Methods and compositions for treating asthma and allergic diseases

Abstract

The present invention relates to the field of allergies. The inventors have obtained preliminary data on wild-type (PCSK9+ / +) or PCSK9-deficient mice (PCSK9- / -), and found that under basal conditions and in the absence of specific stimulation, PCSK9 deficiency significantly increases the percentage of regulatory T cells in the spleen, mesenteric lymph nodes and Peyer's patches. In addition, the inventors have discovered the effects of allergic attack on PCSK9 expression and secretion on primary human bronchial epithelial cells. Interestingly enough, the first results obtained by Q-PCR showed that HDM and LPS increased the level of PCSK9mRNA. Therefore, the present invention relates to PCSK9 inhibitors for the treatment of asthma and / or allergic diseases (such as food allergies).

Description

Technical Field

[0001] The present invention relates to the field of allergies. More specifically, the present invention relates to methods and compositions for treating asthma and allergic diseases such as food allergies. Background Art

[0002] PCSK9 (proprotein convertase subtilisin kexin type 9) is a key regulator of cholesterol metabolism, and PCSK9 inhibitors have been developed to treat hypercholesterolemia and cardiovascular disease.

[0003] PCSK9 was discovered in 2003 in patients with autosomal dominant hypercholesterolemia (1) and quickly established its canonical role as a natural inhibitor of the low-density lipoprotein receptor (LDLR) (2). After autophagy in the endoplasmic reticulum, PCSK9 is secreted into the blood by the liver as a mature form. PCSK9 binds to the extracellular domain of LDLR and interferes with LDLR trafficking by promoting its lysosomal degradation rather than recycling to the membrane. Genetic studies further confirmed that PCSK9 deficiency is associated with reduced LDL-cholesterol (LDL-C) levels and a dramatic reduction in cardiovascular events (3), leading to the development of PCSK9 inhibitors. Recently, studies have shown that human monoclonal antibodies to PCSK9 that block the extracellular form of PCSK9 can significantly reduce major cardiovascular events in patients with hypercholesterolemia (4).

[0004] In addition to this classic role in the liver LDLR pathway, many studies have shown that PCSK9 may play some biological functions in extrahepatic organs (reviewed in (5)). PCSK9 is abundantly expressed in the liver, as well as in the intestine, kidney, lung, and thymus (6). PCSK9 is also secreted from the liver into the blood and can regulate the expression of LDLR in extrahepatic tissues in an endocrine manner (7).

[0005] In addition to its role in lipid metabolism, some studies have suggested that PCSK9 may also be involved in inflammatory responses. For example, hepatic expression of PCSK9 in mice increases in response to lipopolysaccharide (LPS)-induced inflammation (8). Recently, a series of independent studies have highlighted the link between PCSK9 and the innate immune response and septic shock outcomes (reviewed in (9)). PCSK9 deficiency or inhibition has been shown to be associated with improved outcomes of septic shock in humans and mice, possibly due to increased clearance of LPS in an LDLR-dependent manner (9). Recently, a study found a link between hypercholesterolemia, PCSK9 overexpression, and T cell-mediated inflammatory diseases (10). Mice overexpressing a “gain of function” mutant of PCSK9 showed a decrease in blood and spleen regulatory T cells and an increase in T cells (CD3+ / CD45+) in their lungs. Histological analysis also showed increased perivascular and peribronchial inflammation in mice treated with AAV8PCSK9 (10).

[0006] Asthma is a common chronic respiratory disease that affects approximately 300 million people worldwide. Although the prevalence of asthma varies between 1% and 16% of the population in different countries, the prevalence of food allergies is 3% and is increasing. Among the various phenotypes of asthma, the early-onset atopic Th2 type (11) is often associated with allergic conditions and appears to be the final stage of the "atopic march" caused by skin or intestinal inflammation, leading to sensitization to aeroallergens and the development of bronchial asthma (12-14). This chronic inflammatory disease involves many players in immunity (IgE-specific allergens, mast cells, cytokines) and organs (spleen, lymph nodes, intestine, lungs), whose functions can be rapidly altered.

[0007] Several independent groups have recently investigated the effects of PCSK9 on sepsis, but none of them have determined its effects on allergy and / or asthma, which are global health burdens. Therefore, new therapeutic strategies involving immunity are needed to treat asthma and food allergies. Summary of the invention

[0008] The present invention relates to PCSK9 inhibitors for the treatment of asthma and allergic diseases. In particular, the invention is defined by the claims. DETAILED DESCRIPTION

[0009] The inventors have obtained preliminary data for wild-type (PCSK9+ / +) or PCSK9-deficient mice (PCSK9- / -), and found that under basal conditions and in the absence of specific stimulation, PCSK9 deficiency significantly increases the percentage of regulatory T cells in the spleen, mesenteric lymph nodes and Peyer's patches. Interestingly, changes in the activation of these cells in allergies are considered to be crucial in the occurrence of chronic inflammation. In addition, the inventors have discovered the effects of allergic attacks on primary human bronchial epithelial cells on PCSK9 expression and secretion. Very interestingly, the first results obtained by Q-PCR showed that house dust mite (HDM) allergens and LPS increase the level of PCSK9 mRNA. Similarly, HDM causes a strong rise in intracellular PCSK9 protein content. When LPS was used to culture cells, a trend of increased intracellular PCSK9 was also observed (+53%). It was also found that the concentration of PCSK9 increased in the culture medium after HDM or LPS treatment.

[0010] Therefore, the present inventors have found that PCSK9 may be a new potential therapeutic target for the treatment of asthma and allergic diseases (such as food allergies).

