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 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.
Scott
Nathan
N
Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, Exhibition Road, London, SW7 2AZ, UK.
Qazi
Omar
O
Wright
Michael J
MJ
Fairweather
Neil F
NF
Deonarain
Mahendra P
MP
D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology*; D001667:Binding, Competitive; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology; D013744:Tetanus Toxin / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; D013744:Tetanus Toxin