Functional inhibition of β-catenin-mediated Wnt signalling by intracellular VHH antibodies

The Wnt signalling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signalling, including the control of the master transcriptional co-activator β-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against β-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signalling HEK293 reporter bioassay. Further characterisation of one such VHH (named LL3) confirmed that it bound endogenous β-catenin, and that it inhibited the Wnt signalling pathway downstream of the destruction complex, whilst production of a control Ala-substituted CDR3 mutant demonstrated that the inhibition of β-catenin activity by the parent.