Pharmaceutical composition for treating and / or preventing disease in which mast cell exacerbates pathologic condition

Abstract

An antibody or antigen-binding fragment thereof having immunological reactivity with the MCEMP1 protein can be used as an active ingredient of a pharmaceutical composition for treating and / or preventing a disease in which mast cells expressing the MCEMP1 protein on the cell surfaces exacerbate the pathologic condition.

Description

Pharmaceutical composition for treating and / or preventing diseases in which mast cells exacerbate the pathology

[0001] The present invention relates to novel medical uses of antibodies immunologically reactive with MCEMP1 protein or antigen-binding fragments thereof as therapeutic and / or preventive agents for diseases in which mast cells exacerbate the pathology.

[0002] In recent years, various antibody drugs have emerged to treat diseases by targeting antigen proteins on disease-related cells. These antibody drugs have attracted attention because they have demonstrated a certain degree of efficacy as target-antigen-specific therapeutic agents. However, many of the target antigen proteins are also expressed on multiple normal cells. Therefore, antibody administration affects not only disease-related cells but also normal cells that are not involved in the disease, resulting in problematic side effects. Therefore, if we could identify antigens specifically expressed on the surface of disease-related cells and use antibodies targeting them as pharmaceuticals, it would be possible to use antibody drugs with fewer side effects.

[0003] Mast Cell-Expressed Membrane Protein 1 (MCEMP1) is a type II transmembrane protein that has been reported to be specifically expressed on the cell membrane of mast cells, suggesting its possible involvement in mast cell differentiation, immune responses, and allergic reactions (Non-Patent Document 1). Furthermore, it has been suggested that MCEMP1 protein expressed in pulmonary mast cells may promote the exacerbation of asthma (Non-Patent Document 2). It is also known that there are cancers that specifically express MCEMP1 protein on the cell membrane surface, and that antibodies against MCEMP1 protein or antigen-binding fragments thereof can be used as pharmaceutical compositions for treating and / or preventing such cancers (Patent Document 1). However, there have been no reports that antibodies against MCEMP1 protein or antigen-binding fragments thereof are useful for treating or preventing diseases whose pathology is exacerbated by mast cells.

[0004] WO2017 / 170322

[0005] Kang Li. et al. Genomics, 86:68-75 (2005) Youn Jung Choi. et al. Nature Communications, 14:2045 (2023)

[0006] An object of the present invention is to provide a therapeutic and / or prophylactic agent for diseases whose pathology is aggravated by mast cells, which targets mast cells.

[0007] As a result of extensive research, the present inventors have found that the MCEMP1 protein is specifically expressed on the cell surface of mast cells, and have also discovered that diseases whose pathology is exacerbated by mast cells can be treated and / or prevented by damaging mast cells expressing the MCEMP1 protein on their cell surface using antibodies or antigen-binding fragments thereof against the portion of the MCEMP1 protein expressed on the mast cell surface, thereby completing the present invention.

[0008] Therefore, the present invention includes the following aspects (1) to (22).

[0009] (1) A pharmaceutical composition for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising an antibody or its antigen-binding fragment immunologically reactive with MCEMP1 protein as an active ingredient.

[0010] (2) A pharmaceutical composition for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising as an active ingredient an antibody or an antigen-binding fragment thereof that is immunologically reactive with a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, or an amino acid sequence having 80% or more sequence identity to said amino acid sequence, or a fragment thereof containing 7 or more consecutive amino acids.

[0011] (3) A pharmaceutical composition for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising as an active ingredient an antibody or an antigen-binding fragment thereof that is immunologically reactive with a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 10, 12, 14 or 16, or an amino acid sequence having 80% or more sequence identity to said amino acid sequence, or a fragment thereof containing 7 or more consecutive amino acids.

[0012] (4) The pharmaceutical composition according to any one of (1) to (3), wherein the antibody or antigen-binding fragment thereof is a drug-conjugated antibody or antigen-binding fragment thereof.

[0013] (5) The pharmaceutical composition according to any one of (1) to (3), wherein the drug is a cytotoxic agent, an immunomodulator, or a radioisotope.

[0014] (6) The pharmaceutical composition according to any one of (1) to (3), wherein the disease is selected from the group consisting of respiratory diseases, mast cell activation syndrome, cytokine release syndrome, autoimmune diseases, systemic allergic diseases, skin diseases, digestive diseases, cancers that do not express MCEMP1 protein on the cell surface, metabolic diseases, female reproductive diseases, pregnancy, childbirth, and postpartum diseases, sepsis, and graft-versus-host disease.

[0015] (7) The pharmaceutical composition according to (6), wherein the respiratory disease is asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, interstitial lung disease, or allergic rhinitis.

[0016] (8) The pharmaceutical composition according to (6), wherein the autoimmune disease is a collagen disease.

[0017] (9) The pharmaceutical composition according to (8), wherein the collagen disease is rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, polymyositis, dermatomyositis, or giant cell arteritis.

[0018] (10) The pharmaceutical composition according to (6), wherein the systemic allergic disease is anaphylaxis, drug hypersensitivity, food allergy, or poison allergy.

[0019] (11) The pharmaceutical composition according to (6), wherein the skin disease is urticaria, dermatitis, psoriasis, chronic pruritus, or prurigo nodularis.

[0020] (12) The pharmaceutical composition according to (6), wherein the digestive system disease is Crohn's disease, ulcerative colitis, irritable bowel syndrome, eosinophilic esophagitis, eosinophilic duodenitis, or eosinophilic gastritis.

[0021] (13) The pharmaceutical composition according to (6), wherein the cancer that does not express the MCEMP1 protein on the cell surface is a solid cancer.

[0022] (14) The pharmaceutical composition according to (13), wherein the solid cancer is gastric cancer, lung cancer, breast cancer, oral cancer, colorectal cancer, esophageal cancer, small intestine cancer, melanoma, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, prostate cancer, glioblastoma, or thyroid cancer.

[0023] (15) The pharmaceutical composition according to (6), wherein the metabolic disease is diabetes.

[0024] (16) The pharmaceutical composition according to (6), wherein the female reproductive system disease is endometriosis.

[0025] (17) The pharmaceutical composition according to (6), wherein the pregnancy, delivery and postpartum disease is preeclampsia.

[0026] (18) The pharmaceutical composition according to any one of (1) to (17), wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0027] (19) The pharmaceutical composition according to any one of (1) to (18), wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a multispecific antibody.

[0028] (20) A combination drug for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising the pharmaceutical composition according to any one of (1) to (19) and a pharmaceutical composition containing a drug for a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology.

[0029] (21) A method for treating and / or preventing a disease in which mast cells expressing MCEMP1 protein on the cell surface exacerbate the pathology, comprising administering to a subject the pharmaceutical composition according to any one of (1) to (19).

[0030] (22) A method for treating and / or preventing a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising administering to a subject, simultaneously or separately, a pharmaceutical composition according to any one of (1) to (19) and a pharmaceutical composition containing a drug for a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology.

[0031] The antibody against the MCEMP1 protein or an antigen-binding fragment thereof used in the present invention exerts a pharmacological effect on mast cells, and is therefore useful for treating or preventing diseases in which mast cells exacerbate the pathology.

[0032] 1 shows the cytotoxic activity of a polyclonal antibody immunologically reactive with the MCEMP1 protein (anti-MCEMP1 polyclonal antibody) against a human mast cell line (LUVA) that expresses the MCEMP1 protein on its cell surface, under the following conditions: 1: no antibody was added; 2: purified antibody derived from control normal rabbit serum was added; and 3: anti-MCEMP1 polyclonal antibody was added.

[0033] The present invention relates to the use of antibodies or antigen-binding fragments thereof against the MCEMP1 protein or fragments thereof (hereinafter, these may be collectively referred to as "MCEMP1 protein") for the treatment and / or prevention of diseases in which mast cells exacerbate the pathology.

[0034] The present invention relates to a pharmaceutical composition for treating and / or preventing diseases in which mast cells exacerbate the pathology, comprising as an active ingredient an antibody or fragment thereof that is immunologically reactive with an MCEMP1 protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, or an amino acid sequence having 80% or more sequence identity (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 99% or more, for example 99.5% or more) to said amino acid sequence, or a fragment thereof comprising 7 or more consecutive amino acids (7 to the full length of each sequence, preferably 7 to 150, more preferably 7 to 50).

