Membrane-bound IgE Research Articles

Allergenic and Mas-Related G Protein-Coupled Receptor X2-Activating Properties of Drugs: Resolving the Two

Since the seminal description implicating occupation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) in mast cell (MC) degranulation by drugs, many investigations have been undertaken into this potential new endotype of immediate drug hypersensitivity reaction. However, current evidence for this mechanism predominantly comes from (mutant) animal models or invitro studies, and irrefutable clinical evidence in humans is still missing. Moreover, translation of these preclinical findings into clinical relevance in humans is difficult and should be critically interpreted. Starting from our clinical priorities and experience with flow-assisted functional analyses of basophils and cultured human MCs, the objectives of this rostrum are to identify some of these difficulties, emphasize the obstacles that might hamper translation from preclinical observations into the clinics, and highlight differences between IgE- and MRPGRX2-mediated reactions. Inevitably, as with any subject still beset by many questions, alternative interpretations, hypotheses, or explanations expressed here may not find universal acceptance. Nevertheless, we believe that for the time being, many questions remain unanswered. Finally, a theoretical mechanistic algorithm is proposed that might advance discrimination between MC degranulation from MRGPRX2 activation and cross-linking of membrane-bound drug-reactive IgE antibodies.

Direct Inhibition of the Allergic Effector Response by Raw Cow's Milk-An Extensive In Vitro Assessment.

The mechanisms underlying the allergy-protective effects of raw cow’s milk are poorly understood. The current focus is mainly on the modulation of T cell responses. In the present study, we investigated whether raw cow’s milk can also directly inhibit mast cells, the key effector cells in IgE-mediated allergic responses. Primary murine bone marrow-derived mast cells (BMMC) and peritoneal mast cells (PMC), were incubated with raw milk, heated raw milk, or shop milk, prior to IgE-mediated activation. The effects on mast cell activation and underlying signaling events were assessed. Raw milk was furthermore fractionated based on molecular size and obtained fractions were tested for their capacity to reduce IgE-mediated mast cell activation. Coincubation of BMMC and PMC with raw milk prior to activation reduced β-hexosaminidase release and IL-6 and IL-13 production, while heated raw milk or shop milk had no effect. The reduced mast cell activation coincided with a reduced intracellular calcium influx. In addition, SYK and ERK phosphorylation levels, both downstream signaling events of the FcεRI, were lower in raw milk-treated BMMC compared to control BMMC, although differences did not reach full significance. Raw milk-treated BMMC furthermore retained membrane-bound IgE expression after allergen stimulation. Raw milk fractionation showed that the heat-sensitive raw milk components responsible for the reduced mast cell activation are likely to have a molecular weight of > 37 kDa. The present study demonstrates that raw cow’s milk can also directly affect mast cell activation. These results extend the current knowledge on mechanisms via which raw cow’s milk prevents allergic diseases, which is crucial for the development of new, microbiologically safe, nutritional strategies to reduce allergic diseases.

More than one antibody of individual B cells revealed by single-cell immune profiling

Antibodies have a common structure consisting of two identical heavy (H) and two identical light (L) chains. It is widely accepted that a single mature B cell produces a single antibody through restricted synthesis of only one VHDJH (encoding the H-chain variable region) and one VLJL (encoding the L-chain variable region) via recombination. Naive B cells undergo class-switch recombination (CSR) from initially producing membrane-bound IgM and IgD to expressing more effective membrane-bound IgG, IgA, or IgE when encountering antigens. To ensure the “one cell — one antibody” paradigm, only the constant region of the H chain is replaced during CSR, while the rearranged VHDJH pattern and the L chain are kept unchanged. To define those long-standing classical concepts at the single-cell transcriptome level, we applied the Chromium Single-Cell Immune Profiling Solution and Sanger sequencing to evaluate the Ig transcriptome repertoires of single B cells. Consistent with the “one cell — one antibody” rule, most of the B cells showed one V(D)J recombination pattern. Intriguingly, however, two or more VHDJH or VLJL recombination patterns of IgH chain or IgL chain were also observed in hundreds to thousands of single B cells. Moreover, each Ig class showed unique VHDJH recombination pattern in a single B-cell expressing multiple Ig classes. Together, our findings reveal an unprecedented presence of multi-Ig specificity in some single B cells, implying regulation of Ig gene rearrangement and class switching that differs from the classical mechanisms of both the “one cell — one antibody” rule and CSR.

