An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Abstract

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K d = 1.07 × 10−10 M) and antifungal activity was three- to fivefold more efficient (IC50 = 0.46 × 10−6 to 1.17 × 10−6 M) than that for the conventional antibody (K d = 2.91 × 10−8 M and IC50 = 2.14 × 10−6 to 3.78 × 10−6 M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. 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 242:423–426

    Article  CAS  Google Scholar 

  2. Cassone A (2008) Fungal vaccines: real progress from real challenges. Lancet Infect Dis 2:114–124

    Article  Google Scholar 

  3. Cutler JE, Deepe GS Jr, Klein BS (2007) Advances in combating fungal diseases: vaccines on the threshold. Nat Rev Microbiol 1:13–28

    Article  Google Scholar 

  4. de Haard HJ, van Neer N, Reurs A, Hufton SE, Roovers RC, Henderikx P, de Bruïne AP, Arends JW, Hoogenboom HR (1999) A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Biol Chem 274:18218–18230

    Article  Google Scholar 

  5. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, Muyldermans S, Wyns L, Matagne A (2002) Single-domain antibody fragments with high conformational stability. Protein Sci 11:500–515

    Article  CAS  Google Scholar 

  6. Ghahroudi MA, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521–526

    Article  Google Scholar 

  7. Griffiths AD, Williams SC, Hartley O, Tomlinson IM, Waterhouse P, Crosby WL, Kontermann RE, Jones PT, Low NM, Allison TJ, Prospero TD, Hoogenboom HR, Nissim A, Cox JPL, Harrison JL, Zaccolo M, Gherardi E, Winter G (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J 13:3245–3260

    CAS  Google Scholar 

  8. Gruen LC, Kortt AA, Nice E (1993) Determination of relative binding affinity of influenza virus N9 sialidases with the Fab fragment of monoclonal antibody NC41 using biosensor technology. Eur J Biochem 217:319–325

    Article  CAS  Google Scholar 

  9. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448

    Article  CAS  Google Scholar 

  10. Harmsen MM, De Haard HJ (2007) Properties, production, and applications of camelid single domain antibody fragments. Appl Microbiol Biotechnol 77:13–22

    Article  CAS  Google Scholar 

  11. Holliger P, Prospero T, Winter G (1993) “Diabodies”: small bivalent and bispecific antibody fragments. Proc Natl Acad Sci USA 90:6444–6448

    Article  CAS  Google Scholar 

  12. Hoogenboom HR (2002) Overview of antibody phage-display technology and its applications. Methods Mol Biol 178:1–37

    CAS  Google Scholar 

  13. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, Novotný 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 85:5879–5883

    Article  CAS  Google Scholar 

  14. Kabat EA, Wu TT (1991) Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. J Immunol 147:1709–1719

    CAS  Google Scholar 

  15. Kabat EA, Wu TT, Perry H, Gottesman K, Foeller C (1991) Sequences of proteins of immunological interest, 5th edn. National Institutes of Health, Bethesda

    Google Scholar 

  16. Kabir ME, Krishnaswamy S, Miyamoto M, Furuichi Y, Komiyama T (2009) An improved phage-display panning method to produce an HM-1 killer toxin anti-idotypic antibody. BMC Biotechnol 9:99

    Article  Google Scholar 

  17. Kabir ME, Krishnaswamy S, Miyamoto M, Furuichi Y, Komiyama T (2010) Purification and functional characterization of a camelid-like single-domain antimycotic antibody by engineering in affinity tag. Protein Expr Purif 72:59–65

    Article  CAS  Google Scholar 

  18. Kasahara S, Ben Inoue S, Mio T, Yamada T, Nakajima T, Ichishima E, Furuichi Y, Yamada H (1994) Involvement of cell wall beta-glucan in the action of HM-1 killer toxin. FEBS Lett 1:27–32

    Article  Google Scholar 

  19. Komiyama T, Ohta T, Urakami H, Shiratori Y, Takasuka T, Satoh M, Watanabe T, Furuichi Y (1996) Pore formation on proliferating yeast Saccharomyces cerevisiae cell buds by HM-1 killer toxin. J Biochem 4:731–736

    Google Scholar 

  20. Komiyama T, Shirai T, Ohta T, Urakami H, Furuichi Y, Ohta Y, Tsukada Y (1998) Action properties of HYI killer toxin from Williopsis saturnus var. saturnus, and antibiotics, aculeacin A and papulacandin B. Biol Pharm Bull 10:1013–1019

