WO2012077774A1

WO2012077774A1 - Device for prophylaxis and treatment of systemic lupus erythematosus

Info

Publication number: WO2012077774A1
Application number: PCT/JP2011/078506
Authority: WO
Inventors: 保明田村; 昇志佐藤; 俊彦鳥越; 慶太齋藤
Original assignee: 北海道公立大学法人札幌医科大学
Priority date: 2010-12-10
Filing date: 2011-12-09
Publication date: 2012-06-14
Also published as: JP5916135B2; JPWO2012077774A1

Abstract

[Problem] The purpose of the present invention is to provide a device for the prophylaxis and treatment of systemic lupus erythematosus, a method for use as a biomarker of systemic lupus erythematosus, and a method for determining systemic lupus erythematosus. [Solution] The prophylaxis/treatment of systemic lupus erythematosus is characterized by reducing the concentration of Hsp90 in the blood, the serum, or the blood plasma of a subject by using a substance capable of removing Hsp90. The method for use as a biomarker of systemic lupus erythematosus is characterized by using Hsp90 in the blood, the serum, or the blood plasma of a subject as a biomarker. The method for determining systemic lupus erythematosus is characterized by comparing and evaluating the concentration of Hsp90 in the blood, the serum, or the blood plasma of a subject with/against that of a normal control.

Description

Preventive and therapeutic device for systemic lupus erythematosus

The present invention relates to an apparatus for preventing and treating systemic lupus erythematosus, a method for use as a biomarker for systemic lupus erythematosus, and a method for determining systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) (hereinafter also simply referred to as “SLE”) induces an inflammatory reaction by depositing antinuclear antibodies or anti-DNA antibodies or their immune complexes in various organs / tissues It is a systemic autoimmune disease that occurs. Clinical symptoms vary, including fever, anemia, thrombocytopenia, butterfly erythema, erythematous rash, polyarthralgia, serositis, renal symptoms, neurological symptoms, and cardiac symptoms.

Although the details of the cause of SLE are not yet clear, it is thought to develop due to involvement of environmental factors such as ultraviolet rays, viral infection, trauma, surgery, pregnancy, childbirth, and drug treatment in addition to genetic factors. In SLE, high serum interferon α is considered to be one of the causes of disease progression.

Diagnosis of SLE is [1] cheek butterfly erythema, [2] characteristic rash occurring elsewhere, [3] hypersensitivity to sunlight, [4] ulcers in the mouth, [5] arthritis, [6] Water accumulates around lungs, heart, and other organs (serositis), [7] Kidney dysfunction, [8] Decrease in white blood cell count, decrease in red blood cell count due to hemolytic anemia, decrease in platelet count , [9] Brain or nerve dysfunction, [10] Antinuclear antibody reaction positive in blood test, [11] Anti-double-stranded DNA antibody positive in blood test, corresponding to 4 or more items This is done by confirming that However, since some of these diagnostic items include severe symptoms, new diagnostic items have been sought in order to enable a more complete early diagnosis.

For treatment of SLE, non-steroidal anti-inflammatory drugs such as aspirin are used when symptoms are mild, and corticosteroid drugs such as prednisolone are mainly used when symptoms are severe. . Since corticosteroids have anti-inflammatory and immunosuppressive effects, SLE improves the inflammation of organ lesions caused by SLE and suppresses the production of autoantibodies and the action of autoreactive lymphocytes. It is considered to exert therapeutic effects on However, corticosteroids not only reduce the human defense function, but may also worsen high blood pressure, heart failure, diabetes, peptic ulcer, renal failure, osteoporosis, etc. Careful attention is required. In addition, when corticosteroids are used for a long period of time by intravenous injection or internal use, there are also problems that side effects such as hypertension, elevated blood sugar levels, cataracts, osteoporosis, and gastric bleeding occur. Therefore, development of SLE therapeutic agents other than corticosteroids has been underway. For example, Patent Document 1 discloses that an adenine-derived compound having the ability to inhibit PDE4 family enzymes and substituted at positions 2 and 9 and optionally N (6) of adenine can be used for the treatment of SLE. Is disclosed. However, such compounds are premised on use in combination with corticosteroids. Under such circumstances, there has been a demand for a new treatment method for SLE with fewer side-effect problems.

Also, heat shock protein (HSP) is an intracellular protein whose expression increases by heat shock, and is known to increase in response to stresses such as radiation and malnutrition other than heat. The heat shock protein functions as a molecular chaperone that forms complexes with various proteins in the cell, stabilizes the protein, and functions normally, and in particular, the client protein of heat shock protein 90 (Hsp90) Are known to have many signaling molecules that play an important role in cell proliferation and differentiation, such as protein kinases and steroid hormone receptors. It is also known that heat shock protein forms a complex with a peptide and is released from tumor cells and infected cells to the outside when exposed to stress, thereby contributing to an immune response.

