WO2003084991A1 - Atopic dermatitis-inducing proteins - Google Patents

Abstract

Atopic dermatitis-inducing proteins produced by a patient himself/herself are provided as factors causative of atopic dermatitis. Namely, these proteins are atopic dermatitis-inducing proteins which are secreted by the human body and characterized by binding to the own IgE antibody of the human producer and activating mast cells and basophils. More specifically speaking, the above proteins include a salivary acidic proline-rich phosphoprotein having the amino acid sequence represented by SEQ ID NO:2, a prolactin-inducible protein having the amino acid sequence represented by SEQ ID NO:4 and post-translational modifications thereof.

Description

 Description Atopic dermatitis-inducing protein TECHNICAL FIELD The invention of this application relates to an atopic dermatitis-inducing protein secreted by an atopic dermatitis patient himself, and an atopic dermatitis using this protein or its antibody. And a therapeutic agent for desensitization of atopic dermatitis comprising this protein as an active ingredient.

BACKGROUND ART In recent years, the number of patients with atopic dermatitis has increased rapidly, and the proportion of patients with atopic dermatitis in dermatology patients is over 50%, making it one of the main dermatological diseases. Atopic dermatitis is one of the atopic diseases caused by genetic factors that easily produce IgE antibodies against normal antigens.It begins in infancy, and chronically progresses with aging. Many relieve before puberty. The completed lesions are clearly lichenified, pruritus is always severe, often paroxysmal, and progresses and remits repeatedly with some association with other atopic diseases. The pathogenesis of atopic dermatitis has many unclear factors, and there is still a problem in distinguishing it from other skin diseases.Therapeutic methods have not been established yet. Attempts have been made to use topical corticosteroids, to administer antihistamines and chemotransmitter release agents, or to treat diets excluding eggs, milk, and soybeans. However, in the case of medication, the use of the drug in growing children, and for a prolonged period, is problematic in terms of side effects. In the diet, it is difficult except for the full Nia Rerugi one source, there is a problem in that greater mental burden _c In light of this situation, we have recently attempted to raise the therapeutic effect by elucidating the onset mechanism of atopic dermatitis by making full use of genetic engineering knowledge and inhibiting some of the onset mechanism. Research is being done. For example, focusing on the fact that the production of IgE antibodies against antigens is part of the mechanism that causes symptoms as described above, we decided to use substances that suppress the production of IgE antibodies against antigens externally. Thus, it is considered that the symptoms are alleviated (JP-A-7-109290, JP-A-7-109292, JP-A-9-100236). However, administration of a substance that suppresses the production of IgE antibodies against antigens, although effective to some extent, does not essentially block pathogens, and thus may be effective in alleviating symptoms. Not treated. In addition, since all sources of pathogens are required for foreign substances present in food or the environment, there is no correspondence for substances produced in the body. The invention of this application has been made in view of the above circumstances, and has as an object to provide an atopic dermatitis-inducing protein produced by a patient himself as a causative factor of atopic dermatitis. I have. It is another object of the invention of this application to provide a peptide which is a part of the protein and is involved in the antigenicity thereof. Still another object of the invention of the present application is to provide a method for diagnosing atopic dermatitis using the above-mentioned protein and / or peptide or an antibody thereto. Further, another object of the invention of this application is to provide a therapeutic agent for desensitization of atopic dermatitis, which comprises the above-mentioned protein, protein or peptide as an active ingredient.

Disclosure of the invention This application provides the following inventions (1) to (25) as inventions for solving the above problems. (1) An atopic dermatitis-inducing protein, which is a protein secreted from a human body and which binds to a human IgE antibody secreting the protein and activates mast cells and basophils. .

(2) The protein of the invention (1) secreted from salivary glands or sweat glands.

(3) The protein according to the invention (1) or (2), which has two or more identical amino acid repeating sequences each having four or more amino acid residues.

(4) The protein according to the invention (3), wherein the protein is a salivary acidic proline-rich phosphoprotein consisting of the amino acid sequence of SEQ ID NO: 2.

(5) The protein according to the invention (1) or (2), wherein the protein is a Prolactin-inducible protein consisting of the amino acid sequence of SEQ ID NO: 4. (6) The protein according to the invention (1) or (2), wherein at least one of the constituent amino acid residues is post-translationally modified.

(7) The protein according to the invention (6), wherein the modified amino acid residue is one or more amino acid residues selected from the group consisting of lysine, arginine, methionine, alanine, phenylalanine, proline, asparagine, and tyrosine.

(8) The protein according to invention (7), wherein the modifying group is an alkyl group.

(9) The protein according to the invention (8), wherein the protein is a Prolactin-inducible protein consisting of the amino acid sequence of SEQ ID NO: 4. (10) The protein according to the invention (9), wherein at least the 84th tyrosine residue of SEQ ID NO: 4 is methylated.

 (11) A peptide which is a part of the protein of the invention (1) and comprises an amino acid sequence constituting a binding region to an IgE antibody.

(12) The peptide according to the invention (11), which is a part of the protein according to the invention (3) or (4).

(13) The peptide according to the invention (12), which comprises at least four consecutive amino acid sequences of SEQ ID NO: 2.

(14) The peptide of the invention (11), which is a part of the protein of the invention (5).

(15) The peptide according to the invention (11), which is a part of the protein according to any one of the inventions (6) to (10). (16) The peptide according to the invention (14) or the invention (15), which comprises at least four consecutive amino acid sequences of SEQ ID NO: 4.

(17) An antibody prepared using the protein of the invention (1) or the peptide of the invention (11) as an antigen, wherein the antibody binds to the protein of the invention (1).

(18) an antibody prepared using the protein of the invention (3) or (4) or the peptide of the invention (12) or (13) as an antigen, which binds to the protein of the invention (3) or (4) antibody.

(19) An antibody prepared using the protein of the invention (5) or the peptide of the invention (16) as an antigen, wherein the antibody binds to the protein of the invention (5).

