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

Title of Invention: Pharmaceutical composition for treating and / or preventing diseases in which mast cells exacerbate pathological condition Technical Field

[0001] The present invention relates to a novel medical use of antibodies or antigen-binding fragments thereof having immunological reactivity with MCEMP1 protein as therapeutic and / or preventive agents for diseases in which mast cells exacerbate pathological condition. Background

[0002] In recent years, a variety of antibody medicines for diseases treatment that target antigen proteins on cells involved in diseases have come into existence. The antibody medicines used as targeted antigen-specific therapeutic agents exhibit drug efficacy to a certain extent, and thus they have been gaining attention. However, many target antigen proteins are also expressed on multiple normal cells. As a result of antibody administration, not only cells involved in diseases, but also normal cells not involved in diseases can be exposed to pharmacological action, thereby causing side effects, which become problematic. Thus, it is expected that, if it becomes possible to identify antigens that are specifically expressed on the surface of a cell involved in diseases and to use antibodies targeting such antigens as medicaments, then treatment with antibody medicines that cause fewer side effects could be realized.

[0003] It has been reported that Mast Cell-Expressed Membrane Protein 1 (MCEMP1), a type II transmembrane protein, is likely expressed on cell membranes in a manner specific for mast cells, suggesting the possibility that the protein participates in mast cell differentiation, immune response, and allergic response (Non Patent Literature 1). Furthermore, it has been suggested that MCEMP1 protein expressed on lung mast cells may promote asthma exacerbation (Non-Patent Literature 2). In addition, there are cancers that specifically expresses MCEMP1 protein on the cell membrane surface, and it is known that an antibody or antigen-binding fragment thereof against MCEMP1 protein can be used as a pharmaceutical composition for the treatment and / or prevention of the cancer (Patent Literature 1). However, none of the previous reports show that an antibody or antigen-binding fragment thereof against MCEMP1 protein is useful for treatment or prevention of diseases whose pathology is exacerbated by mast cells. Citation List Patent Literature

[0004] [Patent Literature 1]: WO2017 / 170322. Non-Patent Literature

[0005] [Non-Patent Literature 1]: Kang Li. et al. Genomics, 86:68-75 (2005) [Non-Patent Literature 2]: Young Jung Choi. et al. Nature Communications, 14:2045 (2023) Summary Technical Problem

[0006] An object of the present invention is to provide a therapeutic and / or preventive agent targeting mast cells for diseases whose pathology is exacerbated by mast cells. Solution to Problem

[0007] As a result of intensive studies, the present inventors have discovered that MCEMP1 protein is specifically expressed on the cell surface of mast cells. Further, they have discovered that by using an antibody or antigen-binding fragment thereof against the portion of MCEMP1 protein that is expressed on the surface of the mast cells, and damaging the mast cells that express MCEMP1 protein on their cell surface, it is possible to treat and / or prevent diseases whose pathology is exacerbated by mast cells. These findings have led to the completion of the present invention.

[0008] Therefore, the present invention encompasses the following embodiments (1) to (22).

[0009] (1) A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity with MCEMP1 protein, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

[0010] (2) A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity 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 with the amino acid sequence, or with a fragment of the polypeptide comprising 7 or more consecutive amino acids, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

[0011] (3) A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity 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 with the amino acid sequence, or with a fragment of the polypeptide comprising 7 or more consecutive amino acids, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

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

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

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

[0015] (7) The pharmaceutical composition according to (6), wherein 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 autoimmune disease is collagen disease.

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

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

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

[0020] (12) The pharmaceutical composition according to (6), wherein 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 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 metabolic disease is diabetes.

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

[0025] (17) The pharmaceutical composition according to (6), wherein pregnancy, childbirth and postpartum disease is pregnancy-induced hypertensive nephropathy.

[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 pharmaceutical combination for treatment and / or prevention of a disease, comprising the pharmaceutical composition according to any one of claims (1) to (19), and a pharmaceutical composition comprising a therapeutic agent(s) for the disease whose pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

[0029] (21) A method for treating and / or preventing a disease, comprising administering, to a subject, the pharmaceutical composition according to any one of claims (1) to (19), wherein the disease pathology is exacerbated by mast cells expressingMCEMP1 protein on the cell surface.

[0030] (22) A method for treating and / or preventing a disease, comprising administering, simultaneously or separately to a subject, the pharmaceutical composition according to any one of (1) to (19), and a pharmaceutical composition comprising a therapeutic agent(s) for the disease whose pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface. Advantageous Effects of Invention

[0031] The antibody or antigen-binding fragment thereof against MCEMP1 protein used in the present invention exerts a pharmacological effect to mast cells. Therefore, the antibody or antigen-binding fragment thereof against MCEMP1 is useful for treating and / or preventing diseases in which mast cells exacerbate pathology. Brief Description of Drawings

[0032] [Figure 1] This figure shows the cytotoxic activity against human mast cell line (LUVA) that expresses MCEMP1 protein on its cell surface by polyclonal antibodies (anti-MCEMP1 polyclonal antibodies) having immunological reactivity with MCEMP1 protein. 1: Cytotoxic activity against LUVA cells in the condition without antibody addition, 2: condition with the addition of a control purified antibody derived from normal rabbit serum, and 3: condition with the addition of anti-MCEMP1 polyclonal antibodies. Description of Embodiments

[0033] The present invention relates to a use of an antibody or antigen-binding fragment thereof against MCEMP1 protein or a fragment (hereinafter sometimes collectively referred to as “MCEMP1 protein”) thereof for treatment and / or prevention of diseases in which mast cells exacerbate pathology.

[0034] The present invention relates to a pharmaceutical composition for treatment and / or prevention of diseases in which mast cells exacerbate pathology, comprising, as an active ingredient, an antibody or fragment thereof having immunological reactivity with MCEMP1 protein having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, or 8 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably 99% or more, for example, 99.5% or more) sequence identity with the amino acid sequence, or with a fragment thereof comprising 7 or more consecutive amino acids (from 7 amino acids to the full-length of corresponding sequence, preferably from 7 to 150 amino acids and more preferably from 7 to 50 amino acids).

[0035] The present invention also relates to the pharmaceutical composition for treatment and / or prevention of diseases in which mast cells exacerbate pathological condition, comprising, as an active ingredient, an antibody or fragment thereof having immunological reactivity with a fragment of MCEMP1 protein, the fragment being a polypeptide consisting of 7 or more (from 7 amino acids to the full-length of corresponding sequence, preferably from 7 to 40 amino acids, more preferably from 7 to 20 amino acids, for example, from 7 to 12 or from 8 to 11 amino acids) consecutive amino acids of an amino acid sequence shown in any one 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, further preferably 95% or more, and particularly preferably 97% or more) sequence identity with the amino acid sequence.

[0036] The therapeutic effect 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, or fragment thereof used in the present invention can be clarified by evaluating the improvement of pathology aggravated by mast cells or the inhibition of mast cell proliferation in animals into which mast cells have been transplanted or in which mast cells have excessively proliferated in vivo. Alternatively, as described later, the therapeutic effect can be clarified by evaluating death of mast cells mediated by cytotoxic activity of immune cells or complement, the decrease of viable cells, or a reduction in cell function against mast cells expressing the polypeptide in vitro.

[0037] Similarly, the therapeutic effect of an antibody or antigen-binding fragment thereof against a polypeptide of the even-numbered SEQ ID NOS: 10 to 16 or fragment thereof used in the present invention can be clarified by evaluating the improvement of pathology aggravated by mast cells or the inhibition of mast cell proliferation in animals into which mast cells have been transplanted or in which mast cells have excessively proliferated in vivo. Alternatively, as described later, the therapeutic effect can be clarified by evaluating mast cell death mediated by cytotoxic activity of immune cells or complement, a decrease of viable cells, or a reduction in cell function against mast cells expressing the polypeptide in vitro.

[0038] Similarly, the therapeutic effect of a conjugate of an antibody against MCEMP1 or antigen-binding fragment thereof (hereinafter sometimes collectively referred to as "anti-MCEMP1 antibody") and a therapeutic agent(s) can be clarified by evaluating the action of the therapeutic agent in improving pathology aggravated by mast cells or inhibiting mast cell proliferation in animals into which mast cells have been transplanted or in which mast cells have excessively proliferated in vivo. Alternatively, the therapeutic effect can be clarified by evaluating death of mast cells, a decrease of viable cells, or a reduction in cell function against mast cells expressing the polypeptide in vitro.

