Nhe I - - - - - U - - - U - - - - - - AU - Bgd /

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementaritj-determining regions (CDRs) in a single-chain Fv specific for the 0 polysaccharide of SalmoneUla serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10%/1 while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv. The relatively low affinities that are characteristic of carbohydrate binding proteins represent a major challenge in efforts to produce therapeutics designed to intervene in carbohydraterecognition events central to many disease states. Previous mutation studies with an antibody, Se155-4, specific for the 0 polysaccharide of Salmonella serogroup B (1) showed that rational redesign for improved binding and interpretation of altered binding remain difficult even when the three-dimensional structures of the epitope and antibody are known (2, 3). Phage libraries as a source of diversity for in vitro mimicry of the immune system, and its capacity for antigen-driven selection, are a useful alternative to a completely rational approach. The success of phage display in antibody redesign and in the isolation of different binding activities ultimately depends on the composition of the souirce of diversity, namely, the antibody gene library (4). Error-prone PCR and chemical mutagenesis of target DNA followed by PCR have been used to randomize antibody genes (5-7) but these procedures have the disadvantages of uncontrolled mutation frequency and the alteration of residues throughout the structure, some of which may compromise protein stability. Such an approach has been used to obtain a 10-fold improvement in antigen binding by Sel55-4 (5). In well-designed libraries, however, introduction of diversity should be limited to the complementaritydetermining regions (CDRs) and to nearby framework residues that fine tune the conformation of the CDRs. Gene amplification using spiked primers (8, 9) and codon-based mutagenesis approaches (10) have been examined as a means of targeting diversity to specific regions. Barbas et at (11) constructed a semisynthetic library in which a Fab sequence The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. was randomized by replacing the heavy-chain CDR3 gene sequence with randomly synthesized oligonucleotides of the same length. In a similar approach, Hoogenboom and Winter (12) introduced random sequences encoding five or eight heavy-chain CDR3 residues into rearranged gene libraries. In a preliminary report (13), we described a synthetic strategy by using the ligase chain reaction (LCR) for the simultaneous randomization of the three heavy chain variable region (VH) CDRs. Here, we report the randomization of all six CDRs to a degree that resulted in amino acid substitution levels approximating those observed in the in vivo affinity maturation process (14). From these libraries, we isolated single-chain Fv (scFv) mutants with binding properties that were much superior to those previously isolated from libraries in which randomization was introduced throughout the scFv