(20) An antibody prepared using the protein of any one of the inventions (6) to (10) or the peptide of the invention (16) as an antigen, wherein the protein of any one of the inventions (6) to (10) Antibodies that bind to (21) Antibodies that bind to one or more of the proteins of the inventions (1) to (10) or one or more of the peptides of the inventions (11) to (16) are present in the serum of the subject A method for diagnosing atopic dermatitis, characterized in that the test is conducted to determine whether or not the subject has serum antibodies, and that the subject is determined to be a patient with atopic dermatitis or a high-risk atopic dermatitis.

(22) The leukocyte fraction collected from the subject's blood contains one or more of the proteins of the inventions (1) to (10) or one or more of the peptides of the inventions (11) to (16) A method for diagnosing atopic dermatitis, comprising determining from the degree of histamine release a patient with atopic dermatitis or a subject at risk for atopic dermatitis.

(23) A test is performed to determine whether or not a biological sample of a subject contains a protein that binds to one or more of the antibodies according to any one of the inventions (17) to (20). A method for diagnosing atopic dermatitis, comprising determining that the patient is a dermatitis patient or a high-risk person with atopic dermatitis.

(24) An atopy characterized by containing, as an active ingredient, at least one of the proteins of the inventions (1) to (10) or at least one of the peptides of the inventions (11) to (16). For desensitization of atopic dermatitis. -The aspects, terms and concepts in each of the inventions described above are defined in detail in the description of the embodiments of the invention and examples. Various techniques used to carry out the present invention can be easily and reliably implemented by those skilled in the art based on known documents and the like, except for the technique whose source is clearly indicated. For example, the preparation of a drug that can be used for the treatment method of the present invention is described in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990, by genetic engineering and molecular biological techniques. Are described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. Has been described. BEST MODE FOR CARRYING OUT THE INVENTION The atopic dermatitis-inducing protein of the invention (1) is a protein secreted from a human body (particularly a sweat gland or salivary gland) of a human (particularly a patient with atopic dermatitis or a high-risk person thereof). An isolated and purified protein characterized by binding to a human IgE antibody secreting this protein and activating mast cells and basophils. This protein can be isolated and purified by the method of Example 1 described later. The protein of the present invention (1) specifically includes three different proteins. The first is the protein of the invention (3), which is a protein having two or more identical amino acid repetitive sequences consisting of four or more amino acid residues S, and a more specific example thereof is the protein of the invention (4). That is, the protein of the invention (4) is a salivary acidic proline-rich phosphoprotein (hereinafter referred to as “PRP protein”) consisting of the amino acid sequence of SEQ ID NO: 2. This PRP protein has nine QGPP repeats, four QGPPQQGG repeats, five PQGPP repeats, and three QQGPP repeats. Although the existence and structure of this PRP protein were known (for example, J. Biol. Chem. 260: 11123-11130, 1985, etc.), humans who secrete this PRP protein themselves It has not been known at all that it is an attractant protein that causes atopic dermatitis by binding to IgE antibodies and activating mast cells and basophils. A second specific embodiment of the protein of the invention (1) is the protein of the invention (5), that is, a Prolactin-inducible protein consisting of the amino acid sequence of SEQ ID NO: 4 (hereinafter referred to as “PIP protein”). Although the existence and structure of this PIP protein were known (for example, J. Biol. Chem. 262: 15236-15241, 1987, etc.), atopic dermatitis was activated by activating mast cells and basophils. To be a trigger protein It is not known at all. A third specific embodiment of the protein of the invention (1) is the protein of the invention (6) (that is, a protein in which at least one of the constituent amino acid residues is post-translationally modified). Specifically, the modified amino acid residue is one or more amino acid residues selected from the group consisting of lysine, arginine, methionine, alanine, phenylalanine, proline, asparagine, and tyrosine, and the modified group is an alkyl group. It is preferably a group. A specific example of the protein of the present invention (6) is a PIP protein in which the 84th tyrosine residue in the amino acid sequence of SEQ ID NO: 4 is methylated (hereinafter sometimes referred to as “modified PIP protein”). It is. In addition, in this modified PIP protein, any arginine residue in SEQ ID NO: 4 may be methylated. It has never been known that a PIP protein having such modified amino acid residues activates mast cells and basophils, thereby inducing atopic dermatitis.

The PRP protein and PIP protein, or the modified PIP protein can be isolated and purified from human sweat or saliva by the same method as in Example 1 described later. It can also be prepared by a method of chemically synthesizing the amino acid sequences of SEQ ID NOS: 2 and 4, respectively, by a known peptide synthesis method. Peptide synthesis can be performed using various solid phase methods (see, for example, Science 269: 202, 1995; Methods Enzymol. 289: 3-13, 1997), and also, for example, ABI431A peptide synthesizer.

(Perkin Elmer) or the like. In order to produce a PIP protein in which at least the tyrosine residue at the 84th position (and any arginine residue) is methylated, peptide synthesis is one of the preferred methods.

Another method for producing PRP proteins and PIP proteins or modified PIP proteins is to prepare proteins by genetic engineering using known DNA sequences (SEQ ID NOS: 1 and 3, respectively) encoding each. . For example, RNA is prepared by in vitro transcription from a recombinant expression vector having the aforementioned DNA sequence, The protein can be produced in vitro by performing in vitro translation using this as a type III. In addition, when a recombinant expression vector is introduced into prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells to produce transformed cells, DNA sequences from the transformed cells are obtained. Can be prepared. When a protein is expressed by in vitro translation, the above DNA sequence is inserted into a vector having an RNA polymerase promoter to prepare a recombinant expression vector, and this vector is used as an RNA corresponding to the promoter. The protein can be produced in vitro by adding it to an in vitro translation system, such as a persimmon reticulocyte lysate or wheat germ extract containing polymerase. Examples of the RNA polymerase promoter include T7, T3, and SP6. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, ρΤ7 / 319, pBluescript II, and the like. This in vitro transcription / translation method is one of the preferred methods for producing a PIP protein in which at least the 84th tyrosine residue (and any arginine residue) is methylated. When the protein is expressed in a microorganism such as Escherichia coli, the above-described expression vector having an origin, a promoter, a liposome binding site, a DNA cloning site, and a mine that can be replicated in the microorganism is used. A target protein can be obtained from a microorganism by preparing an expression vector in which the DNA sequence is recombined, transforming a host cell with this expression vector, and culturing the resulting transformant. At this time, it can also be expressed as a fusion protein with another protein (for example, a fusion protein with glutatin? S? Transferase? (GST) or green fluorescent protein (GFP)). Examples of expression vectors for Escherichia coli include a pUC system, pBluescript II, a pET expression system, a pGEX expression system, and the like. When the protein is to be expressed in eukaryotic cells, the recombinant vector is inserted by inserting the protein into the expression vector for eukaryotic cells having the DNA sequence, promoter, splicing region, poly (A) addition site, and the like. Once produced and introduced into eukaryotic cells, It can be obtained from transformed eukaryotic cells. Examples of expression vectors include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pcDNA3, pMSG, pYES2, and the like. When pIND / V5-His, pFLAG-CMV-2, pEGFP-Nl, pEGFP-Cl, etc. are used as expression vectors, fusion with various tags such as His-tag, FLAG-tag, myc-tag, HA-tag, GFP etc. The desired protein can also be expressed as a protein. As eukaryotic cells, monkey kidney cells COS7, mammalian cells such as Chinese ovarian ovary cells CHO and the like, budding yeast, fission yeast, silkworm cells, African egg cells and the like are generally used. Any eukaryotic cell can be used as long as it can express E. coli. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.