[0039] Additionally, the nucleotide sequences of the DNA encoding the 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 an amino acid sequence isolated as human MCEMP1 protein. The amino acid sequence represented by SEQ ID NO: 4 is an amino acid sequence isolated as a canine homolog of human MCEMP1. The amino acid sequence represented by SEQ ID NO: 6 is an amino acid sequence isolated as a feline homolog of human MCEMP1. The amino acid sequence represented by SEQ ID NO: 8 is an amino acid sequence isolated as a mouse homolog of human MCEMP1.

[0041] According to the present invention, an antibody that binds to a portion of MCEMP1 protein that is expressed on the surface of mast cells is preferably used. Specifically, a polypeptide comprising an extracellular region ofMCEMP1 protein includes the amino acid sequence shown in SEQ ID NO: 10 (human), 12 (canine), 14 (feline), or 16 (mouse) or fragments thereof (preferably, the fragments each consisting of 7 or more consecutive amino acids of any one of the amino acid sequences), or an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, and particularly preferably 99% or more sequence identity to any one of these polypeptides. Antibodies of the present invention include all antibodies which bind to the above polypeptides and exhibit mast cell death, viable cell decrease, or reduction in cell function.

[0042] The anti-MCEMP1 antibody usable in the present invention may be any types thereof, as long as they can exhibit inhibitory activity against mast cell proliferation. Examples thereof can include monoclonal antibodies, polyclonal antibodies, synthetic antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, and single-chain antibodies (scFv). The antibodies used in the present invention also include antibody fragments, for example, antigen binding fragments such as Fab and F(ab')2. These antibodies and fragments thereof can be prepared by methods known to persons skilled in the art. In the present invention, antibodies or fragments thereof capable of specifically binding to MCEMP1 protein are desirable. Such antibodies are preferably monoclonal antibodies; however, as long as homogeneous antibodies can be stably produced, polyclonal antibodies may also be used. In addition, if the subject is a human, a human antibody or a humanized antibody is desirable in order to avoid or suppress the immune rejection.

[0043] The word "specifically binding to MCEMP1 protein or fragments thereof" as used herein means that an antibody of interest specifically binds to MCEMP1 protein or fragments thereof and does not substantially bind to other proteins.

[0044] The activity of the antibody usable in the present invention against mast cells can be clarified by evaluating the inhibition of mast cell proliferation in animals into which mast cells have been transplanted or in animals in which mast cells have excessively proliferated in vivo. Alternatively, as described later, the activity against mast cells that express the polypeptide can be clarified by evaluating whether or not the antibody exhibits cytotoxic activity mediated by immune cells or complement, or action of a therapeutic agent conjugated with the anti-MCEMP1 antibody in vitro.

[0045] Moreover, in the present invention, the subject animals in need of treatment and / or prevention of diseases in which mast cells exacerbate pathological condition are mammals such as human, pet animals, livestock animals, sport animals, or experimental animals. The preferred subject animal is a human.

[0046] Production of antigens, production of antibodies, and pharmaceutical compositions, related to the present invention, will be explained below.

[0047] <Production of antigens used for antibody production> Proteins or fragments thereof used as sensitizing antigens for obtaining anti-MCEMP1 antibody used in the present invention are not limited with respect to the animal species from which they are derived, including, for example, humans, canines, felines, mice, bovines, horses, rats, and chickens. However, sensitizing antigens are preferably selected so as to have high sequence identity with the antigen expressed by the target cells. Mammal-derived proteins are generally preferable and human-derived proteins are particularly preferable. For instance, if the targeted MCEMP1 protein is human MCEMP1 protein, it is preferable to use human MCEMP1 protein, a fragment thereof, or cells expressing human MCEMP1.

[0048] Nucleotide sequences encoding human MCEMP1 protein and homologs thereof, and amino acid sequences thereof, can be obtained by, for example, accessing the website of GenBank (NCBI, USA) and using an algorithm such as BLAST or FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877,1993; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997).

[0049] According to the present invention, when the amino acid sequence (SEQ ID NO: 2) of full length human MCEMP1 protein is used as a reference sequence, targets are proteins consisting of a sequence having 70% to 100%, preferably 80% to 100%, more preferably 90% to 100%, and further preferably 95% to 100% (e.g., 97% to 100%, 98% to 100%, 99% to 100%, or 99.5% to 100%) sequence identity with the amino acid sequence. The term "sequence identity" as used herein means the ratio of identical amino acids (or nucleotides) to the total number of amino acids (or nucleotides) in the case that two sequences are aligned such that maximum similarity can be achieved with or without introduction of gaps, and is expressed as a percentage (%).

[0050] Fragments of MCEMP1 protein have lengths ranging from the amino acid length of an epitope (or an antigenic determinant), which is the smallest unit of an antigen recognized by an antibody, to less than the full-length of the protein. The epitope refers to a polypeptide fragment having antigenicity or immunogenicity in mammals, preferably in humans. The smallest unit of the epitope consists of approximately 7 to 12 amino acids, and for example, 8 to 11 amino acids. A specific example thereof is a polypeptide consisting of an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more sequence identity with the amino acid sequence of MCEMP1 protein.

[0051] Polypeptides comprising the aforementioned MCEMP1 protein and fragment thereof can be synthesized according to chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method) (the Japanese Biochemical Society (ed.), "Biochemical Experimentation Course (Seikagaku Jikken Koza) 1," Protein Chemistry IV, Chemical Modification and Peptide Synthesis, Kagaku-dojin Publishing Company, Inc. (Japan), 1981). Also, they can be synthesized by general methods using a variety of commercially available peptide synthesizers. In addition, polypeptides of interest can be obtained by preparing DNA encoding the above polypeptides, incorporating each of the DNA into an expression vector and introducing the vector into a host cell, thereby allowing the host cell to produce the polypeptide, using known gene engineering methods (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, etc.).

[0052] DNA encoding the aforementioned polypeptides can be readily prepared by known gene engineering techniques or general methods using commercially available nucleic acid synthesizers. For example, DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 can be prepared by PCR using a human chromosome DNA or cDNA library as a template and a pair of primers designed to enable the amplification of the nucleotide sequence shown in SEQ ID NO: 1. PCR conditions can be appropriately determined. For example, such conditions may comprise conducting 30 cycles of the reaction steps (as one cycle) consisting of: 94°C, 30 seconds (denaturation); 55°C, 30 seconds to 1 minute (annealing); and 72°C, 1 minute (elongation) using a thermostable DNA polymerase (e.g., Taq polymerase) and a Mg2+-containing PCR buffer, followed by reaction at 72°C for 7 minutes after completion of the 30 cycles. However, PCR conditions are not limited to the above-exemplified PCR conditions. PCR techniques and conditions are described in, for example, Ausubel et al., Short Protocols in Molecular Biology, 3rd edition, A Compendium of Methods from Current Protocols in Molecular Biology (1995), John Wiley & Sons (Chapter 15, in particular).

[0053] In addition, desired DNA can be isolated by preparing appropriate probes and primers based on information about the nucleotide and amino acid sequences shown in SEQ ID NOS: 1 to 8 in the Sequence Listing of the present application, and screening a cDNA library of e.g. human with the use of such probes and primers. Preferably, such cDNA library is produced from a cell, organ, or tissue in which the protein represented by SEQ ID NO: 2, 4, 6 or 8 is expressed. Examples of the cell or tissue include, but not limited to, cells or tissues from normal tissues or disease sites, such as bone marrow, peripheral blood mononuclear cell (PBMC), mast cells, mast cell expressing tissues, pathology expressing tissues. Operations such as preparation of probes or primers, construction of cDNA libraries, screening of cDNA libraries, and cloning of genes of interest, as described above, are known to persons skilled in the art, and they can be carried out according to, for example, the methods described in Sambrook et al., Molecular Cloning, the 2nd edition, Current Protocols in Molecular Biology (1989) and Ausbel et al. (ibid.). DNAs encoding human MCEMP1 protein and fragment thereof can be obtained from the thus obtained DNAs.

