Agents for the prevention or treatment of allergic diseases

Abstract

[Problem] The present invention addresses the problem of discovering a molecule that is selectively expressed in mast cells and providing a prophylactic or therapeutic agent for allergic diseases. [Solution] This prophylactic or therapeutic agent is for allergic diseases and comprises an anti-Clec12b antibody or an antibody fragment thereof.

Description

[Technical Field]

[0001] This invention relates to an agent for the prevention or treatment of allergic diseases. [Background technology]

[0002] Mast cells (also called MCs) are found in various organs such as the skin, abdominal cavity, lungs, and intestines, and are involved in the development of allergic diseases and the host's defense against infections. Mast cell precursors are produced in the bone marrow, and then migrate to peripheral tissues to differentiate into tissue-resident mast cells. Mast cells can be classified into connective tissue type and mucosal type, and are found in various peripheral tissues such as the nose, skin, abdominal cavity, lungs, and intestines not only in humans but also in mice (Non-Patent Literature 1, Non-Patent Literature 2). Mast cells have cytoplasmic granules containing inflammatory molecules such as cytokines, histamine, proteases, chemokines, and lipid mediators (Non-Patent Literature 1). It has been suggested that mast cells in each organ have different functions, and that the components of inflammatory molecules contained in the granules also differ (Non-Patent Literature 2, Non-Patent Literature 3). When mast cells are stimulated by allergen-IgE immune complexes, pathogen-associated molecular patterns (PAMPs), injury-associated molecular patterns (DAMPs), or inflammatory molecules, they undergo activation and degranulation, releasing inflammatory molecules into peripheral tissues (Non-Patent Literature 1). This process plays a crucial role in the development of allergic diseases and the host's defense against infection (Non-Patent Literature 2, 4). Excessive activation of mast cells is known to be one of the causes of allergic diseases, but the mechanisms regulating mast cell activation are not yet fully understood. Furthermore, there are few reports on molecules selectively expressed in mast cells, and in particular, very few molecules expressed in mast cells that are tissue- or cell-selective are known. Known preventative or therapeutic agents for allergic diseases include anti-allergic drugs that suppress the release of inflammatory molecules such as histamine from mast cells, antihistamines that inhibit the action of histamine, and steroids that suppress inflammation. However, no approach that targets mast cells themselves is known.

[0003] Incidentally, the Clec12b gene encodes member 12B of the C-type lectin receptor family. The C-type lectin receptor family is a type of immune receptor that binds to sugar chains in a calcium-dependent manner. For example, it has been suggested that it contributes to host defense against fungi by recognizing fungal lectins (Non-Patent Literature 5). It has been shown that Clec12b has an immune receptor tyrosine-based inhibitory motif (ITIM), and that human CLEC12b recruits phosphatases (SHP-1 and SHP-2) to the ITIM (Non-Patent Literature 6), suggesting that Clec12b is an inhibitory receptor. Furthermore, according to Uniprot, its estimated molecular weight is approximately 31 kDa. It has been reported that Clec12b is expressed in vitro in activated macrophages and U937 cell line (derived from human monocytes), but not in human PBMCs (peripheral blood mononuclear cells) (Non-Patent Literature 6). However, the function of Clec12b and the tissues and cell types in which it is expressed are often unknown. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Moon, T., Laurent, C., Morris, K., Marcet, C., Yoshimura, T., Sekar, Y., and Befus, A. Advances in mast cell biology: new understanding of heterogeneity and function. Mucosal Immunology 2010; 3,111-128. [Non-Patent Document 2] Bishoff, S. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 2007; 7, 93-104 [Non-Patent Document 3] Fang, Y., Zhang, T., Lidell, L., Xu, X., Lycke, N., and Xiang, Z. The immune complex CTA1-DD / IgG adjuvant specifically targets connective tissue mast cells through FcγRIIIA and augments anti-HPV immunity after nasal immunization. Mucosal Immunology 2013; 6(6), 1-11 [Non-Patent Document 4] Abraham, S., and John A. Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 2010; 10, 440-452. [Non-Patent Document 5] Hardison, S., and Brown, G. C-type lectin receptors orchestrate antifungal immunity. Nat Immunol. 2012;13(9):817-22. [Non-Patent Document 6] Hoffmann, S., Schellack, C., Textor, S., Konold, S., Schmitz, D., Cerwenka, A., Pflanz, S., and Watzl, C. Identification of CLEC12B, and Inhibitory Receptor on Myeloid Cells. Journal of Biological Chemistry 2007; 282(31): 3 ,22370-22375 [Overview of the project] [Problems that the invention aims to solve]

[0005] The inventors of this invention hypothesized that if a molecule selectively expressed in mast cells could be identified and an antibody that specifically binds to that molecule could be used to eliminate the mast cells, it would be effective in preventing or treating allergic diseases. Therefore, the objective of this invention is to identify a molecule selectively expressed in mast cells and to provide a preventive or therapeutic agent for allergic diseases that targets that molecule. [Means for solving the problem]

[0006] The inventors diligently studied to solve the above problems. As a result, they found that the above problems can be solved by having the following configuration, and thus completed the present invention. The present invention relates, for example, to the following [1] to [8]. [1] An agent for the prevention or treatment of allergic diseases, comprising an anti-Clec12b antibody or a fragment thereof. [2] The anti-Clec12b antibody has cytotoxic activity against Clec12b-expressing cells, wherein the agent for the prevention or treatment of allergic diseases according to [1]. [3] The Clec12b-expressing cell is a mast cell, the preventive or therapeutic agent for allergic diseases as described in [2]. [4] The preventive or therapeutic agent for allergic diseases according to [2] or [3], wherein the Clec12b-expressing cells are cutaneous mast cells. [5] A prophylactic or therapeutic agent for allergic diseases according to any one of [2] to [4], wherein the cytotoxic activity is antibody-dependent cell-mediated cytotoxicity (ADCC) activity. [6] The anti-Clec12b antibody is an antibody that removes Clec12b-expressing cells, the preventive or therapeutic agent for allergic diseases according to any one of [1] to [5]. [7] A preventive or therapeutic agent for allergic diseases according to any of [1] to [6], wherein the allergic disease is a skin allergic disease. [8] The preventive or therapeutic agent for allergic diseases according to [7], wherein the skin allergic disease is at least one selected from atopic dermatitis, contact dermatitis, and urticaria. [Effects of the Invention]

[0007] According to the present invention, an agent for the prevention or treatment of allergic diseases can be provided. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a photograph showing the expression of Clec12b in human cutaneous mast cells. [Figure 2] Figure 2 shows the amino acid sequence of the heavy chain variable region of TX109. [Figure 3] Figure 3 shows the amino acid sequence of the light chain variable region of TX109. [Figure 4] Figure 4 is a graph showing the binding characteristics of TX109 as determined by ELISA. [Figure 5] Figure 5 is a histogram showing the binding characteristics of TX109 to mouse Clec12b, as determined by flow cytometry. [Figure 6] Figure 6 is a histogram showing the isotype analysis results of TX109 by flow cytometry. [Figure 7] Figure 7 shows the results of immunoprecipitation and Western blotting performed using TX109. [Figure 8-1]Figure 8-1 shows a dot plot and histogram illustrating the analysis results of immune system cells expressing mouse Clec12b. [Figure 8-2] Figure 8-2 is a continuation of Figure 8-1. [Figure 8-3] Figure 8-3 is a continuation of Figure 8-2. [Figure 8-4] Figure 8-4 is a continuation of Figure 8-3. [Figure 8-5] Figure 8-5 is a continuation of Figure 8-4. [Figure 9] Figure 9 is a graph showing the results of evaluating the cytotoxic activity of TX109 against RMA cells expressing Clec12b. [Figure 10] Figure 10 shows the experimental schedule for the efficacy of anti-Clec12b in a dermatitis model induced by mite antigen application. [Figure 11] Figure 11 shows micrographs of skin sections stained with hematoxylin eosin or toluidine blue. [Figure 12] Figure 12 is a graph showing epidermal thickness and the number of mast cells per unit area (mm²). [Figure 13] Figure 13 is a graph showing the score for skin inflammation symptoms. The horizontal axis of the graph represents the number of days, and the vertical axis represents the score. [Modes for carrying out the invention]

[0009] Next, the present invention will be described in detail. <Anti-Clec12b antibody or its antibody fragment> (Anti-Clec12b antibody) The origin of Clec12b (C-type lectin domain family 12 member B) in this invention is not particularly limited as long as it is an organism that expresses Clec12b, and includes organisms derived from primate mammals including mice, rats, hamsters, guinea pigs, dogs, pigs, monkeys, or humans. Clec12b is preferably human Clec12b or mouse Clec12b, and more preferably human Clec12b.

