Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.     

Monoclonal antibodies defining epitopes on human IgE.

Twelve monoclonal antibodies (mAb) were isolated that bound to six clusters of epitopes on the constant region of the epsilon chain of human IgE. Four of the mAb bound to the C epsilon 1 or early C epsilon 2 regions; three of these bound to the IgE myeloma protein PS and to serum IgE but not to the IgE myeloma protein ND. These mAb probably recognize an allotypic marker. Another mAb reacted with heat-denatured, but not native IgE. Four of the mAb failed to release histamine; the epitopes recognized by these mAb are in the C epsilon 1, C epsilon 2 and C epsilon 3-4 regions of IgE. Three of these non-histamine releasing mAb did not bind to IgE on the basophil surface. These mAb recognize epitopes in C epsilon 2 and C epsilon 3-4 that are not accessible when IgE is bound to its receptor. Four mAb inhibited IgE binding to basophils; two of these did not release histamine, and two others that bind to epitopes in the C epsilon 2-4 domain, released histamine and therefore blocked IgE binding by steric hindrance. Inhibition of IgE binding by different mAb suggest that the Fc epsilon RI and Fc epsilon RII bind to partly overlapping regions of the IgE molecule although the sites do not appear to be identical. A number of sites on C epsilon 1 and C epsilon 3-4 were accessible when IgE is bound to its basophil receptor. The data support the concept that only part of the Fc portion of IgE is hidden in the receptor and that portions of C epsilon 1-4 are accessible on the cell surface. These mAb should be useful in determining the domains of IgE that are critical for its biological activity.     

Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE

In an attempt to identify the site on IgE which binds with high affinity to the Fc? receptor (Fc?R) on mast cells, we established monoclonal anti-IgE antibodies (mAb) by fusion of myeloma cells with rat splenocytes immunized with purified murine IgE mAb. Six individual mAb were found to react with various IgE mAb of different specificities and not with immunoglobulins of other classes. Three different clusters of epitopes on the Fc? portion could be detected by antibody competition studies. These antigenic determinants were expressed on the Fc? portion and required the two heavy chains in their native conformation. Two groups of mAb and their Fab′ fragments completely inhibited the binding of ¹²⁵I-labeled IgE to rat basophilic leukemia cells (RBL), and one mAb inhibited the specific IgE binding only partially (55–65%). Likewise, the Fab′ fragments of the purified mAb inhibited the antigen-mediated, IgE-dependent, serotonin release of RBL cells. These in vitro findings were confirmed by in vivo experiments, which demonstrated that the anti-IgE mAb could specifically block passive cutaneous anaphylaxis reaction when injected i.d., before challenging with the antigen. The differences in blocking reactivity of the various anti-IgE mAb are discussed in view of heterogeneity in the IgE-Fc?R interaction.     

Soluble IgE receptors--elements of the IgE network

Soluble isoforms of three human IgE Fc receptors, namely Fc?RI, Fc?RII, and galectin-3, can be found in serum. These soluble IgE receptors are a diverse family of proteins unified by the characteristic of interacting with IgE in the extracellular matrix. A truncated form of the alpha-chain of Fc?RI, the high affinity IgE receptor, has recently been described as a soluble isoform (sFc?RI). Multiple soluble isoforms of CD23 (sCD23), the low affinity IgE receptor also known as Fc?RII, are generated via different mechanisms of extracellular and intracellular proteolysis. The second low affinity IgE receptor, galectin-3, only exists as a secretory protein. We here discuss the physiological roles of these three soluble IgE receptors as elements of the human IgE network. Additionally, we review the potential and current use of sFc?RI, sCD23, and galectin-3 as biomarkers in human disease.     

Anti-anti-IgE idiotypic antibodies mimic IgE in their binding to the Fc epsilon receptor

The binding site of some anti-idiotypic antibodies (anti-Id) can appear as a structural image of the antigen and as such may mimic its biologic activity. We raised anti-anti-IgE antibodies in an attempt to obtain anti-Id capable of interacting with the Fc epsilon receptor (Fc epsilon R). Guinea pigs were immunized with purified murine monoclonal antibodies (mAb) that had been found to react with epitopes closely related to the site on the IgE molecule which is recognized by the Fc epsilon R. After only two injections, we could detect in the immune sera anti-Id that inhibited the binding of IgE to the anti-IgE mAb used as immunogens. However, only after 10 immunizations over a period of about 6 months could we detect antibodies that competed efficiently with the binding of IgE to rat basophilic leukemia (RBL) cells. The "IgE-like" anti-Id could be affinity purified from immunosorbents made of the anti-IgE mAb. F(ab')2 and Fab' fragments were as effective inhibitors of IgE binding as the intact anti-anti-Id antibodies. Some of the anti-Id caused RBL degranulation and all of them, like IgE, inhibited the binding of specific anti-Fc epsilon R mAb to RBL cells. In summary, by hyperimmunization with anti-IgE mAb we could obtain anti-Id whose antigen-binding site is recognized by the mast cell receptor specific to the Fc portion of IgE.     

