MXPA98001253A - Allergen fusion proteins -xc - Google Patents

Abstract

Fusion proteins comprising one or more antigens, and one or more fractions that interact with the human Fcy II receptor (FcyRII) (CD3

Description

Porgnoa OF FERIOW OF ALERBEN -3K-D32 The present invention relates to human IgG and antigen / allergen complexes (or a combination of antigens / allergens). It refers to fusion proteins between anti-CD32 molecules and antigen / allergen (or a combination of antigens / allergens). Allergens are defined herein as antigens to which atopic patients respond with allergic reactions. The antigens, as used in the present, may be of different origins, for example environmental allergens (for example, household dust mite, birch pollen, grass pollen, cat antigens, cockroach antigens), or food allergens (e.g., cow's milk, peanut, shrimp, soy), or a combination of both, or non-relevant antigens such as bovine serum albumin (BSA). Allergy is a disease in which IgE antibodies mediate the activation of effector cells (mast cells, basophils, eosinophils), but they also improve the presentation of the antigen through receptors by low and high affinity for IgE (B cells, monocytes, dendritic cells). These igE actions can be counteracted by specific IgG antibodies that interact with CD32 (Fc? RII) on both effector and inducer cells. Therefore, allergy should be considered as a disease with an imbalance of Th2 cells with Th2. The success of IgG antibodies to counteract IgE in vivo depends on the relative concentration of specific IgE on specific IgG, and also on the IgG isotype. All human isotypes have a low affinity for CD32, which can be overcome by the complex formation with allergen, but many allergens do not form sufficiently large complexes with the IgG molecules to allow a stable bond with CD32. To date, two forms of active vaccination using allergens are used. The most common is the so-called "immunotherapy", which depends on frequent immunizations with relatively high concentrations of allergens. This technique is only moderately effective in a minority of allergic diseases, such as allergy to bee stings, and in some cases of rhinitis and conjunctivitis, and recently some reports have shown effectiveness in asthma and atopic dermatitis. Classically, the subcutaneous route is used for the administration of allergens, but this route has recently been compared with oral application, or even with local application, and the results are generally positive but not always consistent. A more recent approach to immunotherapy, the so-called Saint-Remy technique (see, for example, European Patent Numbers EP 0,178,085 and 0,287,361), uses autologous IgG antibodies that complex in vitro with the relevant allergens. This approach allows smaller amounts of allergen to be applied with fewer side effects. The mechanism behind both therapies is unclear. In the classic approach, there seems to be a beneficial effect if therapy induces an increase in specific IgG antibodies, although not every significant increase in specific IgG is correlated with successful immunotherapy. A possible reason for this is the relatively low affinity of IgG antibodies to CD32 on B cells, monocytes, and mast cells. The Saint-Remy approach selects patient-specific IgG antibodies, which are subsequently mixed with relevant allergens in vitro. In this way, they ensure that the allergen can not freely react with cells or other antibody isotypes on cells, such as IgE on mast cells. In addition, presumably anti-idiotypic antibodies are raised against specific IgG molecules, and subsequently prevent allergy, although this has not been confirmed by experimental data. IgG molecules are important for directing the immune response away from B cells, even the monocytes. Therefore, the Saint-Remy approach should work when allergen-specific IgG is used in general, rather than specific IgG for the donor-specific allergen. However, a problem with the Saint-Remy approach is in the low affinity of human IgG antibodies to their Fc? on B cells, in contrast to IgE which binds easily to almost all cells that present antigen, including B cells, leading to an extension of the allergy and to an induction of Th2 cells. The use of complexes should cancel out the problem of low affinity, due to a better avidity of the complex, compared with the simple IgG molecules; however, again, the combination of allergen / IgG in complexes is unfavorable, because the natural number of allergen epitopes to bind to human IgG molecules is less than 5, too little to improve the avidity of the complex by the receptors. With the present invention, the risk factors of classical immunotherapy are reduced, and the problems encountered in the isolation of specific IgG molecules and the low affinity of these IgG antibodies to CD32 are avoided. The binding of IgE with mast cells and basophils induces the release of histamine when cross-linked by the antigen. In addition, IgE can improve antigen presentation by human B cells (Figure 1). Otherwise, in the absence of IgE, B cells are poor cells in the presentation of antigen, with the exception of a very small portion of B cells, which have a specific antigen receptor. The recovery of antigen can occur through pinocytosis, but it takes high antigen concentrations and / or long incubation times. In Figure 2, it is shown that allergen pinocytosis for 5 days can mimic the presentation of IgE-mediated antigen, but that IgG-mediated antigen presentation does not lead to T cell stimulation. In fact, the data indicate the prevention of allergen presentation by complexing the allergen with IgG. In another series of experiments, the influence of these IgG-allergen complexes on the presentation of IgE-mediated antigen or on the recovery of allergen through pinocytosis was studied. In Figure 3, it is shown that the IgG complexes inhibit the IgE-mediated antigen presentation in a dose-dependent manner, and furthermore, antigen presentation is inhibited after pinocytosis. This effect is not specific to the antigen (IgG / BSA complexes inhibit the Der Pl specific stimulus), and monomeric IgG (uncomplexed) does not inhibit antigen presentation. The toxicity of the IgG / allergen complexes is excluded by the experiment shown in Figure 4. The IgG3-Der Pl complexes (pre) incubated with human monocytes, can induce a normal T cell response. This implies that the IgG / allergen complexes inhibit the presentation of allergen by B cells, and at the same time stimulate monocytes and dendritic cells. In a similar manner, the aCD32 antibodies or the accumulated IgG coated on the bottom of the cavities where the stimuli are made, also inhibit the presentation of antigen by the B cells (Figure 5). The efficiency with which CD32 directed molecules inhibit antigen presentation, more likely depends on the amount of crosslinking of the CD32 molecule. However, the direction of the CD32 antigen on B cells, as in the natural IgG / antigen complexes (Figure 2), or as in the antigen / aCD32 fusion proteins according to this invention (Figures 13a and 13b), does not lead to the presentation of antigen in the complex. On the other hand, the same complexes directed to monocytes do induce an antigen-specific T cell response (Figures 4, 13a, and 13b). This phenomenon can be explained by the presence of CD32B on B cells, where monocytes and also dendritic cells express CD32A. Accordingly, the IgG / antigen complexes, as well as the antigen / aCD32 fusion proteins, direct the immune response to the lineage cells of monocytic and dendritic cells, which induce T cell responses, mainly of type B cells. Thl, and other APCs that predominantly express CD32B will not present the antigen in the complex, and therefore, these complexes prevent (in addition) the induction of Th2 cell responses by B cells. The crosslinking interaction between the complex and CD32 does not depend of heavy crosslinking (more than 2 CD32 molecules), with the proviso that the link of the complex is sufficiently strong to allow a stable interaction. In fact, it has been shown that the binding of very large complexes, which induce multiple cross-linking of CD32 (mimicked by accumulated coated IgG) on monocytic cells, leads to up-regulation of co-receptors, such as CD80, important for the specific stimulation of the T cell antigen (Table 1). Apart d? the presentation of the antigen, the IgG / antigen complexes or the antigen / aCD32 fusion proteins according to the invention, also inhibit Ig production by normal human B cells. In Figure 14, it is shown that tonsillar B cells (from the tonsils) stimulated with aCD40 and IL-4 in the presence of antibodies to CD32 or accumulated coated human IgG, which interact efficiently with CD32, are no longer able to produce antibodies. . The treatment with F (ab) fragments of aCD32 is as efficient as the use of complete antibodies (Figure 15), indicating that, for the inhibition of Ig production after the stimulation of aCD40 and IL-4, cross-linking is not necessary. of the receiver. The B cells that are stimulated by the. antigen through its B-cell receptor (BCR) need the cross-linking of the B cell receptor and CD32. The down regulation of Ig production is reversible and depends on time. When antibodies to CD32 are added to B cells 4 days after stimulation with aCD40 plus IL-4, no inhibition of Ig production is seen. In Figure 16, it is shown that also, the antigen-specific induction of antibody production is inhibited by treatment with aCD32 of B cells. In nature, IgG molecules control the response of B cells through the interaction with CD32.

