Phage display of chelating recombinant antibody libraries.

High affinity and discriminating specificity are important parameters for any successful antibody based targeting strategy. We herein describe a system for the construction and subsequent selection of affinity-optimised chelating recombinant antibodies (CRAbs) from a randomised filamentous phage-display inter-scFv linker library. Using a simple, robust and highly degenerate tandem scFv cloning strategy a phage-display library of CRAbs with varied inter-scFv linkers was constructed and characterised. The library consisted of two single-chain Fvs (scFvs) of well characterised anti-lysozyme antibodies D1.3 and HyHEL-10(TF), specific for distinct non-overlapping epitopes, separated by flexible polypeptide linkers of varying lengths and sequences. The use of a stringent affinity-based selection strategy quickly led to the enrichment of CRAbs with a restricted set of linker lengths (16-21 amino acids) which agrees very closely with previously described crystal structure data, affinity measurements and mathematical modelling. This CRAb linker phage-display selection strategy is a widely applicable approach for the selection of very high affinity CRAbs for pairs of scFvs against potentially any target antigen, complementing the more arbitrary affinity maturation approaches based on random mutagenesis.