Synthetic single-framework antibody library integrated with rapid affinity maturation by V-L shuffling

: Brockmann EC, Akter S, Savukoski T, Huovinen T, Lehmusvuori A, Leivo J, Saavalainen O, Azhayev A, Lovgren T, Hellman J, Lamminmaki U

Publisher: OXFORD UNIV PRESS

: 2011

: Protein Engineering, Design and Selection

: PROTEIN ENGINEERING DESIGN & SELECTION

: PROTEIN ENG DES SEL

: 9

: 24

: 9

: 691

: 700

: 10

: 1741-0126

DOI: https://doi.org/10.1093/protein/gzr023

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.