[0011] Methods for assessing or predicting the severity of asthma or allergic disease

[0012] Therefore, the first object of the present invention relates to a method for assessing or predicting the severity of asthma or the severity of an allergic disease in a subject, comprising i) determining the level of PCSK9 in a biological sample obtained from the subject, ii) comparing the level determined in step i) with a predetermined reference value and iii) when the PCSK9 level is higher than the predetermined reference value, concluding that the subject suffers from or is susceptible to severe asthma or a severe allergic disease, or when the PCSK9 level is lower than the predetermined reference value, concluding that the subject does not suffer from or is not susceptible to severe asthma or a severe allergic disease.

[0013] In a specific embodiment, the method is suitable for predicting whether a subject is at risk of having asthma or severe asthma.

[0014] As used herein, the term "allergic disease" or "allergy" refers to the reaction of the immune system, in particular the reaction of specific IgE antibodies. Typically, IgE antibodies and antigens bind to membrane receptors of mast cells and granulocytes, and the antigen-antibody reaction releases inflammatory mediators, dilates blood vessels, makes capillary permeability too high, and causes tissue infiltration such as inflammatory cells. Typically, allergic conditions include allergic inflammation and / or chronic inflammation. For example, allergic conditions include allergic inflammation and are selected from the following: allergic rhinitis, atopic dermatitis, allergic asthma, allergic conjunctivitis, gastrointestinal inflammation, urticaria and / or food allergies. In the context of the present invention, allergic diseases are asthma, severe asthma or food allergies.

[0015] As used herein, the term "asthma severity" refers to the Global Initiative for Asthma (GINA) definition of severe asthma: asthma that requires high doses of inhaled steroids and / or oral steroids in combination with other controllers for control, or that remains uncontrolled despite such treatment.

[0016] As used herein, the term "risk" refers to the probability that an event will occur within a specific time period, such as a severe asthma attack, and can represent the "absolute" risk or "relative" risk of a subject. Absolute risk can be measured by reference to the actual observed results after measurement of a relevant time cohort, or by reference to an indicator value derived from a statistically significant historical data cohort followed during the relevant time period. Relative risk refers to the ratio of a subject's absolute risk to the absolute risk of a low-risk cohort or the average population risk, which may vary depending on the clinical risk factors assessed.

[0017] As used herein, the term "subject" refers to any mammal, such as rodents, cats, dogs and primates. In particular, in the present invention, the subject is a human. In a specific embodiment, the subject is a human suffering from or suspected of suffering from asthma and / or an allergic disease. More particularly, the subject suffers from or is suspected of suffering from severe asthma or food allergies.

[0018] As used herein, the term "biological sample" refers to a sample obtained from a subject, such as blood, saliva, breast milk, urine, semen, plasma, synovial fluid, serum or plasma and bronchoalveolar lavage fluid (BAL). In a specific embodiment, the biological sample is a blood sample. The term "blood sample" refers to any blood sample from a subject. Peripheral blood is preferred, and mononuclear cells (PBMC) are preferred cells. Typically, these cells can be extracted from whole blood using Ficoll (a hydrophilic polysaccharide that separates blood layers), and PBMC forms a cell ring under the plasma layer. In addition, PBMC can be extracted from whole blood using a hypotonic lysis method, which will preferentially lyse red blood cells. Such a procedure is well known to those skilled in the art. In another embodiment, the biological sample is plasma and / or bronchoalveolar lavage fluid (BAL).

[0019] As used herein, the term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9, also known as neuronal apoptosis-regulated convertase (NARC-1), which is a proteinase K-like subtilisin identified as the ninth member of the secretory subtilisin family. PCSK9 is a natural inhibitor of the LDL receptor (LDLR) and plays a key role in cholesterol metabolism. PCSK9 binds to the extracellular domain of LDLR and triggers its intracellular degradation, thereby controlling the level of LDL particles in the blood circulation.

[0020] As used herein, the term "level" refers to the expression level of PCSK9. Generally, the expression level of the PCSK9 gene can be determined by any technique known to those skilled in the art. In particular, the expression level of each gene can be measured at the genome and / or nucleic acid and / or protein level. In a specific embodiment, the expression level of the gene is determined by measuring the amount of the nucleic acid transcript of each gene. In another embodiment, the expression level is determined by measuring the amount of the corresponding protein of each gene. The amount of nucleic acid transcripts can be measured by any technique known to those skilled in the art. In particular, the detection can be performed directly on the extracted messenger RNA (mRNA) sample, or the reverse transcribed complementary DNA (cDNA) prepared from the extracted mRNA can be detected by techniques well known in the art. From the mRNA or cDNA samples, the amount of nucleic acid transcripts can be measured using any technique well known to those skilled in the art, including nucleic acid microarrays, quantitative PCR, microfluidic cards, and hybridization using labeled probes. In a specific embodiment, the expression level is determined by using quantitative PCR. Quantitative or real-time PCR is a well-known and easily available technique to those skilled in the art and does not require precise description. The method for determining the amount of mRNA is well known in the art. For example, first, according to standard methods, for example, using a lyase or chemical solution or according to the instructions of the manufacturer, the nucleic acid contained in the biological sample is extracted by a nucleic acid binding resin. Then, the extracted mRNA is detected by hybridization (for example, Northern blot analysis) and / or amplification (for example, RT-PCR). Preferred quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and amplification based on nucleic acid sequence (NASBA). It is found that the nucleic acid having at least 10 nucleotides and showing sequence complementarity or homology with the mRNA of interest herein can be used as a hybridization probe or amplification primer. It should be understood that such nucleic acids do not need to be identical, but are generally at least about 80% identical to homologous regions with comparable sizes, more preferably 85% identical, and even more preferably 90-95% identical. In certain embodiments, it is advantageous that nucleic acids are used in combination with appropriate means (such as detectable labels) to detect hybridization. Various suitable indicators are well known in the art, including fluorescent, radioactive, enzymatic or other ligands (e.g., avidin / biotin). The probe typically comprises a single-stranded nucleic acid between 10 and 1000 nucleotides in length, such as between 10 and 800, more preferably between 15 and 700, and typically between 20 and 500. The primer is typically a shorter single-stranded nucleic acid, between 10 and 25 nucleotides in length, which is designed to completely or almost completely match the nucleic acid of interest to be amplified.Probes and primers are "specific" for the nucleic acids to which they hybridize, i.e., they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50% formamide, 5x or 6x SCC. SCC is 0.15M NaCl, 0.015M sodium citrate). The nucleic acid primers or probes used in the above-mentioned amplification and detection methods can be assembled into a kit. Such a kit comprises a common primer and a molecular probe. The kit also comprises components necessary to determine whether amplification has occurred. The kit may also comprise, for example, PCR buffer and enzyme; positive control sequence, reaction control primer; and instructions for amplifying and detecting a specific sequence. In a specific embodiment, the method of the present invention comprises the steps of providing total RNA extracted from a biological sample and amplifying and hybridizing the RNA with a specific probe, in particular by quantitative or semi-quantitative RT-PCR. In another embodiment, the expression level is determined by DNA chip analysis. Such DNA chips or nucleic acid microarrays consist of different nucleic acid probes chemically attached to a substrate, which can be beads of microchip, slide or microsphere size. Microchip can be made up of polymer, plastic, resin, polysaccharide, silicon dioxide or silicon dioxide based material, carbon, metal, inorganic glass or nitrocellulose.Probe comprises can be about 10 to about 60 base pairs of nucleic acid, such as cDNA or oligonucleotide.For determining expression level, biological sample from detection subject is marked (described biological sample is optionally reverse transcribed at first), and it is contacted with microarray under hybridization conditions, thereby causes forming between target nucleic acid and the complex complementary to the probe sequence attached to microarray surface.Then, detect the hybridization complex of mark, and can carry out quantitative or semi-quantitative to it.Can realize mark by various methods, for example, by using radioactive or fluorescent label.Many variants of microarray hybridization technology are available to those skilled in the art (referring to for example, Hoheisel, Nature Reviews, Genetics, 2006,7:200-210 summary).

