Interleukin-1 for the treatment and prevention of allergies

Abstract

The present invention relates to interleukin-1 (IL-1) and a composition containing the same for use in the prevention and / or treatment of allergies, particularly in desensitization methods. The present invention also relates to a composition containing IL-1 together with an allergen, a medical device containing the same, and a desensitization kit.

Description

[Technical Field]

[0001] The present invention relates to interleukin-1 (IL-1) and a composition containing the same for use in the prevention and / or treatment of allergies, particularly in desensitization methods. The present invention also relates to a composition containing IL-1 together with an allergen, a medical device containing the same, and a desensitization kit. [Background technology]

[0002] Allergies are one of the fastest growing medical conditions worldwide and a major public health problem. They disrupt the lives of millions of people and can lead to very serious complications. Currently, more than one in four people in Europe suffer from allergies, and the World Health Organization (WHO) predicts that by 2050, the prevalence will reach one in two people worldwide, particularly in industrialized countries.

[0003] Allergies are caused by a deregulated immune system, which overreacts to normally harmless substances. These substances, known as "allergens," can be found in the air (e.g., pollen, cat hair), food (e.g., milk, eggs, peanuts), medicines (e.g., penicillin, curare), and materials with which people come into contact (e.g., latex, nickel). The allergic reactions they induce can lead to a variety of symptoms, including dermatitis, eczema, rhinitis, asthma attacks, edema, or anaphylaxis.

[0004] The immediate symptoms of allergy are primarily due to type I hypersensitivity (also known as immediate hypersensitivity). Type I hypersensitivity is a misguided immune response to nonpathogenic antigens that causes elevated levels of immunoglobulin E (IgE). IgE sensitizes mast cells and basophils via the high-affinity IgE receptor FcεRI, causing the cells to degranulate upon antigen encounter and release proinflammatory mediators. In type I hypersensitivity, IgG antibodies play a key role in regulating IgE activity, either by neutralizing the allergen (anti-allergen IgG), neutralizing IgE (anti-IgE IgG), or directly inhibiting FcεRI signaling in mast cells and basophils by crosslinking FcεRI with the IgG receptor FcγRIIb, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). Both IgG and IgE responses are regulated by T follicular helper cells (TFH) and T follicular regulatory cells (TFR), which control germinal center B cell (GCB) responses.

[0005] The only disease-modifying therapy for allergy patients is allergen-specific immunotherapy (also called desensitization), which is based on repeated administration of increasing doses of allergen, resulting in an increase in regulatory T and B cells and an increase in the IgG:IgE ratio. These characteristics of allergen-specific immunotherapy provide the basis for novel therapeutic approaches to improve allergy immunotherapy.

[0006] However, approximately half of those who undergo desensitization experience mild side effects, such as mild rash, sneezing, watery eyes, mild asthma symptoms, itching, fatigue, and headache. In very rare cases, immunotherapy can trigger an anaphylactic reaction, which can lead to serious problems such as nausea, difficulty breathing, and circulatory problems. In the most severe cases, anaphylactic shock can occur, requiring immediate medical attention, including adrenaline (epinephrine) injections to quickly suppress the body's dangerous overreaction. Furthermore, because the risk of side effects varies depending on the allergen, the applicability of desensitization to all allergens is limited; for example, immunotherapy for peanut or cat allergies is rarely performed. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there remains a real need for new means to treat allergies in allergic individuals and to reduce the side effects of desensitization methods. [Means for solving the problem]

[0008] The present invention is believed to fulfill such a need by providing a new use of interleukin-1 (IL-1) for preventing or treating allergies or reducing their side effects.

[0009] Using a mouse model based on alum / ovalbumin sensitization, we surprisingly found that adding IL-1 during immunization conferred protection from anaphylaxis by increasing the IgG / IgE ratio. Furthermore, IL-1 directly upregulated FcγRIIb on mast cells, reducing their responsiveness to allergen challenge. Furthermore, lack of IL-1R2 in TFR cells significantly reduced the IgG / IgE ratio and enhanced IL-1-dependent proliferation of TFR cells. Additionally, we demonstrated the therapeutic potential of low-dose IL-1 in models of systemic anaphylaxis and food allergy. IL-1 mediates the suppression of allergic responses by regulating follicular T cells and mast cells, and thus has potential as an adjuvant to improve the safety and efficacy of allergy immunotherapy.

[0010] Thus, in one aspect, the present invention relates to interleukin-1 (IL-1) for the prevention and / or treatment of allergies.

[0011] The present invention further relates to a composition comprising IL-1 as an active ingredient for the prevention and / or treatment of allergies.

[0012] The present invention further relates to methods for treating and / or preventing allergies by administering IL-1 or a composition comprising IL-1 to an individual in need thereof.

[0013] The present invention further relates to the use of IL-1 or a composition comprising IL-1 in the manufacture of an external medicament for the treatment and / or prevention of allergies.

[0014] As used herein, the term "interleukin-1" (or "IL-1") refers to interleukin-1α (IL-1α) and interleukin-1β (IL-1β). Both cytokines are agonists and are expressed in multiple cell lineages, including monocytes, macrophages, neutrophils, hepatocytes, and tissue macrophages throughout the body. Despite differing amino acid sequences, IL-1α and IL-1β can both be synthesized as 33 kDa, 271 aa procytokines that are enzymatically cleaved (by calpain) into a biologically active 17 kDa, 159 aa mature segment and a 112 aa prosequence.

[0015] Thus, in the present invention, interleukin-1 can be IL-1α or IL-1β, preferably IL-1β.

[0016] In one embodiment, the interleukin-1 is mammalian IL-1, preferably human IL-1.

[0017] In the context of the present invention, "IL-1" also includes natural or artificial variants of IL-1 having at least about 90% amino acid sequence identity with the sequence of IL-1α or IL-1β. Variants can be generated using classical genetic engineering techniques, for example, by insertion, substitution, deletion, or a combination thereof. Substitutions in the protein sequences of the present invention can be conservative or non-conservative.

[0018] Although not limiting to the present invention, some reference sequences of IL-1α and IL-1β are shown in Table 1.

[0019] TIFF2026506704000001.tif101170

[0020] In one embodiment, the interleukin-1 has an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1 or SEQ ID NO:2.

[0021] In one embodiment, the interleukin-1 has an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1.

[0022] In one embodiment, the interleukin-1 has an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.

[0023] In one embodiment, the interleukin-1 has an amino acid sequence that has at least 95%, preferably at least 99%, more preferably 100% identity to SEQ ID NO:1.

[0024] In one embodiment, the interleukin-1 has an amino acid sequence that has at least 95%, preferably at least 99%, more preferably 100% identity to SEQ ID NO:2.

