Bhoday, Rajinder; (2002) Application of antibody engineering techniques to develop antibodies for detection of inflammatory and malignant disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

It is proposed that antibody engineering techniques can be used to generate clinically useful antibodies. These techniques provide a means of creating new antibodies of defined specificities and tailoring their properties as well as those of hybridoma-derived monoclonal antibodies. In this thesis the potential of antibody engineering to produce reagents for the clinic has been explored. Three approaches have been taken. First, construction of Fab and scFv antibody fragments from an established murine hybridoma. In this case the target antigen was NCA-95, a marker of inflammation. Functional expression of fragments could not be demonstrated so phage display technology with a murine scFv repertoire was successfully applied with this antigen. A second approach, using synthetic carbohydrates, was to select antibodies against tumour associated glycoproteins. Naive human scFv and Fab repertoires and a murine scFv repertoire were tested. A single human Fab fragment with binding to STn, a MUC1 tumour associated carbohydrate antigen, was isolated. Finally, antibody engineering techniques were applied to rescue the binding regions of a human hybridoma which reacted with a (non-defined) tumour antigen on human colorectal cells. Nine different scFv fragments were generated. These were screened by immunohistochemistry against a range of normal and tumour human tissues. Specific reactivity was observed against a number of tumours including carcinoma of the colon, breast and ovary. The work presented in this thesis has demonstrated the feasibility of isolating antibody fragments, against markers of inflammation and malignancy, by selection from antibody repertoires using phage display and rescue of V-genes from hybridomas.

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