[0021] Mendelian randomization studies have been used to validate the relevance of PCSK9 inhibition for the treatment of cardiovascular disease. For example, it has been demonstrated that a 10 mg / dL reduction in LDL-cholesterol associated with a loss-of-function variant in PCSK9 reduces cardiovascular events by 19% (Ference BA, N Eng J Med, 2016; 375: 2144-53).

[0022] Therefore, in a specific embodiment, the loss-of-function variant of PCSK9 can be detected in a patient cohort with asthma or allergic diseases by the above method. Generally, the mutation R46L can be detected by the method described above. As the inventors found in previous studies, the mutation R46L is known to affect about 2% of the French population: Bonnefond A, Diabetologia, 2015, 58: 2051-5.

[0023] When a subject has a loss of function of PCSK9, this means that the loss of function improves the allergic disease, and more particularly, reduces the severity of the allergic disease.

[0024] In another embodiment, the measurement of PCSK9 levels is carried out by immunological detection. Generally, by any method known in the art, at least one antibody specifically bound to PCSK9 is used to achieve immunological detection or quantification of PCSK9 levels. Examples of the method include, but are not limited to, standard electrophoresis and immunodiagnostic techniques, such as western blot, immunoprecipitation assay, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (such as gel agglutination assay, blood agglutination assay, etc.), complement fixation assay, protein A assay, immunoelectrophoresis assay, high performance liquid chromatography, size exclusion chromatography, solid phase affinity, etc. In a specific embodiment, PCSK9 levels are measured by ELISA. In a specific embodiment, intracellular PCSK9 protein and secreted PCSK9 protein levels are measured.

[0025] As used herein, the term "predetermined reference value" refers to a threshold value or a cutoff value. Generally, the "threshold value" or "cutoff value" can be determined experimentally, empirically or theoretically. As will be appreciated by those of ordinary skill in the art, the threshold value can also be arbitrarily selected based on existing experimental and / or clinical conditions. For example, the retrospective measurement results of historical subject samples that are correctly stored can be used to establish a predetermined reference value. The threshold value must be determined so that the best sensitivity and specificity are obtained according to the function of the test and the benefit / risk balance (clinical outcomes of false positives and false negatives). Generally, the receiver operating characteristic (ROC) curve based on experimental data can be used to determine the best sensitivity and specificity (therefore, the threshold value also needs to be determined). For example, after determining the expression level of a selected peptide in a set of references, the expression level determined in the sample to be tested can be statistically processed using an algorithm analysis to obtain a classification standard that is of great significance to the sample classification. The full name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. It is mainly used for clinical biochemical diagnostic testing. The ROC curve is a comprehensive indicator of a continuous variable reflecting the true positive rate (sensitivity) and the false positive rate (1-specificity). It uses an image combination method to reveal the relationship between sensitivity and specificity. A series of different cutoff values ​​(thresholds or critical values, the boundary values ​​between normal and abnormal results of diagnostic tests) are set as continuous variables to calculate a series of sensitivity and specificity values. Then, sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw the curve. The higher the area under the curve (AUC), the higher the accuracy of the diagnosis. On the ROC curve, the point closest to the upper left corner of the coordinate graph is the critical point with both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, as AUC approaches 1, the diagnostic results will get better and better. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is medium. When AUC is higher than 0.9, the accuracy is high. The algorithm method is preferably completed by a computer. The ROC curve can be drawn using existing software or systems in the prior art, such as: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0026] Methods and compositions for treating asthma and / or allergic diseases

[0027] A second object of the present invention relates to a PCSK9 inhibitor for use in treating asthma and / or allergic diseases in a subject in need thereof.