[0025] "Identity," with respect to the percentage of amino acid sequence "identity," is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to those in a target sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Percent sequence identity is determined by conventional methods. Briefly, for example, two amino acid sequences can be aligned using the ClustalW algorithm (Thompson et al., Nuc. Ac. Res. 22:4673-4680, 1994) and the PAM250 weight matrix (Dayhoff et al., "Atlas of Protein Sequence and Structure." National Biomedical Research Foundation. Washington, DC 5:345-358, 1978), and the default parameters provided by the MegAlign program (DNASTAR, Inc., Madison, Wisconsin) to optimize the alignment score. The percent identity is then calculated as [total number of perfect matches x 100] divided by [length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences].

[0026] In one embodiment, interleukin-1 is fused to another protein, particularly a carrier protein and / or another biologically active protein.

[0027] In the present invention, the term "allergy" means any allergy caused by any allergen, preferably an allergy associated with type I hypersensitivity.

[0028] In one embodiment, the allergy is caused by an autoallergen.

[0029] As used herein, the term "autoallergen" refers to a self-antigen that induces inflammation via IgE.

[0030] In one embodiment, the autoallergen is double-stranded DNA (dsDNA), particularly dsDNA associated with systemic lupus erythematosus.

[0031] In one embodiment, the autoallergen is IL-24. In particular, IL-24 is associated with chronic idiopathic urticaria.

[0032] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above for use in a method for desensitizing an allergic individual.

[0033] According to the present invention, the individual is an animal, preferably a human.

[0034] As used herein, the expression "method of desensitization" (or desensitization) refers to known methods of preventing or reducing immediate hypersensitivity by administration of increasing doses of an allergen.

[0035] As used herein, the term "allergen" refers to a molecule capable of inducing an allergic response and any substance, composition, or material containing such a molecule. Allergens can be of various natures, such as lipids, proteins, peptides, polypeptides, chemical compounds, metals, plastics, etc. Allergens can be in their natural state or artificially produced (e.g., by recombinant technology and / or, e.g., enzymatic technology). Allergens can be structurally altered or modified to improve their stability, immunogenicity, etc. Allergens can also be a mixture of several molecules (e.g., an extract). Allergens can be used in various states, such as liquid or dry.

[0036] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, wherein IL-1 is used in combination with at least one allergen.

[0037] As used herein, the phrase "IL-1 is used in combination with at least one allergen" indicates that IL-1 and the at least one allergen are administered to an individual at the same time or at different times. IL-1 and the at least one allergen may be present in the same composition or in different compositions.

[0038] In one embodiment, the present invention relates to a composition comprising IL-1 as defined above, which further comprises at least one allergen.

[0039] In one embodiment, the present invention relates to a composition comprising IL-1 as defined above, wherein said allergen is a food allergen, a contact allergen or a respiratory allergen, or a drug.

[0040] As used herein, the phrase "food allergen" refers to any allergen that may be contained in a food, for example, but not limited to, groundnuts, peanuts, milk, eggs, tree nuts and seeds (such as, but not limited to, hazelnuts, cashews, walnuts, pecans, Brazil nuts, macadamia, chestnuts, pistachios, coconut, almonds, sesame, mustard, etc.), fish, shellfish, crustaceans, grains (such as, but not limited to, wheat, corn, oats, barley, rye, rice, sorghum, spelt, etc.), legumes (such as, but not limited to, soybeans, kidney beans, black beans, common beans, chickpeas, peas, cowpeas, lentils, lupins, etc.), or mixtures thereof.

[0041] As used herein, the phrase "contact allergen" refers to any allergen that may come into contact with the skin of an individual, such as, but not limited to, latex, nickel, gold, fragrance mix, thimerosal, neomycin sulfate, formaldehyde, cobalt chloride, bacitracin, quaternium-15, or mixtures thereof.

[0042] As used herein, the expression "respiratory allergen" refers to any allergen that may be present in the air, including, but not limited to, dust mites, rye, ragweed, cockroaches, pollen, mold, animal dander, animal hair, dust, or mixtures thereof.

[0043] As used herein, the term "drug" refers to any agent that can induce an allergic response, including, but not limited to, antibiotics (e.g., penicillin), painkillers (e.g., aspirin, ibuprofen, and naproxen sodium), chemotherapy drugs, poisons, or drugs for autoimmune diseases, or mixtures thereof.

[0044] In one embodiment, the allergen is egg or a compound contained in egg, particularly ovalbumin.

[0045] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above for use in immunotherapy.

[0046] As used herein, the term "immunotherapy" refers to the administration of an agent to manipulate an individual's immune system. Examples of immunotherapy include, but are not limited to, desensitization, vaccination, part of a cancer cell treatment, or part of a treatment for an immune disease. Immunotherapy can be either active or passive. In active immunotherapy, a molecule that elicits an immune response against that molecule is administered to an individual (e.g., vaccination against a pathogen, desensitization to an allergen, or administration of a tumor antigen). In passive immunotherapy, an immune molecule is administered to a patient who does not produce it themselves (e.g., administration of an antibody specific to a pathogen or tumor). In the present invention, administration of IL-1 is useful for preventing or treating, or at least reducing the side effects of, allergies that may arise during immunotherapy.

[0047] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 for the treatment or prevention of anaphylaxis, in particular anaphylactic shock.

[0048] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, administered by oral, epicutaneous, subcutaneous, transdermal, intralymphatic, intramuscular, intravenous, nasal or rectal route.

[0049] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, administered at least twice, preferably at least three times.

[0050] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, administered to a human individual at a dose of 1 μg or less, preferably 100 ng or less, more preferably 50 ng or less, and even more preferably 10 ng or less of IL-1 per kg of body weight.

[0051] In one embodiment, the invention relates to IL-1 or a composition comprising IL-1 as defined above, administered to a human individual at a dose of 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng or 50 ng of IL-1 per kg of body weight.

[0052] In one embodiment, IL-1 or a composition comprising IL-1 is administered in a dose range of 1 ng to 30 ng, particularly 2 ng to 27 ng, of IL-1 per kg of body weight.

[0053] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, administered to a human individual at a dose of 1 ng or less, preferably 500 pg or less, more preferably 100 pg or less of IL-1 per kg of body weight.

[0054] In one embodiment, the present invention relates to IL-1 or a composition comprising IL-1 as defined above, administered at least twice, preferably at least three times within a five day period.

[0055] In one embodiment, the invention relates to IL-1 or a composition comprising IL-1 as defined above, administered at least twice, preferably at least three times, with one week between each administration.

[0056] In another aspect, the present invention also relates to a composition comprising IL-1 and at least one allergen.

[0057] In one embodiment, the present invention relates to a composition as defined above in the form of a solution, gel, powder, aerosol spray, lotion or foam.

[0058] In one embodiment, the present invention relates to a composition as defined above, wherein said allergen is a food allergen, a contact allergen or a respiratory allergen, or a drug.

[0059] In one embodiment, the present invention relates to a composition as defined above, with the proviso that the allergen is not ovalbumin.

[0060] In another aspect, the present invention also relates to a medical device comprising a composition comprising IL-1 and at least one allergen as defined above, suitable for administering the composition to an individual.

