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Trimolecular Complex Formation of IgE, FcRI, and a Recombinant Nonanaphylactic Single-Chain Antibody Fragment with High Affinity for IgE ¹

Christian Lupinek^*, Kenneth H. Roux, Sylvia Laffer^*, Ingrid Rauter^*, Kavita Reginald^*, Michael Kneidinger, Katharina Blatt, Tanja Ball^*, Ines Pree, Beatrice Jahn-Schmid^*, Jean-Pierre Allam^¶, Natalija Novak^¶, Anja Drescher^||, Franz Kricek^#, Peter Valent, Hakan Englund^** and Rudolf Valenta^2,*

^* Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna, Austria; Department of Biological Science, Florida State University, Tallahassee, FL 32306; Division of Hematology and Hemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria; Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna, Vienna, Austria; ^¶ Department of Dermatology and Allergy, University of Bonn Medical Center, Bonn, Germany; ^|| GE Healthcare Biacore Products, Freiburg, Germany; ^# Novartis Research Center, Vienna, Austria; and ^** Phadia, Uppsala, Sweden

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 β-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 FcRI, and that the ScFv is able to recognize FcRI-bound IgE and to prevent IgE binding to FcRI. 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 FcRI- or FcRII-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, FcRI, 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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by the SFB program F18 and Projects F1815 and F1818 of the Austrian Science Fund.

² Address correspondence and reprint requests to Dr. Rudolf Valenta, Division of Immunopathology, Department of Pathophysiology, Center for Physiology and Pathophysiology, Medical University of Vienna, Vienna General Hospital, Waehringer Guertel 18–20, 3Q, 1090 Vienna, Austria. E-mail address: rudolf.valenta{at}meduniwien.ac.at

³ Abbreviations used in this paper: DC, dendritic cell; HSA, human serum albumin; MFI, mean fluorescence intensity; RU, resonance unit; ScFv, single-chain variable fragment; SPR, surface plasmon resonance.