Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

High affinity targeting of CD23 inhibits IgE synthesis in human B cells.

The low-affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti-CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti-CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up-regulation of CD23 in human peripheral B cells were induced by IL-4 and anti-CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real-time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico-molar range. Anti-CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti-CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein

Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release.

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Natural anti-FcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria.

IgG autoantibodies against the alpha-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the alpha-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti-alpha-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

DARPins against a functional IgE epitope

The monoclonal anti-IgE antibody omalizumab (Xolair is mostly used for the treatment of severe allergic asthma. However, the requirement of high doses and suboptimal cost-effectiveness limits the use of the treatment. Here we propose to use a new drug format based on non-immunoglobulin structures, potentially offering increased clinical efficacy while being more cost-effective. For this purpose, DARPins (designed ankyrin repeat proteins) against the constant heavy chain region of IgE have been isolated. DARPins were binding to IgE with high specificity and affinities in the low nanomolar range. Selected DARPins antagonized the interaction between IgE and its high-affinity receptor in inhibition assays. Furthermore, anti-IgE DARPins were shown to inhibit proinflammatory mediator release from rat basophilic leukemia cells expressing human high-affinity IgE receptors with higher efficacy than the monoclonal anti-IgE antibody omalizumab. DARPins may thus represent promising future drug candidates for the treatment of allergy.

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.

An engineered disulfide bond reversibly traps the IgE-Fc3-4 in a closed, nonreceptor binding conformation

IgE antibodies interact with the high affinity IgE Fc receptor, FcεRI, and activate inflammatory pathways associated with the allergic response. The IgE-Fc region, comprising the C-terminal domains of the IgE heavy chain, binds FcεRI and can adopt different conformations ranging from a closed form incompatible with receptor binding to an open, receptor-bound state. A number of intermediate states are also observed in different IgE-Fc crystal forms. To further explore this apparent IgE-Fc conformational flexibility and to potentially trap a closed, inactive state, we generated a series of disulfide bond mutants. Here we describe the structure and biochemical properties of an IgE-Fc mutant that is trapped in the closed, non-receptor binding state via an engineered disulfide at residue 335 (Cys-335). Reduction of the disulfide at Cys-335 restores the ability of IgE-Fc to bind to its high affinity receptor, FcεRIα. The structure of the Cys-335 mutant shows that its conformation is within the range of previously observed, closed form IgE-Fc structures and that it retains the hydrophobic pocket found in the hinge region of the closed conformation. Locking the IgE-Fc into the closed state with the Cys-335 mutation does not affect binding of two other IgE-Fc ligands, omalizumab and DARPin E2_79, demonstrating selective blocking of the high affinity receptor binding.

Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid

The vitamin A metabolite retinoic acid (RA) plays a fundamental role in cellular functions by activating nuclear receptors. Retinaldehyde dehydrogenase-II (RALDH2) creates localized RA gradients needed for proper embryonic development, but very little is known regarding its regulated expression in adults. Using a human ex vivo model of allergic inflammation by coincubating IgE receptor-activated mast cells (MCs) with blood basophils, we observed prominent induction of a protein that was identified as RALDH2 by mass spectroscopy. RALDH2 was selectively induced in basophils by MC-derived interleukin-3 (IL-3) involving PI3-kinase and NF-kappaB pathways. Importantly, neither constitutive nor inducible RALDH2 expression was detectable in any other human myeloid or lymphoid leukocyte, including dendritic cells. RA generated by RALDH2 in basophils modulates IL-3-induced gene expression in an autocrine manner, providing positive (CD25) as well as negative (granzyme B) regulation. It also acts in a paracrine fashion on T-helper cells promoting the expression of CD38 and alpha4/beta7 integrins. Furthermore, RA derived from IL-3-activated basophils provides a novel mechanism of Th2 polarization. Thus, RA must be viewed as a tightly controlled basophil-derived mediator with a high potential for regulating diverse functions of immune and resident cells in allergic diseases and other Th2-type immune responses.

The basophil activation test in immediate-type drug allergy

Diagnosis of drug allergy involves first the recognition of sometimes unusual symptoms as drug allergy and, second, the identification of the eliciting drug. This is an often difficult task, as the clinical picture and underlying pathomechanisms are heterogeneous. In clinical routine, physicians frequently have to rely upon a suggestive history and eventual provocation tests, both having their specific limitations. For this reason both in vivo (skin tests) and in vitro tests are investigated intensively as tools to identify the disease-eliciting drug. One of the tests evaluated in drug allergy is the basophil activation test (BAT). Basophils with their high-affinity IgE receptors are easily accessible and therefore can be used as indicator cells for IgE-mediated reactions. Upon allergen challenge and cross-linking of membrane-bound IgE antibodies (via Fc-epsilon-RI) basophils up-regulate certain activation markers on their surface such as CD63 and CD203c, as well as intracellular markers (eg, phosphorylated p38MAPK). In BAT, these alterations can be detected rapidly on a single-cell basis by multicolor flow cytometry using specific monoclonal antibodies. Combining this technique with in vitro passive sensitization of donor basophils with patients' serum, one can prove the IgE dependence of a drug reaction. This article summarizes the authors' current experience with the BAT in the diagnostic management of immediate-type drug allergy mediated by drug-specific IgE antibodies.

Ring size of somatostatin analogues (ODT-8) modulates receptor selectivity and binding affinity

The synthesis, biological testing, and NMR studies of several analogues of H-c[Cys (3)-Phe (6)-Phe (7)-DTrp (8)-Lys (9)-Thr (10)-Phe (11)-Cys (14)]-OH (ODT-8, a pan-somatostatin analogue, 1) have been performed to assess the effect of changing the stereochemistry and the number of atoms in the disulfide bridge on binding affinity. Cysteine at positions 3 and/or 14 (somatostatin numbering) were/was substituted with d-cysteine, norcysteine, D-norcysteine, homocysteine, and/or D-homocysteine. The 3D structure analysis of selected partially selective, bioactive analogues (3, 18, 19, and 21) was carried out in dimethylsulfoxide. Interestingly and not unexpectedly, the 3D structures of these analogues comprised the pharmacophore for which the analogues had the highest binding affinities (i.e., sst 4 in all cases).