[0061] In one embodiment, the present invention relates to a medical device as defined above, selected from the group comprising a patch, an inhaler, a nebulizer and a syringe.

[0062] In another aspect, the present invention provides a desensitization kit comprising: a composition comprising IL-1; At least one allergen; The present invention relates to a kit comprising:

[0063] In one embodiment, the present invention relates to a desensitization kit as defined above, with the proviso that the allergen is not ovalbumin.

[0064] The following figures and examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. While the invention has been described with reference to specific embodiments, it will be apparent to those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step(s), to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. [Brief explanation of the drawings]

[0065] [Figure 1] Mice were immunized with OVA, OVA + IL-1, or treated with anakinra (OVA + anakinra), and serum was collected on days 0 and 21. A. Mean ± SEM of OD50 values ​​for OVA-specific IgG. B. Mean ± SEM of total IgE levels (μg / ml). C. Mean ± SEM of OD450 values ​​for OVA-specific IgE. D. Ratio of specific IgG to specific IgE. E. Wild-type or IL-1RaKO mice were immunized with OVA. Mean ± SEM of OD50 values ​​for OVA-specific IgG. F. Mean ± SEM of total IgE levels (μg / ml). G. Mean ± SEM of OD450 values ​​for OVA-specific IgE. H. Ratio of specific IgG to specific IgE. I. On day 28, mice were challenged intravenously with OVA, and body temperature was measured with a 10-minute interval between measurements. Mean ± SEM of the decrease in body temperature compared to baseline is shown in OVA, OVA + IL-1, or OVA + anakinra mice. J. Mean ± SEM reduction in body temperature compared to baseline in wild-type or IL-1RaKO mice. [Figure 2]The effect of D21-immunized serum on OVA binding to effector cells is shown as the mean ± SEM of the relative OVA-A488 MFI fold change compared to naive serum. A. OVA-A488 binding to basophils in the presence of 1:10 serum. B. OVA-A488 binding to BMMCs in the presence of 1:10 serum. C. The effect of D21-immunized serum on effector cell degranulation is shown as the mean ± SEM of the anti-CD63 MFI fold change compared to unchallenged cells (baseline). D. Serum inhibition assay using basophils in the presence of 1:1000 serum. D. Serum inhibition assay using sensitized BMMCs in the presence of 1:1000 serum. E. OVA challenge on day 28 in the presence of anti-FcγRIIb blocking antibody or isotype control antibody. The mean ± SEM of the decrease compared to baseline body temperature is shown. [Figure 3]Mice were immunized with OVA, OVA + IL-1, or OVA + anakinra, and PMCs were examined on day 28. A. Mean ± SEM of PMC frequency is shown. B. Mean ± SEM of anti-FcεRI MFI in PMCs. C. Mean ± SEM of FcγRIIb MFI in PMCs. D. FcγRIIb:FcεRI MFI ratio. E. OVA-immunized mice were injected sc with PBS or IL-1 on day 21. PMCs were examined by flow cytometry on day 22. Mean ± SEM of anti-FcεRI MFI in PMCs is shown. F. Mean ± SEM of FcγRIIb MFI in PMCs is shown. G. Alternatively, mice were challenged with OVA on day 22. Mean ± SEM of decrease compared to baseline body temperature is shown. H. BMMCs were treated with various concentrations of IL-1β for 48 hours. Mean ± SEM of fold change in anti-FcγRIIb and anti-FcεRI MFI compared to unstimulated control cells is shown. I. Representative BMMC raw histograms of anti-FcεRI and anti-FcγRIIb expression in the absence or presence of 0.2 μg / ml IL-1β for 48 hours. J. Sensitized BMMC were primed with 0.2 μg / ml IL-1β for 48 hours. OVA was mixed with serum from D21 OVA-immunized WT mice and added to IL-1β-primed or unprimed BMMC in the presence or absence of additional anti-FcγRIIb. Shown is the mean ± SEM of relative anti-CD63 expression compared to untreated control cells. [Figure 4]A. CD4cre(WT) or CD4creIL-1R1lox mice were immunized sc with OVA / Alum on days 0 and 14, and serum was collected on days 0 and 21. Mean ± SEM of OD50 values ​​for OVA-specific IgG is shown. B. Mean ± SEM of total IgE levels (μg / ml). C. Mean ± SEM of OD450 values ​​for OVA-specific IgE. D. Mean ± SEM of specific IgG to specific IgE ratio. E. FoxP3cre(WT) or FoxP3creIL-1R2lox mice were immunized sc with OVA / Alum on days 0 and 14, and serum was collected on days 0 and 21. Mean ± SEM of OD50 values ​​for OVA-specific IgG is shown. F. Mean ± SEM of total IgE levels (μg / ml). G. Mean ± SEM of OD450 values ​​for OVA-specific IgE. H. Mean ± SEM of specific IgG to specific IgE ratio. I. CD4creIL-1R1lox were challenged iv with OVA on day 28. Mean ± SEM reduction in body temperature compared to baseline is shown. J. FoxP3creIL-1R2lox were challenged iv with OVA on day 28. Mean ± SEM reduction in body temperature compared to baseline is shown. [Figure 5] FoxP3cre (WT) or FoxP3creIL-1R2lox mice were immunized sc with OVA / Alum on days 0 and 14. A. Mean ± SEM of basophil frequency on day 21. B. Mean ± SEM of basophil anti-IgE MFI on day 21. C. Mean ± SEM of basophil anti-CD63 MFI after OVA challenge. D. OVA-A488 binding to basophils in the presence of 1:10 serum. E. Serum inhibition assay using basophils in the presence of 1:1000 serum. F. OVA-A488 binding to BMMCs in the presence of 1:100 serum. G. Serum inhibition assay using BMMCs in the presence of 1:1000 serum. H. Anti-FcγRIIb blocking antibody or isotype control antibody was injected intravenously 1 hour before OVA challenge on day 28. Body temperature was measured 10 minutes apart. Mean ± SEM of decrease compared to baseline body temperature. [Figure 6]WT or foxp3creIL-1R2lox mice were immunized sc with OVA / Alum on days 0 and 14. On day 23, spleens were harvested and restimulated ex vivo for 48 hours with IL-1, OVA, or OVA + IL-1β. A. Gating strategy for proliferating CD4+ cells, defined as FSHhiKI-67+, and PD-1 / Foxp3 expression (lower panel) are shown in unstimulated cells (left panel) versus IL-1 + OVA-stimulated cells (right panel). B. Mean ± SEM frequency of total proliferating follicular T cells among CD4+ cells. C. Mean ± SEM frequency of proliferating TFR cells among CD45+ cells. D. Mean ± SEM frequency of proliferating TFR cells among CD45+ cells. E. Mean ± SEM frequency of proliferating CD19+ cells among CD45+ cells. F. Anakinra was added to the 48-hour restimulation with IL-1, OVA, or OVA + IL-1. Mean ± SEM frequency of proliferating TFR cells among CD4+ cells is shown. [Figure 7]OVA-sensitized mice were treated three times with OVA or OVA + IL-1β in PBS on days 16, 18, and 20. Serum was collected before treatment and on day 40. A. Mean ± SEM of OD50 values ​​of OVA-specific IgG over time. B. Mean ± SEM of total IgE levels (μg / ml) over time. C. Mean ± SEM of OD450 values ​​of OVA-specific IgE over time. D. On day 60, basophils from OVA- or OVA + IL-1β-treated mice were challenged with OVA in the presence of 1:1000 naive, OVA, or OVA + IL-1β serum on day 60. Inhibition of anti-CD63 MFI compared to naive serum is shown. E. On day 41, mice were challenged intravenously with OVA. Mean ± SEM of decrease compared to baseline body temperature is shown. F. A second challenge was administered on day 65, while one group received a booster injection of IL-1β in PBS on day 64. The mean ± SEM of the decrease compared to baseline body temperature is shown. G. PMCs were isolated at the end of the experiment. The mean ± SEM of anti-FcεRI MFI is shown. H. The mean ± SEM of anti-FcγRIIb MFI in PMCs. I. BALB / c mice were sensitized i.p. with OVA or OVA + IL-1β on days 0 and 10, followed by a cycle of poOVA challenge starting on day 20. The mean ± SEM of the maximum body temperature decrease measured for individual mice is shown. J. The mean ± SEM of the food allergy score is shown. K. A second po challenge was administered on day 40, with scPBS or IL-1β added before each challenge. The mean ± SEM of the maximum body temperature decrease measured for individual mice is shown. L. The mean ± SEM of the food allergy score is shown. [Figure 8] FIG. 1 shows the mode of action of IL-1 proposed by the inventors in regulating responses (associated with tolerance to allergens) and in dysregulating responses (associated with anaphylaxis in response to allergens). [Figure 9]Figure 1 shows two different regimens of therapeutic IL-1 + allergen injection (A and B). Mice were sensitized with OVA / Alum. Serum was collected on day 13. OVA + IL-1β diluted in PBS was injected subcutaneously according to two regimens starting on day 14. Mice received 500 ng, 160 ng, or 50 ng of IL-1β mixed with 10 μg of OVA. Control groups received OVA injection only. Serum was collected on day 40. On day 42, mice were challenged intravenously with 2 μg of OVA, and body weight was monitored over 60 minutes to determine systemic anaphylaxis. [Figure 10] Figure 1 shows dose-dependent protection from systemic anaphylaxis. Body temperature was measured at 10-minute intervals to determine systemic anaphylaxis. The mean ± SEM decrease in degrees Celsius compared to baseline body temperature is shown. Regimen A was tested in 4 mice / group, and Regimen B was tested in 5 mice / group. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0066] Materials and Methods Mice. All animals were kept at the Centre d'Experimentation Fonctionnelle animal facility (Paris, France). All procedures were approved by the local animal ethics committee (approval number A751315). Transgenic mouse strains, including B6.129S-IL1rntm1Dih / J (JAX strain no. 004754), B6.Cg-Tg(Cd4-cre)1Cwi / Bflu / J (JAX strain no. 022071), B6.129(Cg)-Il1r1tm1.1Rbl / J (JAX strain no. 028398), and B6.129(Cg)-Foxp3tm4(YFP / icre)Ayr / J (JAX strain no. 016959), were purchased from the Jackson Laboratory (Bar Harbor, USA), except for the C57BL / 6N-Atm1BrdIl1r2tm1a(EUCOMM)Wtsi / Wtsi strain (MGI: 4842437) provided by the Sanger Institute. Seven-week-old BALB / C (AnNR / J) female mice were purchased from Elevage Janvier (Le Genest-Saint-Isle, France). B6.Cg-Tg(Cd4-cre) and B6.129(Cg)-Foxp3tm4(YFP / icre)Ayr / J mice were used as wild-type mice. There were no differences in the IL-1-mediated effects observed here between the two strains (not shown). Wild-type and transgenic male and female mice aged 6 to 15 weeks were used in the experiments. Different groups within each experiment were performed using mice of the same age range and sex, ±10 days apart.

