|Abstract: ||Chelating Recombinant Antibodies (CRAbs) are a form of high affinity tandem single chain antibody (scFv) with two component scFvs which target non‐overlapping epitopes on the same antigen molecule. The optimised inter‐scFv linker allows simultaneous engagement of the scFvs resulting in a synergistic improvement in affinity. High affinity is a desirable characteristic in therapeutic antibodies since it strongly correlates with improved potency. Building upon previous work, this thesis describes the development of a phage‐display based approach toward isolating CRAbs with both the optimal scFv pairing and inter‐scFv linker using tetanus toxin as a model antigen. This would circumvent the need for structural data and complement the traditional approaches used to enhance antibody affinity.
Fifteen scFvs specific for the C‐terminal sub‐domain of the tetanus toxin heavy chain (TT‐Hc) were characterised in terms of overall and soluble expression levels, sequence and bioinformatics features, phage‐display propensity and toxin neutralisation potency. Seven clones were identified for further analysis by affinity measurements, oligomerisation analysis and quantitative toxin neutralisation capacity. These characteristics varied significantly between clones including the affinity (KD) which varied, from 10nM to over 1000nM. ScFv characterisation revealed a strong positive correlation between phage display propensity and overall expression levels as well as a strong correlation between clone affinity and potency. Multiple scFv pairings hypothetically capable of chelation were identified by competition binding analysis and many of these clones exhibited cooperative binding effects when added in combination to TT‐Hc.
A tandem‐scFv library was constructed linking seven of the characterised scFv clones (C1, C2, C4, J2, J4, N4 and N5) in all 49 possible combinations and permutations each with 7 inter‐scFv linker lengths. The inter‐scFv linkers harboured some randomised codons giving a total diversity of 5x107 clones. The library contained a mixture of hypothetically chelating and competing scFv pairings. Carefully optimised affinity driven phage display which included off‐rate selection enriched the anti‐TT‐Hc tandem‐scFv library to up to 90% chelating clones from under 50% after just two‐rounds of selection with a single clone, C4‐N4, dominating around 30% of the output. This clone was classified as hypothetically chelating from the scFv competition binding experiments. Despite some modest biases in the constructed library, the results clearly indicated that phage display can enrich for an optimal and chelating scFv pairing. This approach for isolating CRAbs will allow for the facile isolation of very high affinity antibodies against any target of interest given a sufficiently diverse panel of scFvs circumventing the need for prior scFv characterisation or elucidation of antibody‐antigen structural data.|