SELECTION OF ANTIBODY SINGLE-CHAIN VARIABLE FRAGMENTS WITH IMPROVED CARBOHYDRATE-BINDING BY PHAGE DISPLAY

A single-chain variable fragment (Fv) version of a murine monoclonal a ntibody, 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-ca rbohydrate antibodies. Random single-chain Fv mutant libraries generat ed by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a random ly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, rive of which s howed wild type activity by enzyme immunoassay. Two of these were appa rently selected on the basis of better functional single-chain Fv yiel d in Escherichia coli. A heavy chain mutation (Ile77 --> Thr) in one m utant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic pro duct. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, b y enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding pro perties 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 tri saccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Exami nation of the structure indicated that two mutations in the heavy chai n CDR2 provided improved surface complementarity between the protein a nd the extended epitope encompassing 2 additional hexose residues. How ever, introduction of only the CDR2 mutations into the wild type struc ture failed to confer the improved binding properties of 4B2, indicati ng an indirect effect by the more distant mutations. Panning of random ly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or bind ing properties.