Especially, the effects of IgE antibodies, which generally have a positive effect on B cells (CD23 positive), can be counteracted by IgG molecules. This prevents the organism from having hyper-reactive immune reactions to the antigens. In allergy, in which IgE antibodies are abundantly present, clearly the natural IgG antibodies have failed to control the immune response. It is interesting that in man, IgE and IgG4 are regulated by the same interleukin, that is, IL-4. However, IgG4 has the lowest affinity of all subclasses of human IgG for CD32. The present invention relates to fusion proteins comprising: a) one or more antigens, and b) one or more fractions, such as from antibody molecules, which interact with the human Fcy II receptor (FcyRII) (CD32) , briefly referred to hereinafter as "the fusion proteins according to the invention". The fusion proteins according to the invention overcome the problems due to the low affinity of the human IgG molecules with CD32. By combining an antibody to CD32 having a Ka < 10"6 with antigen, both negative (B cells) and positive effects of natural IgG molecules are obtained, including the selective stimulation of the immune system that leads to the induction of Thl / ThO memory in the absence of antibody production. is innocuous and directs the immune response to antigen-expressing cells that express CD32, whereby cells predominantly expressing CD32A mediate the presentation of antigen, leading to the induction and activation of Thl cells, as a result of IL- 12 produced by the antigen-presenting cells express CD32A The fusion proteins according to the invention preferably do not have heavy cross-linking.The number of binding sites for CD32 is preferably limited, in order to avoid down-regulation of stimulating co-receptors such as CD40, CD80, or CD86. They also abolish the effector function of the mast cells bearing parts specific IgE of the fusion protein. They have the following unique characteristics: 1) silencing the presentation of antigen mediated by B cells (stop the induction of Th2 cells); 2a) stop the induction of IgE switching; 2b) stop the production of Ig in B cells; 3) stimulate the compartment of the T cells through the interaction with monocytic and / or dendritic cells (stimulation of Thl cells); and 4) silencing the mast cells carrying IgE specific for parts of the fusion protein. These comprise: a) one or more antigens, and b) one or more fractions, such as from antibodies, which interact with the human Fcy II receptor. The parts of the fusion protein - which interact with the Fcy II receptors, for example - may be: 1) complete or incomplete human or humanized (modified) IgG antibody fractions, as long as interaction with these receptors is still possible, which implies that all or part of the Fe fragment must be present; or 2) fractions of human or humanized CD32 antibody, or portions thereof, eg, Fab fragments, which still specifically recognize and bind to the FcyRII antigen (CD32), as expressed on B cells, mast cells, monocytes, and dendritic cells, for example, human or humanized manipulated IgG or aCD32 antibody fractions, or portions thereof, that recognize FcyRII (CD32) with a higher affinity than native IgG or aCD32 antibodies. The antigens can be from whole proteins or parts thereof that still have T cell epitopes on the sequences present in the fusion protein. Any antigen to which allergic patients respond with IgE-mediated hypersensitivity reactions, such as allergens in atopic dermatitis, allergic asthma, allergic rhinitis, and allergic conjunctivitis can be used. The most common environmental allergens are: household dust mite, birch pollen, grass pollen, cat, or cockroach. Each of these allergens has one or more "major allergens" (for example, for the dust mite of the home, the main allergen is Der Pl; for birch pollen, the main allergen is Bet VI). However, complete antigens are not necessary, because the fusion protein will normally induce only T cell responses, and T cells respond to small peptides (8 to 12 amino acids long). Accordingly, a selection of T cell epitopes in the fusion protein can be included for each allergen, thereby reducing the size and molecular weight of the complex. Accordingly, fusion proteins can be produced from one or more DNA arrays containing T-cell epitope, rather than from genes for complete antigens. Cross-reactive epitopes overlapping between allergens are preferred. The fusion protein must specifically bind to CD32, and must contain one or more, for example two or more T cell epitopes for one or more antigens / allergens. To allow a correct processing of the antigen, DNA arrays slightly longer than the actual T-cell epitope in the preparation of the fusion proteins should preferably be included. For fusion to the gene to code for the aCD32 antibody, short DNA sequences derived from genes cloned from major allergens are preferably used, such as house dust mite major allergen I (Der Pl), or house dust allergen. birch pollen (Bet V). These short DNA sequences contain the genetic code for one or more T-cell epitopes, which after processing, appear on the surface of the antigen-presenting cells, and consequently, induce an immune response in the allergen-specific T cells. respond For Der Pl, most T-cell epitopes can be found in the sequence at positions 101-143 in the one-letter amino acid code (SEQ ID No. 1): QSCRRPNAQRFGISNYCQIYPPNANKIREALAQPQRYCRHY T 101 110 120 130 140 Especially, the amino acid sequence at positions 101-131 in the one-letter amino acid code (SEQ ID No. 2): QSCRRPNAQRFGISNYCQIYPPNANKIREAL 101 110 120 130 contains when Jggnos three T-cell epitopes, which bind to a number of HLA class II molecules. The fusion between, for example, aCD32 and antigen (s), can be effected either at the level of the protein (chemical fusion), or at the level of the gene (recombinant fusion protein), and the invention also comprises a process for producing fusion proteins as defined above. This is done in a conventional manner, and preferably comprises the use of recombinant genetic techniques or chemical cross-linking. Recombinant fusion proteins are preferred. The preparation using recombinant genetic techniques can be carried out by known methods, for example, in the following manner: a gene segment containing the antigen binding site and parts of the Fe region down to the exon CH2 of a monoclonal antibody to CD32 by example, clone IV.3) is amplified by polymerase chain reaction from, for example, IV.3 cDNA, and cloned. The appropriate RNA is purified as a source for the amplification of the polymerase