A high-affinity monoclonal anti-IgE antibody for depletion of IgE and IgE-bearing cells.

BACKGROUND: We have identified a monoclonal anti-human immunoglobulin E (IgE) antibody, which recognizes FcepsilonRI-bound IgE and prevents binding of IgE to FcepsilonRI. In this study, we assessed the binding kinetics and affinity of monoclonal antibody 12 (mAb12) for IgE and investigated whether mAb12 can be used for depletion of IgE and isolation of IgE-bearing cells from peripheral blood. METHODS: Binding kinetics and affinity for IgE were studied using Biacore surface plasmon resonance technique experiments. IgE antibodies were depleted from serum using sepharose-coupled mAb12 and IgE-bearing cells were enriched from heparinized blood samples with mAb12. The extent and biological relevance of IgE depletion were studied by quantitative IgE measurements and basophil histamine release experiments. Specific binding of mAb12 to IgE-bearing cells (basophils, mast cells, IgE-secreting plasma cells) was demonstrated by FACS. RESULTS: Monoclonal antibody 12 shows rapid association (k(a) = 5.46e5/Ms) with IgE, almost no dissociation (k(d) = 8.8e-5/s) and an affinity for IgE (K(D) = 1.61e-10 M), which is as high as that of FcepsilonRI. Immobilized mAb12 could be used to deplete IgE antibodies and isolate IgE-bearing cells from peripheral blood in a single-step procedure. CONCLUSIONS: Monoclonal antibody 12 is a high affinity anti-human IgE antibody, which efficiently removes IgE and IgE-bearing cells from peripheral blood and may thus be used for extracorporeal depletion of IgE and IgE-bearing cells.

Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to Fc epsilon RI.

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.

A molecular model of type I allergy: identification and characterization of a nonanaphylactic anti-human IgE antibody fragment that blocks the IgE-FcepsilonRI interaction and reacts with receptor-bound IgE.

BACKGROUND: The IgE-mediated activation of effector cells and antigen-presenting cells through the high-affinity receptor for IgE (FcepsilonRI) represents a key pathomechanism in type I allergy and many forms of asthma. OBJECTIVE: We sought to establish an in vitro molecular model for the interaction of human FcepsilonRI, IgE, and the corresponding allergen and to identify monoclonal anti-human IgE antibodies with a therapeutic profile different from previously established anti-IgE antibodies. METHODS: Human FcepsilonRI alpha chain, a human monoclonal allergen-specific IgE antibody (chimeric Bip 1), and the corresponding allergen, the major birch pollen allergen Bet v 1, were produced as recombinant proteins and analyzed by means of circular dichroism and native overlays, respectively. Using this molecular model, as well as negative stain immunoelectron microscopic analysis, and in vitro cultivated human basophils, we characterized mouse anti-human IgE antibodies. RESULTS: We established a molecular model for the interaction of human IgE with FcepsilonRI. Using this molecular model, we identified a nonanaphylactic anti-human IgE antibody fragment (Fab12), which blocked the IgE-FcepsilonRI interaction and reacted with effector cell-bound IgE. CONCLUSION: Fab12 represents a candidate molecule for therapy of atopy and asthma because it can be used for the depletion of circulating IgE antibodies, as well as for the depletion of IgE-bearing cells.

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation.

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.

Allergic manifestations as the results of a conditional autoimmune response.

By means of repertoire cloning we have isolated human anti-IgE antibodies as well as human anti-FcepsilonRI antibodies. Whether the naturally occurring anti-IgE autoantibodies play a pathophysiological role may be disputed, but the beneficial role of recombinant anti-IgE antibodies as a therapeutic agent has been shown. On the other hand, the natural antibodies isolated from an antibody library of a nonallergic individual against the FcepsilonRI alpha-chain are anaphylactogenic, if FcepsilonRI was not occupied. Thus, anti-FcepsilonRI autoantibodies may be part of a conditional autoimmune reaction, leading to urticaria if local IgE is consumed, e.g. after an immediate reaction. Thus, anti-FcepsilonRI antibodies may represent an amplification arm of the late reaction. The normal occurrence of anti-IgE and anti-IgE receptor autoantibodies may suggest that it might also be feasible to induce such autoantibodies by vaccination. In a monkey model using a mimotope of human IgE it was possible to induce a beneficial anti- IgE autoimmune response. The actual epitope of the IgE molecule was then also molecularly reconstructed by generating recombinant anti-idiotypic antibodies. These antibodies also induced effectively an anti-IgE response in monkeys, suggesting that not only mimotopes but also anti-idiotypic antibodies may be used to generate an autoimmune response. Both of our projects suggest that active immunization may be a new form of immunomodulation for the treatment of allergic disease.

Effects of complement inactivation and IgG depletion on skin reactivity to autologous serum in chronic idiopathic urticaria.

