JP2009210420A - Allergosis remedy and therapeutic effect marker - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a certain and easy clinical marker for determination and/or prediction of therapeutic effect in allergosis and a radical allergosis remedy for allergosis therapy in particular. <P>SOLUTION: The clinical marker for determination and/or prediction of therapeutic effect in allergosis uses an antibody recognizable an apolipoprotein A-IV, an allergosis diagnosis uses the marker, and the allergosis remedy contains the apolipoprotein A-IV. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the use of apolipoprotein A-IV as a therapeutic effect determination marker in the treatment of allergic diseases, a method for determining and / or predicting therapeutic effect using the same, and allergic diseases containing apolipoprotein A-IV It relates to therapeutic drugs.

  In recent decades, the number of patients with allergic diseases has rapidly increased in Japan due to changes in lifestyle and living environment, and there has been a problem of lowering QOL and increasing the burden of medical expenses in patients. Allergic diseases are diseases caused by an excessive reaction of the immune system against foreign antigens that should be harmless in nature. Japanese cedar pollinosis is a type of this allergic disease, and its main cause is said to be an antigenic substance in Japanese cedar pollen, that is, a Japanese cedar pollinosis allergen. When cedar pollen scattered in the atmosphere enters the human body, B cells produce immunoglobulin E antibody (IgE) against the cedar pollen allergen. This IgE binds to the high affinity IgE receptor FcεRI present on the cell membranes of mast cells and basophils. When allergens such as cedar pollen enter again and cross-link IgE on these cell membranes, mediators such as histamine and leukotriene are released (degranulation reaction), leading to the development of allergies. Like allergies caused by other causes, such as food allergies, asthma, and atopic dermatitis, this cedar pollinosis has also become increasingly severe in recent years, and this trend is expected to become more serious in the future. .

  Currently, cedar pollinosis is mainly treated by pharmacotherapy using antihistamines as first-line drugs. However, pharmacotherapy has side effects such as sleepiness and malaise, dry mouth, nausea, diarrhea, and constipation. While the patient's condition cannot be kept healthy, it is not curative and can be taken forever. Can only suppress symptoms. When a steroid drug is used, a temporary (very) good therapeutic effect is observed (in some cases), but serious side effects including glaucoma and the like are concerned, and it is difficult to continue for a long time. In the case of surgical treatments such as lower turbinatectomy or laser cauterization of the lower turbinate, the effect continues for about 2 to 5 years.

  The only radical therapy is immunotherapy, which is a therapy that reduces the reactivity of the antigen by subcutaneous injection or sublingual or swallowing of the allergen (antigen). Although the mechanism is still unclear, immunotherapy has been found to be effective in about 60 to 70% of people. In addition, since side effects (systemic anaphylactic reaction) are rare (although severe), they are being established as effective therapies and will be recommended and spread in the future. However, since there is no appropriate clinical marker, the therapeutic effect cannot be predicted at all, and there is a problem that the effectiveness cannot be determined unless treatment is performed for at least 2 years.

  In foreign countries, anti-IgE antibody therapy that attempts to suppress the effect of IgE reacting with antigens with antibodies against IgE may be applied, but it is still not effective in all patients and is extremely expensive. Therefore, application to all patients is unrealistic at present. In addition, there is a concept of inducing harmless substances in the body to treat allergies, but the factors have not been identified.

  In recent years, protein analysis technology has remarkably developed, and it has become possible to comprehensively obtain information on protein amount (variation) in living tissues such as affected individuals. An example in which a novel disease marker is identified using this technique is also known. However, there have been no reports of using this approach in allergic rhinitis and cedar pollinosis, since considerable skill and expensive equipment are required to perform the series of experimental operations and data analysis of the technology. There are no reports related to the effectiveness of treatment. There have been several reports of comprehensive gene expression analysis comparing patient cells and normal cells, but most of the obtained genes were unanalyzed, and none were developed until functional analysis.