chain reaction, for example, from house dust mites, for the amplification of the Der Pl gene. The allergen gene is co-ligated with the segment Heavy chain isolate in an appropriate mammalian expression vector, such as p350. In addition, the corresponding full light chain gene of, for example, IV.3, is isolated and cloned into an appropriate mammalian expression vector, such as p345, which has characteristics similar to p350. Arabian recombinant plasmids are used for co-transfection in, eg, COS cells, to allow release of the resulting recombinant fusion protein in the culture medium. Purification of the product is preferably done by immunoaffinity purification, based on anti-light chain antibody columns, which are already available in large quantities. Alternatively, a CD32-specific single-chain antibody (scFv) derived from an appropriate phage display library, for example, as described by De Kruif et al., Proc. Nati Acad. Sci. USA 92 (1995) 3938-3942, can be used to make a recombinant fusion protein with the cloned allergen genes (Figure 6). The DNA is extracted from the binding phages, and a DNA fragment containing a semisynthetic VHDJH region fused to one of the VL chains is cloned by means of a flexible linker (scFv fragment). Fusions, for example, with the Der Pl gene, are performed using defined synthetic oligonucleotides that code for the T cell epitopes from Der Pl and other major allergens selected for their high cross-reaction potential (T cells), with the object to cover as many patients as possible with a fusion protein. These oligonucleotides are co-ligated with the scFv antibody fragment into an appropriate mammalian expression vector. The recombinant vector, eg, a plasmid, is stably transfected into the appropriate cells such as COS and / or CHO cells, and the resulting fusion protein is purified from the cell supernatant, for example, by immunoaffinity chromatography as described previously. The resulting gene construct can be expressed, for example, in CHO cells, especially for large-scale production; however, other systems for production by recombinant genetic techniques are also suitable, especially when only T-cell epitopes are used, ie, there is no need for glycosylation. The antibodies to CD32 can be obtained, for example, from a human Ig phage display library containing only F (ab) fragments of the natural antibody. However, the source of the genetic material that codes for the antibody or the antigen / allergen is not critical. The preparation by chemical crosslinking can also be carried out by known methods, for example, for a fusion protein between Mc.a-CD32 and Der Pl, by using the methods described by Calsson et al. FBiochem. J. 173 (1978) 723-737], Cumber et al. Enzymol. 112 (1985) 207-224], and Peeters et al [J. Itnmunol Meth. 120 (1989) 133-143]. Briefly stated, Der Pl and Mc.a-CD32 are derived separately with SPDP in molar proportions of 1/20 and 1/5 respectively, to introduce 2-pyridyl disulfide residues. After a gentle reduction and desalination by gel chromatography, the Mc.a-CD32 derivatives with SPDP (whose disulfide groups are reduced to thiol groups) are incubated with Der Pl derived with SPDP in a molar ratio of 1 / 1.6. The resulting Der Pl-Mc.a-CD32 product is then purified, for example, by gel filtration, on, for example, Superose-12, and by anion exchange chromatography on, for example, FPLC Mono-Q. The starting materials are known or can be prepared according to known procedures, or in a manner analogous to known procedures, or in a manner analogous to that described herein, for example, in the Example. The fusion proteins according to the invention are useful for the prevention and / or treatment of allergies, particularly food allergies. It is not uncommon for patients suffering from anaphylactic response to a particular allergen, also suffering from that response to one or more different allergens. By the method of the present invention, it is possible to desensitize that patient with respect to two or more allergens simultaneously, by administering a fusion protein that includes antigens against each of these allergens. Preferably, the fusion proteins according to the invention will be used for the prevention and / or treatment of allergy in newborn babies who are at risk of allergy by food, for example to milk, or of established allergies in patients with allergy against allergens that are included in the particular fusion protein used. For these indications, the appropriate dosages, of course, will vary depending on, for example, the particular fusion protein used, the host, the mode of application, and the intended indication. However, in general, it is indicated that satisfactory results are obtained with one to three vaccines for 1 to 2 years, but if necessary, additional repeated vaccinations can be made. It is indicated that, for these treatments, the fusion proteins of the invention can be administered in dosages and with an application program similar to that conventionally employed. Accordingly, the invention also relates to the use of a fusion protein as defined above, in the prevention and / or treatment of allergies, including food allergies, and to a method for the treatment of allergies, which comprises administering to a subject in need of such treatment, a prophylactically or therapeutically effective amount of a fusion protein as defined above, together with at least one conventional pharmaceutically acceptable carrier or diluent, as well as fusion proteins as defined above, for use as an agent pharmaceutical, especially as an antiallergic agent. The fusion proteins according to the invention can be mixed with conventional pharmaceutically acceptable diluents and vehicles, and optionally, other excipients, and can be administered parenterally, intravenously, or enterally, for example intramuscularly, or subcutaneously. The concentrations of the fusion protein will, of course, vary depending on, among other things, the compound employed, the treatment desired, and the nature of the dosage form. Accordingly, the invention also includes pharmaceutical compositions comprising a fusion protein as defined above, together with at least one pharmaceutically acceptable carrier or diluent. It also relates to a process for the preparation of an anti-allergy medicament, which comprises mixing a fusion protein as defined above, together with a pharmaceutically acceptable carrier or diluent, and to the use of a fusion protein as defined above, for the manufacture of a medicament for the prevention and / or treatment of allergies, including food allergies.