[0054] The above-described host cells may be any cells, as long as they can express the abovedescribed polypeptides. An example of prokaryotic host cell includes, but is not limited to, Escherichia coli. Examples of eukaryotic host cells include, but are not limited to, mammalian cells such as monkey kidney cell (COS1), Chinese hamster ovary cell (CHO), human embryonic kidney cell line (HEK293), and mouse embryonic skin cell line (NIH3T3), yeast cells such as budding yeast and fission yeast cells, silkworm cells, and Xenopus laevis egg cells.

[0055] When prokaryotic cells are used as host cells, an expression vector preferably having an origin replicable in prokaryotic cells, a promoter, a ribosome-binding site, a multicloning site, a terminator, a drug resistance gene, an auxotrophic complementation gene, a reporter gene, or the like can be used. As expression vectors for Escherichia coli, pUC vectors, pBluescriptII, pET expression systems, pGEX expression systems, and the like can be exemplified. A DNA encoding the above polypeptide is incorporated into such an expression vector, a prokaryotic host cell is transformed with the vector, and then the thus obtained transformed cell is cultured, so that the polypeptide encoded by the DNA can be expressed in the prokaryotic host cell. When constructing the expression vector, the polypeptide can also be expressed as a fusion protein by designing fusion of the polypeptides and various tag proteins and / or another protein.

[0056] When eukaryotic cells are used as host cells, expression vectors for eukaryotic cells preferably having a promoter, a splicing region, a poly(A) addition site, or the like can be used. 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. By similar procedures to those mentioned above, a DNA encoding the aforementioned polypeptide is incorporated into such an expression vector, a eukaryotic host cell is transformed with the vector, and then the thus obtained transformed cell is cultured, so that the polypeptide encoded by the above DNA can be expressed in the eukaryotic host cell. When constructing the expression vector, in cases where the polypeptide is engineered by adding various tags and / or other protein, or when pIND / V5-His, pFLAG-CMV-2, pEGFP-N1, pEGFP-C1, or the like is used as an expression vector, the above polypeptide may be expressed as a fusion protein with a tag, such as His tag (e.g., (His)6 to (His) 10), FLAG tag, myc tag, HA tag, or GFP, or with another protein.

[0057] For introduction of an expression vector into a host cell, well known methods can be employed, such as heat shock method, electroporation, a calcium phosphate method, a liposome method, a DEAE dextran method, microinjection, viral infection, lipofection, and binding with a cell-membrane-permeable peptide.

[0058] Isolation and purification of a polypeptide of interest from host cells can be performed using known isolation techniques in combination. Examples of isolation and purification techniques include, but are not limited to, treatment using a denaturing agent such as urea or a surfactant, ultrasonication, enzymatic digestion, salting-out, solvent fractionation and precipitation, dialysis, centrifugation, ultrafiltration, gel filtration chromatography, SDS-PAGE, isoelectric focusing electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

[0059] <Structure of antibody> In general, antibodies are heteromultimeric glycoproteins comprising at least two heavy chains and two light chains. Representative class of antibodies are IgG which is heterotetrameric glycoproteins (approximately 150 kDa) composed of two identical light (L) chains and two identical heavy (H) chains. Typically, light chain is connected to a heavy chain via a single covalent disulfide bond. However, the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Moreover, heavy chain and light chain also have an intrachain disulfide bond(s). Heavy chain has a variable region (VH region) at N-terminus, and constant regions at the opposite C-terminus. Likewise, light chain has a variable region (VL region) at N-terminus, and a constant region at the opposite C-terminus. The domain composition of each region differs as follows: the variable region of the heavy chain, the variable region of the light chain, and the constant region of the light chain each are only one, while the constant region of the heavy chain is multiple. The constant region of a light chain is aligned with the first constant domain of a heavy chain and the light-chain variable domain is aligned with the heavy-chain variable domain. The variable region of the antibody contains regions called "complementarity determining region (CDR),". The regions function as a antigen binding site by exhibiting high variability. A relatively conserved portion, compared to CDR, in a variable region is called a "framework region (FR)." A complete heavy-chain and light-chain variable region each comprises 4 FRs connected to each other via 3 CDRs. Such CDRs are called "CDRH1," "CDRH2," and "CDRH3," respectively, in such order from the N-terminus in a heavy chain. Similarly, for a light chain, they are called "CDRL1," "CDRL2," and "CDRL3," respectively. CDRH3 plays the most important role in terms of antibody-antigen binding specificity. In addition, CDRs in each chain are retained by FR in the state that they are close to each other, and they contribute to the formation of antigen binding sites of an antibody together with CDRs in the other chain. Constant regions do not directly contribute to antibody-antigen binding. However, they contribute various effector functions such as antibody-dependent cellular cytotoxicity (ADCC activity) and phagocytosis through binding to an Fcy receptor, half-life / clearance rate through binding to a neonatal Fc receptor (FcRn), and complement-dependent cytotoxicity (CDC activity) through binding to a C1q component in the complement cascade.

[0060] <Antibody production> The term "anti-MCEMP1 antibody" used in the present invention refers to an antibody having an immunological reactivity with a full-length MCEMP1 protein or a fragment thereof described above.

[0061] The term "immunological reactivity" used herein indicates the characteristics of an antibody binding to an MCEMP1 antigen in vivo or in vitro. The mast cell-damaging function (e.g., death, or growth inhibition) and / or function to deliver a therapeutic agent conjugated to anti-MCEMP1 antibody to mast cells can be expressed as a result of such binding. Specifically, any type of antibody may be used in the present invention as long as the antibody can bind to MCEMP1 protein in order to damage a mast cell which exacerbates the pathology, or to deliver a therapeutic agent to the mast cell.

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

[0063] Antibodies may be further modified via acetylation, formylation, amidation, phosphorylation, or pegylation (PEG), in addition to glycosylation.

[0064] Production examples for a variety of antibodies are described below.

[0065] The polyclonal antibodies that can be used in the present invention can be obtained in a manner described below.

[0066] Small animals such as mice, human antibody-producing mice, or rabbits are immunized with a naturally occurring MCEMP1 protein, a recombinant MCEMP1 protein that has been expressed as a protein fused with GST or the like in a microorganism such as Escherichia coli, or fragment thereof, or cells expressing MCEMP1 on their cell surface (for example, mast cell line LUVA), thereby immune cells capable of producing anti-MCEMP1 antibody are induced , and then, serum comprising anti-MCEMP1 antibody produced by the immune cells is obtained. The serum is purified via ammonium sulfate precipitation, protein A / protein G column chromatography, DEAE ionexchange chromatography, affinity column chromatography with a column to whichMCEMP1 protein or a synthetic peptide is coupled, or the like for preparation of polyclonal antibodies. In the Examples described below, a rabbit polyclonal antibody against a region in the amino acid sequence of MCEMP1 protein that is expressed on mast cell surfaces was produced, and pathology improving effects thereof were confirmed.

[0067] Another example of an antibody usable in the present invention is a monoclonal antibody. Methods for obtaining a monoclonal antibody include, for example, similar to obtaining a polyclonal antibody, a method of immunizing a small animal with a sensitizing antigen to induce immune cells that produce anti-MCEMP1 antibody, and establishing a fusion cell (hybridoma) from the animal, and a method of isolating the immune cells from the animal and cloning the antibody gene. In the method of establishing a hybridoma, the spleen is extracted from the animal, cells are separated, and these cells are fused with myeloma cells. From the resulting fusion cell (hybridoma), a clone that produces an antibody having the activity to inhibit mast cell proliferation is selected. A hybridoma which produces the monoclonal antibody with the activity to inhibit mast cell proliferation is isolated, cultured, and the antibody can be purified from the culture supernatant using a general affinity purification method.

[0068] Also, a monoclonal antibody-producing hybridoma can be produced in a manner described below, for example. First, an animal is immunized with a sensitizing antigen by a known method. In a general method, immunization is carried out by intraperitoneally or subcutaneously injecting a sensitizing antigen into a mammal. Specifically, a sensitizing antigen is diluted with or suspended in PBS (Phosphate-Buffered Saline), physiological saline, or the like to an appropriate resultant amount. If desired, an appropriate amount of a conventional adjuvant (e.g., Complete Freund's adjuvant) is mixed therewith. After emulsification takes place, the resultant is administered to a mammal several times every 4 to 21 days. In addition, an adequate carrier can be used for immunization with a sensitizing antigen.

