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

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...

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Autores principales: Scott, Nathan, Qazi, Omar, Wright, Michael J., Fairweather, Neil F., Deonarain, Mahendra P.
Formato: Texto
Lenguaje:English
Publicado: Pergamon Press 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874839/
https://www.ncbi.nlm.nih.gov/pubmed/20413159
http://dx.doi.org/10.1016/j.molimm.2010.02.020
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author Scott, Nathan
Qazi, Omar
Wright, Michael J.
Fairweather, Neil F.
Deonarain, Mahendra P.
author_facet Scott, Nathan
Qazi, Omar
Wright, Michael J.
Fairweather, Neil F.
Deonarain, Mahendra P.
author_sort Scott, Nathan
collection PubMed
description 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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spelling pubmed-28748392010-06-10 Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding Scott, Nathan Qazi, Omar Wright, Michael J. Fairweather, Neil F. Deonarain, Mahendra P. Mol Immunol Review 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. Pergamon Press 2010-06 /pmc/articles/PMC2874839/ /pubmed/20413159 http://dx.doi.org/10.1016/j.molimm.2010.02.020 Text en © 2010 Elsevier Ltd. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Review
Scott, Nathan
Qazi, Omar
Wright, Michael J.
Fairweather, Neil F.
Deonarain, Mahendra P.
Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title_full Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title_fullStr Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title_full_unstemmed Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title_short Characterisation of a panel of anti-tetanus toxin single-chain Fvs reveals cooperative binding
title_sort characterisation of a panel of anti-tetanus toxin single-chain fvs reveals cooperative binding
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2874839/
https://www.ncbi.nlm.nih.gov/pubmed/20413159
http://dx.doi.org/10.1016/j.molimm.2010.02.020
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