JP2008141995A - Use of gp2 as intestinal canal m cell marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a molecule which can be used as a universal M cell marker, having general-purpose properties that are superior to those of UEA-1 (Ulex europaeus agglutinin-1) which is a conventional M cell marker, and to provide a medical drug that utilizes the molecule and a means, or the like, which is useful for the development of medical drugs. <P>SOLUTION: A method for detecting or identifying the M cell includes a means for measuring the expression of GP2 (glycoprotein 2), wherein a reagent or a kit for detecting or identifying the M cell comprises one or more means of measuring the GP2; a drug delivery medium comprises a GP2 targeting molecule; the medical drug comprises the GP2 targeting molecule and an active ingredient as the pharmaceutical; a regulator of mucosal immunity comprises a substance capable of regulating the expression or the function of the GP2; the M cell, in which the expression of the GP2 is regulated, is provided, or a tissue containing the M cell is provided; a method for evaluating endocytosis in the M cell includes measuring the expression or the function of the GP2, in the M cell or the tissue containing the M cell; a binder for a microorganism comprises the GP2 in a secreted form; a complex comprises the GP2 and the microorganism or a component thereof and a method, and so on, for evaluating the mucosal immunity in a mammal are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention provides an M cell detection or identification method, an M cell detection or identification reagent or kit, a drug delivery vehicle containing a GP2 targeting molecule, a medicament, and the like.

  Peyer's patch (PP) is one of the gut-associated lymphoid tissues (GALT) and is an important starting site of mucosal immunity. Mucosal lymphoid follicles are covered by specialized follicle-associated ephithelium (FAE) containing M cells that mediate luminal antigen uptake and transepithelial transport. Although M cells are thought to play an important role in mucosal immunity by mediating luminal antigen uptake and transepithelial transport, the molecular mechanism of antigen uptake and transport remains unclear. In particular, the identification of endocytic receptors on the apical membrane of M cells is an interesting subject of this problem.

  As a marker molecule for M cells, Ulex europaeus agglutinin-I (UEA-1), which is a kind of lectin that recognizes α-L-fucose expressed in M cells of mouse PP, is known. There is a problem that other GALT such as cecal lymphoid follicles, colonic lymphoid follicles and human M cells cannot be recognized.

  By the way, glycoprotein 2 (GP2) is 1) a GPI-anchored protein localized in the lipid microdomain, and 2) expressed in the secretory granule membrane of acinar cells of the exocrine pancreas. It is known that it has the molecular characteristics of self-oligomerization, and 4) it is secreted from the acinar cells into the digestive tract lumen (Non-Patent Documents 1 to 4). GP2 knockout animals have also been reported, but no particular phenotypic abnormality was observed, and their functions remain unclear (Non-Patent Documents 2 and 5).

  In addition, Tamm-Horsfall protein (THP) secreted from renal epithelial cells, which is the most abundant protein in mammalian urine and has high homology with GP2 (about 51% homology at the amino acid level), It has been reported that it has the ability to bind to E. coli (Non-patent Document 6).

  The present inventors have also conducted transcriptome analysis using follicle-associated epithelium (FAE) and intestinal epithelial cells (IEC) derived from mice (Non-patent Document 7).

However, the above prior art documents that GP2 is a molecule that is specifically expressed in M cells, in particular that GP2 can be used as a specific marker molecule for human M cells, and that GP2 is optional. There is no description or suggestion that it can be expressed in common in M cells in different types of tissues.
Schmidt, K. et al., J. Biol. Chem. 2001, 276, 14315-23. Yu, S. et al., J. Biol. Chem., 2004, 279, 50274-9. Fukuoka, S. et al., Proc. Natl. Acad. Sci. USA., 1992, 89, 1189-93. Beaudoin, AR et al., J. Histochem.Cytochem., 1991, 39, 575-88. Kobayashi, K. et al., Biochem. Biophys. Res. Commun., 2004, 322, 659-64. Pak, J. et a., J. Biol. Chem., 2001, 276, 9924-30. Hase, K. et al., DNA Res., 2005, 12, 127-37.

  One object of the present invention is to provide a molecule that can be used as a pan-M cell marker, which is more versatile than the conventional M-cell marker UEA-1. Another object of the present invention is to provide a drug and a useful means for developing a drug based on the knowledge obtained by elucidating the mechanism of luminal antigen uptake and transepithelial transport in M cells. .

As a result of intensive studies, the present inventors have found that 1) GP2 is specifically expressed in both human and mouse M cells. 2) GP2 is not only M cells in Peyer's patches but also cecal lymphoid follicles. 3) GP2 is localized in the apical plasma membrane of M cells, and 4) GP2 binds to microorganisms. 5) It has been found that GP2 can be involved in transepithelial transport of microbial antigens in M cells. That is, the present inventors have succeeded in identifying for the first time a molecule (GP2) that can be used as a pan-M cell marker that exceeds species (eg, human, mouse) and tissue. The present inventors have also revealed that GP2 can participate in mucosal immunity by incorporating intestinal microorganisms as endocytosis receptors.
Based on the above, the present inventors have completed the present invention. That is, the present invention is as follows.
[1] A method for detecting an M cell, comprising measuring the expression of GP2.
[2] The detection method according to [1] above, which is a method for detecting human M cells.
[3] The detection method according to [1] above, which is a method for detecting mouse M cells.
[4] A method for identifying M cells, comprising measuring GP2 expression in an intestinal mucosa-derived sample containing M cells.
[5] The identification method according to [4] above, wherein the intestinal mucosa-derived sample contains at least Peyer's patch, isolated lymph node, cecal or large intestine lymphoid follicle.
[6] A reagent or kit for detecting or identifying M cells, comprising one or more means for measuring GP2 selected from the group consisting of a primer, a nucleic acid probe, and an antibody.
[7] A drug delivery vehicle comprising a GP2 targeting molecule.
[8] The drug delivery vehicle according to [7], wherein the GP2 targeting molecule is an antibody.
[9] A medicament comprising a GP2 targeting molecule and a pharmaceutical active ingredient.
[10] The medicament of [9] above, wherein the active ingredient is an antigen.
[11] A mucosal immunity regulator comprising a substance capable of regulating the expression or function of GP2.
[12] An M cell in which GP2 expression is regulated or a tissue containing the same.
[13] A method for evaluating endocytosis in M cells, comprising measuring the expression or function of GP2 in M cells or tissues containing the same.
[14] A binding agent for a microorganism, comprising a secreted form of GP2.
[15] A complex comprising GP2 and a microorganism or a component thereof.
[16] A method for evaluating mucosal immunity in a mammal, including any of the following (a) or (b):
(A) subjecting a mammal in which expression of GP2 is regulated to measurement of mucosal immunity; or (b) subjecting the mammal to measurement of expression or function of GP2.