[0010] The anti-Clec12b antibody is not particularly limited as long as it is an antibody that has the activity to specifically bind to Clec12b. A commercially available or known anti-Clec12b antibody may be used, or TX109, an anti-Clec12b antibody established in the examples herein, may be used, or a new antibody may be prepared by various known methods.

[0011] Known anti-Clec12b antibodies include the rabbit anti-human Clec12b polyclonal antibody (NO. ab211452) manufactured by Abcam, and the mouse anti-human Clec12b monoclonal antibody disclosed in Non-Patent Literature 6. TX109 established in the examples of this specification is an anti-Clec12b antibody having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region having the amino acid sequence of SEQ ID NO: 2.

[0012] The activity that specifically binds to Clec12b refers to the activity that specifically binds to the Clec12b protein. Furthermore, "specifically binding" means that it binds to the Clec12b protein but not to any other protein. The binding activity can be measured by known methods, such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), flow cytometry, and pull-down assay.

[0013] The anti-Clec12b antibody may be an antibody that inhibits the binding of Clec12b to its ligand, or an antibody that does not inhibit the binding of Clec12b to its ligand, but an antibody that inhibits the binding of Clec12b to its ligand is preferred. Furthermore, the anti-Clec12b antibody may be an antibody that inhibits or reduces Clec12b signaling (anti-Clec12b neutralizing antibody), or an antibody that does not inhibit or reduce Clec12b signaling, but an antibody that does not inhibit or reduce Clec12b signaling is preferred. The anti-Clec12b antibody may also be an antibody that does not inhibit or reduce Clec12b signaling mediated by ITIM of Clec12b.

[0014] Clec12b consists of an intracellular domain, a transmembrane domain, and an extracellular domain. It is preferable that the anti-Clec12b antibody binds to all or part of the extracellular domain of Clec12b. The extracellular domain of Clec12b is, for example, the amino acids from positions 65 to 276 in the amino acid sequence of human Clec12b (276 amino acids), and the amino acids from positions 65 to 275 in the amino acid sequence of mouse Clec12b (275 amino acids). It is preferable that the anti-Clec12b antibody binds to a site that exhibits high affinity with Clec12b.

[0015] The anti-Clec12b antibody may be any of the following: mouse antibody, rat antibody, guinea pig antibody, hamster antibody, rabbit antibody, monkey antibody, dog antibody, chimeric antibody, humanized antibody, or human antibody.

[0016] The immunized animals used to produce anti-Clec12b antibodies are not particularly limited, but include, for example, mice, rats, guinea pigs, hamsters, rabbits, monkeys, and dogs, and are preferably mice, rats, and rabbits. The immunized animal used to produce anti-Clec12b antibodies may be a genetically modified non-human animal in which the expression of the Clec12b gene is lost due to the introduction of a mutation in the Clec12b gene or the expression regulatory region of the Clec12b gene in the genome, so-called Clec12b knockout non-human animals. If a Clec12b knockout non-human animal is used as the immunized animal, Clec12b from the same animal species can be used as the antigen. For example, if a Clec12b knockout mouse is used as the immunized animal, mouse Clec12b can be used as the antigen to produce mouse anti-mouse Clec12b antibodies.

[0017] Suitable antigens for producing anti-Clec12b antibodies include, for example, cells in which Clec12b is forcibly expressed using an expression vector, Clec12b expression plasmid vectors, Clec12b expression viral vectors (such as adenovirus vectors), and Clec12b recombinant proteins produced using these vectors. Clec12b recombinant proteins may be fused with other proteins or tagged. From the viewpoint of protein stability, it is preferable to fuse Clec12b recombinant proteins with IgGFc or the like to form chimeric proteins. The antigens used to produce anti-Clec12b antibodies may be used individually or in combination of two or more. Cells in which Clec12b is forcibly expressed and Clec12b recombinant proteins may be used in combination.

[0018] The anti-Clec12b antibody may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferred because it is easier to maintain a constant titer.

[0019] Polyclonal antibodies can be produced by known methods. For example, they can be produced by immunizing a suitable animal with an antigen protein or a mixture of an antigen protein and a carrier protein, collecting antibody-containing material against the antigen protein from the immunized animal, and separating and purifying the antibody as needed. Commonly used animals include mice, rats, sheep, goats, rabbits, and guinea pigs. To enhance antibody production, complete or incomplete Freund's adjuvant can be administered together with the antigen protein. Administration is usually done once every two weeks for a total of about 3 to 10 times. Polyclonal antibodies can be collected from the blood, ascites, etc., of animals immunized by the above method. The polyclonal antibody titer in antiserum can be measured by ELISA. Polyclonal antibodies can be isolated and purified according to immunoglobulin isolation and purification methods, such as purification using antigen-binding solid phase or active adsorbents such as protein A or protein G, salting-out, alcohol precipitation, isoelectric focusing, electrophoresis, adsorption / desorption using ion exchangers, ultracentrifugation, and gel filtration.

[0020] Monoclonal antibodies can be produced by known methods. Specifically, immunosensitization is performed by injecting the antigen, along with an adjuvant as needed, subcutaneously, intramuscularly, intravenously, intra-foot pad, or intraperitoneally into mammals, preferably mice, rats, hamsters, guinea pigs, or rabbits, one to several times. The adjuvant is not particularly limited, and can be complete Freund's adjuvant, incomplete Freund's adjuvant, sodium hydroxide, aluminum hydroxide (Alum), calcium phosphate, aluminum phosphate, alum, hepes, carboxyvinyl polymer, etc., but aluminum hydroxide (Alum) is preferred. Typically, immunization is performed 2 to 6 times at intervals of about one month from the initial immunization, and antibody-producing cells can be obtained from the immunosensitized mammals about 3 to 4 days after the final immunization. The number of immunizations and the time intervals can be appropriately changed depending on the properties of the immunogen used.

[0021] The preparation of hybridomas that secrete monoclonal antibodies can be carried out according to the method of Kohler and Milstein et al. (Nature, 1975, vol.256, pp. 495-497) and similar methods. Specifically, hybridomas can be prepared by cell fusion of antibody-producing cells obtained from immunized mammals, such as the spleen, lymph nodes, bone marrow, or tonsils, preferably contained in the spleen, and myeloma cells from mammals such as mice, rats, guinea pigs, hamsters, rabbits, or humans, more preferably from mice, rats, or humans, that do not produce autoantibodies.

[0022] For use in cell fusion, myeloma cells can be derived from mouse cell lines such as P3-U1, NS-1, SP2 / 0, 653, X63, and AP-1.

[0023] Screening for hybridoma clones that produce monoclonal antibodies is performed by culturing hybridomas, for example, in a microtiter plate, and measuring the reactivity of the culture supernatant from wells showing growth to the antigen used in the aforementioned mouse immunization using methods such as RIA, ELISA, and FACS. Clones that produce monoclonal antibodies that specifically bind to the antigen are then selected. Typically, a method is used in which the antigen is immobilized and the antibodies in the culture supernatant that bind to it are detected using secondary antibodies labeled with radioactive materials, fluorescent materials, enzymes, etc. Alternatively, when using antigen-expressing cells, the hybridoma culture supernatant is added to the cells, followed by a reaction with a fluorescently labeled secondary antibody. The fluorescence intensity of the cells is then measured using a fluorescence detection device such as a flow cytometer to detect monoclonal antibodies that can bind to the antigen of the present invention.

[0024] Monoclonal antibodies from selected hybridoma clones can be produced by culturing hybridomas in vitro, or by culturing them in mice, rats, guinea pigs, hamsters, or rabbits, preferably in the ascites fluid of mice or rats, more preferably in the ascites fluid of mice, and then isolating the resulting culture supernatant or mammalian ascites fluid. When culturing in vitro, any known nutrient medium or any nutrient medium derived from known basic media can be used to grow, maintain, and preserve hybridomas and produce monoclonal antibodies in the culture supernatant, depending on various conditions such as the characteristics of the cell type being cultured, the purpose of the research, and the culture method.

[0025] Monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid described above to saturated ammonium sulfate, ion-exchange chromatography (DEAE or DE52, etc.), or affinity column chromatography such as an anti-immunoglobulin column or a protein A column or protein G column.

[0026] As an anti-Clec12b antibody, a recombinant antibody may be used, which is produced by cloning the antibody gene from antibody-producing cells, such as hybridomas, incorporating it into a suitable vector, introducing it into a host, and then using genetic recombination technology.

