An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent
- 197 Downloads
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.
KeywordsPhage-display panning Single-domain antibody fragment Antifungal activity HM-1 killer toxin Killer toxin (HM-1) neutralizing monoclonal antibody
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.
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.
- 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 13:3245–3260Google Scholar
- Hoogenboom HR (2002) Overview of antibody phage-display technology and its applications. Methods Mol Biol 178:1–37Google Scholar
- 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 85:5879–5883CrossRefGoogle Scholar
- 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 147:1709–1719Google Scholar
- Kabat EA, Wu TT, Perry H, Gottesman K, Foeller C (1991) Sequences of proteins of immunological interest, 5th edn. National Institutes of Health, BethesdaGoogle Scholar
- 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 4:731–736Google Scholar
- 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 10:1013–1019Google Scholar
- 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 164:4367–4374Google Scholar
- Majidi J, Barar J, Baradaran B, Abdolalizadeh J, Omidi Y (2009) Target therapy of cancer: implementation of monoclonal antibodies and nanobodies. Hum Antibodies 18:81–100Google Scholar
- Mitchell TG, Perfect JR (1995) Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 8:515–548Google Scholar
- Singh N, Gayowski T, Wagener MM, Marino IR (1997) Clinical spectrum of invasive cryptococcosis in liver transplant recipients receiving tacrolimus. Clin Transplant 11:66–70Google Scholar
- 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 41:575–581Google Scholar
- Van Bockstaele F, Holz JB, Revets H (2009) The development of nanobodies for therapeutic applications. Curr Opin Investig Drugs 10:1212–1224Google Scholar
- 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) 41:398–403Google Scholar