After expressing a PRP protein, a PIP protein, or a modified PIP protein in prokaryotic cells or eukaryotic cells, isolation and purification of the target protein from the culture can be performed by a combination of known separation procedures. . For example, treatment with denaturing agents such as urea or surfactants, sonication, enzyme digestion, salting-out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing Ion chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like. In order to identify a PIP protein in which at least the 84th tyrosine residue (and any arginine residue) is methylated from the proteins isolated and purified in this way, it is necessary to use atopic protein. The serum of the dermatitis patient is allowed to react with the protein, and the protein that binds to the antibody in the serum may be selected. The invention (11) of the present application is a peptide which is a part of the atopic dermatitis-inducing protein of the invention (1) and which comprises an amino acid sequence constituting a binding region (antigenic determinant or epitope) to an IgE antibody. is there. This peptide can be obtained by degrading the protein of the invention (1) with an appropriate protease. This peptide is specifically a peptide (invention (12)) which is a part of the protein of the invention (31 or (4), the protein of the invention (5) A peptide (Invention (4)) which is a part of the protein; and a peptide (Invention (15)) which is a part of the protein of any of Inventions (6) to (10). More specifically, they are PRP proteins and peptides as a part of PIP proteins (Inventions (13) and (16)). In the case of a peptide as a part of a PRP protein or a PIP protein, a peptide having a desired amino acid sequence can be obtained by a known peptide synthesis method based on SEQ ID NOS: 2 and 4, respectively. Further, the coding region of the desired peptide is cleaved from the DNA sequence of SEQ ID NO: 1 or 3, or PCR is amplified, and the peptide is produced from this DNA sequence by the genetic engineering method as described above. You can also. In addition, the proteins of the inventions (1) to (10) bind to two or more antibodies by having two or more epitopes, and thereby exert physiological activities. Therefore, the peptides of the inventions (11), (12), (14 ·), and (16) can include amino acid sequences constituting two or more epitope regions. Since such a peptide has the same physiological activity (ie, atopic dermatitis-inducing activity) as proteins (1) to (10), it is useful, for example, as an active ingredient of the desensitizing therapeutic agent of the invention (24). is there. Further, the peptide of the present invention may have one epitope. Such a peptide can be used as an antigen for producing an antibody or as a material for the diagnostic method of the inventions (21) and (22). The peptide derived from the PRP protein (Invention (13)) is a peptide containing at least the four consecutive amino acid sequences of SEQ ID NO: 2, for example, a region containing the amino acid repeat sequence of SEQ ID NO: 2 (amino acids Nos. 47 to 158). (In the sequence), preferably a continuous 4 amino acid sequence, 5-10 amino acid sequence, 11-30 amino acid sequence, 31-50 amino acid sequence, 51-80 amino acid sequence, 81-11 amino acid sequence . Further, the peptide derived from the PIP protein is preferably a continuous 4-amino acid sequence or more including the 84th tyrosine residue that has been methylated in the amic acid sequence of SEQ ID NO: 4. More preferably, the methylated tyrosine residue at position 84 and any arginine residue that may be methylated (ie, the second, ninth, 33rd, 47th, 106th, 118th, and 136th arginine residues). Invention (17) is an antibody prepared using the protein of invention (1) or the peptide of invention (11) as an antigen. The antibodies of the inventions (18) to (20) are antibodies prepared using the protein of the inventions (3) to (10) or the peptide of the inventions (12) to (16) as antigens, respectively. These antibodies are polyclonal antibodies or monoclonal antibodies, and include all molecules capable of binding to each of the above-mentioned protein epitopes, Fab, F (ab ') ₂ , Fv fragments and the like. For example, in the case of a polyclonal antibody, such an antibody can be obtained from serum after immunizing an animal using an antigen peptide or a partial fragment thereof as an immunogen. Alternatively, it can be prepared by introducing the above expression vector for eukaryotic cells into the muscle or skin of an animal by injection or gene gun, and then collecting serum. As animals, mice, lads, egrets, goats, and chickens are used. Monoclonal antibodies can be prepared by a known monoclonal antibody preparation method (“monoclonal antibody”, written by Kamei Nagamune and Hiroshi Terada, Hirokawa Shoten, 1990; “Monoclonal Antibody”, James W. Goding, third edition, Academic Press, 1996). According to this, for example, it can be manufactured by the following procedure. Preparation of doma cell group

A mammal is immunized with the above-mentioned atopic dermatitis-inducing protein or epitope peptide as an antigen (immunogen), and boosted as necessary to sensitize the animal. Next, antibody-producing cells (lymphocytes or spleen cells) are excised from the animal, and a fusion cell of this and a myeloma (myelotype) cell line is obtained. Then, from these fused cell lines, select the plurality of cells producing a monoclonal antibody of interest, respectively, by culturing, following _t can be obtained eight Ivry dormer cell groups, details of each step described I do. a) Preparation of immunogen

