Hamilton, Stephen; (2001) Targeting melanoma using specific antibody fragments. Doctoral thesis (M.D), UCL (University College London).

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Abstract

Targeting tumours for diagnostic or therapeutic purposes using monoclonal antibodies or their fragments has shown great promise. However, the optimal characteristics of molecules for such interventions have yet to be defined. Melanoma presents an increasing clinical burden and a clear need for improved diagnostic and therapeutic strategies is evident. Numerous anti-melanoma monoclonal antibodies have been developed. Unfortunately, these molecules have failed to reach routine clinical use. In this thesis, the in vitro and in vivo characteristics of a series of anti-melanoma single chain antibody fragments (scFvs) targeting high molecular weight melanoma- associated antigen (HMW-MAA) are investigated. Attempts are made to optimise the production and purification processes of the recombinant antibody fragments. V- domain-swapped and chain-shuffled affinity variants are produced by PCR and antibody phage display techniques to allow study of the effect of these modifications in vitro and in vivo in a murine model. The coadministration of cationic amino acids such as lysine has been shown to reduce renal accumulation of larger tumour-targeting antibody fragments and the efficacy of this strategy with scFv is investigated. These experiments show the V-domain-swapping of the scFv studied increases protein yield by 50% without hindering tumour targeting in vitro or in vivo. Immunoaffinity purification similarly increases protein yield by 50%. Problematic scFv aggregation is noted at higher concentrations and appears independent of buffer conditions. However, the inclusion of imidazole in the storage buffer solution increases scFv solubility considerably. Increasing affinity by means of chain shuffling results in a clear increase in tumour targeting efficacy in vivo and it is likely that further increase in affinity will, in this model, result in further benefit. Coadministration of L-lysine reduces renal scFv accumulation 18 hours post injection by over 80% and could be employed to clarify imaging studies or reduce renal toxicity associated with scFv-derived therapies.

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