The detection or identification method of the present invention is not limited to detection or identification of M cells, but also quantification of M cells, diagnosis of diseases that can be associated with M cells, a mucosal immunity regulator, and a mucosal immunity regulating action. It can also be used for the development of pharmaceuticals. In the detection or identification method of the present invention, for example, specific detection or identification of human M cells can be realized for the first time by using GP2 as a human M cell marker, and / or GP2 is used as an M cell marker. By doing so, it is possible to realize specific detection or identification of M cells in an arbitrary tissue for the first time, and there is a particularly remarkable effect.
The reagent or kit of the present invention can be useful, for example, for simple implementation of the detection or identification method of the present invention.
The drug delivery vehicle or medicament of the present invention may be useful for the modulation of mucosal immunity via M cells, for example by specific targeting to M cells.
The modulator of the present invention can be useful, for example, in the regulation of mucosal immunity.
The cell or tissue of the present invention may be useful for, for example, development of a mucosal immunity regulator, analysis of antigen uptake and transport in M cells, search for endocytosis-related genes, and the like. The present invention also provides a method of using the cell or tissue of the present invention (endocytosis evaluation method).
The binding agents of the present invention may be useful, for example, for labeling microorganisms, promoting in vitro elimination of microorganisms, and innate immunity. The present invention also provides a complex comprising GP2 and microorganisms or components thereof that can be formed thereby.
The method for evaluating mucosal immunity using animals according to the present invention may be useful because it enables the development of mucosal immunity modulators in vivo and ex vivo.

  The present invention provides a method for detecting or identifying M cells.

  The detection or identification method of the present invention can include, for example, measuring the expression of GP2. The M cell can be derived from the intestinal mucosa. Therefore, the detection or identification method of the present invention may be performed using an intestinal mucosa-derived sample containing M cells. Examples of intestinal mucosa-derived samples containing M cells include, for example, M cell-containing tissues collected from animals (eg, small intestine, duodenum, colon, jejunum, ileum, large intestine, cecal mucosa), M cells or tissues containing the same. A sample derived from the culture can be mentioned. The sample used in the present invention may also be a GLT containing M cells or a constituent tissue thereof. Examples of GALT containing M cells or their constituent tissues include Peyer's patches, isolated lymph nodes, large intestine, cecal lymph follicles, and the like. The animal from which the sample or the M cells contained therein are derived can be mammals such as primates and rodents and birds, and examples thereof include humans, monkeys, cows, pigs, mice, rats, rabbits, and chickens. .

  The expression of GP2 can be measured by a method known per se for the transcription product of GP2 gene or GP2 protein. For example, the expression level of the transcription product of the GP2 gene can be determined after preparing total RNA from a biological sample, followed by PCR (eg, RT-PCR, real-time PCR, quantitative PCR), LAMP (Loop-mediated isothermal amplification) (eg, WO00 / 28082), gene amplification methods such as ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids) (see, for example, WO00 / 56877), Northern blotting, microarray and the like. In addition, the expression level of the translation product can be measured by an immunological technique after preparing an extract from a biological sample or a tissue section. Examples of such immunological techniques include enzyme immunoassay (EIA) (eg, direct competition ELISA, indirect competition ELISA, sandwich ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Examples include Western blotting and immunohistochemical staining. Examples of methods other than the above that enable measurement of GP2 expression include mass spectrometry.

  The detection or identification method of the present invention can be used not only for the detection or identification of M cells but also for the quantification of M cells. The detection or identification method of the present invention can also be used for diagnosis of diseases associated with M cells. For example, M cells may be involved in diseases such as inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis).

  The detection or identification method of the present invention can also be used to screen for subjects that can modulate (eg, increase or decrease) the number of M cells or cannot change the number of M cells. This makes it possible to develop a subject that can or can not change the number of M cells, or to identify a subject that can regulate mucosal immunity. Therefore, the detection or identification method of the present invention includes, for example, (a) a step of bringing a test substance into contact with an M cell or a tissue containing the same, and (b) measuring GP2 expression in the M cell or the tissue containing the same. And (c) selecting a substance that can regulate or cannot change the number of M cells. Substances that can regulate the number of M cells include, for example, prevention of infection of bacteria (eg, Salmonella, Yersinia) (eg, Clark, MA et al., Infect Immun 1996, 64, 4363-4368; Clark, MA et al. , Infect. Immun., 1998, 66, 1237-1243.), A substance that can be useful in the regulation of mucosal immunity and cannot change the number of M cells has other actions as a pharmacological action, and the above action May be useful in the development of certain medicines that do not have

  The subject to be subjected to the screening method may be any compound or composition, such as a nucleic acid (eg, nucleoside, oligonucleotide, polynucleotide), carbohydrate (eg, monosaccharide, disaccharide, oligosaccharide, Polysaccharides), lipids (eg, saturated or unsaturated linear, branched and / or cyclic fatty acids), amino acids, proteins (eg, oligopeptides, polypeptides), small organic compounds, combinatorial chemistry techniques Examples thereof include a prepared compound library, a random peptide library prepared by solid phase synthesis or a phage display method, natural components (eg, components derived from microorganisms, animals and plants, marine organisms, etc.), foods, drinking water and the like.

  The present invention also provides a reagent or kit for detecting or identifying M cells. The reagent or kit of the present invention may contain one or more means for measuring GP2. The means for measuring GP2 is not particularly limited as long as GP2 expression can be measured, and examples thereof include primers, nucleic acid probes, and antibodies.

  The primer that can be contained in the reagent or kit of the present invention is not particularly limited as long as it enables amplification and detection of the GP2 gene. For example, the primer size can be about 12 bp or more, preferably about 15-100 bp, more preferably about 16-50 bp, and even more preferably about 18-35 bp. In addition, since the number of primers required differs depending on the type of gene amplification method, the number of primers that can be included in the reagent of the present invention is not particularly limited. For example, the reagent of the present invention includes two or more primers. obtain. Two or more primers may be mixed in advance or may not be mixed. Such a primer can be prepared by a method known per se.