[0028] In particular, the present invention relates to a method of treating asthma and / or allergic diseases in a subject in need thereof, said method comprising the step of administering to said subject a therapeutically effective amount of a PCSK9 inhibitor.

[0029] In a particular embodiment, the PCSK9 inhibitor for use according to the invention is suitable for the treatment of asthma and / or severe asthma.

[0030] In another embodiment, the PCSK9 inhibitor for use according to the invention is suitable for the treatment of allergic diseases, such as food allergies.

[0031] In a specific embodiment, the PCSK9 inhibitor for use according to the present invention comprises the step of predicting whether the subject is at risk of suffering from severe asthma or severe allergic disease by performing the method of the present invention.

[0032] As used herein, the term "treatment" or "treat" refers to prophylactic or preventive treatment, as well as curative or disease-modifying treatment, including treatment of subjects at risk of or suspected of having the disease, and subjects who are ill or diagnosed with a disease or medical condition, including inhibition of clinical relapse. The treatment may be administered to a subject who suffers from a medical condition or may eventually suffer from the condition in order to prevent, cure, delay the onset of the disease, reduce the severity of the disease, reduce one or more symptoms of the disease or recurrence of the disease, or to prolong the subject's survival beyond the expected survival in the absence of such treatment. "Therapeutic regimen" refers to the treatment mode of the disease, for example, the mode of administration used during treatment. The treatment regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of the disease. The overall goal of the induction regimen is to provide a high level of drug to the subject during the initial stage of the treatment regimen. The induction regimen may (in part or in whole) adopt a "loading regimen", which may include administering a higher dose of the drug than the doctor uses during the maintenance regimen, administering the drug at a higher frequency than the doctor uses during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a subject during treatment of a disease, for example, so that the subject is in remission for a longer period of time (months or years). The maintenance regimen can be continuous therapy (e.g., regular administration of medication, e.g., weekly, monthly, annually, etc.) or intermittent therapy (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment after reaching a predetermined criterion [e.g., disease manifestation, etc.]).

[0033] As used herein, the term "subject" refers to any mammal, such as rodents, cats, dogs and primates. In particular, in the present invention, the subject is a human suffering from or suspected of suffering from at least one allergic disease. In a specific embodiment, the subject is a human suffering from or suspected of suffering from food allergy or asthma. In a specific embodiment, the subject suffers from food allergy. In a specific embodiment, the subject suffers from severe asthma.

[0034] As used herein, the term "allergic disease" or "allergy" refers to the reaction of the immune system, in particular the reaction of specific IgE. Typically, IgE antibodies and antigens bind to membrane receptors of mast cells and granulocytes, and the antigen-antibody reaction releases inflammatory mediators, dilates blood vessels, makes capillary permeability too high, and causes tissue infiltration such as inflammatory cells. Typically, allergic conditions include allergic inflammation and / or chronic inflammation. For example, allergic conditions include allergic inflammation and are selected from the following: allergic rhinitis, atopic dermatitis, allergic asthma, allergic conjunctivitis, gastrointestinal inflammation, urticaria and / or food allergies.

[0035] As used herein, the term "food allergy" refers to an adverse reaction to food mediated by an immune mechanism, involving specific IgE (IgE-mediated), cell-mediated mechanisms (non-IgE-mediated), and mechanisms mediated by both IgE and cells (mixed IgE and non-IgE-mediated). The inability of the organism to develop or destroy food tolerance will lead to excessive production of food-specific IgE antibodies, or changes in cellular events leading to allergic reactions. In particular, food allergies are related to eosinophilic esophagitis, eosinophilic gastritis and gastroenteritis, food protein-induced enterocolitis syndrome and allergic primary colitis, food-induced pulmonary hemorrhagic hemosiderosis (Heiner's syndrome), food-induced allergic reactions (acute urticaria, anaphylactic shock and edema), and food-related atopic dermatitis and asthma.

[0036] As used herein, the term "asthma" refers to a chronic respiratory disease defined by the World Health Organization (WHO) as a chronic disease characterized by recurrent episodes of shortness of breath and wheezing, the severity and frequency of which vary from person to person.

[0037] As used herein, the term "PCSK9" refers to proprotein convertase subtilisin / kexin type 9, also known as neuronal apoptosis-regulated convertase (NARC-1), which is a proteinase K-like subtilisin identified as the ninth member of the secretory subtilisin family. PCSK9 is a natural inhibitor of the LDL receptor (LDLR) and plays a key role in cholesterol metabolism. PCSK9 binds to the extracellular domain of LDLR and triggers its intracellular degradation, thereby controlling the level of LDL particles in the blood circulation.

[0038] As used herein, the term "PCSK9 inhibitor" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the activity or expression of PCSK9. Therefore, it refers to any compound that can directly or indirectly reduce the transcription, translation, post-translational modification or activity of PCSK9. It includes intracellular and extracellular PCSK9 inhibitors.

[0039] In a specific embodiment, the inhibitor of PCSK9 activity used in the present invention is an organic small molecule, an aptamer, an antibody or a polypeptide.

[0040] As used herein, the term "organic small molecule" refers to a molecule that is comparable in size to those organic molecules commonly used in medicine. The term does not include biomacromolecules (e.g., proteins, nucleic acids, etc.). Typically, the size range of an organic small molecule can be up to about 5000Da, more preferably up to 2000Da and most preferably up to about 1000Da. In a specific embodiment, the organic small molecule is DS-9001a, which comprises an albumin binding domain fused to an artificial lipoprotein mutant protein (Anticalin protein fused to ABD). This small molecule is developed by Daiichi Sankyo / Pieris Pharmaceuticals (Phase 1 study in progress).

[0041] As used herein, the term "aptamer" refers to a class of molecules that represent alternatives to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or polypeptide sequences that have the ability to recognize virtually any type of target molecule with high affinity and specificity.

