Biomarker, screening method, detection method for biomarker and method for assisting in diagnosis of demyelinating disease

Abstract

To provide a biomarker, a screening method, a biomarker detection method, and a method for assisting in diagnosis of demyelinating disease that are useful for diagnosing atypical CNS demyelinating disease and PMS, which have previously been difficult to diagnose, and determining a treatment strategy, by a discovered new CNS throat antibody for atypical CNS demyelinating disease and PMS, which tends to become severe.SOLUTION: Provided is a biomarker for diagnosing demyelinating disease or assessing effectiveness of treatment, comprising an anti-ATP1A2 antibody or an anti-ATP1A3 antibody. Also, provided is a screening method for detecting cross-reactivity of mouse optic nerve tissue with an antibody obtained from a patient with demyelinating disease to find out an antigen associated with demyelinating disease. Further, provided are a method for detecting a biomarker and a method for assisting in diagnosis of demyelinating disease using the antibody.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a screening method related to the diagnosis and elucidation of the pathology of demyelinating diseases, a method for detecting biomarkers, and a method for assisting the diagnosis of demyelinating diseases. This application claims priority to U.S. provisional patent application Ser. No. 63 / 652,184, filed May 28, 2024, the contents of which are incorporated herein by reference. [Background technology]

[0002] Multiple sclerosis (MS), or central nervous system demyelination, is believed to be an autoimmune disease directed against the myelin sheath of the central nervous system (CNS), but no specific antibodies or responsible antigens have been identified. Eight disease-modifying drugs have been introduced in Japan, and relapses in relapsing-remitting MS (RRMS) have been relatively suppressed. However, even with existing drugs, it is almost impossible to prevent the progression of disability in chronic progressive MS (PMS), such as secondary progressive MS (SPMS), which half of RRMS cases progress to, and primary progressive MS (PPMS), which has a slowly progressive course from the onset of the disease.

[0003] In PMS, anti-CD20 monoclonal antibodies such as ocrelizumab and ofatumumab, as well as siponimod, slow progression by about 20%. However, because it is extremely difficult to clinically detect chronic progression of disability not due to relapse (progression independent of relapse activity, or PIRA), the diagnosis of transition from RRMS to SPMS is said to be delayed by about three years. This leads to delays in switching to SPMS medications. Furthermore, diagnosing PPMS itself is difficult. Therefore, the development of biomarkers that can diagnose PMS early and lead to an understanding of its pathogenesis is an urgent issue.

[0004] Meanwhile, in neuromyelitis optica spectrum disorder (NMOSD), a disease related to MS, relapses have been successfully suppressed in anti-aquaporin 4 (AQP4) antibody-positive patients with monoclonal antibody therapy such as eculizumab. However, in antibody-negative NMOSD, these monoclonal antibody preparations are ineffective, and steroids and immunosuppressants are difficult to suppress relapses, so there is a strong need to clarify the pathogenesis.

[0005] Some of these cases are positive for anti-myelin oligodendrocyte glycoprotein (MOG) antibodies. However, there are also many atypical cases of MS that are negative for anti-AQP and anti-MOG antibodies but have severe optic nerve and spinal cord damage, and these cases cannot be detected by MOG antibodies or other methods. Therefore, there is a strong need for the development of diagnostic biomarkers that will lead to an understanding of the pathogenesis.

[0006] The present inventors have previously discovered antibodies against throat proteins localized at the nodes of Ranvier, such as neurofascin 155 (NF155) and leucine-rich repeat LGI family member 4 (LGI4), in chronic inflammatory demyelinating polyneuropathy (CIDP), a demyelinating disease of the peripheral nervous system (PNS), and combined central and peripheral demyelination (CCPD), which causes demyelination in both the CNS and PNS. The present inventors have demonstrated that patients positive for these antibodies exhibit unique clinical manifestations, and these PNS throat antibody-positive cases are now referred to as autoimmune nodopathy.

[0007] Regarding anti-NF155 antibodies, Patent Document 1 by the present inventors discloses a method for assisting the diagnosis of chronic inflammatory demyelinating polyneuropathy, which comprises the steps of measuring anti-NF155 antibodies, the majority of which are IgG4, contained in a sample and determining whether the anti-NF155 antibody is positive or negative, and in which, compared with anti-NF155 antibody-negative cases, the nerve conduction velocity in the distal peripheral nerves and nerve roots is slower, the thickening of the proximal peripheral nerves and nerve roots is greater, and the cerebrospinal fluid protein level is higher, as well as a kit and biomarker using the same. This technology aims to provide a method for diagnosing CIDP using the antibody, in particular, a diagnostic method for specifically diagnosing a group of CIDPs with a specific pathophysiology, as well as a kit and biomarker for use in the diagnosis.