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.

Post-transplantation Development of Food Allergies.

The development of food allergies is increasingly being recognized as a post-solid organ transplant complication. In this article, we review the spectrum of post-transplant food allergy development and the proposed mechanisms for de novo food allergies and the clinical significance they pose. The development of new food allergies is disproportionately associated with pediatric liver transplants, where it occurs in up to 38% of select populations. The mechanism of food allergy development is not completely understood; however, it is likely promoted by unbalanced immune suppression. De novo food allergy development is a common complication of solid organ transplants with the highest risk occurring in pediatric liver transplant recipients. There are likely multiple mechanisms for food allergy development including passive transfer of membrane-bound IgE and lymphocytes from donor to recipient, as well as loss of food tolerance and active development of new food allergies. The optimal management of food allergies following organ transplants has not been well researched but may include changing the immune suppression regimen if the food allergy does not resolve without intervention.

Treating IgE-mediated diseases via targeting IgE-expressing B cells using an anti-CεmX antibody.

Targeting the IgE pathway is a clinically validated strategy for treating IgE-mediated diseases. Omalizumab, an anti-IgE antibody, which binds to free IgE and prevents the binding of IgE to FcεRI on mast cells and basophils has been approved for severe persistent allergic asthma and chronic spontaneous (idiopathic) urticaria. The therapeutic efficacy of anti-IgE has also been reported in allergic rhinitis, allergic bronchopulmonary aspergillosis, latex allergy, atopic dermatitis, allergic urticaria, anaphylaxis, and others. Anti-CεmX, which binds to membrane-bound IgE (mIgE) on IgE-switched B cells, lyses mIgE-expressing B lymphoblasts and prevents the allergen-induced generation of IgE-producing plasma cells, offers an alternative mechanism of intervening with the IgE inflammatory pathway. Because anti-CεmX does not bind to free IgE, it can modulate the IgE pathway regardless of the serum IgE levels in treated patients. These unique pharmacologic mechanisms potentially enable anti-CεmX to provide different clinical utilities from anti-IgE and serve as a therapeutic and a prophylactic in some IgE-mediated diseases, which are not adequately treated with current medicine.

The discovery of IgE 50 years later

In 1906, Von Pirquet1 used the term supersensitivity without immunity to describe (1) symptoms of inhalant allergy, (2) positive immediate skin test results, and (3) the fact that the results of other tests of immunity were not positive in these patients. The other tests he referred to were precipitin tests and complement fixation. Thus, he opened up the question of serum tests for supersensitivity. In 1919, Ramirez2 reported that a patient who had received a blood transfusion from a horse allergic donor became allergic to horse dander. At this point, Prausnitz and Kustner3 set out to investigate whether the serum of allergic individuals contained a factor or factors that could sensitize the skin. In 1921, they reported that the local injection of serum from a fish allergic individual, Kustner, to an individual who was only allergic to pollen, Prausnitz, would transfer specific sensitivity.3 This transfer of sensitivity came to be known as the P-K test and was used widely to study sensitivity not only to common allergens but also to extracts as diverse as those obtained from schistosomes.4,5 Furthermore, in New York, Cooke et al6 identified that there were other antibodies in the serum that increased during desensitization treatment and could block the skin-sensitizing activity. By the 1950s, it was clear that the transferred sensitivity was specific, that it could be diluted extensively, and that the skin remained locally sensitive for days if not weeks after the injection of serum.7,8 It was also already clear that the ability to sensitize the skin was lost after moderate heating of the serum.7 Several studies had also been reported on the physical properties of P-K activity. Indeed, Heimlich et al9 at Cal Tech reported on the sedimentation properties of skin-sensitizing antibodies in 1960. However, the studies before 1964 had not succeeded in defining the nature of these antibodies.