[0042] As used herein, the term "antibody" refers to any antibody-like molecule with an antigen-binding region, and the term includes antibody fragments comprising an antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DAB or VHH), TandAb dimers, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, mini antibodies, diabodies, bispecific antibody fragments, diabodies, tribodies (respectively, scFv-Fab fusion protein, bispecific or trispecific); sc-diabodies; κ (λ) bodies (scFv-CL fusion); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immune protein, a mini antibody); SMIP ("small modular immunopharmaceutical"); scFv-Fc dimers; DART (ds stabilized diabody "dual affinity ReTargeting"); small antibody mimics comprising one or more CDRs, etc. Preparation and use of various antibody-based constructs and fragments are well known in the art. In particular, in the context of the present invention, the antibody is a single domain antibody. The term "single domain antibody" has its ordinary meaning in the art and refers to a single heavy chain variable domain of an antibody, a type of antibody that can exist in mammals of the genus Camelus, which naturally lack light chains. Such single domain antibodies are also called VHH or For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341(6242):544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. In the context of the present invention, the amino acid residues of a single domain antibody are numbered according to the universal numbering of the VH domain given by the international ImMunoGeneTics information system amino acid numbering (http: / / imgt.cines.fr / ). In particular, in the context of the present invention, an antibody is a single chain variable fragment. The term "single chain variable fragment" or "scFv fragment" refers to a single folded polypeptide comprising the VH and VL domains of an antibody connected by a linker molecule. In such scFv fragments, the VH and VL domains can be VH-linker-VL or VL-linker-VH sequences. In addition to promoting its production, the scFv fragment can also include a tag molecule connected to the scFv by a spacer. Therefore, the scFv fragment includes the VH and VL domains involved in antigen recognition, but does not include the immunogenic constant domains of the corresponding antibody. In a specific embodiment, the inhibitor of PCS9 activity is an endosome specific for PCSK9. As used herein, the term "endosome" generally refers to an intracellular antibody or antibody fragment. Antibodies, particularly single-chain variable antibody fragments (scFv), can be modified for intracellular localization. For example, such modifications may require fusion to a stable intracellular protein, such as, for example, maltose binding protein, or the addition of an intracellular transport / localization peptide sequence, such as, for example, endoplasmic reticulum retention. In some embodiments, the endosome is a single domain antibody.

[0043] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is a monoclonal antibody.

[0044] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is evolocumab, which is marketed by Amgen under the trade name (or AMG 145) and has the following general formula in the art: 6242 H 9648 N 1668 O 1996 S 56 .

[0045] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is Alelimumab, which is sold under the trade name Praluent (REGN727 or SAR2365553) by Sanofi and Regeneron Pharmaceuticals and has the following general formula in the art: 6472 H 9996 N 1736 O 2032 S 42 .

[0046] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is Bococizumab, also known as PF-04950615 or RN316, which is being developed by PFIZER (in Phase 3 studies).

[0047] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is LGT-209, which is being developed by Novartis (Phase 2 studies ongoing).

[0048] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is RG-7652, which is being developed by Roche (in Phase 2 studies).

[0049] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is LY3015014, which is being developed by Eli Lilly (Phase 2 studies ongoing).

[0050] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is MEDI 4166, which is being developed by AstraZeneca and Medimmune. MEDI 4166 is a PCSK9 antibody fused to a GLP1 peptide.

[0051] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is AT04A or AT06A, which is a vaccine developed by AFFiRiS AG (phase 1 study ongoing), Landlinger C et al., Eur Heart J. 2017aug21.

[0052] As used herein, the term "polypeptide" refers to a polypeptide that specifically binds to PCSK9 and can be used as a PCSK9 inhibitor that binds to and chelates PCSK9 proteins to prevent their signal transduction. Polypeptides refer to short peptides of at least 2 amino acid residues and at most 10 amino acid residues in length, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e., a length greater than 100 amino acid residues) and proteins (functional entities containing at least one peptide, oligopeptide or polypeptide, which can be chemically modified by glycosylation, lipidation or containing a prosthetic group). The definition of polypeptides also includes natural forms of peptides / proteins in mycobacteria, as well as recombinant proteins or peptides in any type of expression vector that transforms any type of host, and chemically synthesized peptides. In particular, PCSK9 activity inhibitors are intracellular peptides. Typically, intracellular peptides mainly interfere with the transmission of PCSK9 signals in the cytoplasm, mitochondria and / or nuclei. In a specific embodiment, the anti-PCSK9 active polypeptide is BMS-962476, which is characterized by the amino acid sequence disclosed in WO 2011130354. This polypeptide is also described in Mitchell et al., 2010 (J Pharmacol Exp Ther. 2014 Aug; 350(2):412-24. doi: 10.1124 / jpet.114.214221. Epub 2014 Jun 10.).

[0053] In a specific embodiment, the PCSK9 inhibitor for use in the present invention is an inhibitor of PCSK9 expression.

[0054] As used herein, "an inhibitor of PCSK9 expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene encoding PCSK9. Typically, an inhibitor of PCSK9 expression has a biological effect on one or more of the following events: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing RNA transcripts (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translating RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0055] In some embodiments, inhibitors of PCSK9 expression are antisense oligonucleotides. Antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, will directly prevent the translation of PCSK9 mRNA by binding thereto, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level and activity of PCSK9 protein in cells. For example, antisense oligonucleotides complementary to a unique region of the mRNA transcript sequence encoding PCSK9 can be synthesized, for example, by conventional phosphodiester technology, at least about 15 bases and mRNA transcript sequences encoding PCSK9, and administered by, for example, intravenous injection or infusion. Methods for using antisense technology to specifically reduce gene expression of known genes of sequence are well known in the art (see, for example, U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

[0056] In a specific embodiment, the inhibitor of PCSK9 expression is shRNA. ShRNA is usually expressed using a vector introduced into the cell, wherein the vector utilizes the U6 promoter to ensure that shRNA is always expressed. The vector is usually passed to daughter cells so that gene silencing can be inherited. The shRNA hairpin structure is cut into siRNA by the cell mechanism and then binds to the RNA-induced silencing complex (RISC). The complex binds and cuts the mRNA that matches the siRNA bound to it.