[0069] As described above, after immunization of a mammal and confirmation of an increase to a desired antibody level in serum, immunocytes are collected from the mammal and subjected to cell fusion. Particularly preferable examples of immunocytes are splenocytes.

[0070] Mammalian myeloma cells are used as relevant parent cells subjected to fusion with the above immunocytes. For such myeloma cells, the following various examples of known cell lines are preferably used: P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 15481550), 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. D. H. et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), and R210 (Galfre, G. et al., Nature (1979) 277, 131-133).

[0071] Basically, cell fusion of immunocytes and myeloma cells described above can be carried out according to a known method such as the method of Kohler and Milstein et al. (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0072] More specifically, cell fusion described above is carried out, for example, in the presence of a cell fusion promoter in a conventional nutrients-containing culture solution. Examples of a fusion promoter to be used include polyethylene glycol (PEG) and Sendai virus (HVJ). If desired, an adjuvant such as dimethylsulfoxide may be further added for improvement of fusion efficiency.

[0073] The proportion of immunocytes used to that of myeloma cells used can be arbitrarily determined. For example, the ratio of immunocytes to myeloma cells is preferably 1:1 to 10:1. Examples of a culture solution that can be used for cell fusion described above include an RPMI1640 culture solution and an MEM culture solution adequate for growth of the above myeloma cell lines as well as other conventional culture solutions used for this kind of cell culture. Further, a serum replacement such as fetal calf serum (FBS) can be used in combination therewith.

[0074] For cell fusion, the above immunocytes and myeloma cells are sufficiently mixed at predetermined amounts in the culture solution. A PEG solution (e.g., average molecular weight: approximately 1000 to 6000) that has been previously heated to approximately 37°C is added thereto at a concentration of generally 30% to 60% (w / v), followed by mixing. This results in formation of hybridomas of interest. Subsequently, sequential addition of an appropriate culture solution and removal of the supernatant via centrifugation are repeatedly carried out to remove cell fusion agent(s) and the like that are not preferable for the growth of hybridomas.

[0075] The thus obtained hybridomas are cultured in a conventional selection culture solution such as an HAT culture solution (a culture solution comprising hypoxanthine, aminopterin, and thymidine) for selection. Culture in such an HAT culture solution is continuously carried out for a sufficient time period (generally several days to several weeks) for death of cells (non-fused cells) other than hybridomas of interest. Next, a conventional limiting dilution method is employed to screen for hybridomas producing antibodies of interest and to carry out single cloning.

[0076] In a method for isolating immune cells from antigen-sensitized animals and cloning antibody genes, B cells are identified from peripheral blood or lymphoid tissue and isolated as single cells. The cDNA of the antibody gene is prepared by RT-PCR using an antibody-specific primer and amplified by PCR. The obtained DNA is inserted into an expression vector, expressed by 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 used for cloning of the antibody of interest.

[0077] Further, as well as obtaining the above hybridomas via immunization of non-human animals with antigens, it is also possible to obtain hybridomas that produce human antibodies having a desired activity (e.g., cell growth inhibition activity) by sensitizing human lymphocytes (e.g., 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 myeloma cells having the ability to permanently divide (e.g., U266) (accession no. TIB196).

[0078] Monoclonal antibody-producing hybridomas produced as above can be passaged in a conventional culture solution. In addition, they can be preserved in liquid nitrogen for a long period of time.

[0079] Specifically, immunization is carried out using a desired antigen or cells expressing a desired antigen as sensitizing antigen(s) according to a conventional immunization method. The obtained immunocytes are fused with known parent cells by a conventional cell fusion method. Then, monoclonal antibody-producing cells (hybridomas) are screened for by a conventional screening method. Thus, antibody production can be carried out.

[0080] A known human antibody-producing mouse used herein is, for example, a KM Mouse (Kirin Pharma / Medarex) or a XenoMouse (Amgen) (e.g., WO02 / 43478 and WO02 / 092812). When such mice are immunized with MCEMP1 proteins or fragments thereof, complete human polyclonal antibodies can be obtained from blood. In addition, complete human monoclonal antibodies can be produced by a method of fusing splenocytes collected from immunized mice with myeloma cells.

[0081] Antigen preparation can be carried out in accordance with a method such as a method using animal cells (JP Patent Publication No. 2007-530068) or a method using a baculovirus (e.g., WO98 / 46777). If the immunogenicity of an antigen is low, an antigen is bound to a macromolecule having immunogenicity, such as albumin, and then, the antigen can be used for immunization.

[0082] Further, it is possible to use a gene recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating the gene into an adequate vector, introducing the vector into a host, and using a genetic engineering technique. (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 of a variable region (V region) of an antibody is synthesized from mRNA of a hybridoma with the use of a reverse transcriptase. After DNA encoding a V region of an antibody of interest is obtained, such DNA is ligated to desired DNA encoding an antibody constant region (C region). The resultant is incorporated into an expression vector. Alternatively, DNA encoding an antibody V region may be incorporated into an expression vector comprising DNA of an antibody C region. Such DNA is incorporated into an expression vector so that it is expressed under control of an expression control region such as an enhancer or a promoter. Next, host cells are transformed with such expression vector, thereby allowing the antibody to be expressed.

[0083] Monoclonal antibodies include human monoclonal antibodies and non-human animal monoclonal antibodies (e.g., mouse monoclonal antibodies, rat monoclonal antibodies, rabbit monoclonal antibodies, and chicken monoclonal antibodies). Monoclonal antibodies can be produced by culturing hybridomas obtained via fusion of myeloma cells and splenocytes from non-human mammals (e.g., mice, human antibody-producing mice, or rabbits) immunized with MCEMP1 proteins or fragments thereof.

[0084] A chimeric antibody is an antibody produced by combining sequences from different animals. An example thereof is an antibody consisting of mouse antibody heavy-chain and lightchain variable regions and human antibody heavy-chain and light-chain constant regions. Such a chimeric antibody can be produced by a known method. For example, a chimeric antibody can be obtained by ligating DNA encoding a non-human antibody V region to DNA encoding a human antibody C region, incorporating the resultant into an expression vector, introducing the vector into a host, and allowing the host to produce an antibody.

[0085] Polyclonal antibodies include antibodies obtained by immunizing human antibodyproducing animals (e.g., mice or rabbits) with MCEMP1 proteins or fragments thereof. Polyclonal antibodies comprise antibodies produced from multiple types of antibody-producing cells. An antibody produced from one type of antibody-producing cell isolated therefrom corresponds to monoclonal antibody.

[0086] A humanized antibody is an engineered antibody, and it is sometimes referred to as a "reshaped human antibody." A humanized antibody is constructed by replacing complementarity determining regions (CDRs) of a human antibody with CDRs of an immunized animal-derived antibody. Also, general genetic engineering techniques for this purpose are known.

[0087] Specifically, a DNA sequence designed to ligate mouse antibody CDRs or rabbit antibody CDRs to framework regions (FRs) of a human antibody is synthesized by PCR method using several oligonucleotides prepared to have portions overlapping each other at their ends. A humanized antibody can be obtained by ligating the above obtained DNA to DNA encoding a human antibody constant region, incorporating the resultant into an expression vector, introducing the vector into a host, and allowing the host to produce an antibody production (see EP239400A and WO96 / 02576). Human antibody FRs to be ligated to each other via CDRs are selected, provided that complementarity determining regions derived from immunized animals can form a good antigen binding site. If necessary, amino acids in framework regions of an antibody variable region may be substituted in such a manner that complementarity determining regions in a reshaped human antibody form an appropriate antigen binding site (Sato K. et al., Cancer Research 1993, 53: 851-856). In addition, the framework regions may be substituted with framework regions from various human antibodies (see WO99 / 51743).

[0088] After a chimeric antibody or a humanized antibody is produced, amino acids in a variable region (e.g., FR) or a constant region may be, for example, substituted with different amino acids.

