title: Human anti-self antibodies with high specificity from phage display libraries.
abstract: Recently we demonstrated that human antibody fragments with binding activities against foreign antigens can be isolated from repertoires of rearranged V-genes derived from the mRNA of peripheral blood lymphocytes (PBLs) from unimmunized humans. The heavy and light chain V-genes were shuffled at random and cloned for display as single-chain Fv (scFv) fragments on the surface of filamentous phage, and the fragments selected by binding of the phage to antigen. Here we show that from the same phage library we can make scFv fragments encoded by both unmutated and mutated V-genes, with high specificities of binding to human self-antigens. Several of the affinity purified scFv fragments were shown to be a mixture of monomers and dimers in solution by FPLC gel filtration and the binding kinetics of the dimers were determined using surface plasmon resonance (k(on) = 10(5)-10(6) M-1s-1, k(off) = 10(-2)s-1 and Ka = 10(7) M-1). The kinetics of association are typical of known Ab-protein interactions, but the kinetics of dissociation are relatively fast. For therapeutic application, the binding affinities of such antibodies could be improved in vitro by mutation and selection for slower dissociation kinetics.
authors.affiliation: MRC Centre for Protein Engineering, Cambridge, UK.
authors.forename: A D
authors.initials: AD
authors.lastname: Griffiths
authors.forename: M
authors.initials: M
authors.lastname: Malmqvist
authors.forename: J D
authors.initials: JD
authors.lastname: Marks
authors.forename: J M
authors.initials: JM
authors.lastname: Bye
authors.forename: M J
authors.initials: MJ
authors.lastname: Embleton
authors.forename: J
authors.initials: J
authors.lastname: McCafferty
authors.forename: M
authors.initials: M
authors.lastname: Baier
authors.forename: K P
authors.initials: KP
authors.lastname: Holliger
authors.forename: B D
authors.initials: BD
authors.lastname: Gorick
authors.forename: N C
authors.initials: NC
authors.lastname: Hughes-Jones
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000235:genetics / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics*; D000939:Epitopes / Q000276:immunology; D015698:Genomic Library; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D007135:Immunoglobulin Variable Region