The protein of the invention (1) to (θ) or the peptide of the invention (11) to (16) is appropriately added It can be selected as an immunogen. These immunogens can also be used in the form of fusion proteins with other proteins (for example, Daryuthion-1S-transferase: GST). The use of such a fusion protein is particularly preferable in that the isolation of the target protein from the expression product of the host-vector system and the screening step of eight hybridoma cells described later are easy and reliable. Successful antibody production using a short-chain peptide as an antigen is described in, for example, the following literature (Biochem. J. 266: 497-504, 1990; Biochem. J. 288: 195-205). , 1992, Molecular and Cellular Biology 8: 2159-2163, 1988).

b) Animal immunity

 As the animal to be immunized, a mammal used in a known method for producing eight hybridomas can be used. Specifically, for example, mice, rats, goats, sheep, whales, horses, etc. However, from the viewpoint of availability of myeloma cells to be fused with the extracted antibody-producing cells, it is preferable to use mice or rats as immunized animals. The strains of mice and rats actually used are not particularly limited. In the case of mice, for example, strains A, AKR, BALB / c, BDP, BA, CE, C3H, 57BL, C57BR, C57L, DBA , FL, HTH, HT1, LP, NZB, NZW, RF, REL SJL, SWR, WB, 129, etc., and in the case of rats, for example, Low, Lewis, Spraque, Daley, ACL BN, Fischer etc. Can be used. Of these, the BALB / c strain in mice and the low strain in rats are particularly preferred as animals to be immunized, considering the compatibility with myeloma cells described below. The age of the mouse or rat at the time of immunization is preferably 5 to 12 weeks.

 Immunization of an animal can be performed by administering a solution of the immunogen intradermally or intraperitoneally to the animal. The administration schedule of the immunogen varies depending on the type of the animal to be immunized, individual differences, and the like. The dose of the antigen varies depending on the type of animal, individual differences, and the like, but is generally about 10-100 ig / ml. cj cell fusion

Antibody production from the immunized animal 1 to 5 days after the last immunization date in the above administration schedule Spleen cells or lymph cells, including live cells, are aseptically removed. The antibody-producing cells can be separated from these spleen cells or lymph cells according to a known method.

 Next, the antibody-producing cells and the myeloma cells are fused. The myeloma cells are not particularly limited, and can be appropriately selected from known cell lines and used. However, in consideration of the convenience in selecting eight hybridomas from the fused cells, it is preferable to use an HGPRT-deficient HGPRT-hypoxanthine-guanine phosphoribosyl transferase (defective) strain that has established its selection procedure. That is, mouse-derived X63-Ag8 (X63), NSl-Ag4 / l (NS-l), P3X63-Ag8.Ul (P3Ul), X63-Ag8.653 (X63.653), SP2 / 0-Agl4 ( SP2 / 0), MPC1 1-45.6TG1.7 (45.6TG) FO, S149 / 5XXO, BU.l, etc., 210.RSY3.Ag. l.2.3 (Y3), etc. U266AR (SKO-007), GM 1500 · GTG-A12 (GM 1500), UC729-6> LICR-LOW-HMy2 (HMy2), 8226AR / NIP4-1 (NP41) and the like.

 The fusion of the antibody-producing cell and the myeloma cell can be appropriately performed according to a known method, under conditions that do not extremely reduce the cell viability. As such a method, for example, a chemical method of mixing antibody-producing cells and myeloma cells in a high-concentration polymer solution such as polyethylene glycol or a physical method using electrical stimulation can be used.

 The selection of fused cells and non-fused cells is preferably performed, for example, by a known HAT (hypoxanthine'aminopterin thymidine) selection method. This method is effective when obtaining fusion cells using myeloma cells of an HGPRT-deficient strain that cannot survive in the presence of aminopterin. That is, by culturing the unfused cells and the fused cells in the HAT medium, only the fused cells having aminobuterin resistance can be selectively left and grown. d) Hybridoma screening

Screening of hybridoma cells producing the desired monoclonal antibody can be performed by known enzyme immunoassay (EIA: Enzyme Immunoassay), radioimmunoassay (RIA: Radio Immunoassay), fluorescent antibody method, etc. . When a fusion protein is used as an immunogen, the fusion partner protein By carrying out each of the above-mentioned screening methods in combination, it is possible to screen the hybridoma cells more reliably.

 By such screening, a group of hybridoma cells producing monoclonal antibodies that specifically bind to the proteins of the inventions (1) to (10) can be obtained.

 The hybridoma cells after screening are cloned by a known method such as a methylcellulose method, a soft agarose method, or a limiting dilution method, and used for antibody production.

 The hybridoma cells obtained by the above-mentioned method can be stored in liquid nitrogen or at -80 in the following freezer in a frozen state.

2: Acquisition and purification of monoclonal antibodies

 The desired monoclonal antibody can be obtained by culturing the hybridoma cells prepared in 1 above by a known method.

 The culture may be performed, for example, in the medium having the same composition used in the cloning method described above, or in order to produce a large amount of monoclonal antibodies, inject the hybridoma cells into the mouse intraperitoneal cavity, and use Antibodies may be collected.