[0089] Here, the amino acid substitution is a substitution of, for example, less than 15, less than 10, not more than 8, not more than 7, not more than 6, not more than 5, not more than 4, not more than 3, or not more than 2 amino acids, preferably 1 to 5 amino acids, and more preferably 1 or 2 amino acids. A substituted antibody should be functionally equivalent to an unsubstituted antibody. The substitution is preferably a conservative amino acid substitution, which is a substitution between amino acids having similar characteristics in terms of charge, side chains, polarity, aromaticity, and the like. For example, amino acids having similar characteristics can be classified into the following types: basic amino acids (arginine, lysine, and histidine); acidic amino acids (aspartic acid and glutamic acid); uncharged polar amino acids (glycine, asparagine, glutamine, serine, threonine, cysteine, and tyrosine); nonpolar amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, and methionine); branched-chain amino acids (threonine, valine, isoleucine); and aromatic amino acids (phenylalanine, tyrosine, tryptophan, and histidine).

[0090] Antibodies of the present invention may be modified antibodies. An example of a modified antibody is an antibody bound to a molecule such as polyethylene glycol (PEG). Regarding modified antibodies of the present invention, substances that bind to an antibody are not limited. Such a modified antibody can be obtained by chemically modifying an obtained antibody. A method of such modification has been already established in the field related to the present invention.

[0091] The expression "functionally equivalent" used herein indicates a situation in which an antibody of interest has biological or biochemical activity similar to that of an antibody of the present invention. Specifically, such antibody has a function of damaging mast cells, has a function of delivering therapeutic agents, or causes essentially no rejection reaction when applied to humans. An example of such activity is cell growth inhibition activity or binding activity.

[0092] A known method for preparing a polypeptide functionally equivalent to a given polypeptide that is well known to persons skilled in the art is a method comprising introducing a mutation into a polypeptide. For instance, a person skilled in the art can adequately introduce a mutation into an antibody of the present invention using a site-specific mutagenesis method (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, TA., (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492; or Kunkel (1988) Methods Enzymol. 85, 2763-2766) or the like. Thus, an antibody functionally equivalent to the antibody of the present invention can be prepared.

[0093] An aforementioned antibody capable of recognizing an epitope of MCEMP1 protein recognized by an anti-MCEMPl antibody can be obtained by a method known to persons skilled in the art. For example, it can be obtained by: a method comprising determining an epitope of MCEMP1 protein recognized by an anti-MCEMPl antibody by a general method (e.g., epitope mapping) and producing an antibody using a polypeptide having an amino acid sequence contained in the epitope as an immunogen; or a method comprising determining an epitope recognized by a produced antibody by a general method and selecting an antibody having an epitope identical to an epitope of an anti-MCEMPl antibody. Here, the term "epitope" refers to a polypeptide fragment having antigenicity or immunogenicity in mammals and preferably in humans. The smallest unit thereof consists of approximately 7 to 12 amino acids and preferably 8 to 11 amino acids.

[0094] The affinity constant Ka (association rate constant kon / dissociation rate constant koff) of an antibody of the present invention is preferably at least 107 M-1, at least 108 M-1, at least 5 x 108 M-1, at least 109 M-1, at least 5 x 109 M-1, at least 1010 M-1, at least 5 x 1010 M-1, at least 1011 M-1, at least 5 x 1011 M-1, at least 1012 M-1, or at least 1013 M-1.

[0095] An antibody of the present invention can be in a form of conjugate of antibody and therapeutic agent(s). Binding between an antibody and an therapeutic agent(s) can be carried out via a linker having a group reactive to an amino group, a carboxyl group, a hydroxy group, a thiol group, or the like (e.g., an imidyl succinate group, a formyl group, a 2-pyridyldithio group, a maleimidyl group, an alkoxycarbonyl group, or a hydroxy group).

[0096] A linker refers to a substance capable of binding an antibody and a therapeutic agent. The linker may be either cleavable or non-cleavable linker. A cleavable linker is characterized by comprising a substance that can be cleaved by enzymes, pH, reduction action, or radiation irradiated from outside the body. Preferably, it is designed so that the therapeutic agent does not act on normal cells because the linker is not cleaved in normal sites, but cleaved specifically at disease sites, so that the therapeutic agent acts on mast cells that exacerbate the diseases. Furthermore, the linker may have the effect of improving the solubility and / or stability of the conjugate of antibody and therapeutic agent.

[0097] Examples of therapeutic agent used to compose the conjugate include, but are not limited to, a cytotoxic agent, an immunomodulatory agent, and a radioisotope. When the therapeutic agent is a radioisotope, it is desirable that the radioisotope is effective not only for treating the diseases but also for diagnosing the diseases.

[0098] An antibody of the present invention is preferably an antibody having an immunological reactivity with MCEMP1 protein or an antibody capable of specifically recognizing MCEMP1 protein. Such an antibody is preferably an antibody having a structure that allows a subject animal to which the antibody is administered to almost completely or completely avoid a rejection reaction. If the subject animal is a human, examples of such antibodies include human antibodies, humanized antibodies, chimeric antibodies (e.g., human-rabbit chimeric antibodies), single-chain antibodies, and bispecific antibodies. Such an antibody is a recombinant antibody having human antibody-derived heavy-chain and light-chain variable regions, a recombinant antibody having heavy-chain and light-chain variable regions each consisting of non-human animal antibody- derived complementarity determining regions (CDR1, CDR2, and CDR3) and human antibody-derived framework regions, or a recombinant antibody having non-human animal antibody-derived heavy-chain and light-chain variable regions and human antibody-derived heavy-chain and light-chain constant regions. The first two antibodies are preferable.

[0099] The above recombinant antibody can be produced in the manner described below. DNA encoding a monoclonal antibody against MCEMP1 protein (e.g., a human monoclonal antibody, a mouse monoclonal antibody, a rat monoclonal antibody, a rabbit monoclonal antibody, or a chicken monoclonal antibody) is cloned from an antibody-producing cell such as a hybridoma. DNAs encoding a light-chain variable region and a heavy-chain variable region of the antibody are produced by an RT-PCR method or the like using the obtained clone as a template. Then, the sequences of a light-chain variable region and a heavy-chain variable region or the sequences of CDR1, CDR2, and CDR3 are determined by 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] Further, such DNAs encoding variable regions or DNAs encoding CDRs are produced by a genetic engineering technique (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)) or a DNA synthesizer. Here, the above human monoclonal antibody-producing hybridoma can be produced by immunizing a human antibodyproducing animal (e.g., a mouse or rabbit) with MCEMP1 protein and fusing splenocytes from the spleen removed from the animal with myeloma cells. In addition to the above, if necessary, DNAs encoding human antibody-derived light-chain or heavy-chain variable regions and constant regions are produced by a genetic engineering technique or a DNA synthesizer.

[0101] In the case of a humanized antibody, DNA in which the CDR coding sequences in a DNA encoding a human antibody-derived light-chain or heavy-chain variable region have been substituted with corresponding CDR coding sequences of an antibody from a non-human animal (e.g., a mouse, a rabbit, a rat, or a chicken) is produced. The DNA obtained as above is ligated to the DNA encoding a constant region of a human antibody-derived light chain or heavy chain. Thus, DNA encoding a humanized antibody can be produced.

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

[0103] A single-chain antibody is an antibody in which a heavy-chain variable region and a light-chain variable region are linearly ligated to each other via a linker. DNA encoding a singlechain antibody can be produced by ligating DNA encoding a heavy-chain variable region, DNA encoding a linker, and a DNA encoding a light-chain variable region together. Here, a heavychain variable region and a light-chain variable region are those from a human antibody or those from a human antibody in which CDRs alone have been substituted with CDRs of an antibody from a non-human animal (e.g., a mouse, a rabbit, a rat, or a chicken). In addition, the linker consists of 12 to 19 amino acids. An example thereof is (G4S)3 consisting of 15 amino acids (G. -B. Kim et al., Protein Engineering Design and Selection 2007, 20 (9): 425-432).

[0104] A bispecific antibody is an antibody capable of specifically binding to two different epitopes. DNA encoding a bispecific antibody (herein also referred to as diabody) can be produced by, for example, ligating 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 together in such order (provided that DNA encoding a light-chain variable region B and DNA encoding a heavy-chain variable region B are ligated to each other via DNA encoding a linker described above). Here, both a heavy-chain variable region and a light-chain variable region are those from a human antibody or those from a human antibody in which CDRs alone have been substituted with CDRs of an antibody from a non-human animal (e.g., a mouse, a rabbit, a rat, or a chicken).