  There is a report example of apolipoprotein A-IV as one of proteins derived from living tissue. Apolipoprotein is an important component of lipoprotein, and is supposed to have a role of transporting lipid to a tissue in a soluble state in blood. The apolipoprotein A-IV gene contains three exons separated by two introns and is expressed in intestinal epithelial cells. The primary translation product is a 396-residue protein precursor that, after proteolytic processing, is secreted with milk fat particles in the intestine, the main site of synthesis.

  Although the exact function of this apolipoprotein A-IV is not known, it is said to act as a powerful activator of lecithin-cholesterol acyltransferase in vitro (Non-patent Document 1). Apolipoprotein A-IV knockout mice have decreased high-density lipoprotein levels (Non-Patent Document 2), and the overexpressed transformants show a preventive effect on atherosclerosis under certain conditions. Has also been reported (Non-Patent Document 3).

In addition, apolipoprotein A-IV has been reported to have anti-inflammatory and antioxidant effects in animal models and in vitro studies (Non-patent Document 4). In the gene polymorphism analysis of apolipoprotein A-IV, It has been suggested that there is a significant correlation with gene polymorphisms of genes closely related to atopic disease (Non-patent Document 5). However, until apolipoprotein A-IV shows a significant inverse correlation between its amount in plasma and the presence or absence and extent of the therapeutic effect of allergic diseases, and on its own, until it shows an inhibitory effect on allergic reaction alone I couldn't predict it at all.
J. Biol. Chem. 1994; 269: 29883-29890 J. Lipid Res. 1997; 38: 1782-1794 Science 1996; 273: 966-968 J. Clin. Invest. 2004; 114: 260-269 Circulation 2006; 113: 2193-2200

  The problem to be solved by the present invention is to provide a reliable and easy therapeutic effect determination and / or prediction marker and a means for contributing to the development of a curative allergic disease therapeutic method in the treatment of allergic diseases. is there.

  The present inventors conducted a sublingual immunotherapy double-blind test for cedar pollinosis patients over a long period of time, and conducted comprehensive protein analysis of the plasma of patients who were provided, and the proteins in the active drug group and the placebo group The two-dimensional electrophoresis profiles of were compared. As a result, apolipoprotein A-IV was found as a protein whose amount specifically increases only in the active drug group. Apolipoprotein A-IV was significantly elevated in the plasma of patients with clinical efficacy, and no change was observed in patients with no clinical efficacy or in the placebo group. That is, it was found that there is a significant correlation between the expression level of apolipoprotein A-IV and the clinical effect.

  As a result of examining the action (function) of artificially produced apolipoprotein A-IV, the present inventors have found that histamine release from human basophil cells is suppressed, and can be used as an antiallergic agent. This was confirmed and the present invention was completed. That is, the present invention is as follows.

[1] A clinical marker for determining and / or predicting the therapeutic effect of an allergic disease, comprising an antibody that recognizes apolipoprotein A-IV.
[2] The clinical marker according to [1], wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.
[3] The clinical marker according to [1], wherein the treatment of allergic disease is any one of immunotherapy, drug therapy, and surgical therapy.
[4] The clinical marker according to [3], wherein the immunotherapy is immunotherapy by sublingual administration, swallowing administration, or subcutaneous administration of allergen.
[5] The clinical medicine according to [3], wherein the drug is any one of a mast cell stabilizer, an antihistamine, an anti-leukotriene, an antiprostaglandin D2 / thromboxane A2, a Th2 cytokine inhibitor, or a steroid. marker.
[6] A method for determining and / or predicting the therapeutic effect of allergic diseases, comprising the following steps (A) and (B):
(A) a step of measuring the amount of apolipoprotein A-IV in a biological sample of a subject, and (B) a step of determining and / or predicting the therapeutic effect of allergic disease based on the measurement result of (A).
[7] A method for determining and / or predicting the therapeutic effect of allergic diseases, comprising the following steps (a), (b) and (c):
(A) a step of binding a protein prepared from a biological sample of a subject and the clinical marker according to any one of [1] to [5],
(B) measuring a protein derived from a biological sample bound to the clinical marker using the clinical marker as an index, and (c) determining the therapeutic effect of allergic disease based on the measurement result of (b) and / or Or predicting.
[8] The determination of the presence or absence of the therapeutic effect of the allergic disease in the step (c) is performed by comparing the measurement result obtained for the subject before the start of the treatment with the measurement result obtained for the subject after the start of the treatment. The method according to [7], wherein the method is carried out using as an indicator that the amount of binding to the predictive marker is increased.
[9] The method according to any one of [6] to [8], wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.
[10] The method according to any one of [6] to [8], wherein the treatment of allergic disease is any one of immunotherapy, drug therapy, and surgical therapy.
[11] The method according to [10], wherein the immunotherapy is immunotherapy by sublingual administration, swallowing administration or subcutaneous administration of allergen.
[12] The method according to [10], wherein the drug is any one of a mast cell stabilizer, an antihistamine, an anti-leukotriene, an antiprostaglandin D2 / thromboxane A2, a Th2 cytokine inhibitor, or a steroid. .
[13] The method according to [8], wherein the start of treatment is 6 months before the peak of cedar pollen scattering.
[14] The method according to [8], wherein the treatment is started 6 months to 1 year after the treatment is started.
[15] A therapeutic agent for allergic diseases, comprising apolipoprotein A-IV.
[16] The therapeutic agent according to [15], wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.