Detection of mouse IgE by CELISA

The detection and quantitation of mouse IgE is usually impaired by the difficulty to obtain reliable antibody reagents which are fully specific for the epsilon chain - and reactive enough - to be used in an enzyme-linked immunosorbent assay (ELISA). An ELISA on cells (CELISA) was developed for the detection of mouse IgE, using rat basophilic leukemia (RBL) cells. It is based on the high affinity of the receptors for the Fc of IgE (Fc epsilon R) displayed on the surface of the RBL cells. Since the epsilon chain specific recognition is achieved by the biological receptor of IgE, the detection of cell-bound IgE does not need the use of epsilon chain specific antibodies. Instead, one can use any enzyme-coupled antibody capable to recognize the IgE through its light-chain epitopes. Interestingly, when the IgE bound to the RBL cells has a known specificity, it can be detected through its paratopes using the cognate antigen coupled to an enzyme.     

Membrane-bound IgE on B cells is increased during Clonorchis sinensis infection

A high level of serum IgE is a hallmark of helminthic disease. Secretory IgE can bind FcεRI or FcεRII/CD23. The combination of IgE and FcεRI, a high-affinity interaction, has long received attention and is believed to facilitate helminth control, while the properties of CD23-bound IgE have long been unexplored. Here, we established a Clonorchis sinensis (C. sinensis) infection model with different mouse strains and investigated membrane-bound IgE on B cells during infection. We show that after infection, the increase in CD23 expression on B cells was obvious, even in relatively resistant C57BL/6 mice, as well as in susceptible BALB/c and FVB mice. Although the serum IgE amount was lower in C57BL/6 mice than in BALB/c and FVB mice, the level of IgE binding to peripheral B cells was also elevated. Additionally, the IgE on B cells was soon undetectable in vitro due to dissociable binding. The results of the present study demonstrate the dramatic increase in CD23-bound IgE on B cells after C. sinensis infection. The significance of CD23-bound IgE in Ag transport and presentation has gained consideration in allergy development for its potential ability to promote the Th2 response. Therefore, even though the association of IgE and CD23 is not as substantial as that of IgE and FcεRI, membrane-bound IgE on B cells may be worth further study regarding clonorchiasis and other parasitic infections.     

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcɛRI with FcγRIIb

Background IgE binds to mast cells and basophils via its high‐affinity receptor, Fc?RI, and cross‐linking of Fc?RI‐bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross‐linking of Fc?RI with FcγRIIb, an ITIM‐containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti‐idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to Fc?RI‐bound IgE, via its Fab regions, and the negative regulatory receptor, FcγRIIb, via its Fc region. Objective To assess the ability of human 2G10 to inhibit anti‐IgE and allergen‐driven basophil degranulation through cross‐linking of Fc?RI‐bound IgE with FcγRIIb. Methods 2G10 was assessed for its ability to bind to FcγRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti‐IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL‐4 expression intracellularly, using flow cytometery. Results Human 2G10 was able to bind to FcγRIIb on transfected cells and on purified basophils, and induce a dose‐dependent inhibition of both anti‐IgE and Der p 1‐driven degranulation of basophils. Conclusion The inhibition of basophil degranulation by the human IgG1 anti‐idiotype 2G10 highlights the therapeutic potential of IgE‐reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcγRIIb.     

The human monocyte-like cell line THP-1 expresses Fc gamma RI and Fc gamma RII.