  The nucleic acid probe that can be included in the reagent or kit of the present invention is not particularly limited as long as it enables detection of the GP2 gene. The nucleic acid probe may be either DNA or RNA, but DNA is preferable in consideration of stability and the like. Nucleic acid probes can also be either single stranded or double stranded. The size of the nucleic acid probe is not particularly limited as long as it can specifically hybridize to the transcription product of the GP2 gene, but is, for example, about 15 or 16 or more, preferably about 15 to 1000 bp, more preferably about 50 to 500 bp. The nucleic acid probe may be provided in a form immobilized on a substrate like a microarray. Such a nucleic acid probe can be prepared by a method known per se.

The antibody that can be contained in the reagent or kit of the present invention is not particularly limited as long as it is an antibody that can specifically bind to the GP2 protein. For example, the antibody against GP2 protein may be either a polyclonal antibody or a monoclonal antibody. The antibody may also be a fragment of an antibody (eg, Fab, F (ab ′) ₂ ), a recombinant antibody (eg, scFv). The antibody may be provided in a form immobilized on a substrate such as a plate. The antibody can be produced by a method known per se.

  For example, a polyclonal antibody may be a commercially available adjuvant using GP2 protein or a partial peptide thereof (if necessary, a complex cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin)) as an antigen. (Eg, complete or incomplete Freund's adjuvant) and administered subcutaneously or intraperitoneally 2-4 times every 2 to 3 weeks (measure the antibody titer of partially collected serum by a known antigen-antibody reaction, It can be obtained by collecting the whole blood about 3 to about 10 days after the final immunization and purifying the antiserum. Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

  Monoclonal antibodies can be obtained by cell fusion methods (eg, Takeshi Watanabe, principles of cell fusion methods and preparation of monoclonal antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the factor is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and about 3 days after the final administration, the spleen or lymph node is collected and white blood cells are collected. The leukocytes and myeloma cells (eg, NS-1, P3X63Ag8, etc.) are fused to obtain a hybridoma that produces a monoclonal antibody against the factor. The cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a well-known EIA or RIA method. The hybridoma producing the monoclonal antibody can be cultured in vitro or in vivo, such as mouse or rat, preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively.

  Furthermore, the antibody used in the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. Chimeric antibodies include, for example, “Experimental Medicine (Special Issue), Vol. 6, No. 10, 1988”, Japanese Patent Publication No. 3-73280, and humanized antibodies include, for example, Japanese Patent Publication No. 4-506458, JP-A-62-296890 and the like, human antibodies include, for example, `` Nature Genetics, Vol.15, p.146-156, 1997 '', `` Nature Genetics, Vol.7, p.13-21, 1994 '', JP 4-504365 Publication, International Application Publication No. WO 94/25585 Publication, `` Nikkei Science, June, 40 to 50, 1995 '', `` Nature, Vol.368, p.856-859, 1994 ", Japanese Patent Publication No. 6-500233, and the like. For example, as described below, when the antibody used in the present invention is used as a medicine, a humanized antibody and a human-type antibody are preferable.

The means for measuring GP2 can be provided in a form labeled with a labeling substance, if necessary. Examples of the labeling substance include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, and ¹²³ I, biotin, and streptavidin. And the like, and the like.

  The reagent or kit of the present invention may be provided in a form including additional components in addition to the means for measuring GP2. In this case, each component included in the reagent or kit may be provided in a form isolated from each other, for example, stored in a different container. For example, when the GP2 measurement means is not labeled with a labeling substance, the reagent or kit of the present invention may further contain a labeling substance. The reagent or kit of the present invention may further contain a positive control (eg, a polynucleotide encoding the GP2 gene or a partial nucleotide thereof, a GP2 protein or a partial peptide thereof).

  The present invention also provides a drug delivery vehicle comprising a GP2 targeting molecule.

  The GP2 targeting molecule is not particularly limited as long as it is a substance that can target GP2, and examples thereof include antibodies and aptamers. Examples of the antibody include those described above. From the viewpoint of clinical application, a humanized or human antibody is preferable.

  The drug delivery vehicle used in the present invention is not particularly limited as long as it can bind to the GP2 targeting molecule and can deliver the active ingredient, and examples thereof include liposomes and microspheres. The GP2 targeting molecule can be covalently or non-covalently bound to the drug delivery vehicle. The amount of GP2 targeting molecule to be bound to the drug delivery vehicle can be adjusted accordingly.

  The present invention also provides a medicament comprising a GP2 targeting molecule and an active ingredient.

  The medicament of the present invention may contain, for example, a GP2 targeting molecule covalently or non-covalently bound to the active ingredient. If the GP2 targeting molecule and the active ingredient are covalently bound to each other, they can be bound without a linker or via a suitable linker. The medicament of the present invention may also contain a GP2 targeting molecule and an active ingredient that are not bound to each other. In this case, the medicament of the present invention may be provided in the form of a drug delivery vehicle as described above.

  Examples of the active ingredient that can be included in the medicine include antigens (eg, viral, bacterial, and parasitic antigens that can be used as vaccines), substances that can regulate the expression or function of GP2 described later, and drugs.

  When the medicament of the present invention contains an antigen, it may further contain an adjuvant. The adjuvant is not particularly limited as long as it can enhance immunogenicity. For example, BCG, trehalose dimycolate (TDM), Merck 65, AS-2, aluminum phosphate, aluminum hydroxide, keyhole limpet Examples include hemocyanin, dinitrophenol, dextran, and TLR ligands (eg, lipopolysaccharide (LPS), CpG).

  The medicament of the present invention can be used in the same manner as the agent of the present invention described later. For example, the medicament of the present invention may contain a pharmaceutically acceptable carrier described below.

  The present invention also provides a modulator of mucosal immunity. The agent of the present invention may contain a substance capable of regulating the expression or function of GP2.