  In general, immunotherapy needs to be treated for several years, so the physical burden on the patient is great. If there is no effect in the short term, it is necessary to continue the drug treatment centering on the internal use of antihistamines while performing immunotherapy, and the overall medical cost will be higher than that of drug therapy alone. However, if the effect of immunotherapy can be predicted in a short period of time, the treatment can be discontinued and no drug treatment is required in an effective case. In other words, long-term medical expenses will be reduced. In doing so, it is necessary to establish an indicator of the effect. Once the index is established, it can be used for objective effect determination of other treatments, and the treatment management by numerical values can be performed, so that the physical burden on the patient and medical expenses can be suppressed.

  Apolipoprotein A-IV has a histamine release-inhibiting action on basophils (etc.), so it not only contributes to the provision of a curative treatment for cedar pollinosis, which is a national disease, but it can also be used in allergic diseases in general. Can have a therapeutic effect.

  In the present specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides, etc. are defined by IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. Biochem., 138: 9 (1984). ], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), and conventional symbols in the field.

  In the present specification, when the term “apolipoprotein A-IV gene” or “DNA of apolipoprotein A-IV” is used, a human apolipoprotein represented by a specific base sequence (SEQ ID NO: 1) unless otherwise specified by the SEQ ID NO: It is used to include A-IV genes (DNA) and genes (DNA) encoding homologues, mutants and derivatives thereof. Specifically, the human apolipoprotein A-IV gene (GenBank Accession No. NM 000482) described in SEQ ID NO: 1, its mouse homolog (eg, GenBank Accession No. NM 007468), rat homolog (eg, GenBank Accession No. NM) 012737) and the like.

  As used herein, the term “antibody” includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, Fab fragments, fragments produced by Fab expression libraries, and the like, which have antigen binding properties. Some of the antibodies are included.

  As used herein, “allergic disease” refers to a disease that develops upon antigen stimulation, and examples include hay fever, asthma, atopic dermatitis, allergic rhinitis, food allergy, and the like. Preferably, it is hay fever, in which the increased expression of the apolipoprotein A-IV gene correlates with the therapeutic effect, and more preferably cedar pollinosis.

  In the present specification, “treatment” refers to a medical practice for improving symptoms of allergic diseases, such as immunotherapy, drug therapy, or surgical therapy (inferior turbinate or mucosal mucosa by laser Cauterization). Preferably, it is a curative immunotherapy.

  As used herein, “immunotherapy” refers to desensitization therapy by sublingual administration, swallowing administration or subcutaneous administration of allergen, and immunoreactive treatment using innate immunity ligands, preferably safe and side effects It is immunotherapy by sublingual administration or swallowing administration.

  As used herein, the term “drug” refers to a drug used in drug therapy for improving symptoms of allergic diseases. For example, a mast cell stabilizer, an antihistamine, an anti-leukotriene, an antiprostaglandin D2 Examples include thromboxane A2 drugs, Th2 cytokine inhibitors, steroid drugs, and the like.

