A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Synthetic single-framework antibody library integrated with rapid affinity maturation by V-L shuffling
Tekijät: Brockmann EC, Akter S, Savukoski T, Huovinen T, Lehmusvuori A, Leivo J, Saavalainen O, Azhayev A, Lovgren T, Hellman J, Lamminmaki U
Kustantaja: OXFORD UNIV PRESS
Julkaisuvuosi: 2011
Journal: Protein Engineering, Design and Selection
Tietokannassa oleva lehden nimi: PROTEIN ENGINEERING DESIGN & SELECTION
Lehden akronyymi: PROTEIN ENG DES SEL
Numero sarjassa: 9
Vuosikerta: 24
Numero: 9
Aloitussivu: 691
Lopetussivu: 700
Sivujen määrä: 10
ISSN: 1741-0126
DOI: https://doi.org/10.1093/protein/gzr023
Tiivistelmä
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 Kunkels 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 0.810 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.
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 Kunkels 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 0.810 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.
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