title: Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling.
abstract: Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.
authors.affiliation: Department of Biotechnology, University of Turku, Turku, Finland. eechbr@utu.fi
authors.forename: E-C
authors.initials: EC
authors.lastname: Brockmann
authors.forename: S
authors.initials: S
authors.lastname: Akter
authors.forename: T
authors.initials: T
authors.lastname: Savukoski
authors.forename: T
authors.initials: T
authors.lastname: Huovinen
authors.forename: A
authors.initials: A
authors.lastname: Lehmusvuori
authors.forename: J
authors.initials: J
authors.lastname: Leivo
authors.forename: O
authors.initials: O
authors.lastname: Saavalainen
authors.forename: A
authors.initials: A
authors.lastname: Azhayev
authors.forename: T
authors.initials: T
authors.lastname: Lövgren
authors.forename: J
authors.initials: J
authors.lastname: Hellman
authors.forename: U
authors.initials: U
authors.lastname: Lamminmäki
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000235:genetics*; D032621:DNA Shuffling / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016296:Mutagenesis; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies; D009113:Muramidase