[0067] Injections. All injections, except for the OVA challenge, were performed in a volume of 100 ml. Mice were immunized on days 0 and 14 by subcutaneous injection of 100 μg of OVA (OVA A5503, Sigma-Aldrich) in Imject™ Alum (Thermo Fisher) diluted 1:1 with PBS. Anakinra (Kineret™, Swedish Orphan Biovitrum) was administered subcutaneously at 50 mg / kg twice daily on days 0–5 and 14–16. Mice treated with IL-1β received recombinant murine IL-1β (0.5 μg per mouse; Myltenyi Biotec) mixed with OVA / Alum. For induction of anaphylaxis, mice were intravenously injected with 2 μg of OVA in 200 ml of PBS. Rectal temperature was measured at 10-minute intervals over 1 hour using a rectal thermometer (Biosep Lab Instruments, Vitrolles, France). For FcγRIIb blockade, 100 μg of anti-CD32b antibody (Thermo Fisher, clone AT130-2) was injected intraperitoneally 1 h before OVA challenge. Alternatively, a mouse IgG2a, κ isotype control (Miltenyi Biotec) was injected. For short-term treatment injections, 0.5 μg of IL-1β in PBS or PBS alone was injected subcutaneously. For long-term treatment injections, 10 μg of OVA in PBS or 0.5 μg of IL-1β in PBS plus 10 μg of OVA were injected subcutaneously three times, with an interval of 2 days between each injection. For food allergy sensitization, 10 μg of OVA in 1:1 Alum / PBS was injected intraperitoneally on days 0 and 10. Oral challenge was performed by oral gavage with 20 mg of OVA once every two days for a maximum of five challenges, and body temperature was assessed at 30 to 45 min. Clinical scores were based on the severity of diarrhea rated from 0 to 3 points, and the appearance of hirsute pelage rated from 0 to 2 points.

[0068] Sampling. To assess basophils, blood was collected from the tail vein into EDTA tubes (final concentration 1 mM EDTA). Erythrocytes were lysed using BD Pharm Lyse™ (BD Bioscience) according to the manufacturer's protocol and washed three times with PBS. Peritoneal lavage was performed on sacrificed mice by flushing the peritoneal cavity with 5 ml of ice-cold PBS while constantly massaging. The peritoneum was then incised, and the fluid was collected with a Pasteur pipette. Erythrocytes were lysed, followed by three washes with PBS. Whole spleens were removed from sacrificed mice and transferred to 48-well plates containing RPMI 1640 medium (Sigma). The spleens were then mashed, filtered through a cell strainer (70 μm, Sigma Aldrich), and washed three times with PBS. To generate mouse bone marrow-derived mast cells (BMMCs), the femurs and tibias of the hind legs were isolated from sacrificed mice and bilaterally cut. The bone marrow was flushed with RPMI 1640 medium using a syringe. All isolated cells were centrifuged at 300 g for 5 minutes at 4°C and finally suspended in RPMI 1640, 20% FCS medium (Sigma).