[0008] Regarding anti-LGI4 antibodies, the present inventors' Patent Document 2 discloses a biomarker containing an anti-LGI4 antibody for diagnosing inflammatory demyelinating polyneuropathy or determining the effectiveness of treatment, or for indicating that the inflammatory demyelinating polyneuropathy is autoimmune nodopathy, as well as a diagnostic kit containing the same and a method for assisting diagnosis using the same. This technology aims to provide a biomarker, diagnostic kit, and method for assisting diagnosis that specifically diagnose a group of acute and chronic inflammatory demyelinating peripheral neuropathy with a specific pathophysiology, in which the autoantibodies are unknown. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 6751025 [Patent Document 2] Japanese Patent Application Publication No. 2025-21769 Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, there is a strong need for CNS throat antibodies that can be used to elucidate and diagnose CNS demyelinating diseases such as anti-AQP4 antibody-negative and anti-MOG antibody-negative MS / NMOSD / CCPD. Many autopsy pathology reports have shown that MS also exhibits nodopathy, with extension of the nodes of Ranvier from the initial lesion. More than 10 years ago, it was reported that autoantibodies against NF186 and TAG1, axonal proteins at the nodes of Ranvier, are present in MS, but since then, there have been no reports from other institutions confirming this, and no established CNS throat antibodies exist.

[0011] The present invention was made in consideration of the above-mentioned circumstances, and its purpose is to discover new CNS throat antibodies for atypical CNS demyelinating diseases and PMS, which tend to become severe, and to provide biomarkers, screening methods, methods for detecting biomarkers, and methods to assist in the diagnosis of demyelinating diseases that are useful for diagnosing atypical CNS demyelinating diseases and PMS, which have previously been difficult to diagnose, and determining treatment strategies. [Means for solving the problem]

[0012] In order to solve the above problems, the present invention has the following aspects. [1] Biomarkers for diagnosing demyelinating diseases or determining the effectiveness of treatment, including anti-ATP1A2 antibodies or anti-ATP1A3 antibodies. [2] The biomarker according to [1], wherein the demyelinating disease is a demyelinating disease affecting the central nervous system (CNS) or a demyelinating disease affecting both the CNS and peripheral nervous system (PNS). [3] A screening method for detecting cross-reactivity of antibodies obtained from patients with demyelinating diseases with mouse optic nerve tissue to identify antigens related to demyelinating diseases. [4] The screening method according to [3], wherein an antibody against an antigen related to a demyelinating disease is used as a biomarker for diagnosing a demyelinating disease or determining the effectiveness of a treatment for the demyelinating disease. [5] The screening method according to [3], comprising the steps of injecting serum obtained from a patient with the demyelinating disease into the optic nerve of a mouse in vivo and detecting the crossover by tissue immunohistochemistry (TBA) method. [6] The screening method according to [3], which comprises the step of detecting the cross-linking in vitro using a Western blotting method on an extract of mouse optic nerve tissue. [7] The screening method according to [6], wherein the Western blotting method comprises subjecting an extract of the mouse optic nerve tissue to two-dimensional electrophoresis, followed by two-dimensional Western blotting. [8] A biomarker detection method in which ATP1A2 or ATP1A3 is expressed in cells derived from human neuroblastoma cells, and a sample derived from a subject is added to the cultured cell line, and cross-reaction with the ATP1A2 or ATP1A3 is detected using a cell-based assay (CBA) method to determine whether the sample contains anti-ATP1A3 antibodies. [9] The method for detecting a biomarker according to [8], wherein the cells are SKN cells (cells derived from human neuroblastoma).

[10] The method for detecting a biomarker described in [8], wherein the sample is serum obtained from the subject or an extract thereof.

[11] A method for detecting a biomarker for diagnosing a demyelinating disease or assessing a therapeutic effect, comprising: A method for detecting biomarkers, comprising detecting cross-reactivity between the sample and an extract of cells or tissues in which ATP1A2 or ATP1A3 has been overexpressed by Western blotting, and determining whether or not the sample contains anti-ATP1A2 antibodies or anti-ATP1A3 antibodies.

[12] The method for detecting a biomarker according to

[11] , wherein the cell or tissue extract is an extract from HEK293 (human embryonic kidney cells).

[13] The method for detecting a biomarker according to

[11] , wherein the sample is serum obtained from the subject or an extract thereof.

[14] A method for detecting an anti-ATP1A2 antibody or an anti-ATP1A3 antibody in a sample derived from a subject, A method for assisting in the diagnosis of a demyelinating disease, wherein a significantly higher abundance of the anti-ATP1A2 antibody or anti-ATP1A3 antibody compared to its abundance in healthy individuals indicates that the subject is suffering from a demyelinating disease.

