title: Trimolecular complex formation of IgE, Fc epsilon RI, and a recombinant nonanaphylactic single-chain antibody fragment with high affinity for IgE.
abstract: IgE is a central molecule in allergic disease. We have isolated cDNAs coding for the heavy and light chains of a murine mAb specific to human IgE and expressed a recombinant single-chain variable fragment (ScFv) derived thereof in Escherichia coli. The purified recombinant ScFv has a molecular mass of 28 kDa as measured by mass spectrometry and shows a beta-sheet fold as determined by circular dichroism. In biosensor-based studies it was demonstrated that the ScFv rapidly and stably binds to human IgE with an affinity of K(D) of 1.52 x 10(-10) M, which is almost as high as the affinity of IgE for FcepsilonRI, and that the ScFv is able to recognize FcepsilonRI-bound IgE and to prevent IgE binding to FcepsilonRI. The ScFv reacts specifically with IgE but not with other isotypes, allows the measurement of allergen-specific IgE in serum samples, and specifically targets cells that contain FcepsilonRI- or FcepsilonRII-bound IgE or that secrete IgE. Using negative-stain electron microscopy we demonstrated the formation of bimolecular complexes consisting of two ScFv molecules and one IgE and trimolecular complexes consisting of IgE, FcepsilonRI, and ScFv in which only one ScFv is able to bind to IgE. Accordingly, we found that the ScFv does not cross-link basophil-bound IgE and hence does not induce histamine release or activation of basophils as demonstrated by FACS analysis of CD203c expression and by histamine release experiments. In vivo skin testing confirmed the lack of allergenic activity of the ScFv. The recombinant ScFv may represent a universal tool for the IgE-targeted treatment of allergies.
authors.affiliation: Department of Pathophysiology, Division of Immunopathology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Lupinek
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Roux
authors.forename: Sylvia
authors.initials: S
authors.lastname: Laffer
authors.forename: Ingrid
authors.initials: I
authors.lastname: Rauter
authors.forename: Kavita
authors.initials: K
authors.lastname: Reginald
authors.forename: Michael
authors.initials: M
authors.lastname: Kneidinger
authors.forename: Katharina
authors.initials: K
authors.lastname: Blatt
authors.forename: Tanja
authors.initials: T
authors.lastname: Ball
authors.forename: Ines
authors.initials: I
authors.lastname: Pree
authors.forename: Beatrice
authors.initials: B
authors.lastname: Jahn-Schmid
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Allam
authors.forename: Natalija
authors.initials: N
authors.lastname: Novak
authors.forename: Anja
authors.initials: A
authors.lastname: Drescher
authors.forename: Franz
authors.initials: F
authors.lastname: Kricek
authors.forename: Peter
authors.initials: P
authors.lastname: Valent
authors.forename: Hakan
authors.initials: H
authors.lastname: Englund
authors.forename: Rudolf
authors.initials: R
authors.lastname: Valenta