Establishment of a reliable dual-vector system for the phage display of antibody fragments

초록

To resolve some of the technical limitations in a phage-displayed Fab library, we have designed two dual-vector systems, DVS-I and DVS-II, composed of a set of replicon-compatible plasmid (pLA-1 or pLT-2) for producing soluble L chain fragments and phagenrid (pHflg3T-1 or pHflg3A-2) for expressing Fd (V-H+C-H1)-Delta pIII fusion molecules as well as a genotype-phonotype linkage. Compared to the DVS-I (pLA-1 and pHflg3T-1), the DVS-II (pLT-2 and pHflg3A-2) showed stable transformation efficiency regardless of the order of the vectors introduced into the host cells. In addition, expression of soluble Fab molecules with antigen-binding reactivity, recombinant phage titer and display level of functional Fab-Delta pIII on the phage progenies of the DVS-II were comparable with a conventional phage display system using a single phagemid vector. More importantly, the phage displaying target-specific Fab-ApIII molecules was successfully enriched by panning, which allows isolation of the pHflg3A-2 phagemid encoding antigen-specific Fd molecules. We believe that the DVS-II may provide a valuable tool in the construction of a combinatorial phage-displayed Fab library with large diversity. Furthermore, it can be readily applied to isolation of desired antibody clones if L chain promiscuity of antibodies in determining antigen-binding specificity is considered, or in guided-selection or chain shuffling of mAbs of non-human origin. (C) 2007 Elsevier B.V. All rights reserved.

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