[15] The method for assisting in the diagnosis of a demyelinating disease according to

[14] , wherein the demyelinating disease is a demyelinating disease affecting the central nervous system (CNS) or a demyelinating disease affecting both the CNS and peripheral nervous system (PNS). [Effects of the Invention]

[0013] According to the present invention, new CNS throat antibodies have been discovered for atypical CNS demyelinating diseases and PMS, which tend to become severe, and it is possible to provide biomarkers, screening methods, methods for detecting biomarkers, and methods for assisting in the diagnosis of demyelinating diseases that are useful for diagnosing atypical CNS demyelinating diseases and PMS, which have previously been difficult to diagnose, and determining treatment strategies. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a photographic diagram showing an outline of the procedure for an in vivo experiment in which patient serum IgG was injected into the mouse optic nerve. [Figure 2] FIG. 1 is a photograph of immunostaining showing the binding of patient serum IgG to the nodes of Ranvier (juxta-paranodes) in the optic nerve TBA. [Figure 3] FIG. 1 is a photograph showing the results of Western blot (WB) of patient serum using protein lysate extracted from mouse optic nerve. [Figure 4] FIG. 1 is a photograph showing the results of two-dimensional electrophoresis and Western blotting of mouse optic nerve protein lysate. [Figure 5] FIG. 1 is a photograph showing the results of immunostaining by the CBA method. [Figure 6] FIG. 1 is a photograph showing the results of Western blotting using ATP1A2 or ATP1A3 overexpression lysate prepared in HEK293 cells. [Figure 7] Schematic and photographic representations of typical demyelination lesions in anti-ATP1A2 / 3 antibody-positive cases. [Figure 8] FIG. 1 is a schematic diagram of anti-ATP1A2 / 3 antibody-positive cases. [Figure 9]FIG. 1 is a photograph showing the results of immunostaining following in vivo injection of patient serum IgG into mouse optic nerves. [Figure 10] FIG. 1 is a schematic diagram of a discussion of each antibody and its target in demyelination. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, the biomarker, screening method, biomarker detection method, and method for assisting in the diagnosis of demyelinating diseases according to the present invention will be described with reference to embodiments, although the present invention is not limited to the following embodiments.

[0016] (biomarker) The biomarker of this embodiment is a biomarker for diagnosing a demyelinating disease or determining the therapeutic effect, comprising an anti-ATP1A2 antibody or an anti-ATP1A3 antibody.

[0017] In this embodiment, by detecting a biomarker in a sample or measuring the amount of a biomarker, etc., it is possible to obtain information for diagnosing a demyelinating disease in a subject from whom the sample was derived. Alternatively, the detection or measurement can be used to assess the effectiveness of a treatment. Assessment of the effectiveness of a treatment broadly includes assessing the impact of a treatment when inflammatory demyelinating polyneuropathy is being treated.

[0018] In this embodiment, the demyelinating disease broadly refers to diseases caused by damage to the myelin sheath of myelinated nerves. The demyelinating diseases of this embodiment are preferably demyelinating diseases that affect the central nervous system (CNS) and demyelinating diseases that affect both the CNS and peripheral nervous system (PNS). The demyelinating disease of this embodiment is preferably an anti-AQP4 antibody-negative or anti-MOG antibody-negative demyelinating disease, and is also preferably a CNS demyelinating disease such as MS / NMOSD / CCPD. These demyelinating diseases have been difficult to diagnose or assess the effectiveness of treatment with conventional techniques, but the use of the biomarker of this embodiment makes it possible to effectively diagnose or assess the effectiveness of treatment.

[0019] Here, ATP1A3 is highly expressed in the axonal membrane of the juxta-paranode and regulates Na+ transport after axon excitation. + and K. + ATP1A3 is known to maintain the concentration gradient of ATP1A2. Mutations in the ATP1A3 gene cause alternating hemiplegia of childhood (AHC). Down-regulation of ATP1A3 leads to axonal degeneration. ATP1A2 is highly expressed in the glial cell membrane at the nodes of Ranvier in astroglia and oligodendroglia, and is known to be responsible for K+ clearance after axonal excitation. ATP1A1 is expressed on the cell membrane of almost all cells and mediates the transport of Na + and K. + It is known that they are responsible for maintaining the concentration gradient and potential difference of the The protein RefSeQ for human ATP1A1 is NP_000692, for ATP1A2 is NP_000693, and for ATP1A3 is NP_689509.1 (isoform 1).

[0020] The anti-ATP1A2 antibody or anti-ATP1A3 antibody included in the biomarker of this embodiment can be one that cross-reacts with the above-mentioned human ATP1A2 or ATP1A3. In the following description, ATP1A2 or ATP1A3 (ATP1A2 / A3) refers to human ATP1A2 or human ATP1A3 (human ATP1A2 / A3), and anti-ATP1A2 antibody or anti-ATP1A3 antibody (anti-ATP1A2 / A3 antibody) refers to an antibody that binds to ATP1A2 or ATP1A3. On the other hand, as described below, the present invention can also be applied to antibodies from other animals. For example, to obtain information to assist in the diagnosis of other animals, anti-ATP1A2 antibodies or anti-ATP1A3 antibodies from other animals can also be used.

[0021] The biomarker of this embodiment includes an anti-ATP1A2 antibody or an anti-ATP1A3 antibody and can be used to diagnose demyelinating diseases. The anti-ATP1A2 antibody or the anti-ATP1A3 antibody can be applied in the following ways, for example.

[0022] The biomarker of this embodiment can be used for the differential diagnosis of demyelinating diseases, as well as for selecting an initial treatment method for demyelinating diseases and a method for preventing recurrence. For example, in an individual patient with demyelinating diseases, whether the biomarker is positive or negative can easily tell which treatment or drug is appropriate.

