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Disulfide-Stabilized Fv Fragments

  • Ulrich BrinkmannEmail author
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

The design and generation of disulfide-stabilized Fv fragments (dsFv’s) addresses stability and aggregation problems that are frequently associated with single-chain Fvs. VH and VL of dsFv’s are connected by an interdomain disulfide bond. To generate such molecules, one amino acid each in the framework region of in VH (at position 44) and VL (at position 100) are mutated to a cysteine, which in turn form a stable interchain disulfide bond. The resulting dsFv’s (no linker peptide) or scdsFv (linker as well as interchain disulfide bond) can be easily produced in various expression systems. Disulfide-stabilized Fv’s solve most problems that are frequently associated with Fvs or scFvs; they are very stable and in most instances show full antigen binding activity.

Keywords

Fusion Protein Inclusion Body Antigen Binding Linker Peptide Refold Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

IPTG

Isopropyl-β-d-thiogalactopyranoside

scFv

Single-chain Fv

dsFv

Disulfide-stabilized Fv

VH and VL

Variable region of heavy or light-chain

GuCl

Guanidine chloride

DTE

Dithioerythritol

IB

Inclusion body

IG

Immunoglobulin

References

  1. Adams GP, McCartney JE, Tai MS, Oppermann H, Huston JS, Stafford WF III, Bookman MA, Fand I, Houston LL, Weiner LM (1993) Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single-chain Fv. Cancer Res 53:4026–4034PubMedGoogle Scholar
  2. Bera TK, Onda M, Brinkmann U, Pastan I (1998) A bivalent disulfide-stabilized Fv with improved antigen binding to erbB2. J Mol Biol 281(3):475–483. PMID:9698563PubMedCrossRefGoogle Scholar
  3. Bera TK, Viner J, Brinkmann E, Pastan I (1999) Pharmacokinetics and antitumor activity of a bivalent disulfide-stabilized Fv immunotoxin with improved antigen binding to erbB2. Cancer Res 59(16):4018–4022PubMedGoogle Scholar
  4. Bera TK, Williams-Gould J, Beers R, Chowdhury P, Pastan I (2001) Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer. Mol Cancer Therapeut 1(2):79–84Google Scholar
  5. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS, Whitlow M (1988) Single-chain antigen-binding proteins. Science 242:423–426PubMedCrossRefGoogle Scholar
  6. Brinkmann U, Reiter Y, Jung SH, Lee B, Pastan I (1993) A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proc Natl Acad Sci USA 90:7538–7542PubMedCrossRefGoogle Scholar
  7. Buchner J, Pastan I, Brinkmann U (1992) A method to increase the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Anal Biochem 205:263–270PubMedCrossRefGoogle Scholar
  8. FitzGerald K, Holliger P, Winter G (1997) Improved tumour targeting by disulphide stabilized diabodies expressed in Pichia pastoris. Protein Eng 10(10):1221–1225PubMedCrossRefGoogle Scholar
  9. Glockshuber R, Malia M, Pfitzinger I, Pluckthun A (1990) A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29:1362–1367PubMedCrossRefGoogle Scholar
  10. Hao HJ, Jiang YQ, Zheng YL, Ma R, Yu DW (2005) Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety. Biochimie 87(8):661–667. PMID:15927340PubMedCrossRefGoogle Scholar
  11. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE, Haber E, Crea R, Oppermann H (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA 16:5879–5883CrossRefGoogle Scholar
  12. Huston JS, Mudgett-Hunter M, Tai MS, McCartney J, Warren F, Haber E, Oppermann H (1991) Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol 203:46–88PubMedCrossRefGoogle Scholar
  13. Kleinschmidt M, Rudolph R, Lilie H (2003) Design of a modular immunotoxin connected by polyionic adapter peptides. J Mol Biol 327(2):445–452. PMID:12628249PubMedCrossRefGoogle Scholar
  14. Pastan I, Hassan R, Fitzgerald DJ, Kreitman RJ (2006) Immunotoxin therapy of cancer. Nature Rev Cancer 6(7):559–565CrossRefGoogle Scholar
  15. Rajagopal V, Pastan I, Kreitman RJ (1997) A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: comparison with its single-chain and disulfide-stabilized homologs. Protein Eng 10(12):1453–1459. PMID:9543007PubMedCrossRefGoogle Scholar
  16. Reiter Y, Brinkmann U, Kreitman RJ, Jung SH, Lee B, Pastan I (1994a) Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions. Biochemistry 33(18):5451–5459PubMedCrossRefGoogle Scholar
  17. Reiter Y, Brinkmann U, Jung S-H, Lee B, Kasprzyk PG, King CR, Pastan I (1994b) Improved binding and anti-tumor activity of a recombinant anti-erbB2 immunotoxin by disulfide-stabilization of the Fv fragment. J Biol Chem 269:18327–18331PubMedGoogle Scholar
  18. Reiter Y, Brinkmann U, Jung S-H, Lee B, Pastan I (1994c) Engineering disulfide bonds into conserved framework regions of Fv fragments: recombinant immunotoxins containing disulfide-stabilized Fv with improved biochemical characteristics. Protein Eng 7:697–704PubMedCrossRefGoogle Scholar
  19. Reiter Y, Kreitman RJ, Brinkmann U, Pastan I (1994d) Cytotoxic and antitumor activity of a recombinant immunotoxin composed of disulfide-stabilized anti-Tac Fv fragment and truncated Pseudomonas exotoxin. Int J Cancer 58:142–149PubMedCrossRefGoogle Scholar
  20. Reiter Y, Kurucz I, Brinkmann U, Jung SH, Lee B, Segal DM, Pastan I (1995) Construction of a functional disulfide-stabilized TCR Fv indicates that antibody and TCR Fv frameworks are very similar in structure. Immunity 2(3):281–287. PMID:7697545PubMedCrossRefGoogle Scholar
  21. Reiter Y, Brinkmann U, Lee B, Pastan I (1996) Engineering antibody Fv fragments for cancer detection and therapy: disulfide-stabilized Fv fragments. Nature Biotechnol 14(10):1239–1245. PMID:9631086CrossRefGoogle Scholar
  22. Rodrigues ML, Presta LG, Kotts CE, Wirth C, Mordenti J, Osaka G, Wong WL, Nuijens A, Blackburn B, Carter P (1995) Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug. Cancer Res 55(1):63–70PubMedGoogle Scholar
  23. Schmiedl A, Breitling F, Dübel S (2000) Expression of a bispecific dsFv-dsFv’ antibody fragment in Escherichia coli. Protein Eng 13(10):725–734. PMID:11112512PubMedCrossRefGoogle Scholar
  24. Schmiedl A, Zimmermann J, Scherberich JE, Fischer P, Dübel S (2006) Recombinant variants of antibody 138H11 against human gamma-glutamyltransferase for targeting renal cell carcinoma. Human Antibodies 15(3):81–94. PMID:17065739PubMedGoogle Scholar
  25. Skerra A, Pluckthun A (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240:1038–1041PubMedCrossRefGoogle Scholar
  26. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130PubMedCrossRefGoogle Scholar
  27. Webber KO, Reiter Y, Brinkmann U, Kreitman RJ, Pastan I (1995) Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog. Mol Immunol 4:249–258CrossRefGoogle Scholar
  28. Yokota T, Milenic DE, Whitlow M, Schlom J (1992) Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 52:3402–3408PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2010

Authors and Affiliations

  1. 1.Roche Pharma ResearchBiologics R&DPenzbergGermany

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