MIMICKING SOMATIC HYPERMUTATION - AFFINITY MATURATION OF ANTIBODIES DISPLAYED ON BACTERIOPHAGE USING A BACTERIAL MUTATOR STRAIN

Human antibodies can now be isolated from antibody repertoires display ed on the surface of filamentous bacteriophage in a process that mimic s the primary immune response. Here we have attempted to mimic the sec ondary response, the natural process of affinity maturation of antibod ies occurring in germinal centres, by multiple cycles of random mutati on and selection. Phage displaying a human antibody fragment recognisi ng the hapten 2-phenyl-5-oxazolone were grown in a mutator strain of b acteria (Escherichia coli: mutD5) to generate a large repertoire of an tibodies that should include the majority of possible single nucleotid e point mutations. The repertoire of phage antibody mutants was then s elected by binding to hapten. By multiple rounds of growth in the muta tor strain, and increasingly stringent selection, we succeeded in isol ating mutants with improved binding affinities; furthermore, the distr ibution of mutations and nucleotide substitution preferences strongly resembled those of somatic hypermutation. We then constructed a geneal ogical tree from the sequences of mutants taken at different rounds, a nd identified four sequentially acquired mutations that together impro ve the binding affinity of the antibody by a factor of 100-fold (from K-d 320 nM to 3.2 nM). (C) 1996 Academic Press Limited