[0023] Furthermore, the amount of biomarkers in patient samples can be compared before and after use of the treatment / drug, and since anti-ATP1A2 antibodies or anti-ATP1A3 antibodies are pathogenic, if the amount of the biomarker consisting of anti-ATP1A2 antibodies or anti-ATP1A3 antibodies decreases after administration compared to before administration, it can be determined that the drug is effective for the patient. On the other hand, if the amount of the biomarker before and after administration is the same, or if the amount of the biomarker after administration is higher than before administration, it can be determined that the drug is ineffective for that patient. Furthermore, by confirming that the biomarker decreases during continued treatment, it is possible to reduce the dosage of the therapeutic drug.

[0024] (Screening method) The screening method of this embodiment is a screening method for detecting cross-reactivity of mouse optic nerve tissue with antibodies obtained from patients with demyelinating diseases, thereby finding antigens related to demyelinating diseases.

[0025] A conventional method for detecting cross-reactivity of mouse optic nerve tissue with an antibody obtained from a patient with a demyelinating disease may be used as appropriate. For example, a method of immunostaining mouse optic nerve tissue as an immunostaining sample, or a method of extracting tissue and performing Western blotting (WB) may be used. When identifying an antigen, it is particularly preferable to use multiple methods described above in combination.

[0026] In the screening method of this embodiment, it is preferable to use an antibody against an antigen related to the demyelinating disease as a biomarker for diagnosing the demyelinating disease or determining the therapeutic effect.

[0027] The screening method of this embodiment preferably comprises the steps of injecting serum obtained from a patient with the demyelinating disease into the optic nerve of a mouse in vivo and detecting the crossover by tissue immunohistochemistry (TBA) method. Furthermore, the screening method of this embodiment preferably includes a step of detecting the cross-over in vitro using a Western blot method on an extract of mouse optic nerve tissue. Furthermore, the Western blotting method is also preferably carried out by subjecting an extract of the mouse optic nerve tissue to two-dimensional electrophoresis, followed by two-dimensional Western blotting.

[0028] (Method for detecting biomarkers) The biomarker detection method of this embodiment involves expressing ATP1A3 in cells derived from human neuroblastoma cells, adding a sample from a subject to the cultured cell line, and using a cell-based assay (CBA) method to detect cross-reaction with ATP1A2 or ATP1A3 to determine whether the sample contains anti-ATP1A3 antibodies.

[0029] In the biomarker detection method of this embodiment, the cells are preferably SKN cells (human neuroblastoma-derived cells).

[0030] In the biomarker detection method of this embodiment, the specimen contains antibodies and the like possessed by the subject. Such specimens can be body fluids such as blood and cerebrospinal fluid, or derivatives thereof. Examples of blood-derived substances that can be used include blood extracts such as serum. In this embodiment, the specimen is preferably serum obtained from the subject or an extract thereof. The extract may be a fraction or purified product of serum.

[0031] The biomarker detection method of this embodiment is a biomarker detection method for detecting whether a sample derived from a subject contains a biomarker for diagnosing a demyelinating disease or determining the effectiveness of treatment, and it is also preferable to detect cross-reaction between the sample and an extract of cells or tissues that overexpress ATP1A2 or ATP1A3 by Western blotting to determine whether the sample contains anti-ATP1A2 antibodies or anti-ATP1A3 antibodies.

[0032] In the biomarker detection method of this embodiment, it is also preferable that the cell or tissue extract is an extract from HEK293 (human embryonic kidney cells).

[0033] In the biomarker detection method of this embodiment, it is also preferable that the sample is serum obtained from the subject or an extract thereof.

[0034] (Method for assisting in the diagnosis of demyelinating diseases) The method for assisting in the diagnosis of demyelinating disease of this embodiment includes a step of measuring anti-ATP1A2 antibody or anti-ATP1A3 antibody contained in a sample derived from a subject, and a significantly higher amount of the anti-ATP1A2 antibody or anti-ATP1A3 antibody compared to the amount present in healthy individuals indicates that the subject is suffering from a demyelinating disease.

[0035] Here, the method for assisting in the diagnosis of demyelinating diseases refers to a method for obtaining information that can be used in the diagnosis of demyelinating diseases. For example, by obtaining information on the tissues, cells, etc. of a subject from whom a specimen is derived, information that can be used for diagnosis can be obtained, and information that can be used in the diagnosis of demyelinating diseases can be obtained. Indicating that the subject is suffering from a demyelinating disease broadly refers to information regarding the condition of the demyelinating disease, such as information regarding the severity of the demyelinating disease and the effectiveness of treatment.

[0036] In the method of this embodiment for assisting in the diagnosis of a demyelinating disease, the demyelinating disease is preferably a demyelinating disease that affects the central nervous system (CNS) or a demyelinating disease that affects both the CNS and peripheral nervous system (PNS).

[0037] In the method for assisting in the diagnosis of a demyelinating disease of this embodiment, the subject of diagnosis is any animal that can be affected by a demyelinating disease, such as humans, non-human primates, dogs, cats, rabbits, rats, mice, etc. In the explanation of this embodiment and examples, the subject is mainly humans, but the same applies to other animals.