[0027] Specifically, mRNA encoding the antibody variable region is isolated from hybridomas or immune cells that produce the target antibody, such as sensitized lymphocytes, immortalized with oncogenes. mRNA isolation is performed by preparing total RNA using known methods, such as guanidine ultracentrifugation, and then preparing mRNA using an mRNA Purification Kit (Pharmacia). The cDNA of the antibody variable region is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis can be performed using an AMV Reverse Transcriptase First-strand cDNA Synthesis Kit. Alternatively, the 5'-Ampli FINDER RACE Kit (Clonetech) and the 5'-RACE method using PCR can be used for cDNA synthesis and amplification. The target DNA fragment is purified from the obtained PCR product and ligated to vector DNA. Furthermore, a recombinant vector is prepared from this and introduced into E. coli or other organisms, and colonies are selected to prepare the desired recombinant vector. The base sequence of the target DNA is confirmed by known methods, such as the deoxyribochemical method.

[0028] Once the DNA encoding the variable region of the target antibody is obtained, it can be ligated with the DNA encoding the desired antibody constant region and incorporated into an expression vector. Alternatively, the DNA encoding the antibody variable region may be incorporated into an expression vector containing the DNA of the antibody constant region. To produce the target antibody, the antibody gene is incorporated into an expression vector so that it is expressed under the control of an expression regulatory region, such as an enhancer / promoter. Next, host cells can be transformed using this expression vector to express the antibody.

[0029] Antibody gene expression may involve simultaneously transforming the host by incorporating the heavy chain or light chain of the antibody into separate expression vectors, or by incorporating the DNA encoding both the heavy chain and light chain into a single expression vector to transform the host.

[0030] As a method for producing antibodies, the so-called phage display method (Nature Biotechnology 23, 1105 (2005)) may be used. Specifically, an antibody gene library prepared by a known method using human or animal (e.g., rabbit, mouse, rat, hamster, etc.) B lymphocytes as material, or an antibody gene library completely synthesized by selecting and modifying human or animal germ line sequences, is displayed on the cell surface or ribosome of bacteriophages, E. coli, yeast, animal cells, etc. In this case, examples of antibody forms to be displayed on the cell surface include IgG molecules, IgM molecules, Fab fragments, single-stranded Fv (scFv) fragments, etc.

[0031] The antibody fragment gene obtained in this way can be recombined with the corresponding region of the IgG antibody gene using a known method to obtain the antibody gene. Then, the gene obtained in this way can be incorporated into a suitable vector, introduced into a host, and antibodies can be produced using genetic recombination technology.

[0032] Anti-Clec12b antibodies may be recombinant antibodies such as antibodies containing the human Fc region, antibodies containing the human constant region, human chimeric antibodies (hereinafter also simply referred to as chimeric antibodies), humanized antibodies (also referred to as human CDR transplant antibodies), and human antibodies, in order to reduce immunogenicity in humans.

[0033] A chimeric antibody is an antibody composed of the heavy chain variable region (hereinafter also referred to as VH) and light chain variable region (hereinafter also referred to as VL) of an antibody from an animal other than a human, and the heavy chain constant region (hereinafter also referred to as CH) and light chain constant region (hereinafter also referred to as CL) of a human antibody. The type of animal from which the variable region is derived is not particularly limited, as long as it is an animal capable of producing hybridomas, such as mice, rats, hamsters, and rabbits.

[0034] Human-type chimeric antibodies against Clec12b can be produced by obtaining cDNA encoding VH and VL from an antibody of a non-human animal that specifically binds to human Clec12b, inserting these cDNAs into an expression vector containing the CH and CL genes of a human antibody, respectively, to construct a human-type chimeric antibody expression vector, and then introducing and expressing it in animal cells.

[0035] The CH of the human chimeric antibody is not particularly limited as long as it is a human immunoglobulin (hereinafter also referred to as hIg), but those of the hIgG class are preferred. The CL of the human chimeric antibody is not particularly limited as long as it belongs to hIgG.

[0036] Humanized antibodies are antibodies in which the VH and VL CDRs of antibodies from non-human animals are transplanted to appropriate positions on the VH and VL of human antibodies. Anti-Clec12b humanized antibodies can be produced by constructing a cDNA encoding a variable region (hereinafter also called the V region) obtained by transplanting the VH and VL CDRs of antibodies from non-human animals that specifically bind to Clec12b into the framework (hereinafter also called the FR) of any human antibody, inserting this cDNA into an expression vector containing DNA encoding the CH and CL of a human antibody, constructing a humanized antibody expression vector, and introducing it into animal cells for expression. The amino acid sequences of the VH and VL FR of human antibodies are not particularly limited as long as they are amino acid sequences derived from human antibodies. The CH of humanized antibodies is not particularly limited as long as it is hIg, but hIgG class is preferred. The CL of humanized antibodies is not particularly limited as long as it belongs to hIg.

[0037] Anti-Clec12b antibodies are preferably found to possess effector activity. "Effector activity" refers to activity expressed via the Fc region of the antibody, and is known to include cytotoxic activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cell-mediated cytotoxicity (CDC) activity, as well as antibody-dependent cell-mediated phagocytosis (ADCP) activity by phagocytic cells such as macrophages and dendritic cells. The anti-Clec12b antibody is preferably an antibody that has cytotoxic activity against Clec12b-expressing cells, and more preferably an antibody that has antibody-dependent cell-mediated cytotoxicity (ADCC) activity against Clec12b-expressing cells.

[0038] Anti-Clec12b antibodies, if their isotype is IgG, typically exhibit effector activity, particularly ADCC activity. The strength of effector activity varies depending on the antibody isotype and the species from which the antibody originates, but IgG, especially IgG1, exhibits high activity. The effector activity of the anti-Clec12b antibody may be controlled by known methods.

[0039] Anti-Clec12b antibodies are preferably antibodies that remove Clec12b-expressing cells. Examples of such anti-Clec12b antibodies include antibodies that have activity (also called neutralizing activity or antagonist activity) that suppresses the activation of Clec12b-expressing cells (such as cell proliferation, migration, invasion, and / or degranulation), or antibodies that have apoptosis-inducing activity and / or cytotoxic activity in Clec12b-expressing cells.

[0040] The anti-Clec12b antibody is preferably an antibody that removes Clec12b-expressing cells by cytotoxic activity. The anti-Clec12b antibody may inhibit Clec12b-dependent signaling, thereby causing inhibition of cell proliferation and / or induction of apoptosis in Clec12b-expressing cells, and may also be an antibody that eliminates Clec12b-expressing cells through cytotoxic activity.

[0041] The anti-Clec12b antibody may be a recycling antibody, sweeping antibody, bispecific antibody, or T-cell redirecting antibody modified for purposes such as improving the blood retention of the antibody or antigen, or it may be an antibody modified product conjugated with various molecules such as polyethylene glycol (PEG).

[0042] As the anti-Clec12b antibody, commercially available or known anti-Clec12b antibody variants, chimeric antibodies, humanized antibodies, etc., may be used.

[0043] The anti-Clec12b antibody or its antibody fragment may be mixed with one or more pharmaceutically acceptable carriers to form a pharmaceutical composition containing the anti-Clec12b antibody or its antibody fragment.

[0044] The isotype of the anti-Clec12b antibody is not particularly restricted. The heavy chain constant region of the anti-Clec12b antibody may be any of IgG, IgM, IgA, IgD, or IgE, but it is preferably IgG, and more preferably IgG1, due to its high effector activity. The light chain constant region of the anti-Clec12b antibody may be either an Igκ chain or an Igλ chain, but it is preferably an Igκ chain.

[0045] Anti-Clec12b antibodies may be used individually or in combination of two or more types.

[0046] (Method for evaluating effector activity) The method for evaluating ADCC activity is not particularly limited and can be evaluated using known methods, such as co-existing effector cells (NK cells, PBMCs; peripheral blood mononuclear cells, etc.) with target cells (Clec12b-expressing cells) and detecting cytotoxicity of the target cells by the effector cells. Effector cells may be cells stimulated with IL-2, Poly(I:C), etc. Cell damage to target cells can occur, for example, by introducing radioisotopes (Cr) into the target cells. 51ADCC activity can also be detected by measuring the amount of radioisotopes or fluorescent substances (calcein) released from lysed target cells after they have been introduced into the cells. ADCC activity can also be evaluated by measuring the cytotoxic activity of effector cells, for example, by measuring the expression level of degranulation markers such as CD107a, or the expression level or production level of cytokines such as IFN-γ in effector cells. ADCC activity can also be measured by reporter assays, such as measuring the luminescence signal of firefly luciferase using cells into which a luciferase gene driven by an Fcγ receptor variant and an NFAT (Nuclear Factor of Activated T cells) response element has been introduced, instead of effector cells. For example, if an anti-Clec12b antibody induces cytotoxicity of target cells by effector cells, then the anti-Clec12b antibody can be said to have ADCC activity. Also, for example, if an anti-Clec12b antibody increases the expression level of CD107a or IFN-γ in effector cells in the presence of target cells, then the anti-Clec12b antibody can be said to have ADCC activity.

