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A recombinant immunotoxin containing a disulfide-stabilized Fv fragment
U Brinkmann1, Y Reiter, S H Jung
1Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Summary
Disulfide-stabilized Fv (dsFv) immunotoxins maintain antigen binding and exhibit enhanced stability compared to single-chain Fv (scFv) versions. This disulfide bond strategy offers a versatile approach for developing more stable immunotoxins for therapeutic use.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Recombinant immunotoxins are crucial therapeutic agents.
- The Fv fragment, comprising variable heavy and light chains, is essential for antigen binding.
- Fv heterodimers can be unstable, limiting therapeutic applications.
Purpose of the Study:
- To engineer a stable disulfide-stabilized Fv (dsFv) fragment for the B3 immunotoxin.
- To assess the impact of disulfide stabilization on antigen binding, activity, and stability.
- To evaluate the general applicability of this dsFv construction method.
Main Methods:
- Computer modeling of the B3 Fv structure to predict disulfide bond positions.
- Construction and expression of a disulfide-linked two-chain dsFv-immunotoxin in E. coli.
- Comparison of the activity, specificity, and stability of B3(dsFv) and B3(scFv) in vitro.
Main Results:
- The disulfide-stabilized Fv (dsFv) immunotoxin retained indistinguishable activity and specificity compared to its single-chain Fv (scFv) counterpart.
- The dsFv demonstrated significantly enhanced stability at 37°C in human plasma compared to scFv.
- The disulfide bond was introduced at a conserved framework region, suggesting broad applicability.
Conclusions:
- Disulfide stabilization effectively enhances the stability of Fv fragments without compromising antigen binding or activity.
- The developed dsFv construction method is potentially applicable to other antibodies for immunotoxin development.
- Disulfide-stabilized Fv fragments represent a more versatile and therapeutically promising alternative to scFv fragments.