[0035] The present invention also relates to a pharmaceutical composition for treating and / or preventing a disease in which mast cells exacerbate the pathology, comprising as an active ingredient an antibody or a fragment thereof that is immunologically reactive with a fragment of the MCEMP1 protein, the antibody being a polypeptide consisting of 7 or more consecutive amino acids (7 to the full length of each sequence, preferably 7 to 40, more preferably 7 to 20, for example, 7 to 12 or 8 to 11) of an amino acid sequence represented by any of the even-numbered SEQ ID NOs: 10 to 16, or a polypeptide consisting of an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, particularly preferably 97% or more) sequence identity to said amino acid sequence.

[0036] The pharmacological efficacy of an antibody or antigen-binding fragment thereof against a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 used in the present invention, or a fragment thereof, can be determined by in vivo evaluation of the improvement of pathological conditions aggravated by mast cells or the inhibition of mast cell proliferation in an animal into which mast cells have been transferred or in which mast cells have been over-proliferated, or by in vitro evaluation of the death of mast cells due to cytotoxic activity mediated by immune cells or complement, or the reduction in viable cells or the impairment of cellular function in mast cells expressing the polypeptide, as described below.

[0037] Similarly, the pharmacological efficacy of an antibody or an antigen-binding fragment thereof against a polypeptide or a fragment thereof of an even-numbered SEQ ID NO: 10 to 16 used in the present invention can be determined by in vivo evaluation of an animal into which mast cells have been transferred or an animal in which mast cells have been over-proliferated, to determine whether it improves pathological conditions aggravated by mast cells or suppresses mast cell proliferation; or, as described below, by in vitro evaluation of mast cells expressing the polypeptide, to determine whether it kills the mast cells through cytotoxic activity mediated by immune cells or complement, or whether it reduces viable cells or reduces cell function.

[0038] Similarly, the efficacy of a conjugate of the above-mentioned antibody against MCEMP1 or its antigen-binding fragment (hereinafter, these may be collectively referred to as "anti-MCEMP1 antibody") with a drug can be determined by evaluating in vivo the effect of the drug in improving pathological conditions exacerbated by mast cells or suppressing mast cell proliferation in animals into which mast cells have been transplanted or in animals in which mast cells have been over-proliferated, or by evaluating in vitro the death of mast cells, or the reduction in viable cells, or the impairment of cellular function in mast cells expressing the polypeptide.

[0039] The base sequences of DNA encoding proteins consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 are shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, respectively.

[0040] The amino acid sequence represented by SEQ ID NO: 2 in the sequence listing disclosed in the present invention is the amino acid sequence isolated as human MCEMP1 protein, the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of its canine homologue (homolog), the amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence of its feline homologue, and the amino acid sequence represented by SEQ ID NO: 8 is the amino acid sequence of MCEMP1 isolated as its mouse homologue.

[0041] In the present invention, antibodies that bind to a portion of the MCEMP1 protein that is expressed on the cell surface of mast cells are preferably used. Specifically, examples include polypeptides containing the extracellular domain of the MCEMP1 protein, such as the amino acid sequence represented by SEQ ID NO: 10 (human), 12 (dog), 14 (cat), or 16 (mouse), fragments thereof (preferably consisting of 7 or more consecutive amino acids of these amino acid sequences), or amino acid sequences that share 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more sequence identity with these polypeptides. The antibodies of the present invention include all antibodies that bind to these polypeptides and show mast cell death, a decrease in viable cells, or a decrease in cellular function.

[0042] The anti-MCEMP1 antibody used in the present invention may be any type of antibody as long as it can exert mast cell proliferation inhibitory activity, and may be, for example, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a multispecific antibody (e.g., a bispecific antibody), a human antibody, a humanized antibody, a chimeric antibody, or a single-chain antibody (scFv). The antibody used in the present invention may also be an antibody fragment (e.g., Fab or F(ab')). ２ These antibodies and fragments thereof can also be prepared by methods known to those skilled in the art. In the present invention, antibodies or fragments thereof capable of specifically binding to the MCEMP1 protein are desirable, and monoclonal antibodies are preferred, but polyclonal antibodies may also be used as long as homogeneous antibodies can be stably produced. Furthermore, when the subject is a human, human antibodies or humanized antibodies are desirable in order to avoid or suppress rejection reactions.

[0043] Here, "specifically binds to the MCEMP1 protein or a fragment thereof" means specifically binding to the MCEMP1 protein or a fragment thereof, and not substantially binding to other proteins.

[0044] The activity of the antibodies that can be used in the present invention against mast cells can be determined by assessing the inhibition of mast cell proliferation in animals into which mast cells have been transferred or in which mast cells have been over-proliferated in vivo, or by assessing whether or not the antibodies exhibit immune cell- or complement-mediated cytotoxic activity or the effect of a drug conjugated to an anti-MCEMP1 antibody against mast cells expressing the polypeptide in vitro, as described below.

[0045] Furthermore, subject animals to be treated and / or prevented in the present invention for diseases in which mast cells exacerbate the pathology are mammals such as humans, pet animals, livestock, sports animals, and laboratory animals, with the preferred subject animal being humans.

[0046] The production of antigens, the production of antibodies, and pharmaceutical compositions according to the present invention will be described below.

[0047] <Preparation of antigen for antibody production> The protein or fragment thereof used as a sensitizing antigen for obtaining the anti-MCEMP1 antibody used in the present invention may be derived from any animal species, including human, dog, cat, mouse, cow, horse, rat, and chicken. However, it is preferable to select a sensitizing antigen that has high sequence identity with an antigen expressed by target cells. In general, proteins derived from mammals are preferred, and proteins derived from humans are particularly preferred. For example, when the target MCEMP1 protein is human MCEMP1 protein, it is preferable to use human MCEMP1 protein or a fragment thereof, or cells expressing human MCEMP1.

[0048] The nucleotide sequence and amino acid sequence encoding the human MCEMP1 protein and its homologues can be obtained, for example, by accessing the GenBank (NCBI, USA) website and using algorithms such as BLAST and FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993; Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997).

[0049] In the present invention, when the amino acid sequence of SEQ ID NO: 2, which is the full-length amino acid sequence of human MCEMP1 protein, is used as a reference, a protein having a sequence that has 70 to 100%, preferably 80 to 100%, more preferably 90 to 100%, and even more preferably 95 to 100%, for example, 97 to 100%, 98 to 100%, 99 to 100%, or 99.5 to 100%, identity to this amino acid sequence is used as a target. Here, "sequence identity" is calculated as the ratio of identical amino acids (or bases) to the total number of amino acids (or bases) when the two sequences are aligned to maximize similarity, with or without introducing gaps, and is expressed as a percentage (%).

[0050] A fragment of the MCEMP1 protein has a length that is greater than or equal to the amino acid length of an epitope (antigenic determinant), which is the minimum unit recognized by an antibody, but less than the full-length of the protein. An epitope refers to a polypeptide fragment that is antigenic or immunogenic in mammals, preferably humans, and the minimum unit is a polypeptide consisting of an amino acid sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of the MCEMP1 protein.

[0051] The above-mentioned MCEMP1 protein and polypeptides containing fragments thereof can be synthesized according to chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method) (Japanese Biochemical Society, Biochemical Experiment Course 1, Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Tokyo Kagaku Dojin (Japan), 1981). Alternatively, they can be synthesized by standard methods using various commercially available peptide synthesizers. In addition, known genetic engineering techniques (Sambrook et al., Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology (1989), Cold Spring Harbor Laboratory Press; Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.) may be used. The desired polypeptide can be obtained by preparing DNA encoding the polypeptide using a recombinant vector such as PEG-1 or PEG-2, and then incorporating the DNA into an expression vector, which is then introduced into a host cell, and allowing the host cell to produce the polypeptide.

[0052] DNA encoding the above polypeptide can be easily prepared by known genetic engineering techniques or by a standard method using a commercially available nucleic acid synthesizer. For example, DNA containing the nucleotide sequence of SEQ ID NO: 1 can be prepared by performing PCR using human chromosomal DNA or a cDNA library as a template and a pair of primers designed to amplify the nucleotide sequence described in SEQ ID NO: 1. The PCR reaction conditions can be appropriately set, and for example, a thermostable DNA polymerase (e.g., Taq polymerase) and Mg ２＋Examples of conditions include, but are not limited to, a PCR buffer containing 94° C. for 30 seconds (denaturation), 55° C. for 30 seconds to 1 minute (annealing), and 72° C. for 1 minute (extension), with 30 cycles being one cycle, followed by a reaction at 72° C. for 7 minutes. PCR techniques and conditions are described, for example, in Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons (especially Chapter 15).