[0069] Flow cytometry. All primary antibody staining was performed in 96-well round plates (Thermo Fisher) in PBS for 20 minutes at 4°C. Between all steps, cells were washed twice with 200 μl of PBS. Fixation and permeabilization were performed using the eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher) according to the manufacturer's protocol. Basophils were defined as those negative for anti-mouse CD49b+ (Thermo Fisher, clone DX5), anti-mouse IgE+ (Thermo Fisher, clone 23G3), anti-mouse CD451 (Thermo Fisher, clone 30-F11), and anti-CD117 (Thermo Fisher, clone 2B8) and anti-CD19 (BD Bioscience, clone 1D3). Peritoneal mast cells (PMCs) and bone marrow-derived mast cells (BMMCs) were defined as anti-mouse FcεRI+ (Thermo Fisher, clone MAR-1), anti-mouse IgE+, anti-mouse CD117+, anti-mouse F4 / 80- (Thermo Fisher, clone BM8), and anti-mouse CD49b-. Basophil and mast cell activation was examined with anti-CD63 (Thermo Fisher, clone NVG-2) staining. Germinal center B cells were defined as CD45+, anti-mouse CD19+, or anti-mouse B220+ (Thermo Fisher, clone RA3-6B2), anti-mouse GL-7+ (Thermo Fisher, clone GL-7), anti-mouse IgD- (Thermo Fisher, clone 11-26c), and anti-mouse CD3- (Thermo Fisher, clone 145-2C11). OVA-specific GCB cells were examined by staining with 1 μg / ml Invitrogen™ Ovalbumin, Alexa Fluor™ 488 Conjugate (Thermo Fisher Scientific). Proliferating cells were examined by staining with anti-mouse Ki-67 (Thermo Fisher, clone SolA15).Plasmablasts were defined as CD19+ / B220+, GL-7-, IgD-, and CD138+ (Thermo Fisher, clone 281-2). Follicular T cells were gated with anti-mouse CD3+, anti-mouse CD4+ (Thermo Fisher, clone RM4-5), anti-mouse CXCR5+ (Thermo Fisher, clone 2G8), and anti-mouse PD-1+ (Thermo Fisher, clone RPM1-30) antibodies, and CD19+ or B220+ cells were excluded. Among follicular T cells, TFRs were defined as TFRs with anti-mouse FoxP3- (Thermo Fisher, clone FJK-16s). All flow cytometry was performed on a CytoFLEX (Beckman Coulter, Brea, USA) and analyzed using FLOWJO software (TreeStar Inc, Ashland, OR) or CytEXPERT (Beckman Coulter).

[0070] ELISA. For ELISA, 96-well Nunc Maxisorp ELISA plates (Thermo Fisher Scientific) were coated overnight with reagents in PBS at 4°C. For anti-OVA IgG, 500 ng / ml OVA was coated. For total and specific IgE, rat anti-mouse anti-IgE (BD Biosciences, clone R35-72) was coated at 2 μg / ml. Blocking was performed with Blocker™ Casein solution (Thermo Fisher Scientific) for 2 hours. Serum dilutions were added to the plate and incubated for 2 hours at room temperature. For specific IgG ELISA, serum was initially diluted 1:100, followed by 10-fold dilution steps. For total IgE, the initial dilution was 1:50, followed by 3-fold dilution steps. Purified mouse IgE (Biolegend, clone MEA-36) was used to generate a standard curve. For specific IgE, serum was diluted 1:10. The plates were then washed five times with PBS / 0.05% Tween. Specific IgG was detected with biotin rat anti-mouse IgG (Southern Biotech, Birmingham, AL, USA), biotin rat anti-mouse IgG1 (Southern Biotech), or biotin rat anti-mouse IgG2a (Southern Biotech) for 1 hour at room temperature. Total IgE was detected with biotin rat anti-mouse IgE (Southern Biotech). After three washes with PBS / 0.05% Tween, specific IgG and total IgE ELISAs were further incubated with streptavidin-HRP (Thermo Fisher) for 1 hour at room temperature. Specific IgE was detected by adding 500 ng / ml OVA in a first step for 1 hour at room temperature. The plates were washed five times with PBS-Tween and then incubated with polyclonal anti-OVA-HRP (Thermo Fisher). Finally, all plates were washed five times with PBS / 0.05% Tween, and the ELISA was developed with 1x TMB substrate solution (Thermo Fisher) and stopped with 1M HCl. All ODs were measured at 450 nm to determine the half-maximal antibody titer (OD 50) was defined as the reciprocal of the dilution rate that produced half of the OD measured at saturation.

[0071] Effector Cell Binding and Activation Assay. For the basophil assay, approximately 200 μl of blood from three mice was pooled, lysed, and the cells were resuspended in RPMI 1640 medium (Sigma) containing 10% FBS. For the basophil activation test, cells were first incubated with titrated doses of OVA at 37°C for 1 hour. 5 nM OVA was established as an appropriate dose for comparing basophil activation and was used in all subsequent experiments. However, the basophil assay was later optimized by shortening the incubation time to 20 minutes at 37°C. For BMMCs, activation was performed with 1 nM OVA at 37°C for 20 minutes based on titration. Because IgE-antigen complexes do not induce anaphylaxis, serum was not further purified to minimize loss of material. The presence of mouse BD Fc-Block™ (BD Biosciences) further enabled determination of IgG-dependent binding effects by serum. For basophil activation studies, serum from D21-immunized mice was premixed with 5 nM OVA or 1 nM OVA for BMMCs at room temperature for 20 min before addition to basophils. Binding assays in basophils and BMMCs were performed by premixing 25 nM OVA-A488 with a 1:10 dilution of serum in culture medium at 4°C for 20 min. Activation / inhibition assays were performed using a 1:1000 dilution of serum after initial titration experiments. Essentially, the same protocol was used for BMMC binding and activation assays, except for an additional binding experiment using a 1:100 serum dilution. For all activation assays, instead of Fcγ-block, a more specific blocking antibody, anti-FcγRIIb, was added to the cells at a 1:200 dilution before the addition of the OVA-IgG complex.

[0072] TFH / TFR restimulation assay. Restimulation experiments of TFH / TFR cells were performed using whole splenocytes obtained from immunized mice on day 21. Isolated splenocytes were cultured at 2 million cells per well in 96-well round (U)-bottom plates (Thermo Fisher Scientific). Culture medium was RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% FBS, 20 mM L-glutamine (Thermo Fisher Scientific), and penicillin-streptomycin (100 U / mL, Thermo Fisher Scientific) in a final volume of 200 μl per well. IL-1β, OVA, and anakinra were used at 0.5 μg / ml, 1.2 μg / ml, and 0.5 μg / ml. Splenocytes were cultured at 37°C and 5% CO for 48 hours. Cells were then washed three times with PBS before staining.