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized Fc gamma R expression on this cell line by flow cytometry, radiolabeled IgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-Fc gamma RI, II, and III mAb, and a rabbit anti-Fc gamma RIII F(ab')2 demonstrated that only Fc gamma RI and Fc gamma RII are expressed by these cells. A panel of anti-Fc gamma RIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (Fc gamma RI) and of 42 to 53 kDa (Fc gamma RII). Fc gamma R expression was determined by binding of radioiodinated human IgG1 (to detect Fc gamma RI), mAb IV.3 (to detect Fc gamma RII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 x 10(-9) M) for IgG1 were found on THP-1 cells. Interferon-gamma (IFN gamma) upregulated Fc gamma RI expression by THP-1 cells 2.8-fold, whereas Fc gamma RI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-alpha (TNF alpha), and vitamin D3 had no effect on IgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFN gamma treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFN gamma, TNF alpha, PMA, or vitamin D3 had no effect on Fc gamma RII expression. That Fc gamma RI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human IgG1 as well as mouse IgG2a mAb to Fc gamma RII inhibited immune complex binding by 76 to 84%, whereas mouse IgG1 mAb to Fc gamma RII had minimal effect on immune complex binding. Fc gamma R expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.     

Quantitative and qualitative analysis of the Fc receptor for IgE (Fc epsilon RII) on human eosinophils

In order to characterize the Fc receptor for IgE (Fc epsilon RII) on human eosinophils, we have compared the binding of human IgE myeloma protein to that of a monoclonal antibody (mAb BB10) directed against a common antigenic determinant of the Fc epsilon RII present on eosinophils, platelets and macrophages. Scatchard analysis of the binding to human eosinophils of the BB10 mAb revealed a linear monophasic binding curve, with a binding affinity of 1.17 x 10(7) M-1 and a number of 10(5) binding sites per cell. Biochemical analysis of the human eosinophil Fc epsilon R, performed by immunosorbent chromatography with either BB10 mAb or IgE, showed under nonreducing conditions a major component of 200 kDa. Under reducing conditions, 3 peptide fragments were obtained, with molecular masses of 45-50, 23 and 15 kDa. Finally, comparative analysis suggested that the Fc epsilon RII of human eosinophils and of a human macrophage cell line (U937) are structurally related and differ from the high-affinity Fc epsilon RI present on basophilic granulocytes.     

Inhibition of human interleukin 4-induced IgE synthesis by a subset of anti-CD23/Fc epsilon RII monoclonal antibodies.

Specific monoclonal antibodies (mAb) directed against the CD23 antigen were used to study human interleukin 4 (hIL4)-induced IgE production by blood and tonsillar mononuclear cells. Both peripheral blood and tonsillar mononuclear cells stimulated by hIL4 expressed membrane CD23 as detected by the binding of all anti-CD23 mAb. Nevertheless, two sets of anti-CD23 mAb could be distinguished. The first set, including mAb 25, was able to decrease significantly hIL4-induced IgE synthesis by mononuclear cells. The second set, including EBVCS#1, did not affect hIL4-induced IgE synthesis. All the anti-CD23 mAb were able to bind specifically to a human B cell line expressing recombinant CD23. Inhibition experiments revealed that the two sets of anti-CD23 mAb did not recognize the same epitope on the CD23 antigen. In fact, all the anti-CD23 mAb, except EBVCS#1, were able to inhibit IgE binding to CD23 on RPMI 8866 cells. Moreover, the first set of antibodies, which decreased IgE production, was able to up-regulate membrane CD23 expression on hIL4-stimulated tonsillar mononuclear cells. Conversely, EBVCS#1, which had no effect on IgE production, did not affect hIL4-induced CD23 expression. These results indicate that CD23 plays a key role in human IgE synthesis.     

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI‐mediated basophil activation by a tumour‐specific IgE antibody to evaluate the risk of type I hypersensitivity

Background IgE antibodies, sequestered into tissues and retained locally by the high‐affinity IgE receptor, Fc?RI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross‐link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross‐link FRα‐MOv18‐IgE‐receptor‐Fc?RI complexes on basophils to cause type I hypersensitivity. Objective To assess the propensity for MOv18 used in a therapeutic setting to cause Fc?RI‐mediated type I hypersensitivity. Methods As validated readouts of the potential for MOv18 to cause Fc?RI‐mediated type I hypersensitivity we measured release of a granule‐stored mediator from a rat basophilic leukaemia cell line RBL SX‐38 stably transfected with human tetrameric (αβγ2) Fc?RI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. Results Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX‐38 cell degranulation. Exposure to FRα‐expressing ovarian tumour cells at target‐to‐effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). Conclusion and Clinical Relevance These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, Fc?RI‐mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre‐clinical studies. Cite this as: S. M. Rudman, D. H. Josephs, H. Cambrook, P. Karagiannis, A. E. Gilbert, T. Dodev, J. Hunt, A. Koers, A. Montes, L. Taams, S. Canevari, M. Figini, P. J. Blower, A. J. Beavil, C. F. Nicodemus, C. Corrigan, S. B. Kaye, F. O. Nestle, H. J. Gould, J. F. Spicer and S. N. Karagiannis, Clinical & Experimental Allergy, 2011 (41) 1400–1413.     