The monoclonal antibody thus obtained can be purified by, for example, ammonium sulfate precipitation, gel filtration, ion exchange chromatography, affinity chromatography, or the like. Next, the invention (21) is characterized by using at least one of the proteins of the inventions (1) to (10) or one or more of the peptides of the inventions (11) to (16) to obtain an atopic property. This is a method to diagnose dermatitis. That is, the proteins of the inventions (1) to (10) have been identified as proteins that bind to antibodies (IgE antibodies) in the serum of patients with atopic dermatitis, and the inventions (11) to (16) Is a peptide containing an epitope of these proteins, it is reacted with the serum of the subject, and the serum containing an antibody that binds to these proteins or peptides is used as the serum of a patient with atopic dermatitis or a high-risk patient with atopic dermatitis. Can be determined. For specific diagnosis, for example, the serum of a subject is brought into contact with the above-described protein or peptide, and the protein or peptide is reacted with the IgE antibody in the serum of the subject in a liquid phase. Further, a signal of the labeled anti-IgE antibody may be detected by reacting with a labeled anti-IgE antibody that specifically binds to the IgE antibody in the serum. Enzymes, radioisotopes, and fluorescent dyes can be used as the labeling substance of the labeled anti-IgE antibody. The enzyme is not particularly limited as long as it satisfies conditions such as a high turnover number, stability even when bound to an anti-IgE antibody, and specific coloring of the substrate.For example, peroxidase , / 3-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose 16-phosphoryl dehydrogenase, malate dehydrogenase and the like can also be used. The binding between these enzymes and the anti-IgE antibody can be performed by a known method using a crosslinking agent such as a maleimide compound. As the substrate, a known substance can be used depending on the type of the enzyme to be used. For example, when using peroxidase as an enzyme, use 3,3 ', 5,5'-tetramethylbenzine, and when using alkaline phosphatase, use paranitrophenol or the like. it can. As the radioactive isotope, those used in ordinary RIA such as i ²⁵ I and ³ H can be used. Fluorescent dyes include those used in ordinary fluorescent methods such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC), as well as fluorescent proteins such as green fluorescent protein. You can use any quality. In the case of using an enzyme, the signal is detected by adding a substrate that decomposes and develops color by the action of the enzyme, and measuring the amount of decomposition of the substrate optically to determine the enzyme activity, which is converted to the amount of bound antibody. Then, the amount of the antibody is calculated from the comparison with the standard value. When using radioisotopes, measure the radiation dose emitted by the radioisotope using a scintillation counter. When a fluorescent dye is used, the amount of fluorescence may be measured by a measuring device combined with a fluorescence microscope. Samples for signal detection include Western blot analysis and protein conjugate + serum antibody + labeled anti-IgE antibody conjugate using well-known separation means (chromatography, salting out, alcohol precipitation, enzymatic method). , Solid phase method, etc.) and the signal of the labeled anti-IgE antibody may be detected. The diagnostic method of the invention (21) can also immobilize one or more types of protein peptides on a plate, and test the binding of the subject serum to the antibody on the plate. By immobilizing the protein / peptide on the plate, unbound labeled binding molecules can be easily removed. The method of the present invention (21) enables not only the diagnosis of atopic dermatitis but also the quantification of the amount of an antibody to be detected. By preparing a standard (calibration curve, etc.) in advance, and comparing the amount of antibody obtained from the serum of the subject with a standard, an accurate amount of antibody can be measured, and the degree of disease or occurrence of disease can be measured. The degree of danger can be estimated with high accuracy. The method of the invention (22) is characterized in that the leukocyte fraction collected from the blood of the subject contains at least one of the proteins of the inventions (1) to (10) or the peptide of the inventions (11) to (16) At least one or more is added to distinguish patients with atopic dermatitis or high-risk atopic dermatitis using the degree of histamine release from leukocytes as an index. In other words, the histamine released by the stimulation of the protein of the invention (1) to (10) or the peptide of the invention (11) to (16) causes atopic dermatitis. By measuring the amount, it becomes possible to diagnose with high accuracy the presence or absence of the disease, the degree thereof, or the degree of the risk of development. The amount of histamine can be measured, for example, by a known method (Koro, O. et al. J. Allergy Clin. Immunol., 103, 663-670, 1999). As a criterion for diagnosis, for example, the amount of increase in histamine released when a leukocyte fraction collected from a subject is stimulated with the protein or peptide is 5% or more as compared with that without stimulation. A case can be determined as positive. Invention (23) tests whether a biological sample of a subject contains a protein that binds to one or more antibodies of any of inventions (17) to (2), and examines whether the protein is present in the sample. It is characterized in that a person is determined to be a patient with atopic dermatitis or a person at high risk for atopic dermatitis. That is, the antibodies of the inventions (17) to (20) are antibodies that specifically bind to the proteins of the inventions (1) to (10), and the proteins of the inventions (1) to (10) are atopic dermatitis. Therefore, a biological sample containing a protein that binds to this antibody can be determined as a sample of an atopic dermatitis patient or a high-risk patient thereof. The biological sample may be any sample isolated from a subject, but saliva or sweat is particularly preferred. One embodiment of the diagnostic method of the present invention (23) is a method of binding an antibody and a protein in a liquid phase system. For example, an antibody labeled with a labeling substance as exemplified in the invention (21) is brought into contact with a biological sample to bind the labeled antibody to a target protein, and this conjugate is treated in the same manner as in the invention (21). The labeled signal is recovered in a similar manner. Another method of diagnosis in a liquid phase system involves contacting an antibody (primary antibody) with a biological sample to bind the primary antibody to the target protein, and then binding a labeled antibody (secondary antibody) to the conjugate. Then, the labeled signal in the conjugate of the three is detected. Alternatively, to further enhance the signal, an unlabeled secondary antibody may be first bound to the antibody + protein conjugate, and a labeling substance may be bound to this secondary antibody. Such binding of the labeling substance to the secondary antibody can be performed, for example, by biotinylating the secondary antibody and avidinizing the labeling substance. Or a partial region of the secondary antibody