[0073] Statistics. All results shown are representative of at least three independent experiments, except for Figure 2E (two independent experiments) and Figures 3A–3E (two independent experiments). All data are shown as mean ± standard error. All statistical tests and evaluations were performed using GraphPad PRISM 6.0 (GraphPad Software, Inc., La Jolla, CA). For all experiments, an alpha value of 0.05 was used, and statistical significance of p values ​​is indicated as follows: * is less than or equal to 0.05, ** is less than or equal to 0.01, and *** is less than or equal to 0.001. Comparisons between two groups (e.g., antibody titers, cell frequencies, activation assays, and proliferation assays) were performed using a nonparametric two-tailed Student's t-test. Dose- and time-dependent comparisons were performed using two-way ANOVA followed by Bonferroni correction to obtain individual p values ​​(e.g., IL-1 titration and anaphylaxis experiments on BMMCs).

[0074] Example I - The presence of IL-1 during immunization enhances antibody responses and reduces systemic anaphylaxis The influence of IL-1 on allergic immune responses was investigated. In a first step, IgG and IgE responses were examined in mice immunized with OVA, OVA + IL-1, or IL-1 blocked with daily anakinra treatment (OVA + anakinra). Initial antibody serum kinetics allowed comparison of day 0 (naive) and day 21 sera for all conditions (data not shown). Figure 1A shows that IL-1β significantly increased specific IgG titers, whereas IL-1 blockade slightly reduced specific IgG. Similarly, IL-1β led to an increase in total IgE levels (Figure 1B). Specific IgE levels, while generally very low, were increased in immunized mice compared with the naive group, with an increase observed in the OVA + IL-1 group (Figure 1C). Basophil-presented antibodies and serum IgG subclasses were also examined; all were upregulated in the presence of IL-1 (data not shown). Because the IgG / IgE balance is an important feature in allergy, we determined the IgG:IgE ratio, which showed a significant increase in OVA + IL-1-immunized mice (Figure 1D). In the next step, we investigated the antibody response to OVA sensitization in IL-1RaKO mice. Similar to OVA + IL-1-immunized mice, IL-1RaKO mice exhibited higher IgG and IgE responses and a higher IgG / IgE ratio compared with wild-type mice (Figures 1E–H). Finally, mice immunized with OVA, OVA + IL-1, and OVA + anakinra were compared in terms of the anaphylactic response to OVA challenge on day 28. IL-1β significantly protected mice from systemic anaphylaxis, and IL-1 blockade had a strong exacerbating effect (Figure 1I). Similarly, IL-1RaKO mice were protected from systemic anaphylaxis compared with wild-type mice (Figure 1J). In conclusion, despite elevated IgE levels, excess IL-1 during sensitization increases the IgG:IgE ratio and suppresses the anaphylactic response.

[0075] Example II - IL-1-promoted IgG promotes FcγRIIb-dependent protection from allergic anaphylaxis Since IL-1β has been shown to confer protection against anaphylaxis, we investigated the IgG dependence of this process. We evaluated the effects of OVA, OVA + IL-1, and OVA + anakinra-induced sera on the binding of OVA-IgG immune complexes (ICs) to whole blood basophils (all titrations and IL-1RaKO sera, data not shown). Alternatively, we used in vitro-matured bone marrow-derived mast cells (BMMCs) to address higher cell numbers and purity (phenotypic and functional characterization, data not shown). 1:10 diluted sera (naive, OVA, OVA + IL-1, OVA + anakinra) were premixed with fluorescent OVA-A488 for 30 min at 4°C before adding them to the cells to form ICs. Because OVA can bind to basophils via IgE in the absence of serum, naive serum was used as a baseline for OVA binding. Figure 2A shows that OVA + IL-1 serum promotes IC binding to basophils compared to naive serum. The additional presence of Fcγ-Block completely blocked this enhanced IC binding. In BMMCs, the effect of OVA + IL-1 serum was even more pronounced, as shown in Figure 2B. In the next step, we tested the effects of various sera on basophil and mast cell degranulation. In titration experiments, sera were diluted 1:1000 for activation assays. Furthermore, a specific anti-FcγRIIb blocking antibody was used instead of a generic Fcγ-Block. OVA + IL-1 serum inhibited basophil and BMMC degranulation in an FcγRIIb-dependent manner more effectively than OVA serum compared with naive serum (Figures 2C and 2D). Finally, mice immunized with OVA, OVA + IL-1, or OVA + anakinra were intravenously injected with 100 μg of anti-FcγRIIb antibody or an isotype control 1 h before OVA challenge. OVA- or OVA+IL-1-immunized mice injected with FcγRIIb antibodies showed similar levels of anaphylactic enhancement compared with mice injected with an isotype control antibody (Figure 2F). Thus, antibodies induced by OVA+IL-1 immunization enhance Fcγ-dependent binding to allergic effector cells and reduce their degranulation in an FcγRIIb-dependent manner.

[0076] Example III - IL-1β upregulates FcγRIIb expression and reduces its activity on mast cells IL-1R1 expression on mast cells has been previously described. Since IL-1β has been demonstrated to regulate allergic anaphylaxis via IgG / FcγRIIb, we evaluated whether IL-1β directly affects the expression of IgE / IgG surface receptors on mast cells. To this end, peritoneal mast cells (PMCs) were isolated 28 days after immunization with OVA, OVA + IL-1, and OVA + anakinra. Interestingly, the frequency of peritoneal mast cells in OVA + IL-1-immunized mice was higher than in the naive, OVA, and OVA + anakinra groups (Figure 3A). However, FcεRI was reduced in OVA + IL-1-immunized mice compared with mice immunized with OVA alone (Figure 3B). Interestingly, FcγRIIb expression was lower in OVA + anakinra-treated mice compared with OVA and OVA + IL-1-treated mice (Figure 3C). Overall, the MFI ratio of FcγRIIb:FcεRI in PMCs from OVA + IL-1-immunized mice was significantly increased (Fig. 3D). In the next step, OVA-sensitized mice were injected with IL-1β 1 day after the injection, and PMCs were isolated and challenged with OVA. One day after IL-1 injection, FcγRIIb, but not FcεRI, was upregulated in PMCs (Fig. 3E and 3F). Mice pretreated with IL-1 showed significantly reduced anaphylaxis (Fig. 3G). Mixing IL-1 with anakinra or adding anti-FcγRIIb inhibited protection from anaphylaxis (data not shown). IL-1β was then titrated against BMMCs in vitro for 48 hours, and the relative expression of FcεRI and FcγRIIb was assessed by flow cytometry. In IL-1β-treated BMMCs, a significant upregulation of FcγRIIb was detected, whereas FcεRI expression was more stable (Figures 3H and 3I). We then performed a serum inhibition assay by adding serum from OVA-immunized mice (day 21) to BMMCs primed with IL-1β for 48 hours. As shown in Figure 3J, IL-1β priming reduced the activation of BMMCs in the presence of OVA serum compared with naive serum.Thus, IL-1β not only increases the IgG:IgE antibody ratio but also enhances the FcγRIIb:FcεRI receptor ratio, thereby reducing mast cell activation. In conclusion, the presence of IL-1β during immunization has a dual effect on serum IgG and mast cell FcγRIIb, which contributes to protection from allergic anaphylaxis.

