A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: comparison with its single-chain and disulfide-stabilized homologs.

Abstract

Disulfide-stabilized Fvs (dsFvs) are recombinant proteins composed of a heavy-chain variable domain (VH) of an antibody connected via a disulfide bond to the light-chain variable domain (VL). In single-chain Fvs (scFvs), a peptide connector links VH and VL. The dsFv form of the anti-Tac monoclonal antibody which reacts with the alpha subunit of the IL2 receptor was recently reported to be more stable and to aggregate less during renaturation than anti-Tac(scFv). In addition, it could be produced in a better yield owing to less aggregation. However, the yields are still too low to permit the production of material for clinical trials in which the dsFv will be used to image or treat IL2 receptor (CD25)-containing tumors. To increase the efficiency by which VH and VL associate and form a disulfide bond during renaturation, we have prepared an Fv form of anti-Tac which is both single chain and disulfide stabilized (scdsFv). The recombinant protein is expressed in Escherichia coli, where it accumulates in inclusion bodies. Using inclusion body protein as the reference point, the yield of purified anti-Tac(scdsFv) was 13% compared with 2% for anti-Tac(dsFv). Anti-Tac(scdsFv) has equivalent binding affinity, immunoreactivity after radiolabeling and stability. The results show that a linker between VH and VL facilitates heterodimer formation and leads to disulfide bond formation in a higher percentage of the molecules renatured. Thus anti-Tac(scdsFv) is the preferred form of anti-Tac(Fv) to be used for clinical studies. We anticipate that scdsFvs will be the optimum recombinant form of Fv to produce from bacteria.

• Authors: V Rajagopal, I Pastan, R J Kreitman
• Journal: Protein engineering (Protein Eng) Vol. 10 Issue 12 Pg. 1453-9 (Dec 1997) ISSN: 0269-2139 [Print] England
• PMID: 9543007 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Disulfides
• Immunoglobulin Fragments
• Receptors, Interleukin-2
• Recombinant Proteins
• immunoglobulin Fv

Topics

• Antibody Specificity
• Disulfides (chemistry)
• Drug Stability
• Escherichia coli (genetics)
• Gene Expression
• Hydrogen-Ion Concentration
• Immunoglobulin Fragments (chemistry, genetics, isolation & purification)
• Protein Denaturation
• Receptors, Interleukin-2 (immunology)
• Recombinant Proteins
• Regression Analysis