[0057] In some embodiments, the inhibitor of PCSK9 expression is a small inhibitory RNA (siRNA). The expression of PCSK9 can be reduced by contacting the subject or cell with a small double-stranded RNA (dsRNA) or a vector or construct that causes the production of small double-stranded RNA, thereby specifically inhibiting the expression of PCSK9 (i.e., RNA interference or RNAi). For genes with known sequences, methods for selecting suitable dsRNA or vectors encoding dsRNA are well known in the art (e.g., see Tuschl, T. et al., (1999); Elbashir, SM et al., (2001); Hannon, GJ. (2002); McManus, MT. et al., (2002); Brummelkamp, ​​TR. et al., (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619 and WO 01 / 68836). In a specific embodiment, the siRNA is ALN-PCS02, also known as inclisiran, which is being developed by Alnylam (Phase 3 studies ongoing), Ray KK et al., N Egnl J Med, Apr 2017.

[0058] In some embodiments, the inhibitor of PCSK9 expression is a ribozyme. Ribozymes are enzymatic RNA molecules that can catalyze RNA-specific cutting. The ribozyme mechanism of action involves sequence-specific hybridization of ribozyme molecules with complementary target RNAs, followed by endonuclease cleavage. Therefore, engineered hairpin or hammerhead motif ribozyme molecules that specifically and effectively catalyze the endonuclease cleavage of PCSK9 mRNA sequences are useful within the scope of the present invention. First, the specific ribozyme cleavage site in any potential RNA target is identified by scanning the ribozyme cleavage site of the target molecule, which typically includes the following sequences GUA, GUU and GUC. Once identified, the predicted structural features of the short RNA sequence between about 15 to 20 ribonucleotides corresponding to the target gene region containing the cleavage site can be evaluated, such as secondary structure, which may cause the oligonucleotide sequence to be inappropriate. The suitability of the candidate target can also be evaluated by using, for example, ribonuclease protection assays to detect the accessibility of the candidate target point to the hybridization of complementary oligonucleotides.

[0059] In some embodiments, the inhibitor of PCSK9 expression is an endonuclease. The term "endonuclease" refers to an enzyme that cuts phosphodiester bonds within a polynucleotide chain. Some (such as deoxyribonuclease I) cut DNA relatively non-specifically (regardless of sequence), while many are commonly referred to as restriction endonucleases or restriction endonucleases, and only cut at very specific nucleotide sequences. Endonuclease-based genome inactivation mechanisms usually require a first step of DNA single-strand or double-strand breaks, which can then trigger two different cellular DNA repair mechanisms that can be used for DNA inactivation: error-prone non-homologous end joining (NHEJ) and high-fidelity homology-directed repair (HDR).

[0060] In a specific embodiment, the endonuclease is CRISPR-cas. As used herein, the term "CRISPR-cas" has its general meaning in the art and refers to related clustered regularly spaced short palindromic repeats, which are prokaryotic DNA segments containing short repeat base sequences. In some embodiments, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 B1 and US 2014 / 0068797. In some embodiments, the endonuclease is CRISPR-Cpf1, which is a CRISPR recently characterized by Zetsche et al. from Provotella and Francisella 1 (Cpf1) ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0061] As used herein, the term "administering" or "administration" refers to injecting or otherwise physically delivering a substance (e.g., an inhibitor of PCSK9) present in vitro to a subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, the administration of the substance is usually performed after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is usually administered before the onset of the disease or its symptoms.

[0062] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of a PCSK9 inhibitor to treat a disease (e.g., food allergies and / or asthma) at a reasonable benefit / risk ratio applicable to any drug therapy. It should be understood that the total daily dosage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound used; duration of treatment; drugs used in combination or concomitantly with the specific polypeptide used; and factors well known in the medical field. For example, it is well known to those skilled in the art that the initial dose of the compound should be lower than the dose required to achieve the desired therapeutic effect, and the dose should be gradually increased until the desired effect is achieved. However, the daily dose of the product can vary over a wide range of 0.01 to 1,000 mg per adult per day. Typically, the composition comprises 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of active ingredient for symptomatic adjustment of the dosage of the patient to be treated. The drug typically comprises from about 0.01 mg to about 500 mg of active ingredient, typically from 1 mg to about 100 mg of active ingredient. An effective amount of the drug is typically provided at a dosage level of 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly about 0.001 mg / kg to 7 mg / kg body weight per day.

[0063] The PCSK9 inhibitors as described above may be administered in combination with classical treatments for asthma or allergic diseases.

[0064] Thus, the present invention relates to i) a PCSK9 inhibitor and ii) a classical therapy as a combined preparation for use in treating a subject suffering from asthma or an allergic disease.

[0065] As used herein, the term "classical treatment" refers to any compound, natural or synthetic, used to treat asthma or allergic diseases.

[0066] In a specific embodiment, the conventional treatment of asthma or allergic diseases refers to beta-adrenergic receptor agonists, such as corticosteroids, such as flunisolide, ciclesonide, mometasone, fluticasone, budesonide, beclomethasone and prednisone; bronchodilators, such as epinephrine, racemic epinephrine, indacaterol, salbutamol, levobutol, olodaterol, formoterol, arformoterol, pirbuterol, terbutaline, metanephrine, salmeterol, ipratropium bromide ammonium, albuterol, tiotropium, umeclidinium, glycopyrrolate, and rifacin; methylxanthines, such as theophylline, dyphylline, and aminophylline; antileukotrienes, such as montelukast, zafiralast, and zileuton; interleukin inhibitors, such as reslizumab, dupilumab, benralizumab, mepolizumab, and omalizumab; mast cell stabilizers, such as cromolyn; and allergen immunotherapy, such as Grastek, OralAir, and Ragwitek.