  GP2 can be human GP2 (eg, GenBank accession number: NP_001493) or an ortholog thereof (eg, for mouse GP2, see, for example, Genbank accession number: NP_080265), or a variant thereof. The ortholog of GP2 is not particularly limited, and can be derived from, for example, a non-human mammal (eg, cow, sheep, pig, goat, monkey, rabbit, rat, hamster, guinea pig, mouse). Each GP2 has one or more (for example, 1 to 10, preferably 1 to 5, more preferably 1, 2 or 3) amino acid mutations in the encoded amino acid sequence as long as it can have endocytotic activity. (Eg, deletion, substitution, addition, insertion) (such a protein corresponds to the “mutant” described above).

  In one embodiment, the agent of the present invention may be a mucosal immunity enhancer. In this case, the agent of the present invention may contain a substance that can promote the expression or function of GP2.

  The expression of GP2 means that a translation product (that is, a protein) is produced from a gene encoding GP2, and is localized in its functional site in a functional state. Therefore, the substance that promotes the expression of GP2 may act at any stage of GP2 transcription, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding. As used herein, promotion of GP2 expression includes supplementation of GP2 (protein) itself.

  Examples of substances that promote the expression or function of GP2 include GP2 and its expression vector.

  GP2 can be a natural protein or a recombinant protein. GP2 can be prepared by a method known per se. For example, a) GP2 may be recovered from a biological sample containing GP2, and b) a host cell (eg, Escherichia, Bacillus, yeast, insect cell, insect) A transformant may be prepared by introducing a GP2 expression vector into animal cells), and the components produced by the transformant may be recovered. C) Rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate GP2 may be synthesized by a cell-free system using the above. GP2 is a method that utilizes solubility such as salting out and solvent precipitation; a method that mainly uses a difference in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; A method utilizing charge difference such as exchange chromatography; a method utilizing specific affinity such as affinity chromatography and use of GP2 antibody; a method utilizing hydrophobic difference such as reverse phase high performance liquid chromatography; etc. A method using a difference in isoelectric point such as an electric point electrophoresis method;

  In another embodiment, the agent of the present invention may be an inhibitor of mucosal immunity. In this case, the agent of the present invention may contain a substance that can suppress the expression or function of GP2. The substance that suppresses the expression of GP2 may act at any stage such as GP2 transcription, post-transcriptional regulation, translation, post-translational modification, localization, and protein folding.

  Examples of substances that suppress the expression of GP2 include antisense nucleic acids (eg, DNA, RNA, modified nucleotides, or chimeric molecules thereof), ribozymes, RNAi-inducible nucleic acids (RNAi effects by being introduced into cells) Polynucleotides, preferably RNA (eg, siRNA), aptamers, and expression vectors containing nucleic acids encoding them.

  Examples of the substance that suppresses the function of GP2 include the above-described antibodies, dominant negative mutants or expression vectors thereof, and low molecular compounds.

  The dominant negative mutant of GP2 has reduced activity due to the introduction of a mutation into GP2. The dominant negative mutant can indirectly inhibit its function by competing with natural GP2. Examples of the mutation include an amino acid mutation (eg, deletion, substitution, addition of one or more amino acids) that causes a decrease in the function of the functional site.

  When the substance that regulates the expression or function of GP2 is a nucleic acid molecule or a protein molecule, the agent of the present invention can contain an expression vector containing a nucleic acid molecule encoding the nucleic acid molecule or protein molecule as an active ingredient. In the expression vector, the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in a mammalian cell to be administered. The promoter that can be included in the expression vector of the present invention is not particularly limited as long as it allows the expression of the factor under its control, and may be appropriately selected depending on the type of factor. For example, the polIII promoter (eg, tRNA promoter, U6 Promoter, H1 promoter) and mammalian promoter (eg, CMV promoter, CAG promoter, SV40 promoter). In addition, as a promoter to be used, a promoter specific to M cells (eg, a natural promoter of GP2) may be used.

  The expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule. Furthermore, a selection marker gene for selecting transformed cells (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.) is further included. You can also

  The basic skeleton vector used as the expression vector may be a plasmid or a viral vector, but suitable vectors for administration to mammals such as humans include adenovirus, retrovirus, adeno-associated virus, herpes virus, vaccinia. Examples of the virus vector include viruses, poxviruses, polioviruses, Sindbis viruses, and Sendai viruses.

  The agent of the present invention may contain any carrier, for example, a pharmaceutically acceptable carrier, in addition to the substance that regulates the expression or function of GP2. Examples of pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , Gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Aerosil, Talc, Sodium lauryl sulfate lubricant, Citric acid, Menthol, Glycyllysine / Ammonium salt, Glycine, Orange powder, Fragrance, Sodium benzoate Preservatives such as lithium, sodium hydrogen sulfite, methyl paraben, propyl paraben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methyl cellulose, polyvinyl pyrrolidone, aluminum stearate, dispersants such as surfactants, Examples include, but are not limited to, water, physiological saline, diluents such as orange juice, base waxes such as cacao butter, polyethylene glycol, and white kerosene.

  Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, sachets or tablets containing an effective amount of the substance as solids or granules. A suspension in which an effective amount of a substance is suspended in a suitable dispersion medium, an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium and emulsified, or a powder, a granule or the like.

  Formulations suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local infusion, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants Further, a buffer solution, an antibacterial agent, an isotonic agent and the like may be contained. Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like. The preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials. In addition, the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.

  The dosage of the agent of the present invention includes the activity and type of the active ingredient, the mode of administration (eg, oral and parenteral), the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, body weight, age Usually, it is about 0.001 mg to about 5.0 g as an active ingredient amount per day for an adult, although it may not be unconditionally different depending on etc. The agent of the present invention can be used in the same manner as the aforementioned medicament of the present invention. For example, the medicament of the present invention may contain the above-mentioned adjuvant.

  The agent of the present invention is useful, for example, as a medicine. When the agent of the present invention is used as a medicine, it can be used, for example, for diseases or conditions for which prevention or treatment of diseases accompanied by abnormal mucosal immunity or enhancement of mucosal immunity is desired. Examples of diseases accompanied by abnormal mucosal immunity include inflammatory bowel diseases (eg, Crohn's disease, ulcerative colitis), and food allergies. Examples of diseases or conditions for which enhancement of mucosal immunity is desired include cancer and infections (eg, infections caused by microorganisms such as bacteria, viruses or parasites). A subject to which the agent of the present invention can be applied may be a subject who has or is at risk of developing two or more of the above diseases or conditions.

  The present invention also provides an M cell in which GP2 expression is regulated or a tissue containing the same. The cell or tissue of the present invention can be derived from the mammal described above. The cells of the present invention can be derived from the tissues described above. The tissue of the present invention can be similar to the tissue described above.