  In the present invention, “therapeutic effect determination and prediction marker” refers to a substance for identification that is directly or indirectly used for diagnosing the presence / absence of an effect or the prediction of the effect by a treatment method for allergic diseases. This includes proteins that vary in quantity and antibodies that can specifically recognize and bind to them in living tissues, particularly in intestinal mucosal epithelial cells or plasma in connection with the treatment of allergic diseases. Based on the above properties, this antibody can be effectively used as a probe for detecting the protein expressed in living tissue or cells.

  The “biological tissue” to be diagnosed in the present invention refers to a tissue or a cell in which the amount of apolipoprotein A-IV of the present invention increases with the treatment of allergic diseases caused by antigen stimulation. Specific examples include intestinal mucosal epithelial cells, bronchial mucosa, nasal mucosa, gastric mucosa, intestinal mucosa, oral mucosa, blood and the like.

  The clinical marker for determination and / or prediction of therapeutic effect of the present invention is characterized by containing an antibody recognizing apolipoprotein A-IV.

  The antibody is used to detect the presence or absence of apolipoprotein A-IV in a biological sample of a subject or to quantify the amount of the antibody, thereby determining the effect of treating the allergic disease received by the subject (treatment) It is useful as a clinical marker for effect determination and / or prediction.

  The antibody is also useful as a tool (clinical marker for predicting therapeutic effect) for detecting fluctuations in the amount of apolipoprotein A-IV in the future prediction of the therapeutic effect of allergic diseases described below.

  The form of the antibody is not particularly limited, and may be a polyclonal antibody using apolipoprotein A-IV as an immunizing antigen or a monoclonal antibody thereof. Furthermore, it may be a chimeric antibody, a single chain antibody, a humanized antibody, or a fragment generated by a Fab fragment or a Fab expression library prepared based on the gene encoding the monoclonal antibody.

  Methods for producing these antibodies are known, and the antibodies can also be produced according to conventional methods (Current Protocol in Molecular Biology, Chapter 11.12 to 11.13 (2000)). Specifically, when the antibody of the present invention is a polyclonal antibody, an oligopeptide having a partial amino acid sequence of these proteins using apolipoprotein A-IV expressed and purified in Escherichia coli according to a conventional method or according to a conventional method Can be synthesized to immunize non-human animals such as rabbits, and obtained from the sera of the immunized animals according to a conventional method. On the other hand, in the case of a monoclonal antibody, a non-human animal such as a mouse is immunized with an oligopeptide having a partial amino acid sequence of apolipoprotein A-IV expressed and purified in Escherichia coli according to a conventional method, and the resulting spleen cells and bone marrow It can be obtained from hybridoma cells prepared by cell fusion with tumor cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).

  Apolipoprotein A-IV used as an immunizing antigen for antibody production is based on DNA cloning, construction of each plasmid, transfection into a host, transformant based on the sequence information of the gene provided by the present invention and the like. It can be obtained by culturing and recovering the protein from the culture. These operations are based on methods known to those skilled in the art or methods described in the literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, DM. Glover, IRL PRESS (1985)), etc. Can be done.

  Specifically, a recombinant DNA (expression vector) capable of expressing a gene encoding apolipoprotein A-IV in a desired host cell is prepared, and this is introduced into a host cell for transformation. By culturing and recovering the target protein from the resulting culture, a protein as an immunizing antigen for producing the antibody of the present invention can be obtained. Moreover, the partial peptides of these apolipoproteins A-IV can also be produced by a general chemical synthesis method (peptide synthesis) according to the amino acid sequence information provided by the present invention and the like.

The method for determining and / or predicting the therapeutic effect of an allergic disease of the present invention comprises the following steps (A) and (B):
(A) a step of measuring the amount of apolipoprotein A-IV in a biological sample of a subject, and (B) a step of determining and / or predicting the therapeutic effect of allergic disease based on the measurement result of (A).

  In one aspect, the method for determining and / or predicting the therapeutic effect of an allergic disease of the present invention is characterized by using the clinical marker for determining and / or predicting the therapeutic effect of the present invention. Since the clinical marker has a property of specifically binding to apolipoprotein A-IV, the apolipoprotein A-IV present in the animal tissue can be specifically detected.

