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Title: Generation of Affimer reagents for targeting the IL-7/IL-7R signalling
Author: Perez Witzke, Daniel Alberto
ISNI:       0000 0004 8501 0223
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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Interleukin (IL)-7 is a cytokine involved in the pathogenesis of certain haematological malignancies and several autoimmune diseases. IL-7 bind to the IL-7R, an heterodimer constituted by IL-7Ra and the common-g chains, expressed by CD4 T-cells and induces a survival signal. The IL-7/IL-7R signalling axis is a validated therapeutic target using antibodies directed to IL-7Ra in animal models, reducing the disease symptoms and/or preventing the disease. Moreover, an anti-IL7Ra antibody has been tested in humans showing a blockade of IL-7 signalling without impacting the physiological numbers/levels of lymphocytes. However, no anti-IL7Ra antibody has been approved for human therapeutic. Affimers reagents are small and stable artificial proteins which are an alternative to antibodies. Affimers bind with nanomolar affinities to human proteins and are commonly used for blocking protein-protein interactions. The aim of this work is to generate Affimer reagents that recognise the IL-7Ra and inhibit the IL-7 signalling cascade, as an alternative to antibodies. Affimer libraries were interrogated by phage display using glycosylated IL-7Ra ectodomains. After three panning rounds, 23 Affimers were raised (shown by phage ELISA and DNA sequencing). Affimer binding to IL-7Ra was observed by pull-down (soluble IL-7Ra; n=17) and flow cytometry (membrane IL-7Ra; n=6) assays. Several Affimers (n=10) inhibited the IL-7 signalling cascade in engineered cells using an IL-7/STAT5 reporter assay. A candidate (Affimer 42) inhibited STAT5 phosphorylation and IL-7-induced proliferation in human CD4 T-cells from two healthy donors. Altogether, these results indicate that Affimer 42 appears best, binding to soluble/membrane IL-7Ra and inhibiting the IL-7/IL-7R signalling cascade. This work demonstrates the potential of screening Affimer libraries for a cytokine receptor target, identifying specific cytokine antagonists. This might lead to the generation of new therapeutic agents for haematological malignancies and autoimmune diseases.
Supervisor: Ponchel, Frederique ; Tiede, Christian ; Robinson, James Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available