Developing an antibody targeting CεmX of mIgE for the treatment of IgE-mediated diseases (HYP4P.305)

Abstract CεmX (also referred to as M1’) is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ε heavy chain of membrane-bound IgE (mIgE) on human B lymphocytes. Antibodies that target CεmX are potentially useful in controlling IgE production for treating allergic and other IgE-mediated diseases. Herein we report that an anti-CεmX mAb, 4B12, was shown to be effective in reducing allergen-specific IgE and IL-5 production upon the challenge of the allergen in an asthma model employing CεmX gene knocked-in mice that express mIgE containing human CεmX domain on B cells. 4B12 could also alleviate airway hyper-responsiveness (AHR), allergen-induced eosinophil infiltration, and lung inflammation in those mice. Furthermore, we demonstrated that a humanized 4B12 mAb (referred to as FB825) could inhibit the production of human IgE in mice that had been reconstituted with human peripheral blood mononuclear cells. Based on the abilities of 4B12 to bind to mIgE and to lyse mIgE-expressing B cells by apoptosis, ADCC, and other cytolytic mechanisms and on the above results on animal models, a phase-I human clinical trial of FB825 to investigate its IgE-related biological effects is being performed.

Developing an antibody targeting CεmX of mIgE for the treatment of allergic and other IgE-mediated diseases (VAC6P.954)

Abstract CεmX (also referred to as M1’) is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ε heavy chain of membrane-bound IgE (mIgE) on human B lymphocytes. Antibodies that target CεmX are potentially useful in controlling IgE production for treating allergic and other IgE-mediated diseases. Based on its abilities to bind to mIgE with high affinity and to lyse mIgE-expressing B lymphocytes by apoptosis, ADCC, and other cytolytic mechanisms, humanized 4B12 monoclonal antibody (h4B12 mAb) is now under preclinical development. In an allergic asthma animal model employing genetically modified mice that express mIgE containing human CεmX domain, we have demonstrated that mAb 4B12 was effective in reducing antigen-specific IgE, but not immunoglobulins of other isotypes. 4B12 could also markedly alleviate airway hyperresponsiveness (AHR) to inhaled methacholine in those mice. Furthermore, we have demonstrated that h4B12 could inhibit the production of human IgE in mice that had been reconstituted with human PBMCs. We have now developed a transfected CHO cell line capable of producing h4B12 at a high yield in preparation for human clinical trials.

Two potential therapeutic antibodies bind to a peptide segment of membrane-bound IgE in different conformations

IgE mediates hypersensitivity reactions responsible for most allergic diseases, which affect 20-40% of the population in developed countries. A 52-residue domain of membrane-bound IgE (mIgE) called CεmX is currently a target for developing therapeutic antibodies; however, its structure is unknown. Here we show that two antibodies with therapeutic potential in IgE-mediated allergic diseases, which can cause cytolytic effects on mIgE-expressing B lymphocytes and downregulate IgE production, target different conformations of an intrinsically disordered region (IDR) in the extracellular CεmX domain. We provide an important example of antibodies targeting an extracellular IDR of a receptor on the surface of intended target cells. We also provide fundamental structural characteristics unique to human mIgE, which may stimulate further studies to investigate whether other monoclonal antibodies (mAbs) targeting intrinsically disordered peptide segments or vaccine-like products targeting IDRs of a membrane protein can be developed.