For example, the method for determining and / or predicting the therapeutic effect of an allergic disease of the present invention can be performed by a method including the following steps (a), (b) and (c):
(a) a step of binding a protein prepared from a biological sample of a subject and a clinical marker (antibody) for determination and / or prediction of the therapeutic effect of the present invention,
(b) measuring a protein derived from a biological sample bound to the clinical marker using the clinical marker as an index,
(c) A step of determining and / or predicting the therapeutic effect of allergic disease based on the measurement result of (b).

  Specifically, the method for determining and / or predicting the therapeutic effect of the present invention uses the clinical marker (antibody) for the determination and / or prediction of the therapeutic effect of the present invention as a probe, ELISA method, Western blotting method, It can be carried out by using the detection means such as a radioisotope immunoassay (RIA method), a fluorescent antibody method, an immunohistochemical staining method, etc. as an indicator that the amount of protein binding to the clinical marker is increased.

  Depending on the type of detection means used, a part of the subject's tissue (bronchial mucosa, nasal mucosa, gastric mucosa, intestinal mucosa, oral mucosa, etc.) can be collected with a biopsy, etc. A protein prepared according to a conventional method from a sample obtained by collecting cells or the like present in blood (eg, blood, sputum, nasal discharge, gastric juice), or a protein dissolved in a body fluid can be used.

  More specifically, the method for determining and / or predicting the therapeutic effect of the allergic disease of the present invention uses the clinical marker (antibody) for the determination and / or prediction of the therapeutic effect of the present invention by the ELISA method or the like. This can be carried out by using as an indicator that the amount of apolipoprotein A-IV binding to the marker is increased.

  The ELISA method may be performed using any of a direct adsorption method, a sandwich method, and a competitive method, but is preferably performed using a sandwich method with high detection specificity. The clinical marker of the present invention is used as a capture antibody or primary antibody, and reacted with a sample component to be measured in an appropriate container. Then, a labeled antibody (an antibody that binds to the primary antibody) labeled with an enzyme such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP) is used as the secondary antibody, reacted with the primary antibody, and then the enzyme and its substrate And a specific signal obtained is detected and quantified with a spectrophotometer or a (micro) plate reader.

  For example, in the case of blood tests, the amount of apolipoprotein A-IV in the subject's blood (plasma) is compared with the amount of the protein in the control blood. This can be done by judging. In the case of diagnosis such as determination and / or prediction of the therapeutic effect of immunotherapy in Japanese cedar pollinosis, there is a significant correlation between the variation in the amount of apolipoprotein A-IV and the therapeutic effect of immunotherapy. The apolipoprotein A-IV concentration (protein amount) in the plasma is compared with the control protein concentration (protein amount), and the difference between the two is determined.

  In this case, a biological sample (a sample containing protein) collected from a subject before the start of treatment is necessary as a control. However, a part of the tissue of a patient suffering from an allergic disease before the start of treatment is biopsied or the like. A protein prepared according to a conventional method from a sample obtained by collecting or collecting cells or the like present in a body fluid or a protein dissolved in a body fluid can be used. As used herein, “a person suffering from allergic disease” has at least a subjective symptom of allergic disease, and preferably results of other test methods include, for example, abnormal IgE (RAST) values, various specific IgE (RIST) ) A person who has been diagnosed with an allergic disease due to abnormal values, positive skin test, positive prick test, or positive eosinophils in nasal discharge, sputum, or ear leakage. The “person suffering from allergic disease” may be simply referred to as a patient in this specification.

  Comparison of the amount of protein between the tissue before the start of treatment and the tissue after the start of treatment is performed by measuring the biological sample collected from the subject before the start of treatment and the biological sample collected from the subject after the start of treatment. it can. When comparing the amount of protein in the treatment of Japanese cedar pollinosis, blood is collected 6 months before the peak of annual pollen scattering and used as a sample before the start of treatment, and then the pollen is produced 6 months after the start of treatment. Blood is again collected at the peak of scattering and used as a sample after the start of treatment. Alternatively, regardless of whether the pollen is scattered or not, blood is collected before the start of treatment and 6 months after the start of treatment and after 1 year, and is used as a sample before the start of treatment and after the start of treatment, respectively. A value of apolipoprotein A-IV obtained by measuring them under uniform measurement conditions can be used for comparison.