[0053] Furthermore, the desired DNA can be isolated by preparing appropriate probes and primers based on the information on the nucleotide and amino acid sequences represented by SEQ ID NOs: 1 to 8 in the Sequence Listing herein and using them to screen a cDNA library of a human or other organism. The cDNA library is preferably prepared from cells, organs, or tissues expressing the proteins of SEQ ID NOs: 2, 4, 6, or 8. Examples of such cells and tissues include, but are not limited to, cells or tissues derived from normal or diseased sites, such as bone marrow, peripheral blood mononuclear cells (PBMCs), mast cells, mast cell-expressing tissues, and pathological tissues. The procedures for preparing the above-mentioned probes or primers, constructing a cDNA library, screening the cDNA library, and cloning the target gene are known to those skilled in the art and can be carried out, for example, according to the methods described in Sambrook et al., Molecular Cloning, 2nd Edition, Current Protocols in Molecular Biology (1989), Ausbel et al. (cited above), etc. From the DNA thus obtained, DNA encoding the human MCEMP1 protein or a fragment thereof can be obtained.

[0054] The host cell may be any cell capable of expressing the polypeptide, and examples of prokaryotic cells include Escherichia coli, and examples of eukaryotic cells include mammalian cells such as monkey kidney cells COS1 and Chinese hamster ovary cells CHO, human embryonic kidney cell line HEK293, mouse embryonic skin cell line NIH3T3, yeast cells such as budding yeast and fission yeast, silkworm cells, and Xenopus oocytes, but are not limited to these.

[0055] When prokaryotic cells are used as host cells, the expression vector preferably has an origin replicable in prokaryotic cells, a promoter, a ribosome binding site, a multicloning site, a terminator, a drug resistance gene, an auxotrophy complementing gene, a reporter gene, and the like. Examples of expression vectors for E. coli include the pUC series, pBluescript II, the pET expression system, and the pGEX expression system. By incorporating DNA encoding the above-mentioned polypeptide into such an expression vector, transforming prokaryotic host cells with the vector, and then culturing the resulting transformant, the polypeptide encoded by the DNA can be expressed in the prokaryotic host cells. When constructing an expression vector, the polypeptide can also be expressed as a fusion protein by adding various tags or other proteins to the polypeptide.

[0056] When eukaryotic cells are used as host cells, the expression vector preferably used is a eukaryotic expression vector having a promoter, a splicing region, a poly(A) addition site, etc. Examples of such expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3.1, pSecTag (A, B, C), and pYES2. As described above, by incorporating DNA encoding the polypeptide into such an expression vector, transforming eukaryotic host cells with the vector, and then culturing the resulting transformant, the polypeptide encoded by the DNA can be expressed in the eukaryotic host cells. When constructing an expression vector, if various tags or other proteins are added to the polypeptide, or if pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or the like is used as the expression vector, the polypeptide can be expressed as a fusion protein to which various tags such as a His tag (e.g., (His) to (His)), a FLAG tag, a myc tag, an HA tag, or a GFP, or other proteins, are added.

[0057] The expression vector can be introduced into host cells using well-known methods such as heat shock, electroporation, calcium phosphate, liposome, DEAE-dextran, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

[0058] Isolation and purification of a target polypeptide from host cells can be carried out by combining known separation procedures, including, but not limited to, treatment with a denaturant such as urea or a surfactant, sonication, enzymatic digestion, salting out or solvent fractional precipitation, dialysis, centrifugation, ultrafiltration, gel filtration chromatography, SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reversed-phase chromatography.

[0059] <Antibody Structure> Antibodies are typically heteromultimeric glycoproteins containing at least two heavy chains and two light chains. IgG, a representative class of antibodies, is a heterotetrameric glycoprotein of approximately 150 kDa composed of two identical light (L) chains and two identical heavy (H) chains. Typically, the light chain is linked to the heavy chain by one covalent disulfide bond, but the number of disulfide bonds between heavy chains varies among immunoglobulin isotypes. Each heavy and light chain also has an intrachain disulfide bond. The heavy chain has a variable region (VH region) at its N-terminus and a constant region at its opposite C-terminus. Similarly, the light chain has a variable region (VL region) at its N-terminus and a constant region at its opposite C-terminus. Each region has a domain structure: the heavy chain variable region, light chain variable region, and light chain constant region all contain a single region, while the heavy chain has multiple constant regions. The light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. The variable region of an antibody contains regions called complementarity-determining regions (CDRs), which exhibit particularly high variability and function as antigen-binding sites. The relatively conserved portions of the variable region, compared to the CDRs, are called framework regions (FRs). Complete heavy and light chain variable regions each contain four FRs connected by three CDRs. The three CDRs are designated CDRH1, CDRH2, and CDRH3 in the heavy chain, from the N-terminus, and CDRL1, CDRL2, and CDRL3 in the light chain. CDRH3 is the most important for the antibody's binding specificity to the antigen. The CDRs of each chain are held together in close proximity by FRs and contribute to the formation of the antibody's antigen-binding site together with the CDRs from the other chain. The constant region does not directly contribute to the binding of an antibody to an antigen, but contributes to various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis via binding to Fcγ receptors, half-life / clearance rate via binding to neonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC) via binding to the C1q component of the complement cascade.

[0060] <Preparation of Antibody> In the present invention, the anti-MCEMP1 antibody means an antibody that is immunologically reactive with the full-length MCEMP1 protein or a fragment thereof as described above.

[0061] Here, "immunological reactivity" refers to the property of an antibody binding to an MCEMP1 antigen in vivo or in vitro, and through such binding, the antibody exerts a function of damaging mast cells (e.g., killing or inhibiting proliferation) or delivering a drug conjugated to the anti-MCEMP1 antibody to mast cells. In other words, the antibody used in the present invention may be of any type, as long as it can bind to the MCEMP1 protein to damage mast cells that aggravate the pathology, or can deliver a drug to mast cells.

[0062] Examples of antibodies include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, etc. Antibodies also include, for example, antibody fragments (e.g., Fab and F(ab')). ２ The antibody may also be any class of immunoglobulin molecule, such as IgG, IgE, IgM, IgA, IgD, and IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0063] The antibody may be further modified by glycosylation, as well as acetylation, formylation, amidation, phosphorylation, or pegylation (PEG).

[0064] Examples of the production of various antibodies are given below.

[0065] Polyclonal antibodies that can be used in the present invention can be obtained, for example, as follows.

[0066] Anti-MCEMP1 antibody-producing immune cells are induced by immunizing small animals such as mice, human antibody-producing mice, and rabbits with native MCEMP1 protein, recombinant MCEMP1 protein expressed in a microorganism such as Escherichia coli as a fusion protein with GST, fragments thereof, or cells expressing MCEMP1 on their cell surface (e.g., the mast cell line LUVA). Serum containing the anti-MCEMP1 antibody produced by the immune cells is then obtained. This serum is then purified, for example, by ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, or an affinity column coupled with MCEMP1 protein or a synthetic peptide. In the Examples described below, rabbit polyclonal antibodies against a region of the amino acid sequence of MCEMP1 protein that is expressed on the cell surface of mast cells were produced, and their pathological amelioration effects were confirmed.

[0067] Another example of an antibody that can be used in the present invention is a monoclonal antibody. Methods for obtaining a monoclonal antibody include, for example, immunizing a small animal with a sensitizing antigen to induce immune cells that produce anti-MCEMP1 antibodies, followed by establishing fused cells (hybridomas) from the animal, as in the case of obtaining polyclonal antibodies, or isolating immune cells from the animal and cloning the antibody gene. The method for establishing a hybridoma involves extracting the spleen from the animal, separating the cells, fusing them with myeloma cells, and selecting clones from the resulting fused cells (hybridomas) that produce antibodies that inhibit mast cell proliferation. A monoclonal antibody that inhibits mast cell proliferation can be prepared by isolating a hybridoma that produces the monoclonal antibody, culturing the hybridoma, and purifying the antibody from the culture supernatant using a standard affinity purification method.

[0068] Hybridomas producing monoclonal antibodies can also be prepared, for example, as follows. First, an animal is immunized with a sensitizing antigen according to known methods. A common method is to inject the sensitizing antigen intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount of PBS (Phosphate-Buffered Saline), physiological saline, or the like, and if desired, mixed with an appropriate amount of a conventional adjuvant, such as complete Freund's adjuvant. The resulting mixture is emulsified and administered to the mammal several times every 4 to 21 days. An appropriate carrier can also be used during immunization with the sensitizing antigen.

