title: High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold.
abstract: Phage-displayed synthetic antibody libraries were built on a single human framework by introducing synthetic diversity at solvent-exposed positions within the heavy chain complementarity-determining regions (CDRs). The design strategy of mimicking natural diversity using tailored codons had been validated previously with scFv libraries, which produced antibodies that bound to antigen, murine vascular endothelial growth factor (mVEGF), with affinities in the 100nM range. To improve library performance, we constructed monovalent and bivalent antigen-binding fragment (Fab) libraries, and explored different CDR-H3 diversities by varying the amino acid composition and CDR length. A Fab with sub-nanomolar affinity for mVEGF was obtained from a library with CDR-H3 diversity designed to contain all 20 naturally occurring amino acids. We then expanded the library by increasing the variability of CDR-H3 length and using tailored codons that mimicked the amino acid composition of natural CDR-H3 sequences. The library was tested against a panel of 13 protein antigens and high-affinity Fabs were obtained for most antigens. Furthermore, the heavy chain of an anti-mVEGF clone was recombined with a library of light chain CDRs, and the affinity was improved from low nanomolar to low picomolar. The results demonstrated that high-affinity human antibodies can be generated from libraries with completely synthetic CDRs displayed on a single scaffold.
authors.affiliation: Department of Protein Engineering, Genentech Inc. 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Chingwei V
authors.initials: CV
authors.lastname: Lee
authors.forename: Wei-Ching
authors.initials: WC
authors.lastname: Liang
authors.forename: Mark S
authors.initials: MS
authors.lastname: Dennis
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D001435:Bacteriophages / Q000235:genetics*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007700:Kinetics; D008958:Models, Molecular; D019151:Peptide Library; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library