Whole Antigenic Lysates ofIxodes ricinus, but not Der-p2 Allergen-Like Protein,are Potent Inducers of Basophil Activation in Previously Tick-Exposed Human Hosts

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro. In the current study, we hypothesize that hard tick-derived material contains potent inducers being able to promote basophil stimulation, which correlates with a sensitization immunological response following tick bites. To this end, biological material from two hard tick cell lines (IRE11 and IDE8 - derived from Ixodes ricinus and I.scapularis, respectively) as well as I.ricinus salivary gland and body lysates were used in a human basophil activation test (BAT) to analyse binding and cross-linking capacity of membrane-bound IgE, because basophils are one of the main effector cells of allergic reactions. Additionally, Der-p2 allergen-like gene from I.ricinus was recombinantly expressed as a 15-kDa histidine-tagged fusion protein, purified and included as a stimulus within the setup. Blood was drawn and submitted to BAT screening from a pool of 36 individuals, both bitten and who served solely as negative controls. We have found that seven subjects (19%), all of whom were at least two times tick-bitten, positively reacted to the aforementioned stimuli, whereas the reactivity level of the ones bearing single bites proved to be within the normal range. Moreover, no significant upregulation of the assessed basophil activation marker was detected in the case of Der-p2, except a faint reaction at high dosages. We conclude that at least two tick bites of the human host must occur in order to induce significant basophil activation.

Antibodies binding to discrete segments of CϵmX of mIgE on B lymphocytes (P6004)

Abstract CϵmX is a discrete domain of 52 a.a. residues, located between the CH4 domain and the C-terminal membrane anchor peptide of the ϵ heavy chain of membrane-bound IgE (mIgE) on human B cells. Among the monoclonal antibodies (mAbs) prepared for their ability to bind to CϵmX fragments or CϵmX-containing recombinant proteins in ELISA, those that can bind to native mIgE on B cells can cause ADCC, apoptosis, and other cytolytic mechanisms on the B cells and are potential useful for controlling IgE production for treating allergic diseases. The CϵmX-specific mAbs prepared previously appear to recognize discrete and overlapping peptide segments of CϵmX. A linear peptide epitope of an antibody is defined by a contiguous peptide segment of minimal stretch that confers maximum binding by the antibody. In some instances, while the peptide epitopes recognized by two mAbs may be adjacent to each other and overlap, the two mAbs do not bind to the adjacent epitopes by a substantial degree. This can be illustrated by the pair of humanized mAbs, h4B12 and h47H4. The mAb h4B12 binds to peptide P1-15 (GLAGGSAQSQRAPDR) and h47H4 binds to peptide P7-18 (AQSQRAPDRVLC). Although the two epitopes share a segment AQSQRAPDR, the mAb h4B12 does not bind to P7-18, and h47H4 does not bind to P1-15. A related presentation from our group (Hsing-Mao Chu et al.) will also reveal that the shared peptide segment AQSQRAPDR has two markedly different conformations when it is associated with h4B12 or with h47H4.

Structural analysis of epitopes recognized by two anti-CϵmX mAbs reveals intrinsic disorder of the CϵmX domain (P6002)

Abstract In human membrane-bound IgE (mIgE), a CϵmX domain containing 52 aa residues is located between the CH4 domain and the C-terminal membrane-anchoring segment. Antibodies specific for CϵmX, which can cause lysis of mIgE+ B cells, are being developed to treat allergic diseases and an anti-CϵmX mAb h47H4 is in phase IIb clinical trials. Hence, structural characterization of CϵmX free and complexed with anti-CϵmX mAb is of wide interest and importance. We have solved the crystal structures of (i) free CϵmX fused to IgG1-Fc and (ii) part of CϵmX complexed with an anti-CϵmX mAb, h4B12-Fab, at 1.94 Å resolution. The crystal structure of the IgG1-Fc•CϵmX fusion protein of homodimer exhibits a lack of well-defined electron density for residues 10 to 52 of A subunit and in absence of B subunit in dimeric CϵmX domain. The first 33 residues are also predicted to be intrinsically disordered. The 3D complex structure and epitope mapping by ELISA showed that the anti-CϵmX mAb, h4B12, binds to GLAGGSAQSQRAPDR, which has been reported to bind another anti-CϵmX mAb, h47H4, except for the first 6 residues (in italics). Comparison of the 3-D complex structure of h4B12 and h47H4 revealed different conformations of AQSQRAPDR, a feature characteristic of the intrinsically disordered regions in the CϵmX domain. This is the first report showing that the drug target, CϵmX, contains an intrinsically disordered region that can switch to different conformations upon binding to different anti-CϵmX mAbs.

