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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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Comparative potency measurement for 7 anti-TT-Hc scFvs. The binding of recombinant monomeric TT-Hc to plate coated ganglioside GT1b was measured by ELISA at a fixed concentration (100 nM) both in the absence (“No scFv”) and the presence of the anti-TT-Hc scFvs C1, C2, C4, J2, J4, N4 and N5 at a single fixed concentration of 650 nM. Data is represented as % TT-Hc-binding in comparison to binding in the absence of scFv which is normalised to 100%. Bars represent means of pentuplicate measurements with standard error. Binding in the presence of scFv was compared to binding in their absence using Student's t-test. *p < 0.05; **p < 0.01. No star = not significant.
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fig4: Comparative potency measurement for 7 anti-TT-Hc scFvs. The binding of recombinant monomeric TT-Hc to plate coated ganglioside GT1b was measured by ELISA at a fixed concentration (100 nM) both in the absence (“No scFv”) and the presence of the anti-TT-Hc scFvs C1, C2, C4, J2, J4, N4 and N5 at a single fixed concentration of 650 nM. Data is represented as % TT-Hc-binding in comparison to binding in the absence of scFv which is normalised to 100%. Bars represent means of pentuplicate measurements with standard error. Binding in the presence of scFv was compared to binding in their absence using Student's t-test. *p < 0.05; **p < 0.01. No star = not significant.

Mentions: Clone C1 inhibited binding of TeNT-Hc to the ganglioside in a dose-dependent manner with binding reduced to background levels at the highest concentration of scFv (900 nM). The dose-response curve generated resulted in an IC50 value of 212 ± 17 nM. Neutralising activity of the remaining clones was determined at a single fixed concentration of 650 nM allowing a comparative analysis (Fig. 4). Clone C1 completely ablated binding of TeNT-Hc to ganglioside (p < 0.01) whilst clone C4 diminished binding to around 10% of the levels observed in the absence of scFv (p < 0.01). Clones J2 and J4 reduced toxin–ganglioside binding to around 70% of levels in the absence of scFv (p < 0.05) and clones N4 and N5 reduced binding to around 60% (p < 0.05). Clone C2 did not reduce the binding of TeNT-Hc at the concentration tested.


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)

Comparative potency measurement for 7 anti-TT-Hc scFvs. The binding of recombinant monomeric TT-Hc to plate coated ganglioside GT1b was measured by ELISA at a fixed concentration (100 nM) both in the absence (“No scFv”) and the presence of the anti-TT-Hc scFvs C1, C2, C4, J2, J4, N4 and N5 at a single fixed concentration of 650 nM. Data is represented as % TT-Hc-binding in comparison to binding in the absence of scFv which is normalised to 100%. Bars represent means of pentuplicate measurements with standard error. Binding in the presence of scFv was compared to binding in their absence using Student's t-test. *p < 0.05; **p < 0.01. No star = not significant.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Comparative potency measurement for 7 anti-TT-Hc scFvs. The binding of recombinant monomeric TT-Hc to plate coated ganglioside GT1b was measured by ELISA at a fixed concentration (100 nM) both in the absence (“No scFv”) and the presence of the anti-TT-Hc scFvs C1, C2, C4, J2, J4, N4 and N5 at a single fixed concentration of 650 nM. Data is represented as % TT-Hc-binding in comparison to binding in the absence of scFv which is normalised to 100%. Bars represent means of pentuplicate measurements with standard error. Binding in the presence of scFv was compared to binding in their absence using Student's t-test. *p < 0.05; **p < 0.01. No star = not significant.
Mentions: Clone C1 inhibited binding of TeNT-Hc to the ganglioside in a dose-dependent manner with binding reduced to background levels at the highest concentration of scFv (900 nM). The dose-response curve generated resulted in an IC50 value of 212 ± 17 nM. Neutralising activity of the remaining clones was determined at a single fixed concentration of 650 nM allowing a comparative analysis (Fig. 4). Clone C1 completely ablated binding of TeNT-Hc to ganglioside (p < 0.01) whilst clone C4 diminished binding to around 10% of the levels observed in the absence of scFv (p < 0.01). Clones J2 and J4 reduced toxin–ganglioside binding to around 70% of levels in the absence of scFv (p < 0.05) and clones N4 and N5 reduced binding to around 60% (p < 0.05). Clone C2 did not reduce the binding of TeNT-Hc at the concentration tested.

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