Mrgpre binding agent for use in the treatment of inflammatory and pain disorders

Abstract

The present invention relates to the treatment of inflammation. Here, the inventors generated a mouse model able to trace the expression of MrgprE and to interrogate its function, in vivo. They identified the cells expressing MrgprE notably among leukocytes. The MrgprEMut mice do not present any abnormality especially in the immune and peripheral nervous systems. Interestingly, the ablation of MrgprE expression decrease significantly asthma associated airway hyperresponsiveness and inflammation; symptoms that are restored when MrgprE is expressed by Nav1.8+ sensory fibers. In the context of asthma, an inhibitor of MrgprE could have a dual mode of action preventing both airway hyperresponsiveness and inflammation. Thus, the present invention relates to a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

Description

[0001] MRGPRE BINDING AGENT FOR USE IN THE TREATMENT OF INFLAMMATORY AND PAIN DISORDERS

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] MRGPRs for Mas-Related G Protein-Coupled Receptors correspond to a large family of seven transmembrane domains proteins. This family gathers approximately 50 members across several species among which human and mice. MRGPRs have been discovered for their expression by sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (Dong et al., Cell, 2001). Sensory neurons innervate the entire body and contain diverse specialized subpopulations of neurons that are able to detect a large spectrum of stimuli. In order to organize an adapted answer to environmental attacks, these neurons coordinate behavioral and immune responses. Thus, most MRGPRs are associated to nociception and pruriception (Guan et al., PNAS, 2010). On the other hand, neuro-immune circuits participate to pathogens clearance and tissue repair by orchestrating immune responses (Valentin et al., Annual Review of Immunology, 2018). Interestingly, it has been shown that the expression of MRGPRs is not limited to sensory neurons, and some members of the MRGPRs family have been recently described to be expressed by specialized leukocytes (Dwyer et al., Nature immunology, 2016) The immune and neuronal systems can therefore share receptors and communicate through the release of common messengers. The known endogenous ligands of MRGPRs are for the most peptide in nature. Hence, the expression of MRGPRs by the immune system should favor its control by neuronal messengers like neuropeptides. Although, the literature on MRGPRs is growing, several members of the family, are still orphan receptors with unknown functions.

[0006] The first immune cells characterized for its expression of MRGPRs was mast cells (McNeil et al., Nature, 2015). Mast cells are sentinel immune cells strategically located at the host - environment barrier tissues and in the vicinity of nerves and blood vessels. They express a myriad of receptors that allows them to perceive pathogens and communicate with neurons and further immune cells. Mouse mast cells have been shown to harbor the Mrgprs members a4, bl, b2, x2, xl, b8, bl3 and e (Dwyer et al., Nature immunology, 2016). Among these receptors, Mrgprb2 and its human ortholog MRGPRX2 were the most studied. Mrgprb2 I MRGPRX2 recognize several endogenous molecules termed “basic secretagogues” as well as exogenous molecules notably from pharmaceutical compounds, bacterial quorum sensing and tick saliva (Corbiere et al., Exp Dermatol., 2023). The stimulation of Mrgprb2 triggers mast cell degranulation and the release of pro-inflammatory mediators. Interestingly, the degranulation of mast cells can be fine-tuned depending on the receptor engage to adapt the pattern of degranulation to the threatening cue. Thus, the stimulation of Mrgprb2 compared to that of FceRI (the high affinity receptor for IgE) triggers a faster and locally constrained release of smaller granules that ultimately leads to different consequences on the intensity and the duration of the inflammatory reaction in vivo (Gaudenzio et al., JCI, 2016). The dermal CD301b+dendritic cells have been described to express MRGPRA1 that has for ligand the substance P. The stimulation of MRGPRA1 triggers the migration of dendritic cells to the lymph node, a process that is necessary to initiate an adaptive immune response (Perner et al., Immunity, 2020).

[0007] The immune response is crucial for the survival of organisms. Nevertheless, when exacerbated or developed towards innocuous molecules, inflammation can disrupt the tissue homeostasis, cause irreversible damages and therefore be life threatening. Inflammatory disorders, including allergies and asthma, are diseases caused by a misconducted immune response against harmless compounds. Many FDA-approved drugs (i.e. neuromuscular blocking agents, antibiotics) are cationic and trigger MRGPRX2-dependent mast cell degranulation leading in most cases to an injection site reaction and in rare occurrence to a more severe and systemic reaction called anaphylaxis (Corbiere et al., Exp Dermatol., 2023). Allergy can also take a chronic form when the antigen against which it is directed cannot be avoided (e.i. atopic dermatitis, allergic asthma, .. .). In this case the pathology triggers a remodeling of the involved tissue that can leads to organ dysfunction (e.i. host environment barrier tissue breakdown, fibrosis around bronchi, ...). In the context of atopic dermatitis, nociceptors have been shown to be activated by house dust mite proteases that trigger their release of Substance P, an agonist of Mrgprb2. Mast cells, located in the vicinity of nerve fibers, express Mrgprb2. Upon Substance P activation, these mast cells initiate the type 2 immune response responsible for the development of further atopic dermatitis symptoms (Serhan et al., Nat Immunol. 2019).