[0038] (Effects of this embodiment) According to this embodiment, new CNS throat antibodies have been discovered for atypical CNS demyelinating diseases and PMS, which tend to become severe, and it is possible to provide biomarkers, screening methods, biomarker detection methods, and methods to assist in the diagnosis of demyelinating diseases that are useful for diagnosing atypical CNS demyelinating diseases and PMS, which have previously been difficult to diagnose, and determining treatment strategies.

[0039] The inventors established the gold standard CBA method for antibody measurement by transfecting a human neuroblastoma-derived SKN cell line with an ATP1A3-GFP expression plasmid and using the resulting cell as an antigen. Furthermore, WB analysis using ATP1A2 and ATP1A3 overexpression lysates can confirm reactions to not only ATP1A3 but also ATP1A2. Therefore, anti-ATP1A2 / 3 antibody-positive individuals can be identified with high accuracy by using the TBA method using mouse optic nerves, the CBA method using an ATP1A3-transfected SKN cell line, and the WB method using ATP1A2 and ATP1A3 overexpression lysates.

[0040] Previous reports on ATP1A2 and ATP1A3 have reported the detection of autoantibodies against myocardial Na-K-ATPase (ATP1A) by ELISA in patients with dilated cardiomyopathy (Baba A, et al., J Am Coll Cardiol 2002;40:1153-9), but the significance of this has not been established. The ELISA method using myocardial Na-K-ATPase antigen in this report cannot be considered the gold standard for measuring autoantibodies, and it is thought that there are many nonspecific reactions.

[0041] Although an antibody against the transmembrane portion of ATP1A3 has been reported in one case of paraneoplastic neurological syndrome (Scharf M, et al., Neurology 2015;84:1673-1679), no reports have been published in demyelinating diseases. The study described a CBA method using HEK293 cells transfected with an ATP1A3 expression vector. However, our studies showed that the antibody reacted nonspecifically with ATP1A1 expressed in KEK293 cells, making it impractical. Furthermore, no immunoreactivity data was presented for this antibody, leaving open the question of whether it truly reacted with ATP1A3. Therefore, antibodies against ATP1A and their effects in relation to demyelinating diseases have not been elucidated until now. The present invention provides a novel method for screening ATP1A as an antigen associated with demyelinating diseases, anti-ATP1A antibodies, and antibodies against ATP1A.

[0042] As shown in the Examples below, anti-ATP1A2 / 3 antibodies have been detected in PMS and subtypes of CNS demyelinating diseases of unknown etiology, such as anti-AQP4 antibody-negative and anti-MOG antibody-negative MS / NMOSD / CCPD. Based on the results of passive transfer experiments, these antibodies are considered to be pathogenic. Based on these data, the inventors speculate that CNS nodoantibodies (anti-ATP1A2 / 3 antibodies) induce autoimmune nodopathy via an autoantibody-mediated mechanism, causing demyelination and axonal damage at the nodes of Ranvier. Anti-ATP1A2 / 3 antibodies are deeply involved in the pathology of CNS demyelinating diseases of unknown etiology, and antibody-positive patients are prone to large lesions and severe disorders. They may also present with peripheral nerve demyelination and hyperplasia. Therefore, antibody-positive patients should receive adequate immunotherapy from the early stage, including blood purification therapy, intravenous immunoglobulin therapy, and immunosuppressants. Young antibody-positive patients are prone to seronegative NMOSD, including severe optic nerve damage and spinal cord damage, and therefore require adequate and long-term immunotherapy from the early stage. In the future, if this antibody is positive, it will be desirable to treat it with molecular targeted drugs that reduce the antibody. Therefore, this antibody is thought to be extremely useful in diagnosing CNS demyelinating diseases of unknown cause and determining treatment strategies.

[0043] The diseases for which this antibody can be measured are shown in Table 1. At the onset of these diseases, it is thought that targets for measurement in the differential diagnosis include acute disseminated encephalomyelitis, various myelitis, optic neuritis, CIDP, Guillain-Barré syndrome (GBS), Sjögren's syndrome, and other systemic autoimmune diseases, CNS disorders associated with vasculitis, acute cerebral infarction, and brain tumors.

[0044] [Table 1]

[0045] These diseases and their diagnostic and therapeutic significance are described below. If this antibody is positive at the initial onset of CNS demyelinating diseases (MS / NMOSD / ADEM / encephalomyelitis, etc.), including those of unknown etiology, it indicates a relapsing course rather than a monophasic course like acute disseminated encephalomyelitis, providing evidence for the introduction of immunotherapy to suppress relapses. MS affects approximately 3 million people worldwide and is on the rise, particularly in Europe and the United States. In recent years, the number of cases in Japan has also increased rapidly due to the modernization and Westernization of the environment. Therefore, the discovery and measurement of this antibody are extremely significant. During follow-up of MS, a positive antibody test indicates a high risk of PMS, providing evidence for early consideration of the possibility of PMS and the introduction of treatment.