An antibody (a tertiary antibody) recognizing (for example, an Fc region) may be labeled, and the tertiary antibody may be bound to the secondary antibody. In addition, as the primary antibody and the secondary antibody, both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. Separation of the conjugate from the liquid phase and detection of a signal can be performed in the same manner as in the invention (21). Another embodiment of the diagnostic method of the invention (23) is a method of testing the binding between an antibody and a target protein in a solid phase system. This method using a solid phase system is a preferred method because detection of a trace amount of protein and simplification of the operation are preferable. In other words, in this solid phase method, an antibody (primary antibody) is immobilized on a resin plate or the like, the target protein is bound to the immobilized antibody, and the unbound protein is washed off, and then left on the plate. To bind a labeled antibody (secondary antibody) to the antibody + protein conjugate and detect the signal of this secondary antibody Is the law. This method is a so-called “sandwich method”, and is widely used as “ELISA (enzyme linked immunosorbent assay)” when an enzyme is used as the primary method. As the primary antibody and the secondary antibody, both monoclonal antibodies can be used, or one of the primary antibody and the secondary antibody can be a polyclonal antibody. The detection of the signal can be performed in the same manner as in the invention (21). The method of the present invention (23) enables not only the diagnosis of atopic dermatitis but also the quantification of the amount of atopic dermatitis-inducing protein. By preparing a standard (calibration curve, etc.) in advance and comparing the amount of protein obtained from the subject's biological sample with the standard, accurate protein measurement can be performed, and the degree of disease or the risk of developing the disease Can be estimated with high accuracy. Invention (24) is characterized by containing, as an active ingredient, at least one of the proteins of inventions (1) to (10) and / or at least one of the peptides of inventions (11) to (16). It is a therapeutic agent for desensitization of atopic dermatitis. `` Desensitization treatment '' means that in allergy involving IgE antibodies, a small amount of therapeutic allergen is administered in increasing doses over a certain number of days, so that no allergic reaction occurs even if the allergen enters This is a cure. Since the proteins of the inventions (1) to (10) and the peptides of the inventions (11) to (16) have the activity of inducing atopic dermatitis as described above, they are used in the treatment for desensitization. For allergens. In this case, the peptide is preferably a peptide having two or more epitope regions like a protein and exhibiting the same physiological activity as that of the protein (attractable dermatitis-inducing activity). It may be a peptide having an epitope region (so-called “hapten”). The therapeutic agent for desensitization of the present invention (23) can be formulated by uniformly mixing the above-mentioned protein nopeptide with a pharmacologically acceptable carrier. The carrier can be appropriately selected from a wide range depending on the dosage form of the drug, but the drug of the present invention is desirably in a unit dosage form that can be administered orally or by injection. Oral liquid preparations such as suspensions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, oils such as sesame oil and soybean oil, and preservatives such as alkyl parahydroxybenzoate. It can be manufactured using a flavoring agent such as an agent, stroberberry flavor, peppermint and the like. Powders, pills, capsules and tablets include lactose, glucose, sugar, mannitol and other excipients, starch, disintegrants such as sodium alginate, magnesium stearate, talc, etc. And a binder such as polyvinyl alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. Tablets and capsules are preferred unit dosage forms in the formulations of the present invention in that they are easy to administer. When manufacturing tablets and capsules, a solid pharmaceutical carrier is used. Further, the solution for injection can be formulated using a carrier comprising a salt solution, a glucose solution, a mixture of a saline solution and a glucose solution, various buffers and the like. Alternatively, the composition may be formulated in a powder state, and mixed with the liquid carrier before use to prepare an injection solution. The administration schedule of the desensitizing therapeutic agent of the present invention varies depending on the patient's age, body weight, symptoms, administration route, and the like. The amount of protein Z peptide as a therapeutic allergen is determined by performing an intradermal test in advance, and It is preferable to measure the threshold value at the initial dose and to use the amount before and after the threshold value as the first dose. The administration interval and the amount of increase in allergen after the second administration can be appropriately determined depending on the presence or degree of allergic reaction. Eventually, allergens that are about 10,000 times the threshold dose can be administered. Therefore, for example, in the case of the hyposensitizing drug of this invention, if the initial dose of allergen is formulated as lng / ml X 0.05 ml, the final dose of the drug is 10 _mg / ml X 0.05 ml Can be EXAMPLES Hereinafter, the invention of this application will be described more specifically and in detail with reference to examples, but the invention of this application is not limited to the following examples.

Example 1

 Purification and identification of atopic dermatitis-inducing protein from human sweat.Sweet of healthy subjects, patients with atopic dermatitis and patients with allergic rhinitis were collected and sterilized by filtration. Humans had few positive reactions (11%), but high rates of wheals occurred in patients with atopic dermatitis (85%) and allergic rhinitis (71.4%). Therefore, the substances in the collected sweat were extracted by the following method, and the substances were identified.

Sweat was collected from several healthy persons or patients with atopic dermatitis, and the target antigen protein was purified using histamine release from peripheral blood basophils as a marker for patients with atopic dermatitis. Sweat was collected from several healthy persons or patients with atopic dermatitis in a sauna bath, and the residue was removed with filter paper. The residue was diluted with 20 mM Tris buffer (pH 8.0) and sterilized by filtration with a 0.02 im filter. Using the AKTA system, fractionate sweat diluted with an anion exchange resin column (monoQHR10 / 10, Amersham Pharmacia Biotec), and dilute the fraction containing the target protein with 20 mM acetate buffer (pH 4.75). After adjusting the salt concentration and pH, fractionation was performed using a cation exchange resin column (monoS HR5 / 5, Amersham Pharmacia Biotec). The fraction containing the protein of interest was diluted with 20 mM Tris buffer (pH 8.0) and fractionated on an anion exchange resin column (monoQSP1.6 / 5, Amersham Pharmacia Biotec) using the SMART system. The fraction containing the protein of interest was collected, diluted 10-fold with a 0.1% aqueous solution of trifluoroacetic acid, and further fractionated on a reversed-phase column (RPC C2 / C 18, Amersham Pharmacia Biotec). Thus, the target antigen protein was purified purely. This fraction was subjected to a mass spectrometer Q-TOF (Micromass) to obtain protein The partial sequence of the amino acid was determined. In particular, when a PIP database search was performed, the amino acid sequence ai matched human salivary acidic proline-rich Ohosphoprotein (PRP).

 'When the ConA column-adsorbed fraction of another lot of sweat was purified in the same manner, the modified Proptin-inducible protein (PIP) and the modified PIP in which the 84th tyrosine was methylated were confirmed. It was also confirmed that at least one arginine residue was methylated in this modified PIP. In other words, as a result of analyzing the digested peptide fragment obtained from the purified sweat antigen by Q-TOF, the mass corresponding to tyrosine and arginine contained mass having a mass 14 daltons larger than the theoretical molecular weight of each. Turned out to be.

 Furthermore, a leukocyte histamine release test, etc. using each of the above purified proteins was performed on individuals with an allergic disease or allergic predisposition, and it was confirmed that they reacted with high specificity. .