[0008] Asthma is a heterogeneous and multifactorial disease affecting people of all ages and characterized by a chronic inflammation of the respiratory tract and an airway hyperresponsiveness. According to the Global Asthma Report 2022, 300 million people suffer from asthma worldwide and this figure is only increasing over time. Currently, 50% of patient present symptoms that are still poorly controlled and asthma remains responsible for 1000 daily deaths. The most severe symptom in asthma is the limitation of airflow, particularly linked to bronchoconstriction, edema of the respiratory tract, over production of mucus and remodeling of the airway. The gravity and intensity of the symptoms can change over time and range from simple variable symptoms such as wheezing, shortness of breath, chest tightness and cough, to the death of the patients due to airway obstruction. Thus asthma can be classified according to the severity of the symptoms (mild, moderate or severe), but also depending on the inflammatory mechanisms involved, such as a predominance of type 2 immunity or not. The majority of new therapies in development to treat asthma focus on targeting pro-inflammatory cytokines (e.g., interleukins 4, 5, 13 or even TSLP) using therapeutic antibodies and are intended for patients suffering from severe asthma who represent only 7% of asthmatics. The lungs are innervated by sensory and motor neurons, which participate not only in controlling bronchoconstriction but also serve as powerful regulators of allergic immune response, including within the lungs. Manipulation of neuroimmune circuits allows the fine regulation of inflammatory processes in several organs (including the lungs) and represent new therapeutics options with high potential. Interestingly, sensory neurons from vagal afferent that innervate the lungs can trigger bronchoconstriction through the activation of MrgprCl l by BAM 8-22 (Han et al., nature neuroscience, 2018).

[0009] As Mrgprs are involve in inflammation, allergy, asthma and itch, modulating the activity of these receptors is a promising strategy to alleviate human neurosensory-inflammatory disorders. MRGPRX2 as well as MRGPRX4 are already the subject of therapeutic development by biotech and big pharma, essentially in the US. Hence, a small molecule antagonist of MRGPRX2 is currently under evaluation for the management of chronic urticaria and other mast cell mediated disorders while another small molecule directed against MRGPRX4 is tested for the treatment of cholestatic pruritus.

[0010] SUMMARY OF THE INVENTION:

[0011] Among all Mrgprs, MrgprE is an orphan receptor with unknown function that has not been studied in pathological context. Mouse and human MrgprE are clear orthologs and share a remarkable sequence identity especially in their predicted extracellular binding site, favoring the hypothesis that they have a common activating ligand. Here, the inventors generated a mouse model able to trace the expression of MrgprE and to interrogate its function, in vivo. They identified the cells expressing MrgprE notably among leukocytes. The MrgprEMut(or MrgprE KO) mice do not present any abnormality especially in the immune and peripheral nervous systems. Interestingly, the ablation of MrgprE expression decreases significantly asthma associated airway hyperresponsiveness and inflammation; symptoms that are restored when MrgprE is expressed by Navi.8+ sensory fibers. In addition, lung biopsies from asthmatics stains for MrgprE at the level of nerve fibers and peribronchial immune cells. Therefore, MrgprE participates to a neuroimmune circuit involved in asthma pathology.

[0012] In the context of asthma, an inhibitor of MrgprE could have a dual mode of action preventing both airway hyperresponsiveness and inflammation.

[0013] Thus, the present invention relates to a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

[0014] Particularly, the invention is defined by its claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] The present invention also relates to a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

[0017] As used in the invention, the term “inflammatory disorders” encompasses “allergic inflammatory disorders” and “pulmonary inflammatory disorders”.

[0018] As used herein, the term “allergic inflammatory disorders” include, for example, airway hyperresponsiveness, anaphylactic hypersensitivity, asthma, allergic asthma, allergic rhinitis, atopic dermatitis, vernal conjunctivitis, eczema, IgE-mediated urticarial, chronic spontaneous urticaria, prurigo nodularis, contact dermatitis, food allergies, IgE-mediated anaphylaxis, esophagus eosinophilic or esophagitis.

[0019] As used herein the term “inflammatory disorders” also denotes non allergic inflammation and particularly non allergic pulmonary inflammation like Chronic obstructive pulmonary disease (COPD). Thus, in some embodiments, the pulmonary inflammatory disorder is Chronic Obstructive Pulmonary Disease (COPD).

[0020] As used herein, the term “subject” denotes a mammal, such as a rodent like a mouse, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human and more particularly an human with an inflammatory disorder.

[0021] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc ]).

[0022] As used herein the terms “MRGPRE binding agent” denotes molecules, compounds, viruses, cells or particles including nanoparticles which can bind the MRGPRE protein. The binding agent comprises a “binding domain” as used herein, the term “binding domain”, i.e. an amino acid sequence region or any structure that preferentially binds to the target molecule under physiological conditions. Binding agents include binding chemical molecules, protein such as antibody but as well as any other proteins potentially capable of binding a given target molecule that typically include but are not limited to protein ligands and receptors, aptamer, polypeptide.

[0023] The term “binding” as used herein refers to a direct association between two molecules or agents, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogenbond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of the binding of a binding protein (e.g. an antibody) to a predetermined target molecule (MRGPRE) typically is a binding with an affinity corresponding to a KD of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less, about IO'10M or less, or about 10'11M or even less.

[0024] In a particular embodiment, the MRGPRE binding agent is an inverse agonist, an antagonist, a modulator or an inhibitor of MRGPRE.