[0105] Recombinant DNA produced as above is incorporated into one or a plurality of appropriate vector(s). Each such vector is introduced into a host cell (e.g., a mammal cell, a yeast cell, or an insect cell) for (co)expression. Thus, a recombinant antibody can be produced. See, 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 antibodies preferably have cytotoxic activity.

[0107] In addition, a hybridoma capable of producing a different human antibody or a nonhuman animal antibody (e.g., a rabbit antibody) against MCEMP1 protein is produced. A monoclonal antibody produced by the hybridoma is collected. Then, the antibody is determined whether or not the obtained antibody is an antibody of interest using, as indicators, immunological binding activity to MCEMP1 protein and cytotoxic activity. Thus, a monoclonal antibodyproducing hybridoma of interest is identified. Thereafter, as described above, DNAs encoding heavy-chain and light-chain variable regions of an antibody of interest are produced from the hybridoma and sequenced. The DNAs are used for production of different antibodies.

[0108] Further, the above antibody of the present invention may be any antibodies having a substitution, deletion, or addition of one or several (and preferably, 1 or 2) amino acid(s), particularly in a framework region sequence and / or a constant region sequence, as long as it has the specific property of specifically recognizing MCEMP1. Here, the term "several amino acids" indicates 2 to 5 and preferably 2 or 3 amino acids. Complementarity determining regions (CDRs) encoded by DNAs of the above sequences are regions that determine antibody specificity. Therefore, sequences encoding the other regions (i.e., constant regions and framework regions) in an antibody may be sequences from a different antibody. Here, different antibodies include antibodies from non-human organisms. However, in view of reduction of side effects, human-derived antibodies are preferable. That is to say, in the above case, DNA regions encoding framework regions and constant regions of heavy and light chains preferably comprise nucleotide sequences encoding the relevant amino acid sequences from a human antibody.

[0109] DNA of the present invention can be obtained by, for example, the aforementioned methods or the following methods. First, total RNA is prepared from a hybridoma for an antibody of the present invention using a commercially available RNA extraction kit. Then, cDNA is synthesized with a reverse transcriptase using random primers and the like. Next, cDNA encoding an antibody is amplified by a PCR method using, as primers, oligonucleotides having sequences conserved in variable regions of known rabbit antibody heavy-chain and light-chain genes. Sequences encoding constant regions can be obtained by amplifying known sequences by a PCR method. The nucleotide sequence of the DNA can be determined by a general method involving, for example, incorporation into a plasmid or phage for sequence determination.

[0110] It is thought that pathology improving effects of an anti-MCEMP1 antibody used in the present invention upon MCEMP1-expressing mast cells are exhibited through antibodydependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, or pharmacological action of the therapeutic agent conjugated to an anti-MCEMP1 antibody.

[0111] Therefore, the activity of the anti-MCEMP1 antibody used in the present invention can be evaluated by measuring the ADCC activity or CDC activity, or the action of the therapeutic agent conjugated to the anti-MCEMP1 antibody, against mast cells that express MCEMP1 protein on their cell surface in vitro, as specifically shown in the following examples.

[0112] The anti-MCEMP1 antibody used in the present invention binds to MCEMP1 protein expressed on the cell surface of mast cells, and through the above mentioned activity, it exhibits an action of inhibiting proliferation or the action of a therapeutic agent conjugated to the anti-MCEMP1 antibody. Therefore, it is considered useful for treating or preventing diseases in which mast cells exacerbate pathological condition. In other words, the present invention provides a pharmaceutical composition for treating and / or preventing diseases in which mast cells exacerbate pathological condition, which comprises, as an active ingredient, anti-MCEMP1 antibody. When using anti-MCEMP1 antibody for the purpose of administering it to the human body (antibody treatment), it is preferable to use a human antibody or a humanized antibody to reduce its immunogenicity.

[0113] Furthermore, the higher the binding affinity between the anti-MCEMP1 antibody and MCEMP1 protein on the surface of mast cells, the stronger the therapeutic effect of the anti-MCEMP1 antibody can be expected. Therefore, if an anti-MCEMP1 antibody with higher binding affinity to MCEMP1 protein can be obtained, a stronger pathology-improving effect can be expected. Thus, the antibody will be possible to apply as a pharmaceutical composition for the treatment and / or prevention of diseases in which mast cells exacerbate pathological condition. As mentioned above, a higher binding affinity is preferably defined as a binding constant (affinity constant) Ka (association rate constant kon / dissociation rate constant koff) at least 107 M-1 Preferably, at least 108 M-1, at least 5 X 108 M-1, at least 109 M-1, at least 5 X 109 M-1, at least 1010 M-1, at least 5 X 1010 M-1, at least 1011 M-1, at least 5 X 1011 M-1, at least 1012 M-1, or at least 1013 M-1.

[0114] <Binding to Antigen Expression Cells> The capacity of an antibody to bind to MCEMP1 can be determined via binding assay using, for example, ELISA, a Western blot method, immunofluorescence, or flow cytometry analysis as described in the Examples.

[0115] <Immunohistochemical staining> An antibody that recognizes MCEMP1 protein can be tested in terms of reactivity with MCEMP1 protein by an immunohistochemical method well-known to persons skilled in the art using a frozen tissue section fixed with paraformaldehyde or acetone or a paraffin-embedded tissue section fixed with paraformaldehyde. Such section is prepared from a tissue obtained from a patient during surgery, a bone marrow tissue, lymph node, or peripheral blood cells of a patient, or a tissue obtained from an animal carrying xenograft tissue that has been inoculated with a cell line that expresses MCEMP1 naturally or after transfection thereof.

[0116] For immunohistochemical staining, an antibody immunologically reactive to MCEMP1 protein can be stained by a variety of methods. For example, it can be visualized by reacting with a horseradish peroxidase (HRP) labelled goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody.

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

[0118] In the present invention, the target of the pharmaceutical composition for treating and / or preventing diseases in which mast cells exacerbate pathological condition is not particularly limited as long as the target is mast cells expressing MCEMP1 protein on their cell surface.

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

[0120] Examples of respiratory diseases include asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, interstitial lung disease, and allergic rhinitis.

[0121] Examples of autoimmune diseases are preferably collagen diseases. Examples further include rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma, Sjogren's syndrome, polymyositis, dermatomyositis, and giant cell arteritis.

[0122] Examples of systemic allergic diseases include anaphylaxis, drug hypersensitivity, food allergy, and poison allergy.

[0123] Examples of skin diseases include urticaria, dermatitis, psoriasis, chronic pruritus, and nodular prurigo.

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

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

[0126] Examples of metabolic diseases include diabetes.

[0127] Examples of female reproductive system diseases include endometriosis.

[0128] Examples of pregnancy, childbirth and postpartum diseases include pregnancy-induced hypertensive nephropathy.

[0129] In addition, the subject animal of the present invention is a mammal. Examples thereof include mammals such as primates, pet animals, livestock animals, sport animals, and experimental animals. Humans, dogs, and cats are particularly preferable.

[0130] When an antibody used in the present invention is used as a pharmaceutical composition, it can be formulated by a method known to persons skilled in the art. For instance, it can be parenterally used in the form of a parenteral injection of: an aseptic solution comprising water or a pharmacologically acceptable non-water solution; or a suspension liquid. For example, in one possible case, it can be formulated with the combined use of a pharmacologically acceptable carrier or medium or additive and specifically sterilized water, physiological saline, plant oil, an emulsifier, a suspension, a surfactant, a stabilizer, a flavoring agent, an excipient, a vehicle, a preservative, or a binder in an appropriate manner by mixing in a unit dosage form required for a generally acceptable pharmaceutical formulation. The amount of an active ingredient in a formulation is determined such that an appropriate dosage within the indicated range can be achieved.

[0131] An aseptic composition for injection purposes can be formulated in accordance with general formulation practice using a vehicle such as distilled water for injection purposes.

[0132] Examples of an aqueous solution for injection purposes include physiological saline and isotonic solutions comprising glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. The aqueous solution may be used with an appropriate dissolution aid. Examples of such dissolution aid include alcohols (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80(TM) and HCO-60).

