title: Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions.
abstract: A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.
authors.affiliation: Department of Protein Engineering, Genentech Inc., 1 DNA Way, Mailstop 27, South San Francisco, CA 94080, USA.
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Chen
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Fellouse
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000915:Antibody Affinity; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D042461:Vascular Endothelial Growth Factor A