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 CɛmX, 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 CɛmX peptide or its fragments as immunogens for inducing anti-CɛmX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant CɛmX-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-CɛmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.

CϵmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B cells (53.7)

Abstract 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 CϵmX, 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 the virus-like particles (VLPs) formed by hepatitis B virus core antigen (HBcAg) that harbors the entire CϵmX peptide or its fragments as immunogens for inducing anti-CϵmX antibodies. Mice immunized subcutaneously with some of these immunogens produce antibodies that bind to recombinant CϵmX-containing human IgE.Fc in ELISA and Western blot analysis 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 could cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG mediated ADCC on human mIgE-expressing murine B cell lymphoma A20. These results suggest that HBcAg-CϵmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.

The IgE gene in primates exhibits extraordinary evolutionary diversity

Membrane-bound IgE (mIgE) on B lymphocytes is essential for IgE production. Earlier studies showed that the ε chain of mIgE (mε) on human B cells has a "long" isoform, with an extra "CεmX" domain of 52 amino acid (aa) residues between the CH4 domain and the membrane-anchor segment, as compared to the conventional "short" isoform. Because CεmX provides an antigenic site for targeting IgE-expressing B cells to down-regulate IgE production in patients with allergy, analysis of CεmX in various animals is of great interest. Hence, we analyzed the ε Ig gene, in particular, its membrane exon regions encoding the membrane anchor peptide segment and CεmX domain, of 26 species of the order Primates and 12 species of seven non-Primate orders using data obtained experimentally or retrieved from GenBank. Our analyses reveal the unexpected finding that the genes of three extant tarsier species do not contain the membrane exons for mIgE. Another striking finding is that early evolved Strepsirhini primates such as lemurs and lorises do not have gene segments for the long isoform, whereas New World monkeys such as marmosets and squirrel monkeys allow the transcription of only the long isoform. In Old World monkeys and apes, including humans, the ε gene allows the transcription of both isoforms. This work thus reveals the dramatic differences in the gene segment encoding the mε C terminal region among the four major primate lineages: the Strepsirhini primates, the tarsiers, New World monkeys, and Old World monkeys and apes/humans.

Anti-IgE monoclonal antibodies that bind to IgE bound by CD23 but not to IgE bound by IgE Fc receptors on basophils (86.10)

Abstract IgE is a central mediator responsible for immediate-type hypersensitivity reactions. The anti-IgE monoclonal antibody (mAb), omalizumab, has been shown in numerous clinical trials to be efficacious in the treatment of severe allergic asthma and other allergic diseases. Omalizumab was designed to bind to free IgE in blood and membrane-bound IgE (mIgE) on B cells, but not to IgE bound by the high-affinity IgE Fc receptors (FcϵRI) on basophils and mast cells and by the low-affinity IgE Fc receptors (CD23) on B cells and many other cell types. Additionally, omalizumab can bind to IgE and prevent it from binding to both FcϵRI and CD23. Studies by other groups have also shown that CD23 is involved in the synthesis of IgE and that interfering CD23 function, such as by cross-linking CD23 on B cell surface, can cause the down-regulation of IgE production. We have developed anti-IgE mAbs that mimic omalizumab in various aspects except their ability to bind to IgE on CD23. These newly developed mAbs bind to linear epitopes on IgE. They do not induce the degranulation of IgE-pulsed rat basophilic leukemic cells (RBL SX-38), which had been transfected with the genes of human FcϵRI. The ability of the mAbs to cross-link IgE-occupied CD23 on the cell surface of B cell lines is being tested. The results suggest that these anti-IgE mAbs may render a set of pharmacological mechanisms, which are somewhat different from that of omalizumab, for controlling IgE in patients with allergic diseases.