BACKGROUND: Intradermal injection of autologous serum elicits a wheal-and-flare response in about 60% of patients with chronic idiopathic urticaria (CIU). This reactivity has been attributed to the presence of IgG autoantibodies directed against IgE or the alpha-chain of the high-affinity IgE receptor (FcepsilonRIalpha) expressed on basophils and mast cells, leading to the hypothesis that at least some forms of CIU could be sustained by an autoimmune process. OBJECTIVES: The aim of this study was to investigate the relationship between the presence of anti-IgE or anti-FcepsilonRI antibodies and the ability to induce wheal-and-flare responses in CIU sera selected for the capacity to give a positive skin test response. METHODS: Fifteen patients with CIU and a positive skin test response to autologous serum were injected intradermally with native serum and with serum heated at 56 degrees C for 30 minutes and then adsorbed on Sepharose-protein G to obtain IgG depletion. Serum levels of anti-IgE and anti-FcepsilonRIalpha antibodies were measured by ELISA by using purified IgE and recombinant RIalpha-soluble double-fusion protein RIalpha-human serum albumin-RIalpha, respectively. The histamine-releasing activity of sera was tested by using ELISA with whole human blood from a healthy donor. RESULTS: All patients had positive cutaneous responses to native serum injection. Anti-FcepsilonRIalpha antibodies were present in 14 of 15 native sera, only two of which were able to induce in vitro basophil degranulation. On the contrary, detectable amounts of anti-IgE antibodies were not found in any serum. IgG depletion by protein G resulted in complete (10/14 samples) or considerable (4/14 samples) removal of anti-FcepsilonRIalpha antibodies. The two sera endowed with functional activity lost their capacity to trigger histamine release from basophils after heating and protein G adsorption. Nonetheless, heat-decomplemented/IgG-depleted sera elicited wheal-and-flare reactions comparable with those observed with untreated sera. CONCLUSIONS: These results strongly suggest that skin reactivity to autologous serum could be due to as yet unidentified non-Ig reactants present in the sera of patients with CIU.

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody.

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.

Mimicry of human IgE epitopes by anti-idiotypic antibodies.

According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

An in vitro model for the allergen-IgE-FcARI interaction.

BACKGROUND: The interaction of immune complexes consisting of allergens and allergen-specific IgE with the high-affinity Fcepsilon receptor represents the key event in the induction of symptoms in type I allergic individuals. Immediate-type symptoms result from the release of biological mediators due to allergen-induced cross-linking of FcepsilonRI receptors on mast cells and basophils, whereas FcepsilonRI-mediated presentation of allergen-IgE complexes may contribute to late-phase symptoms through enhanced T cell activation. The interaction of allergens/allergen-specific IgE/FcepsilonRI represents, therefore, an important target for therapeutic intervention strategies in type I allergy. METHODS AND RESULTS: A molecular model of the allergen-IgE-FcepsilonRI interaction was established. It consists of recombinant purified Bet v 1, the major birch pollen allergen, a chimeric Bet v 1 specific monoclonal IgE antibody, and the baculovirus-expressed purified human alpha chain of FcepsilonRI. The chimeric Bet v 1-specific IgE antibody consists of the light chain and the heavy chain variable region of a mouse monoclonal Bet v 1 specific antibody, Bip 1, and the constant region of human IgE. The interaction of rBet v 1, chimeric Bip 1, and human alpha chain was investigated by overlay experiments. Nitrocellulose-immobilized recombinant alpha chains was incubated with chimeric Bip 1 and, for control purposes, with mouse-derived Bip 1. Bound chimeric Bip 1 was detected with 125I-labeled rBet v 1. The specific interaction of rBetv 1, chimeric Bip 1, and recombinant human alpha chain is demonstrated. We thus establish a molecular model of the allergen/IgE/alpha chain interaction. The usefulness of the described in vitro system is exemplified by the identification of a mouse monoclonal antihuman IgE antibody which blocked the IgE-alpha chain interaction. CONCLUSIONS: The module system consisting of rBet v 1, chimeric Bip 1, and recombinant alpha chain may be used for the identification of competitors of the allergic effector reaction by means of high throughput screening of compounds or by combinatorial chemistry.

Mimotope and anti-idiotypic vaccines to induce an anti-IgE response.

We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti-IgE antibody termed BSW17. The selected mimotopes and epitopes within the Cepsilon3 and Cepsilon4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17. The chemically synthesized and KLH-coupled IgE epitopes or mimotopes were used to induce an anti-IgE response in rhesus monkeys. The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen. Furthermore, using the same monoclonal anti-IgE antibody, we also generated an anti-idiotypic antibody that showed sequence homology with the IgE epitope in the Cepsilon3 domain. This anti-idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti-IgE immune response. For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE-homologous structures. Orally immunized mice produced serum anti-IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti-IgE response orally.