[0025] As used herein the term “inverse agonist” denotes a drug that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of the agonist. Notably, the inverse agonist can bind with the constitutively active receptor, stabilize it and reduces its activity.

[0026] As used herein, the term “antagonist” denotes a drug that reduces / blocks the response of a ligand to its receptor.

[0027] In another particular embodiment, the MRGPRE binding agent is a modulator of MRGPRE.

[0028] As used herein, the term “modulator” denotes a drug that binds to and regulates the activity of a receptor. According to the invention, the modulator has an antagonistic activity to the receptor MRGPRE.

[0029] As used herein, the term “MRGPRE” for “Mas-related G protein-coupled receptor E” has its general meaning in the art and refers to a G protein-coupled receptor present on peripheral nerves and certain cells of the immune system. As used herein the term “MRGPRE” also relates to MRGPRE and MRGPRE orthologs. The Ensembl number of the human gene of MRGPRE is ENSG00000184350.

[0030] In a particular embodiment, the MRGPRE binding agent is a MRGPRE inhibitor.

[0031] The terms “MRGPRE inhibitor” denotes molecules or compound which can inhibit the activity of the protein (e g. inhibit the MRGPRE activity) or a molecule or compound which destabilizes the protein.

[0032] The term “MRGPRE inhibitor” also denotes inhibitors of the expression of the gene coding for the protein.

[0033] In order to test the functionality of a putative MRGPRE inhibitor a test is necessary. For that purpose, to identify MRGPRE inhibitors, one can use the following assays: - BRET2 on G alpha q: This assay measures Bioluminescence Resonance Energy Transfer (BRET2) between G alpha q, a subunit of G proteins, and a reporter protein. It provides real-time monitoring of G alpha q activation, enabling the identification of small molecules that modulate this key signaling pathway.

[0034] - Beta-Arrestin: The beta-arrestin assay evaluates the recruitment of beta-arrestin proteins in response to receptor activation. By assessing beta-arrestin binding, it helps identify small molecules that influence G protein-coupled receptor signaling, offering insights into potential antagonist activity.

[0035] - Accumulation of IP1: This assay measures the accumulation of inositol monophosphate (IP1), a downstream signaling molecule in the phosphoinositide signaling pathway. It serves as an indicator of G protein-coupled receptor activation, enabling the screening of small molecules for their impact on this critical signaling cascade.

[0036] - Intracellular Calcium Levels: The intracellular calcium assay gauges changes in intracellular calcium concentrations, a key event in GPCR signaling. Small molecules that modulate GPCR activity, including potential antagonists, can be identified by monitoring alterations in intracellular calcium levels, providing valuable insights into their pharmacological effects.

[0037] In one embodiment, the MRGPRE binding agent or inhibitor according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not), antibody, aptamer, or polypeptide.

[0038] In one embodiment, the inhibitors according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

[0039] The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc ). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da

[0040] In one embodiment, the MRGPRE binding agent according to the invention is an antibody.

[0041] Antibodies directed against MRGPRE can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from camels, pigs, cows, horses, rabbits, goats, sheep, and mice, among others, or organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against MRGPRE can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti- MRGPRE single chain antibodies. Compounds useful in practicing the present invention also include anti- MRGPRE antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to MRGPRE.

[0042] Humanized or human anti-MRGPRE antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of nonhuman (e.g., rodent) chimeric antibodies that contain minimal sequence derived from nonhuman immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397). Human antibodies can be generated by organotypic cultures of primary human cells from tonsils, lymph nodes or peripheral blood (see Wagar Lisa E. et al. Nature Medicine volume 27, pagesl25-135 (2021)).

[0043] In some embodiment, for this invention, neutralizing antibodies of MRGPRE is selected.

[0044] In a particular embodiment, the anti -MRGPRE antibody according to the invention may be the 5 LS-A4245 antibody as sold by LSBio.

[0045] In another embodiment, the antibody according to the invention is a single domain antibody against MRGPRE. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

[0046] The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

[0047] VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion ofB-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

[0048] In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0049] Then, for this invention, neutralizing aptamer of MRGPRE is selected.

[0050] In one embodiment, the compound according to the invention is a polypeptide.

[0051] In a particular embodiment the polypeptide is an antagonist or a reverse agonist of MRGPRE and is capable to prevent the function of MRGPRE. Particularly, the polypeptide can be a mutated MRGPRE protein or a similar protein without the function of MRGPRE. In this case, the mutated version of the MRGPRE protein is used as a decoy receptor.

[0052] In one embodiment, the polypeptide of the invention may be linked to a “cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.

[0053] The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012). The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

[0054] When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

[0055] In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0056] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0057] Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

[0058] Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.). The PEG chains (typically 2000 Daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory halflife of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomerular filtration (e.g., less than 60 kDa).

[0059] In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0060] In another embodiment, the MRGPRE inhibitor according to the invention is an inhibitor of MRGPRE gene expression. Small inhibitory RNAs (siRNAs) can also function as inhibitors of MRGPRE expression for use in the present invention. MRGPRE gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that MRGPRE gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836).

[0061] MicroRNA (miRNA) can also function as inhibitors of MRGPRE expression for use in the present invention. MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules.

[0062] Ribozymes can also function as inhibitors of MRGPRE gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of MRGPRE mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0063] Both antisense oligonucleotides and ribozymes useful as inhibitors of MRGPRE gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphorami di te chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphor othioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0064] Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing MRGPRE. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0065] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

[0066] Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double- stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

[0067] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencap sul ati on .