[0133] Examples of oily liquid include sesame oil and soybean oil. Such oily liquid may be used in combination with a dissolution aid such as benzyl benzoate or benzyl alcohol. In addition, it may be mixed with a buffering agent such as a phosphate buffer solution, a sodium acetate buffer solution, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, or an antioxidant. In general, a formulated injection solution is introduced into an adequate ampule.

[0134] The above pharmaceutical composition is orally or parenterally administered. Preferably, it is parenterally administered. Specific examples of dosage forms include injectable dosage form, intranasally-administered dosage form, transpulmonarily-administered dosage form, and percutaneously-administered dosage form. For example, injectable dosage form can be systemically or locally administered via intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. Alternatively, an antibody of the present invention may be administered directly to a disease site by local administration, such as injection, infusion, or implantation of a sustained-release formulation, to the disease site.

[0135] In addition, the administration method can be appropriately determined depending on patient age, weight, gender, and symptoms. A single dose of a pharmaceutical composition comprising an antibody or a DNA encoding an antibody can be selected within a range of, for example, 0.0001 mg to 1000 mg per kg of body weight. Alternatively, the dose can be selected within a range of, for example, 0.001 to 100000 mg per patient's body; however, it is not necessarily limited thereto. The dose and the administration method are changed depending on patient age, weight, gender, and symptoms. However, persons skilled in the art can appropriately select the dose and the method.

[0136] By administering the antibody or antigen-binding fragment thereof of the present invention, or above-mentioned pharmaceutical composition comprising the same to the subject, the above-mentioned diseases, particularly for example, diseases in which mast cells expressing 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 system diseases, cancers that do not express MCEMP1 protein on the cell surface, metabolic diseases, female reproductive system diseases, pregnancy, childbirth and postpartum diseases, sepsis, and graft-versus-host disease, can be treated and / or prevented.

[0137] Furthermore, the present invention encompasses a method for treating and / or preventing diseases in which mast cells exacerbate pathological condition, comprising administering the pharmaceutical composition (or medicine) of the present invention in combination with a therapeutic agent(s) or a pharmaceutical composition (or medicine) comprising a therapeutic agent(s) as exemplified above to the subject. The subject diseases are the same as described above. The antibody or antigen-binding fragment thereof and the therapeutic agent in the present invention may be administered to the subject simultaneously or separately. When administered separately, any pharmaceutical composition may be administered first or afterward, and the administration interval, doses, administration route, and number of administrations may be appropriately selected by a specialist. When administered simultaneously, the present invention encompasses a pharmaceutical composition in a dosage form obtained by, for example, mixing the antibody or fragment thereof according to the present invention and the therapeutic agent in a pharmacologically acceptable carrier (or medium) for formulation. The description about prescription, formulation, administration routes, doses, diseases, etc. regarding pharmaceutical compositions and dosage forms containing antibodies of the present invention is applicable to all of the pharmaceutical compositions and dosage forms containing the therapeutic agents.

[0138] Therefore, the present invention provides a pharmaceutical combination for treating and / or preventing diseases in which mast cells exacerbate pathological condition, comprising the pharmaceutical composition of the present invention and a therapeutic agent as exemplified above. Further, the present invention provides a method for treating and / or preventing diseases in which mast cells exacerbate pathological condition, comprising administering the pharmaceutical combination. Furthermore, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a therapeutic agent(s) together with a pharmacologically acceptable carrier and / or additive, for the treatment and / or prevention of diseases in which mast cells exacerbate pathological condition. Examples

[0139] The present invention is hereafter described in greater detail with reference to the following examples, although the scope of the present invention is not limited thereto.

[0140] (Example 1) Production of human MCEMP1 protein (1) Construction of the expression vector of the extracellular region of human MCEMP1 protein. The DNA nucleotide sequence encoding the extracellular region of human MCEMP1 protein was cloned using the following method based on the gene set forth in SEQ ID NO: 1.

[0141] PCR was performed by adding 1 ng of human MCEMP1 / pcDNA3.1 produced in Example 1 of WO2017 / 170322, 0.4 gM of two primers comprising Ndel and XhoI restriction enzyme digestion sequences (described in SEQ ID NOS: 17 and 18), 0.2 mM dNTPs, and 1.25 U of PrimeSTAR HS DNA polymerase (Takara Bio) to a total volume of 50 gl with each reagent and buffer. PCR was then performed using a Thermal Cycler (BIO RAD) by repeating a cycle of 10 seconds at 98OC, 15 seconds at 55OC, and 30 seconds at 72OC for 30 cycles. The two primers described above amplify the region encoding the sequence set forth in SEQ ID NO: 10 comprising the amino acid sequence of the extracellular region of MCEMP1 protein within SEQ ID NO: 1. 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 products obtained from the above PCR reaction and pET-30a(+) (ThermoFisher Scientific) were respectively subjected to restriction enzyme by using NdeI (Takara Bio) and XhoI (Takara Bio), and then purified using the Wizard SV Gel and PCR CleanUp System (Promega). The purified amplified product and pET30a(+) were ligated by using Ligation High Ver. 2 (Toyobo). The resultant is an expression vector encoding the human MCEMP1 extracellular region / His fusion protein (hereinafter referred to as hMCEMP1ECD-His). The vector is herein referred to as pET-30a(+)-hMCEMP1ECD-His. Furthermore, sequence analysis using a DNA sequencer confirmed DNA nucleotide sequence encoding hMCEMP1ECD-His. The sequence represented in SEQ ID NO: 19 is the nucleotide sequence encoding hMCEMP1ECD-His, and the sequence represented by SEQ ID NO: 20 is the amino acid sequence of hMCEMP1ECD-His.

[0142] (2) Production of hMCEMP1ECD-His. Expression vector pET-30a(+)-hMCEMP1ECD-His was introduced into BL21 (DE3) (Novagen) according to the attached protocol and spread onto selective medium (LB plate containing Kanamycin 50 pg ml) and cultured. Colonies that grew on the selective medium were picked and inoculated onto LB medium containing 25 pg / ml Kanamycin and shaken overnight at 37OC as a pre-culture. The pre-cultured medium was added to LB medium containing 25 pg / 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 0.6-0.7, isopropyl-P-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce the hMCEMP1ECD-His expression, and the culture was incubated with shaking at 37OC for 3 hours.

[0143] The collected E. coli was suspended in a buffer of 50 mM Tris-HCl (pH 8.0) and 300 mM NaCl, and the E. coli was crushed by sonication. The supernatant of the crushed solution was subjected to Ni Sepharose 6 Fast Flow (GE Healthcare Biosciences), washed with a washing buffer (20 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole (pH 8.0)), and then hMCEMP1ECD-His was eluted with an elution buffer (20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole (pH 8.0)). The eluted fraction was collected and substituted with PBS by dialysis. The dialyzed sample was purified by a gel filtration column, HiLoad 16 / 60 Superdex 200 (GE Healthcare Biosciences), equilibrated with PBS, and the peak fraction was collected. After concentration using Amicon Ultra MWCO 3k (Merck Millipore), the sample was aseptically filtered through HT Tuffryn Acrodisc 0.22 pm (PALL), and filtered sample was used in the following experiments.

[0144] (Example 2) Production of polyclonal antibodies that bind to the extracellular region of MCEMP1 protein (1) Production of polyclonal antibodies against hMCEMP1ECD-His. To obtain antibodies that bind to the extracellular region of the human MCEMP1 protein, 1 mg of hMCEMP1ECD-His produced in Example 1 was used as an antigen. The first immunizing sample was mixed with complete Freund's adjuvant (CFA) solution, and subsequent immunizing samples were mixed with incomplete Freund's adjuvant (IFA) solution. The sample was administered subcutaneously to rabbits five times at three-week intervals. Blood was then collected to obtain antiserum comprising the polyclonal antibodies. Furthermore, this anti-serum was purified using a protein G carrier column (GE Healthcare Biosciences) and substituted with PBS to obtain an IgG-type polyclonal antibody against hMCEMP1ECD-His (hereinafter referred to as "anti-MCEMP1 polyclonal antibody").