[0067] As used herein, the terms "combination therapy," "combination preparation," "combination therapy," or "therapy combination" refer to a treatment using more than one drug. A combination therapy can be a dual therapy or a double therapy.

[0068] The drugs used in the combination therapy according to the present invention are administered to the subject simultaneously, separately or sequentially.

[0069] As used herein, the term "simultaneous administration" refers to administration of two active ingredients by the same route, at the same time or substantially at the same time. The term "separate administration" refers to administration of two active ingredients by different routes, at the same time or substantially at the same time. The term "sequential administration" refers to administration of two active ingredients at different times, with the same or different administration routes.

[0070] PCSK9 inhibitors as described above can be combined with pharmaceutically acceptable excipients, and optionally sustained release matrices (such as biodegradable polymers) to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when properly administered to mammals (especially humans). Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semisolid or liquid fillers, diluents, encapsulating materials or formulation aids. For oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration of the pharmaceutical composition of the present invention, the active ingredient can be administered to animals and humans alone or in combination with another active ingredient as a mixture with conventional pharmaceutical supports in a unit administration form. Suitable unit administration forms include oral forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical composition comprises a pharmaceutically acceptable carrier for a preparation that can be injected. These can be particularly isotonic sterile saline solutions (sodium dihydrogen phosphate or disodium hydrogen phosphate, chlorides of sodium, potassium, calcium or magnesium, etc., or mixtures of such salts), or dried, particularly lyophilized compositions, which can be reconstituted into injectable solutions after adding sterile water or saline as needed. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil or propylene glycol aqueous solutions; and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must have a certain fluidity to achieve the purpose of easy injection. It must remain stable under production and storage conditions and must be preserved to prevent contamination by microorganisms (such as bacteria and fungi). Solutions containing the compounds of the present invention as free alkali or pharmacologically acceptable salts can be prepared by mixing with surfactants (such as hydroxypropylcellulose) in water. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oil. Under conventional storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. The polypeptide (or its encoding nucleic acid) can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with the free carboxyl groups may also be derived from inorganic bases (such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils.For example, appropriate fluidity can be maintained by using a coating (such as lecithin) in the case of a dispersion by maintaining the required particle size and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferred to include an isotonic agent, such as sugar or sodium chloride. The absorption of the injectable composition can be extended by using a delayed absorption agent (e.g., aluminum monostearate and gelatin) in the composition. By incorporating the required amount of active polypeptide into a suitable solvent, and as required, incorporating several other ingredients listed above into a sterile injectable solution, and then filtering and sterilizing. In general, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier, which contains a basic dispersion medium and other ingredients required for those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of active ingredients and any other required ingredients from their previous sterile filtered solutions. After preparation, the solution will be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The preparation is easy to be administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules etc. may also be used. For parenteral use in an aqueous solution, for example, if necessary, the solution should be appropriately buffered, and the liquid diluent should first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, according to the present disclosure, those skilled in the art will know that sterile aqueous media can be used. For example, a dose can be dissolved in 1 mL of isotonic NaCl solution, and added to 1000 mL of subcutaneous infusion liquid or injected at the expected infusion site. Some changes in dosage will inevitably occur, depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for individual subjects.

[0071] Screening methods

[0072] In a third aspect, the present invention relates to a method for screening a drug suitable for inhibiting PCSK9, comprising i) providing a test compound and ii) determining the ability of the test compound to inhibit the expression or activity of PCSK9.

[0073] Any biological assay known in the art is suitable for determining the ability of a test compound to inhibit the activity or expression of PCSK9. In some embodiments, the assay first includes determining the ability of a number of compounds to bind to PCSK9. In some embodiments, a cell population is then contacted and activated to determine the ability of the test compound to inhibit PCSK9 activation or expression. In particular, the effect triggered by the test compound is determined relative to a cell population incubated in parallel in the absence of the test compound or in the presence of a control agent (which are both negative control conditions). As used herein, the term "control substance", "control agent" or "control compound" refers to a molecule that is inert or does not have an activity associated with the ability to regulate biological activity or expression. It should be understood that, as determined using in vitro methods as described herein, a test compound that can inhibit the activity or expression of PCSK9 may exhibit similar regulatory abilities in in vivo applications. Typically, the test compound is selected from the following: a peptide, a peptidomimetic, an organic small molecule, an antibody (e.g., an intrabody), an aptamer or a nucleic acid. For example, a test compound according to the present invention can be selected from a library of previously synthesized compounds, or a library of compounds whose structures have been determined in a database, or a library of de novo synthesized compounds.

[0074] The present invention will be further described by the following figures and examples. However, these examples and figures should not be interpreted as limiting the scope of the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 : Regulatory T cells are significantly more abundant in the spleen, mesenteric lymph nodes, and Peyer's patches of PCSK9- / - mice.

[0076] Figure 2: HDM allergen increases intracellular PCSK9 expression and possibly its secretion. Human primary BECs were cultured with HDM allergen or LPS (10 μg / ml) for 48 hours. (A) PCSK9 mRNA levels (B) intracellular PCSK9 protein and (C) "secreted" PCSK9 protein levels. Protein levels were measured by ELISA. *P<0,05.