[0046] Regarding the initial onset and recurrence of CNS demyelinating diseases, if the antibody is positive in CNS demyelinating diseases of unknown cause, they are likely to develop large lesions and severe damage, so it is necessary to administer sufficient immunotherapy from the early stages. If this antibody is positive in MS, it is thought that the disease is progressing or is likely to progress to PMS, so the selection of treatment drugs must be considered. NMOSD (especially seronegative NMOSD) in young patients who are antibody-positive is likely to progress to the course of seronegative NMOSD, including severe optic nerve damage and spinal cord damage, so sufficient immunotherapy must be administered from the early stages and continued for a long period of time. Furthermore, NMOSD is distributed worldwide, but is more common in Asians, especially Japanese. Although CCPD is rare, it is difficult to diagnose, so the discovery and measurement of this antibody is of great significance. Regarding optic neuritis, if the antibody is positive, there is a high possibility of recurrence and it will affect various parts of the CNS, so it is necessary to work with a neurologist from the early stages to introduce immunotherapy and follow up carefully.

[0047] In cases presenting with tumefactive brain lesions (brain tumor-like lesions), when brain tumors such as gliomas are suspected because tumefactive brain lesions are likely to occur, if this antibody is positive, the condition is due to an autoimmune mechanism and is therefore a candidate for immunotherapy. Regarding PNS demyelinating diseases (CIDP / GBS), if this antibody is positive in cases of peripheral nerve demyelinating diseases such as CIDP or Guillain-Barré syndrome (GBS), the disease may progress to a recurrent course and may develop CNS demyelinating lesions. Therefore, follow-up observation, including cerebrospinal MRI, is necessary, and this provides evidence for the introduction of immunotherapy to prevent recurrence. During follow-up of CNS / PNS demyelinating diseases, this antibody will be positive in cases of CCPD presenting with peripheral nerve hypertrophy. If the antibody is positive, the patient should be monitored closely for the occurrence of peripheral neuropathy, and sufficient immunotherapy should be initiated promptly before irreversible thickening of the peripheral nerves occurs.

[0048] Although the embodiment of the present invention has been described above, the present invention is not limited to the above embodiment and various modifications can be made. [Example]

[0049] The effects of the present invention will be made clearer by the following examples and comparative examples. Note that the present invention is not limited to the following examples, and can be practiced by making appropriate changes within the scope of the present invention.

[0050] (Test Example 1) (Searching for CNS nodule antibodies using immunohistochemical staining of mouse optic nerve tissue) The present inventors aimed to discover new CNS nodo antibodies in CNS demyelinating diseases such as MS / NMOSD / CCPD, which are anti-AQP4 antibody-negative and anti-MOG antibody-negative. First, to search for CNS nodo antibodies, we established a new tissue-based immunofluorescence assay (TBA) method using mouse optic nerves, which have a consistent nerve fiber course and make it easy to identify the nodes of Ranvier. While the TBA method has the disadvantage that optic nerve tissue is difficult to extract and only a small number of sections can be obtained, it has the advantage that the CNS is the only tissue with a consistent nerve fiber course, making it easy to identify the nodes of Ranvier. The present inventors developed and performed a method for injecting IgG into the mouse optic nerve to examine the in vivo effects of antibodies.

[0051] FIG. 1 is a photographic diagram showing an outline of the procedure for an in vivo experiment in which patient serum IgG was injected into the mouse optic nerve. (a) shows the overall view of the mouse restraint and treatment equipment, (b) shows the mouse being immobilized and a small incision made on the outer surface of the eyeball to expose the optic nerve, followed by the injection of a fixed amount of solution using a Hamilton syringe, and (c) is an enlarged view of the same.

[0052] Using this in vivo immunostaining and Western blot analysis of collected optic nerve protein lysate, we screened patient serum CNS nodule antibodies and antigens using mouse optic nerves. FIG. 2 is a photograph of immunostaining showing the binding of patient serum IgG to the juxta-paranodes in the optic nerve TBA. As shown in the rectangular areas in each figure, immunohistochemistry of the optic nerve showed that IgG (serum) from patients with SPMS (secondary progressive MS), PPMS (primary progressive MS), CCPD (combined central peripheral demyelination), and seronegative MMOSD (antibody-negative neuromyelitis optica spectrum disorder) showed localization corresponding to CASPR (node ​​of Ranvier marker).

[0053] Figure 3 is a photograph showing the results of Western blot (WB) of patient serum using protein lysate extracted from mouse optic nerves. In the figure, HC indicates control. Antibodies present in the sera of patients with SPMS, PPMS, CCPD, and NMOSD (two cases) all reacted to a 120-kD protein. This indicates the presence of antibodies against the 120-kD protein expressed in each of the above diseases, including atypical CNS demyelinating diseases and PMS. Regarding the IgG subclass, IgG3, which has complement activation ability and induces strong inflammation, was observed in all cases. These results revealed the presence of a novel autoantibody that reacts with a 120kD membrane protein at the paranode of Ranvier in patients with CNS demyelination, such as MS / NMOSD / CCPD, who are anti-AQP4 antibody-negative and anti-MOG antibody-negative.

[0054] (Test Example 2) (Identification of candidate antigens responsible for novel CNS throat antibodies) Using mouse optic nerve protein lysate, we searched for antigens that cross-react with each of the above antibodies. Figure 4 is a photograph showing the results of two-dimensional electrophoresis and Western blotting of mouse optic nerve protein lysate. (a) shows the results of two-dimensional electrophoresis, and (b) shows the results of Western blotting using the two-dimensional electrophoresis gel with the serum of the patient with PPMS. In the Western blot using the patient serum, a band corresponding to a molecular weight of 120 kD was identified as the antigen protein of interest.

[0055] The spot of this electrophoretic band was excised and subjected to liquid chromatography and mass spectrometry. The top three partial peptides with Mascot scores were identified as Na+ / K+ ATPase alpha subunit alpha 3 (ATP1A3): 544, alpha 1 (ATP1A1): 365, and alpha 2 (ATP1A2): 354. Of these three types of ATP1A, as mentioned above, it is already known that ATP1A3 is highly expressed in the axonal membrane at the nodes of Ranvier, and ATP1A2 is highly expressed in the glial cell membrane at the nodes of Ranvier. ATP1A2 is expressed in the cell membranes of oligodendroglia and astroglia, and ATP1A3 is expressed in the axonal membrane. They are proteins essential for maintaining the Na+ / K+ concentration gradient and axonal excitation. On the other hand, ATP1A1 is expressed in almost all cells. Therefore, we focused on ATP1A2 and ATP1A3, which are thought to be highly expressed at the nodes of Ranvier, as antigenic proteins related to the above-mentioned disease.

[0056] Next, the present inventors developed a cell-based assay (CBA) method using ATP1A3-transfected SKN cell line derived from human neuroblastoma. 5 is a photograph showing the results of immunostaining by the CBA method. Cells obtained by transfecting a human neuroblastoma-derived SKN cell line with ATP1A3 were subjected to immunostaining with ATP1A3 (GFP signal) and each of the patient sera. As shown in the figure, each patient's serum reacted with ATP1A3 expressed on the surface of SKN cells, but such a reaction was not observed in healthy subjects.

[0057] FIG. 6 is a photograph showing the results of Western blotting using ATP1A2 or ATP1A3 overexpression lysate prepared in HEK293 cells. Each signal is indicated by an arrow in the figure. In the antibody-positive cases, a faint band is observed due to cross-reaction with ATP1A1 present in the mock (untransfected HEK293 cells) itself. As shown in the figure, compared to the signal of the monoclonal ATP1A antibody (a), the patient sera (b)-(d) reacted more strongly with ATP1A2, followed by ATP1A3. In addition, they reacted weakly with ATP1A1, which is originally expressed in HEK293 cells. In other words, antibody-positive cases reacted more with ATP1A2 / 3 than with ATP1A1 (Mock). These reactions were not observed in the healthy subject (HC) (e). These results suggest that the patient serum recognizes the extracellular portion common to ATP1A2 and ATP1A3. The antibody positivity rates by CBA and Western blot (WB) are shown in Table 2. IgG subclass IgG3, which activates complement and causes strong inflammation, was observed in all cases.

[0058] [Table 2]

[0059] As shown in Table 1, in the CBA method for anti-ATP1A3 antibodies, 16 out of 38 patients with CNS demyelination, such as MS / NMOSD / CCPD, who were anti-AQP4 antibody-negative and anti-MOG antibody-negative (42%) were antibody-positive, whereas all 28 patients with other neurological diseases, such as various myelitis, and healthy controls were antibody-negative (42% vs. 0%, p < 0.001).

[0060] FIG. 7 shows a schematic diagram and photographs of typical demyelinating lesions in anti-ATP1A2 / 3 antibody-positive cases. FIG. 8 is a schematic diagram of anti-ATP1A2 / 3 antibody-positive cases. Anti-ATP1A2 / 3 antibody-positive cases showed characteristic clinical features, including (1) early onset (10-49 years old, 44% under 20 years old), (2) female predominance (81% female), (3) presence of optic neuropathy (88%, of which 50% bilateral), (4) presence of giant lesions (long spinal cord and long optic nerve lesions, tumefactive brain lesions, etc.) (50%), (5) primary disease type was early-onset NMOSD or CCPD (tumefactive brain lesions accompanied by peripheral nerve thickening) with predominant optic neuropathy such as total blindness, and middle-aged PMS, and (6) low rate of positivity of cerebrospinal fluid oligoclonal bands, characteristic of MS (36%).

[0061] Next, to examine the effect of the anti-ATP1A2 / 3 antibody, the in vivo IgG injection method into the mouse optic nerve used in Test Example 1 was used. 9 is a photograph showing the results of immunostaining following in vivo injection of patient serum IgG into mouse optic nerves. In a so-called passive transfer experiment, staining was performed with human IgG, antibodies to the node of Ranvier marker Caspr1, ATP1A1, and ATP1A3.