[0077] Example IV - FoxP3creIL-1R2lox mice exhibit elevated IgE levels, reduced IgG levels and increased anaphylaxis Because IL-1 has previously been shown to activate TNF-α / GCB cells, we evaluated the role of IL-1 receptors expressed on follicular T cells in allergic responses. To assess the role of IL-1R1-expressing follicular T cells in IL-1β-mediated IgG responses, we immunized wild-type (CD4cre) or CD4creIL-1R1lox mice with OVA or OVA + IL-1 and examined the IgG / IgE responses and anaphylactic responses to OVA challenge. As shown in Figures 4A-4D, CD4creIL-1R1lox mice exhibited reduced IgG responses, a slight enhancement of total IgE responses, a trend toward reduced specific IgE, and a non-significant trend toward a reduced specific IgG to specific IgE ratio. In a next step, we evaluated the role of TNF-expressing IL-1R2 by studying FoxP3creIL-1R2lox mice. FoxP3creIL-1R2 mice showed reduced IgG responses but significantly higher naive total IgE levels, which further increased upon immunization (Figures 4E and 4F). In FoxP3creIL-1R2 mice, specific IgE levels were unchanged, but the specific IgG:specific IgE ratio was dramatically reduced (Figure 4H). Anaphylaxis in CD4creIL-1R1 mice was not significantly different from that in WT mice (Figure 4I). In contrast, FoxP3creIL-1R2 mice were significantly more susceptible to systemic anaphylaxis after intravenous OVA challenge (Figure 4J). In both CD4creIL-1R1 and FoxP3creIL-1R2 strains, the additional presence of IL-1 during OVA immunization elevated IgG levels, albeit to a lower level than in WT mice. Nevertheless, OVA + IL-1 immunization had a protective effect against anaphylaxis compared with OVA alone in both strains (data not shown). Thus, the absence of IL-1R1 on follicular T cells reduces IgG levels but does not alter the IgG / IgE ratio sufficiently to promote anaphylaxis.In contrast, the absence of IL-1R2 in TFR increases the predisposition to an allergic phenotype characterized by dysregulation, decreased IgG / IgE balance, and increased susceptibility to allergic anaphylaxis in response to allergen challenge.

[0078] Example V - Elevated IgE levels and reduced FcγRIIb ligation promote anaphylaxis in FoxP3creIL-1R2lox mice Because dysregulation of immune responses was observed in FoxP3creIL-1R2lox mice, we further investigated the mechanism of anaphylaxis. The elevated serum total IgE levels in FoxP3creIL-1R2lox mice were associated with higher blood basophil frequencies (Figure 5A) and elevated surface IgE levels (Figure 5B) compared with WT mice. In contrast, serum IgG subclasses and basophil-IgG levels were reduced (data not shown). In the next step, we investigated the reactivity of basophils to OVA challenge in the absence of serum. FoxP3creIL-1R2lox-derived basophils degranulated more than WT basophils in response to OVA, suggesting a pure IgE effect (Figure 5C). To assess the influence of serum IgG, we examined the effect of D21 serum from WT or FoxP3creIL-1R2lox mice on effector cell binding and activation. Consistent with previous results using IL-1 or anakinra, inhibition of IgG binding and activation correlated with specific IgG levels. We observed reduced IgG-OVA binding to basophils in serum from OVA-immunized FoxP3creIL-1R2 mice compared with WT mice (Figure 5D). On the other hand, WT OVA serum inhibited basophil degranulation more potently than FoxP3creIL-1R2 OVA serum (Figure 5E). The same effect was observed in BMMCs (Figures 5F and 5G). Next, we examined the effect of FcγRIIb blockade before OVA challenge in FoxP3creIL-1R2 mice compared with WT mice. As shown in Figure 5H, FcγRIIb-mediated protection was not as effective in FoxP3creIL-1R2 mice as in WT mice. In conclusion, higher IgE levels, along with a lower IgG:IgE ratio in FoxP3creIL-1R2lox mice, lead to increased frequencies of IgE+ basophils and a lack of FcγRIIb ligation to basophils and mast cells, resulting in increased allergic anaphylaxis.

[0079] Example VI - FoxP3creIL-1R2lox mice exhibit TFR proliferation in response to IL-1 or antigen restimulation Having demonstrated the mechanism of anaphylaxis in FoxP3creIL-1R2lox mice, we investigated how the lack of IL-1R2 on TFRs causes changes in the IgG / IgE balance. Comparing the splenic TFR / TFR / GCB frequencies in WT and FoxP3creIL-1R2lox mice, we observed a significant decrease in GCB cells, an increase in proliferating TFR levels, and increased B cell apoptosis in FoxP3creIL-1R2lox mice (data not shown). Therefore, we performed in vitro restimulation experiments to evaluate the effects of IL-1, OVA, or IL-1 + OVA restimulation on TFR, TFR, and B cell proliferation. Cells were cultured ex vivo for 48 hours in the presence of OVA and / or IL-1β. Proliferating cells were characterized by flow cytometry as Ki-67+FSChi (gating shown in Figure 6A). Compared with splenocytes from WT mice, follicular T cells in FoxP3creIL-1R2lox mice showed significant proliferative responses to IL-1, OVA, and IL-1 + OVA restimulation (Figure 6B). Proliferating cells were predominantly FoxP3+, and no proliferation was observed in the FoxP3- cell population (Figures 6C and 6D). Enhanced TFR proliferation resulted in increased TFR CD69 and ICOS expression and an increased TFR:TFH ratio (data not shown). In contrast, B cell proliferation was observed only in splenocytes from WT mice, and B cell proliferation in FoxP3creIL-1R2lox mice was completely suppressed (Figure 6E). To confirm the IL-1 dependence of TFR proliferation, assays were performed using FoxP3creIL-1R2lox splenocytes in the absence or presence of anakinra. As shown in Figure 6H, TFR proliferation was blocked by the addition of anakinra, most notably in splenocytes restimulated with OVA alone, suggesting the presence of endogenous IL-1 in the system. Collectively, these results indicate that mice lacking IL-1R2 on FoxP3+ cells exhibit elevated levels of TFR proliferation and reduced B cell proliferation in response to IL-1β and / or antigen restimulation. Dysregulation of this response may disrupt the TFR / GCB response, reduce the IgG:IgE balance, and increase allergic responses.