The following abbreviations are used herein: aCD32 antibody to CD32 (anti-CD32 antibody). Ag Antigen. APC cell presenting antigen. BCR B cell receptor. Bet VI major allergen of birch pollen.

BSA bovine serum albumin. CNBR cyanobromide. Der Pl allergen greater than household dust mite (Dermatophagoides pteronyssinus) DPT antigen from household dust mite. DTT dithioerythritol. EBV Epstein-Barr virus. ELISA enzyme linked immunosorbent assay. FACS cell sorter activated by fluorescence. FcyRII receptor Fcy II human (= CD32) Fig. Figure number. FPLC rapid pressure liquid chromatography. GaM goat antibody against mouse. HAc acetic acid. HLA human leukocyte antigen. HPHT hydroxyapatite. IC immune complex. Ig immunoglobulin. IL-12 interleukin-12. LST lymphocyte stimulus test. min. minutes MR molar ratio. PBS serum regulated with phosphate. PCR polymerase chain reaction. p-NPP p-nitrophenyl phosphate. sd standard deviation. SPDP 3- (2-thiopyridyl) propionate N-succinimidyl.

In the following example, which illustrates the invention without limiting it, all temperatures are in degrees Celsius.

Bl-M-ipifr. Preparation and purification of anti-CD32 monoclonal fusion protin-Der Pl ?) Method: Der Pl is conjugated in a covalent manner with Mc.a-CD32 by using SPDP, a bifunctional coupling reagent. In short, Der Pl and Mc.a-CD32 are derived separately with SPDP in molar ratios of 1/20 and 1/5, respectively, for the introduction of 2-pyridyl disulfide residues. After a gentle reduction and desalination by gel chromatography, the Mc.a-CD32 derivative with SPDP (whose disulfide groups are reduced in thiol groups) is incubated with the derivative Der Pl with SPDP in a molar ratio of 1 / 1.6. . The resulting Der Pl-Mc.a-CD32 conjugate is purified by gel filtration on Superosa-12, and by anion exchange chromatography on FPLC Mono-Q.

B) Materials: a) SPDP: molecular weight 312.4, - 25.61 mM delivery solution (8 milligrams / milliliter) in dimethyl formamide, prepared immediately before coupling. b) Der Pl: purified by immunoaffinity chromatography on Mc.a-Der Pl (4C1 / B8 / 3F8) covalently coupled with CNBR-Sepharose 4B. Molecular weight of 28 kD; isoelectric point of 5.0 (PHAST IEF); sterile filtrate, diluted in serum regulated with phosphate. c) Anti-CD32 (IV.3) monoclonal: isotype: mouse IgG2b-K chain, - molecular weight of 150 kD; isoelectric point of 6.2 (PHAST-IEF); purified from the supernatant of the culture medium on Protein-A and HPHT, and dialyzed against serum regulated with phosphate.

Reaction Mixture A: To 1.2 milliliters of Der Pl solution (0.84 milligrams) are added 23.4 microliters of SPDP supply solution (20 molar excess), and the mixture is stirred in a 2.5 milliliter conical reagent bottle (Pierce ) for 45 minutes at room temperature (22 ° C). The reaction mixture is filled to 2.5 milliliters with phosphate-buffered serum and unconjugated SPDP, and the released N-hydroxysuccinimide is removed by desalination on a 9.1 milliliter Pharmacia PD-10 disposable desalination column (Sephadex G-25) equilibrated with 50 milliliters of serum regulated with phosphate. After the application of the fraction containing Der Pl-activated with 2-pyridyl disulfide, it is eluted and grouped with 3.5 milliliters of phosphate-regulated serum. The concentration is approximately 0.17 milligrams / milliliter, which corresponds to 0.6 milligrams. '' Estimation of substitution srad with 2-pyridyl disulfide: A sample of 100 microliters is filled up to 150 microliters with phosphate-regulated serum, and 50 microliters of 150 mM dithioerythritol diluted in phosphate-buffered serum is added. The concentration of pyridine-2-thione released after the addition of dithioerythritol can be determined by measuring the absorbance at 343 nanometers (molar extinction coefficient: 8080 M ^ cpf1), and corresponds to the disulfide residues of 2- pyridyl introduced. For an increase to 343 measured OD nanometers of 0.074 (corrected for dilution), the degree of substitution is calculated as 1.5 moles of 2-pyridyl disulfide / mol of Der Pl.

Reaction B mixture: To 0.5 milliliters of a solution of Mc.a-CD32 (4.2 milligrams), add 5.5 microliters of SPDP supply solution (5 molar in excess), and the mixture is stirred for 45 minutes at room temperature. room temperature. The reaction mixture is applied to a 2.5 milliliter Pierce GF-5 disposable desalting column (equilibrated with 10 milliliters of NaO.lM / HAc acetate buffer, 0.1M NaCl, pH 4.5). After application, the scruffy fraction is eluted and grouped with 1.5 milliliters of acetate buffer (pH 4.5), and filtered with a MILLEX-GV filtration unit of 0.22 microns (under protein binding).

Introduction of SH Groups: 2-pyridyl disulfide groups bound to protein are converted to thiol groups linked to protein by reduction with dithioerythritol. 1.5 milliliters of disheveled sample are incubated under agitation with 1 milliliter of 62.5 mM dithioerythritol (diluted in acetate buffer) at room temperature for 30 minutes (final concentration of dithioerythritol: 25 mM). Because the 2-pyridyl disulfide increases its electrophilicity at an acid pH, reduction can still be performed at a low pH, where the disulfide bonds of the native protein will not be affected. For the measured OD 343 nanometer increase of 0.447, the degree of substitution is calculated as 7 moles of thiol / mol groups of Mc.a-CD32. The reaction mixture is applied on a Pharmacia PD-10 column (equilibrated with 50 milliliters of phosphate-buffered serum), and after application, the scruffy fraction is eluted and pooled with 3.5 milliliters of phosphate-buffered serum. The sample is concentrated to 0.6 milliliters with a concentration unit CENTRIPLUS-10 (membrane YM10). The concentration is approximately 3.1 milligrams / milliliter, which corresponds to 1.86 milligrams.

C) Coupling procedure: 3.3 milliliters (0.56 milligrams) of Der Pl (activated with pyridine disulfide) and 0.6 milliliters (1.86 milligrams) of Mc.a-CD32 (activated with SH) are mixed and stirred in a reagent jar Pierce of 5 milliliters for 2 hours at room temperature (22 ° C), and for 15 hours at + 4 ° C. The molecular ratio of Der Pl / Mc.a-CD32 in the reaction mixture is 1.6 / 1. The coupling reaction is monitored by measuring the increase of OD 343 nanometers, due to the increase of the liberated pyridine-2-thione: Time OD (343 nm) Start 0.1112 15 min 0.1257 30 min 0.1342 45 min 0.1412 60 min 0.1470 75 min 0.1519 120 min 0.1590 D) Purification of the conjugate: Superosa-12: The reaction mixture (3.9 milliliters) is filtered with a unit of MILLEX-GV filtration of 0.22 microns, and concentrates up to 0.5 milliliters with a Centriplus-10 concentration unit. The final concentration of the conjugate is 3.7 milligrams / milliliter, corresponding to 1.9 milligrams. The sample is applied on a Superosa-12 column (HR 10/30) (Pharmacia) equilibrated with serum regulated with phosphate. Bed volume: 24 milliliters; flow rate: 0. 2 milliliters / minute; volume / fraction: 0.4 milliliters, -speed of the logger diagram: 1.5 millimeters / minute, -pressure: 0.3 MPa; Scanning wavelength: 280 nanometers. The eluted high molecular weight fractions are divided according to the elution profile into three groups: 1.2 milliliters, Group A: Fraction # 21-23 1.2 milliliters, Group B: Fraction # 24-26 1.2 milliliters, Group C: Fraction # 29-31 The groups are sterile filtered (MILLEX-GV 0.22 microns), and stored under sterile conditions at + 4 ° C. The total protein concentration is approximately: Group A: 100 micrograms / milliliter; Group B: 200 micrograms / milliliter; Group C: 390 micrograms / milliliter.