[0069] After immunizing a mammal in this manner and confirming that the desired antibody level is elevated in the serum, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells include spleen cells in particular.

[0070] The other parent cell to be fused with the immune cell is a mammalian myeloma cell. This myeloma cell may be any of various known cell lines, such as P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler.G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. al., Nature (1978) 276, 269-270), F.O. al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I.S.J. Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133), etc. are preferably used.

[0071] The cell fusion between the immune cells and myeloma cells can be basically carried out according to known methods, for example, the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0072] More specifically, the cell fusion is carried out in a conventional nutrient medium in the presence of a cell fusion promoter, such as polyethylene glycol (PEG) or Sendai virus (HVJ), and if desired, an adjuvant such as dimethyl sulfoxide may be added to enhance the fusion efficiency.

[0073] The ratio of immune cells to myeloma cells used can be set as desired. For example, it is preferable to use 1 to 10 times more immune cells than myeloma cells. The culture medium used for the cell fusion may be, for example, RPMI 1640 culture medium, MEM culture medium, or any other conventional culture medium used for this type of cell culture, which are suitable for growing the myeloma cell line. Furthermore, serum supplements such as fetal bovine serum (FBS) may also be used in combination.

[0074] Cell fusion is performed by thoroughly mixing predetermined amounts of the immune cells and myeloma cells in the culture medium, adding a PEG solution (e.g., an average molecular weight of about 1000 to 6000) preheated to about 37°C, usually at a concentration of 30 to 60% (w / v), and mixing to form the desired hybridoma. Subsequently, an appropriate culture medium is successively added, and the mixture is centrifuged to remove the supernatant, thereby repeatedly removing cell fusion agents and other substances that are undesirable for hybridoma growth.

[0075] The hybridomas thus obtained are selected by culturing them in a conventional selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT culture medium is continued for a period of time (usually several days to several weeks) sufficient for cells other than the target hybridoma (non-fused cells) to die. Then, a conventional limiting dilution method is performed to screen for and single-clone hybridomas that produce the target antibody.

[0076] In a method for isolating immune cells from an animal sensitized with an antigen and cloning an antibody gene, B cells are identified and lysed from peripheral blood or lymphoid tissue, and cDNA of the antibody gene is prepared by RT-PCR using antibody-specific primers and amplified by PCR. The resulting DNA is inserted into an expression vector, expressed in a recombinant expression system, and purified to obtain a monoclonal antibody (Zaibao Zhang, et al., Front Immunol 2017, 8:494). The obtained monoclonal antibody is then used to clone the desired antibody.

[0077] In addition to obtaining the above-mentioned hybridomas by immunizing a non-human animal with an antigen, it is also possible to obtain hybridomas that produce human antibodies having a desired activity (e.g., cytostatic activity) by sensitizing human lymphocytes, for example, human lymphocytes infected with EB virus, in vitro with a protein, protein-expressing cells, or a lysate thereof, and fusing the sensitized lymphocytes with human-derived immortal myeloma cells, for example, U266 (Registration No. TIB196).

[0078] The hybridomas producing the monoclonal antibodies thus prepared can be subcultured in a normal culture medium and can be stored for a long period in liquid nitrogen.

[0079] That is, a desired antigen or cells expressing the desired antigen are used as a sensitizing antigen, and the antibody is immunized using a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method, and monoclonal antibody-producing cells (hybridomas) are screened by a conventional screening method to produce the antibody.

[0080] Here, known examples of human antibody-producing mice include KM mice (Kirin Pharma / Medarex) and Xeno mice (Amgen) (see, for example, WO 02 / 43478 and WO 02 / 092812). When such mice are immunized with the MCEMP1 protein or a fragment thereof, fully human polyclonal antibodies can be obtained from the blood. Furthermore, spleen cells can be extracted from the immunized mice and fused with myeloma cells to produce fully human monoclonal antibodies.

[0081] Antigens can be prepared, for example, according to a method using animal cells (JP-A No. 2007-530068) or a method using baculovirus (for example, WO98 / 46777), etc. When the antigen has low immunogenicity, it may be bound to an immunogenic macromolecule such as albumin and then used for immunization.

[0082] Furthermore, recombinant antibodies can be produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, and introducing it into a host using genetic engineering (see, for example, Carl, A.K. Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Specifically, cDNA encoding the variable region (V region) of an antibody is synthesized from hybridoma mRNA using reverse transcriptase. Once DNA encoding the V region of the desired antibody is obtained, it is ligated to DNA encoding the desired antibody constant region (C region), and the resulting product is inserted into an expression vector. Alternatively, DNA encoding the antibody V region may be incorporated into an expression vector containing DNA encoding the antibody C region. The DNA is incorporated into the expression vector so that it is expressed under the control of an expression control region, such as an enhancer or promoter. Host cells can then be transformed with this expression vector to express the antibody.

[0083] Monoclonal antibodies include human monoclonal antibodies, non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, chicken monoclonal antibodies, etc.), etc. Monoclonal antibodies can be prepared by culturing hybridomas obtained by fusing splenocytes and myeloma cells from non-human mammals (e.g., mice, human antibody-producing mice, rabbits, etc.) that have been immunized with the MCEMP1 protein or a fragment thereof.

[0084] A chimeric antibody is an antibody produced by combining sequences derived from different animals, such as an antibody consisting of the variable regions of the heavy and light chains of a mouse antibody and the constant regions of the heavy and light chains of a human antibody. Chimeric antibodies can be produced using known methods, for example, by linking DNA encoding the V region of a non-human antibody with DNA encoding the C region of a human antibody, incorporating the resultant into an expression vector, and introducing the vector into a host for production.

[0085] Polyclonal antibodies include antibodies obtained by immunizing human antibody-producing animals (e.g., mice or rabbits) with the MCEMP1 protein or a fragment thereof. Polyclonal antibodies include antibodies produced from multiple types of antibody-producing cells, and an antibody produced by isolating one type of antibody-producing cell among them is a monoclonal antibody.

[0086] Humanized antibodies are modified antibodies, also known as reshaped human antibodies. Humanized antibodies are constructed by replacing the CDRs of an antibody derived from an immunized animal with the CDRs of a human antibody. General genetic recombination techniques for this purpose are also known.

[0087] Specifically, a DNA sequence designed to link the CDRs of a mouse or rabbit antibody with the framework region (FR) of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the human antibody constant region, then incorporated into an expression vector, which is then introduced into a host for production (see EP 239400 and WO 96 / 02576). The FRs of the human antibody linked via the CDRs are selected from those that form a good antigen-binding site with the CDRs derived from the immunized animal. If necessary, amino acids in the framework region of the variable region of the antibody may be substituted so that the CDRs of the reshaped human antibody form a suitable antigen-binding site (Sato K. et al., Cancer Research 1993, 53:851-856). Alternatively, framework regions derived from various human antibodies may be substituted (see WO 99 / 51743).

[0088] After producing a chimeric antibody or humanized antibody, amino acids in the variable region (for example, FR) or constant region may be substituted with other amino acids.

[0089] Amino acid substitutions are, for example, substitutions of less than 15, less than 10, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less amino acids, preferably 1 to 5 amino acids, more preferably 1 or 2 amino acids, and the substituted antibody should be functionally equivalent to the unsubstituted antibody. Conservative amino acid substitutions are desirable, which are substitutions between amino acids with similar properties, such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties can be classified into, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, tyrosine), nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched-chain amino acids (threonine, valine, isoleucine), and aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine).

[0090] The antibody of the present invention may be a modified antibody. Examples of modified antibodies include antibodies conjugated to various molecules such as polyethylene glycol (PEG). The substance to be conjugated to the modified antibody of the present invention is not limited. Such modified antibodies can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.

[0091] Here, "functionally equivalent" means that the antibody of interest has the same biological or biochemical activity as the antibody of the present invention, specifically, the function of damaging mast cells or the function of delivering a drug, and does not essentially cause a rejection reaction when administered to humans, etc. Examples of such activities include cell proliferation inhibitory activity and binding activity.

