Thermal stabilization of a single‐chain Fv antibody fragment by introduction of a disulphide bond

Abstract

A disulphide bond was introduced into a single-chain Fv form of the anticarbohydrate antibody, Se155-4 by replacing Ala-L57 of the light chain and Asp-H106 of the heavy chain with cysteines, by site-directed mutagenesis. To maintain the saltbridge from the latter residue to Arg-H98, Tyr-107 was also altered to Asp. The resulting ds-scFv was shown to retain full antigen-binding activity, by enzyme immunoassay and surface plasmon resonance analysis of binding kinetics. Compared with the parent scFv, the disulphide bonded form was shown to have enhanced thermal stability, by Fourier transform IR spectroscopy. The Tm was raised from 60°C to 69°C. The ds-scFv form thus combines the stable monomeric form of the disulphide form wifh the expression advantages of the scFv.

Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors

“…To increase the thermal stability of Fv antibody fragments, expression of VH and VL as separate polypeptides and subsequent crosslinking via an arti®cial disul®de bond engineered into the antibody variable regions has been suggested (Reiter et al, 1996). Here to simplify expression and puri®cation procedures we attempted to stabilize the scFv(14E1) antibody domain by incorporating a disul®de bond between VH and VL domains, but retained the format of a single chain polypeptide where VH and VL in addition to the disul®de bond are connected by the original synthetic linker sequence (Luo et al, 1995;Young et al, 1995). By site directed mutagenesis using PCR techniques Gln at position VH105 and Ser at position VL43 in scFv(14E1) were replaced by Cys residues (Reiter et al, 1996) (numbering according to Kabat et al (1991)).…”

Disulfide Bond Introduction for General Stabilization of Immunoglobulin Heavy-Chain Variable Domains

“…6 Other methods introduced disulfide bonds between these domains to produce either disulfide-stabilized Fv or single-chain disulfide-stabilized Fv fragments. [7][8][9] Even then, a selected Fv derivative might regularly benefit from a further increase in stability. One outcome is offered by grafting the antigen-specific loops of the antibody fragments onto a stable scaffold.…”

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

“…However, given the likelihood that introducing disulfides may not be a general solution for stabilizing scFvs, we anticipate that linker length optimization combined with rational and molecular evolution strategies will make it possible to routinely improve scFv stability for producing quality IgG-like bispecific antibodies. 6,9,14,[22][23][24][25] While ongoing studies are addressing whether a threshold thermal transition value can be defined for selecting a suitably stabilized scFv for constructing a stability-engineered IgG-like BsAbs, we surmise that a T 50 value ~65°C or a T m value ≥70°C may be desirable. Presumably, related approaches could be applied for…”