  The cell or tissue of the present invention may be, for example, a cell or tissue in which GP2 expression is enhanced, in other words, a GP2 gene is introduced. The GP2 gene to be introduced is not particularly limited. For example, the GP2 gene and the cell or tissue into which it is introduced can be derived from the same or different mammals, respectively. GP2 can also be non-mutant or mutant. Examples of GP2 mutants include those described above. For example, a fusion with another protein (eg, a fluorescent protein such as GFP), a mutation that is resistant to cleavage of the GP2 protein by a protease. Introduced modified proteins, deletions lacking a given domain (eg, see Examples) are also included. The cell or tissue of the present invention can also be a cell or tissue in which the expression of GP2 is suppressed, for example, the GP2 gene is disrupted or the expression of the GP2 gene is suppressed by a trans-acting factor.

  The cells of the present invention can be isolated and / or purified. The cell of the present invention may be a cell whose endocytic ability is regulated by regulation (eg, promotion, suppression) of GP2 expression. The cell of the present invention is a cell in which the expression of GP2 is transiently regulated, a cell in which the expression is constitutively regulated (eg, homozygous or heterozygous defective cells, or a gene introduced into the genome). Genome-modified cells). The cells of the present invention can be transformants or non-transformants. The cells of the present invention can also be cells that have been or have not been treated with a microorganism, such as a bacterium, or a component thereof.

  The cells of the present invention can be produced, for example, by treating M cells with a substance that can regulate the expression or function of GP2. The cells of the present invention can also be produced by isolating and / or purifying M cells from transgenic animals and gene-deficient (so-called knockout) animals. For example, a GP2 knockout animal (eg, Yu, S. et al., J. Biol. Chem., 2004, 279, 50274-9 .; Kobayashi, K. et al., Biochem. Biophys. Res. Commun., 2004 , 322, 659-64)), the cells and tissues of the present invention can be prepared from such animals in the present invention.

  The cells of the present invention can be produced, for example, by a) development of a preventive, therapeutic and / or ameliorating agent for the above-mentioned diseases or conditions (eg, use in the screening method of the present invention), b) antigen uptake and transport in M cells That is, it may be useful for analysis of endocytosis), c) search for endocytosis-related genes, and the like.

  The present invention also provides a method for evaluating endocytosis in M cells, comprising measuring GP2 expression or function in M cells or tissues containing the same. The measurement of GP2 expression can be performed as described above. The measurement of the function of GP2 can be performed under a fluorescence microscope using, for example, a fluorescently labeled antigen or antigen mimetic (eg, an antibody against GP2) as described in Examples described later. Of course, using methods known in the art for evaluating endocytosis (eg, methods similar to methods for evaluating antigen uptake and transport by receptors such as Fc receptor, C-type lectin receptor, and Toll-like receptor), GP2 It is also possible to measure the function. This evaluation method can be useful, for example, for screening for substances that can or cannot regulate endocytosis in M cells. Thus, the method can be, for example, a method comprising assessing whether a subject can modulate GP2 expression or function in M cells. More specifically, the method comprises (a) contacting a test substance with an M cell or a tissue containing the same, (b) measuring GP2 expression or function in the M cell, (c) in the M cell. The method may include a step of selecting a substance that can or cannot regulate endocytosis. For example, substances that can modulate endocytosis in M cells can be useful in the regulation of mucosal immunity, substances that cannot alter endocytosis in M cells have other pharmacological effects, and It may be useful for the development of certain medicines that do not have mucosal immunomodulatory action.

  The present invention also provides a method for screening a GP2 targeting molecule, comprising measuring whether a test substance (eg, those described above, and a test antigen derived from a microorganism) binds to GP2. Measurement of the binding of the test substance to GP2 can be carried out by a method known per se, such as immunological techniques (eg, immunoprecipitation, ELISA), interaction analysis using surface plasmon resonance, two-hybrid System.

  The present invention also provides a binding agent for a microorganism comprising a secreted form of GP2. It is known that GP2 can be cleaved by a protease to become a secreted form (see, for example, Fritz, B. A. et al., Am. J. Physiol., 1996, 270, G176-83). Accordingly, the binding agent of the present invention may contain such cleaved GP2. The microorganism is not particularly limited as long as GP2 can bind thereto, and examples thereof include bacteria such as Escherichia coli, fungi, and yeast. Moreover, as long as GP2 can be combined, it can be similarly applied not only to microorganisms but also to foreign substances such as viruses and parasites. The binding agents of the present invention may be useful, for example, for labeling microorganisms, promoting in vitro elimination of microorganisms, and innate immunity.

  The present invention also provides a complex comprising GP2 and a microorganism or component thereof. The component of the microorganism that can be included in the complex of the present invention can be one having a binding ability to GP2 (ie, an antigen). Since GP2 has a self-oligomer ability, GP2 may be oligomerized in the complex of the present invention to form a higher-order complex. The complex of the present invention can be prepared, for example, by contacting GP2 with a microorganism or a component thereof to obtain a complex containing GP2 and the microorganism or a component thereof. The present invention also provides a method for producing such a composite.

  The present invention also provides a method for evaluating mucosal immunity using animals. The method can be performed in vivo and ex vivo. The method can include, for example, (a) subjecting an animal with regulated expression of GP2 to a mucosal immunity measurement, or (b) subjecting the animal to a measurement of GP2 expression or function. The animal may be the same as described above, but may be an animal that causes abnormal mucosal immunity (eg, an animal with reduced mucosal immunity). For example, the method can include administering a test to an animal, subjecting the animal to which the test is administered to a mucosal immunity measurement, and assessing whether the test can modulate mucosal immunity. The method can also include applying an external stimulus to the animal, subjecting the animal to which the external stimulus has been applied to measurement of mucosal immunity and assessing whether the external stimulus can modulate mucosal immunity. The external stimulus can be any stimulus other than test subject administration (eg, stimulus related to tactile, auditory, visual, etc.). Measurement of mucosal immunity can be performed, for example, based on measurement of IgA amount, mucin, antibacterial peptide amount, etc. in mucosal tissue. The method can be useful, for example, for screening for subjects or external stimuli that can or cannot regulate mucosal immunity.

