Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity.
Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a beta-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5'-end of the beta-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 x 10(9), greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.
Yang
Hye Young
HY
Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, 120-750, Korea.
Kang
Kyung Jae
KJ
Chung
Julia Eunyoung
JE
Shim
Hyunbo
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology*; D001435:Bacteriophages; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D022801:Complementarity Determining Regions / Q000145:classification / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D001618:beta-Lactamases / Q000235:genetics
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D019151:Peptide Library; D001618:beta-Lactamases