[0145] (2) Analysis of immunological reactivity of anti-MCEMP1 polyclonal antibody to MCEMP1 protein. 100 pl of the hMCEMP1ECD-His protein solution (1 pg ml) produced in Example 1 was added to each well of MaxiSorp (ThermoFisher Scientific) and left to stand at 4°C for 18 hours. After removing the solution, a PBS-T solution containing 3% skim milk was added and left to stand at room temperature for 3 hours. After removing the solution, the polyclonal antibody obtained above was added and left to stand at room temperature for 2 hours. After washing each well three times with PBS-T, an HRP-labeled anti-rabbit IgG (H+L) antibody (Invitrogen) diluted 5000 times with PBS was added and left 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 left to stand for 15-30 minutes to allow for color development. After color development, 1 N sulfuric acid was added to stop the reaction, and the absorbance values at 450 nm and 595 nm were measured using a spectrophotometer. The color intensity was calculated by subtracting the absorbance value at 595 nm from the absorbance value at 450 nm. As a result, it was confirmed that the produced anti-MCEMP1 polyclonal antibody is a polyclonal antibody that exhibits immunological reactivity to MCEMP1 protein.

[0146] (3) Analysis of immunological reactivity of anti-MCEMP1 polyclonal antibody to MCEMP1 protein expressed on the cell surface. Next, we analyzed whether the anti-MCEMP1 polyclonal antibody produced in above (1) exhibits specific reactivity to MCEMP1 expressed on the cell surface. 106 cells of the CHO-human MCEMP1 cells and CHO-emp cells produced in Example 3 of WO2017 / 170322 were centrifuged, and 0.1 pg (5 pl) of anti-MCEMPl polyclonal antibody was added respectively. Then, the cells were suspended in 95 pl of PBS containing 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing by PBS containing 3% fetal bovine serum (FBS), the cells were suspended in PBS containing 0.2 ul of R-Phycoerythrin-labeled goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 ul of 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing by PBS containing 3% fetal bovine serum (FBS), fluorescence intensity was measured using FACSVerse (Becton Dickinson). On the other hand, a similar procedure to the above was performed using a purified antibody derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries) that had not been immunized for the specific antigen, instead of the polyclonal antibody against MCEMP1, and this was used as a control. As a result, it confirmed that the anti-MCEMP1 polyclonal antibody showed higher fluorescence intensity in CHO-human MCEMP1 cells compared to the control and did not react to CHO-emp cells, meaning that the anti-MCEMP1 polyclonal antibody is a polyclonal antibody that specifically reacts to MCEMP1 protein expressed on the cell surface.

[0147] (4) Analysis of MCEMP1 protein expression on the surface of mast cells. Next, we analyzed whether human MCEMP1 protein is expressed on the cell surface of the human mast cell line LUVA (Kerafast). First, 106 cells of the above cell line were centrifuged, and FcR blocking reagent, Human (Miltenyi Biotec) was reacted according to the attached protocol to inhibit nonspecific binding to the Fc receptor. 0.1 ug (5 ul) of the anti-MCEMP1 polyclonal antibody prepared in above (1) was added, and then cells were suspended in 95 ul of PBS containing 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing by PBS containing 3% fetal bovine serum (FBS), cells were suspended in PBS containing 0.2 ul of R-Phycoerythrin-labeled goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 ul of 3% fetal bovine serum (FBS) and left to stand on ice for 1 hour. After washing by PBS containing 3% fetal bovine serum (FBS), fluorescence intensity was measured using FACSVerse (Becton Dickinson). On the other hand, a similar procedure to the above was performed using a purified antibody derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries) that did not react toMCEMP1 protein in (3) instead of the anti-MCEMP1 polyclonal antibody, and this was used as a control. As a result, LUVA cells to which the anti-MCEMP1 polyclonal antibody was added showed 74% increase in average fluorescence intensity compared to the control. Therefore, it was confirmed thatMCEMP1 protein is expressed on the cell membrane surface of the above human mast cell line.

[0148] The above-mentioned increase rate of average fluorescence intensity (MFI value) was calculated using the following formula 1. Formula 1: Percentage increase in average 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 (ADCC activity) of anti-MCEMP1 polyclonal antibodies against mast cells Next, we investigated whether anti-MCEMP1 polyclonal antibodies can damage mast cells that express MCEMP1 on their cell surface. Using the anti-MCEMP1 polyclonal antibodies prepared in Example 2 (1), we evaluated the antibody-dependent cellular cytotoxic activity (ADCC activity). The target cells were the human mast cell line (LUVA) that was confirmed to express MCEMP1 protein on the cell surface in Example 2. 106 LUVA cells were collected in a 50 ml centrifuge tube, 100 pCi of chromium (51Cr) was added, and the cells were incubated at 37°C for 2 hours. Subsequently, cells were washed by RPMI medium containing 10% fetal bovine serum (FBS) three times, and 2 X 103 cells were added to each well of a 96-well V-bottom plate. To this, 1.5 pg of the above-mentioned anti-MCEMP1 polyclonal antibody was added, and then 1.5 X 105 cells of natural killer cells (NK cells) isolated from human peripheral blood were added to each well as effector cells to make a total volume of 150 pl and cultured for 16 hours at 37°C under 5% CO2 conditions. After culture, the amount of chromium (51Cr) in the culture supernatant released from the damaged mast cells was measured, and the ADCC activity of the anti-MCEMP1 polyclonal antibody against the mast cells was calculated. As a result, 24.8% ADCC activity against LUVA cells was confirmed (see Figure 1). On the other hand, as a control, when the similar procedure was performed on the above cell line using a purified antibody derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries), which did not react to MCEMP1-expressing cells in Example 2(3) and (4), and in a medium without the antibody, the activity was clearly lower (see Figure 1). Therefore, it was revealed that anti-MCEMP1 polyclonal antibodies can damage mast cells that express MCEMP1 protein on the cell surface through ADCC activity.

[0150] The cytotoxic activity shown in Figure 1 is the result of measuring the amount of chromium (51Cr) released into the culture medium after mixing antibody, effector cells, and target cells which have been incorporated chromium (51Cr), and then culturing for 16 hours as described above. The result is calculated as the cytotoxic activity against the mast cell line using the following formula 2. Formula 2: Cytotoxic activity (%) = (Amount of 51Cr released from target cell when antibody and effector cell are added - Spontaneous release of 51Cr from target cell) / (Amount of 51Cr released from target cell when 2% "Triton X-100" (MP Biomedicals) are added - Spontaneous release of 51Cr from target cell) X 100.

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

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

Claims

1. A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity with MCEMP1 protein and cytotoxic activity against mast cells expressing MCEMP1 protein on the cell surface,wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

2. A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity 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 with the amino acid sequence, or with a fragment of the polypeptide comprising 7 or more consecutive amino acids and cytotoxic activity against mast cells expressing MCEMP1 protein on the cell surface, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

3. A pharmaceutical composition for treatment and / or prevention of a disease, comprising, as an active ingredient, an antibody or antigen-binding fragment thereof having an immunological reactivity 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 with the amino acid sequence, or with a fragment of the polypeptide comprising 7 or more consecutive amino acids and cytotoxic activity against mast cells expressing MCEMP1 protein on the cell surface, wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is conjugated with a therapeutic agent(s).27064460_1 (GHMatters) P129831.AU

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

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

7. The pharmaceutical composition according to claim 6, wherein 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 autoimmune disease is collagen disease.

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

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

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

12. The pharmaceutical composition according to claim 6, wherein digestive system diseaseis 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 MCEMP1 protein on the cell surface is a solid cancer.

14. The pharmaceutical composition according to 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 according to claim 6, wherein metabolic disease is diabetes.

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

17. The pharmaceutical composition according to claim 6, wherein pregnancy, childbirth and postpartum disease is pregnancy-induced hypertensive nephropathy.

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 according to 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 pharmaceutical combination for treatment and / or prevention of a disease, comprising the pharmaceutical composition according to any one of claims 1 to 3, and a pharmaceuticalcomposition comprising a therapeutic agent(s) for the disease whose pathology is exacerbated by mast cells expressing an MCEMP1 protein on the cell surface,wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

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

22. A method for treating and / or preventing a disease, comprising administering, simultaneously or separately to a subject, the pharmaceutical composition according to any one of claims 1 to 19, and a pharmaceutical composition comprising a therapeutic agent(s) for the disease whose pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface,wherein the disease pathology is exacerbated by mast cells expressing MCEMP1 protein on the cell surface.

Images