  The determination of whether a subject has a therapeutic effect on allergic disease (current and in the future) is based on whether the protein level of apolipoprotein A-IV in the subject's tissue after treatment has started It can be carried out as an index that the number is twice or more, preferably three times or more compared. If the protein level of the subject after the start of treatment is considerably higher than the level before the start of treatment, it can be determined that the subject has (in the future) the therapeutic effect of the allergic disease or is highly likely . Regarding the relationship between the protein level and the therapeutic effect, it can be determined that the higher the level, the higher the therapeutic effect.

  The therapeutic agent of the present invention is not particularly limited as long as it contains apolipoprotein A-IV itself as an active ingredient.

  The present invention provides a therapeutic agent and a therapeutic or preventive method capable of preventing or treating an allergic disease state by applying it to an animal.

  The animals are preferably humans and vertebrates other than humans, and in particular, domestic animals such as cows, horses, pigs, sheep, goats, chickens, dogs, and cats, or pets are preferred.

  The therapeutic agent of the present invention is a therapeutic agent for allergic diseases, preferably a therapeutic agent for Japanese cedar pollinosis, and in particular, treatment of Japanese cedar pollinosis in patients who are not expected to have the effect of immunotherapy or are not expected to be recognized. It can also be used as a medicine.

  The apolipoprotein A-IV contained in the therapeutic agent of the present invention may be an artificially produced protein, and can be produced by the same method as apolipoprotein A-IV used as an immunizing antigen for the production of the above-mentioned antibody. . Human apolipoprotein A-IV (for example, see GenBank / EBI Data Bank Accession Nos. NM 000482 (cDNA) and AY422950 (genomic)) gene when intended for use in humans or human cells What was expressed and refine | purified by introduce | transducing an expression vector into the cell derived from a human is preferable. In addition, the apolipoprotein A-IV of the therapeutic agent is an epitope such as a histidine (His) tag, Flag tag, Myc tag, etc. so that it can be distinguished from natural apolipoprotein A-IV or to facilitate purification. May be apolipoprotein A-IV to which is added.

  The therapeutic agent of the present invention may be the active ingredient as it is, or may contain a known pharmaceutically acceptable carrier and the like. Examples of the carrier include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, arabic Binders such as rubber, polyethylene glycol, sucrose, starch, starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerosil, talc , Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyllysine / ammonium salt, glycine, orange powder, sodium benzoate, sulfurous acid Preservatives such as sodium hydrogen, methylparaben, propylparaben, stabilizers such as citric acid, sodium citrate, acetic acid, suspensions such as methylcellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, water, physiological Examples include diluents such as saline, base waxes such as cacao butter, polyethylene glycol, and white kerosene, but are not limited thereto.

  The therapeutic agent of the present invention can be used in combination with other allergy therapeutic agents or immunosuppressive agents. The combination may be combined in one preparation, or may be administered as separate preparations at the same time or at appropriate intervals.

  Examples of drugs that can be used in combination include mast cell stabilizers, antihistamines, anti-leukotrienes, anti-prostaglandin D2 / thromboxane A2 drugs, Th2 cytokine inhibitors or steroid drugs.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited at all by these Examples.

[Example 1]
Plasma was collected from cedar pollinosis patients (15 active and 9 placebo) who participated in a placebo-controlled double-blind study of cedar-specific sublingual immunotherapy before the trial and 6 months after peaking did. The internal standard plasma (mixed equal volume of all samples), the plasma before the start of the test, and the plasma after 6 months (30 g protein amount) are labeled with Cy2, Cy3, and Cy5, respectively, and Ettan DIGE system (GE healthcare) Was used for two-dimensional electrophoresis in the pH 4-7 range. Decyder software (GE healthcare) was used for the analysis, and changes that were 1.1 times or more after 6 months compared to before the start of the study were defined as being changed. The expression intensity at each spot was obtained by Decyder, and these data were analyzed by GeneSpring software (Silicon Genetics). This study has been approved by the Ethics Committee of Fukui University School of Medicine.

