Phage display of antibody fragments from natural or synthetic antibody libraries with the single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. However, the nature of the artificial single chain constructs results in unstable proteins expressed on the phage surface or as soluble proteins secreted in the bacterial culture medium. The stability of the variable domain structures can be enhanced with interdomain disulfide bond, but the single chain disulfide-stabilized constructs (sc-dsFv) have yet to be established as a feasible format for bacterial phage display due to diminishing expression levels on the phage surface in known phage display systems. In this work, biological combinatorial searches were used to establish that the c-region of the signal sequence is critically responsible for effective expression and functional folding of the sc-dsFv on the phage surface. The optimum signal sequences increase the expression of functional sc-dsFv by 2 orders of magnitude compared with wild-type signal sequences, enabling the construction of phage-displayed synthetic antivascular endothelial growth factor sc-dsFv libraries. Comparison of the scFv and scdsFv variants selected from the phage-displayed libraries for vascular endothelial growth factor binding revealed the sequence preference differences resulting from the interdomain disulfide bond. These results underlie a new phage display format for antibody fragments with all the benefits from the scFv format but without the downside due to the instability of the dimeric interface in scFv. Single chain variable fragment (scFv)2 displayed as a fusion protein amino-terminal to the pIII minor capsid protein on the filamentous phage surface is one of the most prominent methods in antibody engineering. The small size of the scFv construct enables superior tissue-penetrating capabilities over the whole IgG or Fab fragment (1), making scFv an ideal scaffold for designing tumor-homing molecules carrying therapeutic or imaging agents (for reviews see Refs. 2-8). scFv is a single polypeptide chain antibody fragment construct encoding a light chain variable domain and a heavy chain variable domain, with a flexible linkage peptide connecting the two domains (9 -11). The recombinant antibody fragment frequently retains antigen-recognizing capability rivaling that of the parent antibody. Moreover, an scFv library, which could contain more than one billion scFv variants, can be propagated with an Escherichia coli vector of bacterial phage origin (12, 13); the recombinant phages displaying the scFv variants can be selected or screened for antigen binding and re-amplified with the host E. coli. The sequences of the selected scFv can be further diversified with various mutagenesis technologies for affinity maturation (14, 15). As such, the scFv scaffold supports a powerful in vitro technology mimicking the production of high affinity antibodies in mammalian immune responses to foreign antigens, and the re...
Phage-displayed single chain variable fragment (scFv) libraries have been powerful tools in antibody engineering. But the scFv structures are frequently unstable due to the dissociation of the dimeric interface between the two variable domains. One solution is the sc-dsFv construct, where the single chain variable domain fragment is stabilized with an additional interface disulfide bond, leading to stable and homogeneous dimeric interface for the sc-dsFv structure. However, the phagemid system that is capable of effective expression for both sc-dsFv-pIII fusion proteins on phage surface and secreted non-fusion sc-dsFv in bacterial culture medium has not been demonstrated. In this work, a biological combinatorial approach was applied to optimize the signal sequence N-terminal to the sc-dsFv-pIII fusion protein encoded in a phagemid. The optimized sc-dsFv phage display systems were compatible with both the phage-based directed evolution procedure and the high throughput screening of the soluble sc-dsFv. The utility of the phagemid systems was demonstrated in generating anti-VEGF sc-dsFv with VEGF-binding affinity one order of magnitude higher than the corresponding scFv, due only to the interface disulfide bond in the sc-dsFv. Moreover, the protein stability of the sc-dsFv construct was unmatched by the corresponding scFv. These advantages of the sc-dsFv were gained through the interface disulfide bond of the sc-dsFv and the novel signal sequence in the phagemid.
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