[0068] In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e g., a viral promoter, such as CMV promoter or any synthetic promoters.

[0069] In a particular embodiment, an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of MRGPRE.

[0070] Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEI) and the high-fidelity homology-directed repair (HDR).

[0071] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0072] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trap. Dis., Vol 8:e2671 ), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714 ), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi:10.1534 / genetics.113.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, 1. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0073] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0074] In another embodiment, the invention relates to a method for treating inflammatory disorders in a subject in need thereof comprising administering to a subject in need thereof a therapeutically effective amount of a MRGPRE binding agent.

[0075] Pharmaceutic composition

[0076] In another aspect, the invention relates to a pharmaceutic composition comprising a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

[0077] In a particular embodiment, the pharmaceutic composition is a therapeutic composition.

[0078] According to the invention, the MRGPRE binding agent or the pharmaceutic composition of the invention are administrated in a therapeutically effective amount.

[0079] Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutic compositions.

[0080] As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required. For example, the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of the inhibitor or the composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, and for example, the ability of a compound to inhibit inflammatory disorders may, for example, be evaluated in an animal model system predictive of efficacy in human. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. Administration may e.g. be topical, oral, intranasal, parenteral, intravenous, intraocular, intrathecal, epidural, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,

[0081] 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0082] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0083] 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0084] 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0085] Administration may be topical, oral, intranasal, parenteral, intravenous, intrathecal, epidural, intraocular, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of the agent of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of the agent of the present invention in an amount of about 0.1- 100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0086] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0087] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.

[0088] The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

[0089] Particularly, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions In particular, these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.

[0090] In addition, other pharmaceutically acceptable forms include, e g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

[0091] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.

[0092] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0093] Combination and kit of part

[0094] In another aspect of the invention, the MRGPRE binding agent of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising a MRGPRE binding agent according to the invention and a further therapeutic active agent.

[0095] According to the invention, the further therapeutic active agent can be an antiinflammation agent and may be added to the pharmaceutical composition or used in combination with the MRGPRE binding agent of the invention in the case of the treatment of inflammation.

[0096] For example, these anti-inflammation agents can be nonsteroidal anti-inflammatory drugs like aspirin, ibuprofen, and naproxen, P-agonists, corticoids, anti-histaminics, antileukotrienne, antibodies anti-IgE, anti-IL5 or anti-IL4Ra / IL13Ra (like the dupilumab) (see for example Akdis CA, 2012).

[0097] Another aspect of the present invention relates to i) a MRGPRE binding agent, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory disorders in a subject in need thereof.

[0098] As used herein, the term “simultaneous use” denotes the use of a MRGPRE binding agent and at least one therapeutic active agent occurring at the same time.

[0099] As used herein, the term “separate use” denotes the use of a MRGPRE binding agent and at least one therapeutic active agent not occurring at the same time.

[0100] As used herein, the term “sequential use” denotes the use of a MRGPRE binding agent and at least one therapeutic active agent occurring by following an order.

[0101] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

[0102] Figure 1. Elaboration of an MrgprE mutant (MrgprEMut) mice to track MrgprE expression and interrogate its function in vivo. A. MrgprEMutmice have been engineered by inserting a genetic cassette upstream of the coding sequence of mrgpre. This cassette is flanked by loxP sites and encodes the fluorescent protein tdTomato, followed by a stop codon. The gene coding sequences are labeled on the schema, and the loxP sites are indicated by arrowheads. As a result, these mutant mice produce the fluorescent protein tdTomato instead of the MrgprE receptor. The expression of MrgprE can be restored through the action of a Cre recombinase. B. Genotyping of MrgprEMut(Mut), wild type (WT) and heterozygous (Het) mice. The WT band is 525 bp long and the mutant 410 bp.

[0103] Figure 2: mrgpre is expressed by sensory neurons from dorsal root ganglia. A. Sections of dorsal root ganglia were stained for tubulin p3 (left column) and tdTomato (right column) in wild type (WT, top line) and MrgprEMut(Mut, bottom line) mice. Neurons Tubulin P3+ tdTomato+, found exclusively in MrgprEMutmice, indicate the expression of mrgpre in sensory neurons. B. Quantification of tdTomato+ neurons and C. measure of the mean intensity of fluorescence (MFI) in tdTomato of neurons in dorsal root ganglia of WT (n=16) and MrgprEMut(Mut, n=15) mice. ***, p<0.001, **, p<0.01, Mann Whitney test, error bars represent mean + / - SEM.