[0092] A method for introducing mutations into a polypeptide is well known to those skilled in the art as a method for preparing a polypeptide functionally equivalent to a given polypeptide. For example, those skilled in the art will be familiar with site-directed mutagenesis (Hashimoto-Gotoh, T. et al., (1995) Gene 152, 271-275; Zoller, MJ., and Smith, M. (1983) Methods Enzymol. 100, 468-500; Kramer, W. et al., (1984) Nucleic Acids Res. 12, 9441-9456; Kramer, W. and Fritz, HJ., (1987) Methods Enzymol. 154, 350-367; Kunkel, T.A., (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766), or the like, can be used to appropriately introduce mutations into the antibody of the present invention to prepare an antibody that is functionally equivalent to the antibody.

[0093] Antibodies that recognize the epitope of the MCEMP1 protein recognized by the above-mentioned anti-MCEMP1 antibody can be obtained by methods known to those skilled in the art. For example, the epitope of the MCEMP1 protein recognized by the anti-MCEMP1 antibody can be determined by a conventional method (e.g., epitope mapping), and an antibody can be produced using a polypeptide having an amino acid sequence contained in the epitope as an immunogen. Alternatively, the epitope of an antibody produced by a conventional method can be determined, and an antibody having the same epitope as the anti-MCEMP1 antibody can be selected. Here, "epitope" refers to a polypeptide fragment that is antigenic or immunogenic in mammals, preferably humans, and its minimum unit consists of approximately 7 to 12 amino acids, preferably 8 to 11 amino acids.

[0094] Affinity constant K of the antibody in the present invention ａ (binding rate constant k ｏｎ / dissociation rate constant k ｏｆｆ ) is preferably at least 10 ７ M －１ , at least 10 ８ M －１ , at least 5 × 10 ８ M －１ , at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１ , at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or at least 10 １３ M －１ is.

[0095] The antibody of the present invention may be in the form of a so-called conjugate, in which the antibody is bound to a drug. The antibody and drug can be bound via a linker having a group reactive with an amino group, a carboxyl group, a hydroxyl group, a thiol group, etc. (e.g., a succinimidyl group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, a hydroxyl group, etc.).

[0096] A linker refers to a substance capable of binding an antibody to a drug. The linker may be either cleavable or non-cleavable. A cleavable linker is characterized by containing a substance that can be cleaved by an enzyme, pH, a reducing action, or external radiation. It is preferable that the linker is designed so that it is not cleaved at normal sites, preventing the drug from acting on normal cells, but is cleaved specifically at a disease site, allowing the drug to act on mast cells that exacerbate the disease. The linker may also have the effect of improving the solubility or stability of the antibody-drug conjugate.

[0097] The drug conjugate may include, but is not limited to, a cytotoxic agent, an immunomodulator, and a radioisotope. When the drug is a radioisotope, it is desirable that the drug is effective not only for the treatment of a disease but also for the diagnosis of the disease.

[0098] The antibody of the present invention is preferably an antibody immunologically reactive with the MCEMP1 protein or an antibody that specifically recognizes the MCEMP1 protein. The antibody preferably has a structure that minimizes or completely prevents rejection in the target animal to which it is administered. For example, when the target animal is a human, such antibodies include human antibodies, humanized antibodies, chimeric antibodies (e.g., human-rabbit chimeric antibodies), single-chain antibodies, and bispecific antibodies. These antibodies are either recombinant antibodies whose heavy and light chain variable regions are derived from a human antibody, or whose heavy and light chain variable regions consist of complementarity-determining regions (CDR1, CDR2, and CDR3) derived from a non-human animal antibody and framework regions derived from a human antibody, or whose heavy and light chain variable regions are derived from a non-human animal antibody and whose heavy and light chain constant regions are derived from a human antibody. The first two antibodies are preferred.

[0099] These recombinant antibodies can be produced as follows. DNA encoding a monoclonal antibody against the MCEMP1 protein (e.g., a human monoclonal antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a rabbit monoclonal antibody, a chicken monoclonal antibody, etc.) is cloned from antibody-producing cells such as hybridomas, and DNA encoding the light chain variable region and the heavy chain variable region of the antibody is prepared by RT-PCR or the like using this as a template. The sequences of the light chain and heavy chain variable regions or the sequences of each of the CDR1, CDR2, and CDR3 are determined based on the Kabat EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md. (1991)).

[0100] Furthermore, DNA encoding each of these variable regions or DNA encoding each of the CDRs is prepared using genetic recombination techniques (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. Here, the human monoclonal antibody-producing hybridoma can be prepared by immunizing a human antibody-producing animal (e.g., a mouse or rabbit) with the MCEMP1 protein and then fusing splenocytes excised from the immunized animal with myeloma cells. Separately, if necessary, DNA encoding the variable and constant regions of the light or heavy chain derived from a human antibody is prepared using genetic recombination techniques or a DNA synthesizer.

[0101] In the case of a humanized antibody, DNA encoding a humanized antibody can be prepared by replacing the CDR coding sequence in DNA encoding the light chain or heavy chain variable region derived from a human antibody with the corresponding CDR coding sequence of an antibody derived from a non-human animal (e.g., mouse, rabbit, rat, chicken, etc.), and then linking the resulting DNA to DNA encoding the light chain or heavy chain constant region derived from a human antibody, respectively.

[0102] In the case of a chimeric antibody, DNA encoding the light or heavy chain variable region of an antibody derived from a non-human animal (e.g., mouse, rabbit, rat, chicken, etc.) can be linked to DNA encoding the light or heavy chain constant region, respectively, derived from a human antibody, to prepare DNA encoding the chimeric antibody.

[0103] In the case of a single-chain antibody, the antibody has a heavy chain variable region and a light chain variable region linearly linked via a linker, and DNA encoding the single-chain antibody can be prepared by linking DNA encoding the heavy chain variable region, DNA encoding the linker, and DNA encoding the light chain variable region. Here, both the heavy chain variable region and the light chain variable region are derived from a human antibody, or from a human antibody in which only the CDRs have been replaced with CDRs from an antibody derived from a non-human animal (e.g., mouse, rabbit, rat, chicken, etc.). The linker may consist of 12 to 19 amino acids, such as the 15-amino acid (G4S)3 (G.-B. Kim et al., Protein Engineering Design and Selection 2007, 20(9):425-432).

[0104] In the case of a bispecific antibody, this antibody is capable of specifically binding to two different epitopes. For example, DNA encoding a bispecific antibody called a diabody can be prepared by linking, in this order, DNA encoding a heavy chain variable region A, DNA encoding a light chain variable region B, DNA encoding a heavy chain variable region B, and DNA encoding a light chain variable region A (wherein the DNA encoding the light chain variable region B is linked to the DNA encoding the heavy chain variable region B via DNA encoding a linker as described above). Here, both the heavy chain variable region and the light chain variable region are derived from a human antibody, or from a human antibody in which only the CDRs have been replaced by CDRs from an antibody derived from a non-human animal (e.g., mouse, rabbit, rat, chicken, etc.).

[0105] The recombinant DNA prepared as described above can be incorporated into one or more appropriate vectors, which are then introduced into host cells (e.g., mammalian cells, yeast cells, insect cells, etc.) and (co)expressed to produce a recombinant antibody (P.J. Delves, ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY, P. Shepherd and C. Dean, Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; J.W. Goding, Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS).

[0106] The above-mentioned antibody preferably has cytotoxic activity.

[0107] Alternatively, hybridomas capable of producing other human or non-human animal antibodies (e.g., rabbit antibodies) against the MCEMP1 protein are prepared, and the monoclonal antibodies produced by the hybridomas are collected and assessed for their immunological binding to the MCEMP1 protein and cytotoxic activity to determine whether they are the desired antibodies. After identifying the hybridomas producing the desired monoclonal antibodies, DNA encoding the heavy and light chain variable regions of the desired antibody is prepared from the hybridomas and sequenced, as described above, and the DNA is used to produce another antibody.

[0108] Furthermore, as long as the above-described antibodies of the present invention have the specificity to specifically recognize MCEMP1, they may have one or several (preferably one or two) amino acid substitutions, deletions, or additions, particularly in the framework region sequence and / or constant region sequence of the antibody. Here, "several" means two to five, preferably two or three. Because the complementarity-determining regions (CDRs) encoded by the DNA of these sequences are the regions that determine the antibody specificity, the sequences encoding other regions of the antibody (i.e., the constant region and framework region) may be sequences derived from other antibodies. Here, "other antibodies" includes antibodies derived from organisms other than humans, but from the viewpoint of reducing side effects, antibodies derived from humans are preferred. That is, in the above-described DNA, the regions encoding the framework regions and constant regions of the heavy and light chains preferably contain nucleotide sequences encoding the corresponding amino acid sequences derived from a human antibody.