[0047] The method for evaluating CDC activity is not particularly limited and can be evaluated using known methods, such as coexisting complement (human serum-derived complement, etc.) with target cells (Clec12b-expressing cells) and detecting complement-induced cytotoxicity of the target cells. Cytotoxicity of target cells is detected by introducing radioisotopes (Cr) into the target cells. 51 CDC activity can be detected by a method in which a radioisotope or fluorescent substance (calcein) is incorporated into the lysed target cells, and the amount of radioisotope or fluorescent substance released from the cells is measured. CDC activity can also be measured using a reporter assay, which uses cells into which a reporter gene has been introduced instead of effector cells. For example, if an anti-Clec12b antibody induces complement cytotoxic activity against target cells, then the anti-Clec12b antibody can be said to have CDC activity.

[0048] The method for evaluating ADCP activity is not particularly limited and can be evaluated using known methods, such as co-existing effector cells (macrophages, etc.) with target cells (Clec12b-expressing cells) and detecting phagocytosis of target cells by effector cells. Phagocytosis of target cells by effector cells can be evaluated by fluorescently labeling the effector cells and target cells and measuring the uptake of target cells by effector cells using flow cytometry or observing it under a microscope. ADCP activity can also be measured using reporter assays, such as measuring the luminescence signal of firefly luciferase, using cells into which a luciferase gene driven by an Fcγ receptor variant and an NFAT (Nuclear Factor of Activated T cells) response element has been introduced instead of effector cells. For example, if an anti-Clec12b antibody induces phagocytosis of target cells by effector cells, then it can be said that the anti-Clec12b antibody possesses ADCP activity.

[0049] (Fragment of anti-Clec12b antibody) The anti-Clec12b antibody fragments are any of the above-mentioned anti-Clec12b antibody fragments and are not particularly limited as long as they have the activity to specifically bind to Clec12b, i.e., antigen-binding activity. Examples of antibody fragment types include Fab, Fab', F(ab')2, scFv, diabody, dsFv, and peptides containing CDR.

[0050] Fab is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, obtained by treating IgG with papain (a proteolytic enzyme). Anti-Clec12b antibody Fab can be produced by treating the anti-Clec12b antibody with papain, or by inserting the DNA encoding the Fab of the said antibody into an expression vector, and then introducing this vector into a prokaryote or eukaryote for expression.

[0051] F(ab')2 is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 100,000, obtained by treating IgG with pepsin (a proteolytic enzyme). Anti-Clec12b antibody F(ab')2 can be produced by treating anti-Clec12b antibody with pepsin or by attaching Fab' (described later) via a thioether bond or disulfide bond.

[0052] Fab' is an antibody fragment with an antigen-binding activity and a molecular weight of approximately 50,000, obtained by cleaving the disulfide bond in the hinge region of F(ab')2. Fab' of an anti-Clec12b antibody can be produced by treating F(ab')2 of the anti-Clec12b antibody with dithiothreitol, or by inserting the DNA encoding Fab' of the antibody into an expression vector and introducing this vector into a prokaryote or eukaryote for expression.

[0053] scFv is an antibody fragment with antigen-binding activity, consisting of one VH molecule and one VL molecule linked using a suitable peptide linker. The scFv of an anti-Clec12b antibody can be produced by obtaining the cDNA encoding the VH and VL molecules of the anti-Clec12b antibody, constructing the DNA encoding the scFv, inserting this DNA into an expression vector, and then introducing this expression vector into a prokaryote or eukaryote for expression.

[0054] A diabody is an antibody fragment formed by the dimerization of scFv, and is a bivalent antibody fragment with antigen-binding activity. The diabody of an anti-Clec12b antibody can be produced by obtaining the cDNA encoding the VH and VL of the anti-Clec12b antibody, constructing the DNA encoding the diabody, inserting this DNA into an expression vector, and introducing this expression vector into a prokaryote or eukaryote for expression.

[0055] dsFv is an antibody fragment in which a polypeptide with one amino acid residue each in VH and VL is replaced with a cysteine residue and is bound via a disulfide bond between the cysteine residues. The dsFv of the anti-Clec12b antibody can be prepared by obtaining cDNAs encoding VH and VL of the anti-Clec12b antibody, constructing a DNA encoding dsFv, inserting this DNA into an expression vector, and introducing and expressing this expression vector into prokaryotes or eukaryotes.

[0056] The peptide containing a CDR is a peptide containing at least one region or more of the CDR of VH or VL. The peptide containing the CDR of the anti-Clec12b antibody can be prepared by constructing a DNA encoding the CDRs of VH and VL of the anti-Clec12b antibody, inserting this DNA into an expression vector, and introducing and expressing this expression vector into prokaryotes or eukaryotes. Also, the peptide containing the CDR of the anti-Clec12b antibody can be prepared by chemical synthesis methods such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). Preferably, a peptide containing 6 CDRs derived from the anti-Clec12b antibody can be mentioned.

[0057] The fragments of the anti-Clec12b antibody may be used alone or in combination of two or more. Also, they may be used in combination with the anti-Clec12b antibody and its fragments.

[0058] <Clec12b-expressing cells> Clec12b-expressing cells are not particularly limited as long as they express Clec12b. Examples of Clec12b-expressing cells include, for example, immune-related cells, more specifically, Pro-B cells, Pre-B cells, B cells, Pre-T cells, T cells, NK cells, γδT cells, ILC (Innate Lymphoid Cell), dendritic cells, macrophages, monocytes, cells of the mast cell lineage (mast cells and precursors of mast cells (c-Kit - FcεRIα - positive cells)), eosinophils, basophils, etc. Clec12b-expressing cells are preferably Pro-B cells, Pre-B cells, B cells, Pre-T cells, T cells, NK cells, γδT cells, ILC (Innate Lymphoid Cell), dendritic cells, macrophages, monocytes, cells of the mast cell lineage (mast cells and precursors of mast cells (c-Kit - FcεRIα - cells)), eosinophils, or basophils. Since Clec12b is highly expressed in Clec12b-expressing cells, they are preferably cells of the mast cell lineage (mast cells and precursors of mast cells (c-Kit - FcεRIα - cells)), and more preferably mast cells. Also, since Clec12b is highly expressed in Clec12b-expressing cells, they are preferably cells of the cutaneous mast cell lineage (cutaneous mast cells and precursors of cutaneous mast cells (c-Kit - FcεRIα - cells)), and more preferably cutaneous mast cells. Mast cells are classified into two subtypes, mucosal mast cells (mast cells containing only tryptase) and connective tissue mast cells (mast cells containing tryptase, chymase, carboxypeptidase, and cathepsin G-like protease), based on the difference in proteases present in the granules. Since most human cutaneous mast cells are connective tissue mast cells, the cutaneous mast cells are preferably connective tissue mast cells.

[0059] Clec12b-expressing cells may be used alone or in combination of two or more.

[0060] The method for confirming the expression of Clec12b is not particularly limited, and examples include known methods such as detection of the Clec12b gene by PCR or the like, Western blotting, ELISA, and detection of the Clec12b protein by flow cytometry or the like.

[0061] <Agent for preventing or treating allergic diseases> The present invention provides a preventive or therapeutic agent for allergic diseases, comprising an anti-Clec12b antibody or an antibody fragment thereof, and is effective in preventing or treating allergic diseases. As shown in the examples, the inventors have found that Clec12b expression is selective for mast cells among immune-related cells, and particularly selective for cutaneous mast cells.

[0062] Mast cells play a significant role in the onset and exacerbation of allergic diseases. For example, it has been reported that the number of mast cells is increased in the skin of patients with atopic dermatitis (Yoshimichi Okayama, The Role of Mast Cells in Human Skin Allergic Diseases, Veterinary Clinical Dermatology 21 (3): 137-141, 2015). Therefore, if an anti-Clec12b antibody or a fragment thereof binds to Clec12b on the cell membrane of Clec12b-expressing cells, the effector function of the anti-Clec12b antibody can eliminate the Clec12b-expressing cells, thereby preventing or treating allergic diseases. Furthermore, analysis of Clec12b expression suggests that the allergic disease preventive or therapeutic agent of the present invention, which contains an anti-Clec12b antibody or an antibody fragment thereof, has little effect on other immune-related cells and acts selectively on mast cells, particularly cutaneous mast cells. Therefore, it is presumed that the allergic disease preventive or therapeutic agent of the present invention will have few side effects.

[0063] The preventive or therapeutic agent for allergic diseases of the present invention may be any pharmaceutical composition containing an anti-Clec12b antibody or an antibody fragment thereof as an active ingredient, but it is preferable to provide it as a pharmaceutical preparation manufactured by mixing it with one or more pharmaceutically acceptable carriers and using any method well known in the art of pharmaceutical formulation. Examples of pharmaceutically acceptable carriers include physiological saline, phosphate-buffered saline, phosphate-buffered saline glucose solution, and buffered saline. Antibodies may be freeze-dried and reconstituted as needed by adding one of the above-mentioned buffered aqueous solutions.