Non-Patent Document 1 describes that Hsp90 activates interferon γ and interferon α, and that the antiviral effect of interferon γ and interferon α is suppressed by geldanamycin, an inhibitor of Hsp90. Has been. Patent Document 2 describes that Hsp90 activates tumor necrosis factor α (Tumor Necrosis Factor α: TNFα) and interleukin 6 (IL-6), and claim 21 inhibits Hsp90. Methods have been described for reducing TNFα and IL-6 by administering a substance to a patient such as systemic lupus erythematosus (SLE). In addition, Non-Patent Document 2 by the present inventors describes that Hsp90 forms a complex with its own DNA (Hsp90-self DNA complex), thereby suppressing the degradation of self-DNA, and that self-DNA is It is suggested that human plasmacytoid dendritic cells promote interferon alpha production by efficiently incorporating them into plasmacytoid dendritic cells and localizing their own DNA to the early endosomes of plasmacytoid dendritic cells. ing. Further, Non-Patent Document 3 describes that Hsp90 expression is increased in peripheral blood mononuclear cells in SLE patients. However, since Hsp90 is not a secreted protein, its concentration in the serum of SLE patients was not expected to be significantly increased and was not known.

Special table 2009-529023 International Publication No. 2007/077454 Pamphlet

An object of the present invention is to provide a systemic lupus erythematosus prevention / treatment device, a method for use as a biomarker for systemic lupus erythematosus, and a method for determining systemic lupus erythematosus.

The present inventors have previously studied the relationship between SLE and Hsp90, and that Hsp90 inhibitors inhibit interferon α production from plasmacytoid dendritic cells and preventive / therapeutic effects of SLE. (Japanese Patent Application No. 2010-219422). As a result of intensive studies under the circumstances described in the background art described above, the present inventors have found that [a] the Hsp90 concentration is extremely high in the serum of SLE patients at the stage of disease progression, [b ] In the serum of SLE patients in remission after treatment, the concentration of Hsp90 is reduced to the same level as in healthy individuals, and [c] The serum from which Hsp90 has been removed from the serum of SLE patients is human plasmacytoid. The interferon α concentration in the culture supernatant when added to the dendritic cell and cultured is the same as the interferon α in the culture supernatant when the serum of the SLE patient is added to the human plasmacytoid dendritic cell and cultured. The inventors have found that the concentration is remarkably reduced as compared with the concentration, and have completed the present invention.

That is, the present invention is characterized in that (1) a systemic lupus erythematosus prevention / treatment device comprising a removal means for Hsp90, and (2) the removal means for Hsp90 contains a removal substance for Hsp90. The preventive / therapeutic device according to (1) or (2) above, wherein the preventive / therapeutic device according to (1), or (3) a substance for removing Hsp90 is supported on a solid phase carrier. Relates to the device.

The present invention also relates to (4) a method of using Hsp90 in blood, serum or plasma as a biomarker for systemic lupus erythematosus.

Furthermore, the present invention comprises (5) a method for determining systemic lupus erythematosus, comprising the following steps (A) and (B):
(A) a step of measuring in vitro the concentration of Hsp90 in the blood, serum or plasma of a subject;
(B) a step of comparing and evaluating the Hsp90 concentration measured in the step (A) with the Hsp90 concentration in blood, serum or plasma of a normal control;
(6) The determination method according to (5), further comprising the following step (C) or (D):
(C) A step of evaluating the subject as systemic lupus erythematosus when the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control;
(D) The degree of the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control, and the severity of the pathological condition of systemic lupus erythematosus in the subject. The step of evaluating;
About.

According to the present invention, systemic lupus erythematosus can be prevented and / or treated with few side effects. Moreover, according to the present invention, a new biomarker for systemic lupus erythematosus can be provided. Furthermore, according to the present invention, systemic lupus erythematosus can be determined quickly and easily.

It is a figure which shows the Hsp90 density | concentration in the serum sample extract | collected from the SLE patient or the healthy subject. “Healthydonor” represents the result in the case of a healthy person, “before treatment” of “SLE patient 1” represents the result in the case of exacerbation of symptoms in SLE patient 1, and “before 、 treatment” of “SLEpatient 2” “SLE 患者 patient 1” “after treatment” represents the result of the SLE patient 1 after treatment, and “SLE patient 2” “after treatment” "" Represents the result in the post-treatment remission phase of SLE patient 2. It is a figure which shows the Hsp90 density | concentration in the serum sample extract | collected from the SLE patient or the healthy subject. “Healthy donor” represents the result of a healthy person, and Δ in the figure represents the concentration of each person. “Before treatment” represents the result in the case of symptom exacerbation of SLE patients. “After treatment” represents the result in the post-treatment remission period of the SLE patient, and the ◇ in the figure indicates the concentration of each patient. It is a figure which shows the interferon (alpha) density | concentration in the supernatant of the culture solution which added the serum of the SLE patient, etc. to the human plasmacytoid dendritic cell, and was cultured. “Control” represents the result when no serum was added, “stimulated with SLE patient serum” represents the result when the serum of the SLE patient was added, and “stimulatedwith SLE patient serum removed HSP90” The result at the time of adding the serum which removed Hsp90 from the serum of a SLE patient is represented. It is a figure which shows the Hsp90 density | concentration in serum of a SLE onset model mouse. “MRL / lpr mice 21w” represents the results at 21 weeks of age before the onset, and the Δ mark in the figure indicates the concentration of each mouse. “MRL / lpr mice 28w” represents the results of 28 weeks of age at the onset of the onset, and the ○ marks in the diagram indicate the concentration of each mouse. “MRL / lpr mice 37w” represents the results of 37 weeks of age in the late stage of the onset, and the ◇ in the figure indicates the concentration of each mouse.

1. Systemic Lupus Erythematosus Prevention / Treatment Device The “systemic lupus erythematosus prevention / treatment device” of the present invention (hereinafter also simply referred to as “the prevention / treatment device of the present invention”) is a means for removing Hsp90 (hereinafter simply referred to as “prevention / treatment device of the present invention”). As long as it includes “Hsp90 removing means”), there is no particular limitation. Although the details of the mechanism of action of the preventive / therapeutic effect of SLE by the prophylactic / therapeutic device of the present invention are not clear, the prophylactic / therapeutic device of the present invention allows Hsp90 in the blood, serum or plasma of a subject to After removal, the blood, serum, or plasma is returned to the subject to reduce the Hsp90 concentration in the subject's blood, serum, or plasma, thereby interferon of the subject's plasmacytoid dendritic cells It is considered that a prophylactic and / or therapeutic effect on SLE is exhibited through suppression of α production.

The above-mentioned Hsp90 removing means means means capable of removing any mammalian Hsp90 protein, and includes, for example, means containing a Hsp90 removing substance (hereinafter also simply referred to as “Hsp90 removing substance”). can do. Examples of the Hsp90-removing substance include a substance that binds to Hsp90 (hereinafter also simply referred to as “Hsp90-binding substance”). Among them, an Hsp90-binding substance can be preferably exemplified. More preferred examples include antibodies, proteins, peptides, nucleic acids (preferably DNA), and low molecular weight compounds that bind to Hsp90. Among them, antibodies, proteins, peptides, nucleic acids (preferably DNA) that specifically bind to Hsp90. More specifically, among them, antibodies and DNAs that specifically bind to Hsp90 can be more preferably exemplified because of their superior specificity and binding power to Hsp90. Specific binding to Hsp90 due to its excellent specificity and binding force for Hsp90. Antibodies can be particularly preferably exemplified that. Two or more kinds of Hsp90 removing substances may be used in combination.

The aforementioned “Hsp90 binding substance” is not particularly limited as long as it is a substance that binds to any mammalian Hsp90. Examples of such binding modes include hydrogen bonding, electrostatic bonding, van der Waals bonding, and hydrophobic bonding. (Hydrophobic interaction), covalent bond, ionic bond, and one or more bonds selected from coordination bonds can be exemplified, among them hydrogen bond, electrostatic bond, van der Waals bond, In addition, one or more bonds selected from hydrophobic bonds can be preferably exemplified. An antibody that binds to Hsp90 is considered to be bound to Hsp90 due to the combined involvement of hydrogen bonds, electrostatic bonds, van der Waals bonds, and hydrophobic bonds.

Specific examples of the antibody that binds to Hsp90 include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, etc. It can illustrate more preferably in the point of the specificity. The aforementioned antibody that binds to Hsp90 is produced by administering a fragment containing the Hsp90 or epitope or a cell expressing the protein on the membrane surface to an animal (preferably a non-human) using a conventional protocol, For example, for the preparation of monoclonal antibodies, the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4, 72, 1975) resulting in antibodies produced by continuous cell line cultures. 1983) and EBV-hybridoma method (MONOCLONAL
ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used.

Examples of the protein that binds to Hsp90 described above include Akt protein, HIF-1, ErbB1 / EGFR, ErbB2 / Her2, BCR-Abl, Raf1 and the like. Examples of the nucleic acid (preferably DNA) that binds to the aforementioned Hsp90 include CpG-DNA, human DNA, bacteria / virus DNA, and the like. For example, a protein that binds to Hsp90 is isolated from a DNA sequence that encodes a target protein based on known sequence information, integrated into a suitable expression vector, and the resulting expression vector is introduced into a suitable cell. And it can manufacture by culturing such cells and isolating the target protein from such cells. The nucleic acid that binds to Hsp90 is prepared by PCR using, for example, a primer for isolating the target nucleic acid based on known sequence information, and such a primer and a template DNA such as genomic DNA or cDNA. Can be manufactured.

In addition, whether or not a certain substance is an Hsp90-binding substance can be easily confirmed by, for example, contacting the labeled substance with solid-phased Hsp90, washing, and detecting the aforementioned label. be able to.

The suitable degree of the Hsp90 removal effect exhibited by the Hsp90 removal means in the present invention is not particularly limited, but the Hsp90 concentration in the serum when Hsp90 is removed from the serum of the SLE patient by the Hsp90 removal means is the SLE patient. The ratio is preferably 10% or more, more preferably 20% or more, still more preferably 40% or more, more preferably 60% or more, and even more preferably 70% or more as a percentage of the serum Hsp90 concentration can do.

The “mammal” in the present specification, such as “mammal” in Hsp90 of any mammal, is not particularly limited, but human, monkey, mouse, rat, hamster, guinea pig, cow, pig, horse, rabbit , Sheep, goats, cats, dogs and the like can be preferably exemplified, and humans can be more preferably exemplified. The sequences of these mammalian Hsp90s can be easily obtained by accessing a database such as GenBank. As an example of the sequence of Hsp90, the amino acid sequence of the coding region of human Hsp90 is shown in SEQ ID NO: 1. “Mammalian Hsp90” in the present invention comprises an amino acid sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more identity with the amino acid sequence of SEQ ID NO: 1. A protein having Hsp90 activity can be preferably exemplified.

The prophylactic / therapeutic effect on SLE exhibited by the prophylactic / therapeutic device of the present invention is not particularly limited as long as it is a prophylactic effect and / or therapeutic effect on SLE, but in particular, in blood, serum or in a mammalian subject An increase inhibitory effect and a decrease effect of interferon α concentration in plasma can be preferably exemplified, and among these, the decrease effect can be more preferably exemplified.

The suitable degree of the interferon α concentration lowering effect exhibited by the preventive / therapeutic device of the present invention is not particularly limited, but in the following interferon α measurement assay, the preventive / therapeutic device of the present invention uses Hsp90 The interferon α concentration when the serum from which serum is removed is added is preferably 10% or more, more preferably 20% or more, still more preferably 30%, relative to the interferon α concentration when the serum of the same SLE patient is added. %, More preferably 40% or more, and still more preferably 50% or more.
[Method of interferon α measurement assay]
Human plasmacytoid dendritic cells collected from the peripheral blood of healthy individuals are seeded at 5 × 10 ⁴ cells / well in a well plate, and then serum of SLE patients is added at 100 μL / well and cultured for 24 hours. Next, a culture supernatant is prepared from the culture solution, and the interferon α concentration in the culture supernatant is measured. Further, in this method, instead of “sera of SLE patients”, in the culture supernatant obtained using “serum obtained by removing Hsp90 from the serum of this SLE patient with the preventive / therapeutic apparatus of the present invention” Interferon alpha concentration is measured.

The aspect of the Hsp90 removing substance in the preventive / therapeutic apparatus of the present invention is not particularly limited as long as it can remove Hsp90 in the blood, serum or plasma of the subject. Is preferably carried on a solid phase carrier. The solid phase carrier is not particularly limited as long as it is a solid phase carrier to which the Hsp90 removal substance can be fixed, but examples of the material include gold, silver, copper, aluminum, platinum, titanium, nickel and the like. Metals: Alloys such as stainless steel and duralumin; Glass materials such as silicon, glass, quartz glass, ceramics; Plastics such as polyester resin, polystyrene, polypropylene resin, nylon, epoxy resin, and vinyl chloride resin; Gels such as agarose; Dextran, Cellulose, polyvinyl alcohol, chitosan and the like can be exemplified, and examples of the shape include beads, granules, plates and membranes. In addition, when it is in the form of beads or granules, it can be used as a column for prevention / treatment of systemic lupus erythematosus by filling the column.

When the Hsp90-removing substance is supported on a solid phase carrier, the Hsp90-removing substance can be produced by supporting it on the above-mentioned solid phase support. The method for supporting the Hsp90-removing substance on the aforementioned solid phase carrier is not particularly limited, and conventionally known means such as a so-called physical bonding (adsorption) method, a chemical bonding method, or the like can be used. For example, in the case of the physical binding method, the Hsp90 removing substance is immersed in an aqueous solution in which the Hsp90 removing substance is diluted to an appropriate concentration, then washed with a buffer solution, and dried. It can be supported on. In the case of the chemical binding method, for example, the surface of the solid support is coated with biotin, biotin is bound to the Hsp90 removal substance, and both of these biotins are bound via avidin, The surface of the solid support is modified with an appropriate functional group (for example, a carboxyl group, an amino group, or a sulfhydryl group), and the functional group and the Hsp90 removing substance are cross-linked with a crosslinking agent (for example, carbodiimide or N-hydroxysuccinimide). Etc.), the Hsp90 removing substance can be supported on the solid phase carrier.

The preventive / therapeutic apparatus of the present invention is an apparatus further comprising “a means for bringing blood or the like into contact with the Hsp90 removing means” and / or “a means for separating blood or the like from the Hsp90 removing means” in addition to the Hsp90 removing means. Preferably, the apparatus further includes “a means for deriving blood or the like from the blood vessel of the subject” and / or “a means for introducing the blood or the like after separation into the blood vessel of the subject”. Can be exemplified. If these means are further provided, removal of Hsp90 can be performed more easily and quickly. In addition to the Hsp90 removal means, the prevention / treatment apparatus of the present invention may further contain a means for removing substances that can prevent or treat SLE when removed from blood or the like. .

The method for producing the preventive / therapeutic device of the present invention is not particularly limited as long as the device is provided with the Hsp90 removing means so that the Hsp90 removing effect can be exhibited. For example, the Hsp90 removing means is held on the support member. The method of making it can be illustrated. Further, when the preventive / therapeutic device of the present invention is provided with a part or all of the above-mentioned means in addition to the Hsp90 removing means, it can be produced by holding these means on a support member. In addition, when a part or all of the above-described means is provided in addition to the Hsp90 removing means, the blood of the subject is “means for deriving blood etc. from the blood vessel of the subject”, “the blood is Hsp90. "Means for contacting with removal means", "Hsp90 removal means", "means for separating blood and the like from Hsp90 removal means", and "means for introducing blood after separation into the blood vessel of the subject" It is preferable that each means is held by the support member so as to pass through.

As the above-mentioned “means for deriving blood or the like from the blood vessel of the subject” or “means for introducing the separated blood or the like into the blood vessel of the subject”, a means including a needle or a cannula is preferably exemplified. As the above-mentioned “means for bringing blood or the like into contact with the Hsp90 removing means”, a pump or the like for transferring the derived blood or the like to the “Hsp90 removing means” can be preferably exemplified. As the “means for separating the Hsp90 from the Hsp90 removing means”, a pump that sucks blood or the like and separates it from the Hsp90 removing means, a centrifugal separator, or the like can be preferably exemplified.

The method for using the prophylactic / therapeutic device of the present invention is not particularly limited as long as the method includes the following steps A to C.
Step A; contacting blood, serum or plasma (hereinafter collectively referred to as “blood etc.”) taken from the blood vessel of the subject with the Hsp90 removal means in the prevention / treatment apparatus of the present invention;
Step B; a step of separating the Hsp90 removal means and blood, serum or plasma from the preventive / therapeutic device of the present invention;
Step C; returning the separated blood, serum or plasma into the blood vessel of the subject;

By performing such a method, the Hsp90 concentration in the blood, serum or plasma of the subject is reduced, thereby preventing SLE through suppression of interferon α production of the plasmacytoid dendritic cells of the subject. And / or it is considered that a therapeutic effect is exhibited.

As the blood vessel of the subject in the above step A, a blood vessel of the subject's artery can be preferably exemplified. The serum in the above step A can be obtained by separating blood and clots by leaving the blood taken out from the subject, etc., and removing the clots. After adding an anticoagulant to blood taken out from the specimen, it can be obtained as a supernatant from which cell components have been precipitated by centrifugation. In the step A, the method for taking out blood or the like from the blood vessel of the subject is not particularly limited, and a method using a syringe and a method of inserting a cannula into the blood vessel of the subject can be preferably exemplified. . Furthermore, in the above step A, the method of bringing blood or the like into contact with the Hsp90 removing means is not particularly limited, but a method of mixing blood or the like with the Hsp90 removing means can be preferably exemplified.

In the step B, the method for separating the Hsp90 removing means from the blood or the like is not particularly limited, and a method of simply taking out the Hsp90 removing means from the blood or the like may be used, or the Hsp90 removing means and the blood or the like using centrifugation may be used. It is also possible to separate them.

As the blood vessel of the subject in the above step C, a vein of the subject can be preferably exemplified. In this process, the method of returning separated blood or the like into the blood vessel of the subject is not particularly limited, and a method using a syringe and a method of inserting a cannula into the blood vessel of the subject are preferably exemplified. Can do.

The frequency of use of the prophylactic / therapeutic device of the present invention is not particularly limited, but for example, use once a week (preferably twice or more, more preferably 3 times or more, more preferably 5 times or more), It can be suitably exemplified by continuing for 2 weeks or more (preferably 3 weeks or more, more preferably 6 weeks or more and 5 years or less, and further preferably 3 months or more and 5 years or less). When used multiple times in this manner, the onset of SLE can be more effectively suppressed or delayed at the stage before the onset of SLE, and the vicious circle of inflammation caused by SLE can be interrupted at the stage after the onset of SLE. Therefore, it is considered that a superior therapeutic effect for SLE is exhibited. The preventive / therapeutic device of the present invention may be used together with a means for removing substance X other than Hsp90, which can obtain the effect of preventing / treating SLE by removing it from blood or the like. It may be used before or after use.

SLE can be particularly preferably exemplified as a disease to be prevented or treated by the prevention / treatment device of the present invention. Autoimmune hepatitis (AIH) and primary bile are diseases similar to SLE. There is also the possibility of exerting a preventive / therapeutic effect on cirrhosis of the liver (PBC). Mammals can be exemplified as the species of the subject to be prevented and treated by the prevention / treatment apparatus of the present invention.

2. Method for use as biomarker for systemic lupus erythematosus The method for use as a biomarker for systemic lupus erythematosus of the present invention (hereinafter also simply referred to as “method of use of the present invention”) includes blood, serum or plasma. (Hereinafter simply referred to as “in blood” etc.) Hsp90 (preferably Hsp90 concentration) is not particularly limited as long as it is a method of using it as a biomarker for systemic lupus erythematosus. The method for determining systemic lupus erythematosus of the invention can be preferably exemplified. The Hsp90 in the method of use of the present invention may be any mammalian Hsp90 as described above, and among them, the endogenous Hsp90 of any mammal can be preferably exemplified.

3. Method for determining systemic lupus erythematosus The method for determining systemic lupus erythematosus of the present invention (hereinafter also simply referred to as “method of determination of the present invention”) includes (A) Hsp90 in blood, serum or plasma of a subject. Measuring the concentration in vitro; and (B) comparing and evaluating the Hsp90 concentration measured in the step (A) with the Hsp90 concentration in blood, serum or plasma of a normal control. Although not particularly limited, further, (C) when the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control, the subject is treated with systemic lupus erythematosus. Or (D) the degree to which the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in normal control blood, serum or plasma Can be suitably exemplified by a method comprising: evaluating the severity of a disease state of systemic lupus erythematosus of the subject. When the Hsp90 concentration in blood is measured in the step (A), the Hsp90 concentration in blood of the normal control is used in the step (B), and the Hsp90 concentration in serum is measured in the step (A). When the Hsp90 concentration in the serum of the normal control is used in the step (B) and the Hsp90 concentration in the plasma is measured in the step (A), the plasma of the normal control is determined in the step (B). Medium Hsp90 concentration is used.

The method for measuring the Hsp90 concentration in the step (A) in vitro is not particularly limited as long as it is a method capable of measuring the Hsp90 concentration in blood or the like in vitro. For example, Hsp90α, ELISA kit (manufactured by StressGen), etc. A method for measuring using an anti-Hsp90 antibody can be exemplified. Further, the “Hsp90 concentration in blood, serum or plasma of a normal control” in the above step (B) may be measured when measuring the Hsp90 concentration in the blood of a subject or the like, or may be measured in advance. The numerical value may be used. The method for comparison / evaluation in the step (B) is not particularly limited, but when the Hsp90 concentration measured in the step A is higher than the Hsp90 concentration in blood, serum or plasma of a normal control, The method for evaluating the subject as systemic lupus erythematosus (ie, step (C)), and the level of Hsp90 measured in step A is higher than the Hsp90 concentration in normal control blood, serum or plasma, A method for evaluating the severity of the pathological condition of systemic lupus erythematosus of the subject (ie, step (D)) can be preferably exemplified.

A suitable level of the height of Hsp90 concentration measured in the step (A) when evaluating as systemic lupus erythematosus in the step (C) is not particularly limited, but the Hsp90 concentration measured in the step (A) is not particularly limited. However, it is preferably 2 times or more, more preferably 4 times or more, further preferably 6 times or more, more preferably 8 times or more, and still more preferably with respect to the Hsp90 concentration in normal control blood, serum or plasma. It can be suitably exemplified that it is 10 times or more, more preferably 12 times or more.

In addition, as the “condition of systemic lupus erythematosus” in the above step (D), a symptom caused by an increase in interferon α concentration in blood or the like can be preferably exemplified.

As described above, the determination method of the present invention can determine (determine) whether or not the subject is SLE or the severity of the SLE disease state of the subject. In addition to this, the determination method of the present invention can also determine (determine) the possibility that the subject has SLE. In this case, instead of the above-mentioned step (C), (C ′) the level of the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control, A step of evaluating the degree of possibility that the subject has systemic lupus erythematosus is used.

As another aspect of the present invention, in the production of the systemic lupus erythematosus prevention / treatment device of the present invention, the use of an Hsp90-removing substance, the method of using the Hsp90-removing substance as a systemic lupus erythematosus prevention / treatment device, In the prevention and treatment of systemic lupus erythematosus, the use of an Hsp90 removal substance, the step of bringing the Hsp90 removal substance into contact with blood taken out from the blood vessel of the subject, the step of separating the Hsp90 removal substance and blood, etc. A method for preventing and treating systemic lupus erythematosus, which includes step B of returning B and separated blood into the blood vessel of the subject, can also be exemplified. The contents of the words in these uses and methods and the preferred embodiments thereof are as described above.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

[Measurement of Hsp90 concentration in serum of SLE patients]
In order to examine whether the concentration of Hsp90 in the serum of SLE patients is different from that of normal people, and whether the concentration of SLE patients changes in the symptom exacerbation phase and post-treatment remission phase, the following Hsp90 A measurement assay was attempted.

Under the approval of the ethics committee of the Sapporo Medical University School of Medicine, two patients who received the Sapporo Medical University Hospital were selected and targeted for sample provision. In addition, one healthy person was selected as a person who was requested to provide a normal sample. In addition, after obtaining informed consent from these three subjects, serum samples were collected from each subject. For SLE patients, serum samples were collected not only during the exacerbation period but also during the remission period after treatment.

The amount of Hsp90 in 5 types of serum samples, 4 types of serum samples collected from 2 SLE patients (SLE patient 1 and SLE patient 2) and 1 type of serum sample collected from healthy individuals, was expressed as Hsp90α, ELISA. Measured with kit (manufactured by StressGen). The results of producing the Hsp90 concentration (pg / mL) in each serum sample from this measured value are shown in FIG.

As can be seen from FIG. 1, in both cases of SLE patient 1 (“SLE patient 1”) and SLE patient 2 (“SLE patient 2”)
treatment "), the Hsp90 concentration in the serum sample was very high, but after treatment remission (" after "
treatment "), the Hsp90 concentration decreased to the same level as that of a healthy person (" healthy donor ").

Further, the number of healthy persons and the number of patients were increased (6 healthy persons and 8 patients), and the results of the Hsp90 measurement assay as in Example 1 are shown in FIG. As in Example 1, the Hsp90 concentration in the serum was also high in the symptom exacerbation period, and a significant difference was observed between the healthy person and the post-treatment remission period (P <0.01). From this, it was shown that the Hsp90 concentration in serum correlates with the activity of SLE. Therefore, the Hsp90 concentration in serum is considered to be very useful for the indication of the SLE disease state and the diagnosis of SLE. In addition, it is the knowledge which the present inventors discovered for the first time this time that the density | concentration of Hsp90 is rising in the blood of a SLE patient. Since Hsp90 is not a secreted protein, it was unexpected that the concentration could be increased so much in the blood. *

[Interferon α measurement assay using human plasmacytoid dendritic cells]
A high level of interferon α in serum is considered to be one of the causes of SLE pathological deterioration. In addition, the present inventors have previously shown that Hsp90 inhibitors can inhibit interferon α production from plasmacytoid dendritic cells derived from mice and humans, and systemic lupus erythematosus (SLE) model mice. It has been clarified that the disease state can be improved (Japanese Patent Application No. 2010-219422). In order to examine how the production of interferon α from human plasmacytoid dendritic cells is affected by removing Hsp90 from the serum of SLE patients, the following in vitro interferon α measurement assay is performed. Tried.

