title: Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.
abstract: A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
authors.forename: S J
authors.initials: SJ
authors.lastname: Deng
authors.forename: C R
authors.initials: CR
authors.lastname: MacKenzie
authors.forename: J
authors.initials: J
authors.lastname: Sadowska
authors.forename: J
authors.initials: J
authors.lastname: Michniewicz
authors.forename: N M
authors.initials: NM
authors.lastname: Young
authors.forename: D R
authors.initials: DR
authors.lastname: Bundle
authors.forename: S A
authors.initials: SA
authors.lastname: Narang
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D015723:Gene Library; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011135:Polysaccharides, Bacterial / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012475:Salmonella / Q000276:immunology*; D012710:Serum Albumin, Bovine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine