KR100733695B1 - Composition for prevention treatment and diagnosis of chronic inflammatory airway diseases - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention and treatment of chronic inflammatory airway disease containing alpha-enolase protein as an active ingredient.

The present invention also provides a diagnostic composition and diagnostic kit for chronic inflammatory airway disease containing alpha-enolase protein.

In addition, the present invention is a composition for screening a pharmaceutical agent for treating chronic inflammatory airway disease comprising at least one of alpha-enolase protein and autoantibodies to alpha-enolase in patients with chronic inflammatory airway disease, and treatment of chronic inflammatory airway disease using the same A pharmaceutical screening method is provided. The present invention also provides a method for diagnosing, preventing and treating chronic inflammatory airway disease using alpha-enolase protein.

Alpha-enolase, chronic inflammatory airway disease, bronchial asthma, chronic obstructive pulmonary disease

Description

Composition for prevention, treatment and diagnosis of chronic inflammatory airway diseases

1 is a diagram showing human airway epithelial cells (A549 cells) proteins that react with IgG autoantibodies present in serum of normal and severe asthma patients using immunoblot.

Figure 2 is a diagram stained with Coomassie blue after SDS-PAGE of human airway epithelial cells (A549 cells) protein.

3 is a diagram showing human airway epithelial cells (A549 cells) proteins that react with IgG autoantibodies in serum of severe asthma patients using a two-dimensional immunoblot.

4 is a diagram showing the location of alpha-enolase protein in human airway epithelial cell (A549 cell) protein using goat anti-human alpha-enolase antibody using a two-dimensional immunoblot.

5 is a diagram showing the IgG autoantibody response to the recombinant human alpha-enolase protein using the immunoblot using the serum of normal and severe asthma patients.

FIG. 6 is a diagram illustrating human airway epithelial cell (A549 cell) proteins reacting with IgG autoantibodies present in serum of normal and normal lung function emphysema patients and patients with chronic obstructive pulmonary disease using immunoblot.

7 is a diagram showing the IgG autoantibodies to the recombinant human alpha-enolase protein using the immunoblot using the serum of the normal, normal lung function emphysema patients, patients with chronic obstructive pulmonary disease.

FIG. 8 is a diagram illustrating the immune response between IgG autoantibodies in serum and purely isolated recombinant human alpha-enolase protein in normal, severe asthma and chronic obstructive pulmonary disease patients by enzyme-linked immunoassay (ELISA).

9 is a diagram illustrating proteins reacting with IgG autoantibodies against human airway epithelial cells (A549) and mouse liver cancer cell line (Hepa 1-6) proteins using serum samples of two severe asthma patients using immunoblot. .

10A is a diagram illustrating cytotoxicity by treating serum IgG antibody of a severe asthma patient to a human airway epithelial cell line, and FIG. 10B is a diagram illustrating cytotoxicity by treating serum IgG antibody of a patient with chronic obstructive pulmonary disease on a human airway epithelial cell line. 10C is a diagram illustrating cytotoxicity by treating a goat airway epithelial cell line with a goat IgG antibody or a normal goat IgG antibody against alpha-enolase.

11 is a diagram showing that the cytotoxicity caused by IgG autoantibodies to human airway epithelial cell line is suppressed when IgG antibodies of patients with severe asthma and chronic obstructive pulmonary disease are previously adsorbed using recombinant human alpha-enolase protein.

Figure 12a is a measure of the amount of interleukin-8 secretion from airway epithelial cells treated with IgG antibody in normal control and bronchial asthma patients.

12B is a diagram showing that the interleukin-8 secretion from airway epithelial cells is suppressed when IgG antibody of bronchial asthma is adsorbed with alpha-enolase protein.

The present invention relates to a composition for the prevention, treatment and diagnosis of chronic inflammatory airway diseases.

In the present invention, chronic inflammatory airway disease is a generic disease name that collectively refers to various diseases characterized by chronic inflammatory reactions of the trachea, bronchus, bronchioles, and alveoli resulting from damage and changes in airway tissues (Wardlaw A Et al., Clin Exp Allergy 2005; 35: 1254-62). Chronic inflammatory airway disease, especially bronchial asthma and chronic obstructive disease, may be defined differently, but there are a large number of patients who have these two components at once and it is difficult to classify as either of them (Guerra S, Curr Opin). Pulm Med 2005; 11: 7-13). These two diseases exist in the present invention because there is a so-called "Dutch hypothesis" (Sluiter HJ, et al., Eur Respir J 1991; 4: 479-89), which is another phenotype of one disease caused by the same pathogenesis. The disease is also included in chronic inflammatory airway disease.

Bronchial asthma is a chronic disease that causes airway hyperresponsiveness and intermittent airway contractions due to chronic inflammation of the airways, causing respiratory distress, and there is no known cure for it yet (Global Initiative for Asthma.NIH Publication No. 02-3659, 2002). Severe asthma accounts for about 5-10% of all bronchial asthma, and the current drug therapy is known to increase the risk of death from asthma because the disease is not effectively controlled.

Traditionally, bronchial asthma is caused by allergens (allergens) such as house dust mites and pollen in the outside environment, and inflammation of airway mucosal tissues occurs due to excessive immune responses (allergic reactions) to these substances. As a result, it is believed that asthma symptoms such as coughing and dyspnea occur (Lemanske RF Jr, et al. JAMA 1997; 278: 1855-1873; Global Initiative for Asthma.NIH Publication No. 02-3659, 2002). However, a significant proportion (30-50%) of bronchial asthma patients do not find evidence of allergic reactions to foreign substances present in the environment, and the cause and pathogenesis of the disease are unclear. (Global Initiative for Asthma.NIH Publication No. 02-3659, 2002, p50-66).

Histopathologically, the target of inflammatory responses in bronchial asthma is airway epithelial cells (Montefort S et al., Clin Exp Allergy 1992 22: 511-520), and severe asthma is an airway epithelial cell that develops above airway epithelial cells. It has been claimed to be a disease (Chanez P. Eur Respir J 2005 Jun; 25: 945-6). Thus, in addition to allergic mechanisms known to date, what are the pathogenesis mechanisms that cause chronic inflammatory reactions of airway tissues, and especially severe asthma, have been suggested as a major research subject to be identified in the future (Global Initiative for Asthma.NIH Publication No. 02-3659, 2002, p50-66).

In the previous report, rat and immunized rat serum and normal rat serum, which were injected intravenously into normal rabbits and finally inhaled into the respiratory tract, showed no significant response to lung tissue antigens compared to normal rat serum. In rabbits receiving immunized rat serum, acute respiratory distress and aerobic dysfunction appropriate for human bronchial asthma were observed and confirmed by auscultation, confirming that the antibodies against lung tissue may cause asthma, Histopathological examinations showed that both airway constriction for bronchial asthma, pathologic findings for emphysema, and inflammation of the blood vessels of small arteries in the lung (Karol SA et al., Vrach Delo 1966; 4). : 28-32). In addition, examination of airway tissues of asthmatic patients who died of severe asthma attacks reported deposition of IgG antibodies and complement in airway mucosal tissues (Callerame ML et al., N Engl J Med 1971; 284: 459-64). Biopsies of airway tissues using bronchoscopes in the affected asthma patients reported IgG antibody and complement deposition in airway epithelial cells (Molina C et al., Clin Allergy 1977; 7: 137-45). Based on these reports, recent claims have been made that bronchial asthma is an autoimmune disease (Rottem M, Shoenfeld Y. Int Arch Allergy Immunol 2003; 132: 210-4).

However, there has been no logical causal relationship between autoimmunity and bronchial asthma, which has been clinically significant and has not been specifically identified as a meaningful autoantigen that can be logically associated with chronic inflammatory responses in airways.

Recently, the inventors have found that airway epithelial cell autoantigens associated with non-allergic asthma are cytokeratin 18 protein (Nahm DH, et al. Am J Respir Crit Care Med 2002; 165: 1536-1539). However, autoantibodies have not yet been used for the diagnosis or classification of bronchial asthma, as the autoantigen proteins associated with severe asthma have not yet been identified.

In particular, life-threatening asthma patients with asthma are less likely to respond to conventional treatment medications and are treated with severe asthma or other nonsteroidal anti-inflammatory drugs, including aspirin. A simple test that makes it easy to detect, classify, and diagnose phenotypes of certain bronchial asthma, such as asthma (ie aspirin-sensitive asthma) with high risk of developing severe acute exacerbation of asthma. It doesn't exist yet.

In addition, effective prophylactic, prophylactic and therapeutic agents, or treatments have not yet been developed to treat patients with bronchial asthma, such as severe asthma or aspirin-sensitive asthma, to fundamentally improve and prevent death from asthma. It is not.

On the other hand, the current definition of Chronic Obstructive Pulmonary Disease (COPD) is defined as chronic bronchitis, or pathological histologically, with persistent cough, sputum, and respiratory distress, which are mainly defined by clinical symptoms. Two major underlying diseases, including emphysema, which show irreversible destruction of the airway wall of the airways below the bronchiole and end up with clinically progressive respiratory distress, progressing irreversible obstruction of the airways. This is a chronic disease seen. Chronic obstructive pulmonary disease is the fourth most common cause of death in the United States and Europe and is known to die from complications such as respiratory failure or infection (GOLD workshop summary. Am J Respir Crit Care Med 2001; 163: 1256-1276).

Some patients who have had asthma for a long time are known to have an irreversible airway obstruction that is indistinguishable from chronic obstructive pulmonary disease and can progress to chronic obstructive pulmonary disease (Celli BR et al., Eur Respir J 2004; 23: 932-946). ). Observing short- and long-term response to treatment drugs in a large number of patients with chronic obstructive pulmonary disease that meets current universal diagnostic criteria (GOLD workshop summary. Am J Respir Crit Care Med 2001; 163: 1256-1276). In all cases, it has been shown that respiratory obstruction significantly improves after various types of bronchodilators and steroid inhalation therapy. Therefore, many patients who are consistent with the definition of chronic obstructive pulmonary disease and bronchial asthma are known (Guerra S, Curr). Opin Pulm Med 2005; 11: 7-13).

Currently, COPD is considered to be the most important risk factor for smoking. In addition, various inflammatory-inducing substances (IL-8) from airway epithelial cells are caused by stimulation such as pollutants or chronic bacterial and viral infections. It is estimated that the disease is caused by the secretion of the airway tissue and chronic inflammation of airway tissue (GOLD workshop summary. Am J Respir Crit Care Med 2001; 163: 1256-1276). However, little is known about why the acute inflammatory response caused by these external stimuli has not disappeared and persists chronically. Therefore, it is speculated that various etiologies may be involved in addition to smoking (O'Byrne PM, Postma DS.Am J Respir Crit Care Med 1999; 159: S41-S66). In particular, the reasons why airway inflammatory reactions and airway epithelial cell death persist even after quitting smoking in chronic obstructive pulmonary disease (Hodge S et al., Eur Respir J 2005; 25: 447-54) . Therefore, chronic obstructive pulmonary disease has not been known to the exact cause and pathogenesis until now, it is known that the underlying treatment is difficult, and the risk of death is high when the disease progresses significantly reduced lung function (GOLD workshop summary. Am J Respir Crit Care Med 2001; 163: 1256-1276).

In patients with chronic obstructive pulmonary disease, autoantibodies to antigens present in human airways or lung tissue have been reported (Wagner V et al., Acta Allergol 1965; 20: 1-9). Based on this, the autoimmune hypothesis that chronic obstructive pulmonary disease will occur through autoimmune mechanism has been suggested (Agusti A et al., Thorax 2003; 58: 832-834), but autoantibodies still exist in the blood of patients with chronic obstructive pulmonary disease. This hypothesis has not been clearly demonstrated because autoantigen proteins that react with do not have been identified. On the other hand, it has recently been demonstrated that emphysema may occur in animals by an autoimmune mechanism for vascular endothelial cell autoantigens in relation to the pathogenesis of emphysema, one of the major underlying diseases causing chronic obstructive pulmonary disease (Taraseviciene-Stewart L, Et al., Am J Respir Crit Care Med. 2005; 171: 734-42). Specifically, the results of the experiment showed that when immunizing human vascular endothelial extract antigen to rats to induce an autoimmune reaction, it causes emphysema, and when CD4 + cells are separated from the spleen of emphysema induced rats and transferred to other rats manually. In other rats, emphysema develops, and mice are reported to develop emphysema in mice only when antibodies to vascular endothelial cells obtained from the serum of rats immunized with vascular endothelial cells are injected (Taraseviciene-Stewart). L, et al., Am J Respir Crit Care Med. 2005; 171: 734-42). In other words, although the target autoantigen protein involved in disease development has not yet been identified, autoantibodies to vascular endothelial cell antigens can be used to prove chronic obstructive pulmonary disease.

Meanwhile, in the present invention, alpha-enolase, which has been identified as a target autoantigen protein of chronic inflammatory airway disease, is present in the cytoplasm as a major glycolytic enzyme protein and is also expressed on the cell surface. It exists in the cytoplasm and cell surface of various hematopoietic, epithelial and endothelial cells and has the ability to act as a plasminogen receptor, meaning it plays an important role in the fibrinolytic system (Pancholi V. Cell Mol Life Sci. 2001; 58: 902-920). In addition, the role of alpha-enolase protein as a target autoantigen protein in various chronic inflammatory diseases (Pancholi V., Cell Mol Life Sci. 2001; 58: 902-920) is known, but bronchial asthma or chronic obstructive pulmonary disease is still known. No target autoantigen protein associated with chronic inflammatory airway disease such as disease has been identified. Human alpha-enolase protein consists of 434 amino acids, the sequence of which is described in Gialongo et al. (Giallongo A, et al., Proc. Natl. Acad. Sci. USA 1986; 83: 6741-6745). It is known that the homology of amino acid sequences between mammalian alpha-enolase proteins is very high (Pancholi V., Cell Mol Life Sci. 2001; 58: 902-920).

The inventors have determined that autoantigen proteins involved in the development of bronchial asthma and chronic obstructive pulmonary disease may exist in airway epithelial cells based on the contents described in the documents reported in the past and the results of the deductive reasoning of the inventors. Using the cultured human airway epithelial cell protein, autoantigen proteins that react with IgG autoantibodies were analyzed using immunoblot.

The present inventors have found autoantibodies to airway epithelial cells in the serum of bronchial asthma patients and patients with chronic obstructive pulmonary disease, and have identified that the autoantigens in the airway epithelial cells that react with the autoantibodies are alpha-enolase protein. , By adsorbing autoantibodies of bronchial asthma and chronic obstructive pulmonary disease with alpha-enolase protein, can suppress airway epithelial cytotoxicity by autoantibodies and inhibit the secretion of inflammatory cytokines from airway epithelial cells by autoantibodies The present invention has been completed by confirming that it can.

The present invention is to provide a pharmaceutical composition for the prevention and treatment of chronic inflammatory airway disease containing alpha-enolase protein as an active ingredient.

The present invention also provides a diagnostic composition for chronic inflammatory airway disease containing alpha-enolase protein.

In addition, the present invention is to provide a diagnostic composition for chronic inflammatory airway disease containing alpha-enolase protein and cytokeratin 18 protein at the same time.

In addition, the present invention is to provide a composition for screening a therapeutic agent for chronic inflammatory airway disease comprising at least one of alpha-enolase protein, antibodies thereto and autoantibodies to alpha-enolase in patients with chronic inflammatory gastrointestinal disease. .

The present invention provides a pharmaceutical composition for the prevention and treatment of chronic inflammatory airway disease containing alpha-enolase protein as an active ingredient.

In the present invention, the alpha-enolase protein used for the diagnosis of chronic inflammatory airway disease, therapeutic pharmaceutical composition, and therapeutic screening is derived from a mammal including humans, mice, rats, rabbits, cattle, pigs, goats, and the like. It can be done.

The alpha-enolase protein used in the present invention may be an amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or an alpha-enolease polypeptide fragment exhibiting equivalent physiological activity.

Polypeptide fragment in the present invention means a polypeptide comprising a minimal epitope that recognizes and binds autoantibodies to autoantigens.

SEQ ID NO: 1 is the amino acid sequence of alpha-enolase of human ( Homo sapiens ) (NCBI accession no. P06733), SEQ ID NO: 2 is the amino acid sequence of alpha-enolase of mouse ( Mus musculus ) (NCBI accession no. P17182) )to be.

In addition, the alpha-enolase protein may be one or more selected from among proteins expressed from one selected from the DNA sequence of SEQ ID NO: 3 or the base sequence including the polymorphism of SEQ ID NO: 3.

SEQ ID NO: 3 is an mRNA sequence (NCBI accession no. M14628) comprising a CDS (cDNA) sequence of alpha-enolase of human ( Homo sapiens ).

In addition, the alpha-enolase protein of the present invention can be obtained by separation and purification from mammalian cells, tissues, or microorganisms, or DNA sequences encoding the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2 or fragments thereof, It can be obtained by isolation and purification from a cell, tissue or microorganism using a genetic recombination technique with a selected one of the DNA sequence of SEQ ID NO: 3 or the base sequence including the polymorphism of SEQ ID NO: 3.

The term "chronic inflammatory airway disease" used in the present invention is a generic name for a variety of diseases characterized by chronic inflammatory reactions and tissue damage of airway tissues leading to organs, bronchus, bronchioles, and alveoli. In the present invention, chronic inflammatory airway disease includes bronchial asthma and chronic obstructive pulmonary disease. Also in the present invention the bronchial asthma includes mild, moderate, severe asthma, aspirin-sensitive asthma.

The present invention is based on the fact that the autoantigen of airway epithelial cells that reacts with IgG autoantibodies present in the blood of bronchial asthma patients, especially patients with severe asthma and chronic obstructive pulmonary disease, is an alpha-enolase protein. Based.

According to the existing literature reports and the results of the present invention, autoantibodies in the blood of patients with bronchial asthma and patients with chronic obstructive pulmonary disease react with the alpha-enolase protein present in the airway epithelial cells. It may induce toxicity or form an immune complex consisting of autoantibodies-autoantigens, resulting in airway epithelial cytotoxicity and chronic inflammation of airway tissues through secondary complement activation and chemotaxis of inflammatory cells. The chronic inflammatory response of the airways may cause airway constriction, airway hypersensitivity, and irreversible structural changes in airway tissues, leading to clinical symptoms of bronchial asthma and chronic obstructive pulmonary disease.

In an embodiment of the present invention, the treatment of human airway epithelial cell line with IgG antibody isolated from blood of bronchial asthma and chronic obstructive pulmonary disease patients induces cytotoxic response and inflammatory cytokine secretion. In addition, by administering the alpha-enolase protein of the present invention to the airway epithelial cytotoxicity model by the IgG antibody, autoantibodies in the blood of patients with bronchial asthma and chronic obstructive pulmonary disease are pre-adsorbed to inhibit the cytotoxicity of airway epithelial cells, Inhibits the secretion of inflammatory cytokines.

Therefore, the pharmaceutical composition containing the alpha-enolase of the present invention can be usefully used as a medicament for the prevention, reduction and treatment of chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease.

A pharmaceutical composition containing the alpha-enolase protein of the present invention as an active ingredient may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include an excipient, a disintegrant, and a sweetener. Solubilizers such as binders, coatings, expanding agents, lubricants, lubricants or flavoring agents can be used.

A pharmaceutical composition containing the alpha-enolase protein of the present invention as an active ingredient may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration. .

Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible with saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

Pharmaceutical formulation forms of pharmaceutical compositions containing the alpha-enolase protein of the present invention as an active ingredient include granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions. And sustained release formulations of the active compounds.

Pharmaceutical compositions containing the alpha-enolase protein of the present invention as an active ingredient are intravenous, intraarterial, intraperitoneal, intramuscular, intraperitoneal, sternum, transdermal, nasal, inhaled, topical, rectal, oral, intraocular Or via the intradermal route.

The dosage of the pharmaceutical composition containing the alpha-enolase protein of the present invention as an active ingredient means an amount required to achieve an effect of inhibiting or treating an airway epithelial tissue damage and an inflammatory response caused by autoantibodies. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type and formulation of the formulation and the age, weight, general health, sex and diet, time of administration, route of administration and composition of the patient It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. In adults, it is preferable to administer the alpha-enolase protein at a dose of 0.01 mg / kg to 100 mg / kg once or several times a day.

The present invention also provides a method for preventing and treating chronic inflammatory airway disease, characterized by administering an alpha-enolase protein.

The chronic inflammatory airway disease is preferably bronchial asthma, including severe asthma and aspirin overactive asthma, chronic obstructive pulmonary disease.

The present invention is to suppress the cytotoxicity of airway epithelial cells by autoantibodies by adsorbing autoantibodies in serum of patients with chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease by administering alpha-enolase protein, It has an effect of inhibiting secretion.

In addition, the method of preventing or treating chronic inflammatory airway disease, characterized by administering the alpha-enolase protein of the present invention is intravenous, intraarterial, intraperitoneal, intramuscular, intraperitoneal, intrasternal, Administration may be in conventional manner via the transdermal, nasal, inhaled, topical, rectal, oral, intraocular or intradermal route.

In addition, the dosage in the method for preventing or treating chronic inflammatory airway disease, characterized by administering the alpha-enolase protein of the present invention, It is an effective amount for suppressing or treating the inflammatory response of airway epithelial cytotoxicity by autoantibodies or the secondary airway tissue following inflammatory cytokine secretion from airway epithelial cells or the formation of an immune complex consisting of autoantigen-autoantibodies. In this case, when the alpha-enolase protein is administered once to several times a day, it is preferable to administer at a dose of 0.01 mg / kg to 100 mg / kg. Dosage is based on the type of disease, the severity of the disease, the type and amount of active and other ingredients contained in the composition, the type of formulation and the age, body weight, general health, sex and diet, sex and diet, time of administration, route and composition of the patient. It can be adjusted according to various factors including the secretion rate of the drug, the duration of treatment, and the drug used simultaneously.

The present invention also provides the use of the alpha-enolase protein for the manufacture of a medicament for chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease.

The present invention also provides a composition for the diagnosis of chronic inflammatory airway disease, such as bronchial asthma and chronic obstructive pulmonary disease containing alpha-enolase protein.

The alpha-enolase protein included in the diagnostic composition of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or an alpha-enolase polypeptide fragment showing an equivalent physiological activity, and is a human, rat, mouse And mammals, including rabbits, cattle, pigs, goats, and the like.

The diagnostic composition containing the alpha-enolase protein of the present invention is positive in response to biological samples obtained from patients with bronchial asthma or patients with chronic inflammatory airway disease such as chronic obstructive pulmonary disease having autoantibodies to the alpha-enolase protein. As a result, it can be usefully used for the diagnosis of chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease.

The present invention also provides a diagnostic composition containing alpha-enolase protein and cytokeratin 18 protein simultaneously.

The present invention also provides a composition for diagnosing chronic inflammatory airway disease containing alpha-enolase protein and cytokeratin 18 protein simultaneously.

The alpha-enolase protein included in the diagnostic composition of the present invention is the same as that used in the diagnostic composition, and the cytokeratin 18 protein may be a cytokeratin 18 polypeptide fragment exhibiting SEQ ID NO: 4 or equivalent physiological activity. . SEQ ID NO: 4 is the amino acid sequence of cytokeratin 18 of human ( Homo sapiens ) (NCBI accession no. P05783).

The cytokeratin 18 protein may be derived from mammals including humans, mice, rats, rabbits, cattle, pigs, goats and the like. In the present inventors' paper (Nahm DH, et al. Am J Respir Crit Care Med 2002; 165: 1536-1539), bovine cytokeratin 18 protein exhibits similar antigenicity to human cytokeratin 18 protein and the serum of bronchial asthma patients. It has been reported to react with IgG antibodies.

In addition, the cytokeratin 18 protein may be at least one selected from a protein expressed from one selected from the DNA sequence of SEQ ID NO: 5 or the base sequence including the polymorphism of SEQ ID NO: 5.

SEQ ID NO: 5 is an mRNA sequence (NCBI accession no. NM_199187) comprising a CDS (cDNA) sequence of cytokeratin 18 of human ( Homo sapiens ).

In addition, the cytokeratin 18 protein may be obtained by isolation and purification from a mammalian cell, tissue, or microorganism, or the DNA sequence encoding the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, the DNA sequence described in SEQ ID NO: 5 Alternatively, the gene may be obtained by separation and purification from a cell, tissue or microorganism using a genetic recombination technique with a selected one of the nucleotide sequences including the polymorphism of SEQ ID NO.

Such a diagnostic composition containing the alpha-enolase protein and cytokeratin 18 protein of the present invention is a chronic inflammatory airway disease such as severe asthma patients, aspirin-sensitive asthma patients and chronic obstructive pulmonary disease as shown in the embodiment of the present invention Is very useful for detecting, diagnosing and classifying

In addition to the protein, the diagnostic composition of the present invention may include a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. In addition, to maintain stability, it may be provided in a powder state, dissolved in a suitable buffer or maintained at 4 ℃.

The present invention provides a kit for diagnosing chronic inflammatory airway disease, comprising a composition for diagnosing chronic inflammatory airway disease containing the alpha-enolase protein.

The present invention also provides a kit for diagnosing chronic inflammatory airway disease, comprising a composition for diagnosing chronic inflammatory airway disease simultaneously containing alpha-enolase protein and cytokeratin 18 protein.

The diagnostic kit of the present invention may include, in addition to the alpha-enolease or cytokeratin 18 protein, a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer or maintained at 4 ° C.

The diagnostic kit of the present invention is a polyclonal antibody or monoclonal antibody against alpha-enolease or cytokeratin 18 protein obtained from a mammal for positive control in addition to the alpha-enolase protein or cytokeratin 18 protein. Antibodies, or biological samples from humans with positive autoantibodies to alpha-enolase protein or cytokeratin 18 protein, and the like, and may contain other antibodies or buffers for negative control results. It may further include other components required for the reaction for detecting autoantibodies to alpha-enolase protein or cytokeratin 18 protein in the biological sample of the subject as needed.

Other components required for the immunodetection reaction include anti-human IgG antibodies, colorants, and buffers produced in rats, mice, rabbits, cows, pigs, goats, etc. as secondary antibodies.

The secondary antibody may be a combination of alkaline phosphatase (AP), horse radish peroxidase (HRP), may be used to combine biotin, rhodamine (rhodamine ), A combination of fluorescent materials such as Texas Red, fluorescein, and phycoerythrin may be used.

The biotin-conjugated secondary antibody uses AP or HRF enzyme-linked avidin, the AP-conjugated secondary antibody is BCIP / NBT as a colorant, and HPR-conjugated secondary antibody is used as a colorant as DAB (diaminobenzidine). The color can be diagnosed by the presence of autoantibodies. In the case of a secondary antibody conjugated with a fluorescent substance, the presence of autoantibodies can be diagnosed by observing under a fluorescence microscope.

In addition, immunodetection reactions commonly known in the art may be used in the diagnostic kit of the present invention. Detection methods used in the kit may include fluorescence immunoassay, enzyme-substrate coloration, enzyme immunization, immunoblotting, and immunoblotting. And antigen-antibody reactions such as sedimentation, antigen-antibody aggregation, spectroimmunity, radioisotope immunity, cell flux, complement fixation, and the like.

The kit of the present invention may further include a tube, a well plate, an instruction sheet describing a method of use, etc., which will be used to mix each component as necessary.

The present invention provides a method for diagnosing chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease using a diagnostic composition containing alpha-enolase as an active ingredient.

The diagnostic method includes the steps of: (a) obtaining a biological sample from a subject suspected of having a chronic inflammatory airway disease, such as bronchial asthma and chronic obstructive pulmonary disease; (b) contacting the biological sample with an alpha-enolase protein to form an immunocomplex; (c) diagnosing chronic inflammatory airway disease by detecting the formation of the immunocomplex and confirming the presence of an autoantibody against the alpha-enolase protein.

The present invention provides a method for diagnosing chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease using a diagnostic composition comprising alpha-enolase and cytokeratin 18 protein as an active ingredient.

The diagnostic method includes the steps of: (a) obtaining a biological sample from a subject suspected of having a chronic inflammatory airway disease, such as bronchial asthma and chronic obstructive pulmonary disease; (b) contacting the biological sample with alpha-enolase and cytokeratin 18 protein to form an immunocomplex; (c) diagnosing chronic inflammatory airway disease by detecting the formation of the immunocomplex and confirming the presence of autoantibodies against alpha-enolase and cytokeratin 18 protein.

In the diagnostic method (a) step of the present invention, the biological sample is collected by separating from the human body such as blood, serum, plasma, urine, tears, saliva, sputum, non-secretion, bronchial secretions, bronchial lavage, lung secretions, alveolar lavage Contains all liquids that can be.

In the diagnostic method (b) step of the present invention, the immunocomplex formation may use a method such as immunoblotting, enzyme-immunoassay (ELISA), and can use a method for detecting antigen-antibody reactions common in the art. have.

In the diagnostic method (c) step of the present invention, to detect whether the immune complex is formed by causing a color reaction to the immunocomplex or detecting the fluorescence of the immunocomplex, for detecting antigen-antibody reactions common in the art. Method can be used. At this time, the result of the presence of the immune complex means that the subject has chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease. Wherein the polyclonal or monoclonal antibody, or alpha-enolease protein and cy, to any one or more of alpha-enolase and cytokeratin 18 protein obtained from a mammal instead of the biological sample of the subject for positive control. Biological samples of humans having positive autoantibodies to any one or more of the tokeratin 18 proteins may be used, and other antibodies other than antibodies to any one or more of alpha-enolease and cytokeratin 18 protein for negative control results. Alternatively, the buffer can be used to diagnose.

By using the diagnostic composition and diagnostic method of the present invention, it is possible to easily obtain accurate results of bronchial asthma or chronic obstructive pulmonary disease, which can be efficiently used in large-scale clinical studies, and to treat bronchial asthma or chronic obstructive pulmonary disease. It can be usefully applied to screening and therapeutic research.

The present invention also provides a composition for detecting autoantibodies, a kit for detection, and a method for detecting alpha-enolase protein and cytokeratin 18 protein simultaneously.

The present invention provides a composition for detecting autoantibodies simultaneously containing alpha-enolase protein and cytokeratin 18 protein.

The alpha-enolase protein included in the composition for detecting autoantibodies of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or an alpha-enolase polypeptide fragment showing equivalent physiological activity, cytokeratin 18 The protein may be a cytokeratin 18 polypeptide fragment exhibiting SEQ ID NO: 4 or equivalent physiological activity.

The alpha-enolase and cytokeratin 18 protein may be derived from mammals including humans, mice, rats, rabbits, cattle, pigs, goats and the like.

In addition, the cytokeratin 18 protein may be at least one selected from a protein expressed from one selected from the DNA sequence of SEQ ID NO: 5 or the base sequence including the polymorphism of SEQ ID NO: 5.

In addition, the cytokeratin 18 protein may be obtained by isolation and purification from a mammalian cell, tissue, or microorganism, or the DNA sequence encoding the amino acid sequence of SEQ ID NO: 4 or a fragment thereof, the DNA sequence described in SEQ ID NO: 5 Alternatively, the gene may be obtained by separation and purification from a cell, tissue or microorganism using a genetic recombination technique with a selected one of the nucleotide sequences including the polymorphism of SEQ ID NO.

The composition for detecting autoantibodies simultaneously containing the alpha-enolease protein and cytokeratin 18 protein of the present invention is to detect autoantibodies with higher sensitivity in a subject including chronic inflammatory patients as shown in the examples of the present invention. Very useful.

The composition for detecting autoantibodies of the present invention may include, in addition to the protein, a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. In addition, to maintain stability, it may be provided in a powder state, dissolved in a suitable buffer or maintained at 4 ℃.

The present invention also provides a kit for detecting autoantibodies containing both alpha-enolase protein and cytokeratin 18 protein.

In addition to alpha-enolase and cytokeratin 18 protein, the kit for detecting autoantibodies of the present invention may include a buffer or a reaction solution that stably maintains the structure or physiological activity of the protein. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer or maintained at 4 ° C.

The kit for detecting autoantibodies of the present invention is a polyclonal antibody to an alpha-enolase or cytokeratin 18 protein obtained from a mammal for positive control, in addition to the alpha-enolase protein and cytokeratin 18 protein, or Monoclonal antibodies, or biological samples from humans with positive autoantibodies to alpha-enolase protein or cytokeratin 18 protein, and the like, and may contain other antibodies or buffers for negative control results. It may further include other components required for the reaction for detecting autoantibodies to alpha-enolase protein or cytokeratin 18 protein in the biological sample of the subject as needed.

Other components required for the immunodetection reaction include anti-human IgG antibodies, colorants, and buffers produced in rats, mice, rabbits, cattle, pigs, goats, and the like as secondary antibodies.

The secondary antibody may be a combination of alkaline phosphatase, holesradyperoxidase, biotin may be combined, and a combination of fluorescent materials such as rhodamine, tex red, floresin, phycoerythrin, etc. Can also be used.

The biotin-coupled secondary antibody uses AP or HRF enzyme-coupled avidin, and the AP-coupled secondary antibody is developed by BCIP / NBT, HPR-coupled secondary antibody using DAB, etc. The presence of an antibody can be detected. In the case of a secondary antibody combined with a fluorescent material, the presence of autoantibodies can be detected by observing under a fluorescence microscope.

In addition, immunodetection reactions commonly known in the art may be used in the kit for detecting autoantibodies of the present invention, and the detection methods used in the kit may include fluorescence immunoassay, enzyme-substrate coloration, enzyme immunization, immunoblotting, and immunization. And antigen-antibody reactions such as sedimentation, antigen-antibody aggregation, spectroimmunity, radioisotope immunity, cell flux, complement fixation, and the like.

The kit of the present invention may further include a tube, a well plate, an instruction sheet describing a method of use, etc., which will be used to mix each component as necessary.

The present invention provides a method for detecting autoantibodies using a composition for detecting autoantibodies containing alpha-enolease and cytokeratin 18 as an active ingredient.

The method for detecting autoantibodies includes: (a) obtaining a biological sample from a subject; (b) contacting the biological sample with alpha-enolase and cytokeratin 18 protein to form an immunocomplex; (c) detecting the presence of autoantibodies by detecting the formation of the immunocomplex and confirming the presence of autoantibodies against alpha-enolase and cytokeratin 18 protein.

In the method (a) of the autoantibody detection method of the present invention, the biological sample is separated from the human body such as blood, serum, plasma, urine, tears, saliva, sputum, secretion, bronchial secretion, bronchial lavage, lung secretion, alveolar lavage, and the like. Contains all liquids that can be collected.

In the method of detecting autoantibodies (b) of the present invention, the immunocomplex formation may be performed using immunoblotting, enzyme-immunoassay (ELISA), and the like. It is available.

In the method (c) of detecting an autoantibody of the present invention, a color reaction is generated on the immunocomplex or the fluorescence of the immunocomplex is detected to detect whether the immunocomplex is formed, thereby detecting an antigen-antibody reaction which is common in the art. A method for doing so can be used. At this time, the result of the presence of the immune complex means that the subject has autoantibodies to any one or more of alpha-enolase and cytokeratin 18 protein. Wherein the polyclonal antibody or monoclonal antibody, or alpha-enolease protein and cy, to any one or more of alpha-enolease and cytokeratin 18 protein obtained from the mammal instead of the biological sample of the subject for positive control. Biological samples of humans having positive autoantibodies to any one or more of the tokeratin 18 proteins may be used, and other antibodies other than antibodies to any one or more of alpha-enolease and cytokeratin 18 protein for negative control results. Or using a buffer.

Using the composition for detecting autoantibodies, the kit for detection and the detection method of the present invention, it is possible to easily obtain accurate results on whether or not autoantibodies are retained for any one or more of alpha-enolase and cytokeratin 18 protein. It can be effectively used in clinical research and can be usefully applied to the study of diseases related to autoantibodies.

The present invention is for screening a therapeutic agent for chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease, comprising at least one of alpha-enolase protein, antibodies thereto and autoantibodies to alpha-enolase in patients with chronic inflammatory airway disease. A composition and a method for screening a therapeutic agent using the same are provided.

The alpha-enolase protein included in the composition for screening a therapeutic drug of the present invention has an amino acid structure of SEQ ID NO: 1 or SEQ ID NO: 2, or a base sequence of SEQ ID NO: 3, including a polymorphism of SEQ ID NO: 3 At least one selected from among alpha-enolase polypeptide fragments exhibiting physiological activity equivalent to alpha-enolase.

Antibodies to the alpha-enolase protein included in the composition for therapeutic drug screening of the present invention are those prepared with antibodies to the alpha-enolase protein, but those commercially available or commercially available in the art can be used.

The composition for screening a therapeutic drug of the present invention maintains the structure or physiological activity of the protein in addition to the alpha-enolase protein, the antibody to the alpha-enolase protein, and the autoantibody to the alpha-enolase in patients with chronic inflammatory airway disease. It may include a buffer or a reaction solution. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer or maintained at 4 ° C.

The therapeutic drug screening composition of the present invention expresses a substance or alpha-enolase protein that inhibits binding between autoantibody and alpha-enolase protein to alpha-enolase in a biological sample obtained from a patient with severe asthma or chronic obstructive pulmonary disease. Screening of substances capable of inhibiting the cytotoxic response by the autoantibodies to the cells or secretion of inflammatory mediators including inflammatory cytokines can be selected as a therapeutic agent for chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease. .

In addition, the present invention is screening for a therapeutic agent for chronic inflammatory airway disease using a composition comprising one or more of the alpha-enolase protein, an antibody thereto and autoantibodies against alpha-enolase in a patient with chronic inflammatory airway disease Provide a method.

The therapeutic drug screening method for chronic inflammatory airway disease of the present invention comprises the steps of: (a) separating autoantibodies from biological samples obtained from patients with chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease; (b) contacting the test substance with the autoantibodies isolated in step (a); (c) the test substance may inhibit binding of the autoantibodies to alpha-enolase protein or alpha-enol The method comprises selecting a therapeutic agent for chronic inflammatory airway disease by determining whether the cytotoxic response of autoantibodies to the raised protein expressing cells or the secretion of inflammatory mediators including inflammatory cytokines by autoantibodies can be suppressed.

In the therapeutic drug screening method of the present invention, the test substance is estimated to have the potential as an inhibitor of chronic inflammatory airway disease according to a conventional selection method or randomly selected individual nucleic acids, proteins, other extracts or natural products, compounds, etc. Can be.

In the therapeutic drug screening method of the present invention, the reaction between the screening composition and the test substance may be checked by using conventional methods used to confirm whether the protein-protein reaction including the antigen-antibody reaction and the reaction between the protein and the compound are used. have.

For example, a method of measuring activity after reacting an alpha-enolase protein or autoantibody with a test substance, a yeast two-hybrid, and a phage display peptide clone that binds to an alpha-enolase protein ( search for phage-displayed peptide clones, high throughput screening (HTS) using natural and chemical libraries, drug hit HTS, cell-based screening, or DNA arrays A screening method using a (DNA array) can be used.

Matters related to genetic engineering techniques in the present invention are more clearly described by Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)). Done.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1 Identification of Alpha-Enolease Protein as Autoantigen in Bronchial Asthma Patients

(1-1) Target patient

We collected and tested serum samples from 78 bronchial asthma (severe asthma) patients and 58 healthy normal controls.

All patients with bronchial asthma had a typical clinical history appropriate for bronchial asthma and had a 20% reduction in FEV ₁ compared to baseline after inhalation of less than 8 mg of methacholine per ml on lung function tests or FEV ₁ after inhalation of bronchodilators. The increase was over 15% over this baseline. In addition, all bronchial asthma patients underwent skin terminal tests using 50 common allergens (Bencard Co. Brentford, UK) and defined as atopic dermatitis when the mean diameter of swelling was greater than 3 mm for one or more inhalable allergens.

History of asthma patients have received standard asthma medications consistent with GINA's Global Initiative for Asthma.NIH Publication No. 02-3659, 2002, p50-66. Severe asthma was defined as a patient with a history of steroid intravenous treatment who visited the emergency room or was admitted to a hospital due to asthma exacerbation.

In addition, there has been a typical history of acute exacerbation of bronchial asthma after taking aspirin or other analgesic agents in asthma patients, or a method reported by lysine-aspirin that has been nebulized in a laboratory (Park HS. Clin Exp Allergy 1995). (25: 28-40), clinically defined as aspirin-sensitization only when incidence of an acute asthma reaction was identified.

Serum samples from all bronchial asthma patients were collected and stored at -20 ° C.

(1-2) Cell Culture

A549 cells (ATCC CCL-185; Giard et al., J Natl Cancer Inst 1973; 51: 1417-23), a human airway epithelial cell line, were purchased from the American Type Culture Collection (VA, USA) in the United States and cultured as recommended by ATCC. It was.

(1-3) Cell protein extraction and immunoblot analysis

Immunoblot analysis was performed with human airway epithelial cells and serum of normal and bronchial asthma patients of (1-1) to identify human airway epithelial protein binding to IgG autoantibodies in serum.

First, in order to extract the protein from human airway epithelial cell A549 of (1-2), the cultured cells were 10 mM Tris / HCl, pH 7.2, 2% sodium dodecyl sulfate (SDS), 158 mM NaCl, 10 mM Lysis buffer containing DTT was added and dissolved to extract the protein.

The prepared human airway epithelial cell proteins were separated by discontinuous sodium dodecyl sulfate / polyacrylamide electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transposed to polyvinylidine difluoride (PVDF); Bio-Rad Laboratories, Hercules, CA. After translocation, the PVDF membrane is first reacted with Tris buffered saline (TBS) containing 5% bovine serum, 10% skim milk powder and 0.1% Tween 20 for at least 1 hour to block nonspecific protein binding to PVDF, and then the membrane is 4 mm wide strip Cut into pieces. Next, the cleaved PVDF membrane strip was reacted with serum of a patient diluted 1: 100 (v / v) in the same buffer for 2 hours at room temperature. After washing, the membrane was reacted with alkaline phosphatase-attached goat anti human IgG antibody (Sigma Chemical Co., St. Louis, Mo.) for 2 hours at room temperature. After the final wash, the membrane was reacted with BCIP / NBT (5-bromo-4-chloro-3-indoyl phosphate / nitro blue tetrazolium; Sigma Chemical Co, St. Louis, MO) for 5 minutes and developed to develop serum and serum. The reacted protein was confirmed.

In order to minimize the technical error in the detection rate of autoantibodies between experiments, each test included a positive reference serum and a negative control serum, which are the criteria of positive determination. At least two of them independently read the test results visually, and showed a significantly stronger reaction band for specific airway epithelial cell proteins than the negative control serum. It is defined as autoantibodies detected when the two readings are equal to or greater than.

Mouse monoclonal antibodies (clone CK5; Sigma chemical Co) and human alpha-enol against cytokeratin 18 protein to determine the location of reactive autoantiproteins in reading autoantibody detection results for airway epithelial cell proteins Goat antibodies against the raised protein (Santa Cruz Biotech Inc., Santa Cruz, Calif.) And secondary antibodies with alkaline phosphatase for each were purchased and used for the experiment.

Using the above-described SDS-PAGE and immunoblot method, the location of airway epithelial cells that reacted with IgG autoantibodies in bronchial asthma patients was confirmed by BCIP / NBT coloration and the airway epithelial cells were separated by SDS-PAGE. Later, after transfer to PVDF membrane, the staining pattern, molecular weight, etc. of the proteins appearing in the result of staining with Coomassie blue (coomassie blue) was confirmed, the location of the target autoantigen protein was confirmed.

1 is a result of immunoblot analysis by reacting IgG autoantibodies and airway epithelial cells (A549 cells) proteins present in the serum of normal, bronchial asthma patients. Lanes 1 to 3 are normal human serum, lanes 4 to 9 serum of severe asthma patients, lane 10 is goat antibody against human alpha-enolase, lane 11 is mouse monoclonal antibody to cytokeratin 18 protein, and lane 12 shows the result of reacting only dilution buffer as a negative control. The arrows after * indicate the positions of human alpha-enolase proteins identified by goat antibodies.

IgG autoantibodies in each patient sera were negative in three normal controls and positive in five of six severe asthma patients (FIG. 1).

2 is a diagram stained with Coomassie blue by SDS-PAGE of human airway epithelial cell proteins. Lane 1 is recombinant human cytokeratin 18 protein and 2 is airway epithelial cell (A549) extract protein. As shown in FIG. 1, the position of autoantigen protein was indicated by comparative analysis with the position of airway epithelial cell protein reacting with serum IgG antibody of severe asthma patients. The arrows after the asterisks indicate the location of the 52-kDa autoantigen protein that reacts with IgG antibodies in the serum of severe asthma patients.

As a result, airway epithelial cell autoantigens reacted with IgG autoantibodies in serum of patients with severe asthma, and their size was about 52-kDa.

(1-4) Detection of Airway Epithelial Cell Autoantigens in Bronchial Asthma Patients

The presence of autoantibodies to airway epithelial cell autoantigens in the serum of 78 patients with bronchial asthma and 58 normal subjects of (1-1) was confirmed by the method (1-3).

As a result, autoantibodies against 52-kDa airway epithelial cell proteins were detected in 32 of 78 patients (41%) of total bronchial asthma patients (severe asthma patients) and of 2 of 58 healthy patients (3%). The IgG autoantibody detection rate was significantly higher than that of the chi-square test (p <0.001).

Table 1 compares the detection rate of IgG autoantibodies against 52-kDa airway epithelial protein in serum of normal and bronchial asthma patients.

number % IgG autoantibody detection against 52-kDa protein p value * Control group (normal) 58 2 (3%) <0.001 Bronchial Asthma Patients Group (Severe Asthma Patients) 78 32 (41%) * The statistical significance of the difference between the two groups was calculated by chi-square test.

As described above, it was confirmed that the IgG autoantibody detection rate for the 52-kDa airway epithelial cell protein was significantly higher in bronchial asthma patients compared to normal people.

(1-5) Identification of autoantigen proteins reacted with serum of bronchial asthma

The airway epithelial cell 52-kDa autoantigen protein in response to IgG autoantibodies in serum of the severe bronchial asthma patient was identified by the following method.

The airway epithelial cell extracted proteins were separated by SDS-PAGE and stained using Coomassie blue staining (FIG. 2). Lane 1 is recombinant human cytokeratin 18 protein and 2 is airway epithelial cell (A549) extract protein. The arrows after the asterisks indicate the location of the 52-kDa autoantigen protein that reacts with IgG antibodies in the serum of severe asthma patients.

Protein bands corresponding to the positions of autoantigen proteins were cut from the gel and subjected to enzymatic in-gel digestion using trypsin.

Trypsin-cleaved peptide fragments were subjected to liquid chromatography-electrospray tandem mass spectrometry (LC-MS / MS) analysis using Quadrupole time-of-flight mass spectrometry (QTOF2), Micro mass, Beverly, Mass.

The results were analyzed using NCBI (Besthesda, MD) non-redundant protein database and Mascot program (Matrix Science, U.K.).

The 52-kDa airway epithelial cell autoantigen protein was treated with trypsin to analyze the mass and amino acid permutation of the peptide fragments. It was found to be consistent and confirmed that the 52-kDa autoantigen protein was human alpha-enolase (Table 2).

Matching peptide no. Mass (M r ) measured Peptide sequences (+ modifications) 1 2 3 4 5 6 7 8 9 898.22 1142.17 1311.03 1405.14 1424.15 1555.13 1803.21 1959.14 2031.23 TIAPALVSK IGAEVYHNLK LMIEMDGTENK (+2 Oxidations of Methionine) GNPTVEVDLFTSK YISPDQLADLYK VVIGMDVAASEFFR (+ Oxidation of Methionine) AAVPSGASTGIYEALELR DATNVGDEGGFAPNILENK FTASAGIQNPVKD

(1-6) Reconfirmation of alpha-enolase as autoantigen

In order to reconfirm that the 52-kDa autoantigen identified above was alpha-enolase, two-dimensional immunoblot analysis was performed. For airway epithelial proteins to be used for two-dimensional immunoblot analysis, IPG loading buffer containing 9M urea (IPG loading) as recommended by an IPG strip (Amersham Pharmacia Biotech, Piscataway, NJ) Protein was extracted by directly lysing human airway epithelial cells (A549 cells) in a buffer). For two-dimensional immunoblot analysis, after performing the IEF (isoelectrical focusing) using a linear immobilized pH gradient strip (Amersham Pharmacia Biotech, Piscataway, NJ) was subjected to SDS-PAGE and immunoblot as described above. The primary antibody was reacted with serum IgG autoantibodies in severe asthma patients or goat antibodies against human alpha-enolase proteins (Santa Cruz Biotech Inc., Santa Cruz, CA). In addition, after reacting a goat anti-human IgG antibody or a rabbit anti-chlorine antibody (Sigma Chemical Co., St. Louis, MO) to which alkaline phosphatase is bound as a secondary antibody conjugate, BCIP / NBT was used as described above. Antibody reaction protein was developed.

Figure 3 shows the analysis of airway epithelial cell proteins that react with IgG autoantibodies in the serum of patients with severe asthma using a two-dimensional immunoblot, autoantigen has a molecular weight of 52-kDa and isoelectric point (pI value) This protein corresponds to about 7 (FIG. 3).

As a result of confirming the location of the alpha-enolase protein in the airway epithelial cell protein using goat anti-human alpha-enolase antibody using a two-dimensional immunoblot in FIG. 4, it reacts with the IgG autoantibody in the serum of the severe asthma patient of FIG. 3. It was confirmed that the protein has the same molecular weight and the same isoelectric point (pI value) as the autoantigen. Therefore, it was confirmed that the airway epithelial cell autoantigen that reacts with the IgG autoantibody in the serum of severe asthma patients was alpha-enolase protein. .

(1-7) Detection of autoantibodies in blood of severe asthma patients using recombinant human alpha-enolase protein

According to a previously reported method (Lee KH, et al. Arthritis Rheum. 2003; 48: 2025-2035), human alpha-enolase protein from E. coli transformed by transfection of cDNA of human alpha-enolase protein. Was synthesized and purified.

Using the produced alpha-enolase protein, it was confirmed by immunoblot analysis whether autoantibodies reacting with this protein were detected in the serum of severe asthma patients.

FIG. 5 shows the results of detecting IgG autoantibodies against recombinant human alpha-enolase protein using immunoblot using serum from normal and severe asthma patients. Lanes 1 to 3 are normal human serum, lanes 4 to 9 serum of severe asthma patients, lane 10 is goat antibody against human alpha-enolase, lane 11 is mouse monoclonal antibody to cytokeratin 18 protein, and lane 12 shows the result of reacting only dilution buffer as a negative control. The arrows after * indicate the positions of human alpha-enolase proteins identified by goat antibodies. Serum samples used in Figure 5 and the arrangement order of the serum samples in the experiment is the same as in FIG.

IgG autoantibodies in serum of each patient were negative in three normal controls and positive in five of six severe asthma patients (FIG. 5).

These experimental results are in good agreement with the results of IgG autoantibody detection for 52-kDa autoantigens using the total airway epithelial protein of FIG. 1, wherein the autoantigens in 52-kDa airway epithelial cells are human alpha-enolase protein. It could be confirmed again (Fig. 5).

(1-8) IgG Autoantibody Detection Against Alpha-Enolease in Aspirin Sensitized Asthma

Serum samples of 22 asthma patients with aspirin-sensitization classified by the criteria described in (1-1) above and 139 asthma patients without history of hypersensitivity to aspirin or other analgesic agents The detection of IgG autoantibodies was confirmed by the immunoblot described in (1-3) and (1-7).

As a result, 13 out of 22 (59.1%) sera of 22 asthmatic patients with aspirin-sensitive hypersensitivity, and 28 (20.1%) of 139 asthmatics without history of hypersensitivity to aspirin or other analgesic agents. IgG autoantibodies to enolase were detected. This shows a significantly higher detection rate compared to the detection rate of 2 of 58 normal patients (3%) of Table 1 (p <0.05), and in asthma patients (aspirin-sensitive asthma patients) with aspirin-sensitization The detection rate of IgG autoantibody against alpha-enolase was significantly higher than that of normal or asthmatic patients without aspirin-sensitization (p <0.05). These results show that IgG autoantibodies against alpha-enolase can be detected in asthma patients to predict the presence of aspirin-hypersensitivity.

Example 2 Identification of IgG Autoantibodies to Alpha-Enolase Protein in Serum Samples of Chronic Obstructive Pulmonary Disease

(2-1) target patient

We examined serum samples from nine patients with chronic obstructive pulmonary disease, two patients with emphysema with normal lung function, and 58 healthy controls. The diagnosis of chronic obstructive pulmonary disease was in accordance with the recently published NHLBI / WHO GOLD Workshop summary (Am J Respir Crit Care Med 2001; 163: 1256-1276). Two patients with emphysema had a history of smoking more than 10 pack years, and clinically complained of respiratory distress and had adequate findings of emphysema on chest X-ray, but FEV1 and FVC measurements exceeded 80% of the predicted values. Showed normal findings. A total of nine patients with chronic obstructive pulmonary disease had a clinical history of cough, sputum, and respiratory distress, which were equivalent to those of typical chronic obstructive pulmonary disease. The ratio of aerobic volume / spiral (FEV1 / FVC) measurements for less than 1% and 1 second was less than 70%. All patients with chronic obstructive pulmonary disease had a history of smoking for more than 10 years, and chest X-ray showed no evidence of tuberculosis or other lung disease. Serum samples from patients with chronic obstructive pulmonary disease were stored at -20 ° C.

(2-2) Cell protein extraction and immunoblot analysis

In order to identify human airway epithelial cell proteins binding to IgG autoantibodies in serum of patients with chronic obstructive pulmonary disease, the serum of the human airway epithelial cells of (1-2) and chronic obstructive pulmonary disease of (2-1) was used. Immunoblot analysis was performed as in (1-3) above.

Figure 6 shows the results of analyzing the airway epithelial cells (A549 cells) proteins that react with the IgG autoantibodies present in the serum of normal, normal lung function, emphysema patients, patients with chronic obstructive pulmonary disease. Lanes 1 and 2 are serum of normal individuals, lanes 3 and 4 are serum of emphysema patients with normal lung function, lanes 5 to 13 are serum of patients with chronic obstructive pulmonary disease, lane 14 is serum of bronchial asthma patients, lane 15 The result of reacting the mouse monoclonal antibody with tokeratin 18 protein is shown. And lane 16 shows the result of the reaction of the goat antibody to alpha-enolase.

6, IgG autoantibodies to 52-kDa airway epithelial cell proteins were detected in 4 out of 9 patients (44.4%) with chronic obstructive pulmonary disease, and 2 out of 58 normal controls (3.4%). The detection rate was significantly higher than the detection rate (chi-square test, p <0.05). In addition, it was confirmed that 52-kDa airway epithelial cell autoantigens to which IgG autoantibodies in serum of patients with chronic obstructive pulmonary disease corresponded to the positions of alpha-enolease proteins identified by anti-human alpha-enolase antibody (FIG. 6).

In eight out of nine patients with chronic obstructive pulmonary disease (89%), IgG autoantibodies were detected for the protein band corresponding to the position of cytokeratin 18 protein identified by mouse monoclonal antibody. The detection rate was significantly higher than that of 5 control groups (8.6%) (chi-square test, p <0.05) (FIG. 6).

(2-3) Detection of autoantibodies in blood of patients with chronic obstructive pulmonary disease using recombinant human alpha-enolase protein

Immunoblot analysis was performed with the same sample arrangement as in FIG. 6 using the same patient sera as (2-2) and purely isolated human alpha-enolase protein produced by the same genetic recombination as (1-7). IgG autoantibodies were detected and the results are shown in FIG. 7.

Figure 7 shows the results of the detection of IgG autoantibodies against recombinant human alpha-enolase protein using immunoblot using the serum of normal, normal lung function emphysema patients, patients with chronic obstructive pulmonary disease. Lanes 1 and 2 are normal serum, lanes 3 and 4 are serum of emphysema patients showing normal lung function, lanes 5 to 13 are serum of chronic obstructive pulmonary disease patients, lane 14 is the result of reaction of serum of asthma patients.

7, four of nine patients with chronic obstructive pulmonary disease (44.4%) detected IgG autoantibodies against recombinant alpha-enolase protein, which was significantly higher than four of 58 normal controls (6.9%). High detection rate was shown (chi-square test, p <0.05).

As shown in FIG. 6 and FIG. 7, it can be seen that in patients with chronic obstructive pulmonary disease, IgG autoantibodies are detected by recombinant alpha-enolase or airway epithelial cell alpha-enolase protein.

Therefore, it was confirmed that the autoantigen to which serum IgG autoantibodies respond to chronic obstructive pulmonary disease was alpha-enolase protein (FIG. 7).

Example 3 Identification of IgG Autoantibodies in Serum of Chronic Inflammatory Airway Disease Patients with Recombinant Human Alpha-Enolase Protein Using Enzyme Immunoassay

Enzyme-linked immunosorbent assay (ELISA) was used to detect IgG autoantibodies against human alpha-enolase protein present in the serum of patients with bronchial asthma and chronic obstructive pulmonary disease.

In a 96 well microtiter plate, the human alpha-enolase protein produced by the above recombination method was diluted to a concentration of 5 µg / ml with 0.1 M carbonate buffer (pH 9.6) and 50 µl per well was added. The reaction was carried out at 16 ° C. for 16 hours. Only negative 0.1 M carbonate buffer (pH 9.6) was reacted in the negative control wells.

After the reaction, the cells were washed three times with phosphate buffered saline (PBST) containing 0.05% Tween 20, and 350 µl of PBST (10% BS-PBST) containing 10% bovine serum was added and reacted at room temperature for 1 hour. .

Dilution (1:20, 1:40, 1:80 ratio) of serum from each patient to be examined (normal, severe asthma and two patients with chronic obstructive pulmonary disease) using 10% BS-PBST; v / v 50 μl was dispensed into each well, followed by reaction at room temperature for 2 hours. After washing three times with PBST again, 50 μl of a goat anti human IgG antibody (Sigma) to which alkaline phosphatase was attached was added and reacted for 2 hours. After three washes, 100 μl of p-nitrophenylphosphate (pNPP) color developing solution (Sigma Chemical Co, Saint Louis, MO, USA) was added to each well. After 10 minutes, the reaction was stopped by adding 100 μl of 0.1 N NaOH solution to each well, and the absorbance was measured with a microplate reader.

The measurement results showed that the absorbance obtained by reacting each diluted serum sample with wells to which human alpha-enolase protein was attached was reacted with the same sample reacted with a carbonate buffer-free well without antigen protein. The mean value of the value was expressed as a corrected absorbance value.

The experimental results were obtained by means of four separate experiments (quadruplicate) for each experimental sample, and the results were expressed as mean and standard deviation.

Figure 8 shows the IgG autoantibodies and recombinant human alpha- in serum of two normal patients (control 1, control 2), two patients with severe asthma (patient 1, 2) and two patients with chronic obstructive pulmonary disease (patient 3, 4). The result of detecting the reaction with the enolase protein is shown by the enzyme immunoassay.

The results showed statistically significant absorbance values in two patients with severe asthma and two patients with chronic obstructive pulmonary disease (t-test, p <0.05) compared to the measurements of two normal serum samples (FIG. 8).

Example 4 Immunoblot Analysis for Mouse Alpha-Enolase Protein

Hepa, a mouse liver cancer cell line, indicates whether IgG autoantibodies in serum of patients with severe asthma and chronic obstructive pulmonary disease may respond selectively to human alpha-enolase protein or to other mammalian cell alpha-enolase protein. Immunoblot analysis was performed using 1-6 cell lines.

Hepa 1-6 cell line (ATCC CRL-1830; VA, USA; Darlington GJ et al., J Nat'l Cancer Inst 1980; 64: 809-819), a mouse liver cancer cell line, was purchased and cultured according to the recommendation of ATCC. Hepa 1-6 cell protein was prepared by the protein extraction method of 3).

The immunoblot analysis was performed using the serum of two severe asthma patients with proteins of human airway epithelial cells (A549) and mouse liver cancer cell lines (Hepa 1-6).

9 is a comparative analysis of proteins reacting with IgG autoantibodies against human airway epithelial cells (A549) and mouse liver cancer cell lines (Hepa 1-6) proteins using serum samples from two patients with severe asthma. Show the results. Lanes 1, 3 and 5 are the results of electrophoresis of cultured human airway epithelial cell proteins and lanes 2, 4 and 6 of mouse liver cancer cell line proteins. Lanes 1, 2 and 3, 4 used in this experiment responded to sera from other patients with severe asthma, and lanes 5 and 6 showed the results of the reaction of goat antibodies to human alpha-enolase.

Using the serum of two severe asthma patients, IgG autoantibodies against alpha-enolase in these serums were identified in mouse hepatocellular carcinoma cells of the same molecular weight as human alpha-enolase. It also reacted with the protein corresponding to the position, and the goat antibody against alpha-enolase was confirmed to be consistent with the position of the protein to react. These results were obtained from human alpha-enolase protein (SEQ ID NO: 1) and mouse alpha-enolase protein (SEQ ID NO: 1), as known in the literature (Pancholi V., Cell Mol Life Sci. 2001; 58: 902-920). No. 2) consists of all 434 amino acids and is considered to be a reasonable result with the report that the homology of the nucleotide sequence is very high (FIG. 9).

As shown above, the results of the autoantigen-autoantibody test using human airway epithelial cells and mouse hepatocytes were consistent, and the alpha-enolase protein of the mouse was used to detect the autoantibodies in the serum of patients with severe asthma and It was confirmed that it could be detected.

Example 5 Confirmation of Cytotoxic Inhibitory Effects of Airway Epithelial Cells by IgG Autoantibodies upon Administration of Alpha-Enolease Protein

(5-1) Confirmation of cytotoxicity against airway epithelial cells by IgG autoantibodies

A method previously reported by researchers (Martin S, et al .; Tissue Antigens 1991; 37: 152-155) was modified to airway epithelial cells by IgG autoantibodies using a microcytotoxicity method using a terasaki tray. Cytotoxicity was measured.

In this experiment, IgG antibodies were isolated from plasma of two patients with severe asthma, two patients with chronic obstructive pulmonary disease, and one control group with IgG autoantibodies against human alpha-enolase protein.

Two patients with severe asthma and two normal controls were separated by about 90% purity using ethanol precipitation and ultrafiltration according to the method for preparing intravenous IgG antibodies (Lebing W et al. Vox Sang 2003; 84: 193-201), and patients with chronic obstructive pulmonary disease were isolated by affinity chromatography using Protein A column with purity greater than 95%. The purity of the isolated IgG antibody was confirmed using SDS-PAGE and protein staining. In addition, as a result of quantitatively measuring the amount of endotoxin in the separated IgG antibody solution using LAL (Limulus amebocyte lysate), contamination of endotoxin was not detected.

In addition, two types of intravenous human IgG antibodies (Livgamma, Dongshin Pharmaceutical Co., Korea; IVglobulin, Green Cross Pharmaceutical Co., Korea) isolated from plasma of a large number of healthy donors were purchased. It was used as a control IgG antibody.

First, the human IgG antibodies were diluted using DMEM / F12 medium to concentrations of 5 mg / ml, 1 mg / ml, and 0.2 mg / ml, respectively, and then quadruplicate 1 μl per well in a 96 well Terasaki tray per sample. Four wells were dispensed in advance. In addition, only four DMEM / F12 medium was dispensed as a negative control.

Cultured human airway epithelial cell line A549 was isolated by treatment with Trypsin / EDTA, and then aliquoted at 1 μl to 2000 cells per well. At this time, 5 μl of mineral oil was dispensed so that the solution did not evaporate, and then reacted at room temperature for 2 hours 30 minutes. Again, 5 μl of 5% Eosin Y dye (Sigma Chemical Co.) per well was used to stain the cells, and 5 μl of formalin solution per well was used to fix the stained cells. The cytotoxicity was measured by counting the number of cells having a cell membrane covered with, and not damaged by light microscopy.

The airway epithelial cytotoxicity by the IgG antibody was compared with the average cell number of the wells reacted with the sample and the average cell number of the negative control wells containing only DMEM / F12 medium. .

Cytotoxicity (% cytolysis) = [(average cell number of negative control well-average cell number of sample wells) / average cell number of negative control well] x100

Goat IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) against alpha-enolase protein in positive control experiment and normal goat IgG antibody (reagent grade; Sigma Chemical Co., St. Louis, MO) as negative control antibody Airway epithelial cell toxicity was measured by the same method as described above.

The experimental results were obtained by means of four separate experiments (quadruplicate) for each test sample, and the results were expressed as mean and standard deviation.

10A and 10B, the test results of two patients with severe asthma compared to plasma control (healthy control 1) or intravenous IgG antibody Livgamma (healthy controls 2) and IVglobulin (healthy controls 3) of normal control group IgG antibodies isolated from plasma of patients 1 and 2) and 2 patients with chronic obstructive pulmonary disease (patients 3 and 4) showed significantly high cytotoxicity at conditions of 1 μg per well and 5 μg per well. (t-test, p <0.05).

In addition, the results of the experiments of FIG. 10C show that goat IgG antibodies against alpha-enolase protein (specific IgG) were 12.5 ng per well to 100 ng per well when compared to the normal goat IgG antibody (control IgG) used as a negative control. All of the conditions showed significantly higher airway epithelial cytotoxicity (t-test, p <0.05).

(5-2) Confirmation of cytotoxic inhibitory effect on airway epithelial cells by IgG autoantibodies

Serum alpha for chronic inflammatory airway disease Patients In order to adsorb autoantibodies to the enol raised, agarose beads (Sepharose 4B ^TM bead, Amersham Pharmacia Biotech, Piscataway, NJ), a recombinant prepared by (1-6) How the Human alpha-enolase protein or bovine serum albumin was attached to 1 ml each of 1 ml of agarose beads as instructed by the agarose beads manufacturer.

100 µl of the two types of beads and 100 µg of IgG antibody isolated from plasma of two severely asthmatic patients diluted to 5 mg / ml in DMEM / F12 medium were mixed and reacted at 4 ° C for 16 hours. Thereafter, the supernatant was collected by centrifugation, and cytotoxicity to human airway epithelial cells was measured in the same manner as in Example 4.

In addition, 50 μg of recombinant human alpha-enolase protein with glutathione-S-transferase (GST) attached as prepared in (1-6) and 50 μg of GST protein in negative control were included. Cut the PVDF membrane strip into small pieces in an eppendorf tube, and then react with 50 μg of IgG antibody at 4 ° C. for 16 hours in a patient with chronic obstructive pulmonary disease diluted to 5 mg / ml in DMEM / F12 medium. The supernatant was collected by centrifugation and tested.

The experimental results were obtained by means of four separate experiments (quadruplicate) for each test sample, and the results were expressed as mean and standard deviation.

Referring to FIG. 11, when IgG antibodies of patients with severe asthma (patient 1 and 2) and patients with chronic obstructive pulmonary disease (patient 3) were previously adsorbed using recombinant human alpha-enolase protein (B of each graph), the control term It was confirmed that the cytotoxicity of airway epithelial cells by IgG autoantibodies was significantly reduced compared with the case of adsorption with the same amount of bovine serum albumin or GST protein (A in each graph) (t-test, p <0.05). .

Table 3 below shows the mean ± standard deviation of airway epithelial cytotoxicity (% cytolysis) when IgG antibody of patients with severe asthma and chronic obstructive pulmonary disease was adsorbed with alpha-enolase protein and when adsorbed with control antigen. Value.

When adsorbed with the alpha-enolase protein, compared to the case of adsorbing with the control protein, it was confirmed that significantly lower percentage of cytolysis.

patient Adsorption with Control Protein (% Cytolysis) Adsorption with alpha-enolase protein (% cytolysis) Patient 1 73.9 ± 2.8 * 9.4 ± 4.9 Patient 2 38.8 ± 5.6 *  4.9 ± 4.5 Patient 3  41.5 ± 9.7 *  14.9 ± 13.5    *: P <0.01

These results suggest that alpha-enolase protein can be used to suppress cytotoxic responses against airway epithelial cells of IgG autoantibodies against alpha-enolase protein present in blood in patients with severe asthma or chronic obstructive pulmonary disease. Indicates.

Example 6 Confirmation of Improved Diagnosis Rate of Chronic Inflammatory Airway Disease by Simultaneous Measurement of IgG Autoantibodies against Alpha-Enolease and Cytokeratin 18

The presence of autoantibodies against alpha-enolase and cytokeratin 18 identified as autoantigens of bronchial asthma and chronic obstructive pulmonary disease in patients with bronchial asthma (severe asthma), patients with chronic obstructive pulmonary disease and non-smokers. Confirmed.

The experimental method was performed using the airway epithelial cell A549 cell protein and immunoblot analysis method of (1-3), and immunized with serum from 78 patients with severe asthma, 9 patients with chronic obstructive pulmonary disease, and 58 non-smokers. Blot analysis was performed.

Antibodies to alpha-enolase use goat anti human alpha-enolase antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and antibodies against cytokeratin 18 are anti human cytokeratin 18 monoclonal antibody (Clone CK5, Sigma chemical Co., st. Louis, MO).

Table 3 shows the results of immunoblotting assay for IgG autoantibodies against alpha-enolase protein, cytokeratin 18 protein and recombinant alpha-enolase present in airway epithelial cells in serum samples of severe asthma and normal controls. .

As a result, it can be seen that when measuring the presence of IgG autoantibodies against alpha-enolase, the severely asthmatic patients showed significantly higher detection rate (p <0.05) compared to the normal control group.

In addition, when comprehensively determining the presence or absence of IgG autoantibodies against alpha-enolase and cytokeratin 18, the detection rate of IgG autoantibodies against one or more of the two autoantigen proteins is normal in severe asthma. It was significantly higher than the control group (p <0.05) (Table 4).

In addition, 21.8% higher diagnosis rate can be obtained when IgG autoantibodies against alpha-enolase and cytokeratin 18 protein are simultaneously measured compared to the diagnosis of severe asthma with IgG autoantibodies against alpha-enolase alone. (Table 4).

Number of patients (%) IgG autoantibody detection rate Severe Asthma (n = 78) Normal control (n = 58) Alpha-Enolase 32 (41.0) * 2 (3.4) Cytokeratin18 25 (32.1) * 5 (8.6) Alpha-Enolease + Cytokeratin 18 49 (62.8) * 7 (12.1) Recombinant Alpha-Enolase 31 (39.7) * 4 (6.9) * P <0.01 for the comparison with healthy controls. ‡ Either of autoantibodies to alpha-enolase or cytokeratin 18 were positive.

In addition, FIG. 6 shows that the detection rate of chronic obstructive pulmonary disease is 44.4% (4 out of 9) when only IgG autoantibodies to alpha-enolase present in airway epithelial cells are detected. Simultaneous detection of IgG autoantibodies against cytokeratin 18 protein resulted in the detection of IgG autoantibodies against one or more of the two autoantigen proteins in all 9 patients with chronic obstructive pulmonary disease (100%). It can be seen that the detection rate was significantly higher than the detection rate of 12.1% at (Table 4) (p <0.05).