[0064] The route of administration of the allergic disease prevention or treatment agent of the present invention is not particularly limited and can be administered by intravenous injection, intraperitoneal, oral, nasal, mucosal, intramuscular or subcutaneous, intranasal, intratracheal, skin, transdermal, or intradermal route. Intravenous or intraperitoneal administration is preferred. The dosage form of the agent for the prevention or treatment of allergic diseases of the present invention is not particularly limited and includes, for example, tablets, granules, sprays, capsules, syrups, emulsions, suppositories, injections, ointments, tapes, and the like.

[0065] Capsules, tablets, powders, granules, etc., can be manufactured using excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropyl cellulose, and gelatin; surfactants such as fatty acid esters; and plasticizers such as glycerin as additives.

[0066] Liquid preparations such as emulsions and syrups can be manufactured using water, sugars such as sucrose, sorbitol, and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil, and soybean oil; preservatives such as p-hydroxybenzoic acid esters; and flavors such as strawberry and peppermint as additives.

[0067] The injectable preparation may be used as an additive to water, sucrose, sorbitol, xylose, trehalose, fructose, and other sugars; sugar alcohols such as mannitol, xylitol, and sorbitol; buffers such as phosphate buffer, citrate buffer, and glutamate buffer; and surfactants such as fatty acid esters.

[0068] The dosage (effective dose) of the allergic disease preventive or therapeutic agent of the present invention is not particularly limited, but preferably 0.1 to 100 mg, more preferably 0.5 to 50 mg, and even more preferably 1 to 10 mg of anti-Clec12b antibody per kg of body weight is administered once to several times a day in a single dose. The total number of doses and the frequency of administration are also not particularly limited and may be appropriately selected depending on the type of allergic disease, the severity of symptoms, the recipient (age, sex, weight, etc.), the route of administration, etc. Administration may be a single dose, may be administered daily for several days in a row, or may be administered over a period of one to several weeks with intervals of several days between doses.

[0069] The allergic diseases treatable by the preventive or therapeutic agent for allergic diseases of the present invention are not particularly limited and include all allergic diseases in general. Examples of such allergic diseases include anaphylactic shock, bronchial asthma, allergic rhinitis, allergic conjunctivitis, drug and insect allergies, food allergies, contact dermatitis, urticaria, atopic dermatitis, and allergic rhinitis including hay fever.

[0070] The allergic diseases treatable by the allergic disease preventive or therapeutic agent of the present invention are preferably allergic diseases involving mast cells, given that Clec12b expression is abundant in mast cells. Examples of allergic diseases involving mast cells include anaphylactic shock, bronchial asthma, allergic rhinitis, allergic conjunctivitis, drug allergies, food allergies, urticaria, contact dermatitis, and atopic dermatitis.

[0071] Furthermore, since Clec12b expression is abundant in cutaneous mast cells, the allergic diseases that can be treated with the allergic disease preventive or therapeutic agent of the present invention are preferably cutaneous allergic diseases involving mast cells. Examples of cutaneous allergic diseases involving mast cells include atopic dermatitis, contact dermatitis, and urticaria.

[0072] Furthermore, "treatment" includes not only curing a disease or symptom but also alleviating (improving) it, and "prevention" includes not only preventing a disease or symptom from occurring in the first place but also preventing its recurrence after it has been cured.

[0073] The target recipients of the preventive or therapeutic agent for allergic diseases of the present invention may be humans or non-humans (e.g., mammals such as mice), but are preferably humans, and more preferably humans who are suffering from or at risk of suffering from an allergic disease.

[0074] The allergic disease preventive or therapeutic agent of the present invention can be used in combination with other agents, such as other allergic disease preventive or therapeutic agents. When the allergic disease preventive or therapeutic agent of the present invention is used in combination with other allergic disease preventive or therapeutic agents, the preventive or therapeutic effect of the allergic disease is more likely to be increased than when the allergic disease preventive or therapeutic agent of the present invention is used alone. The types of other allergic disease preventive or therapeutic agents are not limited and include, for example, antihistamines, chemical mediator release inhibitors, anti-leukotriene drugs, thromboxane A2 inhibitors, Th2 cytokine inhibitors, steroid nasal sprays, vasoconstrictors, β2 receptor agonists: LABA, β2 receptor agonists: SABA, steroid inhalants, theophylline preparations, anticholinergics, immunosuppressants, and anaphylaxis adjuncts (adrenaline). The other allergic disease preventive or therapeutic agents may be used individually or in combination of two or more.

[0075] One embodiment of the present invention is a method for preventing or treating an allergic disease, comprising administering an effective amount of an anti-Clec12b antibody or a fragment thereof to a subject. Furthermore, one embodiment of the present invention is a method for preventing or treating an allergic disease, comprising administering an effective amount of an anti-Clec12b antibody or an antibody fragment thereof, which has cytotoxic activity against Clec12b-expressing cells, to a subject. Furthermore, one embodiment of the present invention is a method for preventing or treating a skin allergic disease, comprising administering an effective amount of an anti-Clec12b antibody or an antibody fragment thereof having cytotoxic activity against skin mast cells to a subject.

[0076] One embodiment of the present invention is an anti-Clec12b antibody or an antibody fragment thereof for use in the prevention or treatment of allergic diseases. Furthermore, one embodiment of the present invention is an anti-Clec12b antibody or an antibody fragment thereof having cytotoxic activity against Clec12b-expressing cells for use in the prevention or treatment of allergic diseases. Furthermore, one embodiment of the present invention is an anti-Clec12b antibody or an antibody fragment thereof having cytotoxic activity against cutaneous mast cells for use in the prevention or treatment of skin allergic diseases. [Examples]

[0077] The present invention will now be described in more detail with reference to examples, but the present invention is not limited thereto. All experiments were conducted in accordance with the guidelines of the Animal Ethics Committee of the Research Animal Resource Center, University of Tsukuba.

[0078] [Example 1] Expression of Clec12b in human cutaneous mast cells (method) In accordance with the Declaration of Helsinki, written informed consent was obtained from subjects prior to the study. This study was also conducted with the approval of the Ethics Committee of the University of Tsukuba School of Medicine. Human skin specimens obtained from healthy volunteers (University of Tsukuba Hospital) were stained with an Opal 4-Color Automation IHC Kit (PerkinElmer) using control mIgG, or anti-human β-tryptase monoclonal antibody (Abcam, NO. ab2378, clone AA1) and anti-human Clec12b polyclonal antibody (Abcam, NO. ab211452) as primary antibodies, and observed under a fluorescence microscope.

[0079] (result) The results are shown in Figure 1. The scale bar is 100 μm. The staining image with anti-human Clec12b polyclonal antibody (green, hCLEC12B) overlapped with the staining image with anti-human β-tryptase monoclonal antibody (red, β-tryptase), indicating that the same cells were stained. β-tryptase is an enzyme within mast cell granules that is specifically expressed in human dermal mast cells. Although human skin tissue contains many more macrophages and other cells than mast cells, almost all cells stained with anti-human Clec12b polyclonal antibody were cells stained with anti-human β-tryptase monoclonal antibody, and there were almost no cells that were not stained with anti-human β-tryptase monoclonal antibody. In other words, it became clear that hClec12b is not expressed in cells other than mast cells (e.g., macrophages) in human skin, or its expression level is extremely low. That is, it became clear that hClec12b is selectively expressed in mast cells in human skin.

[0080] [Example 2] Establishment of anti-mouse Clec12b monoclonal antibody (TX109) (1) Mouse C57BL / 6N mice were purchased from CLEA Japan, Inc. (Tokyo) and reared at the University of Tsukuba Animal Resource Center under conditions free of specific pathogens.

[0081] (2) Production of RMA cells that stably express Flag-tagged mouse Clec12 A sequence encoding a flag tag was added to the 3' end of mouse Clec12b (mClec12b) cDNA and inserted into the retroviral vector pMx (Cell Biolabs, Inc.) to construct a flag-tagged mouse Clec12 gene expression pMx vector. This flag-tagged mouse Clec12 gene expression pMx vector was transfected into RMA cells. Drug selection was performed using ampicillin. RMA cells that stably express flag-tagged mouse Clec12b (mouse Clec12b with a flag tag attached to the C-terminus) were designated as mouse Clec12b-expressing RMA cells. Untransfected RMA cells were designated as parental RMA cells.

[0082] (3) Production of fusion protein (Clec12b-Fc) A chimeric protein (Clec12b-Fc) was created by fusing the extracellular portion of mouse Clec12b (amino acids 65-275 out of the 275 total amino acids of mouse Clec12b) with the N-terminal Fc portion of human IgG, following the method described in the literature (Kanemaru et al, Science Immunol 4: aax6908, 2019).