  The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

(Method)
1) Animals BALB / c mice were purchased from CLEA Japan and maintained under specific pathogen-free conditions in RIKEN animal facilities until they were used in experiments at 6-10 weeks of age. All animal experiments were approved by the Animal Research Committee of RIKEN Yokohama Institute.

2) cDNA clone and plasmid construction To produce mouse and human GP2 recombinant proteins, cDNA corresponding to C-terminal transmembrane region or ZP domain deleted GP2 (GP2 (ΔTM) or GP2 (ΔZP), respectively) Amplified from RIKEN FANTOM clone or human FAE, respectively. Polymerase chain reaction (PCR) was performed using specific primers for each gene. Primer sequences are as follows.
Mouse GP2 (ΔTM) (mGP2 (ΔTM))
5'-GAAGATCTACCATGAAAAGGATGGTGGGTT-3 '(forward: SEQ ID NO: 1)
5'-CCGCTCGAGTGTAGTGTGGGGAGTCCCC-3 '(Reverse: SEQ ID NO: 2)
Human GP2 (ΔTM) (hGP2 (ΔTM))
5'- CGCGGATCCACCATGGAAAGGATGGTGGGC-3 '(forward: SEQ ID NO: 3)
5'-CCGCTCGAGTCCATTCATGACACCGGGAGA-3 '(reverse: SEQ ID NO: 4)
mGP2 (ΔZP)
5'-GAAGATCTACCATGAAAAGGATGGTGGGTT-3 '(forward: SEQ ID NO: 5)
5′-CCGCTCGAGCAGAAGAGGCTGCAAACTCTG-3 ′ (reverse: SEQ ID NO: 6)
The resulting cDNA fragment, containing the fragment encoding the Fc segment of human IgG _1, was inserted into BamH1 / XhoI cloning sites of modifications of pcDNA3 expression vector (Invitrogen) (pcDNA3-Fc) . N-terminal sequences of mouse and human GP2 (GP2-N) were prepared in the same way as GP2 (ΔTM) using specific primers for each gene. Primer sequences are as follows.
mGP2
5'-GAAGATCTCGATTCATCAGTTGCATGGTG-3 '(forward: SEQ ID NO: 7)
5'-CCGCTCGAGGAAACCCATACCTCCCAGCA-3 '(reverse: SEQ ID NO: 8)
hGP2
5'-CGCGGATCCGCGATAAAAACATGAGCGGC-3 '(forward: SEQ ID NO: 9)
5'- CCGCTCGAGCTCTCCAGAATGTTCCTGCAG-3 (Reverse: SEQ ID NO: 10)
The resulting cDNA fragment was inserted into the BamHI / XhoI cloning site of the pcDNA3 expression vector.

3) Preparation of recombinant human or mouse GP2 protein Human embryonic kidney (HEK) 293T cells were transiently transfected with GP2 (ΔTM) or GP2 (ΔZP) / pcDNA3-Fc plasmid vector and 5% CO ₂ incubator Incubated at 37 ° C for about 10 days. Secreted recombinant protein in the culture medium was purified by HiTrap® Protein A affinity column (GE Healthcare).

4) Isolation of FAE and IEC FAE and IEC were isolated as described in previous reports (Hase, K. et al., DNA Res., 2005, 12, 127-37). Briefly, PP was exfoliated from the small intestine and immersed in HBSS containing 30 mM EDTA (pH 7.2). After incubation for 20 minutes at room temperature, FAE was isolated by manipulation with a fine needle under a stereomicroscope (MZ12.5; Leica Microsystems). IEC was also isolated in a similar manner from small intestine pieces after PP removal.

5) Quantitative PCR analysis Mouse and human GP2 gene expression was measured using SYBR® Premix Ex Taq® (TaKaRa) and previous reports (Hase, K. et al., DNA Res., 2005, 12, 127- Investigated by real-time PCR using specific primers as described in 37):
mGP2
5'-GATACTGCACAGACCCCTCCA-3 '(forward: SEQ ID NO: 11)
5'-GCAGTTCCGGTCATTGAGGTA-3 '(reverse: SEQ ID NO: 12)
hGP2
5'-GTTGAGTCCGTGCTGTATGTGG-3 '(forward: SEQ ID NO: 13)
5'-TCTGAACTGAGAACCGGCTTTC-3 '(reverse: SEQ ID NO: 14)
The expression level of the GP2 gene was normalized by the expression of the GAPDH gene in each tissue, and the data were expressed as a change in FAE over IEC.

6) In situ hybridization (ISH)
Digoxigenin labeled RNA probes were prepared by in vitro transcription with T7 or SP6 RNA polymerase (Roche) using mouse and human GP2-N / pcDNA3 plasmid vectors digested with XhoI or BamHI as templates, respectively. ISH is automated for Discovery® according to the manufacturer's instructions (Ventana Japan) as described by previous reports (Hase, K. et al., DNA Res., 2005, 12, 127-37). Performed with ISH system and RiboMap kit. Briefly, 4% buffered formalin-fixed sections of PP were deparaffinized, treated with protease and hybridized with 50 ng of mGP2-specific antisense probe or control sense probe for 6 hours at 65 ° C. The sections were then incubated with biotin-labeled anti-digoxigenin Ab (Jackson ImmunoResearch Laboratories) for 20 minutes at 37 ° C followed by alkaline phosphatase-conjugated streptavidin for 16 minutes at 37 ° C. Signal was detected with BlueMap NBT / BCIP substrate kit (Ventana Japan) and sections were counterstained with Nuclear Fast Red.
For ISH analysis of human GP2 (hGP2), a non-inflammatory site at the end of the ileum containing lymphoid follicles of human subjects obtained from endoscopic biopsies or surgical sections was used. Biopsy samples were fixed with 4% buffered formalin and hybridized using hGP2-specific antisense probes or control sense probes as described above for mouse samples.

7) Antibody production a) Rabbit anti-GP2 polyclonal antibody Rabbit immunized with synthetic peptide (154th to 169th positions in mouse GP2 amino acid sequence: CSEELGEYHVYKLQGT (SEQ ID NO: 15)), and purified from the obtained serum using protein A column went.
b) Rat anti-mouse GP2 monoclonal antibody A fusion protein of the amino acid terminal 224 amino acids (SEQ ID NO: 16) of mouse GP2 and human IgG Fc region is expressed in cultured cells (HEK293T cells), and the protein is purified using a protein A column. As a result, a large amount of antigen protein was prepared. After immunizing rats with this antigen, hybridomas producing mouse GP2-specific antibodies were established by fusing spleen-derived B lymphocytes and myeloma cells. Large-scale preparation of the monoclonal antibody was performed by injecting the hybridoma into the abdominal cavity of nude mice and then purifying the obtained ascites with a protein G column.