FPLC-Mono 0: 1 milliliter of Group C (390 micrograms) is dialyzed against 20 mM ethanolic amine / HCl, 0.01% NaN3 buffer, pH of 9.0, and purified by ion exchange chromatography on anion exchanger FPLC-Mono Q (HR5 / 5). Starting buffer A: 20 mM ethanolic amine / HCl, 0.01% NaN3, pH 9.0; limiting buffer B: A + 0.5 M NaCl, pH of 9.0; flow rate: 1 milliliter / minute, -volume / fraction: 1 milliliter; recorder diagram speed: 5 mm / minute; Pressure: 1.2 Mpa; Scanning wavelength: 280 nanometers. After application, a linear gradient program is started for 30 minutes (conductivity: 50 - 1260 μS / cm). The relevant peak is eluted in fraction # 8 (at 340 μS / cm). The protein concentration is approximately 45 micrograms / milliliter (OD 280 nanometers, E% : 14.0). The sample is dialyzed against serum regulated with phosphate, and sterile filtered. The protein concentration of the final purified material is 20 micrograms / milliliter (OD 280 nanometers).

E) Analytical Determinations: a) Total protein concentration: Total protein concentrations are estimated according to Bradford, using the BIO-RAD I Protein Assay Kit; standard: bovine IgG. b) Determination of Der Pl, of Mc.a-CD32, and of conjugate of Der Pl-Mc.a-CD32 by means of assay i ™? -? t? r -linked fiber < n - »p» -n - »t Enzyme-linked immunosorbent assays are performed on solid phase in PVC microtiter plates. The coating regulator of NaHC30.1 M / Na2C03, 0.01 percent NaN3, pH 9.6, serum phosphate buffered with 0.05 percent Tene-20, is used as a wash solution, and fetal calf serum to 2 percent in washing regulator as a diluent for the samples, and biotin and enzyme conjugates. The substrate is 1 milligram / milliliter of p-NPP diluted in lM dietanolic amine / HCl buffer, pH of 9.8. The stop solution is 2 M NaOH. All incubation steps are done in a humidified chamber. The equipment for sample processing and OD reading at 405 nanometers is the Beckman Biomek-1000 laboratory workstation. Quantitative evaluation: Beckman immuno-adjustment, - curve fitting: four-parameter logic. b) 1) Determination of Der Pl (Figure 7): Coating: 100 microlitres / Mc.a-Der Pl (5H8) cavity, 10 micrograms / milliliter overnight at + 4 ° C. After washing, add 100 microliters / sample cavity: a) Der Pl (starting material for coupling) (250-0.49 nanograms / milliliter) as the standard. b) Superosa-12, Group A (4000-7.81 nanograms / -mililiter). c) Superosa-12, Group B (4000-7.81 nanograms / -mililiter). d) Superosa-12, Group C (4000-7.81 nanogra os / -mililitro).

Incubation for 2 hours at + 37 ° C. After washing, add 100 microliters / cavity of Mc.a-Der Pl conjugate. (4Cl / B8 / 3F8) -Biotine diluted to 1/500, and incubated for 2 hours at + 37 ° C. After washing, 50 microliters / cavity of streptavidin-alkaline phosphatase conjugate diluted to 1/1000 is added and incubated for 1 hour at + 37 ° C. After washing, add 100 microliters / cavity of substrate, incubate for 60 minutes at 37 ° C, and stop. Quantitative evaluation from the standard curve: Superosa-12 ,, Group A: 16.1 micrograms / milliliter. Superosa-12, Group B: 54.9 micrograms / milliliter. Superosa-12, Group C: 135 micrograms / milliliter. b) 2) Determination of Mc.a-CD32 (mouse IaG2b) (Fiaura 8): Coating: 100 microlitres / cavity of goat Pea-mouse IsG2b (Southern), at 5 micrograms / milliliter overnight at +4 ° C. After washing, add 100 microliters / sample cavity: a) Mc.a-CD32 (starting material for coupling) (1000-1.96 nanograms / milliliter) as the standard. b) Superosa-12, Group A (8000-15.63 nanograms / - ililiter). c) Superosa-12, Group B (8000-15.63 nanograms / -mililiter). d) Superosa-12, Group C (8000-15.63 nanograms / -mililiter).

Incubation for 2 hours at + 37 ° C. After washing, 50 microliters / cavity of goat Pea conjugate-mouse IgG2b-alkaline phosphatase (Southern) diluted 1/1000 are added and incubated for 2 hours at + 37 ° C. After washing, add 100 microliters / cavity of substrate, incubate for 15 minutes at room temperature, and stop. Quantitative evaluation from the standard curve: Superosa-12, Group A: 17.7 micrograms / milliliter. Superosa-12, Group B: 58.8 micrograms / milliliter. Superosa-12, Group C: 190 micrograms / milliliter. Evaluation of the molar ratio of Der Pl / Mc.a-CD32 in the conjugate according to the enzyme-linked immunosorbent assay: Superosa-12, Group A: 4.9 / 1 Superosa-12, Group B: 5.0 / 1 Superosa-12, Group C: 3.8 / 1 b) 3) Detection of Der Pl-Mc.a-CD32 conjugate: b) 3) l) Coating: Mc.a-Der Pl (mouse IgG2a) (Figure 9): Coating: 100 microliters / cavity of Mc.a-Der Pl (5H8), at 10 micrograms / milliliter overnight at + 4 ° C.

After washing, add 100 microliters / sample cavity: a) Der Pl (starting material for coupling) (4000-7.81 nanograms / milliliter). b) Superosa-12, Group A (4000-7.81 nanograms / -mililiter). c) Superosa-12, Group B (4000-7.81 nanograms / -mililiter). d) Superosa-12, Group C (4000-7.81 nanograms / -mililiter).

Incubation for 2 hours at + 37 ° C. After washing, 50 microliters / cavity of goat Pea conjugate-mouse IgG2b-alkaline phosphatase (Southern) diluted to 1/1000 is added and incubated for 2 hours at + 37 ° C. After washing, add 100 microliters / cavity of substrate, incubate for 30 minutes at room temperature, and stop. The Der Pl-Mea-CD32 conjugate bound to Mc.a-Der Pl is detected by the Mea-CD32 division in each group of the purified fraction of Superose-12. b) 3) 2) Coating: mouse Pc.a-IgG2b (Figure 10): Coating: 100 microlitres / goat Pea cavity-mouse IgG2b (Southern), at 5 micrograms / milliliter overnight at + 4 ° C. After washing, 100 microliters / sample cavity is added. a) Der Pl (starting material for coupling) (1000-1.95 nanograms / milliliter). b) Superosa-12, Group A (8000-15.63 nanograms / -mililiter). c) Superosa-12, Group B (8000-15.63 nanograms / -mililiter). d) Superosa-12, Group C (8000-15.63 nanograms / -mililiter). Incubation for 2 hours at + 37 ° C. After washing, add 100 microliters / cavity of Mc.a-Der Pl conjugate (4C1 / B8 / 3F8) -Biotin (mouse IgGl) diluted to 1/500, and incubate for 2 hours at + 37 ° C. . After washing, 50 microliters / cavity of streptavidin-alkaline phosphatase conjugate diluted to 1/1000 is added and incubated for 1 hour at + 37 ° C. After washing, add 100 microliters / cavity of substrate, incubate for 60 minutes at 37 ° C, and stop. The Der Pl-Mea-CD32 conjugate bound to mouse Pea-IgG2b is detected by dividing Der Pl in each group of the purified fraction of Superosa-12. o) Dafcarminaa-L? n dal paao molaoular of the conjugate of Dar Pl- Me.a-CD32 madianfca alact-ro-Eoraaia ^ gradient of the native polyacillic era; The molecular weight of the conjugates of Der Pl-Mc.a-CD32 in the groups of the purified fraction of Superosa-12, is estimated with a gradient of 4 to 15 percent of native PHAST gel (Pharmacia) (separation scale: 1000 kD - 150 kD), comparing with the native high molecular weight standard proteins (kit, Pharmacia). Detection: silver-stained (silver-stained case, Pharmacia) (Figure 11): Evaluation: Super-12, Group A: band with a molecular weight of 700 kD. Superosa-12, Group B: band at a molecular weight of 460 kD, 330 kD. Superosa-12, Group C: band at a molecular weight of 330 kD, 170 kD.