[0083] (4) Establishment of hybridomas Clec12b-Fc (100 μg in 500 μL of PBS) is mixed with ALUM (aluminum hydroxide gel adjuvant), and Clec12b - / -Mice (obtained from mice established by Professor Akira Yamazaki of Osaka University) were injected intraperitoneally. Subsequently, intraperitoneal injections of Clec12b-Fc alone, without ALUM, were administered once a month for a total of four times. Three days after the last injection, spleen cells were collected from the three immunized mice and fused with SP2 / 0 myeloma cells to obtain hybridomas. The hybridomas were cultured in HAT medium (Sigma-America, NO. H0137-10VL), colonies were picked, and they were grown in medium E (Stem Celll Technologies). Mouse Clec12b-expressing RMA cells or parental RMA cells were stained with the culture supernatant of the hybridomas and analyzed by flow cytometry. Hybridomas producing antibodies that specifically bind to mClec12b were screened, and the selected clone was designated as clone TX109. The antibody produced by clone TX109 is referred to as TX109. Anti-Clec12b antibodies were purified from the culture supernatant of clone TX109 or from ascites fluid obtained by inoculating clone TX109 into the peritoneal cavity of mice using HiTrap Protein G (Merck, NO.GE17-0404-01) (also referred to as purified TX109).

[0084] (5) Determination of the TX109 sequence The DNA sequences of the variable regions of the heavy and light chains of TX109 were determined by PCR. The details of the method are described in Thomas Tiller et al., Journal of Immunological Methods, 350, 2009, 183-193. The amino acid sequences of the variable regions of the heavy and light chains of TX109 (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) are shown in Figures 2 and 3.

[0085] [Example 3] Characterization of TX109 (1) Evaluation of TX109 binding characteristics by ELISA method (method) A chimeric protein (Clec12a-Fc) was created by fusing the extracellular portion of mouse Clec12a (amino acids 65-267 of the 267 total amino acids of mouse Clec12a) with the N-terminal Fc portion of human IgG, according to the method described in the literature (Kanemaru et al, Science Immunol 4: aax6908, 2019). A chimeric protein (Clec1b-Fc) was created by fusing the extracellular portion of mouse Clec1a (amino acids 74-280 out of the 280 total amino acids of mouse Clec1a) with the N-terminal Fc portion of human IgG, following the method described in the literature (Kanemaru et al, Science Immunol 4: aax6908, 2019). A chimeric protein (Clec1b-Fc) was created by fusing the extracellular portion of mouse Clec1b (amino acids 65-275 out of the 275 total amino acids of mouse Clec1b) with the N-terminal Fc portion of human IgG, following the method described in the literature (Kanemaru et al, Science Immunol 4: aax6908, 2019). Human IgG (Cosmo Bio, NO.EVHM-C02-100) or chimeric protein (Clec12b-Fc, Clec12a-Fc, Clec1a-Fc, or Clec1b-Fc, 1 μg in 200 μL of PBS) was added to 96-well plates and coated overnight at 4°C. Then, the plates were washed with 0.05% Tween diluted in PBS, and purified TX109 (0 μg, 0.1 μg, 0.01 μg, or 0.001 μg in 100 μL of PBS) was added, followed by incubation at room temperature for 2 hours. Next, wasabi peroxidase (HRP)-labeled anti-human IgG1 (Jackson Immuno Research Laboratories, NO.109-035-008) or wasabi peroxidase (HRP)-labeled anti-mouse IgG1 secondary antibody (Cytiva, NO.NA931V) was added, and incubation at room temperature for 1 hour. After washing each well with 0.05% Tween diluted in PBS, ABTS TMThe 1-Component Microwell Peroxidase Substrate Kit (Ceracare, NO. 5120-0041) was added, and the OD405 value was measured. The experiment was conducted with n=3.

[0086] (result) The results are shown in Figure 4. From Figure 4, TX109 did not bind to Human IgG (hIgG), Clec12a-Fc (Clec12a), Clec1a-Fc (Clec1a), or Clec1b-Fc (Clec1b), but bound only to Clec12b-Fc (Clec12b). Furthermore, TX109 bound to Clec12b-Fc (Clec12b) in a manner dependent on the amount of TX109 added. It was revealed that TX109 specifically binds to Clec12b.

[0087] (2) Evaluation of the binding characteristics of TX109 to mouse Clec12b by flow cytometry (method) 1 x 10 5 Mouse Clec12b-expressing RMA cells (Flag-mClec12b-RMA) or parental RMA cells (RMA) were treated with 0.5 μg of purified TX109 or anti-Flag antibody (SIGMA, NO. F1804) and incubated on ice for 30 minutes. After washing with PBS, PE-anti-mouseIgG (BioLegend, NO. 405307) was added, incubated on ice for 30 minutes, stained, and analyzed by flow cytometry.

[0088] (result) The results are shown in Figure 5. Representative data from two or three independent experiments are shown. In the upper histogram, the vertical axis represents cell number and the horizontal axis represents anti-Flag. In the lower histogram, the vertical axis represents cell number and the horizontal axis represents TX-109. TX109 did not bind to parental RMA cells, but specifically bound to mouse Clec12b-expressing RMA cells. This revealed that TX109 specifically binds to mouse Clec12b.

[0089] (3) Isotype analysis of TX109 by flow cytometry (method) 1 x 10 5 Mouse Clec12b-expressing RMA cells from cells were treated with 0.5 μg of purified TX109 and incubated on ice for 30 minutes. After washing with PBS, anti-mIgG1 antibody (BD Bioscience, NO. 555749), anti-mIgG2a antibody (BD Bioscience, NO. 553390), anti-mIgG2b antibody (BD Bioscience, NO. 553395), anti-mIgG3 antibody (BD Bioscience, NO. 553403), anti-mIgκ antibody (BD Bioscience, NO. 550003), or anti-mIgλ antibody (BD Bioscience, NO. 553434) were added, incubated on ice for 30 minutes, stained, and analyzed by flow cytometry.

[0090] (result) The results are shown in Figure 6. Representative data from two or three independent experiments are shown. In each histogram, the vertical axis represents the number of cells, and the horizontal axis represents IgG1, IgG2a, IgG2b, IgG3, Igκ, and Igλ, respectively. It was revealed that the subtype of TX109 is IgG1 and that it possesses a κ-type light chain.

[0091] (4) Use of TX109 for immunoprecipitation and Western blotting (method) (Immunoprecipitation) Protein G beads (Thermo Fisher, NO.10004D) conjugated with purified TX109 or anti-Flag antibody (SIGMA, NO.F1804) were incubated with mouse Clec12b-expressing RMA cells or parental RMA cell lysates at 4°C for 2 hours and washed with lysis buffer. Non-reducing SDS buffer was added to the protein G beads, and they were boiled. The supernatant was used as a Western blotting sample (Non-Reducing). In addition, when reducing SDS buffer was added to the protein G beads and they were boiled, the supernatant was used as a reducing sample.

[0092] (Western blotting) The samples were electrophoresed on a 10% TGX gel and then transferred to a polyvinylidene difluoride membrane. After transfer, the membrane was incubated at room temperature for 1 hour with Tris-buffered saline containing 3% BSA and 0.1% Tween 20 to block it. Then, anti-Flag antibody (Sigma, NO. F7425-2MG) was added, and the membrane was incubated at room temperature for 1 hour. After washing, HRP-labeled anti-rabbit IgG antibody (Signa-Aldrich, NO. MFCD00162788) was added and the membrane was incubated at room temperature for 45 minutes. Purified TX109 was biotinylated using a biotin labeling kit (Dojindo, No. LK03) to obtain biotinylated TX109. In Figure 7B, membranes incubated with biotinylated anti-Flag antibody (Sigma, NO. F9291-1MG) or biotinylated TX109 at room temperature for 1 hour were washed and then incubated with SA-HRP (Cytiva, NO. RPN1231V) at room temperature for 45 minutes. After washing, the membranes were treated with SuperSignal TM The West Pico PLUS Luminal / Enhancer solution (Thermo Fisher, NO. 34579) was reacted with a mixture of a stable peroxide solution (Thermo Scientific, NO. 1863096). Fluorescence was detected using LAS4000.