Genetic variations in the CεmX domain of human membrane-bound IgE

The epsilon chain of membrane-bound IgE (mIgE) is expressed predominantly as a "long" isoform, containing an extra segment of 52 amino acid (a.a.) residues, referred to as C epsilon mX, between the CH4 domain and the C-terminal membrane-anchoring transmembrane peptide. C epsilon mX results from an alternative splicing of the epsilon RNA transcript at 156-bp upstream of the splicing acceptor site used by the "short" isoform. Here, based on an analysis of the C epsilon mX genomic DNA sequences of 320 subjects residing in Taiwan, we report that single-nucleotide polymorphisms have been found at two positions, namely, G/T at #46 and A/G at #93 (along the 156 bp of C epsilon mX), with the former creating an amino acid change from Val to Leu at #16 (along the 52 a.a. of C epsilon mX) and the latter resulting in no change (Gly). Among the 640 C epsilon mX sequences identified, the previously known 46G93A allelic form appeared 293 times, the newly discovered 46T93A allelic form (GeneBank accession no. GU208817) 26 times, and the 46G93G allelic form (GU208818) 321 times. No 46T93G allelic form was found. Serum IgE measurements showed that the polymorphisms did not correlate with the levels of serum IgE. The anti-C epsilon mX monoclonal antibody, 4B12, could bind equally well to mIgE.Fc(L)(16V) and mIgE.Fc(L)(16L). While genetic variation of C epsilon mX of broader populations should also be investigated, these newly discovered genetic variants of C epsilon mX in the Taiwanese population do not seem to affect the feasibility of using an anti-C epsilon mX mAb, such as 4B12, to target mIgE-expressing B cells.

Basophil activation tests in food allergy

Basophil activation tests in food allergy

Anti-CmεX monoclonal antibodies potentially applicable for targeting mIgE-expressing B cells (140.20)

Abstract On B lymphocytes, membrane-bound IgE (mIgE) is part of BCR and essential for generating isotype-specific IgE response. On mIgE+ B cells in man, the membrane-bound ε chain (mε) exists predominantly in the long form (mεL), containing 52-a.a. CεmX domain between CH4 and the C-terminal membrane-anchoring segment; the conventional form of mε (mεS) exists in minor proportions. CεmX thus provides an attractive site for immunological targeting of mIgE+ cells. Here we have shown that 9 newly prepared CεmX-specific monoclonal antibodies (mAbs), as well as previously reported a20, can bind to mIgE.FcL-expressing CHO cells, while only 4B12 and 26H2 can bind to mIgE.FcL-expressing B cell line Ramos cells. In epitope mapping studies using synthetic peptides representing different parts of CεmX, 4B12 was shown to bind to the N-terminal part, 26H2 the middle part, and all others the C-terminal part of CεmX. These results suggest that the C-terminal part of CεmX is sterically hindered for mAb binding by associated components in the BCR. The chimerized mAbs c4B12 and c26H2, when cross-linked by secondary antibodies, could lyse mIgE.FcL-expressing Ramos cells by apoptosis through a BCR-dependent caspase pathway. Using PBMCs as the source of effector cells, c4B12 and c26H2 could cause ADCC against mIgE.FcL-expressing Ramos cells in a does-dependent fashion. These results indicate that anti-CεmX mAbs, 4B12 and 26H2, can bind to native mIgE on B cells and are potentially useful for targeting mIgE+ B cells for controlling IgE production.