The molecular weight of the final conjugate of FPLC-Der Pl purified with Mono-Q-Mea-CD32 is estimated at 330 kD (Figure 12).

F) Test Results: a) Specific stimulus of the antigen of the T cell clone CFTS4: 3.1 with a Der Pl fusion protein of CD32: The chemically linked aCD32 anterior (Medarex clone IV.3) with purified Der Pl is used to stimulate the specific T cell clone of Der Pl CFTS4: 3.1 in different concentrations, using a standard LST. In a first set of experiments, Group A and Group B are used from the chemically fused preparation, Group A containing a single band of 700 kD (two aCD32 molecules fused with 10 Der Pl molecules), and the Group consisting of B in two bands, at 460 kD and 330 kD (one molecule aCD32 with 10 Der Pl, and one molecule aCD32 with 5 Der Pl, respectively). In a second set of experiments, the purified protein is used from Group C, which consists of a single band of approximately 300 kD (an aCD32 molecule fused with five Der Pl molecules). Monocytes and B cells are pre-incubated for 1 hour at room temperature with the different fractions indicated in Figures 13a and 13b. As the control stimulus, 100 micrograms / milliliter of house dust mite antigen is added to the T cells, plus antigen presenting cells during the entire culture period. B cells are able to stimulate CFTS4: 3.1 with home dust mite antigen but not with Group A, Group B, or Group C, whereas monocytes can stimulate CFTS4: 3.1 with mite antigen from dust home with Group A, Group B, and Group C. This confirms the above findings with the natural IgG allergen complexes referred to above. In addition, this indicates that, for the stimulation of the antigen by CD32 monocytes, cross-linking is not necessary, since all fractions are stimulating the T cells equally well. b) Inhibition of Sintaig to Igfi; Purified human tonsillar (of the amygdala) B cells are stimulated with aCD40 and IL-4 in the presence and absence of commercially available antibodies to CD32, aggregated at the concentrations indicated (Figure 14). After 9 days, the culture supernatants are tested for the IgE and IgGl content. All aCD32 antibodies inhibit the production of IgE as well as IgGl in a dose-dependent manner. In Figure 15, it is shown that even Fab fragments from the Medarex IV.3 clone can inhibit the synthesis of IgE. This indicates that B cells that are not activated by means of their B cell receptor can be blocked in their production of antibodies by the monomeric interaction with CD32. For B cells that are stimulated in a specific manner of the antigen, co-crosslinking between CD32 and the B-cell receptor is necessary. This implies that, for example, in the allergy where B cells recover antigen (e.g. Der Pl) through igE and CD23, and subsequently become stimulated Der Pl specific Th2 cells that lead to the production of IgE by B cells, these B cells can be deactivated by interacting with CD32 on the surface of the cells B. Actually, even in the known interaction of B cells - T cells, antibodies to CD32 block the synthesis of antibodies (Figure 16). Table 1 Monocytes (91 percent CD14 positive) derived from a normal donor by elution, are incubated with 100 units / milliliter of IFN-7 for 24 hours in the presence or absence of 5 micrograms / milliliter of accumulated human IgG ( coated in the cavities, overnight at 4 ° C), and / or 10 micrograms / milliliter of monoclonal mouse anti-human aCD32 (Medarex IV.3), and subsequently stained by the markers indicated for the activated cell sorter analysis by fluorescence. The upregulation induced by IFN-7 of CD80 and CD40 can be inhibited by heavy cross-linking of FC7 receptors by accumulated human IgG on monocytes. The previous incubation with aCD32 counteracts the inhibition by accumulated human IgG, indicating that: 1) inhibition by accumulated human IgG is mediated through CD32, and 2) the mere link with CD32 does not cause down regulation of the co-receptors.

The expression of HLA-DR is not influenced by the treatments.

Explanation of the Figures; Figure 1 »In lace» * r «-V ?? m dfl De-C-Pl- (31HIP CQ? 1 GD23 end relation with the anti-gong preaag-ton; Panel A = fluorescence; Panel B = proliferation B cells were boosted from Epstein-Barr virus with previously formed immune complexes, consisting of a constant concentration of IgE (? 7.5 micrograms / milliliter, • . 0 micrograms / milliliter, ßi 2.5 micrograms / milliliter), and variable concentrations of Der P1- (3) NIP. In Panel A, the binding of previously formed immune complexes with B cells is shown. Immune complexes with an identical molar ratio are connected with a line, and the real molar ratio (0.1, 0.25, 0.5, 1, 7) it is indicated in Arabic numbers next to the line. The IgE link reached a plateau at Der Pl concentrations of about 0.2 micrograms / milliliter. The IgE link in the absence of Der P1- (3) NIP is shown on the dotted box on the lower part of the right Y-axis. In Panel B, the presentation of the antigen is shown by irradiated Epstein-Barr virus B cells boosted with the same immune complexes. Here, the connecting lines indicate complexes made with identical concentrations of IgE, to emphasize that the presentation of the antigen was not influenced by the molar ratio of the immune complexes (correlation coefficient = 0.96). Monomeric complexes [highest concentration of Der P1- (3) NIP of each line] were also efficiently presented to the T cells. No stimulation of T cells was observed with any of the concentrations of Der P1- (3) NIP used (area shaded) in the absence of IgE molecules. The data show the incorporation of 3H-thymidine at day 5 (average + standard deviation of the triplicated cavities).