[0104] Figure 3: mrgpre is expressed by specialized subset of immune cells, MrgprEMutmice present the same number and distribution of immune cells than their WT littermate and are able to establish an adequate immune response. A-D. Identification by flow cytometry of specialized immune cells subset and histogram comparing the tdTomato MFI of WT to MrgprEMutmice. A. B cells from the blood (CD45+, CD19+), B. mast cells from the peritoneal cavity (CD45+, CD117+, FceRIa+), C. alveolar macrophages from the bronchoalveolar space (CD45+, CD1 lc+, SiglecF+, MerTK+), and D. basophils from the blood (CD45+, CD117-, FceRIa+). tdTomato+ expression, found exclusively in MrgprEMutmice, indicate the expression of mrgpre by B cells, mast cells, alveolar macrophages and basophils. E. Spleen weight, F. leukocytes count in the spleen, G. leukocytes count in the peritoneal cavity and H. leucocyte count in the blood of WT and MrgprEMutmice. Frequency of the different immune cell types I. in the spleen, J. in the peritoneal cavity and K. in the blood of MrgprE WT and mutant (Mut) mice. WT and MrgprEMutmice present an equivalent number and distribution of leukocytes in the spleen, peritoneal cavity and blood. L. Measurement of ear swelling across time following a passive cutaneous anaphylaxis (PCA) in WT and MrgprEMut mice. In the PCA model, mice were challenged by means of ear intradermal (i d.) injection of 1 nmol of substance P in 20 ul of PBS, and control mice were mock-injected i.d. with 20 ul of PBS. Ear swelling was then measured every 15 minutes for a period of 2 hours. MrgprE depletion has no effect on substance P-depend mast cell activation. M. Measurement of body temperature loss across time following a passive systemic anaphylaxis (PSA) in WT and MrgprEMutmice. In the PSA model, mice were sensitized by means of intraperitoneal injection (i.p.) of 10 ug of mouse DNP-HAS-specific IgE in 200ul PBS, and control mice were mock- injected i.p. with 200ul of PBS. 16 hours later, sensitized or non-sensitized control mice were injected i.p. with 500ug of DNP-HSA and rectal temperature was measured every 10 minutes during a period of 2 hours. MrgprE depletion has no impact on IgE-dependent Mast cell activation. N. Measurement of clinical score and O. spleen weight following atopic dermatitis model in WT and MrgprEMutmice. In the atopic dermatitis model a gauze pad filled with 10 ug of D. farinae and 500 ng of Staphylococcal Enterotoxin B (or PBS) was placed on the shaved back of the mice and occluded with a Tegaderm™ Transparent Dressing each week for three weeks to induce the development allergic skin inflammation. MrgprE depletion do not modify the development of atopic dermatitis.

[0105] Figure 4: MrgprEMutmice are protected from the development of chronic HDM- induced asthma. A. Protocol for inducing HDM (D. pteronyssinus) sensitization and chronic HDM-induced airway pathology. MrgprEMutand WT control mice were sensitized with three intranasal (i n.) administrations of 100 ug of HDM followed by 10 weekly i.n. challenges with 20ug of HDM; control mice were mock-sensitized with PBS, followed by 10 weekly intranasal (i n.) challenges with PBS. B. Changes in total lungs resistance (Rrs) induced by aerosolized methacholine, C. numbers of leukocytes in the lung, 24 hours after the tenth challenge with HDM (or PBS) in WT and MrgprEMutmice. MrgprEMutmice are protected from the development of airway hyper responsiveness and inflammation in a model of chronic HDM- dependent asthma.

[0106] Figure 5: MrgprEMutmice are protected from the development of acute HDM- induced asthma. A. Protocol for inducing HDM (D. pteronyssinus) sensitization and acute HDM-induced airway pathology. MrgprEMutand WT control mice were sensitized with intranasal (i.n.) instillation of 100 ng of LPS (or PBS as a control) on day 1; followed by the administration i.n. of 40 ug of D. pteronyssinus on day two. Seven days later, mice were challenged by i.n. instillation of 10 ug D. pteronyssinus (or PBS). Three days after the challenge, we measured B. the changes in Newtonian resistance (Rn) induced by aerosolized methacholine, the lung total C. leukocytes D. eosinophils and E. neutrophils count by flow cytometry. MrgprEMutmice are protected from the development of airway hyper responsiveness and lung inflammation in a model of acute HDM-induced asthma.

[0107] Figure 6: The expression of MrgprE by sensory neurons is necessary to develop asthma induced airway hyperresponsiveness. A. Sections of dorsal root ganglia were stained for tubulin P3 (left column) and tdTomato (right column) in MrgprEMut(Mut ere-, top line) and MrgprEMut, Navi.8 ere (Mut cre+, bottom line) mice. B. Quantification of tdTomato+ neurons in dorsal root ganglia of MrgprEMut(Mut ere-) and MrgprEMut, Navi.8 ere (Mut cre+) mice. The diminution of tdTomato+ neurons in dorsal root ganglia from MrgprEMut, Navi .8 cre+ mice indicate the excision of the tdTomato - STOP cassette in these neurons and the re-expression of MrgprE. C. We measured the changes in Newtonian resistance (Rn) induced by aerosolized methacholine, after the completion of the acute asthma model in MrgprEMutand MrgprEMut, Navi .8 Cre mice. The re-expression of MrgprE solely in sensory neurons restored the asthmatic phenotype.

[0108] Figure 7: MRGPRE is expressed by peribronchial neuronal fibers and immune cells in asthmatic and COPD human samples. Sections of asthmatic (A) and COPD (B) human lungs were stained for MRGPRE (left column) and DAPI (right column) or MRGPRE (left column) and CD45 (right column). We can observe MRGPRE nerve fibers close to the bronchi and MRGPRE4alveolar cells in asthmatic patients while MRGPRE4CD454cells are observed in human COPD lungs. In human as in mice, MRGPRE is expressed following the same pattern.

