title: Optimal design features of camelized human single-domain antibody libraries.
abstract: We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Tanha
authors.forename: P
authors.initials: P
authors.lastname: Xu
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: F
authors.initials: F
authors.lastname: Ni
authors.forename: H
authors.initials: H
authors.lastname: Kaplan
authors.forename: S A
authors.initials: SA
authors.lastname: Narang
authors.forename: C R
authors.initials: CR
authors.lastname: MacKenzie
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library