title: A novel bispecific DARPin targeting FcγRIIB and FcεRI-bound IgE inhibits allergic responses.
abstract: BACKGROUND
  Binding of allergen-specific IgE to its high-affinity receptor FcεRI on basophils and mast cells is a central event in the development of allergies. Exposure of these cells to allergens induces the release of soluble mediators causing allergic symptoms. The inhibitory low-affinity IgG Fc-receptor FcγRIIB is co-expressed on allergic effector cells and has been implicated in negative regulation of immediate hypersensitivity responses. In order to harvest the inhibitory function of this receptor, we aimed to select specific binders against FcγRIIB and to generate a bispecific molecule simultaneously targeting FcγRIIB and FcεRI-bound IgE on the surface of allergic effector cells.
  
  
  METHODS
  We selected FcγRIIB-specific binding molecules from a library of designed ankyrin repeat proteins using ribosome display technology. The bispecific binding modality was generated by molecular cloning and recombinant protein expression. We determined binding characteristics on molecular and cellular levels using SPR, ELISA, and flow cytometry. The inhibitory potential of the newly described molecules was assessed in different cellular degranulation assays ex vivo and in a mouse model of passive systemic anaphylaxis.
  
  
  RESULTS
  We demonstrate that the selected DARPin®proteins recognize FcγRIIB with high affinity. Furthermore, the bispecific binding protein successfully interferes with allergen-induced cell degranulation and efficiently inhibits systemic anaphylaxis in vivo. Mechanistically, we report that FcγRIIB-mediated inhibition of effector cell activation requires direct ligation to an activating FcεRI receptor.
  
  
  CONCLUSIONS
  The described bispecific DARPin protein has the ability to co-ligate FcγRIIB with FcεRI-bound IgE on allergic effector cells and represents an efficient dual-modality to interfere with allergic hypersensitivity reactions.
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern, Switzerland.
authors.forename: F
authors.initials: F
authors.lastname: Zellweger
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Gasser
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern, Switzerland.
authors.forename: D
authors.initials: D
authors.lastname: Brigger
authors.affiliation: Institute of Immunology, University of Bern, Bern, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Buschor
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Vogel
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland.|Department of Rheumatology, Immunology and Allergology, University Hospital Bern, Bern, Switzerland.
authors.forename: A
authors.identifier: http://orcid.org/0000-0001-8746-3339
authors.initials: A
authors.lastname: Eggel
mesh_terms: D000818:Animals; D001491:Basophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D015550:Cell Degranulation / Q000187:drug effects / Q000276:immunology; D003429:Cross Reactions / Q000276:immunology; D004195:Disease Models, Animal; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology* / Q000378:metabolism*; D007073:Immunoglobulin E / Q000276:immunology* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding / Q000187:drug effects; D017455:Receptors, IgE / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C577317:DARPin E2 79; C499173:FCGR2B protein, human; D007074:Immunoglobulin G; D017455:Receptors, IgE; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E
keywords: FcγRIIb; FcεRIα; IgE; allergy; bispecific DARPin