  Mass spectrometry was performed on spots showing statistically significant (P <0.05) changes. After two-dimensional electrophoresis of plasma for mass spectrometry analysis, the gel was stained with silver (DodecaSilver Stain Kits, BioRad), and the target spot was cut out with Ettan Spot Picker (GE healthcare). After destaining and trypsin treatment, analysis was performed using MALDI-TOFMS: AXIMA-CFR plus (Shimadzu), and the resulting peptide mass fingerprint (PMF) was analyzed using the Mascot database. The identification was made when the result obtained by Mascot and the Swiss-2DPAGE data matched or when the result was reproduced by Mascot analysis after multiple MALDI-TOFMS. As a result, there were 8 proteins (8 spots) that significantly changed only in the active drug group, 3 proteins (3 spots) that changed only in the placebo group, and 5 that changed in both groups. It was protein (5 spots) (FIG. 1). Numbers were assigned to a total of 16 spots, and protein identification of each spot was attempted. As a result, 14 spots of protein could be identified.

  When the plasma concentration of apolipoprotein A-IV among the proteins significantly increased only in the active drug group was measured by ELISA, the rate of variation showed a significant inverse correlation with the clinical score at the peak of cedar scattering. (Table 1). In other words, in cases where sublingual immunotherapy was effective, apolipoprotein A-IV levels were increased.

  To quantify apolipoprotein A-IV in plasma, anti-human apolipoprotein A-IV antibody (A4-18A3, BML Inc, Kawagoe, Japan) was coated on the plate, and the sample or recombinant apolipoprotein A-IV was plated. This was performed by an ELISA system for detection with a biotinylated anti-human apolipoprotein A-IV antibody (A4-11G12, BML Inc). A lower symptom score indicates better symptoms with less sneezing, runny nose and nasal congestion.

[Example 2]
DNA was amplified by PCR from a human liver cDNA library. The primer in that case is
F primer CAG TCG ACG ATG TTC CTG AAG GCC GTG GTC
R primer GGG ATC CCA GCT CTC CAA AGG GGC CA
Met.

  The amplified DNA and histidine-tagged protein expression plasmid vector pBudCE4.1 (Invitrogen Corporation, Tokyo) were cleaved with restriction enzymes SalI and BamHI, respectively, and each was subjected to electrophoresis on an agarose gel to extract DNA. Apolipoprotein A-IV was ligated with pBudCE4.1 at Ligationhigh (TAKARA; Takara Shuzo, Kyoto). This was cultured in LB medium together with E. coli DH5α. Thereafter, DH5α was seeded on LB agar medium to form colonies. In particular, this colony was further cultured in LB medium supplemented with zeocin, to which pBudCE4.1 is resistant. Centrifugation gave an E. coli pellet, and the plasmid vector was purified by QIA prep spin (QIAGEN; Qiagen, Tokyo). The obtained plasmid vector was confirmed to have a cDNA sequence of apolipoprotein A-IV by a sequencer.

  COS7 cells in which the resulting vector was cultured were transfected using effectene (QIAGEN). After culturing for 2 days, a cell lysate was prepared, and the centrifuged supernatant was passed through a column of Ni-NTA Fast start kit (QIAGEN) to obtain apolipoprotein A-IV with a histidine tag. Furthermore, the eluted apolipoprotein A-IV was placed in a cellulose dialysis tube and suspended in PBS for about 18 hours to obtain a PBS solution of apolipoprotein A-IV. It was confirmed that apolipoprotein A-IV was purified by Coomassie staining and Western blotting.