Therefore, the simultaneous determination of the presence of two types of autoantibodies against alpha-enolase and cytokeratin 18 protein may be useful for the diagnosis and classification of patients with severe asthma and chronic obstructive pulmonary disease.

In addition, 161 bronchial asthma patients were screened for skin reactions using 50 common allergens (Bencard Co. Brentford, UK) such as (1-1) to determine atopic dermatitis. Similarly, the presence of aspirin-sensitization was confirmed.

Autoantibodies to alpha-enolase and / or cytokeratin 18 were identified in atopic, aspirin-sensitive patients classified in this manner.

The results in Table 5 below show that asthma patients without aspirin hypersensitivity in asthmatic patients (or aspirin-sensitive asthmatic patients) with aspirin-sensitization, when measuring the presence of autoantibodies against alpha-enolase In comparison, it was confirmed that the detection rate was significantly higher (p <0.05).

In addition, IgG autoantibodies against alpha-enolase and cytokeratin 18 simultaneously detected IgG autoantibodies against one or more of the two autoantigen proteins in aspirin-sensitive asthma patients compared to asthma patients without aspirin hypersensitivity. It can be seen that the detection rate is significantly high (p <0.05) (Table 5).

In addition, when detecting two autoantibodies simultaneously, the sensitivity of diagnosing aspirin-sensitive asthma can be improved by 18.2% compared to the case of detecting only IgG autoantibodies against alpha-enolase autoantigen protein. (Table 5).

Association between IgG Autoantibody Detection Rate for Airway Epithelial Cell Autoantigen Protein and Presence of Atopy or Aspirin-Sensitization in Patients with Bronchial Asthma atopy Aspirin-hypersensitivity voice positivity voice positivity Number of patients 49 112 139 22 IgG autoantibody detection rate The number of patients (%) The number of patients (%) Alpha-Enolase 15 (30.6) 26 (23.2) 28 (20.1) 13 (59.1) * Cytokeratin18 20 (40.8) † 28 (25.0) 41 (29.5) 7 (31.8) Alpha-Enolease + Cytokeratin 18 30 (61.2) † 47 (42.0) 60 (43.2) 17 (77.3) § Recombinant Alpha-Enolase 14 (28.6) 29 (25.9) 33 (23.7) 10 (45.5) ¶ * P <0.001 for the comparison with asthmatic patients without aspirin-hypersensitivity. † P = 0.04 for the comparison with atopic asthmatic patients. ‡ Either of autoantibodies to alpha-enolase or cytokeratin 18 were positive. §P = 0.003 for the comparison with asthmatic patients without aspirin-hypersensitivity. ¶P = 0.03 for the comparison with asthmatic patients without aspirin-hypersensitivity.

Therefore, the simultaneous determination of the presence of two types of autoantibodies, alpha-enolease and cytokeratin 18, may be useful in diagnosing aspirin-sensitive asthma patients with higher sensitivity.

Example 7 Confirmation of Inhibition of Inflammatory Cytokine IL-8 (interleukin-8) in Patients with Bronchial Asthma by Alpha-Enolase-Containing Composition of the Present Invention

Previous reports have shown that the administration of IL-8 to guinea pigs causes neutrophil inflammation of the lower airways and airway hypersensitivity, a common characteristic of bronchial asthma and chronic obstructive pulmonary disease (Xiu Q et al., Clin Exp Allergy). 1995; 25: 51-9). In addition, in patients with chronic obstructive pulmonary disease, monoclonal antibodies against IL-8 neutralized the effects of IL-8, significantly improving respiratory distress (Mahler DA, Chest 2004; 126: 926-934). . It is also known that monoclonal antibodies against IL-8 can be used for the treatment of bronchial asthma (U.S. Pat. No. 5,874,080).

Based on this, the degree of secretion of IL-8 by autoantibodies in bronchial asthma patients was observed, and the change in the amount of IL-8 secretion was confirmed when the autoantibodies of bronchial asthma patients were treated with alpha-enolase of the present invention. .

(7-1) Secretion of Inflammatory Cytokine IL-8 by IgG Autoantibodies in Bronchial Asthma Patients

Trypsin / EDTA treated human airway epithelial cells (A549) cells were diluted in 100 μl of medium per well to 20,000 cells per well in 96-well culture plates, and then attached for 16 hours in an incubator. IgG antibody of one bronchial asthma patient (Patient 1, patient whose IgG was confirmed against alpha-enolase in serum samples by immunoblot) after the culture medium was removed in the same manner as in (5-1). And IgG antibodies (Livgamma, Dongshin Pharmaceutical Co., Korea) isolated from a number of healthy healthy subjects were diluted in DMEM / F12 medium and quadruplicate into four wells for each sample at 200 μl per well. Then, 200 μl of DMEM / F12 medium or 1% Triton X-100 containing DMEM / F12 medium was quadruplicate into four wells, and then reacted with the incubator for 16 hours. The 96 well-culture plates were centrifuged and supernatants were collected from each well. The concentration of IL-8 in the supernatant of the culture was measured using an ELISA kit (Endogen, Woburn, MA), and the results are shown in FIG. 12A.

12A, the IgG antibody of the bronchial asthma patient (Patient 1) at the IgG antibody treatment concentration of 0.1 mg / ml to 2.5 mg / ml was significantly higher than the IgG antibody of the control group (Controls 1). Induced secretion of IL-8 (p <0.05).

Therefore, it was confirmed that IgG autoantibodies of bronchial asthma stimulate the airway epithelial cells and induce inflammation through IL-8 secretion.

(7-2) Inhibition of Inflammatory Cytokine IL-8 Secretion by Autoantibodies in Bronchial Asthma Patients Using Alpha-Enolase Protein of the Present Invention

In addition, after diluting the IgG antibody of the bronchial asthma patient (Patient 1) to a concentration of 2.5 mg / ml, human serum albumin (human serum) with agarose bead attached with human alpha-enolase prepared by genetic recombination and a negative control protein Agarose beads with albumin) attached were adsorbed for 16 hours as indicated in (5-2). After that, the supernatant was taken, treated with human airway epithelial cells (A549) cells in the same manner as in (7-1) and reacted, and then the amount of IL-8 in the airway epithelial cell culture was measured.

As a result of FIG. 12B, airway epithelial cells due to IgG antibody stimulation were detected when IgG antibody of bronchial asthma patient (Patient 1) was adsorbed with alpha-enolease protein (B) compared with the case where human serum albumin was adsorbed (A). IL-8 secretion was found to be significantly suppressed (p <0.05).

 The experimental results showed that IgG autoantibodies against alpha-enolase protein present in the blood of bronchial asthma patients promote IL-8 secretion from airway epithelial cells, and the IgG autoantibodies of bronchial asthma patients with alpha-enolase protein. It can be shown to inhibit IL-8 secretion from airway epithelial cells.

Therefore, alpha-enolase protein of the present invention is administered to patients with chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease, thereby inhibiting inflammatory response of airway by inhibiting IL-8 secretion from airway epithelial cells by IgG autoantibodies. can confirm.

As described above, in the present invention, the alpha-enolase protein was identified as an autoantigen to which autoantibodies react in serum of patients with chronic inflammatory airway diseases such as severe asthma and chronic obstructive pulmonary disease. The pharmaceutical composition of the present invention as an ingredient inhibits the toxicity of airway epithelial cells by autoantibodies in serum of patients with severe asthma and chronic obstructive pulmonary disease, and inhibits the secretion of inflammatory cytokines such as bronchial asthma and chronic obstructive pulmonary disease. It can be usefully used as a medicine for the prevention, reduction and treatment of chronic inflammatory airway disease.

In addition, the diagnostic composition containing alpha-enolase of the present invention can be usefully used for the diagnosis of chronic inflammatory airway diseases such as bronchial asthma and chronic obstructive pulmonary disease. In addition, the diagnostic composition containing the alpha-enolease and cytokeratin 18 of the present invention is useful for the detection, diagnosis, and classification of chronic inflammatory airway diseases such as severe asthma, aspirin-sensitive asthma, and chronic obstructive pulmonary disease. Can be used.

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Claims (30)

A pharmaceutical composition for the prevention and treatment of chronic inflammatory airway disease containing alpha-enolase protein as an active ingredient. The pharmaceutical composition of claim 1, wherein the alpha-enolase is derived from a mammal selected from the group consisting of human, mouse, rat, rabbit, pig, cow and goat. The pharmaceutical composition according to claim 2, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 1. The pharmaceutical composition according to claim 2, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 2. The pharmaceutical composition of claim 1, wherein the chronic inflammatory airway disease is bronchial asthma or chronic obstructive pulmonary disease. 6. The pharmaceutical composition of claim 5, wherein the bronchial asthma is severe asthma or aspirin-sensitive asthma. A diagnostic composition for chronic inflammatory airway disease containing alpha-enolase protein. The method of claim 7, wherein the alpha-enolase is human, mouse, rat, rabbit, pig, A diagnostic composition derived from a mammal selected from the group consisting of bovine and goat. The diagnostic composition according to claim 8, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 1. The diagnostic composition of claim 8, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 2. 9. The diagnostic composition according to claim 7, wherein the chronic inflammatory airway disease is bronchial asthma or chronic obstructive pulmonary disease. The diagnostic composition of claim 11, wherein the bronchial asthma is severe asthma or aspirin-sensitive asthma. delete delete delete delete delete delete delete delete A diagnostic composition for chronic inflammatory airway disease containing alpha-enolase protein and cytokeratin 18 protein simultaneously. The diagnostic composition of claim 21, wherein the alpha-enolase is derived from a mammal selected from the group consisting of human, mouse, rat, rabbit, pig, cow and goat. The diagnostic composition according to claim 22, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 1. The diagnostic composition according to claim 22, wherein the alpha-enolase protein has the amino acid sequence set forth in SEQ ID NO: 2. The diagnostic composition of claim 21, wherein the cytokeratin 18 protein is derived from a mammal selected from the group consisting of human, mouse, rat, rabbit, pig, cow and goat. The diagnostic composition according to claim 25, wherein the cytokeratin 18 protein has the amino acid sequence set forth in SEQ ID NO: 4. The diagnostic composition according to claim 21, wherein the chronic inflammatory airway disease is bronchial asthma or chronic obstructive pulmonary disease. 28. The diagnostic composition of claim 27, wherein the bronchial asthma is severe asthma or aspirin-sensitive asthma. delete delete

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