    Google Scholar 

  21. Lendvai N, Qu XW, Hsueh W, Casadevall A (2000) Mechanism for the isotype dependence of antibody-mediated toxicity in Cryptococcus neoformans-infected mice. J Immunol 164:4367–4374

    CAS  Google Scholar 

  22. Levitz SM (1991) The ecology of Cryptococcus neoformans and the epidemiology of cryptococcosis. Rev Infect Dis 13:1163–1169

    Article  CAS  Google Scholar 

  23. Lu D, Shen J, Vil MD, Zhang H, Jimenez X, Bohlen P, Whitte L, Zhu Z (2003) Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity. J Biol Chem 278:43496–43507

    Article  CAS  Google Scholar 

  24. Majidi J, Barar J, Baradaran B, Abdolalizadeh J, Omidi Y (2009) Target therapy of cancer: implementation of monoclonal antibodies and nanobodies. Hum Antibodies 18:81–100

    CAS  Google Scholar 

  25. Mitchell TG, Perfect JR (1995) Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 8:515–548

    CAS  Google Scholar 

  26. Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R (1994) Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng 7:1129–1135

    Article  CAS  Google Scholar 

  27. Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163

    Article  CAS  Google Scholar 

  28. Rahbarizadeh F, Rasaee MJ, Forouzandeh-Moghadam M, Allameh AA (2005) High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli. Protein Expr Purif 44:32–38

    Article  CAS  Google Scholar 

  29. Selvakumar D, Miyamoto M, Furuichi Y, Komiyama T (2006) Inhibition of fungal β-1,3-glucan synthase and cell growth by HM-1 killer toxin single-chain anti-idiotypic antibodies. Antimicrob Agents Chemother 50:3090–3097

    Article  CAS  Google Scholar 

  30. Shoham S, Levitz SM (2005) The immune response to fungal infections. Br J Haematol 129:569–582

    Article  Google Scholar 

  31. Singh N, Gayowski T, Wagener MM, Marino IR (1997) Clinical spectrum of invasive cryptococcosis in liver transplant recipients receiving tacrolimus. Clin Transplant 11:66–70

    CAS  Google Scholar 

  32. Takasuka T, Komiyama T, Furuichi Y, Watanabe T (1995) Cell wall synthesis specific cytocidal effect of Hansenula mrakii toxin-1 on Saccharomyces cerevisiae. Cell Mol Biol Res 41:575–581

    CAS  Google Scholar 

  33. Van Bockstaele F, Holz JB, Revets H (2009) The development of nanobodies for therapeutic applications. Curr Opin Investig Drugs 10:1212–1224

    Google Scholar 

  34. Vaughan TJ, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, McCafferty J, Hodits RA, Wilton J, Johnson KS (1996) Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol 14:309–314

    Article  CAS  Google Scholar 

  35. Ward ES, Güssow D, Griffiths AD, Jones PT, Winter G (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341:544–546

    Article  CAS  Google Scholar 

  36. Yamamoto T, Imai M, Tachibana K, Mayumi M (1986) Application of monoclonal antibodies to the isolation and characterization of a killer toxin secreted by Hansenula mrakii. FEBS Lett 195:253–257

    Article  CAS  Google Scholar 

  37. Yamamoto T, Uchida K, Hiratani T, Miyazaki T, Yagiu J, Yamaguchi H (1988) In vitro activity of the killer toxin from yeast Hansenula mrakii against yeasts and molds. J Antibiot (Tokyo) 41:398–403

    CAS  Google Scholar 

  38. Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) Bacterial expression and characterization of a picloram-specific recombinant Fab for residue analysis. J Agric Food Chem 46:4457–4463

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors’ contributions

M.E. Kabir and T. Komiyama conceived and designed the experiments. M.E. Kabir and S. Krishnaswamy performed the experiments. M.E. Kabir, M. Miyamoto, Y. Furuichi, and T. Komiyama analyzed the data. M.E. Kabir wrote the paper.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Tadazumi Komiyama.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kabir, M.E., Krishnaswamy, S., Miyamoto, M. et al. An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent. Appl Microbiol Biotechnol 90, 553–564 (2011). https://doi.org/10.1007/s00253-011-3123-8

Download citation

Keywords

  • Phage-display panning
  • Single-domain antibody fragment
  • Antifungal activity
  • HM-1 killer toxin
  • Killer toxin (HM-1) neutralizing monoclonal antibody