Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv

Abstract

Using molecular modeling technology, we have recently identified two positions in conserved framework regions of antibody Fv fragments (Fvs) that are distant from CDRs, and potentially can be used to make recombinant Fv fragments in which the unstable VH and VL heterodimer is stabilized by an interchain disulfide bond inserted between structurally conserved framework positions. A disulfide bond has been introduced at one of these positions, VH44-VL105, and shown to stabilize various Fvs that retain full binding and specificity. Recombinant immunotoxins, e.g. B3(dsFv)-PE38KDEL in which this disulfide-stabilized Fv moiety is connected to a truncated form of Pseudomonas exotoxin (PE; PE38KDEL) which contains the translocation and ADP ribosylation domains, are indistinguishable in binding and specificity from its single-chain immunotoxin counterparts. We have now analyzed the alternative position, (VH111-VL48), predicted by the modeling methodology, for disulfide stabilization of mAb B3(Fv) by producing a recombinant immunotoxin with such disulfide-stabilized (ds) Fv. This immunotoxin was also very active and retained full specificity to B3 antigen-positive cells. However, it was 2- to 3-fold less active than the VH44-VL105 dsFv-molecule. We also tested various biochemical features of VH44-VL105 and VH111-VL48 dsFv immunotoxins and compared them with the corresponding single-chain immunotoxin. We found the dsFv immunotoxins were more stable in human serum and more resistant to thermal and chemical denaturation than the single chain (sc) Fv immunotoxin. Because dsFv immunotoxins and dsFvs have full activity and specificity and improved stability, they may be more useful than scFv immunotoxins as therapeutic and diagnostic agents.

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

“…Known methods for stabilizing Fv fragments are as singlechain Fvs (scFvs) (1,27,28) and as disulfide-stabilized Fvs (dsFvs) (29)(30)(31)). Yet, scFvs still are often unstable (29,32,33) and can have lower affinities compared with Fabs and whole IgG because the linker interferes with binding or does not sufficiently stabilize the heterodimer (30,34,35). Although the dsFvs generally are more stable and do not have a linker, they require the incorporation of a disulfide into the library construction.…”

Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

“…Unfortunately, many scFv fragments are not stable for longer periods. Even when provided with superb antigen-binding properties, they o�en fail to show convincing effects during practical applications, because they tend to denature and aggregate under the conditions faced in practice (Reiter et al, 1994;Willuda et al, 1999). It can be shown, however, that this is not an intrinsic drawback of the scFv format, but rather a property of particular scFv coding sequences, which can be corrected by engineering, reviewed by Worn and Plueckthun (2001).…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…Spontaneous and transient opening of the interface between the V L and V H chains has been suggested to result in exposure of normally hidden hydrophobic patches, leading to aggregation [111]. The introduction of an interdomain disulfide bond (between the V L and V H chains) [112], and variations in the length and flexibility of the V L -V H linker [113] have been shown to increase interdomain stability by limiting the opening of the V L -V H interface.…”