title: Improved FcγRIIb targeting functionally translates into enhanced inhibition of basophil activation.
abstract: BACKGROUND
  Mast cells and basophils express the high-affinity IgE receptor, FcεRI, as well as the low-affinity IgG receptor, FcγRIIb. While FcεRI is responsible for IgE-dependent degranulation upon coaggregation with allergens, FcγRIIb has been shown to downregulate degranulation through cross-linking with FcεRI. A previously developed fusion protein consisting of an anti-IgE DARPin linked to the human IgG1-Fc part (DE53-Fc) has been shown to simultaneously target FcεRI and FcγRIIb with low affinity and to thereby prevent basophil activation. The affinity of a ligand for its receptor is known to be critical for the functional consequences of the binding. So we generated two mutated DE53-Fc molecules with either an improved (DE53-Fc mut+) or a reduced (DE53-Fc mut-) binding to FcγRIIb and assessed their potential to inhibit IgE-dependent basophil activation.
  
  
  METHODS
  DE53-Fc was modified by introducing single site-directed point mutations in the Fc part. The mutated constructs were used to assess kinetic parameters as well as the inhibitory capacity on basophil activation and the production of leukotriene C4 (LTC4) and IL-13.
  
  
  RESULTS
  DE53-Fc mut+ showed increased affinity for FcγRIIb as well as an enhanced potential to inhibit IgG1 binding to FcγRIIb, resulting in improved efficacy in functional assays. Furthermore, DE53-Fc mut+ decreased de novo-synthesized LTC4 as well as the cytokine IL-13, suggesting that it might be an inhibitor of the allergic late-phase reaction.
  
  
  CONCLUSIONS
  Our data suggest that improved binding to FcγRIIb at constant low-affinity binding to IgE leads to more efficient coaggregation of FcεRI-FcγRIIb and results in the enhanced inhibition of basophil activation.
authors.affiliation: Institute of Immunology, University of Bern, Inselspital, Bern, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Buschor
authors.forename: Alexander
authors.initials: A
authors.lastname: Eggel
authors.forename: Fabian
authors.initials: F
authors.lastname: Zellweger
authors.forename: Beda M
authors.initials: BM
authors.lastname: Stadler
authors.forename: Monique
authors.initials: M
authors.lastname: Vogel
mesh_terms: D000915:Antibody Affinity; D001491:Basophils / Q000276:immunology*; D015550:Cell Degranulation / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D017455:Receptors, IgE / Q000276:immunology; D017452:Receptors, IgG / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000494:pharmacology*
chemical_list: C577317:DARPin E2 79; C499173:FCGR2B protein, human; D017455:Receptors, IgE; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D007073:Immunoglobulin E