| Summary: | The Wnt signalling pathway is of central importance in embryogenesis and adult tissue homeostasis, as well as numerous cancers and other diseases. Despite the developmental and potential therapeutic significance of this pathway many aspects of Wnt signalling, including the role of the master transcriptional co-activator, J3-catenin, remain poorly understood. This thesis describes the derivation of VHH intracellular antibodies, with specificity for β-catenin, and their application to the study of the canonical Wnt signalling pathway. Intracellular antibodies, intrabodies, are engineered antibody fragments capable of binding and modifying the function of intracellular proteins, and represent valuable tools for the study of intracellular interactions. The aim of the work was to further the understanding of the regulation of β-catenin, and potentially to use the intrabodies as tools to identify sites on β-catenin for future therapeutic intervention. A diverse immune llama VIM phagemid library was constructed, from which five antibodies with 3-6nM affinity for β-catenin were selected, and demonstrated function-modifying intracellular activity in a luciferase based Wnt signalling reporter bioassay. Control Ala-substituted CDR3 mutants were produced for each intrabody, and used to demonstrate the specific inhibition of β-catenin activity by the parent intrabodies. Further characterisation of intrabody LL3 demonstrated that it bound endogenous 13- catenin specifically and inhibited the Wnt signalling pathway downstream of the destruction complex. The binding location of LL3 on β-catenin Was shown to reside between residues 164 and 390, which overlaps with the binding domains of several transcription factors on β-catenin. This work is thought to represent the first report of functional inhibition of β-catenin-mediated co-activation of transcription by an intrabody, and further study of the method of action of function-modifying VHH intrabodies to β-catenin may enable the identification of novel sites for therapeutic intervention on the Wnt signalling pathway.
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