Example 2

 Preparation of Recombinant PRP Protein by Escherichia coli cDNA encoding human PRP (DNA sequence of SEQ ID NO: 1) was cloned from cDNA library of human cultured cell line 'ZR75-1' (ATCC strain No. CRL-1500), and pQE vector (QIAGEN ), And the resulting expression vector (pQE-PRP) was used to transform E. coli strain M15. After culturing it in large amounts, 2 mM IPTG was added to induce protein expression. The cultured E. coli cells were collected by centrifugation, and the cells were suspended in 8 M urea buffer and sonicated to solubilize the protein. The insoluble fraction was removed by centrifugation, and the recombinant PRP was purified using an affinity column for histidine tag (Ni-NTA agarose).

Example 3

Production of recombinant PIP using Escherichia coli CDNA encoding human PIP (DNA sequence of SEQ ID NO: 3) was cloned from the cDNA library of human cultured cell line ZR75-1, and used in the same manner as in Example 2 except that E. coli JM109 strain was used as a host cell. The recombinant PIP was purified by the method.

Example 4

 の Preparation of a heron anti-PRP polyclonal antibody

Recombinant PRP 100 / ig prepared in Example 2 was subcutaneously administered with Freund's complete adjuvant to the back skin of 4-week-old NZW Pergum, and three weeks later, recombinant PRP lOO jtig was subcutaneously administered with Freund's incomplete adjuvant. . Three weeks later, recombinant PRP100 g was subcutaneously administered together with Freund's complete adjuvant, and three days later, blood was collected from the auricular artery. The IgG fraction was purified using a protein G column, and the antibody titer was measured by immunoassay using an immobilized recombinant PRP immobilized plate to prepare a rabbit heron anti-PRP polyclonal antibody.

Example 5

Preparation of mouse anti-PRP monoclonal antibody The recombinant PRP prepared in Example 2 was mixed with Freund's complete adjuvant and administered intraperitoneally to the mouse.Two weeks later, the recombinant PRP was mixed with Freund's incomplete adjuvant. The mice were administered intraperitoneally. Two weeks later, recombinant PRP was intraperitoneally administered, and three days later, immunized mice were sacrificed by transection of the vertebrae, and the spleen was removed.The removed spleen was crushed on a nylon mesh, and RPMI culture solution was used. Cell suspension _{c c} Separately prepared mouse myeloma cell line Sp2 / 0-Agl4 cells were fused with splenocytes and polyethylene glycol and cultured in HAT medium. The fused cells (Eighty-brown dorma) were selectively cultured on a micro-tie plate. When colony formation can be confirmed under an inverted microscope, a part of the culture supernatant is collected, The binding to the antigen was confirmed by immunoassay. Pairs of cells in which binding to the antigen was confirmed were recovered, subjected to limiting dilution, and cultured in a microplate. Similarly, the cells of the gel showing positive results in immunoassay were collected, and the antibody-producing cells were collected again as a single clone by limiting dilution. This monoclonal cell line was cultured in large quantities, the culture supernatant was applied to a Tin G column, and the IgG fraction was affinity purified. The binding to PRP was confirmed by ELISA and Western blotting, and an anti-PRP monoclonal antibody was prepared.

Example 6

 Preparation of Mouse Anti-PIP Monoclonal Antibody A method similar to that of Example 5 except that the recombinant PIP prepared in Example 3 was used as an immunogen. Thus, a mouse anti-PIP antibody was prepared.

Example 7

 Preparation of Recombinant PRP Using Mammalian Cells The same PRP cDNA as in Example 2 was recombined into a mammalian expression vector (pEF-BOS; Nucleic Acids Res. 18 (17): 5322, 1990), and the resulting plasmid was obtained. (PEF-PRP) was transfected into CHO cells, and the insoluble fraction was removed by centrifugation from the culture supernatant obtained by culturing the cells (CHO-PRP) in large quantities. Recombinant PRP was purified by affinity column equipped with anti-PRP monoclonal antibody.

Example 8

Production of recombinant PIP by mammalian cells The PIP cDNA (PIPmat DNA) excluding the signal sequence was introduced into a mammalian expression vector (pME18S; Proc Natl Acad Sci US A. 89 (12): 5206-52 10, 1992) to obtain The resulting plasmid (pME18S-PIPmat) was transfected into COS-7 cells. Subsequently, a large amount of the recombinant cells (COS-PIPmat) were cultured, and the insoluble fraction was removed from the culture supernatant by centrifugation to obtain a fraction containing the recombinant PIPmat. Similarly, only the pME18S vector was transfected into COS-7 cells as a control, and the supernatant (imock) was prepared.

Example 9

 Measurement of specific IgE antibody in subject serum against PRP The recombinant PRP prepared in Example 2 was diluted to 1-5 ig / ml with phosphate buffer buffer (PBS), and the solution was added to all wells of a microtiter plate. Noted. After shaking at room temperature for 2 hours, the plate was washed twice with 0.02% Tween / PBS (T-PBS). 3% BSA / PBS solution was added to block nonspecific binding sites, and the mixture was shaken at room temperature for 2 hours. After washing twice with T-PBS, 100 / il of the serum of the subject diluted to various concentrations with 3% BSA / PBS was added and shaken for 2 hours. After washing twice with T-PBS, an HRP-labeled anti-human IgE antibody solution was added, shaken at room temperature for 2 hours, and washed twice with T-PBS. A few minutes after adding the chromogenic substrate, it was confirmed that the color had developed, a stop solution was added, and the HRP signal was detected with a plate reader.

 As a result, when sera from patients with atopic dermatitis were tested, the presence of specific IgE antibodies was detected, but IgE antibodies to which PRP binds were not detected in sera from healthy subjects.

Example 10

PIP quantification in sweat The anti-PIP monoclonal antibody prepared in Example 6 was diluted with PBS and —Dispensed to After leaving at room temperature for 2 hours, the plate was washed twice with T-PBS, then a 3% BSA / PBS solution was added for blocking nonspecific binding sites, shaken at room temperature for 2 hours, and washed twice with PBS. Add the sweat sample to each well, shake for 2 hours at room temperature, wash twice with Τ-PBS, add anti-PRP ゥ sagi polyclonal antibody diluted with T-PBS, shake for 2 hours at room temperature, Washed twice with T-PBS. Add HRP-labeled anti-mouse IgG antibody, shake for 2 hours at room temperature, wash twice with T-PBS, add chromogenic substrate, and after a few minutes, confirm that the color has developed, add stop solution, and add plate HRP signal was detected by the reader.