Aspergillus oryzae lectin induces anaphylactoid oedema and mast cell activation through its interaction with fucose of mast cell-bound non-specific IgE

We investigated whether Aspergillus oryzae lectin (AOL), a fucose-specific lectin, induces anaphylactoid reactions and mast cell activation. The injection of AOL into footpads of mice produced a dose-related acute paw oedema. The AOL-induced oedema was attenuated by predose of histamine H1 receptor blocker or pretreatment of the lectin with fucose before injection and was not observed in SCID and mast cell-deficient WBB6F1-W/Wv mice. These results suggested that the AOL-induced anaphylactoid reaction was mediated by histamine released from mast cells. In addition, the activation of mast cells was seemed to be induced by the crosslinking of IgE on the cell surface following the binding of AOL to fucose residues in IgE. Consistent with the in vivo results, AOL induced the degranulation of the rat mast cell line RBL2H3 sensitized with monoclonal IgE. As AOL induced the increase in intracellular Ca(2+) concentration of IgE-sensitized RBL2H3 cells as well as antigen stimulation, AOL could input signals from FcεRI. The degranulation of IgE-sensitized RBL2H3 cells by AOL was diminished by pretreatment of AOL with fucose. Defucosylated IgE did not induce degranulation of RBL2H3 cells in response to AOL stimulation, in spite of its ability to induce degranulation by antigen stimulation as intact IgE. These results indicated that AOL bound to fucose residue of IgE causing antigen-independent IgE-mediated mast cell activation and anaphylactoid reactions in vitro and in vivo, respectively. AOL bound to human IgE as well as to mouse IgE, suggesting the possible implication of AOL in the allergic response to Aspergillus oryzae in humans.     

Thy-1-mediated activation of rat basophilic leukemia cells does not require co-expression of the high-affinity IgE receptor

The glycosylphosphatidylinositol (GPI)-anchored protein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL-2H3. Antibody-mediated aggregation of Thy-1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (Fc?RI). To examine whether there is any relationship between Thy-1- and Fc?RI-mediated activation, we have isolated from mutagenized RBL-2H3 cells a variant cell line deficient in the expression of surface Fc?RI, and analyzed its ability to be activated by an antibody to Thy-1. Northern and immuno-blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for Fc?RI γ subunit. The cells did not respond by release of secretagogues and protein-tyrosine phosphorylation to IgE and antigen and anti-Fc?RI monoclonal antibody (mAb) but their response to anti-Thy-1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned Fc?RI γ subunit into the variant cells restored the surface expression of Fc?RI and responsiveness to both the antigen and anti-Fc?RI mAb but had no effect on responsiveness to anti-Thy-1 mAb. The combined data indicate that aggregation of surface Thy-1 glycoproteins activates a metabolic pathway which is independent of the presence of Fc?RI γ subunit and surface expression of Fc?RI.     

Inhibition of human cord blood-derived mast cell responses by anti-Fc epsilon RI mAb 15/1 versus anti-IgE Omalizumab

Aggregation of the alpha-chain of the high affinity IgE receptor (Fc epsilon RI alpha) on mast cells or basophils after cross-linking of receptor-bound IgE by its antigen or an anti-IgE antibody results in cell activation and release of inflammatory mediators. Omalizumab (Xolair), Novartis Pharmaceuticals; Genentech Inc.) is a recombinant humanized anti-IgE mAb developed for the treatment of severe allergic asthma. It complexes with free serum IgE, which prevents its binding to Fc epsilon RI and thereby interrupts the allergic cascade. Administration of an inhibitory anti-Fc epsilon RI alpha mAb may represent an alternative strategy to neutralize IgE-mediated receptor activation. In the present report, for the first time, we have performed direct side of side comparison between the inhibitory anti-Fc epsilon RI alpha mAb designated 15/1 and Omalizumab for their effects on human cord blood-derived mast cells. We provide the first evidence that both 15/1 mAb and Omalizumab efficiently inhibit Fc epsilon RI-mediated human mast cell responses in vitro (degranulation, activation, release of IL-8 and IL-13, phosphorylation of Akt) and that mAb 15/1 is a non-anaphylactogenic antibody, which compared to Omalizumab, displays markedly higher inhibitory potency in the presence of high IgE levels.     

