Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

Department of Biochemistry, Niigata University of Pharmacy and Applied Life Sciences, Japan.
Analytical Biochemistry (Impact Factor: 2.22). 09/2009; 395(1):16-24. DOI: 10.1016/j.ab.2009.08.003
Source: PubMed


Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1+HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC(50) values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40microM and 2.20microM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K(d) value of 3.09x10(-11)M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.


Available from: M. Enamul Kabir
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    • "Taking advantage of its immunegenicity and a potent ß-1,3-glucan synthase inhibitor, HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) has been produced. To produce a new antifungal drug, we used nmAb-KT as an immunogen to generate recombinant anti-idotypic scFv antibodies that share structural and functional similarities with the active site of HM-1 (Selvakumar et al., 2006b; Kabir et al., 2009; Krishnaswamy et al., 2009). "
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    ABSTRACT: Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.
    Journal of Molecular Recognition 07/2011; 24(4):631-41. DOI:10.1002/jmr.1075 · 2.15 Impact Factor
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    • "In our laboratory using various approaches we identified several recombinant anti-idiotypic scFv antibodies of HM-1 killer toxin those were highly effective against different fungal species growth (Selvakumar et al. 2006a, b, c; Krishnaswamy et al. 2009; Kabir et al. 2009). To identify and compare the effectiveness of some small peptides as antifungal agent, thirteen overlapping synthetic HM-1 peptides (P1 -P13) were prepared from the N-terminus of the primary structure of HM-1 (Yamamoto et al. 1986a) and also six peptides (SP1 – SP6) belonging to the primary structure of scFv-A1 (Selvakumar et al. 2006a) were designed and synthesized with special regard to CDR regions (V H -CDR 1–3 and V L -CDR 1–3) and also a scramble peptide (peptide with altered amino acid positions) of SP6 (Sc-SP6: SVTIVCSAKT) were synthesized from Sigma Genosys Co. Ltd., Japan (Fig. 1a and b). "
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    ABSTRACT: Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a β-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall β-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.
    Applied Microbiology and Biotechnology 06/2011; 92(6):1151-60. DOI:10.1007/s00253-011-3412-2 · 3.34 Impact Factor
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    ABSTRACT: We reported previously competitive panning elution with PBS (pH 7.0) that contains HM-1 killer toxin (HM-1) and Candida albicans membrane fraction (CaMF) to release phages bound with CaMF as an antigen. Here, as an alternative strategy, we isolated high-binding affinity recombinant single-chain fragment variables (scFvs) with in vitro anti-fungal activity from an scFv phage library. The competitive panning elution contained acidic, neutral and basic pH buffers with original antigen HM-1 or HM-1 peptide 6 used to release phages bound with HM-1-neutralizing monoclonal antibody (nmAb-KT). For neutral pH eluted conditions, 87.5% of clones showed high-binding affinity against nmAb-KT by using ELISA, but was 16% and 26% for acidic and basic eluted conditions, respectively. After nucleotide sequencing, we obtained seven different anti-idiotypic antibodies from the different selection procedures. The clone expression and purification by using a HisTrap HP column, showed that clones scFv S3, S4 and S7 had in vitro antifungal activity against Saccharomyces cerevisiae, Candida albicans. The purified scFvs showed strong binding affinity with nmAb-KT by using ELISA. These results showed that changing the buffer pH with competing elements plays important role in elution of bound phages to targeted antigen and also in identification of positive scFv phages.
    Journal of Biochemistry 05/2010; 147(5):723-33. DOI:10.1093/jb/mvq006 · 2.58 Impact Factor
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