8) Immunofluorescent staining For immunofluorescent staining of frozen sections of PP, sections were fixed with paraformaldehyde (BD Pharmingen) and incubated with 5% normal goat serum / phosphate buffered saline (PBS) for 30 minutes at room temperature. did. GP2 expression was determined by overnight 2.5 μg / ml rat anti-mouse GP2 mAb (clone 2F11-C3) at 4 ° C., followed by 0.5 μg / ml Alexa 488 conjugate anti-rat IgG Ab (1 hour at room temperature). Invitrogen). M cells were stained with 10 μg / ml rhodamine labeled Ulex europaeus agglutinin-1 (UEA-1) (Vector Laboratories) and finally counterstained with DAPI. For immunofluorescent staining of paraffin sections, isolated paraffin, cecal lymphoid follicles, colonic lymphoid follicles of 1% zinc sulfate / 4% formalin (Richard-Allan Scientific) fixed sections (5 μm) were deparaffinized and rehydrated. Treatment with 3% H ₂ O ₂ in PBS for 20 minutes at room temperature blocked endogenous peroxidase activity. Sections were incubated with 5% normal goat serum in PBS and 0.5% blocking buffer (Roche) for 30 minutes at room temperature and then with anti-mouse GP2 mAb at 4 ° C. overnight. Primary antibody binding was visualized with tyramide-fluorescein isothiocyanate (FITC) (PerkinElmer). The sample was analyzed with a fluorescence microscope (OLYMPUS BX51). For whole mount staining, PP was excised from the intestine and fixed with paraformaldehyde (BD Pharmingen) for 1 hour at 4 ° C. and 0.1% saponin / 0.2% bovine serum albumin (BSA) / PBS for 1 hour at 4 ° C. And permeabilized. GP2 expression was detected in the same manner as staining frozen sections.

9) Bacterial binding assay For bacterial binding assay, 0.065-200 μg / ml recombinant mouse and human GP2 (ΔTM) -Fc protein was added and incubated at 4 ° C. in a 96-well polystyrene microtiter plate (NAGEL). Incubated overnight. After washing, the microtiter plate was blocked with 1% bovine serum albumin (BSA) in PBS for 2 hours and then the immobilized recombinant protein was washed with 1 × 10 ⁶ E. coli strain in 0.1% NaN ₃ in PBS. Incubated with C25 or 2 hours at room temperature. After 5 washes with PBS, genomic DNA was extracted from the bound DNA using the QIAamp DNA kit (QIAGEN). Copy number of 16S ribosomal DNA (rDNA) was determined using SYBR® Premix Ex Taq® (TaKaRa) and specific primers for universal 16S rDNA (Greisen K. et al., J Clin. Microbiol., 1994). , 32, 335-351).
Universal 16S rDNA
5'-AACTGGAGGAAGGTGGGGAT-3 '(forward: SEQ ID NO: 17)
5'-AGGAGGTGATCCAACCGCA-3 '(Reverse: SEQ ID NO: 18)

10) Ligature loop assay Mice were anesthetized with avatine (5 g tribromoethanol in 2.5 ml t-amyl alcohol; 200 mg / kg animal weight) and kept warm on a 37 ° C warm pad during the assay. 1 × 10 ⁶ FITC-labeled E. coli strain C25 was injected into the ligament loop. One hour after injection, PP was excised from the intestine and immunostained as described in the method of whole mount staining. Samples were observed with a DeltaVision deconvolution microscope (SEKI Technotron Corp).

Example 1: Expression of the GP2 gene in M cells In the intestine, CX3CR1-expressing dendritic cells (DC) that reside in the lamina propria of M cells and the mouse small intestine and form transepithelial dendrites that directly collect luminal antigens Is an important sampling site for luminal antigens (Niess, JH et al., Science, 2005, 307, 254-8). The mechanism of receptor (eg, Fc receptor, C-type lectin receptor, and Toll-like receptor) mediated antigen uptake in DC has been well analyzed in previous studies, but that of M cells remains unclear. The present inventors have known that such a gene that is specifically expressed in M cells is expressed in the secretory granule membrane of the acinar cells of the exocrine pancreas and is secreted into the intestinal tract by pancreatic juice. A glycoprotein 2 (GP2) gene (Beaudoin, AR et al., J. Histochem. Cytochem., 1991, 39, 575-88), which is a type membrane protein, was identified. To confirm the specific expression of the GP2 gene in M cells, we examined quantitative PCR (qPCR) and in situ hybridization (ISH). The GP2 gene is exclusively expressed in FAE (FIG. 1) and co-stained with Ulex europaeus agglutinin-I (UEA-1), a lectin that recognizes α-L-fucose expressed in M cells of mouse PP. (FIG. 2). Similar to mice, limited expression of the GP2 gene in M cells was also confirmed in human PP (FIG. 3). Furthermore, when the mRNA expression level in tissues such as brain, tongue, liver, lung, salivary gland, kidney, heart, testis, thymus, spleen, stomach and FAE was confirmed by real-time PCR, GP2 mRNA was expressed in these tissues. GP2 mRNA expression in FAE was significant compared to expression in other tissues.