Isolation of human Fab antibodies specific for the low-affinity IgE receptor (CD23) by selecting a hierarchical antibody library system against B lymphoblastic IM-9 cells

The development of human antibodies specific for certain B cell markers is required to generate therapeutic antibody leads with improved therapeutic indices against B-cell lymphomas. To meet this demand, we selected a primary human antibody library, HuDVFab-8L, against human B lymphoblastic IM-9 cells via a ‘Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)’ cell panning approach. Six Fab clones that specifically bound to IM-9 cells were successfully isolated. Among these clones, two clones (IM-L6-E and IM-L8-G), were found to be specific for CD23 (Fc?RII). Affinity maturation of these Fab clones was then performed in a hierarchical manner by constructing secondary antibody libraries through combining heavy (H) chains of two Fabs with the human kappa L chain sublibrary HuNL-D3 followed by biopanning against the CD23 antigen. Clone IM-L6-5, one of the affinity maturated Fab derivatives from IM-L6-E, has a binding affinity of k_D ≈ 30 nM to soluble CD23. In addition, IM-L6-5 Fab is able to bind to an inducible form of CD23 expressed on U937 cells upon IL-4 stimulation, and inhibits binding of human IgE to CD23. Since the Fab IM-L6-5 is derived from a fully human naïve origin, we believe that IM-L6-5 can be utilized for the development of a therapeutic mAb which may have an improved therapeutic index over lumiliximab, a primatized anti-CD23 mAb, for the treatment of CLL or allergic diseases.     

Conformational differences of human IgE on hydrophobic and hydrophilic solid phases detected by monoclonal antibodies

Very sensitive assays of IgE are required for determining prevalence of allergic reactions in children. In order to develop a sensitive two-site IRMA two kinds of mAb were produced. Antibodies specific for D epsilon 1 determinants were derived from immunization with a 40 kDa papain Fc fragment. They bound equally native and 56 degrees C heated IgE. D epsilon 2 specific mAb were obtained after immunization with IgE anti-D epsilon 1 complex and were selected on the basis of their inability to bind heated IgE. In a two-site assay on plastic plates, D epsilon 1 specific mAb led to the binding of IgE but always prevented further binding of anti-D epsilon 1 mAb, anti-human kappa chain mAb or allergen on bound IgE. This was not true when CNBr activated cellulose was used. The influence of the nature of the solid phase disappeared when D epsilon 2 specific mAb were coated on plastic tubes. In this case, the binding of a second mAb with identical or different fine specificity was observed. The best matched pair was E 164 (anti-D epsilon 2) on the solid phase and 6H10 (anti-D epsilon 1) as a tracer. As little as 0.2 UI/ml of IgE could be detected in a 2 hr test.     

The role of CD23 in the regulation of allergic responses

IgE, the key molecule in atopy has been shown to bind two receptors, FcεRI, the high-affinity receptor, and FcεRII (CD23), binding IgE with lower affinity. Whereas cross-linking of IgE on FcεRI expressed by mast cells and basophils triggers the allergic reaction, binding of IgE to CD23 on B cells plays an important role in both IgE regulation and presentation. Furthermore, IgE-immune complexes (IgE-ICs) bound by B cells enhance antibody and T cell responses in mice and humans. However, the mechanisms that regulate the targeting of the two receptors and the respective function of the two pathways in inflammation or homeostasis are still a matter of debate. Here, we focus on CD23 and discuss several mechanisms related to IgE binding, as well as the impact of the IgE/antigen-binding on different immune cells expressing CD23. One recent paper has shown that free IgE preferentially binds to FcεRI whereas IgE-ICs are preferentially captured by CD23. Binding of IgE-ICs to CD23 on B cells can, on one hand, regulate serum IgE and prevent effector cell activation and on the other hand facilitate antigen presentation by delivering the antigen to dendritic cells. These data argue for a multifunctional role of CD23 for modulating IgE serum levels and immune responses.     

CɛmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B lymphocytes

Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of C?mX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore virus-like particles formed by hepatitis B virus core antigen (HBcAg) that harbors the entire C?mX peptide or its fragments as immunogens for inducing anti-C?mX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant C?mX-containing human IgE.Fc in ELISA and Western blot analyses, and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice were able to cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG could mediate ADCC in human mIgE-expressing A20 murine B-cell lymphoma. These studies suggest that HBcAg-C?mX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.