[0080] Example VII - IL-1β protects sensitized mice from systemic and oral allergen challenge Because IL-1 was observed to regulate both mast cells and follicular T cells and suppress allergic responses, we evaluated whether three subcutaneous injections of IL-1 + OVA diluted in PBS had a desensitizing effect in OVA-sensitized mice. Serum antibody levels were examined on day 14 before treatment and on days 40, 60, and 80 after treatment. Consistent with previous results, IL-1 + OVA immunotherapy significantly increased specific IgG and total IgE levels, but no changes in specific IgE levels were observed (Figures 7A-7C). On day 80, a basophil activation test was performed in the presence of serum. Basophils from the OVA or OVA + IL-1 groups were compared in the presence of 1:1000 naive, OVA, or OVA + IL-1 serum (day 80). Although no differences in basophil activation were observed between the OVA or OVA + IL-1 groups, OVA + IL-1 serum significantly reduced basophil activation (Figure 7D). The first challenge was performed on day 41, and IL-1 + OVA-treated mice were protected from systemic anaphylaxis compared with OVA-treated mice (Figure 7E). One day before the second challenge on day 65, one group received a booster injection of IL-1 in PBS. Figure 7F shows that IL-1 + OVA mice were still significantly protected compared with OVA-treated mice on day 65, and the addition of IL-1 had an additive effect, resulting in a complete absence of anaphylaxis. PMCs were isolated at the end of the experiment on day 80. Figure 7G shows that IL-1 + OVA therapy reduced FcεRI expression and increased FcγRIIb expression. Finally, the therapeutic potential of IL-1 was confirmed in a second allergy model. For this model, BALB / C mice were orally challenged with OVA by oral gavage. In our model in C57BL / 6 mice, the presence of IL-1 during sensitization exerted a preventative antiallergic effect, reducing body temperature and allergy scores (Figures 7I and 7J). Similarly, IL-1 treatment of sensitized mice during OVA challenge correspondingly reduced anaphylaxis and allergy scores (Figures 7K and 7L), thus demonstrating the short- and long-term therapeutic potential of IL-1 in various applications, different mouse strains, and different types of allergen challenge.

[0081] The mechanism of action of IL-1 proposed by the present inventors is summarized in FIG.

[0082] Example VIII. Dose-Response Relationships and Regimen Having demonstrated the efficacy of IL-1 in suppressing allergic anaphylaxis in mice, the dose-response relationship was investigated.

[0083] For this purpose, mice were injected with three doses of recombinant IL-1β per mouse: 500 ng (previously used), 160 ng, and 50 ng, mixed with the same antigen concentration (10 μg of OVA).

[0084] We hypothesized that spacing out IL-1β injections might have a beneficial effect, and therefore tested these three doses in two different immunization regimens (Figure 9).

[0085] In both regimens, mice were sensitized with OVA on day 0 and challenged with OVA on day 42. For regimen A, IL-1+OVA was injected three times within 5 days. For regimen B, three IL-1+OVA injections were separated by a week.

[0086] Regimen A reduced systemic anaphylaxis after OVA challenge in a dose-dependent manner (Figure 10). Similarly, OVA-specific serum IgG showed a dose-dependent increase. Interestingly, Regimen B was superior to Regimen A, as 160 ng completely protected mice from anaphylaxis, and even 50 ng had a positive effect.

[0087] The dose-dependent nature of this treatment demonstrates a direct link between IL-1 levels and the suppression of allergic responses in mice. Reducing IL-1 doses to non-toxic levels while maintaining efficacy remains a major challenge.

[0088] Example IX. Proposed IL-1 Doses for Human Application Several lessons can be learned from previous clinical trials of IL-1β for estimating effective doses in humans.

[0089] In a phase 1 study, the effects of recombinant human interleukin-1β (IL-1β) were investigated in patients with gastrointestinal cancer, both alone and in combination with myelosuppressive doses of 5-fluorouracil (Crown et al., Blood 78(6):1420-1427, 1991). The doses used were 0.002 μg / kg, 0.027 μg / kg, 0.068 μg / kg, and 0.1 μg / kg. Interestingly, cellular changes, e.g., in circulating platelets or neutrophils, were observed even at low doses, which were completely free of toxicity, with toxicity occurring only at the higher dose (0.1 μg / kg).

[0090] The role of IL-1 in generating antibody responses was not understood at the time and was not relevant to this study. It is now speculated that lower doses (0.002 μg / kg and 0.027 μg / kg) may also be sufficient to activate antibody responses, as they have similar biological effects on immune cells.

[0091] Several lessons can also be learned from a recent Phase 1b study of nine cancer patients receiving the RNA-LPX vaccine, which observed an increase in circulating IL-1β after intravenous injection (Tahtinen et al., Nat Immunol. 23(4):532-542, 2022). This response peaked 4-6 hours after injection, consistent with our hypothesis that IL-1 mediates an antiviral, non-allergic immune response.

[0092] Furthermore, this study highlights the highly relevant findings of current studies showing that mice are far more resistant (1000-fold) to IgG antibody induction by RNA vaccines, a phenomenon linked to IL-1, as mice rapidly upregulate the IL-1-neutralizing IL-1 receptor antagonist IL-1Ra, a mechanism of IL-1 inhibition.

[0093] Considering low-dose IL-1 therapy, we conclude that the biological activity of IL-1 on antibody responses is much higher in humans than in mice, suggesting that the dose must be lowered in humans. Therefore, the range previously demonstrated to be safe in humans (2 ng / kg to 27 ng / kg) is expected to have a very good effect on IgG responses and similarly good effects on suppressing allergies.

Claims

1. Interleukin-1 (IL-1) for the prevention and / or treatment of allergies.

2. A composition for preventing and / or treating allergies, comprising IL-1 as an active ingredient.

3. 10. The composition of claim 2 for use in a method for desensitizing an allergic individual.

4. The composition according to claim 2 or 3 for immunotherapy.

5. The composition according to any one of claims 2 to 4, wherein said IL-1 is used in combination with at least one allergen.

6. The composition according to any one of claims 2 to 5, further comprising at least one allergen.

7. The composition according to any one of claims 2 to 6, wherein the allergen is a food allergen, a contact allergen, or a respiratory allergen, or a drug.

8. 8. The composition of any one of claims 2 to 7, wherein the composition is administered by oral, epicutaneous, subcutaneous, transdermal, intralymphatic, intramuscular, intravenous, intranasal or rectal route.

9. A composition comprising IL-1 and at least one allergen.

10. 10. The composition of claim 9, in the form of a solution, gel, powder, aerosol spray, lotion or foam.

11. 11. A medical device comprising a composition according to claim 9 or 10, the medical device being suitable for administering the composition to an individual.

12. 12. The medical device of claim 11, selected from the group comprising a patch, an inhaler, a nebulizer and a syringe.

13. A desensitization kit comprising: a composition comprising IL-1; at least one allergen; Includes a kit.

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