Inhibition of histamine release From human peripheral blood mononuclear cells, isolated basophils in a 96-well plate, 5 × 10 ⁴ cells per well, 10% FBS-added RPMI 180 μl, autoserum 20 μl at 37 ° C., 30 minutes After warming, 1 μg / ml of apolipoprotein A-IV was added. After culturing at 37 ° C. for 30 minutes, 0.1 μg / ml of cedar pollen extract Cryj1 was added. After further incubation at 37 ° C. for 30 minutes, rapid cooling was performed to stop histamine release. The difference in the histamine release rate was determined by the presence or absence of apolipoprotein A-IV addition from the histamine concentration in the culture, the cytoplasm when the cytoplasm was disrupted by ultrasound, and the total amount of histamine including the culture (Table). 2). The histamine concentration was quantified by measuring the absorbance at 430 nm using ELISA for histamine measurement. An average of 43.3% inhibition of histamine release was observed.

  The amount of histamine released when no apolipoprotein A-IV was added and when 1 μg / ml apolipoprotein A-IV was added was p <0.01 by non-parameteirc test, and a significant difference was observed.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the parameter | index for the treatment effect determination and / or prediction of allergic diseases, such as a cedar pollinosis, and it can perform an easy and exact diagnosis of the said disease, and apolipoprotein A -Development of new therapeutic agents for allergic diseases based on the mechanism of action of -IV.

Images obtained by performing two-dimensional electrophoresis on proteins derived from plasma of subjects before and 6 months after the start of the test, and acquiring and analyzing data. The spot (Spot) considered that there was a significant fluctuation | variation in an active drug group, a placebo group, and the both groups is shown. Spot 10 represents apolipoprotein A-IV. The figure which made the result of Table 1 into a graph.

Claims (16)

  A clinical marker for determining and / or predicting the therapeutic effect of an allergic disease, comprising an antibody that recognizes apolipoprotein A-IV.   The clinical marker according to claim 1, wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.   The clinical marker according to claim 1, wherein the treatment of allergic disease is any one of immunotherapy, drug therapy, and surgical therapy.   The clinical marker according to claim 3, wherein the immunotherapy is immunotherapy by sublingual administration, swallowing administration or subcutaneous administration of allergen.   The clinical marker according to claim 3, wherein the drug is any one of a mast cell stabilizer, an antihistamine, an anti-leukotriene, an antiprostaglandin D2 / thromboxane A2, a Th2 cytokine inhibitor, or a steroid. A method for determining and / or predicting the therapeutic effect of allergic diseases, comprising the following steps (A) and (B):
(A) a step of measuring the amount of apolipoprotein A-IV in a biological sample of a subject, and (B) a step of determining and / or predicting the therapeutic effect of allergic disease based on the measurement result of (A). A method for determining and / or predicting the therapeutic effect of allergic diseases, comprising the following steps (a), (b) and (c):
(A) a step of binding a protein prepared from a biological sample of a subject and the clinical marker according to any one of claims 1 to 5;
(B) measuring a protein derived from a biological sample bound to the clinical marker using the clinical marker as an index, and (c) determining the therapeutic effect of allergic disease based on the measurement result of (b) and / or Or predicting.   The determination of the presence or absence of the therapeutic effect of allergic disease in step (c) compares the measurement result obtained for the subject before the start of treatment with the measurement result obtained for the subject after the start of treatment, and determines and / or predicts the therapeutic effect. The method according to claim 7, wherein the method is performed using as an indicator that the amount of binding to the marker is increased.   The method according to any one of claims 6 to 8, wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.   The method according to any one of claims 6 to 8, wherein the treatment of allergic diseases is any of immunotherapy, drug therapy, and surgical therapy.   The method according to claim 10, wherein the immunotherapy is an immunotherapy by sublingual administration, swallowing administration or subcutaneous administration of allergen.   The method according to claim 10, wherein the drug is any one of a mast cell stabilizer, an antihistamine, an anti-leukotriene, an anti-prostaglandin D2 / thromboxane A2, a Th2 cytokine inhibitor, or a steroid.   The method according to claim 8, wherein the start of treatment is 6 months before the peak of cedar pollen scattering.   The method according to claim 8, wherein the treatment is started 6 months to 1 year after the treatment is started.   A therapeutic agent for allergic diseases containing apolipoprotein A-IV. The therapeutic agent according to claim 15, wherein the allergic disease is hay fever, allergic rhinitis, allergic conjunctivitis, bronchial asthma, atopic dermatitis or food allergy.