Biochemistry and Pharmacology - Research Publications

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    The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities
    Dhagat, U ; Hercus, TR ; Broughton, SE ; Nero, TL ; Shing, KSCT ; Barry, EF ; Thomson, CA ; Bryson, S ; Pai, EF ; McClure, BJ ; Schrader, JW ; Lopez, AF ; Parker, MW (TAYLOR & FRANCIS INC, 2018-01-01)
    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.
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    EPO does not promote interaction between the erythropoietin and beta-common receptors (vol 8, 12457, 2018)
    Shing, KSCT ; Broughton, SE ; Nero, TL ; Gillinder, K ; Ilsley, MD ; Ramshaw, H ; Lopez, AF ; Griffin, MDW ; Parker, MW ; Perkins, AC ; Dhagat, U (NATURE PUBLISHING GROUP, 2019-05-21)
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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    EPO does not promote interaction between the erythropoietin and beta-common receptors
    Shing, KSCT ; Broughton, SE ; Nero, TL ; Gillinder, K ; Ilsley, MD ; Ramshaw, H ; Lopez, AF ; Griffin, MDW ; Parker, MW ; Perkins, AC ; Dhagat, U (NATURE PUBLISHING GROUP, 2018-08-20)
    A direct interaction between the erythropoietin (EPOR) and the beta-common (βc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and βc receptor in tissue protection while others have shown no involvement of the βc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the βc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and βc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the βc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the βc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the βc receptor do not directly interact and that the IRR is not involved in anaemic stress.
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    CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common beta chain of the IL-3, GM-CSF and IL-5 receptors
    Panousis, C ; Dhagat, U ; Edwards, KM ; Rayzman, V ; Hardy, MP ; Braley, H ; Gauvreau, GM ; Hercus, TR ; Smith, S ; Sehmi, R ; McMillan, L ; Dottore, M ; McClure, BJ ; Fabri, LJ ; Vairo, G ; Lopez, AF ; Parker, MW ; Nash, AD ; Wilson, NJ ; Wilson, MJ ; Owczarek, CM (TAYLOR & FRANCIS INC, 2016-04-02)
    The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).
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    A dual role for the N-terminal domain of the IL-3 receptor in cell signalling
    Broughton, SE ; Hercus, TR ; Nero, TL ; Kan, WL ; Barry, EF ; Dottore, M ; Shing, KSCT ; Morton, CJ ; Dhagat, U ; Hardy, MP ; Wilson, NJ ; Downton, MT ; Schieber, C ; Hughes, TP ; Lopez, AF ; Parker, MW (NATURE PUBLISHING GROUP, 2018-01-26)
    The interleukin-3 (IL-3) receptor is a cell-surface heterodimer that links the haemopoietic, vascular and immune systems and is overexpressed in acute and chronic myeloid leukaemia progenitor cells. It belongs to the type I cytokine receptor family in which the α-subunits consist of two fibronectin III-like domains that bind cytokine, and a third, evolutionarily unrelated and topologically conserved, N-terminal domain (NTD) with unknown function. Here we show by crystallography that, while the NTD of IL3Rα is highly mobile in the presence of IL-3, it becomes surprisingly rigid in the presence of IL-3 K116W. Mutagenesis, biochemical and functional studies show that the NTD of IL3Rα regulates IL-3 binding and signalling and reveal an unexpected role in preventing spontaneous receptor dimerisation. Our work identifies a dual role for the NTD in this cytokine receptor family, protecting against inappropriate signalling and dynamically regulating cytokine receptor binding and function.