[0109] EXAMPLE:

[0110] Material & Methods

[0111] Mice

[0112] MrgprEMutmice were generated by Cyagen. Mice were bred and housed in the animal facility of CREFRE (Toulouse, France, and littermate control mice were used in all experiments. For the experiments using MrgprEMutand WT mice, mice were bred using heterozygous trio to generate homozygous WT and MrgprEMutmice. For the experiments using MrgprEMut, Navi.8 cre mice, breeders were kept homozygous for MrgprE mutation while one breeder was heterozygous for the cre allele and the other WT.

[0113] Genotyping

[0114] Genotyping of MrgprEMutmice was performed using samples harvest for mice identification (phalanges) and the kit Phire Tissue Direct PCR Master Mix (Thermoscientific) according to the manufacturer instructions. For MrgprEMutmice, the annealing temperature was 60°C and different primers were used. The WT band is 525 bp and the mutant band is 410 bp. For Navi.8 ere mice, the annealing temperature was 65°C. The WT band is 400 bp and the mutant band is 750 bp.

[0115] Immunohisto chemistry

[0116] Dorsal root ganglia

[0117] Dorsal root ganglia were harvest in HBSS without calcium magnesium and then place 1 hour in paraformaldehyde 4%, rinse in PBS and included in OCT. OCT blocs were sliced at 25 um of thickness. Tubulin b3 was identified using the mouse antibody clone TUJ 1 conjugated to Alexa Fluor 647 (BioLegend) at a concentration of 5 ug / mL. tdTomato was identified using an unconjugated rabbit recombinant polyclonal antibody (710530, Invitrogen) at a concentration of 5 ug / mL and detected using a goat anti-rabbit IgG antibody conjugated to Alexa Fluor Plus 594 (Invitrogen) at a concentration of 1 ug / mL. Primary antibodies were incubated over night at 4°C and the secondary antibody was incubated for 4 hours at room temperature. Image were acquired on a Leica SP8 microscope.

[0118] Human lung samples

[0119] Human lung samples were collected by the Pr. Laurent Guilleminault at Larrey hospital, Toulouse. Samples were rinsed in PBS, incubated 1 hour in paraformaldehyde 4%, rinse in PBS and included in OCT. OCT blocs were sliced at 25 um of thickness. MRGPRE was identified using a primary unconjugated rabbit polyclonal antibody (alomone labs) at a concentration of 5 ug / mL and detected using a secondary goat anti-rabbit IgG antibody conjugated to Alexa Fluor Plus 594 (Invitrogen) at a concentration of 1 ug / mL. Primary antibodies were incubated over night at 4°C and the secondary antibody was incubated for 4 hours at room temperature. Image were acquired on a Leica SP8 microscope.

[0120] Flow cytometry

[0121] Broncho alveolar lavage fluid (BALF)

[0122] To collect BALF, a canula was inserted in the trachea of mice and 2 ml of PBS containing counting beads was injected in the broncho alveolar space and collected.

[0123] Lung

[0124] To obtain single-lung-cell suspensions, lungs were perfused with 10 ml of PBS through the right ventricle of the heart, cut into small pieces and digested in RPMI containing 0.25 mg ml of Liberase (Roche), 0.5 mg ml of DNase I (Roche) and counting beads using the miltenyi octo cell dissociator with heaters and the program 37C_LDK_1.

[0125] Spleen Spleen was harvested in PBS containing EDTA (lOOmM), meshed in a 70 um cell strainer then, cells were resuspended in red blood cell lysis buffer (eBioscience) twice for 5 min at room temperature). Then, counting beads were added to the cell suspension.

[0126] Peritoneal lavage

[0127] 2 ml of PBS containing counting beads were injected in the peritoneal cavity and collected.

[0128] Blood

[0129] 500 ul of blood were collected in a tube containing EDTA (1 OOmM) and counting beads. Red blood cells were lysed with red blood cell lysis buffer (eBioscience).

[0130] Dorsal root ganglia neurons

[0131] Dorsal root ganglia were harvest in HBSS without calcium magnesium, rinse 3 times in HBSS containing calcium magnesium and digested in HBSS containing calcium magnesium supplemented with Img / ml of Collagenase (Roche) and 4 mg / ml of Dispase II (Roche) for 30 min at 37°C under agitation. The cell suspension was rinse with DMEM supplemented with 10% FBS, 1% antibiotics and 500 U of DNAse I (Roche).

[0132] Staining procedure

[0133] Cells were resuspended at a density of 106cells / ml in FACS buffer containing 1% BSA and 2mM EDTA in PBS. Staining mix was prepared with each antibodies diluted at 1 ug / mL in FACS buffer containing 1 ug / ml of Fcblock (TruStain FcX™, anti-mouse CD16 / 32, Biolegend). Staining was performed at 4°C in the dark for 30 min. The cells were then washed in FACS buffer and resuspended in Sytox blue (Invitrogen) following the manufacturer’s instruction to identify dead cells.

[0134] Panels

[0135] Samples were recorded on a Symphony cytometer with Diva software. The analysis was performed using the software FlowJo.

[0136] Passive cutaneous anaphylaxis

[0137] The left ear pinnae of mice were sensitized by means of an intradermal (i.d.) injection with mouse Substance P (1 nmol in 20 ul PBS) or received in the right ear an i.d. injection of 20 ul PBS (control). Ear swelling was measured at different time points during a period of 120 min.