[0109] The DNA of the present invention can be obtained, for example, by the method described above or the following method. First, total RNA is prepared from a hybridoma related to the antibody of the present invention using a commercially available RNA extraction kit, and cDNA is synthesized using reverse transcriptase with random primers or the like. Next, cDNA encoding the antibody is amplified by PCR using oligonucleotides with conserved sequences in the variable regions of known rabbit antibody heavy chain genes and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA can be determined by conventional methods, such as by incorporating it into a sequencing plasmid or phage.

[0110] The pathological improvement effect of the anti-MCEMP1 antibody used in the present invention on MCEMP1-expressing mast cells is thought to occur through antibody-dependent cellular cytotoxicity (ADCC activity), complement-dependent cytotoxicity (CDC activity), or the action of a drug conjugated to the anti-MCEMP1 antibody.

[0111] Therefore, the activity of the anti-MCEMP1 antibody used in the present invention can be evaluated by measuring the above-mentioned ADCC activity or CDC activity, or the effect of a drug conjugated to the anti-MCEMP1 antibody, on mast cells expressing MCEMP1 protein on their cell surface in vitro, as specifically shown in the examples below.

[0112] The anti-MCEMP1 antibody used in the present invention binds to the MCEMP1 protein expressed on the cell surface of mast cells, and due to the above activity, exhibits a proliferation-inhibitory effect or the effect of a drug conjugated to the anti-MCEMP1 antibody, and is therefore thought to be useful in the treatment or prevention of diseases in which mast cells exacerbate the pathology. Specifically, the present invention provides a pharmaceutical composition for the treatment and / or prevention of diseases in which mast cells exacerbate the pathology, which comprises an anti-MCEMP1 antibody as an active ingredient. When the anti-MCEMP1 antibody is to be administered to the human body (antibody therapy), it is preferably a human antibody or a humanized antibody in order to reduce immunogenicity.

[0113] Furthermore, the higher the binding affinity between an anti-MCEMP1 antibody and the MCEMP1 protein on the surface of mast cells, the stronger the medicinal effect of the anti-MCEMP1 antibody can be expected. Therefore, if an anti-MCEMP1 antibody having high binding affinity with the MCEMP1 protein can be obtained, a stronger effect of improving pathology can be expected, and it can be applied as a pharmaceutical composition for the treatment and / or prevention of diseases in which mast cells exacerbate the pathology. As described above, high binding affinity is defined as a binding constant (affinity constant) K ａ (binding rate constant k ｏｎ / dissociation rate constant k ｏｆｆ ) is preferably at least 10 ７ M －１ , at least 10 ８ M －１ , at least 5 × 10 ８ M －１ , at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１, at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or at least 10 １３ M －１ It is desirable that:

[0114] Binding to Antigen-Expressing Cells The ability of antibodies to bind to MCEMP1 can be determined using binding assays such as those described in the Examples, using ELISA, Western blotting, immunofluorescence and flow cytometry analysis.

[0115] Immunohistochemical Staining Antibodies that recognize MCEMP1 protein can be tested for reactivity with MCEMP1 protein by immunohistochemistry using methods well known to those skilled in the art, using paraformaldehyde- or acetone-fixed frozen sections or paraformaldehyde-fixed paraffin-embedded tissue sections from tissue obtained from a patient during surgery, from the patient's bone marrow tissue, lymph nodes, or peripheral blood cells, or from tissue obtained from an animal bearing xenograft tissue inoculated naturally or after transfection with a cell line expressing MCEMP1.

[0116] For immunohistochemical staining, antibodies immunologically reactive with the MCEMP1 protein can be stained by various methods, for example, by reacting with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody.

[0117] <Pharmaceutical Composition> The present invention provides a pharmaceutical composition (or medicament) comprising the antibody of the present invention, i.e., the above-described anti-MCEMP1 antibody, as an active ingredient. The pharmaceutical composition (or medicament) of the present invention usually contains the above-described anti-MCEMP1 antibody in an effective amount.

[0118] The target of the pharmaceutical composition of the present invention for treating and / or preventing diseases in which mast cells exacerbate the pathology is not particularly limited, as long as it is a mast cell that expresses the MCEMP1 protein on the cell surface.

[0119] The target diseases of the present invention are diseases whose pathology is exacerbated by mast cells expressing a gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8, or a partial sequence thereof consisting of 7 or more consecutive amino acids, preferably diseases whose pathology is exacerbated by mast cells expressing such a polypeptide on their cell surface. The target diseases of the present invention are preferably respiratory diseases, mast cell activation syndrome, cytokine release syndrome, autoimmune diseases, systemic allergic diseases, skin diseases, digestive diseases, cancers that do not express MCEMP1 protein on their cell surface, metabolic diseases, female reproductive diseases, pregnancy, childbirth, and postpartum diseases, sepsis, and graft-versus-host disease.

[0120] Respiratory diseases include, for example, asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, interstitial lung disease, and allergic rhinitis.

[0121] The autoimmune disease is preferably a collagen disease, and examples thereof include rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, polymyositis, dermatomyositis, and giant cell arteritis.

[0122] Systemic allergic diseases include, for example, anaphylaxis, drug hypersensitivity, food allergy, and poison allergy.

[0123] Skin diseases include, for example, urticaria, dermatitis, psoriasis, chronic pruritus, and prurigo nodularis.

[0124] Examples of digestive system diseases include Crohn's disease, ulcerative colitis, irritable bowel syndrome, eosinophilic esophagitis, eosinophilic duodenitis, and eosinophilic gastritis.

[0125] Cancers that do not express MCEMP1 protein on the cell surface are preferably solid cancers, such as gastric cancer, lung cancer, breast cancer, oral cancer, colorectal cancer, esophageal cancer, small intestine cancer, melanoma, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, prostate cancer, glioblastoma, and thyroid cancer.

[0126] Metabolic diseases include, for example, diabetes.

[0127] Diseases of the female reproductive system include, for example, endometriosis.

[0128] Pregnancy, childbirth and postpartum diseases include, for example, preeclampsia.

[0129] The target animals are mammals, including, for example, primates, pet animals, livestock, sports animals, and laboratory animals, with humans, dogs, and cats being particularly preferred.

[0130] When the antibody used in the present invention is used as a pharmaceutical composition, it can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution or suspension in water or other pharmaceutically acceptable liquid for injection. For example, it can be formulated by appropriately combining it with pharmacologically acceptable carriers or vehicles or additives, specifically, sterile water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc., and mixing it in a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that an appropriate dose within the indicated range can be obtained.

[0131] Sterile compositions for injection can be prepared according to conventional pharmaceutical practice using a vehicle such as distilled water for injection.

[0132] Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and these may be used in combination with an appropriate solubilizing agent, such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, or nonionic surfactants such as polysorbate 80™ and HCO-60.

[0133] Examples of oily liquids include sesame oil and soybean oil, and they may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. They may also contain buffers such as phosphate buffer and sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants. The prepared injection solution is usually filled into suitable ampoules.

[0134] Administration may be oral or parenteral, preferably parenteral, and specific examples include injections, intranasal administration, pulmonary administration, transdermal administration, etc. Examples of injections include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., which may be used for systemic or local administration. The antibody of the present invention may also be administered directly to the diseased site by local administration to the diseased site via injection, infusion, implantation of a sustained-release formulation, etc.

[0135] Furthermore, an appropriate administration method can be selected depending on the patient's age, body weight, sex, symptoms, etc. The dosage of a pharmaceutical composition containing an antibody or DNA encoding an antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values. The dosage and administration method vary depending on the patient's body weight, age, sex, symptoms, etc., but can be appropriately selected by one skilled in the art.

[0136] By administering the antibody or antigen-binding fragment thereof of the present invention, or the above-mentioned pharmaceutical composition containing the same to a subject, it is possible to treat and / or prevent the above-mentioned diseases, particularly diseases in which mast cells expressing the MCEMP1 protein on their cell surface exacerbate the pathology, preferably respiratory diseases, mast cell activation syndrome, cytokine release syndrome, autoimmune diseases, systemic allergic diseases, skin diseases, digestive diseases, cancers that do not express the MCEMP1 protein on their cell surface, metabolic diseases, female reproductive system diseases, pregnancy, childbirth, and postpartum diseases, sepsis, and graft-versus-host disease.

