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Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding.

Scott N, Qazi O, Wright MJ, Fairweather NF, Deonarain MP - Mol. Immunol. (2010)

Bottom Line: We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity.This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells.For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK.

ABSTRACT
An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

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Related in: MedlinePlus

Anti-tetanus scFv epitope binning analysis. Each of the 14 phage-scFv fusions C1-C5, J1-J5 (excluding J3) and N1-N5 were assayed for binding to TT-Hc systematically at pre-determined fixed dilutions in the absence of soluble scFv and in the presence of each of the 14 scFvs in soluble non-phage-fused form (3–5 μM). Each phage-scFv was assayed against its non-fused form as an intrinsic positive control for competitive measurement. Each condition was measured in triplicate and bars are means with standard error bars. One-way ANOVA was used to determine whether there was significant global variation between phage-binding in the absence of scFvs and in the presence of scFvs for each clone. Significant variation was confirmed in each case (p < 0.01). A post-ANOVA pair-wise multi-comparative test was performed using the Dunnett algorithm which compares mean individual binding measurements with the positive binding control for each clone taking into account global background variation. Significant differences to the no scFv control are summarised in Table 1 and are denoted in this figure with significance of *p < 0.05 or **p < 0.01.
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fig2: Anti-tetanus scFv epitope binning analysis. Each of the 14 phage-scFv fusions C1-C5, J1-J5 (excluding J3) and N1-N5 were assayed for binding to TT-Hc systematically at pre-determined fixed dilutions in the absence of soluble scFv and in the presence of each of the 14 scFvs in soluble non-phage-fused form (3–5 μM). Each phage-scFv was assayed against its non-fused form as an intrinsic positive control for competitive measurement. Each condition was measured in triplicate and bars are means with standard error bars. One-way ANOVA was used to determine whether there was significant global variation between phage-binding in the absence of scFvs and in the presence of scFvs for each clone. Significant variation was confirmed in each case (p < 0.01). A post-ANOVA pair-wise multi-comparative test was performed using the Dunnett algorithm which compares mean individual binding measurements with the positive binding control for each clone taking into account global background variation. Significant differences to the no scFv control are summarised in Table 1 and are denoted in this figure with significance of *p < 0.05 or **p < 0.01.

Mentions: Fig. 2 and Table 1 show the results for all 14 scFvs that were assayed indicating the competition status in both orientations for each scFv. A one-way ANOVA was conducted followed by a Dunnett multiple comparison analysis used to identify statistically significant differences in phage-binding in the presence of each scFv compared to a control consisting of phage-scFv alone (100% binding). With few exceptions, the data revealed that all HcJ and HcN clones compete for binding to TeNT-Hc, both within and between groups. All HcC clones compete with each other for binding. With the exception of pairings C1 + N5 and C2 + (J4/J5/N4/N5), no soluble HcC clone was able to reduce binding of any HcJ or HcN phage fusion and vice versa indicating that HcC clones were able to simultaneously bind to Hc with HcJ or HcN clones except stated parings.


Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding.

Scott N, Qazi O, Wright MJ, Fairweather NF, Deonarain MP - Mol. Immunol. (2010)

Anti-tetanus scFv epitope binning analysis. Each of the 14 phage-scFv fusions C1-C5, J1-J5 (excluding J3) and N1-N5 were assayed for binding to TT-Hc systematically at pre-determined fixed dilutions in the absence of soluble scFv and in the presence of each of the 14 scFvs in soluble non-phage-fused form (3–5 μM). Each phage-scFv was assayed against its non-fused form as an intrinsic positive control for competitive measurement. Each condition was measured in triplicate and bars are means with standard error bars. One-way ANOVA was used to determine whether there was significant global variation between phage-binding in the absence of scFvs and in the presence of scFvs for each clone. Significant variation was confirmed in each case (p < 0.01). A post-ANOVA pair-wise multi-comparative test was performed using the Dunnett algorithm which compares mean individual binding measurements with the positive binding control for each clone taking into account global background variation. Significant differences to the no scFv control are summarised in Table 1 and are denoted in this figure with significance of *p < 0.05 or **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874839&req=5

fig2: Anti-tetanus scFv epitope binning analysis. Each of the 14 phage-scFv fusions C1-C5, J1-J5 (excluding J3) and N1-N5 were assayed for binding to TT-Hc systematically at pre-determined fixed dilutions in the absence of soluble scFv and in the presence of each of the 14 scFvs in soluble non-phage-fused form (3–5 μM). Each phage-scFv was assayed against its non-fused form as an intrinsic positive control for competitive measurement. Each condition was measured in triplicate and bars are means with standard error bars. One-way ANOVA was used to determine whether there was significant global variation between phage-binding in the absence of scFvs and in the presence of scFvs for each clone. Significant variation was confirmed in each case (p < 0.01). A post-ANOVA pair-wise multi-comparative test was performed using the Dunnett algorithm which compares mean individual binding measurements with the positive binding control for each clone taking into account global background variation. Significant differences to the no scFv control are summarised in Table 1 and are denoted in this figure with significance of *p < 0.05 or **p < 0.01.
Mentions: Fig. 2 and Table 1 show the results for all 14 scFvs that were assayed indicating the competition status in both orientations for each scFv. A one-way ANOVA was conducted followed by a Dunnett multiple comparison analysis used to identify statistically significant differences in phage-binding in the presence of each scFv compared to a control consisting of phage-scFv alone (100% binding). With few exceptions, the data revealed that all HcJ and HcN clones compete for binding to TeNT-Hc, both within and between groups. All HcC clones compete with each other for binding. With the exception of pairings C1 + N5 and C2 + (J4/J5/N4/N5), no soluble HcC clone was able to reduce binding of any HcJ or HcN phage fusion and vice versa indicating that HcC clones were able to simultaneously bind to Hc with HcJ or HcN clones except stated parings.

Bottom Line: We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity.This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells.For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK.

ABSTRACT
An approach for enhancing antibody affinity is to engineer Chelating Recombinant Antibodies (CRAbs) which consist of two tandemly linked single-chain Fvs (scFvs) that bind to distinct non-overlapping epitopes on the antigen molecule leading to a synergistic decrease in K(D). In order to develop this technology, the aim of this present study was to identify scFvs which can simultaneously bind to the tetanus toxin heavy chain C-terminal sub-domain (H(c)), characterise their bio-physical properties and determine their functional efficacy. Over 50 antibodies specific for Hc were isolated from a human scFv phagemid library and found to bind specifically to the C-terminal sub-domain of H(c) (H(c)C clones), the N-terminal sub-domain (HcN clones) or junctional epitopes on the whole Hc fragment only (HcJ clones). Fifteen clones were assayed in a pairwise competition binding study. The revealed, with few exceptions, that H(c)C clones were able to simultaneously bind to the toxin with H(c)N or H(c)J clones. All other combinations competed for binding. Interestingly, we also observed cooperative binding with many non-competing scFv pairings which may impact upon the binding mechanism of CRAbs. We found that 14/15 clones neutralised toxin activity in a ganglioside binding assay and this effect was strongly related to affinity. This included clones that did not bind to the H(c)C sub-domain which is responsible for direct interaction with gangliosides on nerve cells. For 7 scFvs that underwent further characterisation we found broad variations in propensity for multimerisation, affinity and potency. The diverse array of clones characterised in this paper can be used to construct CRAbs and will prove useful in further characterisation of toxin biology and in measuring the effects of polyclonal antibody therapy.

Show MeSH
Related in: MedlinePlus