Human anti-FcepsilonRIalpha autoantibodies isolated from healthy donors cross-react with tetanus toxoid.

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the alpha-subunit of the high-affinity receptor for IgE (FcepsilonRIalpha) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcepsilonRIalpha auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcepsilonRIalpha Ab from an IVIg preparation using recombinant FcepsilonRIalpha. Interestingly, these anti-FcepsilonRIalpha auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcepsilonRIalpha. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcepsilonRIalpha auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.

Interaction of human IgE with Fc epsilon RI alpha exposes hidden epitopes on IgE.

BACKGROUND: Binding of human IgE via the heavy-chain constant region domain 3 (Cepsilon3) to the alpha-chain of its high affinity receptor (FcepsilonRIalpha) is a key event in mediating allergic reactions. We wanted to identify epitopes within Cepsilon3 that are stable to denaturation and to evaluate whether such structures are involved in receptor binding. The existence of stable epitopes would facilitate the generation of compounds that inhibit the IgE-FcepsilonRIalpha interaction. METHODS: Monoclonal anti-human IgE-antibodies against recombinant bacterially synthesized Cepsilon3, which is known to be partly misfolded, were raised in mice. These antibodies were probed for binding to native, immobilized and receptor-bound IgE, respectively, providing tools for the identification of the indicated stable epitopes. RESULTS: Two of the generated antibodies (8E7, 3G9) discriminate between IgE in solution and IgE attached to FcepsilonRIalpha, pointing towards a steric rearrangement within Cepsilon3 induced upon receptor binding. The described antibodies represent tools for studying the mechanism of the Fcepsilon-FcepsilonRIalpha interaction and may be of diagnostic value since serum IgE from various human donors was differently recognized by 8E7, which is indicative for naturally occurring IgE molecules with different steric conformation. CONCLUSION: The presented data support the hypothesis of a conformational change within IgE Cepsilon3 upon receptor binding by showing that monoclonal antibodies raised against recombinant Cepsilon3 differently recognize soluble and receptor-bound IgE. The presence of an IgE portion in sera of human donors that is recognized by 8E7 indicates the existence of IgE molecules in different steric conformations in human blood, which may be related to pathologic parameters.

Epitope-specific antibody response to IgE by mimotope immunization.

We have previously described a mouse monoclonal anti-human IgE antibody (BSW17) capable of recognizing receptor-bound IgE without inducing mediator release from human basophils or mast cells. Moreover, immune complexes of IgE and BSW17 are not able to bind to the IgE receptor. An initial attempt to map the precise epitope recognized by this mAb by using Fc epsilon-derived peptides of variable length was unsuccessful. However, by screening random peptide phage display libraries we isolated circular nona- and octapeptides specifically recognized by BSW17. These constrained peptides mimic at least a part of a conformational epitope and are thus called mimotopes. These mimotopes, either phage displayed or synthetically synthesized, did not react with any other anti-human IgE antibody tested, but efficiently inhibited the binding of human IgE to BSW17 only. The use of Rhodol-Green-labeled free cyclic peptide proved that these interactions were not carrier dependent. Immunization of rabbits with phage clones displaying the specific peptides on the surface induced an anti-human IgE response specific for the epitope of BSW17. Therefore, we conclude that such mimotopes or mimotope-derived peptides might be used for vaccination to induce in vivo a beneficial anti-IgE response as a novel immunotherapy.

Antigen interaction and heat inactivation expose new epitopes on human IgE.

It is well established that heat-denatured IgE is no longer capable of binding to FcepsilonRI. We have found an antibody that interacts with heat-denatured IgE. Interestingly, this antibody can also be used to detect some serum IgE, but not IgE synthesized de novo in vitro. However, native IgE can be transformed into an IgE that is recognized by this antibody, if antigen is added. Our data indicate that physiological mechanisms exist that biologically inactivate IgE which might still be mistaken for 'functional' IgE by assays based on polyclonal antibodies.

The membrane-proximal part of FcepsilonRIalpha contributes to human IgE and antibody binding--implications for a general structural motif in Fc receptors.

