulfide bond stabilization strategy can be incorporated into antibody library design and should lead to hyperstabilized single-domain antibodies (VHHs, VHs), and possibly Fabs and scFvs, if selection pressures such as denaturants or proteases are introduced during antibody selection.
authors.affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. greg.hussack@nrc-cnrc.gc.ca
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.forename: C Roger
authors.initials: CR
authors.lastname: Mackenzie
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D002161:Camelids, New World; D004220:Disulfides / Q000737:chemistry*; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D059748:Proteolysis; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D013696:Temperature
chemical_list: D001427:Bacterial Toxins; D004220:Disulfides; D061905:Single-Domain Antibodies