Figure 2: Comparison of presentation of the antigen with non-ixL B cells yi ff? ^ F? < The Der P1- (2) free NIP that remained in the culture medium during the entire stimulation period, induced a dose-dependent stimulation of the T cells. B cells of autologous Epstein-Barr virus (irradiated) were used as cells that present antigen. The IgE present in the complexes did not significantly improve the presentation of the antigen, but both the IgG1 and the IgG3 present in the immune complexes prevented the presentation of the antigen of Der Pl- (2) NIP for the T cells, comparing with Der P1- (2) Free NIP. The results are presented as the incorporation of 3H-thymidine at day 5 (average + standard deviation of the triplicated cavities).

Figure 3: Inhibition of antigen presentation through the use of IgG3-P1 complexes (3iNIP: In the absence of IgG3, a good stimulation of T cells is found when using IgG3-Der P1- complexes ( 3) NIP The titration of preformed IgG3-Der P1- (3) NIP complexes in the same ratio (1: 1) leads to a dose-dependent inhibition of IgE-mediated antigen presentation. to have this effect, the IgG3 complexes need to be present in the culture medium at the time of T-cell stimulation. The pre-incubation or "boost" of the B cells with the IgG3 complexes is unsuccessful due to the low affinity of IgG3 for CD32 on B cells. Similar inhibition is seen with the presentation of the antigen in the absence of IgE, and also with the complexes of IgG3-BSA- (3) NIP. -figure 4; graaentagion of antiqano mediated by Iqg3 by aonogitoa lnaanon -Tre-K-og. hands with preformed IgG3-Der P1- (3) NIP complexes (ratio of 1: 1) for 1 hour on ice in the absence or in the presence of accumulated non-relevant human IgG. Subsequently driven monocytes are washed and mixed with HLA-DP-coupled T cell clones specific for Der Pl, and after 5 days, proliferation is measured as the incorporation of ^ -thymidine. The IgG3 complexes presented by human monocytes induce an allergen-specific T cell response, which can be blocked by aggregated IgG, indicating the specificity of the interaction of IgG3 with monocytes. The data is shown as the mean + standard deviation of tripled cavities.

Figure 5: Proliferation of cellulose specifies the anticline of Acaro da ppl? P feK ho-gar: influence of an aCD32 or a Tqg tM-naana ss-ffizlada - a-oi-irs - a - presentation - ----- to the antiqen; B cells of human Epstein-Barr virus are propelled with (solid bars) or without (hollow bars) 250 micrograms / milliliter of home dust mite antigen overnight. After irradiation, the B cells are mixed with T cells from a specific T cell clone of Der Pl, and the cells are allowed to proliferate for 5 days. In the presence of different concentrations of aCD32 antibodies cross-linked by GaM, a dose-dependent inhibition of proliferation is observed. The same effect is observed when coating a non-specific accumulated human IgG or CD32 in the culture cavities. and r-r »t¡: Eiam loa gives protainaß of racotabinantaa antra fusion -Craqaent (g) da lEabl da aCD32 ggF? pri ado .a. a The Fab fragments can be obtained from a phage display library, as described by De Kruif et al., Proc. Nati Acad. Sci. USA 92 (1995) 3938-3942. However, any molecule (or part of a molecule) that efficiently interacts with human CD32 can replace the aCD32 Fab fragment. The allergen can be any protein or combination of proteins that cause allergy. In addition, allergens can be replaced by fragments of the allergens containing T-cell epitopes. The recombinant protein can be produced in any available expression system, independently of glycosylation and other modifications after translation. These fusion proteins can also be used for diseases that are characterized by an (over) production of unwanted Ig molecules, such as in rheumatoid arthritis, graft-versus-host disease, or any other disease where auto-antibodies play a role . In these cases, the allergens can be replaced by a simple undefined linker, to combine the two fractions of aCD32, however, the best results will be obtained when the allergen is replaced by the "auto-antigen" that causes the disease.

Fiqjfl 7; Determination of Give Pl groups of the fraction of the conjugate fraction of Dar Pl-Mte.a-CD32 purified by Suparoaa-12. By immunosorbent assay anlazadr- ^^ - ^ HfTTlHt Figure 8 na *? Awi «a.3lAn da Mc .a-0-t32 an group »gives the framed A-.1 with Dar-Pl-Me.a-CD32 purified with Suparoaa-12. madianta ananvn itwmnosorbanta linked non anitlma and ewparedada ^ - Figure 9; Detection of the conjugate of Per gl-Mc.a-CP32 in crupog of the purified fraction of guperoga-12 By means of an immunoabsorbent assay bound with antipi-a? sandwich: Coating: Mc.a-Der Pl.

Figure 10: Detection of Der Pl-Mc.a-CD32 conjugate in m By means of Bybogo -fr test in aj with epri mg and emulsion: Coating: mouse Pc.a-IgG2b.

Figure 11: Silver staining of franations from Groups A. B. and C: Track 1: 350 nanograms from Der Pl. Track 2: 50 nanograms from Mc.a-CD32. Lane 3: 500 nanograms of high molecular weight markers. Track 4: 500 nanograms of starting material for Superosa-12.

Lane 5: 100 nanograms of Superosa-12, Group A. Track 6: 100 nanograms of Superosa-12, Group B. Track 7: 200 nanograms of Superosa-12, Group C. Track 8: 250 nanograms of high molecular weight markers .

High molecular weight markers: 669 kD: thyroglobulin. 440 kD: ferritin. 232 kD: catalase 140 kD: lactate dehydrogenase. 67 kD: bovine serum albumin.

Figure 12; Silver-stained fusion protein purified from Group C; Clue l: 250 nanograms of high molecular weight markers. Lane 2: 45 nanograms of Mono-S purified material by rapid pressure liquid chromatography.

Figures 13a and 13b; Proliferation of T cells specific for Ao; Figure 13a: APC-Mo 250495 and CFB4: 2 Figure 13b: Influence of monomeric purified CD32 / Der Pl.

The chemically bound aCD32 (Medarex IV.3 clone) with purified Der Pl, was used to stimulate the specific Der T-cell clone, CFTS4: 3.1, in different concentrations, using standard LST [Van Reij sen, F.C. et al. (1992) J. Allersy Clin. Immunol. 90 184]. In a first set of experiments (Figure 13a), group A and group B were used from the chemically fused preparation: group A contained a single band of 700 kD (which is 2 aCD32 molecules fused with 10 molecules from Der Pl), and group B consisted of two bands of 460 kD and 330 kD (which is 1 molecule of aCD32 fused with 10 molecules of Der Pl, and 1 molecule of aCD32 with 10 molecules of Der Pl, respectively). In a second set of experiments (Figure 13b), the purified protein (CP230595) was used, consisting of a single band of approximately 330 kD (1 molecule of aCD32 fused with 5 molecules of Der Pl). Monocytes and B cells were previously incubated for 1 hour at room temperature with the different fractions indicated in the figure. As a control stimulus, 100 micrograms / milliliter of house dust mite antigen was added to the T cells plus the antigen presenting cells during the entire culture period. The stimulation of the T cells was measured as the incorporation of -thymidine (average + standard deviation of 4 cavities) after 5 days, as described (Van Reij sen et al., Supra).

