BASIS FOR SELECTION OF IMPROVED CARBOHYDRATE-BINDING SINGLE-CHAIN ANTIBODIES FROM SYNTHETIC GENE LIBRARIES

A technique is described for the simultaneous and controlled random mu tation of all three heavy or light chain complementarity-determining r egions (CDRs) in a single-chain Fv specific for the O polysaccharide o f Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar sp iking with A, G, C, and T at a level of 10% while the antisense strand s 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 dim ers 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 wi th residues in heavy-chain CDR2, oligomerization resulted in functiona l affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.