[0138] Passive systemic anaphylaxis

[0139] We used the passive systemic anaphylaxis (PSA) model to induce a systemic IgE- dependent reaction. Mice were sensitized by mean of intraperitoneal injection (i.p.) of 10 ug of mouse DNP-HSA-specific IgE in 200 ul PBS, and control mice were mock-injected i.p. with 200 ul of PBS. 16 hours later, sensitized or non-sensitized control mice were injected i.p. with 500 ug of DNP-HSA and rectal temperature was measured at different time points during a period of 120 min.

[0140] Atopic dermatitis

[0141] To induce allergic skin inflammation, the back skin of mice was shaved and a solution of 500 ng of Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich) and of 10 pg of Dermatophagoides farinae extract (HDM, Stallergen Greer) in PBS was applied on a gauze pad placed on the shaved back and occluded with a Tegaderm™ Transparent Dressing (3M Healthcare). Three days later, the gauze pads were replaced. Mice were monitored on a daily basis and if a mouse removed the bandage, a new dressing was immediately applied on this mouse and all of the other mice within the same experiment (so that they received the same treatment and equal amounts of antigens). Four days later, dressings were removed and mice were kept without treatment for the next week. This "3 + 4 days” pattern of treatment was repeated two more times, so that the mice were subjected to three cycles of such treatment. Chronic asthma model

[0142] Mice were immunized by three i.n. challenges of 100 ug HDM from D. pteronyssinus (Greer) in 30 ul PBS on days 1, 4 and 7. Starting on day 12, mice were challenged i.n. with 20 ug HDM in 30 ul PBS weekly for 10 weeks; control mice received i.n. challenges with PBS on the same schedule. The day after the last HDM challenge, we estimated bronchial hyperresponsiveness to methacholine by assessing dynamic airway resistance in anesthetized animals subjected to increased doses of methacholine with a FlexiVent small animal ventilator (SCIREQ) system.

[0143] Acute asthma model

[0144] Mice were instilled i n. with 100 ng of LPS (from Escherichia coli O55:B5, Sigma- Aldrich) diluted in 50 ul of PBS. One day later (day 1), mice were administered i.n. with 40 pg of HDM (HDM pteronyssinus, Greer Laboratories) in 50 pl of PBS. Seven days later (day 8), mice were challenged by i.n. instillation of 10 pg of HDM in 50 pl of PBS. Control mice received i.n. challenges with PBS on the same schedule. Three days after the HDM challenge (day 11), we estimated bronchial hyperresponsiveness to methacholine by assessing dynamic airway resistance in anesthetized animals subjected to increased doses of methacholine with a FlexiVent small animal ventilator (SCIREQ) system.

[0145] Results:

[0146] Development of a transgenic mouse to study MRGPRE in vivo.

[0147] The laboratory has generated a new mouse model, the MrgprEMutmouse, allowing to localize the expression of Mrgpre as well as to interrogate its function, in vivo (Fig. 1). The characterization of this mouse model showed that the expression of MRGPRE is shared by sensory neurons from the dorsal root ganglia (Fig. 2) and subsets of immune cells notably involved in allergic diseases: basophils, lung alveolar macrophages, B lymphocytes and mast cells (Fig. 3A-D).

[0148] MrgprEMutmice present a normal immune system and are able to generate an appropriate immune response.

[0149] Although immune cells express MrgprE, its genetic ablation in the MrgprEMutmice do not triggers a modification in the number and distribution of leukocytes populations in the blood, peritoneal cavity and spleen (Fig. 3E-K). In addition, the absence of MrgprE do not impair the establishment of a local and systemic passive allergic reaction (Fig. 3L-M) and in the context of atopic dermatitis, MrgprEMutmice showed a similar phenotype than their WT littermate (Fig. 3N-O).

[0150] MRGPRE is a key receptor in the development of allergic asthma.

[0151] In order to study the involvement of MRGPRE in asthma, we submitted the MrgprEMutmice with two complementary models of asthma that mimic pathological aspects found in some patients. A so-called “chronic” model which summarizes the characteristics of an asthma depend on type 2 immunity with tissue remodeling and mucus production exacerbated (Fig. 4), and a shorter asthma model that recapitulates the characteristics of asthma less dependent on type 2 immunity and in which there is a strong eosinophilic and neutrophilic infiltrate (Fig. 5). For both models we analyzed two pathological components key: the reactivity of the respiratory tract and the inflammatory state, particularly via infiltration leukocyte.

[0152] At the end of the first model of so-called “chronic” asthma (Fig. 4), we observed that the mice MrgprEMutwere fully protected from the development of bronchial hyperresponsiveness and exacerbated mucus production in the lungs. The absence of MrgprE was also associated with a significant reduction in pathogenic leukocyte infiltration.

[0153] In the second asthma model (Fig. 5), we observed the same phenotype, namely that the MrgprEMutmice were fully protected against the pathological features of asthma and also showed a strong decrease in leukocyte infiltration, including eosinophils and neutrophils, in the lung, almost similar to the levels observed in non-asthmatic control mice.

[0154] The re-expression of MRGPRE, solely in the peripheral sensory neurons, is sufficient to restore the asthma associated airway hyperresponsiveness.