[0077] Figure 3: PCSK9 deficiency attenuates allergic responses. PCSK9+ / + (white circles) and PCSK9- / - mice (black circles) were sensitized by intraperitoneal injection of vehicle (white bars) or gliadin (black bars) and then challenged twice by gavage with water or gliadin. One hour after the second oral allergic challenge, body weight (A), plasma PCSK9 concentration (B), and ear thickness (C) were measured and presented as mean ± SEM in dot plots and histograms. One hour after the second oral allergic challenge, spleen weight (D), percentage of splenic CD19+IgE+ cells (E), and plasma gliadin-specific IgE concentration (F) were measured and presented as mean ± SEM in dot plots and histograms. Use Statistical significance was analyzed by nonparametric Mann-Whitney test (Graphpad Software). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; NS: not significant.

[0078] Example

[0079] Our laboratory has access to primary human bronchial epithelial cells (primary human BECs) from a multicenter lung transplant cohort (COLT–NCT00980967 study / CPP 2009–A00036-51). Briefly, these cells were dissociated from lung donor trachea or bronchi by incubation with type XIV collagenase in HEPES-buffered RPMI at 4°C overnight. Human BECs were then cultured on human type IV collagen-coated plates in cnT17 medium containing penicillin and streptomycin (19). Given the expression of PCSK9 in the gut and lung and its potential role in immunity, we hypothesized that PCSK9 may play a role in food allergy and the progression to respiratory complications such as asthma. Preliminary data obtained in our laboratory using wild-type (PCSK9+ / +) or PCSK9-deficient mice (PCSK9- / -) indicate that PCSK9 deficiency significantly increases the percentage of regulatory T cells in the spleen, mesenteric lymph nodes, and Peyer's patches under basal conditions and in the absence of specific stimulation ( Figure 1 Interestingly, altered activation of these cells in allergy is thought to be critical in the development of chronic inflammation. Our driving hypothesis was that PCSK9 deficiency protects against food allergy and asthma.

[0080] Very interestingly, our first results obtained by Q-PCR showed that HDM and LPS increased the level of PCSK9 mRNA. Similarly, HDM caused a strong rise in intracellular PCSK9 protein content. When cells were cultured with LPS, we also observed a trend of insignificant increase in intracellular PCSK9 (+53%). We also found an increase in PCSK9 concentration in the culture medium after HDM or LPS treatment (Figure 2). These initial results once again ensure the relevance of the use of this cell model and show that PCSK9 is expressed in bronchial epithelial cells and is regulated during inflammation.

[0081] Allergy induction was then performed. The allergy induction protocol was routinely set up at l'Institut du Thorax and has been described in detail previously (17). We used PCSK9+ / + and PCSK9- / - male and female mice that were fed a gluten-free regular mouse chow since weaning. To induce sensitization, 5-week-old mice received 3 consecutive intraperitoneal injections of vehicle or deamidated wheat prolamin (10 μg) on ​​days 0, 10, and 20. The mice were then challenged twice by oral administration of 20 mg of deamidated prolamin. The results shown in Figure 3 were obtained 1 h after the second allergic challenge. We did observe that PCSK9+ / + mice lost weight under allergic conditions (diet allergy, AA) compared to control PCSK9+ / + mice (Figure 3A). We also observed a small trend of weight loss in PCSK9- / - under allergic conditions that did not reach significant levels (Figure 3A). Interestingly, in PCSK9+ / + mice, plasma PCSK9 levels were significantly increased after allergy induction (Figure 3B). In both PCSK9+ / + and PCSK9- / - mice, prolamin attack increased spleen weight (Fig. 3D) and IgE+ plasma cell% (Fig. 3E). In contrast, in allergic PCSK9+ / + mice rather than in PCSK9- / - mice, plasma prolamin-specific IgE+ concentrations significantly increased (Fig. 3F), indicating that PSCK9 deficiency weakened allergic reactions. To characterize allergic symptoms, we measured ear thickness with a micrometer after oral attack. In the control group, vehicle solvent gavage did not affect ear thickness in both PCSK9+ / + and PCSK9- / - mice. We found that in PCSK9+ / + mice sensitized and attacked with deamidated prolamin, ear thickness increased significantly after oral attack (Fig. 3C). In contrast, no effect was observed in PCSK9- / - mice under the same conditions (Fig. 3C).

[0082] References

[0083] Throughout this application, various references describe the state of the art in the field to which the invention pertains. The disclosures of these references are incorporated into the present disclosure by reference.

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Claims

1. Use of a reagent for determining the level of PCSK9 in a biological sample obtained from a subject in the preparation of a kit for assessing the severity of an allergic disease in the subject, wherein the assessment comprises i) determining the level of PCSK9 in the biological sample obtained from the subject, ii) comparing the level determined in step i) with a predetermined reference value and iii) when the PCSK9 level is higher than the predetermined reference value, concluding that the subject suffers from or is susceptible to a severe allergic disease, or when the PCSK9 level is lower than the predetermined reference value, concluding that the subject does not suffer from or is not susceptible to a severe allergic disease, wherein the allergic disease is selected from food allergy, allergic rhinitis or asthma.

2. The use according to claim 1, wherein the biological sample is a blood sample and / or a BAL sample.

3. The use according to claim 1 or claim 2, wherein the biological sample is a PBMC sample.

4. The use according to claim 1, wherein the PCSK9 level is measured by ELISA.

5. Use of a PCSK9 inhibitor in the preparation of a medicament for treating an allergic disease in a subject in need thereof, wherein the allergic disease is selected from food allergy, allergic rhinitis or asthma and wherein the PCSK9 inhibitor is alirocumab, evolocumab or bococizumab. The use according to claim 5 , wherein the allergic disease is food allergy.

7. The use according to claim 5, wherein the allergic disease is asthma.

8. Use according to any one of claims 5 to 7, wherein the treatment comprises the step of assessing whether the subject is at risk of suffering from a severe allergic disease by performing the assessment mentioned in claim 1.

9. The use according to any one of claims 5 to 8, wherein the PCSK9 inhibitor is combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition.

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