Development of specific antibody fragments for the detection and treatment of melanoma

The incidence of Malignant Melanoma continues to rise. Melanoma is potentially curable by surgery if caught early. However once the disease has spread the response to adjuvant therapy is poor. The combination of high prevalence and therapeutic resistance has led to an interest in other methods for detecting melanoma at earlier time points, when treatments may affect patient outcome and survival. Immunoscintigraphy is the use of radiolabelled tumour-specific antibodies to detect disease. Previous work has centred on the use of the whole monoclonal antibody LHM2, against the high molecular weight melanoma-associated antigen (HMW-MAA). Single chain Fv fragments (scFv) have been developed from this molecule. In this thesis optimal conditions for storage and stability of the scFvs were explored. Some of the scFvs were selected by rounds of phage display. The clones seemed to have better properties with each round of selection on phage, and the role of Darwinian "selection of the fittest" was explored. Some scFvs were genetically modified by the addition of cysteine residues to form multimeric constructs for improved tumour targeting. We were unable to show improved tumour targeting in vivo when the multimers were compared to their monomeric equivalents, and possible reasons for this are discussed. One of the antibody fragments RAFT3 was modified for therapy by conjugation with Staphyllococcal protein A. This clone was shown to retarget IgG and complement to melanoma in vitro. In vivo the construct behaved more like the larger LHM2 antibody than the parent (RAFT3 scFv), consistent with IgG recruitment in the murine model. Finally the expression of HMW-MAA was studied in a number of melanocytic lesions on paraffin-embedded slides, to determine the role of this antigen for routine immunohistochemical studies. The antibody LHM2 was able to stain all melanomas as reliably as S100 protein (the standard marker highly expressed in melanomas). It was able to detect desmoplastic melanoma in three specimens. HMW-MAA expression in primary and metastatic melanomas was also explored, and did not appear to be a prognostic marker for cutaneous melanoma