[0093] (result) The results are shown in Figure 7. Representative data from two or three independent experiments are shown. Left (A) shows the results using TX109 for immunoprecipitation (IP) and an anti-Flag antibody (αFlag) for immunoblotting (IB). Right (B) shows the results using an anti-Flag antibody (αFlag) for immunoprecipitation (IP) and a biotinylated anti-Flag antibody (αFlag-bio) or biotinylated TX109 (TX109-bio) for immunoblotting (IB). The cells used are indicated as Tf (mouse Clec12b-expressing RMA cells) and P (parental RMA cells). When protein from mouse Clec12b-expressing RMA cells was immunoprecipitated (IP) with TX109 and immunoblotted (IB) with an anti-Flag antibody, a specific band around 60 kDa was observed under both non-reducing and reducing conditions (Figure 7A). This result suggests that the molecular weight of mouse Clec12b is approximately 60 kDa. Furthermore, when mouse Clec12b-expressing RMA cells were immunoprecipitated (IP) with TX109 and immunoblotted (IB) with either an anti-Flag antibody or TX109, a specific band with the same molecular weight was detected (Figure 7B). These results demonstrate that TX109 is an antibody that can be used for both immunoprecipitation and immunoblotting.

[0094] [Example 4] Analysis of immune-related cells expressing Clec12b (1) Isolation of immune-related cells from tissue (method) (Back Skin) Skin tissue (~12cm) taken from the back of a wild-type mouse after shaving. 2 The ) was cut into small pieces and digested at 37°C for 1 hour in RPMI Medium (cRPMI) containing 2 mg / mL collagenase D (Sigma, NO. 11088882001), 1 μL / mL deoxyribonuclease I (Worthington, NO. LS002139), and 10% fetal bovine serum (FBS).

[0095] (Spleen) The spleen was removed from wild-type mice, the capsule was detached in RPMI Medium (cRPMI), and the cells were extracted to form a single-cell suspension.

[0096] (peritoneal exudate cells (PEC)) The peritoneal cavity of wild-type mice was washed with 1 mL of PBS, and peritoneal effusion cells (PECs) were collected.

[0097] (Colon LP) Tissue from the colon of wild-type mice was cut into small pieces, digested with 0.5 M EDTA at 37°C for 15 minutes, and the epithelial layer was removed. After removal of the epithelial layer, the tissue was further treated with cRPMI containing 2 mg / mL collagenase D and 1 μL / mL deoxyribonuclease I at 37°C for 1 hour, and then filtered through a nylon mesh. Digestion and filtration were repeated at least two times to obtain a single-cell suspension.

[0098] (Heart) Cardiac tissue collected from wild-type mice was reperfused with PBS, immediately washed with Hank's balanced salt solution (HBSS), cut into pieces, and digested at 37°C for 1 hour in cRPMI containing 2 mg / mL collagenase D, 1 μL / mL deoxyribonuclease I, and 100 μM chromolin sodium salt (Sigma, NO. C0399-1G). The mixture was then filtered through a 100 μm nylon mesh filter and centrifuged. The pellet was resuspended in 100 μM chromolin sodium salt solution, and then recovered by density gradient centrifugation with 40% Percoll.

[0099] (2) Analysis by flow cytometry To analyze the endogenous expression of Clec12b, cells extracted from skin, spleen, peritoneal exudate, colonic mucosa lamina propria, and heart were stained with antibodies against cell surface markers, biotinylated TX109, or biotinylated mIgG1 (BD Biosciences, NO. 553441) as primary antibodies, and then stained with streptavidin-conjugated APCas secondary antibody (Abcam, NO. ab243099).

[0100] Antibodies against cell surface markers were used, specifically the following anti-mouse antibodies from BioLegend. The clone names are shown in parentheses: B220 (RA3-6B2), FcεRIα (MAR1), CD49b (DX5), F4 / 80 (BM8), CD11c (N418), mCD11b (M1 / 70), MHCII (M5 / 114.15.2), c-Kit (2B8), CD45.2 (104), CD64 (X54-5 / 7.1), Ly6C (HK1.4), and CD200R (OX-110). The anti-mouse CD3ε antibody used was clone 145-2C11 from BD Bioscience.

[0101] Gating was performed as shown below. Back Skin DC;CD45.2 + CD64 - MHCII + Macrophage (MP); CD45.2 + CD64 + MHCII + T cell;CD45.2 + CD64 - MHCII - CD3ε + Monocyte;CD45.2 + CD64 - MHCII - CD3ε - Ly6C + Mast Cell; CD45.2 + CD64 - MHCII - ckit + FcεRIα +

[0102] A) Spleen Dendritic cell (DC); CD11c + MHCII + Basophilia; CD49 +FcεRIα + T cell;CD3ε + B cell;B220 +

[0103] B)PEC DC;CD11c + MHCII + large peritoneal macrophage(LPM);CD11b hi F4 / 80 hi MHCII + small peritoneal macrophage(SPM);CD11b hi F4 / 80 mid MHCII + T cells;CD3ε + B cells;B220 + Mast cell;CD11b lo F4 / 80 lo MHCII - ckit + FcεRIα +

[0104] C)Colon LPLy6C+ Monocyte(Mo);CD45.2 + MHCII - Ly6C + DC;CD45.2 + MHCII + Macrophage(MP);CD45.2 + MHCII + CD11c - CD11b + Mast Cell;CD45.2 + MHCII - CD11c - ckit + FcεRIα +

[0105] D) Heart CD11b + ;CD45.2 + CD11b + T cell;CD45.2 + CD11b - ckit - FcεRIα - CD3ε + Mast Cell;CD45.2 + CD11b - ckit + FcεRIα +

[0106] (Results) The results in the skin are shown in Figure 8-1, the results in the spleen are shown in Figure 8-2, the results in the peritoneal exudate cells (PEC) are shown in Figure 8-3, the results in the colon lamina propria (Colon LP) are shown in Figure 8-4, and the results in the heart are shown in Figure 8-5. Representative data from 2 or 3 independent experiments are shown. The vertical axis of each histogram is the cell count, and the horizontal axis is Clec12b.

[0107] Clec12b was highly expressed in the skin in the cell populations of CD45.2 + , CD64 - , CD3 - , c-Kit + , FcεRIα - (c-Kit + FcεRIα - ), and in the cell populations of CD45.2 + , CD64 - , CD3 - , c-Kit + , FcεRIα + (c-Kit + FcεRIα + ) (Figure 8-1). Clec12b-positive cells were 83% of c-Kit + FcεRIα - and 84% of c-Kit + FcεRIα + . c-Kit + FcεRIα- and c-Kit + FcεRIα + Since these cells also express CD200R, which is selectively expressed in mast cells, it was suggested that these cell populations are cutaneous mast cells. CD200R-positive cells are c-Kit + FcεRIα - 89% of c-Kit + FcεRIα + It was 100%. + FcεRIα - It is thought to be a precursor of mast cells in the skin.

[0108] Clec12b was not expressed in any cell population in the spleen, colon LP, or heart (Figures 8-2, 8-4, and 8-5). As shown in Figure 8-3, Clec12b was slightly expressed in mast cells of peritoneal effusion cells (PECs), but Clec12b-positive cells accounted for only 39.7% of PEC mast cells.

[0109] From these results, it became clear that Clec12b is highly expressed on cutaneous mast cells and also slightly expressed on peritoneal effusion cells (PECs) mast cells. However, the proportion of mast cells expressing Clec12b (Clec12b-expressing mast cells / mast cells) is greater in the skin than in PECs. In other words, it was revealed that Clec12b is selectively expressed in cells of the cutaneous mast cell lineage, and that the majority of cells in the cutaneous mast cell lineage express Clec12b. It was revealed that Clec12b is selectively expressed in mast cells in mouse skin, just as it is in human skin.

[0110] [Example 5] ADCC activity of TX109 against mouse Clec12b-expressing RMA cells (method) (1) Isolation of mouse NK cells Mouse NK cells were isolated from mouse spleen cells by negative selection using biotinylated anti-CD4 antibody (BioLegend, clone GK1.5), biotinylated anti-CD5 antibody (BioLegend, clone 53-7.3), biotinylated anti-CD8a antibody (BioLegend, clone 53-6.7), biotinylated anti-CD19 antibody (BioLegend, clone 1D3), biotinylated anti-Gr-1 antibody (BioLegend, clone 1D3), biotinylated anti-Ter-119 antibody (BioLegend, clone RB6-8C5), and Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). The isolated mouse NK cells were incubated with recombinant human IL-2 (1000 U / mL, BD Biosciences, NO. 54603) for 1 day to obtain IL-2-stimulated NK cells (IL-2-primed NK). Furthermore, NK cells isolated using the method described above from mice injected with 500 μg of Poly(I:C) one day prior to the assay were designated as Poly(I:C)-stimulated NK cells (Poly(I:C)-primed NK).

