Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

Abstract

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...

Probing the stability-limiting regions of an antibody single-chain variable fragment: a molecular dynamics simulation study

“…This indicates that the high stability of the Gln38L -Gln39H hydrogen bonds was unable to prevent the disruption at the domain interface. This finding may explain the recent experimental result that adding a disulfide bond between these two sites did not increase the phage display expression level of the protein (Huang et al, 2010), although phase display expression level is not necessarily linked to protein stability (Kipriyanov et al, 1997). The Gln38L -Gln39H hydrogen-bonding interaction, although highly conserved in VL and VH domains, may not significantly contribute to the stability of the proteins, as also demonstrated earlier by Tan et al (1998) for the S5 scFv fragment.…”

“…The stability of scFv fragments depends on the intrinsic stability of the VL and VH domains and the dimer interface and, in principle, can be improved through protein engineering. The widely employed strategies include conversion to consensus sequence for the VL and VH domains (Steipe et al, 1994;Willuda, et al, 1999;Monsellier and Bedouelle, 2006), disulfide bond linkage between the VL and VH domains (McCartney et al, 1995;Reiter et al, 1996;Huang et al, 2010), and complementarity-determining regions (CDRs) grafting to stable framework (Jung and Pluckthun, 1997;Ewert et al, 2004;Kugler et al, 2009), as well as the combinations of these strategies (see reference Worn and Pluckthun (2001) for a comprehensive review on scFv stability engineering methods). Identification and knowledge of the stability-limiting factors can assist the rational design and engineering of stable scFvs.…”

Loop-Sequence Features and Stability Determinants in Antibody Variable Domains by High-Throughput Experiments

“…Protein engineering is critical for developing protein-based therapeutics and diagnostics. Single-chain antibody variable domain fragments (scFvs) are important pharmaceutical molecules (Chan and Carter, 2010;Holliger and Hudson, 2005;Huang et al, 2010;Miller et al, 2010;Nelson and Reichert, 2009;Weatherill et al, 2012). Because the scFv structure is not stabilized by the constant domains, as in intact immunoglobulin, investigators have applied sequence fitness-searching principles based on random mutagenesis and screening (Jermutus et al, 2001;Jespers et al, 2004;Jung et al, 1999) or rational design with consensus-sequence profiles (Demarest and Glaser, 2008;Ewert et al, 2003;Honegger, 2008;Jordan et al, 2009;Kü gler et al, 2009;Miller et al, 2010;Monsellier and Bedouelle, 2006;Plü ckthun, 1998, 2001) to stability engineering of scFvs.…”

Toward Drug-Like Multispecific Antibodies by Design

“…In addition to the added complexities associated with multispecific analysis, disulfide bond scrambling is another major area where antibody fragments and/or multispecifics present unique challenges. These challenges are especially prominent in multispecifics, particularly those composed of scFvs, because they can also contain additional (engineered) intrachain disulfide bonds that promote domain pairing [ 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 ]. Mispairing of disulfide bonds can result in complex mixtures and aggregates with reduced activity and/or potential immunogenicity.…”