[0062] As shown in the figure, human IgG deposition at the nodes of Ranvier was observed in both NF155 antibody-positive CIDP patient IgG and ATP1A2 / 3 antibody-positive PPMS patient IgG, but not in healthy control IgG. As indicated by the arrow in the figure, Caspr1 shedding was more pronounced in ATP1A2 / 3 antibody-positive PPMS patient IgG. In ATP1A2 / 3 antibody-positive patient IgG, as indicated by the arrowhead in the figure, ATP1A2 was shedding, but ATP1A3 shedding was almost nonexistent. The reason for the greater shedding of ATP1A2 from the glial membrane than ATP1A3 from the axonal membrane is that antibodies have easier access to the glial cell membrane than the axonal membrane. Injection of serum IgG from a patient positive for anti-ATP1A2 / 3 antibodies resulted in the loss of ATP1A2 and disruption of the nodes of Ranvier, suggesting that these antibodies are pathogenic.

[0063] FIG. 10 is a schematic diagram of a study of each antibody and its target in demyelination. The results of passive transfer experiments into the optic nerve suggested that anti-ATP1A2 / 3 antibodies were pathogenic. The immunohistochemistry showed that ATP1A2 was more severely lost from glial membranes than from axonal membranes. This is thought to be because the nodes of Ranvier in the CNS are covered with astroglial and oligodendroglial processes, as shown in the figure, making glial membranes more accessible than axonal membranes.

[0064] Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Industrial Applicability]

[0065] According to the present invention, new CNS antibodies have been discovered for atypical CNS demyelinating diseases and PMS, which tend to become severe, and it is possible to provide biomarkers, screening methods, methods for detecting biomarkers, and methods for assisting in the diagnosis of demyelinating diseases that are useful for diagnosing atypical CNS demyelinating diseases and PMS, which have previously been difficult to diagnose, and determining treatment strategies.

Claims

1. A biomarker for diagnosing a demyelinating disease or determining the effectiveness of treatment, comprising an anti-ATP1A2 antibody or an anti-ATP1A3 antibody.

2. 2. The biomarker of claim 1, wherein the demyelinating disease is a demyelinating disease affecting the central nervous system (CNS) and a demyelinating disease affecting both the CNS and peripheral nervous system (PNS).

3. A screening method for detecting cross-reactivity of mouse optic nerve tissue with antibodies obtained from patients with demyelinating diseases, thereby identifying antigens related to demyelinating diseases.

4. The screening method according to claim 3, wherein an antibody against an antigen related to a demyelinating disease is used as a biomarker for diagnosing a demyelinating disease or determining the effectiveness of a treatment for the demyelinating disease.

5. The screening method according to claim 3, comprising the steps of injecting serum obtained from a patient with the demyelinating disease into the optic nerve of a mouse in vivo and detecting the crossover by tissue immunohistochemistry (TBA) method.

6. 4. The method of claim 3, further comprising detecting said cross-reactivity in vitro by Western blotting on an extract of mouse optic nerve tissue.

7. 7. The screening method according to claim 6, wherein the Western blotting method comprises subjecting an extract of the mouse optic nerve tissue to two-dimensional electrophoresis, followed by two-dimensional Western blotting.

8. A biomarker detection method in which ATP1A2 or ATP1A3 is expressed in cells derived from human neuroblastoma cells, a sample derived from a subject is added to the cultured cells, and cross-reactivity with the ATP1A2 or ATP1A3 is detected by a cell-based assay (CBA) method to determine whether the sample contains an anti-ATP1A3 antibody.

9. The method for detecting a biomarker according to claim 8, wherein the cells are SKN cells (human neuroblastoma-derived cells).

10. The method for detecting a biomarker according to claim 8, wherein the sample is serum obtained from the subject or an extract thereof.

11. A biomarker detection method for detecting whether a sample derived from a subject contains a biomarker for diagnosing a demyelinating disease or determining a therapeutic effect, comprising: A method for detecting biomarkers, comprising detecting cross-reactivity between the sample and an extract of cells or tissues in which ATP1A2 or ATP1A3 is overexpressed by Western blotting, and determining whether the sample contains anti-ATP1A2 antibodies or anti-ATP1A3 antibodies.

12. The method for detecting a biomarker according to claim 11, wherein the cell or tissue extract is a HEK293 (human embryonic kidney cell) extract.

13. The method for detecting a biomarker according to claim 11, wherein the sample is serum obtained from the subject or an extract thereof.

14. measuring anti-ATP1A2 antibodies or anti-ATP1A3 antibodies contained in a sample derived from a subject; A method for assisting in the diagnosis of a demyelinating disease, wherein a significantly higher abundance of the anti-ATP1A2 antibody or anti-ATP1A3 antibody compared to its abundance in healthy individuals indicates that the subject is suffering from a demyelinating disease.

15. 15. The method for assisting in the diagnosis of a demyelinating disease according to claim 14, wherein the demyelinating disease is a demyelinating disease affecting the central nervous system (CNS) or a demyelinating disease affecting both the CNS and peripheral nervous system (PNS).

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