The high affinity receptor for human IgE (FcepsilonRI) on tissue mast cells and blood basophils is responsible for immediate hypersensitivity reactions. Binding of human IgE (hIgE) to FcepsilonRI has been shown to be mediated via three independent regions in the extracellular part of the alpha-subunit of FcepsilonRI (ecFcepsilonRIalpha). By site-directed mutagenesis we investigated the contribution of amino acids within the ecFcepsilonRIalpha FG loop (residues Lys154-Leu165) to binding to hIgE and two monoclonal anti-FcepsilonRIalpha antibodies (15/1, 5H5/F8). The mutated receptors were expressed and secreted from eukaryotic cells as amino-terminal fusion to HSA. We show that the proposed loop region contributes partly to hIgE binding and that the epitope of mAb 15/1, which inhibits hIgE/FcepsilonRIalpha interaction, maps to this region whereby a single W156A mutation results in complete loss of mAb 15/1 binding. In contrast, hIgE binding is not affected by the W156A mutation indicating that different amino acid residues within the loop are recognized by the mAbs 15/1 and hIgE. MAb 5H5/F8 does not recognize a receptor mutant truncated to Ile170. By screening a random dodecapeptide library displayed on bacterial flagella the epitope for mAb 5H5/F8 was mapped to P173REKY177 whereas one of the 15/1 binding clones displayed a peptide with an amino acid sequence homologous to Leu158-lle167. Based on the epitopes identified for the inhibitory mAb 15/1 and the non-inhibitory mAb 5H5F8 and on binding data obtained with polyclonal antisera raised against two ecFcepsilonRIalpha peptides, we propose a structural element in the membrane proximal part of ecFcepsilonRIalpha which forms a 3D structure which might facilitate specific and efficient attachment of hIgE.

Characterization of monoclonal antibodies directed against the alpha-subunit of the human IgE high-affinity receptor.

A panel of monoclonal antibodies (8H10/D11, 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, 8A4/G12/F9, and 8H10/F12) was raised in mice against the recombinant 20-kDa extracellular part of the alpha-chain of the human IgE high affinity receptors (ecFc epsilon RIalpha) produced in insect cells. The antibodies secreted by hybridomas were selected for specific binding to ecFc epsilon RIalpha, by enzyme-linked immunosorbent assay (ELISA). The selected clones were further characterized in surface plasmon resonance (SPR) experiments with ecFc epsilon RIalpha covalently immobilized on the surface of a sensor chip. The generated hybridomas can be divided into three groups. Hybridoma supernatants 8A4/G12/F9 and 8H10/F12 inhibited binding of human IgE to immobilized ecFc epsilon RIalpha in SPR (Group 1). Isotyping revealed that 8A4/G12/F9 and 8H10/F12 were of the IgE/kappa type. Antibodies present in the remaining supernatants were noninhibitory and bound to ecFc epsilon RIalpha in ELISA with intensities comparable to each other. Isotype analysis of antibodies secreted by these hybridomas showed that the antibodies 6F9/H8, 6F9/G9, 5F2/F8/H11, 5F2/F8/G10, and 8H10/D11 were IgG1/kappa. The hybridoma supernatants were purified via protein A chromatography. In a SPR experiment, ecFc epsilon RIalpha, displayed by immobilized human IgE, was still recognized by 6F9/H8 and 6F9/G9 (Group 2) as expected for noninhibitory antibodies. Surprisingly, 8H10/D11, 5F2/F8/H11, and 5F2/F8/G10 (Group 3) did not bind to this complex although they do not inhibit the binding of human IgE to ecFc epsilon RIalpha. All purified monoclonal antibodies gave positive signals in Western blotting.

Can active immunization redirect an anti-IgE immune response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.

Suppression of in vivo IgE and tissue IL-4 mRNA induction by SDZ 280.636, a synthetic muramyl dipeptide derivative.

Modulation of IgE isotype expression on B cells is one of the numerous effects of muramyl peptides on the regulation of the immune system. A non toxic diacyl glycerol derivative of muramyl dipeptide (MDP), in which the L-alanine is replaced by L-threonine (MDP-Threo-GDP; SDZ 280.636), is currently under investigation as lead compound for the development of an anti-allergic drug. In this report, the modulatory effect of orally administered SDZ 280.636 in a murine model on polyclonally induced IgE levels is described. In this model, mice are injected i.v. with goal anti mouse IgD (GAMD) and challenged three to four weeks later with goal IgG (GIG). Both the primary and secondary immune responses lead to an increase of serum IgE levels. We demonstrate the efficacy of this muramyl dipeptide derivative in selectively inhibiting a polyclonal IgE response in GAMD-primed, GIG challenged mice without affecting the levels of other immunoglobulin classes. It is further shown that the induction of interleukin 4 (IL-4) gene transcript levels in lymphoid organs, which is observed as a consequence of boosting GAMD pretreated mice with GIG, is selectively suppressed in gut associated lymphoid tissues (GALT) and mesenteric lymph nodes but not in spleen. In contrast, interleukin 13 (IL-13) mRNA levels are not affected by SDZ 280.636. The findings that SDZ 280.636 inhibits polyclonal IgE responses and suppresses IL-4, but not IL-13 mRNA expression point towards differences in the regulatory pathways of IL-4 and IL-13 gene transcription in lymphoid organs. Thus the mechanism of action appears to involve a specific suppression of IL-4 gene transcription in cells occurring in Peyer's patches and mesenteric lymph nodes which are among the first constituents of the immune system encountered by an orally administered drug.