[0137] Furthermore, the present invention also encompasses a method for treating and / or preventing a disease in which mast cells exacerbate the pathology, comprising administering to a subject the pharmaceutical composition (or medicament) of the present invention in combination with a drug or a pharmaceutical composition (or medicament) containing the drug, as exemplified above. The target diseases are the same as those described above. The antibody or antigen-binding fragment thereof and the drug of the present invention can be administered to a subject simultaneously or separately. When administered separately, either pharmaceutical composition can be administered first or second, and the administration interval, dosage, administration route, and number of administrations can be appropriately selected by a specialist. When administered simultaneously, for example, a pharmaceutical composition in a pharmaceutical dosage form obtained by mixing the antibody or fragment thereof of the present invention and the drug in a pharmacologically acceptable carrier (or medium) and formulating the mixture is also encompassed. Furthermore, the descriptions of the formulation, formulation, administration route, dosage, disease, etc., for the pharmaceutical composition and dosage form containing the antibody of the present invention can be applied to both of the above-mentioned drug-containing pharmaceutical compositions and dosage forms.

[0138] Therefore, the present invention also provides a combined pharmaceutical product for treating and / or preventing a disease whose pathology is aggravated by mast cells, which comprises the pharmaceutical composition of the present invention and a pharmaceutical composition containing an agent as exemplified above, and a method for treating and / or preventing a disease whose pathology is aggravated by mast cells, which comprises administering the combined pharmaceutical product.The present invention also provides a pharmaceutical composition for treating and / or preventing a disease whose pathology is aggravated by mast cells, which comprises the antibody or antigen-binding fragment thereof of the present invention and an agent, together with a pharmacologically acceptable carrier and / or additive.

[0139] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these specific examples.

[0140] Example 1 Preparation of Human MCEMP1 Protein (1) Construction of Expression Vector for Extracellular Domain of Human MCEMP1 Protein The DNA base sequence encoding the extracellular domain of human MCEMP1 protein was cloned based on the gene of SEQ ID NO: 1 by the following method.

[0141] PCR was performed using 1 ng of human MCEMP1 / pcDNA3.1 prepared in Example 1 of WO 2017 / 170322, 0.4 μM each of two primers (described in SEQ ID NOS: 17 and 18) containing NdeI and XhoI restriction enzyme cleavage sequences, 0.2 mM dNTP, and 1.25 U of PrimeSTAR HS DNA polymerase (Takara Bio) in a total volume of 50 μl, along with the accompanying buffer. PCR was performed using a Thermal Cycler (BIO RAD) by repeating 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 30 seconds. The two primers were used to amplify the region of SEQ ID NOS: 10, which contains the amino acid sequence of the extracellular domain of the MCEMP1 protein. After PCR, the amplified DNA was electrophoresed on a 2% agarose gel, and a DNA fragment of approximately 0.3 kbp was purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The amplified product obtained by the above PCR reaction and pET-30a(+) (ThermoFisher Scientific) were each digested with restriction enzymes NdeI and XhoI (both Takara Bio), and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The purified amplified product and pET30a(+) were ligated using Ligation High Ver. 2 (Toyobo) to obtain an expression vector encoding a human MCEMP1 extracellular domain / His fusion protein (hereinafter referred to as hMCEMP1ECD-His) (hereinafter referred to as pET-30a(+)-hMCEMP1ECD-His). Sequence analysis using a DNA sequencer confirmed that this was the DNA base sequence encoding hMCEMP1ECD-His. The sequence represented by SEQ ID NO: 19 represents the base sequence encoding hMCEMP1ECD-His, and the sequence represented by SEQ ID NO: 20 represents the amino acid sequence of hMCEMP1ECD-His.

[0142] (2) Preparation of hMCEMP1ECD-His The expression vector pET-30a(+)-hMCEMP1ECD-His was introduced into BL21(DE3) (Novagen) according to the attached protocol, and then plated onto a selective medium (LB plate containing 50 μg / ml kanamycin) and cultured. Colonies that grew on the selective medium were picked and inoculated into LB medium containing 25 μg / ml kanamycin, followed by overnight shaking at 37°C as a preculture. The precultured medium was added to LB medium containing 25 μg / ml kanamycin and cultured at 37°C with shaking at 132 rpm. The absorbance was measured sequentially, and when the absorbance at 600 nm reached 0.6 to 0.7, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce expression of hMCEMP1ECD-His, followed by shaking culture at 37°C for 3 hours.

[0143] The collected E. coli was suspended in a buffer containing 50 mM Tris-HCl (pH 8.0) and 300 mM NaCl, and then disrupted by sonication. The resulting centrifuged supernatant was applied to Ni Sepharose 6 Fast Flow (GE Healthcare Biosciences). After washing with a washing buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 5 mM imidazole (pH 8.0), hMCEMP1ECD-His was eluted with an elution buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 500 mM imidazole (pH 8.0). The eluted fraction was collected and replaced with PBS by dialysis. The dialyzed sample was purified by applying it to a gel filtration column HiLoad 16 / 60 Superdex 200 (GE Healthcare Biosciences) equilibrated with PBS, and the peak fraction was collected. After concentration using an Amicon Ultra MWCO 3k (Merck Millipore), sterile filtration was performed using an HT Tafflin Acrodisc 0.22 μm (PALL), and this was used in the following experiments.

[0144] Example 2: Preparation of Polyclonal Antibodies Binding to the Extracellular Domain of MCEMP1 Protein (1) Preparation of Polyclonal Antibodies Against hMCEMP1ECD-His To obtain antibodies binding to the extracellular domain of human MCEMP1 protein, 1 mg of hMCEMP1ECD-His prepared in Example 1 was used as the antigen. The initial immunization sample was mixed with a complete Freund's adjuvant (CFA) solution, and subsequent immunization samples were mixed with an incomplete Freund's adjuvant (IFA) solution. This solution was then administered subcutaneously to rabbits five times every three weeks. Blood was then collected to obtain antisera containing polyclonal antibodies. This antisera was further purified using a Protein G carrier column (GE Healthcare Biosciences) and substituted with PBS to obtain IgG-type polyclonal antibodies against hMCEMP1ECD-His (hereinafter referred to as "anti-MCEMP1 polyclonal antibodies").

[0145] (2) Analysis of Immunological Reactivity of Anti-MCEMP1 Polyclonal Antibody to MCEMP1 Protein 100 μl of 1 μg / ml hMCEMP1ECD-His protein solution prepared in Example 1 was added per well of a Maxisorp (ThermoFisher Scientific) plate and allowed to stand at 4°C for 18 hours. The solution was removed, and a PBS-T solution containing 3% skim milk was added and allowed to stand at room temperature for 3 hours. The solution was removed, and the polyclonal antibody obtained above was added and allowed to stand at room temperature for 2 hours. After washing each well three times with PBS-T, HRP-labeled anti-rabbit IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added and allowed to stand at room temperature for 1 hour. After washing the wells three times with PBS-T, TMB substrate solution (ThermoFisher Scientific) was added and the wells were left to stand for 15 to 30 minutes for color development. After color development, the reaction was stopped by adding 1 N sulfuric acid, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorption spectrometer. The absorbance value at 595 nm was subtracted from the absorbance value at 450 nm to calculate the color intensity. As a result, it was confirmed that the prepared anti-MCEMP1 polyclonal antibody is a polyclonal antibody that exhibits immunological reactivity with the MCEMP1 protein.

[0146] (3) Analysis of immunological reactivity of anti-MCEMP1 polyclonal antibodies to MCEMP1 protein expressed on the cell surface Next, we analyzed whether the anti-MCEMP1 polyclonal antibodies prepared in (1) above exhibit specific reactivity to MCEMP1 expressed on the cell surface. 100 cells of CHO-human MCEMP1 cells and 100 cells of CHO-emp cells prepared in Example 3 of WO 2017 / 170322 were used. ６ The cells were centrifuged, and 0.1 μg (5 μl) of anti-MCEMP1 polyclonal antibody was added. The cells were then suspended in 95 μl of PBS containing 3% fetal bovine serum (FBS) and allowed to stand on ice for 1 hour. After washing with PBS containing 3% FBS, the cells were suspended in 0.2 μl of R-Phycoerythrin-conjugated goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 μl of PBS containing 3% FBS and allowed to stand on ice for 1 hour. After washing with PBS containing 3% FBS, the cells were measured for fluorescence intensity using a FACSVerse (Becton Dickinson). A control was prepared by the same procedure as above, using purified antibodies (Fujifilm Wako Pure Chemical Industries) derived from normal rabbit serum that had not been immunized with a specific antigen, instead of the polyclonal antibody against MCEMP1. As a result, the anti-MCEMP1 polyclonal antibody showed higher fluorescence intensity in CHO-human MCEMP1 cells compared to the control but did not react with CHO-emp cells, confirming that it is a polyclonal antibody that specifically reacts with the MCEMP1 protein expressed on the cell surface.