Example 2: Expression of GP2 protein and intracellular localization in M cells In order to confirm the expression of GP2 protein, the present inventors produced polyclonal and monoclonal antibodies against mouse GP2 (mGP2). In immunoblotting and immunofluorescence staining, about 75 kDa mGP2 was expressed only in FAE (FIG. 4), and was the most immunostained in UEA-1-positive M cells (FIG. 5, left side) as in the ISH results. Series of panels). Furthermore, GP2 was localized to the apical plasma membrane of M cells, as expected from acinar cell results. These findings demonstrate for the first time that GP2 is an M cell specific marker molecule in both human and mouse PP. PP is intestinal associated lymphoid tissue (GALT), but other GALTs such as isolated lymph nodes, cecal lymphoid follicles, colonic lymphoid follicles also exist. However, these GALTs have already shown the presence of M cells and transepithelial transport of luminal antigens (Gebert, A. et a., Anat. Rec., 1995, 241, 487-95 .; Liebler, EM et al., Am. J. Vet. Res., 1995, 56, 725-30.), But it is unknown whether M cells express common molecules in different GALTs. Furthermore, although UEA-1 has been used previously as an M cell marker, UEA-1 has been unable to distinguish M cells from cecal lymph follicles, colon lymph follicles and normal intestinal cells on human PP. This problem is caused by the different glycosylation of the membrane in mouse PP. To address these questions, immunostaining was performed with anti-GP2 antibody and observed with a fluorescence microscope. GP2 was also expressed in FAEs of isolated lymph nodes, large intestine lymph follicles, and cecal lymph follicles (FIG. 5, right panel), and co-staining with UEA-1 was confirmed in the isolated lymph nodes. In large intestine and cecal lymphoid follicles, it was confirmed that CCL9 (unpublished data) of chemokine, which has no specificity as in GP2, but has been confirmed in Peyer's patch M cells, is expressed in GP2-positive cells. These results indicate that GP2 is a pan-M cell marker molecule across tissues and species.

Example 3: GP2 has the ability to bind bacteria . GP2 expression on the luminal surface of M cells suggests a potential role of antigen receptors for several substances. Thus, the inventors speculated that intestinal microorganisms are potential candidates as ligands for GP2. In support of this hypothesis, Tamm-Horsfall, secreted from renal epithelial cells, is the most abundant protein in mammalian urine and has high homology with GP2 (approximately 51% homology at the amino acid level). It has already been shown that protein (THP) has the ability to bind E. coli (Pak, J. et al., J. Biol. Chem., 2001, 276, 9924-30). Based on this property, it has been suggested that THP has a role in removing bacteria that have invaded through the ureter. To examine the interaction between GP2 and bacteria, recombinant GP2 at a concentration of 0.065-200 μg / ml immobilized on a microtiter plate was incubated with E. coli C25 and based on the amount of bacterial 16S ribosomal DNA (rDNA). The number of bacteria was quantified by qPCR. As a result, the number of E. coli binding increased in a dose-dependent manner up to 6.5 μg / ml mGP2, but E. coli could not bind to all concentrations of control human IgG-Fc protein (FIG. 6). These results suggest that GP2 on the luminal surface of M cells binds to bacteria.

Example 4: GP2 was examined by an intestinal loop assay to investigate the role of GP2 on the endocytic receptor, an endocytic receptor of bacterial components in M cells . E. coli C25 was labeled with fluorescent isothiocyanate (FITC) and injected into the lumen of the ligation intestinal loop containing Peyer's patches, followed by visualization of M cells by whole-mount staining. As a result, a substance thought to be a cell membrane fraction of E. coli C25 was taken up and transcytosed to the basal side of M cells. It was further transported into the M cell pocket space. In the cytoplasmic region, the C25 cell membrane fraction co-localized with GP2 in the vesicles. Endocytosis via GP2 was confirmed by loop assay using anti-GP2 monoclonal antibody (mAb) and its incorporation. The mAb injected into the lumen bound to the luminal surface of GP2, and partly undergoes endocytosis and was observed as vesicles in the cytoplasmic region, similar to E. coli C25. The cross-linking of GP2 that occurs during antibody treatment is considered to be caused in the same way when bacteria bind to GP2. These results suggest that GP2 is a new endocytic receptor for bacterial components in M cells.

It is a figure which shows the specific expression of mouse | mouth GP2 mRNA in FAE. Quantitative PCR (qPCR) was performed. FAE: follicle-associated ephithelium IEC: intestinal epithelial cell It is a figure which shows co-staining by in situ hybridization of GP2 mRNA and UEA-1 (a known M cell marker) in a mouse | mouth intestinal mucosa origin sample. It is a figure which shows the dyeing | staining by in situ hybridization of GP2 mRNA in a human Peyer's board (PP). It is a figure which shows the immunoblotting which confirms the expression of mouse | mouth GP2 protein in FAE. It is a figure which shows the immuno-staining of mouse | mouth GP2 protein. Upper left: GP2 staining on mouse Peyer's plate Second from upper left: Stained with UEA-1 in the same section as above Third from left in upper left: Staining of nucleus in the same section Lower left: Upper right image in which the above three are superimposed : Second staining from upper right of GP2 and UEA-1 staining in mouse isolated lymph node: Lower right of GP2 staining in colonic lymph follicle Lower right: GP2 staining in cecal lymph follicle It is a figure which shows the coupling | bonding with respect to colon_bacillus | E._coli of mouse | mouth GP2 protein. ■: Mouse GP2 protein ◆: Control human IgG-Fc protein

Claims (16)

  A method for detecting an M cell, comprising measuring the expression of GP2.   The detection method according to claim 1, which is a method for detecting human M cells.   The detection method according to claim 1, which is a method for detecting mouse M cells.   A method for identifying an M cell, comprising measuring GP2 expression in a sample derived from an intestinal mucosa containing the M cell.   The identification method according to claim 4, wherein the sample derived from the intestinal mucosa includes at least Peyer's patch, isolated lymph node, or cecal or large intestine lymphoid follicle.   A reagent or kit for detecting or identifying M cells, comprising one or more means for measuring GP2 selected from the group consisting of a primer, a nucleic acid probe, and an antibody.   A drug delivery vehicle comprising a GP2 targeting molecule.   8. The drug delivery vehicle according to claim 7, wherein the GP2 targeting molecule is an antibody.   A medicament comprising a GP2 targeting molecule and an active ingredient as a medicament.   The medicament according to claim 9, wherein the active ingredient is an antigen.   An agent for regulating mucosal immunity comprising a substance capable of regulating the expression or function of GP2.   An M cell in which GP2 expression is regulated or a tissue containing the same.   A method for evaluating endocytosis in an M cell, comprising measuring the expression or function of GP2 in the M cell or a tissue containing the M cell.   A binding agent for microorganisms comprising a secreted form of GP2.   A complex comprising GP2 and a microorganism or a component thereof. A method for evaluating mucosal immunity in a mammal, including any of the following (a) or (b):
(A) subjecting a mammal in which expression of GP2 is regulated to measurement of mucosal immunity; or (b) subjecting the mammal to measurement of expression or function of GP2.