title: Creation of the large and highly functional synthetic repertoire of human VH and Vκ domain antibodies.
abstract: This protocol describes a method for creation of a highly diverse and functional synthetic phage-displayed repertoire of fully human domain antibodies (dAbs). The repertoire is based on two human frameworks (one VH and one Vκ) that express well in bacteria and are frequently used in human antibodies. To achieve this, we first build dAb libraries, containing full synthetic diversity at key positions in the complementarity-determining regions (CDRs). We then use an antigen-independent preselection of this primary dAb repertoire on generic ligands of the VH and the Vκ scaffolds (namely, the bacterial superantigens, protein A and L) to enrich for folded dAbs. Finally, the CDRs of these preselected dAbs are randomly recombined to further expand genetic diversity. The resulting phage repertoire is in excess of 10(10) clones and is largely populated by correctly folded (over 50%) functional dAbs.
authors.affiliation: Biopharm R&D, GlaxoSmithKline, Cambridge, UK. olga.x.ignatovich@gsk.com
authors.forename: Olga
authors.initials: O
authors.lastname: Ignatovich
authors.forename: Laurent
authors.initials: L
authors.lastname: Jespers
authors.forename: Ian M
authors.initials: IM
authors.lastname: Tomlinson
authors.forename: Ruud M T
authors.initials: RM
authors.lastname: de Wildt
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D061505:Cell Surface Display Techniques / Q000379:methods*; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D023061:Gene Order; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D013205:Staphylococcal Protein A / Q000276:immunology / Q000378:metabolism; D013698:Templates, Genetic; D014169:Transformation, Bacterial
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009841:Oligonucleotides; D019151:Peptide Library; D061905:Single-Domain Antibodies; D013205:Staphylococcal Protein A