[0155] MrgprEMutmice have been crossed to Navi ,8Cre mice in order to restore the expression of MRGPRE in the peripheral sensory nervous system. When the expression of MRGPRE is restored the concerned cells loose the tdTomato fluorescent tracer. Also, in order to access the success of the recombination of mrgpre locus we measure the number of tdTomato cells in DRG from MrgprEMutCre" and MrgprEMutCre+mice. We observe a reduction of tdTomato+ cells in MrgprEMutCre+ mice compare to their Cre" littermate that confirm the efficiency of the recombination. These mice have been submitted to a model of short neutrophilic asthma and the Cre+mice showed an airway hyperresponsiveness that was absent in the Cre" mice. Conclusively, the expression ofMrgprE, especially in sensory neurons, is sufficient to establish this asthma associated symptom. Clinical relevance of the results obtained and patient analysis.

[0156] We had access to biopsies from patients with bronchial hyper-reactivity with an intermediate profile between severe asthma and chronic obstructive pulmonary disease (COPD with eosinophils). We used the confocal laser scanning microscopy in order to analyze in 3D on thick sections the presence of MRGPRE within biopsies. Interestingly, we were able to show by immunostaining that the human MRGPRE receptor is very highly expressed in patients by the neurons innervating the bronchi and the immune cells present within the alveoli pulmonary (Fig- 7).

[0157] Conclusion:

[0158] The inventors have created a new transgenic mouse MrgprEMut, which makes it possible to both trace the expression of the receptor and examine its function in vivo. The MrgprEMutmouse has a completely normal immune and sensory system. They have generated robust data showing that: a) MRGPRE is expressed both by the sensory system and by mast cells, B lymphocytes and alveolar macrophages of the lung; b) the selective absence of MRGPRE in sensory neurons protects against both the development of airway hyperresponsiveness and pulmonary inflammation in several models of asthma; and c) lung biopsies from asthmatic patients show strong expression of MRGPRE at the level of neuronal fibers and peribronchial immune cells that resemble the distribution observed in the mouse model. These results showed that MRGPRE is a relevant target to treat inflammatory disorders and particularly asthma and allergic asthma.

[0159] REFERENCES:

[0160] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0161] Corbiere et al. MRGPRX2 sensing of cationic compounds — A bridge between nociception and skin diseases? Exp Dermatol., 2023.

[0162] Dong et al . A Diverse Family of GPCRs Expressed in Specific Subsets of Nociceptive Sensory Neurons. Cell, 2001.

[0163] Dwyer et al. Expression profiling of constitutive mast cells reveals a unique identity within the immune system. Nature immunology, 2016. Gaudenzio et al. Different activation signals induce distinct mast cell degranulation strategies. JCI, 2016.

[0164] Guan et al. Mas-Related G Protein-Coupled Receptors and the Biology of Itch Sensation, Meixiong and Dong, Annu. Rev. Genet., 2017. Guan et al. Mas-related G-protein-coupled receptors inhibit pathological pain in mice.

[0165] PNAS, 2010.

[0166] Han et al. Mrgprs on vagal sensory neurons contribute to bronchoconstriction and airway hyper-responsiveness, nature neuroscience. 2018.

[0167] McNeil et al. Identification of mast-cell-specific receptor crucial for pseudo-allergic drug reaction. Nature, 2015.

[0168] Pemer et al. Substance P Release by Sensory Neurons Triggers Dendritic Cell Migration and Initiates the Type-2 Immune Response to Allergens. Immunity, 2020.

[0169] Serhan et al. House dust mites activate nociceptor-mast cell clusters to drive type 2 skin inflammation. Nat Immunol. 2019. Valentin et al. Molecular and Functional Neuroscience in Immunity. Annual Review of

[0170] Immunology, 2018.

Claims

CLAIMS;1. A MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

2. The MRGPRE binding agent for use according to the claim 1 wherein inflammatory disorders are an allergic inflammatory disorders or pulmonary inflammatory disorders.

3. The MRGPRE binding agent for use according to the claim 2 wherein allergic inflammatory disorders include anaphylactic hypersensitivity, asthma, allergic asthma, allergic rhinitis, atopic dermatitis, vernal conjunctivitis, eczema, IgE-mediated urticarial, chronic spontaneous urticaria, prurigo nodularis, contact dermatitis, food allergies, IgE-mediated anaphylaxis, esophagus eosinophilic or esophagitis.

4. The MRGPRE binding agent for use according to the claim 2 wherein allergic inflammatory disorders are asthma or allergic asthma.

5. The MRGPRE binding agent for use according to the claim 2 wherein the pulmonary inflammatory disorder is Chronic Obstructive Pulmonary Disease (COPD).

6. The MRGPRE binding agent for use according to the claims 1 to 5 wherein the MRGPRE binding agent is an inverse agonist, an antagonist, a modulator or an inhibitor of MRGPRE.

7. A method for treating inflammatory disorders in a subject in need thereof comprising administering to a subject in need thereof a therapeutically effective amount of a MRGPRE binding agent.

8. A pharmaceutic composition comprising a MRGPRE binding agent for use in the treatment of inflammatory disorders in a subject in need thereof.

9. A MRGPRE binding agent for use or a pharmaceutic composition for use in the treatment of inflammatory disorders wherein said agent or said composition comprise a further therapeutic active agent10. A i) MRGPRE binding agent, and ii) at least one further therapeutic active agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of inflammatory disorders in a subject in need thereof.

Images