[0147] (4) Analysis of MCEMP1 protein expression on the surface of mast cells Next, the human mast cell line LUVA (Kerafast) was analyzed to determine whether human MCEMP1 protein was expressed on the cell surface. ６The cells were centrifuged and reacted with FcR blocking reagent, Human (Miltenyi Biotec) according to the attached protocol to suppress nonspecific binding to Fc receptors. 0.1 μg (5 μl) of the anti-MCEMP1 polyclonal antibody prepared in (1) above was added, and the cells were suspended in 95 μl of PBS containing 3% fetal bovine serum (FBS) and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in 0.2 μl of R-Phycoerythrin-labeled goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 μl of PBS containing 3% fetal bovine serum (FBS), and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), fluorescence intensity was measured using a FACSVerse (Becton Dickinson). Separately, the same procedure as above was repeated using a purified antibody derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries) that did not react with MCEMP1 protein in (3) as a control, instead of the anti-MCEMP1 polyclonal antibody. As a result, LUVA cells treated with anti-MCEMP1 polyclonal antibody showed a 74% increase in mean fluorescence intensity compared to the control. This confirmed the expression of MCEMP1 protein on the cell membrane surface of the above human mast cell line.

[0148] The rate of increase in mean fluorescence intensity (MFI value) was calculated using the following formula 1: Rate of increase in mean fluorescence intensity (%) = (MFI value of cells reacted with anti-MCEMP1 polyclonal antibody - MFI value of cells reacted with control antibody) / (MFI value of cells reacted with control antibody) x 100.

[0149] Example 3 Cytotoxic Activity of Anti-MCEMP1 Polyclonal Antibody Against Mast Cells (ADCC Activity) Next, we investigated whether anti-MCEMP1 polyclonal antibodies can damage mast cells expressing MCEMP1 on the cell surface. Antibody-dependent cellular cytotoxicity (ADCC activity) was evaluated using the anti-MCEMP1 polyclonal antibody prepared in Example 2 (1). The human mast cell line (LUVA), whose cell surface expression of MCEMP1 protein was confirmed in Example 2, was used as the target cell. LUVA was injected at 10６ The pieces were collected in a 50 ml centrifuge tube and 100 μCi of chromium ( ５１ Cr) was added and incubated at 37°C for 2 hours. After that, the cells were washed three times with RPMI 1640 medium containing 10% fetal bovine serum (FBS), and then plated in a 96-well V-bottom plate at 2 x 10 ３ To this, 1.5 μg of the anti-MCEMP1 polyclonal antibody was added, and 1.5 × 10 natural killer cells (NK cells) isolated from human peripheral blood were added as effector cells. ５ Each was added to a total volume of 150 μl and incubated at 37°C, 5% CO ２ After the incubation, the chromium ( ５１ The amount of Cr was measured, and the ADCC activity of the anti-MCEMP1 polyclonal antibody against mast cells was calculated. As a result, 24.8% ADCC activity was confirmed against LUVA cells (see Figure 1). On the other hand, when the same procedure was performed on the above cell line using a purified antibody (Fujifilm Wako Pure Chemical Industries, Ltd.) derived from normal rabbit serum that did not react with MCEMP1-expressing cells in Examples 2(3) and (4) as a control, and a medium without antibody, the activity was clearly low (see Figure 1). Therefore, it was demonstrated that the anti-MCEMP1 polyclonal antibody can damage mast cells that express MCEMP1 protein on their cell surface through its ADCC activity.

[0150] The cytotoxic activity in Figure 1 was determined by the combination of antibodies, effector cells, and chromium ( ５１ The target cells were mixed with chromium ( ５１ The results show the cytotoxic activity against mast cell lines, calculated using the following formula 2: Formula 2: Cytotoxic activity (%) = (removal of cytotoxicity from target cells when antibody and effector cells are added) ５１ Cr release amount - from target cells ５１ Spontaneous release of Cr) / (2% Triton X-100 (MP Biomedicals) from target cells ５１ Cr release amount - from target cells ５１Spontaneous release of Cr) × 100.

[0151] The pharmaceutical composition of the present invention is useful for treating and / or preventing diseases in which mast cells exacerbate the pathology.

[0152] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for the treatment and / or prevention of diseases in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising as an active ingredient an antibody or an antigen-binding fragment thereof that is immunologically reactive with MCEMP1 protein.

2. A pharmaceutical composition for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising as an active ingredient an antibody or antigen-binding fragment thereof that is immunologically reactive with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, 4, 6, or 8, or an amino acid sequence having 80% or more sequence identity to said amino acid sequence, or a fragment thereof containing 7 or more consecutive amino acids.

3. A pharmaceutical composition for the treatment and / or prevention of a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising as an active ingredient an antibody or antigen-binding fragment thereof that is immunologically reactive with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 10, 12, 14 or 16, or an amino acid sequence having 80% or more sequence identity to said amino acid sequence, or a fragment thereof containing 7 or more consecutive amino acids.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof conjugated with a drug.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the drug is a cytotoxic agent, an immunomodulator, or a radioisotope.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the disease is selected from the group consisting of respiratory diseases, mast cell activation syndrome, cytokine release syndrome, autoimmune diseases, systemic allergic diseases, skin diseases, digestive diseases, cancers that do not express MCEMP1 protein on the cell surface, metabolic diseases, female reproductive diseases, pregnancy, childbirth, and postpartum diseases, sepsis, and graft-versus-host disease.

7. The pharmaceutical composition according to claim 6, wherein the respiratory disease is asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, interstitial lung disease or allergic rhinitis.

8. The pharmaceutical composition according to claim 6, wherein the autoimmune disease is a collagen disease.

9. The pharmaceutical composition according to claim 8, wherein the collagen disease is rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjogren's syndrome, polymyositis, dermatomyositis, or giant cell arteritis.

10. The pharmaceutical composition according to claim 6, wherein the systemic allergic disease is anaphylaxis, drug hypersensitivity, food allergy or poison allergy.

11. The pharmaceutical composition according to claim 6, wherein the skin disease is urticaria, dermatitis, psoriasis, chronic pruritus or prurigo nodularis.

12. The pharmaceutical composition according to claim 6, wherein the digestive system disease is Crohn's disease, ulcerative colitis, irritable bowel syndrome, eosinophilic esophagitis, eosinophilic duodenitis, or eosinophilic gastritis.

13. The pharmaceutical composition according to claim 6, wherein the cancer that does not express the MCEMP1 protein on its cell surface is a solid cancer.

14. The pharmaceutical composition of claim 13, wherein the solid cancer is gastric cancer, lung cancer, breast cancer, oral cancer, colorectal cancer, esophageal cancer, small intestine cancer, melanoma, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, prostate cancer, glioblastoma, or thyroid cancer.

15. The pharmaceutical composition of claim 6, wherein the metabolic disease is diabetes.

16. The pharmaceutical composition according to claim 6, wherein the female reproductive system disease is endometriosis.

17. The pharmaceutical composition according to claim 6, wherein the pregnancy, delivery and postpartum disease is preeclampsia.

18. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

19. The pharmaceutical composition of any one of claims 1 to 3, wherein the antibody is a human antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, or a multispecific antibody.

20. A combination drug for treating and / or preventing a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising the pharmaceutical composition according to any one of claims 1 to 3 and a pharmaceutical composition containing a drug for treating a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology.

21. A method for treating and / or preventing a disease in which mast cells expressing MCEMP1 protein on their cell surface exacerbate the pathology, comprising administering to a subject the pharmaceutical composition according to any one of claims 1 to 19.

22. A method for treating and / or preventing a disease exacerbated by mast cells expressing MCEMP1 protein on their cell surface, comprising simultaneously or separately administering to a subject the pharmaceutical composition according to any one of claims 1 to 19 and a pharmaceutical composition containing a drug for treating a disease exacerbated by mast cells expressing MCEMP1 protein on their cell surface.

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