Peripheral blood was collected from the aforementioned healthy individuals, and human plasmacytoid dendritic cells were purified from the aforementioned peripheral blood using a human plasmacytoid dendritic cell isolation kit (Miltenyi Biotec). After seeding human plasmacytoid dendritic cells at 5 × 10 ⁴ cells / well in a 96-well plate, 100 μL / well of the serum of the aforementioned SLE patient of Example 1 was added and cultured for 24 hours. Next, a culture supernatant was prepared from the culture solution, and the interferon α concentration in the culture supernatant was measured by ELISA. Moreover, it replaced with the serum of the above-mentioned SLE patient, and the interferon alpha density | concentration in a culture supernatant was measured by the same method except having added the serum which removed Hsp90 from the serum of this SLE patient. Further, the interferon α concentration in the culture supernatant was measured by the same method except that none of the aforementioned sera was added. The removal of Hsp90 from the serum was performed using protein G beads to which the Fc site of the anti-Hsp90 antibody was bound. Specifically, such beads and serum were mixed well to bind the anti-Hsp90 antibody on the beads and Hsp90 in the serum, and then the beads were recovered to remove Hsp90 from the serum. The results of measuring the interferon α concentration in the culture supernatant by the above-described methods are shown in FIG.

As can be seen from FIG. 3, when no serum was added (“control”), the interferon α concentration was very low, and when the serum of the SLE patient was added (“stimulated with SLE patient serum”) The α concentration was significantly increased, but when the serum from which the Hsp90 was removed was added from the serum of this SLE patient (“stimulated with SLE patient serum removed HSP90”), compared with the case where the serum of the SLE patient was added, The interferon α concentration was significantly reduced to about 50%. This result indicates that a molecule that promotes interferon α production of human plasmacytoid dendritic cells is contained in the serum of SLE patients. It has been reported so far that molecules that stimulate interferon α production of human plasmacytoid dendritic cells are human DNA and human RNA, and that anti-DNA antibodies are detected at a high rate in SLE patients. Therefore, the present inventors believe that anti-DNA antibody-self DNA complex and Hsp90-self DNA complex promote interferon α production of human plasmacytoid dendritic cells. In fact, when Hsp90 was removed from the serum of SLE patients, interferon α production of human plasmacytoid dendritic cells was reduced to about 50%. That is, the fact that a large amount of Hsp90 is detected in the serum of SLE patients is a finding found for the first time by the experiment of Example 1 of the present application, but as we have already reported, (Okuya, Tamura, J. Immunology) , June 2010; 184: 7092-7099) この, this Hsp90 forms a complex with its own DNA (Hsp90-self DNA complex), thereby suppressing the degradation of the self DNA, and the self DNA is converted into human plasma cells. It was considered that the human plasmacytoid dendritic cell production of interferon α was promoted by efficiently incorporating it into the dendritic cell and localizing its own DNA to the early endosome of the plasmacytoid dendritic cell. In addition, from the results of this interferon α measurement assay, it was shown that SLE can be treated by removing Hsp90 from the serum and blood of SLE patients.

[Measurement of serum Hsp90 concentration in SLE model mice]
In order to investigate whether the serum concentration of Hsp90 increases in SLE-onset model animals as well as in humans, the following measurements were performed.

First, MRL / lpr mice (Jackson Laboratory, Inc.), which are SLE model mice, are prepared, before SLE develops (21 weeks old), when SLE begins to develop (28 weeks old), and after SLE develops The concentration of Hsp90 in the serum at 37 weeks of age was measured in the same manner as in Example 1 using Hsp90α, ELISA kit (manufactured by StressGen). The result is shown in FIG.

As can be seen from FIG. 4, there was a statistically significant difference in the serum concentration of Hsp90 between the onset and early stage (P <0.05), the early stage and late stage (P <0.01). It was. From this, it was confirmed that the Hsp concentration in the serum increased with the onset of SLE in the model animals as in the case of humans.

The present invention can be suitably used in the field of prevention / treatment of systemic lupus erythematosus, the field of biomarkers of systemic lupus erythematosus, and the field of determination of systemic lupus erythematosus.

Claims

A system for preventing and treating systemic lupus erythematosus characterized by comprising means for removing Hsp90.
2. The preventive / therapeutic apparatus according to claim 1, wherein the removal means for Hsp90 contains a removal substance for Hsp90.
The preventive / therapeutic device according to claim 1 or 2, wherein a substance for removing Hsp90 is supported on a solid phase carrier.
A method of using Hsp90 in blood, serum or plasma as a biomarker for systemic lupus erythematosus.
A method for determining systemic lupus erythematosus comprising the following steps (A) and (B);
(A) a step of measuring in vitro the concentration of Hsp90 in the blood, serum or plasma of a subject;
(B) a step of comparing and evaluating the Hsp90 concentration measured in the step (A) with the Hsp90 concentration in normal control blood, serum or plasma;
The determination method according to claim 5, further comprising the following step (C) or (D):
(C) A step of evaluating the subject as systemic lupus erythematosus when the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control;
(D) The degree of the Hsp90 concentration measured in the step (A) is higher than the Hsp90 concentration in blood, serum or plasma of a normal control, and the severity of the pathological condition of systemic lupus erythematosus in the subject. The step of evaluating;