 As a result, the protein (PIP) binding to the anti-PIP monoclonal antibody was detected at a higher concentration in the sweat samples of patients with atopic dermatitis than in the sweat samples of healthy subjects.

Example 10

 Histamine-releasing activity of recombinant PRP and PIP The recombinant PRP prepared in Example 2 and the recombinant PIP prepared in Example 3 were further purified by reversed-phase columns (C2 / C18) using the FPLC system (SMART). did. As a result, the main peak appeared in the same fraction as the elution site of hissamine release activity when the sweat component was purified by the same column. In addition, histamine releasing activity from peripheral blood basophils of atopic dermatitis patients was detected in the fraction. The fraction was electrophoresed on SDS-PAGE and detected as a single band at 17 kD.

 From the above results, it was confirmed that recombinant PRP and recombinant PIP produced in Escherichia coli had an activity to induce histamine release from peripheral blood basophils of patients with atopic dermatitis.

Example 11

Measurement of histamine release activity of recombinant PIP and PRP expressed in mammals The recombinant PIPmat and mock obtained in Example 8 were concentrated by ultrafiltration, respectively, and reacted with basophils of atopic dermatitis patients. The amount of histamine released outside the cell and the amount of histamine in the cell by this reaction were measured, and the histamine release rate was examined.The release rate of mock was 10.3%, whereas that of recombinant PIPmat was 40.9%, a high rate. Met.

 Similarly, it was confirmed that the recombinant PRP prepared in Example 7 also had histamine releasing activity.

INDUSTRIAL APPLICABILITY As described in detail above, according to the invention of this application, as a causative factor of atopic dermatitis, an atopic dermatitis-inducing protein produced by a patient himself, and a part of this protein. Peptides involved in its antigenicity are provided. The present invention also provides a method for diagnosing atopic dermatitis using the above-mentioned protein and Z or a peptide, and a therapeutic agent for desensitization of atopic dermatitis using the same as an active ingredient. This makes it possible to diagnose with high accuracy the degree of the symptoms and the risk of developing atopic dermatitis, and to make effective treatment possible.

Claims

The scope of the claims 1. An atopic dermatitis-inducing protein, which is a protein secreted from the human body, which binds to its own IgE antibody and activates mast cells and basophils. . 2. The protein of claim 1, which is secreted from salivary glands or sweat glands. 3. The protein according to claim 1, which has two or more identical amino acid repeating sequences each consisting of four or more amino acid residues. 4. The protein according to claim 3, wherein the protein is a salivary acidic proline-rich phosphoprotein having the amino acid sequence of SEQ ID NO: 2. 5. The protein according to claim 1, wherein the protein is a Prolactin-inducible protein consisting of the amino acid sequence of SEQ ID NO: 4. 6. The protein of claim 1, wherein at least one of the constituent amino acid residues is post-translationally modified. ' 7. The protein according to claim 6, wherein the modified amino acid residue is at least one amino acid residue selected from the group consisting of lysine, arginine, methionine, alanine, phenylalanine, proline, asparagine, and tyrosine. 8. The protein according to claim 7, wherein the modifying group is an alkyl group. 9. The protein according to claim 8, wherein the protein is a Prolactin-inducible protein consisting of the amino acid sequence of SEQ ID NO: 4. 10. At least the 84th tyrosine residue of SEQ ID NO: 4 is methylated and modified. 10. The protein of claim 9, wherein 1 1. A peptide which is a part of the protein of claim 1 and which comprises an amino acid sequence constituting a binding region to an IgE antibody. 12. The peptide of claim 11 which is part of the protein of claim 3 or 4. 13. The peptide of claim 12, comprising at least the four consecutive amino acid sequences of SEQ ID NO: 2. 14. The peptide of claim 11, which is part of the protein of claim 5. 15. The peptide of claim 11, which is part of the protein of any of claims 6 to 10, 16. The peptide of claim 14 or 15, comprising at least the contiguous 4 amino acid 'sequence of SEQ ID NO: 4. 17. An antibody prepared using the protein of claim 1 or the peptide of claim 11 as an antigen, wherein the antibody binds to the protein of claim 1. 18. An antibody prepared using the protein of claim 3 or 4 or the peptide of claim 12 or 13 as an antigen, wherein the antibody binds to the protein of claim 3 or 4. 19. An antibody prepared using the protein of claim 5 or the peptide of claim 16 as an antigen, wherein the antibody binds to the protein of claim 5. 20. An antibody prepared using the protein according to any one of claims 6 to 10 or the peptide according to claim 16 as an antigen, wherein the antibody binds to the protein according to any one of claims 6 to 10. 21. Test for the presence of an antibody in the serum of the subject that binds to at least one of the proteins of claims 1 to 10 or to at least one of the peptides of claims 11 to 16. A method for diagnosing atopic dermatitis, comprising determining a subject whose serum antibody is present as a patient with atopic dermatitis or a person at high risk for atopic dermatitis. 22. Addition of at least one of the proteins of claims 1 to 10 or at least one of the peptides of claims 11 to 16 to the leukocyte fraction collected from the subject's blood, and the degree of histamine release is atopic. A method for diagnosing atopic dermatitis, comprising determining that the patient is a dermatitis patient or a high-risk atopic dermatitis patient. 23. A test is performed to determine whether or not a biological sample of a subject contains a protein that binds to one or more of the antibodies according to any one of claims 17 to 20, and the subject in which the protein is present in the sample is a patient with atopic dermatitis Alternatively, a method for diagnosing atopic dermatitis, characterized in that the subject is determined to be a person at high risk of atopic dermatitis. 24. A desensitizing treatment for atopic dermatitis, comprising as an active ingredient at least one of the proteins of claims 1 to 10 or at least one of the peptides of claims 11 to 16. medicine.