B cells were able to stimulate CFTS4: 3.1 with home dust mite antigen, but not with group A, group B, or group C, whereas monocytes could stimulate CFTS4: 3.1 with the dust mite antigen of the home and with group A, group B, and group C. This corresponds to the previous findings with the natural IgG allergen complexes [Bheekha Escura, R. et al., Immunology (1995) £ - £ 343]. In addition, this indicates that, for the stimulation of the antigen by monocytes, cross-linking of more than two CD32 molecules is not necessary, since all fractions were equally effective in stimulating T cells.

Figure 14: Inhibition of the ginetia of IsE_x • • • • • • • "g" and IoGl: Purified human tonsillar cells (tonsils) were stimulated with aCD40 and IL-4, as described in Armerding, D. and collaborators Immunobiologie 188 (1993) 259-273, in the presence and absence of commercially available aCD32 antibodies aggregates at the indicated concentrations.After 9 days, the culture supernatants were tested for the IgE and IgGl content as It is described (Armerding, D. and colleagues, supra.) The results are shown as the production of antibodies from three pooled fractions of 9 replicates (mean + standard deviation) All the aCD32 antibodies inhibited the production of IgE as well as IgGl in a manner Dose-dependent inhibition of IgM and IgA was comparable.

Figure 15: inhibition of IgG glyantee; Purified human tonsillar (tonsillar) B cells were stimulated with aCD40 and IL-4 as described in Armerding, D. et al., = Iiß £ a, in the presence and absence of a commercially available antibody to CD32.

(Medarex IV.3), or a Fab fragment thereof, added at the indicated concentrations. After 9 days, the culture supernatants were tested for the IgE and IgGl content as described (Armerding, D. et al., Supra. The results are shown as the production of antibodies from three pooled fractions of 9 replicates ( average ± standard deviation.) Both the complete fragments and Fab from the Medarex IV.3 clone were able to inhibit the synthesis of IgE (IgGl, IgM, and IgA). jgyr? $: induction e? gg egpeoiflo de antigeno d 'acro de polYO home? influence of an aCP32; Human tonsillar (human tonsil) B cells were propelled with (solid bars) or without (hollow bars) 250 micrograms / milliliter of house dust mite antigen at night. After irradiation, B cells were mixed with T cells from a specific Der-T cell clone (CFTS4: 3.1), and the cells were allowed to proliferate and produce antibodies for 9 days. After 9 days, the culture supernatants were tested for the IgE and IgGl content as described in Armerding, D. et al., Supra. The results are shown as the production of antibodies from three pooled fractions of 9 replicates (mean ± standard deviation). In the presence of different concentrations of antibodies to CD32, a dose-dependent inhibition of IgE production is seen. This indicates that, even in the known interaction of B-cell T cells, aCD32 antibodies block the synthesis of antibodies.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Sandoz Ltd. (B) STREET: Lichtstrasse 35 (C) CITY: Basel (E) COUNTRY: Switzerland F) POSTAL CODE (ZIP): CH-4002 (G) TELEPHONE: 61- 324 52669 (H) TELEFAX: 61-322 7532 (A) NAME: Mudde, Geert C. (B) STREET: Ruzickagasse 88-104 / Haus 39 (C) CITY: Vienna (E) COUNTRY: Austria (F) ZIP CODE: A-1230 (ii) TITLE OF THE INVENTION: FUSION PROTEINS, (iii) NUMBER OF SEQUENCES: 2 (iv) LEGIBLE FORM BY COMPUTER: (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM : Patentln Reeléase # 1.0 Version # 1.25 (EPO) (v) CURRENT APPLICATION DATA: NUMBER OF APPLICATION: WO PCT / EP96 / (vi) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: GB 9516760.7 (B) DATE OF SUBMISSION: August 16, 1995 (2) INFORMATION FOR SEQ. ID. NO: l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iii) ) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Dermatophagoides pteronyssinus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Gln Ser Cys Arg Arg Pro Asn Wing Gln Arg Phe Gly lie Ser Asn Tyr 1 10 15 Cys Gln lie Tyr Pro Pro Asn Wing Asn Lys lie Arg Glu Wing Leu Wing 20 25 30 Gln Pro Gln Arg Tyr Cys Arg His Tyr Trp Thr 35 40 (2) INFORMATION FOR SEQ ID NO. : 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Dermatophagoides pteronyssinus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gln Ser Cys Arg Arg Pro Asn Ala Gln Arg Phe Gly lie Ser Asn Tyr 1 5 10 15 Cys Gln lie Tyr Pro Pro Asn Wing Asn Lys lie Arg Glu Wing Leu 20 25

Claims (13)

BEIVIMDICACIOligS

1. A fusion protein, which comprises: a) one or more antigens, and (b) one or more fractions that interact with the human Fcy II receptor (FcyRII) (CD32).

2. A fusion protein according to claim 1, wherein the antigens are allergens in atopic dermatitis, allergic asthma, allergic rhinitis, or allergic conjunctivitis.

3. A fusion protein according to claim 1, which is produced from one or more elongations of DNA containing the epitope of the T cells, rather than from the genes for the complete antigens.

4. A fusion protein according to any of claims 1 to 3, wherein the fractions that interact with FcyRII are human or humanized CD32 antibodies, or parts of these antibodies that still specifically recognize and bind to the FcyRII antigen ( CD32).

5. A fusion protein according to any of claims 1 to 3, wherein the fractions that interact with Fc? RII are human or humanized IgG antibodies, or parts of these antibodies that still interact with the Fc? RII antigen ( CD32).

6. A fusion protein according to any of claims 1 to 3, wherein the fractions that interact with FcyRII are human or humanized manipulated antibodies to CD32 or IgG, or portions thereof, which recognize FcyRII (CD32) with a further affinity. high than the native aCD32 or IgG antibodies.

7. A pharmaceutical composition comprising a fusion protein according to claim 1, together with at least one pharmaceutically acceptable carrier or diluent.

8. A process for the production of a fusion protein according to claim 1, which comprises the use of recombinant genetic techniques or chemical cross-linking.

9. The use of a fusion protein according to claim 1, in the prevention and / or treatment of allergies (including food allergies).

10. The use of a fusion protein according to claim 1, for the manufacture of a medicament for the prevention and / or treatment of allergies (including food allergies).

11. a fusion protein according to claim 1, for use as a pharmaceutical product.

12. A method for the treatment of allergies, which comprises administering to a subject in need of such treatment, a prophylactically or therapeutically effective amount of fusion protein according to claim 1, together with at least one conventional pharmaceutically acceptable carrier or diluent. .

13. A process for the preparation of an allergy medicament, which comprises mixing a fusion protein according to claim 1, together with a pharmaceutically acceptable carrier or diluent. Fusion proteins comprising one or more antigens, and one or more fractions that interact with the human Fcy II receptor (FcyRII) (CD32). * * * * *