[0111] (2) ADCC activity evaluation Effector cells were IL-2-stimulated NK cells or Poly(I:C)-stimulated NK cells, and target cells were mouse Clec12b-expressing RMA cells established in Example 2 or parental RMA cells. The ADCC activity of TX109 was evaluated by the following method. Mouse Clec12b-expressing RMA cells or parental RMA cells were pretreated with purified TX109 (100 μg / mL, labeled as TX109 in the graph) or control mouse IgG1,κ (BioLegend, clone: ​​MOPC-21, 100 μg / mL, labeled as Isotype in the graph), and then co-cultured with IL-2-stimulated NK cells or Poly(I:C)-stimulated NK cells for 5 hours. The expression of CD107a and interferon-γ (IFN-γ) in IL-2-stimulated NK cells or Poly(I:C)-stimulated NK cells was analyzed by flow cytometry. The anti-CD107a antibody used was BioLegend, clone: ​​1D4B, and the anti-interferon-γ (IFN-γ) antibody used was BioLegend, clone: ​​XMG1.2.

[0112] (result) The results are shown in Figure 9. The vertical axis of the graph represents the percentage of IFN-γ-positive cells in NK cells, or the percentage of CD107a-positive cells in NK cells. TX109 significantly enhanced the cytotoxic activity of NK cells against mouse Clec12b-expressing RMA cells, whether using IL-2-stimulated NK cells (IL-2 primed NK) or Poly(I:C)-stimulated NK cells (Poly(I:C)-primed NK). TX109 was found to possess ADCC activity. This indicates that anti-Clec12b antibodies such as TX109 can kill Clec12b-expressing cells, thereby potentially preventing or treating allergic diseases.

[0113] [Example 8] Efficacy of anti-Clec12b in a dermatitis model induced by mite antigen application (method) A schematic of the experimental schedule is shown in Figure 10. The body hair on the back of the necks of wild-type BALB / c mice (obtained from CREA Japan Co., Ltd., wild-type, female, 10-13 weeks old, 6 mice per group) was shaved with an electric shaver and then removed with depilatory cream (Kracie "Epilat Depilatory Cream"). This day was designated as Day-1. The day after hair removal (Day 0), use TX109 or a control antibody ("Ultra-LEAF"). TM Purified Mouse IgG1, κ Ctrl Antibody (clone: ​​MOPC-1), BioLegend, Cat. No. 400197, was administered intradermally at a rate of 2 μg / 20 μL to each auricle, and then at a rate of 5 μg / 50 μL to each of the four corners of a 2 cm x 2 cm area in the upper back of the neck (i.e., a total application volume of 20 μg / 200 μL to the four locations within the 2 cm x 2 cm area in the upper back of the neck). PBS was used as the solvent for the antibody. Furthermore, on the following day (Day 1), 100 mg of mite antigen (ointment containing components derived from house dust mites (Dermatophagoides farinae), atopic dermatitis inducing reagent "Biosta AD", manufactured by Biosta Co., Ltd., Cat. No. 303-34131) was applied to the entire back of the neck. The mite antigen was applied once daily for a total of five times using the same method as on Day 1, and the antibody was administered intradermally every other day for a total of three times using the same method as on Day 0.

[0114] From Day 0 onward, skin inflammation symptoms were scored daily. The scoring was performed using the following method: 5 evaluation items were assessed: 1) Itch score, 2) Redness / bleeding score, 3) Face edema score, 4) Abrasion / erosion score, and 5) Crust formation / drying score. Each item was scored on a scale of 0 for asymptomatic, 1 for mild, 2 for moderate, and 3 for severe. The sum of these five scores was used as the Total score. Itching was evaluated based on the number of scratchings during the 5-minute period 10 minutes after habituation. 0-3 scratchings were scored 0 points, 4-6 scratchings 1 point, 7-9 scratchings 2 points, and 10 or more scratchings 3 points. Redness and bleeding were evaluated based on the degree of redness and the number of bleeding sites in the auricle and cervix, excluding non-coagulated bleeding. Edema was assessed based on the degree of facial swelling. Abrasions and erosions were counted and evaluated on the skin behind the auricle and on the back of the neck. Scabbing and dryness were evaluated by counting the degree of dryness and the number of scabs on the skin of the face and neck.

[0115] Skin samples were collected from the neck and back on Day 6. The collected skin samples were fixed and then embedded in paraffin. Sections 4 μm thick were prepared using a microtome, and stained with hematoxylin eosin and toluidine blue using the following methods.

[0116] (Hematoxylin-eosin staining) As staining reagents, we used hematoxylin solution (Modified Mayer's) (Fujifilm Wako Pure Chemical Industries, Ltd., #131-09665) and 0.25% Eosin Y solution (prepared by diluting 0.5% Eosin-Y solution (Fujifilm Wako Pure Chemical Industries, Ltd., #054-06505) with 99.5% EtOH in a 1:1 ratio). The slide glass with the sections attached was heated at 60°C for 10 minutes to make the sections difficult to detach. Next, the sections were deparaffinized (xylene for 5 minutes x 3 times), rehydrated (99.5% EtOH for 3 minutes x 2 times, 90% EtOH for 1 minute x 2 times, 70% EtOH for 1 minute x 2 times), immersed in 200 mL of hematoxylin solution, incubated at room temperature for 5 minutes to stain the nuclei. Next, the sections were washed with running water for 5 minutes, then immersed in 200 mL of 0.25% Eosin Y solution for 5 minutes to stain the cytoplasm. After lightly washing with tap water, the sections were dehydrated (70% EtOH for 1 minute, 90% EtOH for 1 minute, 99.5% EtOH for 3 minutes x 2 times), cleared (xylene for 5 minutes x 3 times), mounted with MountQuick, and subjected to microscopic observation.

[0117] (Toluidine blue staining) A 0.5% toluidine blue solution (Fujifilm Wako Pure Chemical Industries, Ltd., #209-14545) was used as the staining reagent. The slide glass with the sections attached was heated at 60°C for 10 minutes to make the sections less likely to detach. Next, the sections were deparaffinized (xylene for 5 minutes x 3 times), rehydrated (99.5% EtOH for 2 minutes x 2 times, 90% EtOH for 1 minute, 70% EtOH for 1 minute, dH2O for 1 minute), and then immersed in 200 mL of 0.5% toluidine blue solution and incubated at room temperature for 60 minutes to stain. Next, the sections were lightly washed with dH2O, dehydrated (lightly with 70% EtOH, lightly with 90% EtOH, 99.5% EtOH for 1 minute x 2 times), cleared (xylene for 5 minutes x 3 times), mounted with MountQuick, and subjected to microscopic observation.

[0118] Hematoxylin-eosin stained skin sections were observed under a microscope, measuring 1 mm. 2 Five locations were randomly selected in the epidermal layer, and the thickness of the epidermal layer was measured using Keyence BZ-X analyzer. The average value of the five locations was used as the thickness of the epidermal layer. Toluidine blue-stained skin sections were observed under a microscope, with each field of view measuring 1 mm. 2 The number of mast cells exhibiting metachromosis was measured.

[0119] (result) Microscopic images of skin sections stained with hematoxylin eosin (HE) or toluidine blue (Toluidine blue) are shown in Figure 11. Three representative images from each group are shown. Epidermal thickness and unit area (mm²) are also shown. 2 The number of mast cells per 10 When mite antigens are applied to induce dermatitis, the epidermis thickens and more mast cells are found in the dermis. However, when TX109 is administered, the epidermal thickness and area per unit area (mm²) are improved compared to when a control antibody (cIg) is administered. 2 The number of mast cells per unit area had improved (Figures 11 and 12). Furthermore, administration of TX109 resulted in improvement in dermatitis scores across all five categories compared to control antibody administration: itch score, face edema score, redness / bleeding score, abrasion / erosion score, crust formation / drying score, and the total score of these five categories. It has been revealed that TX109 reduces mast cells in the skin and alleviates dermatitis symptoms. These results indicate that anti-Clec12b antibodies are useful as a preventive or therapeutic agent for allergic diseases.

Claims

1. An agent for the prevention or treatment of allergic diseases comprising an anti-Clec12b antibody or a fragment thereof, The anti-Clec12b antibody or its antibody fragment binds to all or part of the extracellular region of Clec12b, A preventive or therapeutic agent for allergic diseases, wherein the allergic disease is a skin allergic disease involving mast cells.

2. The anti-Clec12b antibody has cytotoxic activity against Clec12b-expressing cells, wherein the agent for the prevention or treatment of allergic diseases according to claim 1.

3. The preventive or therapeutic agent for allergic diseases according to claim 2, wherein the Clec12b-expressing cells are mast cells.

4. The preventive or therapeutic agent for allergic diseases according to claim 2 or 3, wherein the Clec12b-expressing cells are cutaneous mast cells.

5. The preventive or therapeutic agent for allergic diseases according to any one of claims 2 to 4, wherein the cytotoxic activity is antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

6. The anti-Clec12b antibody is an antibody that removes Clec12b-expressing cells, as described in any one of claims 1 to 5, for the prevention or treatment of allergic diseases.

7. The preventive or therapeutic agent for an allergic disease according to any one of claims 1 to 6, wherein the skin allergic disease is at least one selected from atopic dermatitis, contact dermatitis, and urticaria.

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